Patrice Codogno

Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/Beclin 1-1, Atg10, or Atg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways.

The lysosomal degradation pathway known as autophagy has an essential homeostatic role in controlling the quality of the cytoplasm. However, this pathway has also been implicated in the pathology of an array of human disorders. Here, Rubinsztein and colleagues provide an overview of the mechanisms and regulation of autophagy, discuss the role of this pathway in disease and highlight potential strategies for therapeutic modulation. Autophagy is an essential, conserved lysosomal degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles. It begins when double-membraned autophagosomes engulf portions of the cytoplasm, which is followed by fusion of these vesicles with lysosomes and degradation of the autophagic contents. In addition to its vital homeostatic role, this degradation pathway is involved in various human disorders, including metabolic conditions, neurodegenerative diseases, cancers and infectious diseases. This article provides an overview of the mechanisms and regulation of autophagy, the role of this pathway in disease and strategies for therapeutic modulation.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997)Eur. J. Biochem. 243, 240–246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3,4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4,5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC50 5–15 nm). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Gαi3 protein suggesting that both class III PI3K and trimeric Gi3 protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling. 3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997)Eur. J. Biochem. 243, 240–246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3,4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4,5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC50 5–15 nm). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Gαi3 protein suggesting that both class III PI3K and trimeric Gi3 protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling. Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells.Journal of Biological ChemistryVol. 275Issue 16PreviewPage 994, Fig. 2, panel B: The quantitity of IL-13 used is indicated in molarity (nm) instead of concentration (ng/ml). It should read “[IL-13] ng/ml.” Full-Text PDF Open Access phosphatidylinositol 3-kinase dipalmitoyl phosphatidylinositol 3-phosphate dipalmitoyl phosphatidylinositol 4-phosphate 4)P2, dipalmitoyl phosphatidylinositol 3,4-bisphosphate 5)P2, dipalmitoyl phosphatidylinositol 4,5-bisphosphate 4,5)P3, dipalmitoyl phosphatidylinositol 3,4,5-triphosphate interleukin-13 lactate dehydrogenase 3-methyladenine protein kinase B, PtdIns(3)P, phosphatidylinositol 3-phosphate phosphatidylinositol 4-phosphate 4)P2, phosphatidylinositol 3,4-bisphosphate 5)P2, phosphatidylinositol 4,5-bisphosphate 4,5)P3, phosphatidylinositol 3,4,5-triphosphate wortmannin reverse transcriptase-polymerase chain reaction Src homology domain 2 Macroautophagy is a major intralysosomal catabolic process conserved during the evolution of eukaryotic cells (1.Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 2.Seglen P.O. Bohley P. 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Cell Res. 1995; 221: 504-519Crossref PubMed Scopus (116) Google Scholar and references therein). After acidification the early autophagosome is transformed in a late autophagosome. Fusion between late autophagosomes and lysosomes triggers the degradation of sequestered material into autolysosomes. Material from the endocytic pathway can be imported in autophagic vacuoles at different steps of their maturation (7.Liou W. Geuze H.J. Geelen M.J.H. Slot J.W. J. Cell Biol. 1997; 136: 61-70Crossref PubMed Scopus (227) Google Scholar, 8.Berg T.O. Fengsrud M. Stromhaug P.E. Berg T. Seglen P.O. J. Biol. Chem. 1998; 273: 21883-21892Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Recently, studies have shed light on the intracellular mechanisms that control membrane flow along the autophagic/lysosomal pathway in mammalian cells (reviewed in Refs. 9.Blommaart E.F.C. Luiken J.J.F.P. Meijer A.J. Histochem. J. 1997; 29: 365-385Crossref PubMed Scopus (217) Google Scholar and 10.Codogno P. Ogier-Denis E. Houri J.J. Cell. Signal. 1997; 9: 125-130Crossref PubMed Scopus (36) Google Scholar). It has been suggested that the phosphoinositide 3-kinase (PI3K)1 signaling cascade controls macroautophagy (11.Blommaart E.F.C. Krause U. Schellens J.P.M. Vreeling-Sindelárová H. Meijer A.J. Eur. J. Biochem. 1997; 243: 240-246Crossref PubMed Scopus (741) Google Scholar) and that 3-MA, an inhibitor of macroautophagic sequestration (12.Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1889-1892Crossref PubMed Scopus (1199) Google Scholar), behaves as a PI3K inhibitor. PI3K belongs to a family of enzymes that phosphorylates the 3′-hydroxyl group on the inositol ring of phosphoinositides (reviewed in Refs.13.Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 14.Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1235) Google Scholar, 15.De Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (664) Google Scholar, 16.Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1329) Google Scholar). These phospholipids are involved in a large array of signal transduction pathways controlling mitogenic responses, differentiation, apoptosis, cytoskeletal organization, and membrane flow along the secretory and endocytic pathways (reviewed in Ref. 17.Martin T.J.F. Annu. Rev. Cell Dev. Biol. 1998; 14: 231-264Crossref PubMed Scopus (453) Google Scholar). PI3Ks are classified into three classes. Class I enzymes are composed of catalytic p110 subunits and p85 adaptors. The SH2 motif contained in p85 adaptors bind to phosphorylated Tyr residues thereby linking the catalytic subunit to the Tyr kinase signaling pathway (class IA). A member of the class I PI3K family (class IB, p110γ/p101) is activated by subunits of trimeric G proteins (18.Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sterweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 19.Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-692Crossref PubMed Scopus (643) Google Scholar). Class I PI3Ks phosphorylate PtdIns, PtdIns(4)P, PtdIns(4,5)P2 but in vivo PtdIns(4,5)P2 is likely to be the favorite substrate. Class II enzymes are large enzymes (>200 kDa) characterized by a C-terminal containing a C2 domain (20.MacDouglas L.K. Domin J. Waterfield M.D. Curr. Biol. 1995; 5995: 1404-1414Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 21.Molz L. Chen Y.W. Hirano M. Williams L.T. J. Biol. 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The complex Vps15p·Vps34p is of fundamental importance in controlling vesicular transport to the yeast vacuole (reviewed in Ref. 26.Wurmser A.E. Gary J.D. Emr S.D. J. Biol. Chem. 1999; 274: 9129-9132Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The human homologue of Vps34p has been shown to be associated with a p150 myristylated protein kinase (27.Panaretou C. Domin J. Cockcroft S. Waterfield M.D. J. Biol. Chem. 1997; 272: 2477-2485Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The goal of the present study was to identify PI3Ks involved in the control of macroautophagy. For this purpose we have used the human colon cancer HT-29 cells which have been demonstrated to be an appropriate model to investigate the signaling pathway that controls macroautophagy (28.Ogier-Denis E. Couvineau A. Maoret J.J. Houri J.J. Bauvy C. De Stefanis D. Isidoro C. Laburthe M. Codogno P. J. Biol. Chem. 1995; 270: 13-16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29.Petiot A. Ogier-Denis E. Bauvy C. Cluzeaud F. Vandewalle A. Codogno P. Biochem. J. 1999; 337: 289-295Crossref PubMed Google Scholar). In the present study we show that macroautophagic sequestration is distinctly controlled by class I and class III PI3K. Stimulation of class I PI3K activity inhibits macroautophagy at the sequestration step whereas the activity of class III PI3K is required to trigger autophagic sequestration. HT-29 and Q204L-expressing cells were cultured as described previously (30.Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Cells were maintained at 37 °C under 10% CO2/air atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin (100 units/ml). 3-MA, phosphoinositides (PtdIns, PtdIns(4)P, and PtdIns(4,5)P2), NADH, metrizamide, and other chemicals were purchased from Sigma. Adenosine was from ICN, histone 2B was from Roche Molecular Biochemicals (Germany). Enzymes, synthetic oligonucleotides, cell culture reagents such as, Geneticin (G418) and LipofectAMINE were from Life Technologies, Inc. (Eragny, France). Nitrocellulose membranes were from Schleicher and Schuell (Dassel, Germany). The phosphoinositides PtdIns(3,4)P2, PtdIns(3,4,5)P3, and rabbit antibodies against p85 and p110α subunits of the class I PI3K were from TEBU (Le Perray en Yvelines, France). Goat anti-PKB polyclonal antibody was from Santa Cruz Biotechnology. The bicinchoninic acid (BCA) kit was from Pierce (Rockford, IL). IL-13 and cDNA encoding for the p150 adaptor were kindly provided by A. Minty (Sanofi Elf BioRecherche, France) and Dr. M. D. Waterfield and C. Panaretou (Ludwig Institute for Cancer Research, London), respectively. Rediprime random primer labeling kit, N+-Hybond membranes, and the radioisotopes [γ-32P]ATP (specific activity 10 mCi/mmol), [32P]dCTP (specific activity 10 mCi/mmol), andl-[U-14C]valine (specific activity 288.5 mCi/mmol) were from Amersham Pharmacia Biotech (Les Ulis, France). Cells were gently washed three times with phosphate-buffered saline (pH 7.4) and then twice with homogenization buffer (50 mm potassium phosphate, pH 7.5, 1 mm phenylmethylsulfonyl fluoride, 300 mmsucrose, 100 μg/ml bovine serum albumin, 0.01% Tween 20). Cells were homogenized in cold homogenization buffer by 13 strokes in a glass/Teflon homogenizer on ice. A post-nuclear supernatant was prepared by centrifugation at 300 × g for 10 min at 4 °C. Post-nuclear material was layered on the top of a 4-ml density cushion of buffered metrizamide/sucrose (10% sucrose, 8% metrizamide, 1 mm EDTA, 100 μg/ml bovine serum albumin, 0.01% Tween 20, pH 7.5) and centrifuged at 7,000 × g for 60 min (31.Kopitz J. Kisen G.Ø. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (201) Google Scholar). Finally, the pellet was washed once with homogenization buffer and resuspended in buffer containing 2 mm Tris-HCl (pH 7.4), 50 mm mannitol, 1 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml aprotinin, and 0.7 μg/ml pepstatin. The suspension was sonicated (VibraCell sonicator, model 72434; power setting 3, microtip, for 20 s at 20% charge) and centrifuged for 10 min at 10,000 × g at 4 °C. The LDH activity was determined by measuring the oxidation of NADH with pyruvate as substrate at 340 nm. When used, WT, LY294002, synthetic lipids, and IL-13 were exposed to the cells for a period of 4 h. In order to maintain PI3K inhibition by WT, fresh WT was added every hour for a total incubation time of 4 h. Protein degradation was determined as previously reported (30.Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). HT-29 cells were incubated for 18 h at 37 °C with 0.2 μCi/ml of l-[14C]valine. Unincorporated radioisotope was removed by three rinses with phosphate-buffered saline (pH 7.4). Cells were then incubated in nutrient-free medium (without amino acids and in absence of fetal calf serum) supplemented with 0.1% bovine serum albumin and 10 mm cold valine throughout the chase period. When required 3-MA (10 mm) was added at the beginning of the chase period. After the first hour of incubation, at which time short-lived proteins were being degraded, the chase medium was replaced with the appropriate fresh medium and the incubation continued for an additional 4 h. Cells were scraped into 0.5 ml of phosphate-buffered saline and the radiolabeled proteins present in the 4-h media and cells were precipitated with 10% trichloroacetic acid, 1% phosphotungstic acid (v/v) at 4 °C. The precipitated proteins were separated from the soluble radioactivity by centrifugation at 600 × g for 10 min then dissolved in 1 ml of Soluene 350 (Packard) and the associated radioactivity was measured. The rate of protein degradation was calculated as acid soluble radioactivity recovered from both cells and medium. When used, WT, LY294002, synthetic lipids, and IL-13, were added at the beginning of the chase period. In order to maintain PI3K inhibition by WT, we added fresh WT every hour for a total incubation time of 4 h. PKB activity was determined after immunoprecipitation of HT-29 cell lysates with the goat anti-PKB polyclonal antibody (1/250) using histone 2B as substrate as described previously (32.Franke T.F. Yang S.-I. Chang T.O. Datta K. Kazlauskas A. Morrison D. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1843) Google Scholar). Briefly, cells grown on 25-cm2 plates were washed once in cold phosphate-buffered saline (pH 7.4) and lysed with 1 ml of lysis buffer (1% Triton X-100, 10% glycerol, 137 mmNaCl, 20 mm Tris-HCl, pH 7.5, 10 μg/ml aprotinin, and leupeptin, 1 mm phenylmethylsulfonyl fluoride, 20 mm sodium fluoride, 1 mm disodium pyrophosphate, and 1 mm Na3VO4). After preclearing, lysates were immunoprecipitated with goat anti-PKB polyclonal antibody using binding protein A-Sepharose to sediment the complexes. After three washes with kinase buffer (20 mmHepes, pH 7.4, 1 mm MgCl2, 1 mmMnCl2), reactions were performed for 30 min at 25 °C under continuous agitation in kinase buffer containing 0.05 mg/ml histone 2B, 5 μm ATP, 1 mm dithiothreitol, and 10 μCi of [γ-32P]ATP. The products of the kinase reactions were separated on 15% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and exposed. Resulting autoradiograms were analyzed. When necessary the same membranes were analyzed subsequently by Western blot using goat anti-PBK to visualize the endogenous protein. Bands were developed by an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) using secondary antibody coupled to horseradish peroxidase. The p110α, p85, p150, and villin probes were 32P-labeled to a specific activity of 109 cpm/μg by random priming. Hybridization was performed at 45 °C in the presence of formamide and high stringency washes were performed in 0.1 × SSC, 0.1% SDS at 60 °C. cDNA was synthesized from mRNA isolated from HT-29 cells and was used to amplify a full-length cDNA encoding p110α and class III PI3K subunits, using RT-PCR. The forward PCR primers were: 5′-GACACTATTGTGTAACTATGGGGG-3′ for p110α, 5′-GATCTCTGTACCTTCTTGATATGGAG-3′ for class III PI3K, and 5′-GCAGATGGTTTCTGTAG-3′ for p150. The reverse PCR primers were: 5′-GTGTTAGGATATCTTGGGGTAAA-3′ for p110α, 5′-TTTGAAGAATAAGTTGATCTTGAGG-3′ for class III PI3K, and 5′-GAATGGAGGGGTACTGATGC-3′ for p150. cDNA (1 μg/ml) encoding the p150 was introduced into exponentially growing HT-29 cells by the LipofectAMINE method. Following transfection (48 h), cells were used for different experiments. Phosphorothioate oligonucleotide was synthesized so as to be anticomplementary to the sequence of class III PI3K (5′-TCCCCCCATCGCACCGTCTGC-3′) (33.Siddhanta U. McIlroy J. Shah A. Zhang Y.T. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Crossref PubMed Scopus (139) Google Scholar). Class III PI3K antisense was introduced into exponentially growing HT-29 cells by the LipofectAMINE method. Following transfection cells were used. Synthetic phosphatidylinositide phosphate (0.1 mg/ml) and phosphatidylserine (0.1 mg/ml) were solubilized in a mixture of chloroform/methanol, 1:1 (v:v), dried under N2, and dispersed by sonication for 15 min at 20 °C in buffer containing 25 mm HEPES (pH 7.4) and 1 mmEDTA. Samples were centrifuged at 6000 × g for 15 min at 4 °C and the supernatant was added at cells for an incubation of 4 h in a nutrient-free medium. PI3K activity was determined by the modified method of Burgering et al. (34.Burgering B.M. Medema R.H. van de Wetering M.L. van der Eb A.J. McCormick F. Bos J.L. EMBO J. 1991; 10: 1103-1109Crossref PubMed Scopus (215) Google Scholar). Briefly, immunoprecipitates and the supernatant of the immunoprecipitation were resuspended in 80 μl of buffer containing 30 mm Hepes and 300 μm adenosine. To both samples, 50 μl of lipid mixture (synthetic lipid and phosphatidylserine) was added for 20 min. The assay of class I PI3K activity was performed on the immunoprecipitate using diC16PtdIns(4,5)P2 as substrate in the presence of Mg2+, whereas class III PI3K activity was performed on the supernatant of a two rounds immunoprecipitation, using phosphatidylinositol as substrate in the presence of Mn2+(18.Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sterweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (552) Google Scholar). The reaction was initiated by the addition of 20 μCi of [γ-32P]ATP, 100 μm ATP, 100 μm MgCl2+, or MnCl2+ and terminated after 15 min by the addition of 80 μl of 1 mHCl and 200 μl of chloroform/methanol, 1:1 (v:v). After vigorous mixing and centrifugation, the organic layer was removed, dried under N2, and resuspended in chloroform/methanol. Extracted lipids were separated by TLC in developing solvents: isopropyl alcohol/H2O/acetic acid, 65:34:1 (v:v:v), and then exposed to hyperfilm. Unlabeled standards (1 mg/ml) were revealed by exposure to iodine vapor. Lipids were identified by comparison with unlabeled standards. Recently it has been shown that the PI3K inhibitors WT and LY294002 stop the macroautophagic pathway at the sequestration step in rat hepatocytes (11.Blommaart E.F.C. Krause U. Schellens J.P.M. Vreeling-Sindelárová H. Meijer A.J. Eur. J. Biochem. 1997; 243: 240-246Crossref PubMed Scopus (741) Google Scholar). In order to investigate the role of PI3K in the control of macroautophagy in HT-29 cells, we have studied the effect of WT and LY294002 on the degradation of [14C]valine-labeled long-lived proteins (Fig. 1) and on the rate of sequestration of the cytosolic enzyme LDH into autophagic vacuoles (Table I). WT and LY294002 impaired protein degradation in a dose-dependent manner with IC50 values in the range of those previously reported for rat hepatocytes (11.Blommaart E.F.C. Krause U. Schellens J.P.M. Vreeling-Sindelárová H. Meijer A.J. Eur. J. Biochem. 1997; 243: 240-246Crossref PubMed Scopus (741) Google Scholar). Results reported in Table I show that 10 nm WT and 10 μm LY294002 are as potent as 10 mm 3-MA as inhibitors of autophagic sequestration of the cytosolic enzyme LDH.Table IAutophagic sequestration in HT-29 cells treated with different drugsTreatmentAutophagic sequestration of LDH%/hUntreated cells4.0 ± 0.6aValues are the mean ± (S.D.) (n= 5).3-MA (10 mm)1.6 ± 0.3WT (10 nm)1.7 ± 0.3LY294002 (10 μm)1.5 ± 0.2IL-13 (10 nm)2.0 ± 0.2PtdIns(3)P (10 μm)6.5 ± 0.7PtdIns(4)P (10 μm)3.5 ± 0.2PtdIns(3,4)P2 (10 μm)2.0 ± 0.1PtdIns(4,5)P2 (10 μm)3.7 ± 0.5PtdIns(3,4,5)P3 (10 μm)2.0 ± 0.2a Values are the mean ± (S.D.) (n= 5). Open table in a new tab In order to explore the mechanism by which PI3K controls the macroautophagic pathway, we have first examined the effect of PI3K products administered to HT-29 cells in liposome form using phosphatidylserine as a carrier (see “Experimental Procedures”). Results reported in Fig.2 A, show that diC16PtdIns(3,4)P2 and diC16PtdIns(3,4,5)P3inhibited the degradation of long-lived proteins in a dose-dependent manner. In contrast, diC16PtdIns(4,5)P2 had no inhibitory effect on protein degradation over the concentration range used. The inhibitory effect of diC16PtdIns(3,4)P2 and diC16PtdIns(3,4,5)P3 on the macroautophagic pathway was confirmed by the assay of the sequestration of the cytosolic enzyme LDH (Table I). The relevance of the effect induced by the synthetic lipids in a cellular environment was studied by measuring the activity of the kinase Akt/PKB which is a well established target of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (35.Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1898) Google Scholar, 36.Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 37.Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1318) Google Scholar). A stimulation of Akt/PKB activity using the histone 2B as a substrate was observed in cells loaded with diC16PtdIns(3,4)P2 and diC16PtdIns(3,4,5)P3 (Fig.3). In contrast, no significant stimulation of PKB activity was detected in cells fed with diC16PtdIns(4,5)P2. From the results reported above we reasoned that an increase in PtdIns(3,4)P2 and PtdIns(3,4,5)P3 by stimulating the HT-29 cells PI3K activity should lead to an effect similar to that obtained with synthetic diC16PtdIns(3,4)P2, and diC16PtdIns(3,4,5)P3. It has been demonstrated that the pleiotropic cytokine IL-13 stimulates PI3K activity and the production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in HT-29 cells (38.Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). As shown in Fig. 2 B and Table I, IL-13 inhibited the macroautophagic pathway in HT-29 cells. Accordingly stimulation of the PI3K activity by IL-13 (Ref. 38.Wright K. Ward S.G. Kolios G. Westwick J. J. Biol. Chem. 1997; 272: 12626-12633Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar and data not shown) was associated with the activation of Akt/PKB (Fig. 3). The bulk of these results seem contradictory because inhibitors and products of PI3K have a similar inhibitory effect on the macroautophagic pathway. However, several classes of PI3K producing different types of phosphoinositides are expressed in the same cell. Thus we next investigated the expression of class I and class III PI3Ks in HT-29 cells. We focused our study on these two families because the class II PI3K does not produce PtdIns(3,4,5)P3 from PtdIns(4,5)P2 and the widely expressed class II isoform PI3K-C2α is poorly sensitive to WT (13.Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 22.Virbasius J.V. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 13304-13307Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 23.Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar). These characteristics make the involvement of class II PI3K in the control of macroautophagy unlikely. RT-PCR and Northern blot analyses were used to characterize the different classes of PI3K present in HT-29 cells (Fig.4 A). Two different mRNAs of catalytic subunits of PI3K were detected corresponding to the class I p110α and the class III PI3K, respectively. The presence of other class I PI3K catalytic subunits was not considered. Class I and class III PI3Ks were also identified by their enzymatic activities (Fig. 4 B). After specific immunoprecipitation of the class I PI3K from HT-29 homogenates enzymatic activities were determined in the pellet (class I activity) and supernatant (class III activity). Using PtdIns(4,5)P2 as a substrate we observed production of PtdIns(3,4,5)P3 in the pellet derived from IL-13-treated cells whereas the basal activity of class I PI3K was very low in unstimulated HT-29 cells (Fig. 4 B). In the supernatant, using PtdIns as a substrate, we observed the production of PtdIns(3)P when Mn2+ was present in the assay whereas the activity was barely detectable when Mg2+ was used instead of Mn2+ (data not shown). This cation dependence has been shown to be a hallmark of class III PI3K (39.Volinia S. Dhand R. Vanhaesebroeck B.S. MacDougall L.K. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (309) Google Scholar). Furthermore, and in contrast to the class I PI3K, the activity of class III PI3K was not dependent on IL-13 treatment. In order to determine the role of class III PI3K in the control of macroautophagy, we have tested the effect of PtdIns(3)P, the only product of class III PI3K (24.Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (849) Google Scholar, 39.Volinia S. Dhand R. Vanhaesebroeck B.S. MacDougall L.K. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (309) Google Scholar). For this purpose, autophagic parameters were determined in cells fed with diC16PtdIns(3)P (Fig.5 and Table I). Results reported on Fig.5 A show that diC16PtdIns(3)P-stimulated protein degradation in a dose-dependent manner whereas its isomer diC16PtdIns(4)P had no effect on protein degradation. According to these data diC16PtdIns(3)P but not diC16PtdIns (4.Mortimore G.E. Kadowaki M. Ciechanover A.J. Schwartz A.L. Cellular Proteolytic Systems. Wiley-Liss, New York1994: 65-87Google Scholar)P also s

Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Autophagy maintains cell, tissue and organism homeostasis through degradation. Complex post-translational modulation of the Atg (autophagy-related) proteins adds additional entry points for crosstalk with other cellular processes and helps define cell-type-specific regulations of autophagy. Beyond the simplistic view of a process exclusively dedicated to the turnover of cellular components, recent data have uncovered unexpected functions for autophagy and the autophagy-related genes, such as regulation of metabolism, membrane transport and modulation of host defenses--indicating the novel frontiers lying ahead.

Macroautophagy is a vacuolar, self-digesting mechanism responsible for the removal of long-lived proteins and damaged organelles by the lysosome. The discovery of the ATG genes has provided key information about the formation of the autophagosome, and about the role of macroautophagy in allowing cells to survive during nutrient depletion and/or in the absence of growth factors. Two connected signaling pathways encompassing class-I phosphatidylinositol 3-kinase and (mammalian) target of rapamycin play a central role in controlling macroautophagy in response to starvation. However, a considerable body of literature reports that macroautophagy is also a cell death mechanism that can occur either in the absence of detectable signs of apoptosis (via autophagic cell death) or concomitantly with apoptosis. Macroautophagy is activated by signaling pathways that also control apoptosis. The aim of this review is to discuss the signaling pathways that control macroautophagy during cell survival and cell death.

Review23 February 2015free access Autophagy in malignant transformation and cancer progression Lorenzo Galluzzi Corresponding Author Lorenzo Galluzzi Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Federico Pietrocola Federico Pietrocola Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author Ravi K Amaravadi Ravi K Amaravadi Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Eric H Baehrecke Eric H Baehrecke Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Francesco Cecconi Francesco Cecconi Cell Stress and Survival Unit, Danish Cancer Society Research Center, Copenhagen, Denmark IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Patrice Codogno Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France Institut Necker Enfants-Malades (INEM), Paris, France INSERM, U1151, Paris, France CNRS, UMR8253, Paris, France Search for more papers by this author Jayanta Debnath Jayanta Debnath Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author David A Gewirtz David A Gewirtz Department of Pharmacology, Toxicology and Medicine, Virginia Commonwealth University, Richmond, Virginia, VA, USA Search for more papers by this author Vassiliki Karantza Vassiliki Karantza Merck Research Laboratories, Rahway, NJ, USA Search for more papers by this author Alec Kimmelman Alec Kimmelman Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Sharad Kumar Sharad Kumar Centre for Cancer Biology, University of South Australia, Adelaide, SA, Australia Search for more papers by this author Beth Levine Beth Levine Center for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Maria Chiara Maiuri Maria Chiara Maiuri Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author Seamus J Martin Seamus J Martin Department of Genetics, Trinity College, The Smurfit Institute, Dublin, Ireland Search for more papers by this author Josef Penninger Josef Penninger Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Mauro Piacentini Mauro Piacentini Department of Biology, University of Rome Tor Vergata, Rome, Italy National Institute for Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy Search for more papers by this author David C Rubinsztein David C Rubinsztein Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Hans-Uwe Simon Hans-Uwe Simon Institute of Pharmacology, University of Bern, Bern, Switzerland Search for more papers by this author Anne Simonsen Anne Simonsen Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Search for more papers by this author Andrew M Thorburn Andrew M Thorburn Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA Search for more papers by this author Guillermo Velasco Guillermo Velasco Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University of Madrid, Madrid, Spain Instituto de Investigaciones Sanitarias San Carlos (IdISSC), Madrid, Spain Search for more papers by this author Kevin M Ryan Kevin M Ryan Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Guido Kroemer Guido Kroemer Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author Lorenzo Galluzzi Corresponding Author Lorenzo Galluzzi Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Federico Pietrocola Federico Pietrocola Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author Ravi K Amaravadi Ravi K Amaravadi Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Eric H Baehrecke Eric H Baehrecke Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Francesco Cecconi Francesco Cecconi Cell Stress and Survival Unit, Danish Cancer Society Research Center, Copenhagen, Denmark IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Patrice Codogno Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France Institut Necker Enfants-Malades (INEM), Paris, France INSERM, U1151, Paris, France CNRS, UMR8253, Paris, France Search for more papers by this author Jayanta Debnath Jayanta Debnath Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author David A Gewirtz David A Gewirtz Department of Pharmacology, Toxicology and Medicine, Virginia Commonwealth University, Richmond, Virginia, VA, USA Search for more papers by this author Vassiliki Karantza Vassiliki Karantza Merck Research Laboratories, Rahway, NJ, USA Search for more papers by this author Alec Kimmelman Alec Kimmelman Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Sharad Kumar Sharad Kumar Centre for Cancer Biology, University of South Australia, Adelaide, SA, Australia Search for more papers by this author Beth Levine Beth Levine Center for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Maria Chiara Maiuri Maria Chiara Maiuri Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author Seamus J Martin Seamus J Martin Department of Genetics, Trinity College, The Smurfit Institute, Dublin, Ireland Search for more papers by this author Josef Penninger Josef Penninger Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Mauro Piacentini Mauro Piacentini Department of Biology, University of Rome Tor Vergata, Rome, Italy National Institute for Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy Search for more papers by this author David C Rubinsztein David C Rubinsztein Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Hans-Uwe Simon Hans-Uwe Simon Institute of Pharmacology, University of Bern, Bern, Switzerland Search for more papers by this author Anne Simonsen Anne Simonsen Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Search for more papers by this author Andrew M Thorburn Andrew M Thorburn Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA Search for more papers by this author Guillermo Velasco Guillermo Velasco Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University of Madrid, Madrid, Spain Instituto de Investigaciones Sanitarias San Carlos (IdISSC), Madrid, Spain Search for more papers by this author Kevin M Ryan Kevin M Ryan Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Guido Kroemer Guido Kroemer Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Search for more papers by this author Author Information Lorenzo Galluzzi 1,2,3,4,‡,‡, Federico Pietrocola1,2,3,‡, José Manuel Bravo-San Pedro1,2,3,‡, Ravi K Amaravadi5, Eric H Baehrecke6, Francesco Cecconi7,8, Patrice Codogno4,9,10,11, Jayanta Debnath12, David A Gewirtz13, Vassiliki Karantza14, Alec Kimmelman15, Sharad Kumar16, Beth Levine17,18,19, Maria Chiara Maiuri1,2,3, Seamus J Martin20, Josef Penninger21, Mauro Piacentini22,23, David C Rubinsztein24, Hans-Uwe Simon25, Anne Simonsen26, Andrew M Thorburn27, Guillermo Velasco28,29, Kevin M Ryan30,‡ and Guido Kroemer1,2,4,31,32,‡ 1Equipe 11 labellisée pas la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 2INSERM, U1138, Paris, France 3Gustave Roussy Cancer Campus, Villejuif, France 4Université Paris Descartes, Sorbonne Paris Cité, Paris, France 5Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA 6Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA 7Cell Stress and Survival Unit, Danish Cancer Society Research Center, Copenhagen, Denmark 8IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome Tor Vergata, Rome, Italy 9Institut Necker Enfants-Malades (INEM), Paris, France 10INSERM, U1151, Paris, France 11CNRS, UMR8253, Paris, France 12Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA 13Department of Pharmacology, Toxicology and Medicine, Virginia Commonwealth University, Richmond, Virginia, VA, USA 14Merck Research Laboratories, Rahway, NJ, USA 15Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 16Centre for Cancer Biology, University of South Australia, Adelaide, SA, Australia 17Center for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 18Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA 19Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA 20Department of Genetics, Trinity College, The Smurfit Institute, Dublin, Ireland 21Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria 22Department of Biology, University of Rome Tor Vergata, Rome, Italy 23National Institute for Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy 24Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK 25Institute of Pharmacology, University of Bern, Bern, Switzerland 26Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 27Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA 28Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University of Madrid, Madrid, Spain 29Instituto de Investigaciones Sanitarias San Carlos (IdISSC), Madrid, Spain 30Cancer Research UK Beatson Institute, Glasgow, UK 31Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 32Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France ‡These authors contributed equally to this work ‡These authors share senior co-authorship *Corresponding author. Tel: +33 1 44277661; E-mail: [email protected] The EMBO Journal (2015)34:856-880https://doi.org/10.15252/embj.201490784 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy. Introduction Macroautophagy (herein referred to as autophagy) is a mechanism that mediates the sequestration of intracellular entities within double-membraned vesicles, so-called autophagosomes, and their delivery to lysosomes for bulk degradation (He & Klionsky, 2009). Autophagosomes derive from so-called phagophores, membranous structures also known as ‘isolation membranes’ whose precise origin remains a matter of debate (Lamb et al, 2013). Indeed, the plasma membrane, endoplasmic reticulum (ER), Golgi apparatus, ER-Golgi intermediate compartment (ERGIC), and mitochondria have all been indicated as possible sources for phagophores (Lamb et al, 2013). Upon closure, autophagosomes fuse with lysosomes, forming so-called autolysosomes, and their cargo is exposed to the catalytic activity of lysosomal hydrolases (Mizushima & Komatsu, 2011). The degradation products of the autophagosomal cargo, which includes sugars, nucleosides/nucleotides, amino acids and fatty acids, can be transported back to the cytoplasm and presumably re-enter cellular metabolism (Fig 1) (Rabinowitz & White, 2010; Galluzzi et al, 2013). Of note, the molecular machinery that mediates autophagy is evolutionary conserved, and several components thereof have initially been characterized in yeast (He & Klionsky, 2009). Figure 1. General organization of autophagic responsesAutophagy initiates with the progressive segregation of cytoplasmic material by double-membraned structures commonly known as phagophores or isolation membranes. Phagophores nucleate from the endoplasmic reticulum (ER), but several other membranous organelles have been shown to contribute to their elongation, including the Golgi apparatus, ER-Golgi intermediate compartment (ERGIC), plasma membrane, mitochondria and recycling endosomes. Completely sealed phagophores, which are known as autophagosomes, fuse with lysosomes to form autolysosomes. This promotes the activation of lysosomal hydrolases and hence causes the breakdown of the autophagosomal cargo. The products of these catabolic reactions reach the cytosol via transporters of the lysosomal membrane and are recycled by anabolic or bioenergetic circuitries. Download figure Download PowerPoint In physiological scenarios, autophagy proceeds at basal levels, ensuring the continuous removal of superfluous, ectopic or damaged (and hence potentially dangerous) entities, including organelles and/or portions thereof (Green et al, 2011). Baseline autophagy mediates a key homeostatic function, constantly operating as an intracellular quality control system (Mizushima et al, 2008; Green et al, 2011). Moreover, the autophagic flux can be upregulated in response to a wide panel of stimuli, including (but not limited to) nutritional, metabolic, oxidative, pathogenic, genotoxic and proteotoxic cues (Kroemer et al, 2010). Often, stimulus-induced autophagy underlies and sustains an adaptive response to stress with cytoprotective functions (Kroemer et al, 2010; Mizushima & Komatsu, 2011). Indeed, the pharmacological or genetic inhibition of autophagy generally limits the ability of cells to cope with stress and restore homeostasis (Mizushima et al, 2008; Kroemer et al, 2010). This said, regulated instances of cell death that causally depend on the autophagic machinery have been described (Denton et al, 2009; Denton et al, 2012b; Liu et al, 2013b; Galluzzi et al, 2015). The detailed discussion of such forms of autophagic cell death, however, is beyond the scope of this review. Autophagy is tightly regulated. The best characterized repressor of autophagic responses is mechanistic target of rapamycin (MTOR) complex I (MTORCI) (Laplante & Sabatini, 2012). Thus, several inducers of autophagy operate by triggering signal transduction cascades that result in the inhibition of MTORCI (Inoki et al, 2012). Among other effects, this allows for the activation of several proteins that are crucial for the initiation of autophagic responses, such as unc-51-like autophagy-activating kinase 1 (ULK1, the mammalian ortholog of yeast Atg1) and autophagy-related 13 (ATG13) (Hosokawa et al, 2009; Nazio et al, 2013). A major inhibitor of MTORCI is protein kinase, AMP-activated (PRKA, best known as AMPK), which is sensitive to declining ATP/AMP ratios (Mihaylova & Shaw, 2011). Besides inhibiting the catalytic activity of MTORCI, AMPK directly stimulates autophagy by phosphorylating ULK1 as well as phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, best known as VPS34) and Beclin 1 (BECN1, the mammalian ortholog of yeast Atg6), two components of a multiprotein complex that produces a lipid that is essential for the biogenesis of autophagosomes, namely phosphatidylinositol 3-phosphate (Egan et al, 2011; Zhao & Klionsky, 2011; Kim et al, 2013). Autophagy also critically relies on two ubiquitin-like conjugation systems, both of which involve ATG7 (Mizushima, 2007). These systems catalyze the covalent linkage of ATG5 to ATG12 and ATG16-like 1 (ATG16L1), and that of phosphatidylethanolamine to proteins of the microtubule-associated protein 1 light chain 3 (MAP1LC3, best known as LC3) family, including MAP1LC3B (LC3B, the mammalian ortholog of yeast Atg8) (Mizushima, 2007). A detailed discussion of additional factors that are involved in the control and execution of autophagic responses can be found in Boya et al (2013). Importantly, autophagosomes can either take up intracellular material in a relatively non-selective manner or deliver very specific portions of the cytoplasm to degradation, mainly depending on the initiating stimulus (Weidberg et al, 2011; Stolz et al, 2014). Thus, while non-selective forms of autophagy normally develop in response to cell-wide alterations, most often of a metabolic nature, highly targeted autophagic responses follow specific perturbations of intracellular homeostasis, such as the accumulation of permeabilized mitochondria (mitophagy), the formation of protein aggregates (aggrephagy), and pathogen invasion (xenophagy) (Okamoto, 2014; Randow & Youle, 2014). Several receptors participate in the selective recognition and recruitment of autophagosomal cargoes in the course of targeted autophagic responses (Rogov et al, 2014; Stolz et al, 2014). The autophagy receptor best characterized to date, that is, sequestosome 1 (SQSTM1, best known as p62), recruits ubiquitinated proteins to autophagosomes by virtue of an ubiquitin-associated (UBA) and a LC3-binding domain (Pankiv et al, 2007). Owing to its key role in the preservation of intracellular homeostasis, autophagy constitutes a barrier against various degenerative processes that may affect healthy cells, including malignant transformation. Thus, autophagy mediates oncosuppressive effects. Accordingly, proteins with bona fide oncogenic potential inhibit autophagy, while many proteins that prevent malignant transformation stimulate autophagic responses (Morselli et al, 2011). Moreover, autophagy is involved in several aspects of anticancer immunosurveillance, that is, the process whereby the immune system constantly eliminates potentially tumorigenic cells before they establish malignant lesions (Ma et al, 2013). However, autophagy also sustains the survival and proliferation of neoplastic cells exposed to intracellular and environmental stress, hence supporting tumor growth, invasion and metastatic dissemination, at least in some settings (Kroemer et al, 2010; Guo et al, 2013b). Here, we discuss the molecular and cellular mechanisms accounting for the differential impact of autophagy on malignant transformation and tumor progression. Autophagy and malignant transformation In various murine models, defects in the autophagic machinery caused by the whole-body or tissue-specific, heterozygous or homozygous knockout of essential autophagy genes accelerate oncogenesis. For instance, Becn1+/− mice (Becn1−/− animals are not viable) spontaneously develop various malignancies, including lymphomas as well as lung and liver carcinomas (Liang et al, 1999; Qu et al, 2003; Yue et al, 2003; Mortensen et al, 2011), and are more susceptible to parity-associated and Wnt1-driven mammary carcinogenesis than their wild-type counterparts (Cicchini et al, 2014). Similarly, mice lacking one copy of the gene coding for the BECN1 interactor autophagy/beclin-1 regulator 1 (AMBRA1) also exhibit a higher rate of spontaneous tumorigenesis than their wild-type littermates (Cianfanelli et al, 2015). Mice bearing a systemic mosaic deletion of Atg5 or a liver-specific knockout of Atg7 spontaneously develop benign hepatic neoplasms more frequently than their wild-type counterparts (Takamura et al, 2011). Moreover, carcinogen-induced fibrosarcomas appear at an accelerated pace in autophagy-deficient Atg4c−/− mice (Marino et al, 2007), as do KRASG12D-driven and BRAFV600E-driven lung carcinomas in mice bearing lung-restricted Atg5 or Atg7 deletions, respectively (Strohecker et al, 2013; Rao et al, 2014). The pancreas-specific knockout of Atg5 or Atg7 also precipitates the emergence of KRASG12D-driven pre-malignant pancreatic lesions (Rosenfeldt et al, 2013; Yang et al, 2014). Several mechanisms can explain, at least in part, the oncosuppressive functions of autophagy. Proficient autophagic responses may suppress the accumulation of genetic and genomic defects that accompanies malignant transformation, through a variety of mechanisms. Reactive oxygen species (ROS) are highly genotoxic, and autophagy prevents their overproduction by removing dysfunctional mitochondria (Green et al, 2011; Takahashi et al, 2013) as well as redox-active aggregates of ubiquitinated proteins (Komatsu et al, 2007; Mathew et al, 2009). In addition, autophagic responses have been involved in the disposal of micronuclei arising upon perturbation of the cell cycle (Rello-Varona et al, 2012), in the degradation of retrotransposing RNAs (Guo et al, 2014), as well as in the control of the levels of ras homolog family member A (RHOA), a small GTPase involved in cytokinesis (Belaid et al, 2013). Finally, various components of the autophagic machinery appear to be required for cells to mount adequate responses to genotoxic stress (Karantza-Wadsworth et al, 2007; Mathew et al, 2007; Park et al, 2014). This said, the precise mechanisms underlying such genome-stabilizing effects remain elusive, implying that the impact of autophagy on DNA-damage responses may be indirect. Further investigation is required to shed light on this possibility. Autophagy is intimately implicated in the maintenance of physiological metabolic homeostasis (Galluzzi et al, 2014; Kenific & Debnath, 2015). Malignant transformation generally occurs along with a shift from a predominantly catabolic consumption of glycolysis-derived pyruvate by oxidative phosphorylation to a metabolic pattern in which: (1) glucose uptake is significantly augmented to sustain anabolic reactions and antioxidant defenses, (2) mitochondrial respiration remains high to satisfy increased energy demands; and (3) several amino acids, including glutamine and serine, become essential as a means to cope with exacerbated metabolic functions (Hanahan & Weinberg, 2011; Galluzzi et al, 2013). Autophagy preserves optimal bioenergetic functions by ensuring the removal of dysfunctional mitochondria (Green et al, 2011), de facto counteracting the metabolic rewiring that accompanies malignant transformation. Moreover, the autophagic degradation of p62 participates in a feedback circuitry that regulates MTORCI activation in response to nutrient availability (Linares et al, 2013; Valencia et al, 2014). Autophagy appears to ensure the maintenance of normal stem cells. This is particularly relevant for hematological malignancies, which are normally characterized by changes in proliferation or differentiation potential that alter the delicate equilibrium between toti-, pluri- and unipotent precursors in the bone marrow (Greim et al, 2014). The ablation of Atg7 in murine hematopoietic stem cells (HSCs) has been shown to disrupt tissue architecture, eventually resulting in the expansion of a population of bone marrow progenitor cells with neoplastic features (Mortensen et al, 2011). Along similar lines, the tissue-specific deletion of the gene coding for the ULK1 interactor RB1-inducible coiled-coil 1 (RB1CC1, best known as FIP200) alters the fetal HSC compartment in mice, resulting in severe anemia and perinatal lethality (Liu et al, 2010). Interestingly, murine Rb1cc1−/− HSCs do not exhibit increased rates of apoptosis, but an accrued proliferative capacity (Liu et al, 2010). The deletion of Rb1cc1 in murine neuronal stem cells (NSCs) also causes a functional impairment that compromises postnatal neuronal differentiation (Wang et al, 2013). However, this effect appears to stem from the failure of murine Rb1cc1−/− HNCs to control redox homeostasis, resulting in the activation of a tumor protein p53 (TP53)-dependent apoptotic response (Wang et al, 2013). Finally, Becn1+/− mice display an expansion of progenitor-like mammary epithelial cells (Cicchini et al, 2014). Of note, autophagy also appears to be required for the preservation of normal stem cell compartments in the human system. Indeed, human hematopoietic, dermal, and epidermal stem cells transfected with a short-hairpin RNA (shRNA) specific for ATG5 lose their ability to self-renew while differentiating into neutrophils, fibroblasts, and keratinocytes, respectively (Salemi et al, 2012). It has been proposed that autophagy contributes to oncogene-induced cell death or oncogene-induced senescence, two fundamental oncosuppressive mechanisms. The activation of various oncogenes imposes indeed a significant stress on healthy cells, a situation that is normally aborted through the execution of a cell death program (Elgendy et al, 2011), or upon the establishment of permanent proliferative arrest (cell senescence) that engages the innate arm of the immune system (Iannello et al, 2013). The partial depletion of ATG5, ATG7 or BECN1 limited the demise of human ovarian cancer cells pharmacologically stimulated to express HRASG12V from an inducible construct (Elgendy et al, 2011). Similarly, shRNAs specific for ATG5 or ATG7 prevented oncogene-induced senescence in primary human melanocytes or human diploid fibroblasts (HDFs) expressing BRAFV600E or HRASG12V (Young et al, 2009; Liu et al, 2013a). Accordingly, the overexpression of the ULK1 homolog ULK3 was sufficient to limit the proliferative potential of HDFs while promoting autophagy (Young et al, 2009). Moreover, both pharmacological inhibitors of autophagy and small-interfering RNAs targeting ATG5, ATG7 or BECN1 prevented spontaneous senescence in HDFs while preventing the degradation of an endogenous, dominant-neg

A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study. A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.

Review30 August 2021Open Access Autophagy in major human diseases Daniel J Klionsky Daniel J Klionsky orcid.org/0000-0002-7828-8118 Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Giulia Petroni Giulia Petroni Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Ravi K Amaravadi Ravi K Amaravadi Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Eric H Baehrecke Eric H Baehrecke Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Andrea Ballabio Andrea Ballabio orcid.org/0000-0003-1381-4604 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Department of Molecular and Human Genetics, Baylor College of Medicine, and Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA Search for more papers by this author Patricia Boya Patricia Boya orcid.org/0000-0003-3045-951X Margarita Salas Center for Biological Research, Spanish National Research Council, Madrid, Spain Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Faculty of Medicine, Department Section of Physiology, Complutense University of Madrid, Madrid, Spain Center for Networked Biomedical Research in Neurodegenerative Diseases (CIBERNED), Madrid, Spain Search for more papers by this author Ken Cadwell Ken Cadwell Kimmel Center for Biology and Medicine at the Skirball Institute, New York University Grossman School of Medicine, New York, NY, USA Department of Microbiology, New York University Grossman School of Medicine, New York, NY, USA Division of Gastroenterology and Hepatology, Department of Medicine, New York University Langone Health, New York, NY, USA Search for more papers by this author Francesco Cecconi Francesco Cecconi orcid.org/0000-0002-5614-4359 Cell Stress and Survival Unit, Center for Autophagy, Recycling and Disease (CARD), Danish Cancer Society Research Center, Copenhagen, Denmark Department of Pediatric Onco-Hematology and Cell and Gene Therapy, IRCCS Bambino Gesù Children's Hospital, Rome, Italy Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Search for more papers by this author Augustine M K Choi Augustine M K Choi Division of Pulmonary and Critical Care Medicine, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA New York-Presbyterian Hospital, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Mary E Choi Mary E Choi New York-Presbyterian Hospital, Weill Cornell Medicine, New York, NY, USA Division of Nephrology and Hypertension, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Charleen T Chu Charleen T Chu orcid.org/0000-0002-5052-8271 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Search for more papers by this author Patrice Codogno Patrice Codogno orcid.org/0000-0002-5492-3180 Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Paris, France Université de Paris, Paris, France Search for more papers by this author Maria Isabel Colombo Maria Isabel Colombo Laboratorio de Mecanismos Moleculares Implicados en el Tráfico Vesicular y la Autofagia-Instituto de Histología y Embriología (IHEM)-Universidad Nacional de Cuyo, CONICET- Facultad de Ciencias Médicas, Mendoza, Argentina Search for more papers by this author Ana Maria Cuervo Ana Maria Cuervo orcid.org/0000-0002-0771-700X Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, USA Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Vojo Deretic Vojo Deretic Autophagy Inflammation and Metabolism (AIM, Center of Biomedical Research Excellence, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Ivan Dikic Ivan Dikic orcid.org/0000-0001-8156-9511 Institute of Biochemistry II, School of Medicine, Goethe University, Frankfurt, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Zvulun Elazar Zvulun Elazar Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Eeva-Liisa Eskelinen Eeva-Liisa Eskelinen Institute of Biomedicine, University of Turku, Turku, Finland Search for more papers by this author Gian Maria Fimia Gian Maria Fimia orcid.org/0000-0003-4438-3325 Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy Department of Epidemiology, Preclinical Research, and Advanced Diagnostics, National Institute for Infectious Diseases ‘L. Spallanzani’ IRCCS, Rome, Italy Search for more papers by this author David A Gewirtz David A Gewirtz orcid.org/0000-0003-0437-4934 Department of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA Search for more papers by this author Douglas R Green Douglas R Green Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Malene Hansen Malene Hansen Sanford Burnham Prebys Medical Discovery Institute, Program of Development, Aging, and Regeneration, La Jolla, CA, USA Search for more papers by this author Marja Jäättelä Marja Jäättelä orcid.org/0000-0001-5950-7111 Cell Death and Metabolism, Center for Autophagy, Recycling & Disease, Danish Cancer Society Research Center, Copenhagen, Denmark Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Terje Johansen Terje Johansen orcid.org/0000-0003-1451-9578 Department of Medical Biology, Molecular Cancer Research Group, University of Tromsø—The Arctic University of Norway, Tromsø, Norway Search for more papers by this author Gábor Juhász Gábor Juhász Institute of Genetics, Biological Research Center, Szeged, Hungary Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary Search for more papers by this author Vassiliki Karantza Vassiliki Karantza Merck & Co., Inc., Kenilworth, NJ, USA Search for more papers by this author Claudine Kraft Claudine Kraft orcid.org/0000-0002-3324-4701 Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Guido Kroemer Guido Kroemer orcid.org/0000-0002-9334-4405 Centre de Recherche des Cordeliers, Equipe Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Nicholas T Ktistakis Nicholas T Ktistakis Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Sharad Kumar Sharad Kumar orcid.org/0000-0001-7126-9814 Centre for Cancer Biology, University of South Australia, Adelaide, SA, Australia Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia Search for more papers by this author Carlos Lopez-Otin Carlos Lopez-Otin orcid.org/0000-0001-6964-1904 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Oviedo, Spain Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain Search for more papers by this author Kay F Macleod Kay F Macleod The Ben May Department for Cancer Research, The Gordon Center for Integrative Sciences, W-338, The University of Chicago, Chicago, IL, USA The University of Chicago, Chicago, IL, USA Search for more papers by this author Frank Madeo Frank Madeo Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed-Graz, Graz, Austria Field of Excellence BioHealth – University of Graz, Graz, Austria Search for more papers by this author Jennifer Martinez Jennifer Martinez Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA Search for more papers by this author Alicia Meléndez Alicia Meléndez Biology Department, Queens College, City University of New York, Flushing, NY, USA The Graduate Center Biology and Biochemistry PhD Programs of the City University of New York, New York, NY, USA Search for more papers by this author Noboru Mizushima Noboru Mizushima orcid.org/0000-0002-6258-6444 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Christian Münz Christian Münz orcid.org/0000-0001-6419-1940 Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland Search for more papers by this author Josef M Penninger Josef M Penninger Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Rushika M Perera Rushika M Perera orcid.org/0000-0003-2435-2273 Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA Department of Pathology, University of California, San Francisco, San Francisco, CA, USA Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Mauro Piacentini Mauro Piacentini orcid.org/0000-0003-2919-1296 Department of Biology, University of Rome “Tor Vergata”, Rome, Italy Laboratory of Molecular Medicine, Institute of Cytology Russian Academy of Science, Saint Petersburg, Russia Search for more papers by this author Fulvio Reggiori Fulvio Reggiori orcid.org/0000-0003-2652-2686 Department of Biomedical Sciences of Cells & Systems, Molecular Cell Biology Section, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands Search for more papers by this author David C Rubinsztein David C Rubinsztein Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK UK Dementia Research Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Kevin M Ryan Kevin M Ryan Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Junichi Sadoshima Junichi Sadoshima Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Laura Santambrogio Laura Santambrogio Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Sandra and Edward Meyer Cancer Center, New York, NY, USA Caryl and Israel Englander Institute for Precision Medicine, New York, NY, USA Search for more papers by this author Luca Scorrano Luca Scorrano orcid.org/0000-0002-8515-8928 Istituto Veneto di Medicina Molecolare, Padova, Italy Department of Biology, University of Padova, Padova, Italy Search for more papers by this author Hans-Uwe Simon Hans-Uwe Simon Institute of Pharmacology, University of Bern, Bern, Switzerland Department of Clinical Immunology and Allergology, Sechenov University, Moscow, Russia Laboratory of Molecular Immunology, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia Search for more papers by this author Anna Katharina Simon Anna Katharina Simon The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, UK Search for more papers by this author Anne Simonsen Anne Simonsen orcid.org/0000-0003-4711-7057 Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital Montebello, Oslo, Norway Search for more papers by this author Alexandra Stolz Alexandra Stolz orcid.org/0000-0002-3340-439X Institute of Biochemistry II, School of Medicine, Goethe University, Frankfurt, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis orcid.org/0000-0002-5253-1466 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece Department of Basic Sciences, School of Medicine, University of Crete, Heraklion, Crete, Greece Search for more papers by this author Sharon A Tooze Sharon A Tooze orcid.org/0000-0002-2182-3116 Molecular Cell Biology of Autophagy, The Francis Crick Institute, London, UK Search for more papers by this author Tamotsu Yoshimori Tamotsu Yoshimori orcid.org/0000-0001-9787-3788 Department of Genetics, Graduate School of Medicine, Osaka University, Suita, Japan Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Japan Search for more papers by this author Junying Yuan Junying Yuan Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Zhenyu Yue Zhenyu Yue Department of Neurology, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Qing Zhong Qing Zhong orcid.org/0000-0001-6979-955X Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, China Search for more papers by this author Lorenzo Galluzzi Corresponding Author Lorenzo Galluzzi [email protected] orcid.org/0000-0003-2257-8500 Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Sandra and Edward Meyer Cancer Center, New York, NY, USA Caryl and Israel Englander Institute for Precision Medicine, New York, NY, USA Department of Dermatology, Yale School of Medicine, New Haven, CT, USA Université de Paris, Paris, France Search for more papers by this author Federico Pietrocola Corresponding Author Federico Pietrocola [email protected] orcid.org/0000-0002-2930-234X Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden Search for more papers by this author Daniel J Klionsky Daniel J Klionsky orcid.org/0000-0002-7828-8118 Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Search for more papers by this author Giulia Petroni Giulia Petroni Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Ravi K Amaravadi Ravi K Amaravadi Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Eric H Baehrecke Eric H Baehrecke Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Andrea Ballabio Andrea Ballabio orcid.org/0000-0003-1381-4604 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Sciences, Section of Pediatrics, Federico II University, Naples, Italy Department of Molecular and Human Genetics, Baylor College of Medicine, and Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA Search for more papers by this author Patricia Boya Patricia Boya orcid.org/0000-0003-3045-951X Margarita Salas Center for Biological Research, Spanish National Research Council, Madrid, Spain Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Faculty of Medicine, Department Section of Physiology, Complutense University of Madrid, Madrid, Spain Center for Networked Biomedical Research in Neurodegenerative Diseases (CIBERNED), Madrid, Spain Search for more papers by this author Ken Cadwell Ken Cadwell Kimmel Center for Biology and Medicine at the Skirball Institute, New York University Grossman School of Medicine, New York, NY, USA Department of Microbiology, New York University Grossman School of Medicine, New York, NY, USA Division of Gastroenterology and Hepatology, Department of Medicine, New York University Langone Health, New York, NY, USA Search for more papers by this author Francesco Cecconi Francesco Cecconi orcid.org/0000-0002-5614-4359 Cell Stress and Survival Unit, Center for Autophagy, Recycling and Disease (CARD), Danish Cancer Society Research Center, Copenhagen, Denmark Department of Pediatric Onco-Hematology and Cell and Gene Therapy, IRCCS Bambino Gesù Children's Hospital, Rome, Italy Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Search for more papers by this author Augustine M K Choi Augustine M K Choi Division of Pulmonary and Critical Care Medicine, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA New York-Presbyterian Hospital, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Mary E Choi Mary E Choi New York-Presbyterian Hospital, Weill Cornell Medicine, New York, NY, USA Division of Nephrology and Hypertension, Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Charleen T Chu Charleen T Chu orcid.org/0000-0002-5052-8271 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Search for more papers by this author Patrice Codogno Patrice Codogno orcid.org/0000-0002-5492-3180 Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Paris, France Université de Paris, Paris, France Search for more papers by this author Maria Isabel Colombo Maria Isabel Colombo Laboratorio de Mecanismos Moleculares Implicados en el Tráfico Vesicular y la Autofagia-Instituto de Histología y Embriología (IHEM)-Universidad Nacional de Cuyo, CONICET- Facultad de Ciencias Médicas, Mendoza, Argentina Search for more papers by this author Ana Maria Cuervo Ana Maria Cuervo orcid.org/0000-0002-0771-700X Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, USA Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Vojo Deretic Vojo Deretic Autophagy Inflammation and Metabolism (AIM, Center of Biomedical Research Excellence, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Search for more papers by this author Ivan Dikic Ivan Dikic orcid.org/0000-0001-8156-9511 Institute of Biochemistry II, School of Medicine, Goethe University, Frankfurt, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Zvulun Elazar Zvulun Elazar Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Eeva-Liisa Eskelinen Eeva-Liisa Eskelinen Institute of Biomedicine, University of Turku, Turku, Finland Search for more papers by this author Gian Maria Fimia Gian Maria Fimia orcid.org/0000-0003-4438-3325 Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy Department of Epidemiology, Preclinical Research, and Advanced Diagnostics, National Institute for Infectious Diseases ‘L. Spallanzani’ IRCCS, Rome, Italy Search for more papers by this author David A Gewirtz David A Gewirtz orcid.org/0000-0003-0437-4934 Department of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA Search for more papers by this author Douglas R Green Douglas R Green Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Malene Hansen Malene Hansen Sanford Burnham Prebys Medical Discovery Institute, Program of Development, Aging, and Regeneration, La Jolla, CA, USA Search for more papers by this author Marja Jäättelä Marja Jäättelä orcid.org/0000-0001-5950-7111 Cell Death and Metabolism, Center for Autophagy, Recycling & Disease, Danish Cancer Society Research Center, Copenhagen, Denmark Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Terje Johansen Terje Johansen orcid.org/0000-0003-1451-9578 Department of Medical Biology, Molecular Cancer Research Group, University of Tromsø—The Arctic University of Norway, Tromsø, Norway Search for more papers by this author Gábor Juhász Gábor Juhász Institute of Genetics, Biological Research Center, Szeged, Hungary Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary Search for more papers by this author Vassiliki Karantza Vassiliki Karantza Merck & Co., Inc., Kenilworth, NJ, USA Search for more papers by this author Claudine Kraft Claudine Kraft orcid.org/0000-0002-3324-4701 Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Guido Kroemer Guido Kroemer orcid.org/0000-0002-9334-4405 Centre de Recherche des Cordeliers, Equipe Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Nicholas T Ktistakis Nicholas T Ktistakis Signalling Programme, Babraham Institute, Cambridge, UK Search for more papers by this author Sharad Kumar Sharad Kumar orcid.org/0000-0001-7126-9814 Centre for Cancer Biology, University of South Australia, Adelaide, SA, Australia Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia Search for more papers by this author Carlos Lopez-Otin Carlos Lopez-Otin orcid.org/0000-0001-6964-1904 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Oviedo, Spain Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain Search for more papers by this author Kay F Macleod Kay F Macleod The Ben May Department for Cancer Research, The Gordon Center for Integrative Sciences, W-338, The University of Chicago, Chicago, IL, USA The University of Chicago, Chicago, IL, USA Search for more papers by this author Frank Madeo Frank Madeo Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed-Graz, Graz, Austria Field of Excellence BioHealth – University of Graz, Graz, Austria Search for more papers by this author Jennifer Martinez Jennifer Martinez Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA Search for more papers by this author Alicia Meléndez Alicia Meléndez Biology Department, Queens College, City University of New York, Flushing, NY, USA The Graduate Center Biology and Biochemistry PhD Programs of the City University of New York, New York, NY, USA Search for more papers by this author Noboru Mizushima Noboru Mizushima orcid.org/0000-0002-6258-6444 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Christian Münz Christian Münz orcid.org/0000-0001-6419-1940 Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland Search for more papers by this author Josef M Penninger Josef M Penninger Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Rushika M Perera Rushika M Perera orcid.org/0000-0003-2435-2273 Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA Department of Pathology, University of California, San Francisco, San Francisco, CA, USA Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Mauro Piacentini Mauro Piacentini orcid.org/0000-0003-2919-1296 Department of Biology, University of Rome “Tor Vergata”, Rome, Italy Laboratory of Molecular Medicine, Institute of Cytology Russian Academy of Science, Saint Petersburg, Russia Search for more papers by this author Fulvio Reggiori Fulvio Reggiori orcid.org/0000-0003-2652-2686 Department of Biomedical Sciences of Cells & Systems, Molecular Cell Biology Section, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands Search for more papers by this author David C Rubinsztein David C Rubinsztein Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK UK Dementia Research Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Kevin M Ryan Kevin M Ryan Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Junichi Sadoshima Junichi Sadoshima Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Laura Santambrogio Laura Santambrogio Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Sandra and Edward Meyer Cancer Center, New York, NY, USA Caryl and Israel Englander Institute for Precision Medicine, New York, NY, USA Search for more papers by this author Luca Scorrano Luca Scorrano orcid.org/0000-0002-8515-8928 Istituto Veneto di Medicina Molecolare, Padova, Italy Department of Biology, University of Padova, Padova, Italy Search for more papers by this author Hans-Uwe Simon Hans-Uwe Simon Institute of Pharmacology, University of Bern, Bern, Switzerland Department of Clinical Immunology and Allergology, Sechenov University, Moscow, Russia Laboratory of Molecular Immunology, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia Search for more papers by this author Anna Katharina Simon Anna Katharina Simon The Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, UK Search for more papers by this author Anne Simonsen Anne Simonsen orcid.org/0000-0003-4711-7057 Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Centre for Cancer Cell Reprogramming, Institute of Clinical Medicine, University of Oslo, Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital Montebello, Oslo, Norway Search for more papers by this author Alexandra Stolz Alexandra Stolz orcid.org/0000-0002-3340-439X Institute of Biochemistry II, School of Medicine, Goethe University, Frankfurt, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis orcid.org/0000-0002-5253-1466 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece Department of Basic Sciences, School of Medicine, University of Crete, Heraklion, Crete, Greece Search for mor

The recent period has witnessed progress in the understanding of the lysosomal autophagic pathway. The discovery of a family of genes conserved from yeast to humans, and involved in the formation of autophagosomes, has unraveled new protein-conjugation systems and has shed light on the importance of autophagy in physiology and pathophysiology. The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of autophagy during cell death. As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) as well as intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of autophagy. The mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals. In addition, autophagy, which is characterized by a flux of membrane from the formation of the autophagosome to the fusion with the lysosome, is regulated by GTPases, similarly to the vesicular transport along the exocytic/endocytic pathway. The aim of the present review is to give an overview of autophagy and to discuss its regulation by activators and effectors of mTOR and GTPases.

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway.Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells.Here we demonstrate that the wild-type PTEN is expressed in this cell line.Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy.This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity.The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase ( Myr PKB).Under these conditions a dramatic inhibition of macroautophagy was observed.By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB.These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.

Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGy-related genes has greatly increased our knowledge about the mechanism responsible for autophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.

Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

Activation of NF-κB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-κB activation mediates repression of autophagy in tumor necrosis factor-α (TNFα)-treated Ewing sarcoma cells. This repression is associated with an NF-κB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-κB activation, TNFα treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-κB-competent cells by the addition of H₂O₂. Small interfering RNA-mediated knockdown of <i>beclin 1</i> and <i>atg7</i> expression, two autophagy-related genes, reduced TNFα- and reactive oxygen species-induced apoptosis in cells lacking NF-κB activation and in NF-κB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-κB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-κB.

Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK. In the present study, carried out with hepatocytes, HT-29 cells, and HeLa cells, we have reexamined the possible role of AMPK in the control of mammalian autophagy. Inhibition of AMPK activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICAR is not related to its ability to activate AMPK. We conclude that in mammalian cells, as in yeast, AMPK is required for autophagy. Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK. In the present study, carried out with hepatocytes, HT-29 cells, and HeLa cells, we have reexamined the possible role of AMPK in the control of mammalian autophagy. Inhibition of AMPK activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICAR is not related to its ability to activate AMPK. We conclude that in mammalian cells, as in yeast, AMPK is required for autophagy. During macroautophagy (hereafter referred to as autophagy), a small part of the cytoplasm is sequestered by a double isolation membrane, presumably derived from the endoplasmic reticulum, to form an autophagosome. This autophagosome then fuses with a lysosome and the sequestered macromolecular material, including proteins, is degraded. The process is activated by various stress situations, including nutrient depletion. Under these conditions, autophagy provides the constituents required to maintain the metabolism essential for survival (1Codogno P. Meijer A.J. Cell Death. Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (952) Google Scholar, 2Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2196) Google Scholar). An example is that of the mammalian liver in which autophagy is accelerated during fasting to provide amino acids for gluconeogenesis to meet the energy requirements of the brain and of erythrocytes. In many cell types, including liver cells, autophagy is inhibited by amino acids, in synergy with insulin, and this inhibition is mediated, at least in part, by mTOR 3The abbreviations used are: mTOR, mammalian target of rapamycin; AICAR, AICA riboside, imidazole-4-carboxamide-1-β-ribofuranoside; ZMP, AICAR monophosphate; 3-MA, 3-methyladenine; AMPK, AMP-activated protein kinase; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; p70S6K, p70S6 kinase; MDC, monodansylcadaverine; CC, compound C; ACC, acetyl-CoA carboxylase; eIF4E, eukaryotic initiation factor 4E; GFP, green fluorescent protein; gdw, gram dry weight of cells; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; Met, metformin. -dependent signaling (1Codogno P. Meijer A.J. Cell Death. Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (952) Google Scholar). Depending on the cell type and the conditions, other signaling pathways, such as the ras/raf/MAPK signaling pathway, may also participate in amino acid control of autophagy (1Codogno P. Meijer A.J. Cell Death. Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (952) Google Scholar). In addition, autophagy is controlled by phosphatidylinositol phospholipids. The process is inhibited by PtdIns(3,4,5)P3, the product of PI3K class I, a lipid kinase located upstream of mTOR in the insulin signaling pathway. By contrast, PI(3)P, the product of PI3K class III, is essential for autophagy (1Codogno P. Meijer A.J. Cell Death. Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (952) Google Scholar, 3Kihara A. Kabeya Y. Ohsumi Y. Yoshimori T. EMBO Rep. 2001; 2: 330-335Crossref PubMed Scopus (728) Google Scholar, 4Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). This requirement for PI(3)P explains why PI3K inhibitors are also autophagy inhibitors. Indeed, the classical autophagy inhibitor 3-methyladenine (5Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1889-1892Crossref PubMed Scopus (1181) Google Scholar) turned out to be a PI3K inhibitor (1Codogno P. Meijer A.J. Cell Death. Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (952) Google Scholar). After the original observation in 1995 that amino acids can stimulate mTOR-dependent signaling (6Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar), it is now generally accepted that the mTOR pathway acts as a sensor of amino acids (7Jacinto E. Hall M.N. Nat. Rev. Mol. Cell Biol. 2003; 4: 117-126Crossref PubMed Scopus (515) Google Scholar). A few years ago we, and others, discovered that mTOR can also sense changes in the cellular energy state via AMP-activated protein kinase (AMPK). Activation of this protein kinase inhibits mTOR-dependent signaling and inhibits protein synthesis (8Meijer A.J. Dubbelhuis P.F. Biochem. Biophys. Res. Commun. 2004; 313: 397-403Crossref PubMed Scopus (179) Google Scholar), which is consistent with AMPK function of switching off ATP-dependent processes (9Hardie D.G. J. Cell Sci. 2004; 117: 5479-5487Crossref PubMed Scopus (965) Google Scholar). Inhibition of mTOR by AMPK, like that caused by addition of rapamycin (1Codogno P. Meijer A.J. Cell Death. Differ. 2005; 12: 1509-1518Crossref PubMed Scopus (952) Google Scholar, 2Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2196) Google Scholar), may be expected to increase autophagy. However, in the literature there is controversy on this issue. In yeast, activation of AMPK stimulates autophagy (10Wang Z. Wilson W.A. Fujino M.A. Roach P.J. Mol. Cell Biol. 2001; 21: 5742-5752Crossref PubMed Scopus (234) Google Scholar). By contrast, activation of AMPK by addition of the cell-permeable nucleotide analogue AICA riboside (AICAR) in hepatocytes strongly inhibits autophagy (11Samari H.R. Seglen P.O. J. Biol. Chem. 1998; 273: 23758-23763Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In the present study, using different mammalian cell types, we have examined the possible role of AMPK in the control of autophagy in more detail. Our data indicate that AMPK, like in yeast, is required for autophagy. Cell culture products were from Invitrogen. Insulin, rapamycin, AICAR (AICA riboside, imidazole-4-carboxamide 1-β-ribofuranoside), the chemicals for enhanced chemiluminescence (ECL), BCA kits, Ponceau red, metformin and protease inhibitors mixture were from Sigma. Compound C was a gift of Merck Sharp & Dohme BV (Haarlem, The Netherlands). Plasmid purification kit Nucleobond AX and nitrocellulose membranes were from Macherey-Nagel (Düren, Germany). The FuGENE 6™ transfection kit was from Roche Applied Science (Basel, Switzerland) and the Lipofectamine™ 2000 was from Invitrogen. l-[U-14C]Valine was from PerkinElmer Life Sciences and l-valine was from Merck (Darmstadt, Germany). Ultima Gold™ scintillation fluid was from Packard Biosciences. Phosphospecific antibodies against protein kinase B (Ser473), AMPK (Thr172), and acetyl-CoA carboxylase (Ser79) were from Cell Signaling Technology Inc. (Leusden, The Netherlands). Rabbit anti-p70S6 kinase, anti-eIF4E, and mouse anti-c-Myc (9E10) were from Santa Cruz Biotechnology. Mouse anti-actin was from Chemicon. Mouse anti-FLAG (2EL-1B11) was from Euromedex (Souffelweyersheim, France). Goat anti-rabbit horseradish peroxidase was from Bio-Rad. All other chemicals and enzymes were obtained from either Roche Applied Science or Sigma. [14C]Chloroquine was from PerkinElmer Life Sciences. Interleukin 13 (IL-13) was kindly provided by Dr. A. Minty (Sanofi Elf Biorecherche, Labege, France). Rapamycin and compound C were dissolved in dimethyl sulfoxide (Me2SO). The final Me2SO concentration did not exceed 0.25% (v/v), which did not affect the processes that were studied. cDNAs encoding the c-Myc-tagged constitutively active AMPK-α1312 (T172D) (AMPKCA) and the c-Myc-tagged dominant-negative-AMPK-α1 (K45R) (AMPKDN) were kindly provided by Dr. D. Carling (Cellular Stress Group, Hammersmith Hospital, London, UK). cDNA encoding for the GFP-tagged LKB1 was a generous gift from Dr M. Billaud (CNRS UMR 5201, Lyon, France). cDNA encoding for FLAG-tagged STRAD (12Baas A.F. Boudeau J. Sapkota G.P. Smit L. Medema R. Morrice N.A. Alessi D.R. Clevers H.C. EMBO J. 2003; 22: 3062-3072Crossref PubMed Scopus (299) Google Scholar) was a generous gift from Dr. H. C. Clevers (Utrecht, The Netherlands). Hepatocytes were isolated from male Wistar rats (250–300 g) starved for 16–20 h by collagenase perfusion (6Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). Hepatocytes (5 mg dry weight/ml) were incubated for the indicated times at 37 °C in minimal medium (Krebs-Henseleit bicarbonate buffer plus 10 mm Na+-Hepes, pH 7.4, and 20 mm glucose) plus the components as indicated in the legends. The final incubation volume was 2 ml. The gas atmosphere was O2/CO2 (19:1, v/v). At the end of the incubations, hepatocytes were collected for gel analysis by centrifugation in 5 volumes of an ice-cold solution of 150 mm NaCl plus 10 mm sodium Hepes (pH 7.4) for 5 s in an Eppendorf centrifuge. For the SDS-PAGE procedures, the pellet was lysed by addition of Laemmli sample buffer and subsequently incubated at 95 °C for 5 min. For determination of ATP, lactate, and amino acids, an aliquot of the incubated cell suspension was acidified with HClO4 (final concentration, 3%, m/v). After removal of the precipitated protein by centrifugation in a microcentrifuge (1 min; 10,000 × g), the clear supernatant was neutralized to pH 7 with a small volume of a mixture of 2 m KOH plus 0.3 m MOPS. HT-29 human colon cancer cells and HeLa cells were maintained in Dulbecco's modified Eagle's medium 4.5 g/liter glucose supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. Medium was replaced three times per week, and cells were passaged at confluency. The cells were grown in a humidified atmosphere of 10% CO2, 90% air at 37 °C. Cells were plated and grown to 50–80% confluency before treatment for different times with vehicle or adequate concentrations of amino acids (4×), rapamycin (100 nm), metformin (1–10 mm), 3-methyladenine (3-MA) (10 mm), IL-13 (30 ng/ml), and AICAR (0.1–1 mm). ATP was determined fluorimetrically with NADP+, glucose, hexokinase, and glucose-6-phosphate dehydrogenase (13Williamson J.R. Corkey B.E. Methods Enzymol. 1979; 55: 200-222Crossref PubMed Scopus (126) Google Scholar). Lactate was measured spectrophotometrically with NAD+ and lactate dehydrogenase (13Williamson J.R. Corkey B.E. Methods Enzymol. 1979; 55: 200-222Crossref PubMed Scopus (126) Google Scholar). AICAR and ZMP were measured by HPLC as described by Samari and Seglen (11Samari H.R. Seglen P.O. J. Biol. Chem. 1998; 273: 23758-23763Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The expression constructs pEGFP-C1-LKB1wt, pcDNA3-FLAG-STRAD, pcDNA3-Myc-AMPKCA, pcDNA3-Myc-AMPKDN, and control vectors were introduced into HT-29 cells and HeLa cells using Lipofectamine™ 2000 and the FuGENE 6™ Reagent transfection kit, respectively. Transfected cells were cultured in complete medium for 48 h before use for the different experiments. Expression levels of each construct were determined by SDS-PAGE analysis using the relevant Tag antibody. After SDS-PAGE resolution, the proteins were transferred on nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk in TBST (10 mm Tris-HCl, pH 8; 100 mm NaCl; 0.1% Tween 20) for 1 h at room temperature and then incubated with appropriate primary antibody overnight at 4 °C (diluted in TBST-5% BSA), followed by incubation with horse-radish peroxidase-conjugated secondary antibody at 1:5000 dilution in TBST-5% nonfat dry milk for 1 h at room temperature. The anti-actin was used at 1:2500 dilution in TBST-5% BSA. All other total antibodies were used at 1:1000 dilution in TBST-5% BSA, and anti-phosphoantibodies were diluted at 1:1000 in TBST-1% BSA. The anti-c-Myc were used at 1:200 dilution in TBST-5% BSA. The anti-FLAG was used at 1:1000 dilution. To quantify the different spots of immunoblotting, we used the freeware Scion Imaging. Amino Acid Analysis—Amino acids were analyzed with HPLC exactly as described by (14Wu G.Y. Bazer F.W. Tuo W.B. Flynn S.P. Biol. Reprod. 1996; 54: 1261-1265Crossref PubMed Scopus (26) Google Scholar). Of the branched chain amino acids, the valine peak in the amino acid spectrum was contaminated with a compound of unknown origin, and was therefore not used. [14C]Chloroquine Accumulation—Accumulation of the divalent weak base chloroquine, which monitors changes in the pH of intracellular acidic compartments, mainly lysosomes, was measured exactly as described elsewhere (15Luiken J.J. Aerts J.M. Meijer A.J. Eur. J. Biochem. 1996; 235: 564-573Crossref PubMed Scopus (43) Google Scholar). In these experiments, the hepatocyte concentration was 1 mg dw/ml, the concentration of chloroquine was <1 μm and the amount of radioactivity was 0.025 μCi/ml of incubation medium. Measurement of the Degradation of Long-lived Proteins— Proteolysis was determined as described previously (16Pattingre S. Bauvy C. Codogno P. J. Biol. Chem. 2003; 278: 16667-16674Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Briefly, cells were incubated for 24 h at 37 °C with 0.05 μCi/ml of l-[U-14C]valine. Unincorporated radioisotope was removed by three rinses with phosphate-buffered saline (PBS). Cells were incubated in complete medium, supplemented with 10 mm cold valine throughout the pre-chase period. After 1 h of pre-chase, the medium was removed by three rinses with PBS containing 0.9 mm CaCl2 and 0.5 mm MgCl2 (PBS+) and a complete mixture of amino acids in which each amino acid was present at four times its concentration in the portal vein of a 24-h-fasted rat (for composition, see (6Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar)), rapamycin (100 nm), metformin (2 mm), AICAR (250 μm), 3-MA (10 mm), or IL-13 (30 ng/ml) in nutrient-free medium (without amino acids and in the absence of fetal calf serum), HBSS or Hanks balanced saline solution, supplemented with 0.1% bovine serum albumin and 10 mm cold valine were added at the beginning of the chase period. During the prechase, the short-lived proteins were being degraded. The chase continued for 4 h. Cells and radiolabeled proteins from the 4-h chase medium were precipitated in trichloroacetic acid at a final concentration of 10% (w/v) at 4 °C overnight. After centrifugation, pellets were dissolved in 0.2 n NaOH. Radioactivity was determined by liquid scintillation counting. Protein degradation was calculated by dividing the acid-soluble radioactivity recovered from both cells and medium by the radioactivity contained in the precipitated proteins from both cells and medium. Monodansylcadaverine (MDC) Staining—MDC staining was performed as previously described (17Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Briefly, cells were seeded on microscope coverglasses. After 3 days, cells were treated for 2 h in appropriate mediums, and during the last 10 min 0.1 μm MDC was added. Cells were then washed by three rinses with PBS+ and fixed with a solution of 3% paraformaldehyde for 20 min. The microscope cover glasses were washed again by three rinses with PBS+, and put on microscope slides with Mowiol. The slides were incubated overnight at 4 °C and observed in the epifluorescence microscope. Only cells expressing GFP-LKB1 were counted for MDC staining. Statistics—Data were summarized as mean ± S.E. Statistical significance was determined using Student's t test (p < 0.05). Effect of Pharmacological Modulators of AMPK Activity on Autophagic Proteolysis in Isolated Rat Hepatocytes—When hepatocytes are incubated in the absence of amino acids, autophagic proteolytic flux is maximal. As previously reported in Ref. 11Samari H.R. Seglen P.O. J. Biol. Chem. 1998; 273: 23758-23763Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, addition of the AMPK activator AICAR under these conditions strongly inhibited autophagy, as indicated by the large reduction in the appearance of the branched chain amino acids isoleucine and leucine (which are not further catabolized in rat liver) (Fig. 1, A and B). This was also accompanied by reductions in the major gluconeogenic amino acids aspartate, glutamate, glutamine, and alanine (Fig. 1, C–F). It should be stressed that these experiments were carried out in the presence of a low concentration of cycloheximide (10 μm) to prevent reincorporation of the proteolytically formed amino acids into protein. The inhibition of proteolysis by AICAR was similar to that observed with the autophagy inhibitor 3-methyladenine and also to that observed with the acidotropic agent methylamine, which inhibits lysosomal function by raising the lysosomal pH (Fig. 1, A and B). Administration of the antidiabetic agent metformin is another way to stimulate AMPK activity (18Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar). The effects of AICAR and metformin on AMPK phosphorylation are compared in Fig. 2. In the presence of AICAR (250 μm), phosphorylation of AMPK was rapid but decreased after 40 min, presumably because continuous intracellular accumulation of ZMP results in ZMP levels high enough to inhibit AMPK (19Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1036) Google Scholar). By contrast, in the presence of 2 mm metformin, AMPK phosphorylation was initially slow (at 40 min it was similar to that seen with AICAR at 20 min) and increased with time to a maximum at 80 min. Even though 2 mm metformin was more potent than AICAR in stimulating AMPK phosphorylation, the ability of metformin to inhibit autophagic production of the branched-chain amino acids was less than that observed with AICAR (Fig. 1, A and B). Furthermore, production of glutamine, glutamate, and aspartate was not significantly affected by metformin while that of alanine was even substantially increased (Fig. 1, C–F), presumably because glycolytic flux increased in the presence of metformin (see below). While AICAR and metformin are AMPK activators, we also wanted to study the effect of inhibition of AMPK activity on autophagy. Compound C has been reported as a specific inhibitor of AMPK (18Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar), and we examined its efficacy in this regard. Metformin-stimulated phosphorylation of AMPK was reversed by compound C (Fig. 3). Compound C did not inhibit AICAR-stimulated AMPK phosphorylation unless amino acids were also present (Fig. 4) (see “Discussion”). The same was true for acetyl-CoA carboxylase (ACC), which is a substrate for AMPK (Fig. 4). It was shown previously that compound C, in addition to its action as an inhibitor of AMPK (18Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar), can also inhibit AMPK activity by competing with AICAR transport across the plasma membrane, thus affecting intracellular conversion of AICAR to ZMP (20Fryer L.G. Parbu-Patel A. Carling D. FEBS Lett. 2002; 531: 189-192Crossref PubMed Scopus (72) Google Scholar). This was confirmed in Table 1 showing that 40 μm compound C partly inhibited both AICAR consumption and ZMP production. Interestingly, ZMP formation was also inhibited by amino acid addition, but in this case without significant effect on AICAR consumption so that the amino acid effect must have been intracellular rather than at the level of AICAR transport. The presence of both compound C and amino acids inhibited ZMP production by about 90% (Table 1). This accounts for the low AMPK phosphorylation and AMPK activity, as indicated by acetyl CoA carboxylase phosphorylation (Fig. 4), under these conditions (see further “Discussion”).FIGURE 4Effect of AICAR, compound C, and rapamycin on the phosphorylation of AMPK, ACC, p70S6k, and protein kinase B, in the absence and presence of amino acids in hepatocytes. Hepatocytes were incubated with 20 mm glucose for 100 min in the absence or presence of 250 μm AICAR, 100 nm rapamycin, or 40 μm CC, or combinations thereof. A complete mixture of amino acids (21Dubbelhuis P.F. Meijer A.J. FEBS Lett. 2002; 521: 39-42Crossref PubMed Scopus (71) Google Scholar) (AA), if present, was added at 30 min. Insulin (10–7 m), if present, was added 5 min before the end of the incubation. eIF4E was used to check for the loading of the gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Inhibition by compound C and by amino acids of ZMP production from AICAR in hepatocytes-ΔAICARZMPμmol/gdwμmol/gdwControl37.1 ± 1.824.2 ± 1.44AA40.5 ± 2.110.6 ± 1.0ap < 0.05 versus control.CC25.5 ± 1.8ap < 0.05 versus control.11.6 ± 0.8ap < 0.05 versus control.4AA + CC27.9 ± 2.3ap < 0.05 versus control.3.6 ± 0.4ap < 0.05 versus control.a p < 0.05 versus control. Open table in a new tab AICAR inhibited amino acid-stimulated, rapamycin-sensitive, phosphorylation of p70S6 kinase, in agreement with our earlier observations (21Dubbelhuis P.F. Meijer A.J. FEBS Lett. 2002; 521: 39-42Crossref PubMed Scopus (71) Google Scholar). This effect was largely prevented by 40 μm compound C (Fig. 4). AICAR alone slightly stimulated p70S6 kinase phosphorylation (Fig. 4), in agreement with the data of Moller et al. (22Moller M.T. Samari H.R. Seglen P.O. Toxicol. Sci. 2004; 82: 628-637Crossref PubMed Scopus (10) Google Scholar) who showed that this phosphorylation was rapamycin-insensitive. Having thus established that compound C can inhibit AMPK activity in intact hepatocytes, whether by a direct effect on the enzyme or indirectly by competing with AICAR for transport into the cells (20Fryer L.G. Parbu-Patel A. Carling D. FEBS Lett. 2002; 531: 189-192Crossref PubMed Scopus (72) Google Scholar), we tested its effect on autophagy. If AMPK activation inhibits autophagy as suggested (11Samari H.R. Seglen P.O. J. Biol. Chem. 1998; 273: 23758-23763Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), one would expect compound C to be able to reverse the inhibition of autophagy by metformin. Unexpectedly, however, compound C inhibited autophagic proteolysis when added alone and the effect was not additive with either that of metformin, AICAR, or 3-methyladenine (Fig. 1, A and B). To rule out the possibility that compound C inhibited the lysosomal proton pump, we tested its effect on the intracellular accumulation of [14C]chloroquine, a divalent weak base, which when present at low concentrations greatly accumulates in acidic intracellular compartments, mainly lysosomes (15Luiken J.J. Aerts J.M. Meijer A.J. Eur. J. Biochem. 1996; 235: 564-573Crossref PubMed Scopus (43) Google Scholar). As a control, the effect of 5 mm methylamine was also tested. Chloroquine accumulation was not affected by compound C (Fig. 5), but methylamine greatly reduced it, as expected. AICAR significantly decreased chloroquine accumulation although not to the same extent as methylamine, whereas metformin had no significant effect. We also examined whether variations in glycolysis were in some way associated with the observed changes in autophagic proteolysis, but this was not the case. Omission of glucose did not affect autophagic proteolysis (Fig. 1, A and B) and lactate was not formed under these conditions (Fig. 1G). ATP levels and AMPK phosphorylation were not affected by glucose depletion (not shown), presumably because mitochondrial oxidation of endogenous fatty acids provided sufficient energy. Metformin stimulated, while both AICAR and 3-methyladenine strongly inhibited production of lactate; compound C, on the other hand, had no effect on lactate formation (Fig. 1G). The effect of metformin on the production of lactate is consistent with the ability of this compound to act as a weak inhibitor of the mitochondrial respiratory chain (23Leverve X.M. Guigas B. Detaille D. Batandier C. Koceir E.A. Chauvin C. Fontaine E. Wiernsperger N.F. Diabetes Metab. 2003; 29: 6S88-6S94Crossref PubMed Google Scholar, 24Owen M.R. Doran E. Halestrap A.P. Biochem. J. 2000; 348: 607-614Crossref PubMed Scopus (1659) Google Scholar). Indeed, we observed a decrease in intracellular ATP levels from 11.2 ± 0.9 to 6.2 ± 0.2 μmol/g dry weight of cells after 90 min of incubation in the presence of 2 mm metformin (n = 5; p < 0.05) (data not shown). The inhibition of glycolysis by AICAR and 3-methyladenine is in agreement with previous observations in hepatocytes (25Caro L.H. Plomp P.J. Wolvetang E.J. Kerkhof C. Meijer A.J. Eur. J. Biochem. 1988; 175: 325-329Crossref PubMed Scopus (114) Google Scholar, 26Vincent M.F. Bontemps F. Van den Berghe G. Biochem. J. 1992; 281: 267-272Crossref PubMed Scopus (68) Google Scholar). In the course of our experiments, we noted that AICAR inhibited insulin-stimulated phosphorylation of protein kinase B, which was prevented by amino acids but not by compound C (Fig. 4). These data indicate that, in addition to activating AMPK, AICAR may have other effects (see “Discussion”). Inhibition of AMPK Activity Blocks Autophagic Proteolysis in Human Cell Lines—We have previously shown that autophagic proteolysis is stimulated when the human colon carcinoma HT-29 cells are incubated in nutrient-free medium (4Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). Pilot experiments showed that metformin and AICAR were able to activate AMPK in HT-29 cells, as determined by its phosphorylation at position Thr172 and phosphorylation of its substrate ACC at position Ser79, with a maximal effect at 250 μm AICAR and 2 mm metformin (Fig. 6A and data not shown). Whatever the concentration used, cell viability was greater than 95% under the experimental conditions used in this study. Following on with these results, we next investigated the effect of AICAR and metformin on the degradation of long-lived [14C]valine-labeled proteins. Both compounds inhibited the degradation of [14C]valine-labeled proteins in nutrient-free medium to the same extent (Fig. 6B). However, only a partial inhibition of proteolysis was observed when compared with 3-MA, amino acids or interleukin 13, known inhibitors of autophagic proteolysis in HT-29 cells (4Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar) (Fig. 6B). To correlate these findings with the state of AMPK activation, HT-29 cells were transfected with a constitutively active form of AMPK (AMPKCA). As previously shown (27Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarchand P. Ferre P. Foufelle F. Carling D. Mol. Cell Biol. 2000; 20: 6704-6711Crossref PubMed Scopus (360) Google Scholar), AMPK activity was increased, as determined here by the phosphorylation of the AMPK substrate ACC, in cells expressing AMPKCA (Fig. 7A). Next, we have analyzed the rate of degradation of long-lived [14C]valine-labeled proteins in cells expressing AMPKCA. As shown in Fig. 7B, the rate of degradation of long-lived [14C]valine-labeled proteins and its sensitivity to autophagy inhibitors were similar to that observed in untransfected HT-29 cells (Fig. 6B) and in cells transfected with an empty vector (data not shown) when incubated in nutrient-free medium. From these findings we reasoned that the inhibition of AMPK activity should inhibit the rate of autophagic proteolysis in cells incubated in nutrient-free medium. For this purpose, we used two approaches. In a first approach, cells were transfected with the cDNA encoding a dominant-negative form of AMPK (AMPKDN). In a second approach, cells were treated with compound C. According to previous results (27Woods A. Azzout-Marniche D. Foretz M. Stein S.C. Lemarch

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C2-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B1. Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C2-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C2-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C2-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes. The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C2-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B1. Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C2-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C2-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C2-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes. Macroautophagy or autophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long lived proteins and organelles (1Seglen P.O. Bohley P. Experientia (Basel). 1992; 48: 158-172Google Scholar, 2Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Google Scholar, 3Klionsky D.T. Ohsumi Y. Annu. Rev. Cell Dev. 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Lullmann-Rauch R. von Figura K. Trends Mol. Med. 2001; 7: 37-39Google Scholar).A family of autophagy-related genes discovered in yeast and almost integrally conserved in all eucaryotic phyla controls the formation of the autophagosome (25Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Google Scholar). Two conjugation systems (Atg5p-Atg12p and Atg8p lipidation) are involved in the formation of the autophagosome (26Ohsumi Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 211-216Google Scholar) together with a class III phosphatidylinositol 3-kinase (class III PI3K, 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; C2-Cer, C2-ceramide, C2-DHCer, C2-dihydroceramide; DGK, diacylglycerol kinase; DMEM, Dulbecco's modified Eagle's medium; FB1, fumonisin B1; HBSS, Hanks' balanced salt solution; IL-13, Interleukin-13; 3-MA, 3-methyladenine; MDC, monodansylcadaverine; Myrio, Myriocin; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PKB, protein kinase B; RT-PCR, reverse transcriptase-PCR; PCD, programmed cell death; TAM, tamoxifen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; C2-Cer, C2-ceramide, C2-DHCer, C2-dihydroceramide; DGK, diacylglycerol kinase; DMEM, Dulbecco's modified Eagle's medium; FB1, fumonisin B1; HBSS, Hanks' balanced salt solution; IL-13, Interleukin-13; 3-MA, 3-methyladenine; MDC, monodansylcadaverine; Myrio, Myriocin; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PKB, protein kinase B; RT-PCR, reverse transcriptase-PCR; PCD, programmed cell death; TAM, tamoxifen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. the homologue of the yeast Vps34) complex (27Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell. Biol. 2001; 152: 519-530Google Scholar, 28Kihara A. Kabeya Y. Ohsumi Y. Yoshimori T. EMBO Rep. 2001; 2: 330-335Google Scholar). In this complex, the protein beclin 1 (the orthologue of the yeast Atg6, see Ref. 29Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Google Scholar) is a tumor suppressor gene product (30Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. Cattoretti G. Levine B. J. Clin. Investig. 2003; 112: 1809-1820Google Scholar, 31Yue Z. Jin S. Yang C. Levine A.J. Heintz N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15077-15082Google Scholar). The drug 3-MA commonly used to inhibit the autophagic pathway (32Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1889-1892Google Scholar) interferes with the activity of class III PI3K to interrupt autophagy at the sequestration step (33Blommaart E.F.C. Krause U. Schellens J.P.M. Vreeling-Sindelárová H. Meijer A.J. Eur. J. Biochem. 1997; 243: 240-246Google Scholar, 34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar). Alternatively, the stimulation of the class I PI3K/PKB signaling pathway by growth factors and cytokines has an inhibitory effect on autophagy in many cell types (15Melendez A. Tallòczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Google Scholar, 34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar, 35Arico S. Petiot A. Bauvy C. Dubbelhuis P.F. Meijer A.J. Codogno P. Ogier-Denis E. J. Biol. Chem. 2001; 276: 35243-35246Google Scholar, 36Kanazawa T. Taneike I. Akaishi R. Yoshizawa F. Furuya N. Fujimura S. Kadowaki M. J. Biol. Chem. 2004; 279: 8452-8459Google Scholar). This inhibitory effect is probably due to the class I PI3K/PKB-dependent activation of the kinase target of rapamycin which down-regulates autophagy (37Blommaart E.F.C. Luiken J.J.F.P. Blommaart P.J.E. Vanwoerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Google Scholar, 38Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Google Scholar, 39Abeliovich H. Dunn W.A. Kim J. Klionsky D.J. J. Cell. Biol. 2000; 151: 1025-1033Google Scholar, 40Abeliovich H. Zhang C. Dunn Jr., W.A. Shokat K.M. Klionsky D.J. Mol. Biol. Cell. 2003; 14: 477-490Google Scholar).Ceramide is a sphingolipid mediator with an essential role in cell growth, cell death, proliferation, and stress response (41Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Google Scholar, 42Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Google Scholar, 43Levade T. Malagarie-Cazenave S. Gouazé V. Ségui B. Tardy C. Betito S. Andrieu-Abadie N. Cuvillier O. Neurochem. Res. 2002; 27: 601-607Google Scholar). As all of these different situations are correlated with modulation of autophagy (14Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Google Scholar), we have investigated the potential role of ceramide in regulating autophagy.Ceramide can be generated and consumed by different metabolic routes (41Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Google Scholar, 42Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Google Scholar, 44Merrill A.H.J. J. Biol. Chem. 2002; 277: 25843-25846Google Scholar, 45Riboni L. Viani P. Bassi R. Prinetti A. Tettamenti G. Prog. Lipid Res. 1997; 36: 153-195Google Scholar). Ceramide is produced by de novo synthesis in the endoplasmic reticulum or by the hydrolysis of sphingomyelin by acid sphingomyelinases, localized in acidic compartments and neutral sphingomyelinases, and localized in the plasma membrane and mitochondria. Ceramide is engaged the biosynthesis of glucosylceramide (and other complex glycosphingolipids) and of sphingomyelin. Ceramide can also generate ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. Sphingosine 1-phosphate is a second messenger that often has an opposite effect to ceramide on biological outcomes (46Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Google Scholar).Here we show that, in two different cell lines, ceramide stimulates autophagy by two non-exclusive mechanisms. In human colon cancer HT-29 cells, C2-Cer, a cell-permeable ceramide analogue, stimulates autophagy by increasing the intracellular pool of long chain ceramides. Ceramide reverts the inhibition of the class I PI3K signaling pathway on autophagy by interfering with the IL-13-dependent activation of protein kinase B (PKB) (34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar) and stimulates the expression of beclin 1. Ceramide also mediates the TAM-dependent accumulation autophagic vacuoles observed in human breast cancer MCF-7 cells (47Bursch W. Hochegger K. Torok L. Marian B. Ellinger A. Hermann R.S. J. Cell Sci. 2000; 113: 1189-1198Google Scholar). The ceramide-dependent expression of beclin 1 in TAM-treated cells was impaired in the presence of Myrio, an inhibitor of the serine palmitoyltransferase, the rate-limiting enzyme of de novo synthesis of ceramide. By contrast, the expression of beclin 1 was stimulated when MCF-7 cells were treated with PDMP, an inhibitor of glucosylceramide synthase. In addition, tamoxifen and C2-Cer inhibit the activation of protein kinase B, whereas myriocin antagonizes the inhibitory effect of tamoxifen. These data suggest a novel function for ceramide in controlling a major lysosomal pathway and provide a novel molecular link between autophagy and cell response to stress.EXPERIMENTAL PROCEDURESReagents—C2-Cer and C2-DHCer were from Calbiochem and were dissolved in ethanol before use. FB1, Myrio, TAM, 3-MA, MDC were purchased from Sigma. Cell culture medium and fetal bovine serum were from Invitrogen. Nitrocellulose membranes were from Schleicher & Schüll. Ceramide from porcine brain, used for internal standard, was from Avanti Polar Lipids Inc. (Alabaster, AL). The radioisotopes l-[U-14C]valine (256 mCi/mmol) and [γ-32P]ATP (3 Ci/μmol), the ECL™ Western blotting detection kit, and the donkey anti-rabbit antibody were purchased from Amersham Biosciences. The polyclonal rabbit anti-phospho-PKB Ser473 was from Cell Signaling, and goat anti-PKB was from Santa Cruz Biotechnology. IL-13 was kindly provided by Adrian Minty (Sanofi-SyntheLabo, France). The rabbit anti-beclin 1 was kindly provided by Tamotsu Yoshimori (National Institute of Genetics, Shizuoka, Japan). The monoclonal mouse anti-actin was from Chemicon International Inc. (Temecula, CA). Goat anti-mouse and swine anti-goat antibodies were obtained from Bio-Rad and Caltag (Burlingame, CA), respectively.Cell Culture and Viability—Human breast cancer cell line MCF-7 was maintained at 37 °C in 10% CO2 in DMEM, supplemented with 5% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin. Human colon cancer cell line HT-29 was maintained at 37 °C in 10% CO2 in DMEM, supplemented with 10% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin as reported previously (34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar). Cell viability was determined by the trypan blue exclusion test.Analysis of Protein Degradation—HT-29 cells were incubated for 24 h at 37 °C with 0.2 μCi/ml of l-[14C]valine. Three hours before the end of the radiolabeling period, cells were treated with increasing concentrations of C2-Cer or C2-DHCer, and when required 100 μm FB1 was added at the same time. At the end of radiolabeling period, cells were washed three times with phosphate-buffered saline, pH 7.4, and then incubated in nutrient-free medium (HBSS plus 0.1% of bovine serum albumin and 10 mm cold valine). After 16 h of incubation, at which time short lived proteins were being degraded, the medium was replaced with fresh nutrient-free medium, and when required 10 mm 3-MA or 30 ng/ml of IL-13 was added, and the incubation was continued for an additional 4-h period. Cells and radiolabeled proteins from the 4-h chase medium were precipitated in trichloroacetic acid at a final concentration of 10% (v/v) at 4 °C. The precipitated proteins were separated from the soluble radioactivity by centrifugation at 600 × g for 10 min and then dissolved in 0.5 ml of 0.2 n NaOH. Radioactivity was determined by liquid scintillation counting. Protein degradation was calculated by dividing the acid-soluble radioactivity recovered from both cells and medium by the radioactivity contained in precipitated proteins from both cells and medium (34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar).MDC Staining of Autophagic Vacuoles—MCF-7 cells were incubated for 4 days with 1 μm of TAM dissolved in Me2SO/EtOH (1:1, v/v). When required 100 nm Myrio dissolved in methanol was added together with TAM. Alternatively, cells were incubated for 4 days with 20 μm PDMP in the absence of TAM. MDC staining was carried out essentially as described previously (48Biederbick A. Kern H.F. Elsasser H.P. Eur. J. Cell. Biol. 1995; 66: 3-14Google Scholar). An MDC stock solution (0.1 m in Me2SO) was diluted 1:1000 in DMEM and applied to the cells for 30 min at 37 °C. After washing with phosphate-buffered saline, cells were examined by fluorescence microscopy (Axioplan, Zeiss).Electron Microscopy and Morphometry—Electron microscopy and morphometric analysis were performed as described previously (49Petiot A. Ogier-Denis E. Bauvy C. Cluzeaud F. Vandewalle A. Codogno P. Biochem. J. 1999; 337: 289-295Google Scholar). Cells were fixed for 30 min with 2.5% glutaraldehyde in 0.1 m cacodylate buffer, embedded in Epon, and processed for transmission electron microscopy by standard procedures. For morphometric analysis, 20 micrographs per condition were used.Quantification of Endogenous Ceramide—Total endogenous ceramide levels were measured using the DGK assay as described previously (50Perry D.K. Hannun Y.A. Trends Biochem. Sci. 1999; 24: 226-227Google Scholar, 51Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Google Scholar). Cells were collected, and lipids were extracted according to Bligh and Dyer (52Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). The organic phase was divided in 1/2 and 1/6 aliquots, dried, and used for ceramide measurement and total phospholipids measurement (53Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Google Scholar), respectively. Briefly, 30 nmol of extracted lipids were incubated at room temperature for 45 min in the presence of β-octyl glucoside/dioleoylphosphatidylglycerol micelles, 2 mm dithiothreitol, 6 μg of proteins of DGK containing membranes (Calbiochem), and 1 mm ATP mixed with [γ-32P]ATP (13 μCi/ml) in a final volume of 0.1 ml. At the end of the reaction, lipids were extracted according to Bligh and Dyer (52Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). [32P]Ceramide 1-phosphate was determined by TLC separation in chloroform/acetone/methanol/acetic acid/water, (10:4:3:2:1 by volume). The radioactivity associated with ceramide 1-phosphate spots was determined after scraping and counting in a scintillator counter. Ceramide levels were referred to the level of total phospholipids.Immunoblotting—Detection of total and phospho-PKB in HT-29 cells was performed as described previously (54Arico S. Pattingre S. Bauvy C. Gane P. Barbat A. Codogno P. Ogier-Denis E. J. Biol. Chem. 2002; 277: 27613-27621Google Scholar). After being resolved by 10% SDS-PAGE, proteins were transferred onto nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBST (10 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 0.1% Tween 20) for 1 h at room temperature and then incubated with appropriate primary antibody (polyclonal rabbit anti-phospho-PKB Ser473 at 1:1000 and polyclonal goat anti-PKB 1:1000) overnight at 4 °C in 1% bovine serum albumin, 0.1% TBST. After three washes in TBST, the membrane was incubated for 1 h at room temperature with the appropriate horseradish peroxidase-labeled secondary antibody (donkey anti-rabbit at 1:5000 and swine anti-goat at 1:2000). Bound antibodies were detected by ECL.For beclin 1 detection, cells were solubilized in 2× Laemmli buffer as described in Ref. 29Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Google Scholar. Proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membrane as described above. Immunoblotting was performed using a rabbit polyclonal anti-beclin 1 (1:3000), and after stripping the membrane was reprobed with a mouse monoclonal anti-actin (1:5000). Primary antibodies were detected using an horseradish peroxidase-labeled anti-rabbit (1:5000) and horseradish peroxidase-labeled anti-mouse (1:5000). Bound antibodies were detected by ECL. Fluorographs were quantitatively scanned using the NIH image software.Semi-quantitative RT-PCR of Beclin 1—RNA from 1.5 × 106 cells was extracted using the Tri-Reagent (Sigma) and then incubated with DNase I (Ambion) for 30 min at 37 °C. First strand cDNA synthesis was performed with 4 μg of RNA by using the SuperScript First Strand Synthesis System™ (Invitrogen). The following primers were used to amplify beclin 1 cDNA (5′-ccaggatggtgtctctcgca-3′/5′-ctgcgtctgggcataacgca-3′) and GAPDH cDNA (5′-cggagtcaacggatttggtcgtat-3′/5′-agccttctccatggtggtgaagac-3′). The thermal cycle conditions were 1 cycle for 2 min at 94 °C, followed by 27 cycles of 1 min at 94 °C, 1 min at 63 °C, and 1 min at 72 °C and terminated by 1 cycle for 5 min at 72 °C. Under these conditions PCR products were amplified in the linear range. GAPDH primers were added to the amplification mixture after the beginning of the seventh cycle, during the denaturation phase. Aliquots of each PCR were subjected to electrophoresis at 100 mV on a 2% agarose gel in the presence of 0.5 μg/ml ethidium bromide. The products of the amplification were revealed under UV light and photographed on Kodak paper. Photographs were quantified using the NIH Image software.Statistical Analysis—Statistical analysis of the differences between the groups was performed using Student's t test. p < 0.05 was considered statistically significant.RESULTSC2-Cer Has a Stimulatory Effect on Autophagy in HT-29 Cells—We have shown previously (55De Stefanis D. Reffo P. Bonelli G. Baccino F.M. Sala G. Ghidoni R. Codogno P. Isidoro C. Biol. Chem. Hoppe-Seyler. 2002; 383: 989-999Google Scholar) that hallmarks of apoptosis are detectable after 8 h of incubation of HT-29 cells in the presence of 100 μm C2-Cer. In order to investigate the effect of C2-Cer on the control of autophagy in viable cells, HT-29 were incubated with an increasing amount of C2-Cer (from 25 to 100 μm) for a limited time. Viability was minimally affected when cells were cultured for 3 h in complete medium with increasing concentrations of C2-Cer and then transferred to a nutrient-free medium in the presence or absence of 3-MA (Fig. 1A, upper panel). In addition, no signs of apoptosis were detectable (changes in nuclear morphology or activation of caspases, data not shown) under these conditions. A dose-dependent increase in the rate of protein degradation was observed in C2-Cer pre-treated cells (Fig. 1A, lower panel). In contrast, no significant change in proteolysis was detected when the same experiment was performed in cells pre-treated with the inactive analogue C2-DHCer. The increase in proteolysis induced by C2-Cer was essentially due to the augmentation in the fraction of protein degradation sensitive to 3-MA (Fig. 1A). This result suggested that C2-Cer stimulates autophagy in HT-29 cells. This conclusion was supported by morphometric analysis showing that the fractional volume occupied by autophagic vacuoles is significantly increased in cells treated with C2-Cer and then incubated either in nutrient-free medium or in complete medium (Fig. 1B).The Endogenous Ceramide Is Responsible for the Stimulatory Effect of C2-Cer on Autophagy in HT-29 Cells—Next we wanted to characterize the mechanism responsible for the effect of C2-Cer on autophagy. The first step toward this goal was to investigate whether the conversion of C2-Cer into endogenous long chain ceramides is critical to stimulate autophagy. It has been shown recently that short chain ceramides can be converted into long chain ceramides via the sequential deacylation of ceramide analogues into sphingosine and the subsequent reacylation with long chain fatty acids by ceramide synthase (56Ogretmen B. Pettus B. Rossi J. Wood R. Usta J. Szule Z. Bielawska A. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: 12960-12969Google Scholar). In a first set of experiments we have analyzed changes in the endogenous pool of long chain ceramides in response to C2-Cer treatment. To this end, HT-29 cells were incubated for 3 h in complete medium with 100 μm C2-Cer or 100 μm C2-DHCer in the presence or absence of 100 μm fumonisin (FB1), an inhibitor of the activity of ceramide synthase (44Merrill A.H.J. J. Biol. Chem. 2002; 277: 25843-25846Google Scholar). Thereafter, cells were incubated in nutrient-deprived medium with 10 mm 3-MA when required. Finally, the level of endogenous long chain ceramides was analyzed by the DGK assay. C2-Cer treatment induced a 1.5-fold increase in the endogenous pool of ceramide, whereas no change was observed in C2-DHCer-treated cells (Fig. 2A). The C2-Cer-dependent increase in endogenous long chain ceramides was totally blocked in the presence of FB1, whereas the autophagic inhibitor 3-MA has no effect on the accumulation of ceramide. From these results we concluded that C2-Cer induces an accumulation of endogenous long chain ceramides in HT-29 cells, and this accumulation is dependent on the recycling of the sphingosine moiety and the activity of ceramide synthase.Fig. 2The effect of C2-Cer on autophagy is mediated by an increase of the endogenous pool of long chain ceramides. A, endogenous long chain ceramides were quantified by the DGK assay as described under “Experimental Procedures.” The amount of ceramide (6.2 pmol of Cer/nmol of Pi) in HT-29 cells cultured in complete medium was set to 1. HT-29 cells were cultured for 3 h in complete medium with 100 μm C2-Cer in the presence or absence of 100 μm FB1 or with 100 μMC2-DHCer. 3-MA (10 mm) was added during the chase period in nutrient-free medium. After 20 h of incubation in nutrient-free medium, cells were harvested, and the ceramide content was analyzed by the DGK assay as described under “Experimental Procedures.” Values are the mean ± S.D. of four independent experiments. B, the rate of degradation of [14C]valine-labeled long lived proteins was measured in cells incubated in nutrient-free medium (HBSS) in the presence or absence of 10 mm 3-MA. C2-Cer (100 μm) or C2-DHCer (100 μm) was added during the last 3 h of incubation in complete medium. FB1 (100 μm) was added together with C2-Cer in complete medium. Values reported are the mean ± S.D. of four independent experiments. *, p < 0.05 versus untreated cells.View Large Image Figure ViewerDownload (PPT)In order to investigate whether the accumulation of long chain ceramides is responsible for the C2-Cer-induced autophagy, we have evaluated the effect of FB1 on protein degradation in C2-Cer-treated cells. We observed that in cells exposed to 100 μm C2-Cer for 3 h, the rate of proteolysis was increased by 30–35% when compared with untreated cells or to cells treated with C2-DHCer (Fig. 2B). The increase of protein degradation was totally blocked by 100 μm FB1 in C2-Cer-treated cells (Fig. 2B). In fact, FB1 was as potent as 3-MA in inhibiting proteolysis. In contrast to FB1, the inhibitory effect of 3-MA on proteolysis was achieved independently of the accumulation of endogenous long chain ceramides (Fig. 2, A and B). Both drugs have no cumulative effect on the inhibition of proteolysis (data not shown), suggesting that 3-MA, which interferes with the activity of class III PI3K (34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar), acts downstream FB1 in the same pathway to inhibit autophagy.Ceramide Interferes with the Class I PI3K Signaling Pathway to Stimulate Autophagy in HT-29 Cells—C2-Cer is able to stimulate the accumulation of autophagic vacuoles, whereas HT-29 cells were cultured in complete medium (Fig. 1B, b) suggesting that ceramide can relieve the inhibitory effect induced by growth factors. We have shown previously (34Petiot A. Ogier-Denis E. Blommaart E.F.C. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Google Scholar, 35Arico S. Petiot A. Bauvy C. Dubbelhuis P.F. Meijer A.J. Codogno P. Ogier-Denis E. J. Biol. Chem. 2001; 276: 35243-35246Google Scholar) that the stimulation of the class I PI3K/PKB signaling pathway by IL-13 has a strong inhibitory effect on autophagy. In addition, ceramide is known to interfere with the activation of PKB in different cell types (57Zhou H. Summers S.A. Birnbaum M.J. Pittman R. J. Biol. Chem. 1998; 273: 16568-16575Google Scholar, 58Summers S.A. Garza L.A. Zhou H. Birnbaum M.J. Mol. Cell. Biol. 1998; 18: 5457-5464Google Scholar, 59Schubert K.M. Scheid M.P. Duriono V. J. Biol. Chem. 2000; 275: 13330-13335Google Scholar, 60Bourbon N.A. Sandirasegarane

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signalling motivated us to explore the possible functional interactions between this signalling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signalling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. Whereas abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signalling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie some common ciliopathies.

Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed 'mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1(+)) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.

Defects in autophagy have been linked to a wide range of medical illnesses, including cancer as well as infectious, neurodegenerative, inflammatory, and metabolic diseases. These observations have led to the hypothesis that autophagy inducers may prevent or treat certain clinical conditions. Lifestyle and nutritional factors, such as exercise and caloric restriction, may exert their known health benefits through the autophagy pathway. Several currently available FDA-approved drugs have been shown to enhance autophagy, and this autophagy-enhancing action may be repurposed for use in novel clinical indications. The development of new drugs that are designed to be more selective inducers of autophagy function in target organs is expected to maximize clinical benefits while minimizing toxicity. This Review summarizes the rationale and current approaches for developing autophagy inducers in medicine, the factors to be considered in defining disease targets for such therapy, and the potential benefits of such treatment for human health.

Autophagy is a self-eating catabolic pathway that contributes to liver homeostasis through its role in energy balance and in the quality control of the cytoplasm, by removing misfolded proteins, damaged organelles and lipid droplets. Autophagy not only regulates hepatocyte functions but also impacts on non-parenchymal cells, such as endothelial cells, macrophages and hepatic stellate cells. Deregulation of autophagy has been linked to many liver diseases and its modulation is now recognized as a potential new therapeutic strategy. Indeed, enhancing autophagy may prevent the progression of a number of liver diseases, including storage disorders (alpha-1 antitrypsin deficiency, Wilson's disease), acute liver injury, non-alcoholic steatohepatitis and chronic alcohol-related liver disease. Nevertheless, in some situations such as fibrosis, targeting specific liver cells must be considered, as autophagy displays opposing functions depending on the cell type. In addition, an optimal therapeutic time-window should be identified, since autophagy might be beneficial in the initial stages of disease, but detrimental at more advanced stages, as in the case of hepatocellular carcinoma. Finally, identifying biomarkers of autophagy and methods to monitor autophagic flux in vivo are important steps for the future development of personalized autophagy-targeting strategies. In this review, we provide an update on the regulatory role of autophagy in various aspects of liver pathophysiology, describing the different strategies to manipulate autophagy and discussing the potential to modulate autophagy as a therapeutic strategy in the context of liver diseases.

The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers--even those who work in the field--to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C(2)-ceramide and C(6)-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr(69), Ser(70), and Ser(87), located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.

Autophagy is a versatile degradation system for maintaining cellular homeostasis whereby cytosolic materials are sequestered in a double-membrane autophagosome and subsequently delivered to lysosomes, where they are broken down. In multicellular organisms, newly formed autophagosomes undergo a process called 'maturation', in which they fuse with vesicles originating from endolysosomal compartments, including early/late endosomes and lysosomes, to form amphisomes, which eventually become degradative autolysosomes. This fusion process requires the concerted actions of multiple regulators of membrane dynamics, including SNAREs, tethering proteins and RAB GTPases, and also transport of autophagosomes and late endosomes/lysosomes towards each other. Multiple mechanisms modulate autophagosome maturation, including post-translational modification of key components, spatial distribution of phosphoinositide lipid species on membranes, RAB protein dynamics, and biogenesis and function of lysosomes. Nutrient status and various stresses integrate into the autophagosome maturation machinery to coordinate the progression of autophagic flux. Impaired autophagosome maturation is linked to the pathogenesis of various human diseases, including neurodegenerative disorders, cancer and myopathies. Furthermore, invading pathogens exploit various strategies to block autophagosome maturation, thus evading destruction and even subverting autophagic vacuoles (autophagosomes, amphisomes and autolysosomes) for survival, growth and/or release. Here, we discuss the recent progress in our understanding of the machinery and regulation of autophagosome maturation, the relevance of these mechanisms to human pathophysiology and how they are harnessed by pathogens for their benefit. We also provide perspectives on targeting autophagosome maturation therapeutically.

The modulation of macroautophagy is now recognized as one of the hallmarks of cancer cells. There is accumulating evidence that autophagy plays a role in the various stages of tumorigenesis. Depending on the type of cancer and the context, macroautophagy can be tumor suppressor or it can help cancer cells to overcome metabolic stress and the cytotoxicity of chemotherapy. Recent studies have shed light on the role of macroautophagy in tumor-initiating cells, in tumor immune response cross-talk with the microenvironment. This review is intended to provide an up-date on these aspects, and to discuss them with regard to the role of the major signaling sub-networks involved in tumor progression (Beclin 1, MTOR, p53 and RAS) and in regulating autophagy.

With regard to cell biology, one area of focus that has shifted back and forth over the years has been the relative emphasis on catabolic versus anabolic processes: the breakdown of glucose, the synthesis of DNA, the oxidation of pyruvate, the biogenesis of membranes, protein degradation, and protein synthesis. Historically, the majority of studies concerned with degradation dealt with the production of energy; however, the analysis of the ubiquitin-proteasome system revealed the importance of protein degradation for controlling various aspects of cell physiology. The ubiquitin-proteasome system is limited primarily to targeting individual proteins for destruction, but cells also have to deal with larger structures that are damaged, potentially toxic or superfluous, and these substrates, including entire organelles, are the purview of autophagy. As a general definition, autophagy encompasses a range of processes in which the cell degrades parts of itself within the lysosome (or the analogous organelle, the vacuole, in yeast and plants), followed by the release and reuse of the breakdown products. Thus, autophagy is in part a mechanism for cellular recycling, but such a definition belies the importance of the different autophagic processes in cell and organismal function and homeostasis. Indeed, defects in autophagy are associated with many human diseases and metabolic disorders. Here, we provide a brief overview of the mechanism of autophagy and some of the physiological roles in which this process is involved.

Summary Both at a basal level and after induction (especially in response to nutrient starvation), the function of autophagy is to allow cells to degrade and recycle damaged organelles, proteins and other biological constituents. Here, we focus on the role microtubules have in autophagosome formation, autophagosome transport across the cytoplasm and in the formation of autolysosomes. Recent insights into the exact relationship between autophagy and microtubules now point to the importance of microtubule dynamics, tubulin post-translational modifications and microtubule motors in the autophagy process. Such factors regulate signaling pathways that converge to stimulate autophagosome formation. They also orchestrate the movements of pre-autophagosomal structures and autophagosomes or more globally organize and localize immature and mature autophagosomes and lysosomes. Most of the factors that now appear to link microtubules to autophagosome formation or to autophagosome dynamics and fate were identified initially without the notion that sequestration, recruitment and/or interaction with microtubules contribute to their function. Spatial and temporal coordination of many stages in the life of autophagosomes thus underlines the integrative role of microtubules and progressively reveals hidden parts of the autophagy machinery.

Significance Atherosclerotic plaques tend to develop preferentially in areas of the vasculature exposed to low and disturbed shear stress (SS), but the mechanisms are not fully understood. In this study, we demonstrate that inefficient autophagy contributes to the development of atherosclerotic plaques in low-SS areas. Defective endothelial autophagy not only curbs endothelial alignment with the direction of blood flow, but also promotes an inflammatory, apoptotic, and senescent phenotype. Furthermore, genetic inactivation of endothelial autophagy in a murine model of atherosclerosis increases plaque burden exclusively in high-SS areas that are normally resistant to atherosclerotic plaque development. Altogether, these findings underline the role of endothelial autophagic flux activation by SS as an atheroprotective mechanism.

Phosphatidylinositol‐3‐phosphate ( PI 3P) is a key player in membrane dynamics and trafficking regulation. Most PI 3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI 3P synthesis, VPS 34 and proteins such as Beclin1 and ATG 14L that regulate PI 3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI 3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI 3P‐binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI 3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI 3P during autophagy, notably during the autophagosome biogenesis sequence.

Article26 May 2017free access Transparent process ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis Anna Chiara Nascimbeni Anna Chiara Nascimbeni Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Francesca Giordano Francesca Giordano orcid.org/0000-0002-5942-1753 Institut Jacques Monod, CNRS UMR 7592, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Daniel Grasso Daniel Grasso Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Maria I Vaccaro Maria I Vaccaro Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Patrice Codogno Patrice Codogno Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Etienne Morel Corresponding Author Etienne Morel [email protected] orcid.org/0000-0002-4763-4954 Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Anna Chiara Nascimbeni Anna Chiara Nascimbeni Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Francesca Giordano Francesca Giordano orcid.org/0000-0002-5942-1753 Institut Jacques Monod, CNRS UMR 7592, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Daniel Grasso Daniel Grasso Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Maria I Vaccaro Maria I Vaccaro Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Search for more papers by this author Patrice Codogno Patrice Codogno Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Etienne Morel Corresponding Author Etienne Morel [email protected] orcid.org/0000-0002-4763-4954 Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Author Information Anna Chiara Nascimbeni1,2, Francesca Giordano3,4, Nicolas Dupont1,2, Daniel Grasso5, Maria I Vaccaro5, Patrice Codogno1,2 and Etienne Morel *,1,2 1Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, Paris, France 2Université Paris Descartes-Sorbonne Paris Cité, Paris, France 3Institut Jacques Monod, CNRS UMR 7592, Paris, France 4Université Paris Diderot-Sorbonne Paris Cité, Paris, France 5Department of Pathophysiology, Institute of Biochemistry and Molecular Medicine, National Council for Scientific and Technological Research, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina *Corresponding author. Tel: +33 172606474; Fax: +33 172606399; E-mail: [email protected] The EMBO Journal (2017)36:2018-2033https://doi.org/10.15252/embj.201797006 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. Synopsis Early autophagic markers are recruited to endoplasmic reticulum-plasma membrane (ER-PM) contact sites established by tethering factors extended synaptotagmins, allowing for local phosphatidylinositol-3-phosphate synthesis and autophagosome biogenesis. Autophagy induction is accompanied by ER-PM contact site mobilization. E-Syt2, a major tethering protein of ER-PM contact sites, forms a complex with VMP1 and Beclin1, two regulators of PI3KC3 complex activity. Local autophagosome biogenesis is initiated by local PI3P synthesis via the targeting of PI3KC3 complex at ER-PM contact sites. Introduction Macro-autophagy (hereafter referred to as autophagy) is a highly regulated intracellular degradation pathway necessary for cellular homeostasis (Boya et al, 2013). Autophagy is initiated by the formation of a specific double-membrane organelle called the autophagosome. The biogenesis of autophagosome is orchestrated by multiple signalling pathways and complexes that regulate membrane dynamics that contain autophagy-related (ATG) proteins. Autophagy initiates with biogenesis of a pre-autophagosomal double-membrane structure, termed the phagophore, which emanates from the omegasome, a subdomain of the endoplasmic reticulum (ER) membrane positive for PI3P (phosphatidyl-inositol-3-phosphate) and PI3P-binding proteins (Axe et al, 2008). The PI3P pool engaged in autophagosome biogenesis is synthesized by the class 3 PI3kinase complex (PI3KC3), comprised of VPS34, VPS15, ATG14L, Beclin1, and regulating adaptors, such as VMP1, NRBF2 and Ambra1, and is dependent on ULK1 complex signalling (Nascimbeni et al, 2017). The phagophore then elongates and is close to form a mature autophagosome that will latter fuse with the lysosome. Although the ER membrane requirement is well established, other membrane sources, like the Golgi apparatus, endosomes, the mitochondria and the plasma membrane (PM), have been proposed to participate, directly, indirectly or partially, in autophagosome biogenesis (Molino et al, 2017), from phagophore generation to growth of the organelle (Ravikumar et al, 2010a,b; Rubinsztein et al, 2012). The ER is a dynamic and complex membranous network that extends throughout the cell impacting a multiplicity of cellular functions (Friedman & Voeltz, 2011). There is growing evidence that close appositions between the ER and the membranes of virtually all other organelles play major roles in cell physiology (Helle et al, 2013). Notably, ER–mitochondria contact sites actively participate in autophagosome biogenesis via the regulation of PI3KC3 complex (Hamasaki et al, 2013). ER-PM contact sites are important for lipid metabolism and transport, notably of phosphoinositides, and these domains have the potential to affect membrane trafficking and signalling events that occur at the PM (Stefan et al, 2013). In higher eukaryotes, three ER-localized proteins, the extended synaptotagmins (E-Syts 1, 2 and 3), play crucial roles in tethering the ER to the PM and are thus considered as key regulators, as well as precise markers, of ER-PM tethering zones (Giordano et al, 2013). Because both ER and PM have been directly associated with autophagy regulation and because ER tethering could be important for membrane remodelling, we hypothesized that the ER cooperates with plasma membrane during the very first steps of the autophagosome biogenesis via the establishment of ER-PM specialized contact sites. Indeed, we show here that stress situations that induce autophagy lead as well to ER-PM contact site mobilization, highlighting a connection between ER-PM tethering and the autophagy machinery. We observed local recruitment of autophagic and pre-autophagic markers at E-Syts domains of the cortical ER during autophagy initiation. Further, autophagy was enhanced in E-Syt-overexpressing cells, whereas inhibition of E-Syts expression reduced autophagosome biogenesis. Finally, we demonstrated that ER-PM contact sites are required for local PI3P synthesis by the PIK3C3 complex. We found that the PIK3C3 complex at ER-PM contact sites is mobilized at the ER membrane via the binding of VMP1 (Molejon et al, 2013a), the ER partner of Beclin1, the major regulator of autophagy-associated PI3P synthesis. Results We first studied the behaviour of ER-PM contact sites in conditions that promote autophagy using a HRP-myc-KDEL reporter that allows indirect visualization of ER lumen by electron microscopy (EM; Giordano et al, 2013). We analysed and quantified ER-PM contact zones in control and in HeLa cells starved to induce autophagy. We observed a massive increase in the number of ER-PM contact sites compared to control situation (Fig 1A and B), and this increase correlated with autophagy induction (Fig 1C). Levels of E-Syt2 and 3 proteins increased after 1 and 4 h of starvation, whereas levels of calnexin (an ER marker), syntaxin 17 [STX17, previously identified as a marker of an autophagy-related ER–mitochondria contact sites (Hamasaki et al, 2013)] or PTPIP51 [an ER–mitochondria tethering protein recently shown to participate in autophagy regulation (Gomez-Suaga et al, 2017)] were not affected (Fig 1D and E). The increase in E-Syt2 was also induced by mechanical stress in KEC cells (Fig EV1A), a condition that promotes autophagy (Orhon et al, 2016), and by serum starvation or mTOR chemical inhibition (Fig EV1B). Figure 1. Starvation increases ER-PM contact sites density A. Electron micrographs of HeLa cells grown under complete medium conditions (control, ctrl) or starved for 1 h (1 h STV). HeLa cells were transfected with the ER luminal marker ssHRP-myc-KDEL (which enables ER identification via an electron-dense (dark) HRP reaction) to allow detection of ER-PM contact sites (black arrows). Scale bar, 2 μm. Representative images from one of three independent experiments are shown. B. Quantification of ER-PM contact sites visualized in electron micrographs with 20 cells analysed per condition in each of three experiments. Means ± s.e.m. are plotted. ***P < 0.001, unpaired two-tailed t-test. C–E. HeLa cells were grown under control and starvation conditions for 1 and 4 h. Representative Western blots of lysates for (C) p62 and lipidated LC3 and (D) E-Syts, calnexin (CLNX), STX17, and PTPIP51 are shown. Actin was used as a loading control. (E) Quantification of Western blots from three independent experiments. Means ± s.e.m. are plotted. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Multiple autophagy stimuli induce E-Syt2 expression Mechanical stress: Western blots and protein quantifications of protein lysates from KEC cells under basal (ctrl) and mechanical stress (flow) conditions for 4–72 h (n = 3). Plotted are mean ± s.d. mTOR inhibition and serum starvation: Western blots and protein quantifications (compared to ctrl) of protein lysates from HeLa cells grown under basal (ctrl) and autophagy-inducing conditions. Cells were cultured with 1.5 μM Torin1, an mTOR inhibitor, for 2 h or in a medium without serum for 1 h, respectively (n = 3). Plotted are mean ± s.d. Download figure Download PowerPoint Since our data suggested that autophagy and formation of ER-PM contact sites are stimulated in the same time frame, we next investigated whether autophagosomal markers could be detected at the cortical ER, near the PM. Indeed, in HeLa cells, we detected the phagophore, autophagosome and autolysosome marker LC3 near the cell boundary as early as 15 min post-starvation, a time at which most of LC3 was associated with the ER marker Sec61β (Fig 2A). The presence of LC3 in the vicinity of the plasma membrane was further confirmed by total internal reflection fluorescence (TIRF) microscopy (Fig 2B). The number of LC3 puncta in the TIRF zone increased with time under starvation conditions (Fig 2B and C), showing that LC3 autophagic structures appear at the immediate vicinity of the PM during starvation-induced autophagy. A similar LC3 pattern was observed in MDCK cells under mechanical stress (Orhon et al, 2016; Fig 2D). These results indicated that in different cell types treated with different autophagy inducers, LC3 staining was detected very close to the PM, suggesting that these autophagic structures might be associated with ER-PM contact sites. Figure 2. Early autophagic structures are detected in the vicinity of the plasma membrane HeLa cells were transfected with RFP-Sec61β (an ER marker) and immunostained for the autophagosome marker LC3. The two markers co-distribute in the vicinity of the plasma membrane (empty arrowheads) after 15 min of starvation (STV 15 min) as shown by confocal microscopy. TIRF analysis after 0 (control), 15 (STV 15 min) and 60 (STV 60 min) min of starvation. Quantification of LC3 puncta per TIRF section (n = 3; 20 cells analysed per condition). **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Means ± s.e.m. are plotted. Representative confocal microscopy images and 3D reconstructions of MDCK cells immunostained for LC3, Na/K-ATPase and DAPI under mechanical stress conditions. Arrowheads indicate LC3 puncta at vicinity of plasma membrane. Data information: Scale bars, 10 and 4 μm (magnified area in A). Download figure Download PowerPoint To analyse further whether ER-PM contact zones are sites of autophagosome formation, we used tagged E-Syt2 and E-Syt3 proteins as markers of ER-PM contact sites: while E-Syt1 can be detected on perinuclear as well as cortical ER structures, E-Syt2 and E-Syt3 localizations are restricted only to cortical ER engaged in ER-PM contact sites (Appendix Fig S1A and Fernández-Busnadiego et al (2015); Giordano et al (2013)). In HeLa cells starved for 15 min, we observed co-distribution of LC3 with the ER markers Sec61βRFP and E-Syt2GFP by confocal microscopy (Fig EV2A and C) at the basal level of the cells and by super-resolution two-colour stimulated emission depletion (STED) microscopy (Fig 3A). 3D reconstructions showed that LC3 was often directly connected to the ER membrane via E-Syt2-positive ER domains (Fig 3B) and sometimes appeared within a membranous niche positive for Sec61βRFP and E-Syt2GFP (Fig EV2A). Similar results were obtained when we used an antibody to ATG16L1 (Fig EV2B and C), a regulator of autophagosome biogenesis known to participate in the early events of LC3 recruitment to omegasome/phagophore structures (Wilson et al, 2014). The LC3-positive structures that were in the vicinity of the PM were negative for Rubicon (Appendix Fig S2), excluding the possibility of a non-autophagy-related LC3-associated phagocytosis (Levine et al, 2015). Using immunogold EM, we clearly observed E-Syt2myc and LC3GFP co-distribution on ER-PM contact sites in HeLa cells starved for 60 min (Fig EV2D); these are likely the same autophagic structures that we observed directly by electron microscopy in the immediate vicinity of cortical ER and PM in the same conditions (Fig 3C). Click here to expand this figure. Figure EV2. LC3 and ATG16L1 reside at ER-PM contact sites under starvation conditions A, B. Confocal microscope images and 3D reconstructions of HeLa cells co-transfected with GFP-E-Syt2 and RFP-Sec61β and immunostained for LC3 or ATG16L1 and DAPI. Arrowheads denote LC3 or ATG16L1 puncta near the E-Syt2-positive niche of the ER. Scale bars, 5 and 2.5 μm (magnified areas). C. Quantification of LC3 and ATG16L1 co-distribution with E-Syt2 at basal plan of the cell n = 80 cells. Mean ± s.e.m. shown. D. Immunogold electron micrographs of starved HeLa cells, showing co-distribution of the myc-E-Syt2 and anti-LC3 antibody at ER (empty arrowheads) and PM (black arrowheads) juxtaposition sites. Scale bar, 250 nm. Download figure Download PowerPoint Figure 3. Autophagosomes can form at ER-PM contact sites STED images of the basal plane of a HeLa cell co-transfected with vectors for expression of the ER marker Sec61βGFP and the ER-PM contact marker mCherry-E-Syt3 and immunostained for the autophagosome marker LC3. Scale bars, 5 μm. Arrowheads indicate autophagic structures (LC3) arising from ER niches (Sec61β-positive) at ER-PM contact sites (E-Syt3-positive). Three-dimensional reconstruction from confocal microscope images of HeLa cells co-transfected with vectors for expression of Sec61βRFP and E-Syt2GFP and immunostained for LC3. Scale bar, 2.5 μm. The arrowheads indicate the LC3-positive structures. Electron micrographs of HeLa cells starved 1 h and transfected with a vector for expression of the ER luminal marker ssHRP-myc-KDEL, showing early autophagic structures in the proximity of the PM. Scale bar, 400 nm. Download figure Download PowerPoint We next analysed distribution of the omegasome-marker (Axe et al, 2008) DCFP1GFP in HeLa cells after a short time of starvation, to maximize detection of autophagosome biogenesis-related events. We clearly observed co-distribution of DFCP1 with membranes positive for E-Syts, LC3 and Sec61βRFP by confocal microscopy (Fig 4A and B) and by time-lapse microscopy (Fig 4C). These results strongly suggest that at least some autophagosome biogenesis occurs at ER-PM contact sites. We then quantified the E-Syt2-positive omegasome structures and the omegasome structures at ER-mitochondria contact sites identified by mitochondrial protein TOM20 (Hamasaki et al, 2013). Our results indicate that, within 15 min of autophagy induction, approximately 30% of DFCP1-positive structures were associated with E-Syt2-positive domains (Fig EV3A and C), a ratio very close to the one we observed for DFCP1-positive ER–mitochondria contact sites (Fig EV3B and C). These results were further confirmed by electron microscopy analyses (Fig EV3D). Together, these data demonstrate that autophagosome assembly at ER-PM contact sites domains accounts for approximately 30% of total autophagosomes observed after a 15-min starvation of HeLa cells. Figure 4. LC3 and DFCP1 are present at ER-PM contact sites under starvation conditions Representative confocal microscopy images taken in the basal plane of a HeLa cell expressing myc-E-Syt2, RFP-Sec61β and GFP-DFCP1 and immunostained for LC3. Scale bars, 10 and 3 μm (magnified areas). 3D reconstructions of representative HeLa cell expressing myc-E-Syt2, RFP-Sec61β and GFP-DFCP1 and immunostained for LC3. Arrowheads denote DFCP1 and LC3 puncta connected with E-Syt2-positive niches of the ER. Scale bar, 5 μm. Time-lapse confocal images of HeLa cells expressing mCherry-E-Syt3 and GFP-DFCP1 after cells were starved for 15 min. Two channels were observed simultaneously using two cameras. Arrowheads denote DFCP1 puncta in E-Syt2-positive niches of the ER. Scale bar, 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Early autophagic structures form at ER-PM and ER–mitochondria contact sites A, B. Confocal microscope images of HeLa cells co-transfected with GFP-DFCP1 (an omegasome marker), RFP-Sec61β (an ER marker) and myc-E-Syt2 (an ER-PM contact sites marker) or immunostained for TOM20 (a mitochondria marker) under starved conditions (15 min). Arrowheads denote omegasome formation at (A) ER-PM or (B) ER–mitochondria contact sites. Scale bars, 10 μm. C. Quantification of percent DFCP1-positive structures at ER-PM (Sec61β/E-Syt2 interface) and ER–mitochondria (Sec61β/TOM20 interface) contact sites. Plotted are mean ± s.e.m., n = 80. D. Electron microscopy images of HeLa cells transfected with the ER luminal marker ssHRP-myc-KDEL and starved for 1 h showing autophagic structures adjacent (within 1 μm) to ER-PM contact sites (ER-PM), ER-mitochondria contact sites (ER-mito) and to neither organelle (cytoplasm). The quantification of these autophagic structures is shown as well. Plotted are means ± s.e.m., n = 80 cells. Download figure Download PowerPoint As previously reported (Giordano et al, 2013), overexpression of E-Syt2 or E-Syt3 stabilized and increased the density of ER-PM contact sites (Appendix Fig S2). In cells overexpressing E-Syts, the lipidation of LC3 was increased and more LC3-positive structures were observed both in fed and starved conditions compared to mock-transfected cells (Fig 5A and B). Interestingly, LC3 puncta were significantly increased in the vicinity of the PM in E-Syt3-overexpressing cells (Fig 5C). Electron microscopy analyses showed twice as many autophagic structures in HeLa cells overexpressing E-Syt2 as in control cells (Fig EV4A). In functional tests monitoring long-lived protein degradation, which depends on autophagy, we observed a significant increase of protein degradation in E-Syt2- and E-Syt3-overexpressing cells, as compared to control cells (Fig EV4B). Thus, the observed autophagic structures originating from the ER-PM contact zones appear to be functional. Figure 5. Overexpression of E-Syt2 and E-Syt3 induces autophagosome formation Western blot analysis of the autophagic flux in cell lysates from control (mock) and GFP-E-Syt2 or GFP-E-Syt3-expressing HeLa cells, under complete medium and starvation (1 h EBSS) conditions, without or with Bafilomycin A1 (+BAF. A1). HeLa cells expressing GFP-E-Syt2 or GFP-E-Syt3 were immunostained for LC3. Compared to control (mock), transfected cells showed a dramatic increase in LC3 puncta, in both basal (complete medium) and starved (1 h) conditions, as evidenced by counting of LC3 puncta (n = 3; 20 cells per condition). The increase in LC3 puncta observed in cells overexpressing E-Syt3GFP (similar results were obtained with E-Syt2GFP, data not shown) involves mainly peripheral rather than perinuclear cellular regions (n = 3). Arrowheads indicate peripheral LC3 puncta (n = 3; 20–70 cells per condition). Data information: Means ± s.e.m. are plotted. NS, non-significant, ***P < 0.001, unpaired two-tailed t-test. Scale bars, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Overexpression of E-Syt2 and E-Syt3 enhances autophagy An electron micrograph of GFP-E-Syt2-expressing HeLa cells starved for 1 h, showing an increased number of autophagic structures (arrowheads) compared to starved control cells. Scale bar, 1 μm. Wortmannin (wort, 100 nM) was used as a negative control, and autophagic structures were counted in 15 μm2 areas (n = 10 cells). Proteolysis analysis showing an increased protein degradation rate in GFP-E-Syt2- and GFP-E-Syt3-expressing cells (n = 3). 3-methyladenine (3-MA) was used at 10 mM. Data information: Means ± s.e.m. are plotted. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Download figure Download PowerPoint We then sought to test how impairing ER-PM contact sites formation would influence autophagy. To do this, we inhibited expression of the three E-Syt proteins (E-Syt1, E-Syt2 and E-Syt3) simultaneously using siRNAs targeting the mRNAs encoding each of these proteins (Fig 6A and B). Interestingly, in the E-Syt-deficient cells the total number of LC3GFP structures was decreased compared to control cells (Fig 6C and D). The difference was even more striking when we quantified the peripheral to perinuclear ratio of LC3 puncta in cells treated with Bafilomycin A1 (a V-ATPase inhibitor preventing fusion between autophagosome and lysosome) to maximize the number of autophagic structures (Fig 6C and D). The decrease observed in siE-Syt-treated cells was primarily due to decreases in numbers of peripheral puncta rather than to decreases in perinuclear LC3. Figure 6. Autophagosome biogenesis is reduced in E-Syt-deficient cells A, B. HeLa cells were treated with control siRNA (siCTRL) or with siRNAs targeting mRNAs encoding each of the E-Syts (siE-Syts). (A) Western blots of cell lysates. Actin is used as a loading control. (B) E-Syts mRNAs were quantified. Means ± s.d. are plotted (n = 3). C. HeLa cells stably transfected with GFP-LC3 and treated with siE-Syts or siCTRL were not treated (−BAF. A1) or were treated with Bafilomycin A1 (+BAF. A1). Representative images are shown. Empty arrowheads indicate peripheral LC3 puncta. Scale bars, 10 μm. D. Quantification of experiments shown in panel (C) (n = 3; 20 cells per condition). Means ± s.e.m. are plotted. E. HeLa cells treated with control siCTRL or with siE-Syts were grown in complete medium or were starved for 1 or 4 h, and cells lysates were subjected to Western blot for indicated proteins. F. Quantification of Western blot shown in panel (E), with 20 cells analysed per condition (n = 5). Data information: NS, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. Download figure Download PowerPoint Western blot analyses performed following a time course of starvation-induced autophagy revealed a decrease in LC3 lipidation as well as decreases of the amounts of the autophagosome biogenesis regulators ATG16L1 and ATG5—ATG12 in E-Syt-deficient cells (Fig 6E and F). Moreover, using the LC3GFP-RFP tandem dye, which is widely used to measure autophagic flux (Klionsky et al, 2016), we observed that the GFP/RFP ratio was not modified in the E-Syt-deficient cells compared to control cells (Fig EV5). Together, these data suggest that, although the number of autophagic structures was diminished when ER-PM contact sites were reduced, the maturation and transport to lysosomes of the remaining autophagosomes were not altered. Click here to expand this figure. Figure EV5. Autophagosome maturation is not affected in E-Syt-deficient cells A, B. HeLa cells stably transfected with mRFP-GFP-LC3 and treated with siE-Syts have fewer autophagosomes, but autophagosomes have normal functionality, as evidenced by (A) microscopy and (B) by RFP+/GFP+ and RFP+/GFP− LC3-puncta counting. Cells treated with Bafilomycin A1 (+BAF. A1) and siCTRL were used as a positive functionality control and showed decreased autophagosome maturation (i.e. reduced RFP+/GFP− LC3-puncta compared to siCTRL-treated cells) as expected (n = 3). ***P < 0.001, unpaired two-tailed t-test. Scale bar, 10 μm. Plotted are mean ± s.e.m. Download figure Download PowerPoint One of the major molecular events responsible for autophagosome biogenesis is the synthesis of PI3P at the omegasome on the ER membrane (Axe et al, 2008; Lamb et al, 2013; Roberts & Ktistakis, 2013). PI3P is synthesized not only at the omegasome membrane but also on early endosomes as well (Di Paolo & De Camilli, 2006; Marat & Haucke, 2016). We observed that the omegasome-marker and PI3P binding protein DFCP1 co-distributed with E-Syt2 domains on the ER (Fig 4). Therefore, we looked directly for PI3P lipid in proximity to these ER-PM contact sites during short-term starvation. Interestingly, we observed PI3P-positive structures [detected by FYVEGST/fluorescent anti-GST antibody indirect staining (Khaldoun et al, 2014)] in the immediate vicinity of ER membrane regions positive for E-Syt3 and LC3 but not in regions stained by endosomal marker EEA1 after 15 min of starvation (Appendix Fig S3A and B). We obtained similar results using 2x-FYVEGFP dye to stain for PI3P (Appendix Fig S3C) and when cells were stained using ATG16L1 and VPS35 (Seaman et al, 1998) to mark early autophagic structures and early endosomes, respectively (Appendix Fig S3D). Our results suggest that E-Syts directly or indirectly participate in autophagosome biogenesis. Because we observed PI3P at E-Syts domains after autophagy induction, we speculated that these proteins are involved in regulation of PI3P synthesis at ER-PM contact site-associated autophagosome biogenesis. To assess this hypothesis, we quantified PI3P puncta in control cells and cells deficient in all three E-Syt proteins under both fed and starved conditions. We used wort

Age-related declines in cognitive fitness are associated with a reduction in autophagy, an intracellular lysosomal catabolic process that regulates protein homeostasis and organelle turnover. However, the functional significance of autophagy in regulating cognitive function and its decline during aging remains largely elusive. Here, we show that stimulating memory upregulates autophagy in the hippocampus. Using hippocampal injections of genetic and pharmacological modulators of autophagy, we find that inducing autophagy in hippocampal neurons is required to form novel memory by promoting activity-dependent structural and functional synaptic plasticity, including dendritic spine formation, neuronal facilitation, and long-term potentiation. We show that hippocampal autophagy activity is reduced during aging and that restoring its levels is sufficient to reverse age-related memory deficits. Moreover, we demonstrate that systemic administration of young plasma into aged mice rejuvenates memory in an autophagy-dependent manner, suggesting a prominent role for autophagy to favor the communication between systemic factors and neurons in fostering cognition. Among these youthful factors, we identify osteocalcin, a bone-derived molecule, as a direct hormonal inducer of hippocampal autophagy. Our results reveal that inducing autophagy in hippocampal neurons is a necessary mechanism to enhance the integration of novel stimulations of memory and to promote the influence of systemic factors on cognitive fitness. We also demonstrate the potential therapeutic benefits of modulating autophagy in the aged brain to counteract age-related cognitive impairments.

Abstract Background Although both circular RNAs (circRNAs) and autophagy are associated with the function of breast cancer (BC), whether circRNAs regulate BC progression via autophagy remains unknown. In this study, we aim to explore the regulatory mechanisms and the clinical significance of autophagy-associated circRNAs in BC. Methods Autophagy associated circRNAs were screened by circRNAs deep sequencing and validated by qRT-PCR in BC tissues with high- and low- autophagic level. The biological function of autophagy associated circRNAs were assessed by plate colony formation, cell viability, transwells, flow cytometry and orthotopic animal models. For mechanistic study, RNA immunoprecipitation, circRNAs pull-down, Dual luciferase report assay, Western Blot, Immunofluorescence and Immunohistochemical staining were performed. Results An autophagy associated circRNA circCDYL was elevated by 3.2 folds in BC tissues as compared with the adjacent non-cancerous tissues, and circCDYL promoted autophagic level in BC cells via the miR-1275-ATG7/ULK1 axis; Moreover, circCDYL enhanced the malignant progression of BC cells in vitro and in vivo. Clinically, increased circCDYL in the tumor tissues and serum of BC patients was associated with higher tumor burden, shorter survival and poorer clinical response to therapy. Conclusions circCDYL promotes BC progression via the miR-1275-ATG7/ULK1-autophagic axis and circCDYL could act as a potential prognostic and predictive molecule for breast cancer patients.

ABSTRACT Autophagy is a catabolic pathway by which cellular components are delivered to the lysosome for degradation and recycling. Autophagy serves as a crucial intracellular quality control and repair mechanism but is also involved in cell remodelling during development and cell differentiation. In addition, mitophagy, the process by which damaged mitochondria undergo autophagy, has emerged as key regulator of cell metabolism. In recent years, a number of studies have revealed roles for autophagy and mitophagy in the regulation of stem cells, which represent the origin for all tissues during embryonic and postnatal development, and contribute to tissue homeostasis and repair throughout adult life. Here, we review these studies, focussing on the latest evidence that supports the quality control, remodelling and metabolic functions of autophagy during the activation, self-renewal and differentiation of embryonic, adult and cancer stem cells.

•Autophagy is defective in liver endothelial cells from patients with non-alcoholic steatohepatitis.•This defect is mirrored by low levels of inflammatory mediators in the portal blood of patients with metabolic syndrome.•Deficient autophagy induces inflammation, features of endothelial-to-mesenchymal transition and apoptosis.•Deficiency in endothelial autophagy promotes liver inflammation, liver cell apoptosis and liver perisinusoidal fibrosis. Background & AimsPrevious studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis.MethodsWe analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively.ResultsPatients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis.ConclusionsA defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis.Lay summaryAutophagy is a physiological process controlling endothelial homeostasis in vascular beds outside the liver. This study demonstrates that autophagy is defective in the liver endothelial cells of patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis at early stages of non-alcoholic steatohepatitis, but also at advanced stages of chronic liver disease. Previous studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis. We analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively. Patients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis. A defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis.

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr308 and Ser473. However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1A280V) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1K114G) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC50 = 3.5 μm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells. Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr308 and Ser473. However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1A280V) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1K114G) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC50 = 3.5 μm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells. nonsteroidal anti-inflammatory drug protein kinase B adenomatous polyposis coli cyclooxygenase glycogen synthase kinase-3β hemagglutinin polyhistidine interleukin-13 myristoylated PKB 3-phosphoinositide-dependent protein kinase-1 propidium iodide phosphatidylinositol 3-kinase serum- and glucocorticoid-regulated kinase wild type fluorescein isothiocyanate benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone Numerous reports, which include epidemiological studies, animal studies, and in vitro cell culture experiments, indicate that nonsteroidal anti-inflammatory drug (NSAID)1 use can significantly reduce the risk for colorectal cancer (1Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2251) Google Scholar, 2Levy G.N. FASEB J. 1997; 11: 234-247Crossref PubMed Scopus (284) Google Scholar) and, to lesser extent, the risk of breast (3Harris R.E. Namboodiri K.K. Farrar W.B. Epidemiology. 1996; 7: 203-205Crossref PubMed Scopus (342) Google Scholar), esophagus (4Funkhouser E.M. Sharp G.B. Cancer. 1995; 76: 1116-1119Crossref PubMed Scopus (288) Google Scholar), prostate (5Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. J. Biol. Chem. 2000; 275: 11397-11403Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar), and stomach (6Thun M.J. Namboodiri M.M. Calle E.E. Flanders W.D. Heath C.W., Jr. Cancer Res. 1993; 53: 1322-1327PubMed Google Scholar) cancers. Although the precise mechanisms for the chemopreventive effects of NSAIDs are not yet known, the ability of these drugs to induce inhibition of cell proliferation, potentiation of immune response, inhibition of angiogenesis, and induction of apoptosis has been reported in recent years (7Shiff S.J. Rigas B. Gastroenterology. 1997; 113: 1992-1998Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 8Elder D.J.E. Paraskeva C. Apoptosis. 1999; 4: 365-372Crossref PubMed Scopus (23) Google Scholar). The most characterized target for NSAIDs is cyclooxygenase (COX), which catalyzes the synthesis of prostaglandins from arachidonic acid (9Williams C.S. Smalley W. DuBois R.N. J. Clin. Invest. 1997; 100: 1325-1329Crossref PubMed Scopus (241) Google Scholar). There are two known COX isoforms, COX-1 and COX-2, with distinct expression patterns and biological activities (1Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2251) Google Scholar, 7Shiff S.J. Rigas B. Gastroenterology. 1997; 113: 1992-1998Abstract Full Text PDF PubMed Scopus (179) Google Scholar). COX-1 is a constitutively expressed enzyme found in most tissues and remains unaltered in colorectal cancer, while COX-2 expression can be up-regulated by a variety of cytokines, hormones, phorbol esters, and oncogenes in colorectal adenomas and adenocarcinomas (10Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar). The molecular basis of the chemopreventive effects of NSAIDs for colon cancer has been attributed mainly to inhibition of COX-2 by induction of the susceptibility of cancer cells to apoptosis (11Rigas B. Shiff S.J. Med. Hypotheses. 2000; 54: 210-215Crossref PubMed Scopus (78) Google Scholar). Consistent with this, null mutation of COX-2 in APCΔ716 knockout mice, a murine model of familial adenomatous polyposis, restored apoptosis and reduced the size and the number of colorectal adenomas (12Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2296) Google Scholar). Similar regression of adenomas has been observed by treatment of Min mouse with the NSAID sulindac (13Labayle D. Fischer D. Vielh P. Drouhin F. Pariente A. Bories C. Duhamel O. Trousset M. Attali P. Gastroenterology. 1991; 101: 635-639Abstract Full Text PDF PubMed Google Scholar). However, observations relating to the proapoptotic effect of NSAIDs lead to contradictory conclusions and demonstrate that they act via COX-dependent and COX-independent mechanisms (for a review, see Ref. 11Rigas B. Shiff S.J. Med. Hypotheses. 2000; 54: 210-215Crossref PubMed Scopus (78) Google Scholar). For example, the addition of exogenous prostaglandins to a colon cancer cell line that lacks COX activity cannot reverse the proapoptotic effect of sulindac sulfide, a metabolite derived from sulindac (14Hanif R. Pittas A. Feng Y. Koutsos M.I. Qiao L. Staiano-Coico L. Shiff S.I. Rigas B. Biochem. Pharmacol. 1996; 52: 237-245Crossref PubMed Scopus (599) Google Scholar). In the same way, sulindac sulfone, another sulindac metabolite that does not inhibit COXs, affects tumor growth in animal models (15Piazza G.A. Alberts D.S. Hixson L.J. Paranka N.S., Li, H. Finn T. Bogert C. Guillen J.M. Brendel K. Gross P.H. Sperl G. Ritchie J. Burt R.W. Ellsworth L. Ahnen D.J. Pamukcu R. Cancer Res. 1997; 57: 2909-2915PubMed Google Scholar) and induces apoptosis in cultured cancer cells expressing or not expressing COXs (16Piazza G.A. Rahm A.L. Krutzsch M. Sperl G. Paranka N.S. Gross P.H. Brendel K. Burt R.W. Alberts D.S. Pamukcu R. Ahnen D.J. Cancer Res. 1995; 55: 3110-3116PubMed Google Scholar, 17Tsujii M. DuBois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2150) Google Scholar). These results suggest that molecular targets of NSAIDs other than COXs might exist and thereby would provide a link between the chemoprotective effect of NSAIDs on cancer cells and their level of COX expression. Recent studies have identified a series of new molecular targets for NSAIDs mainly involved in signaling pathways including 15-lipoxygenase-1 (18Shureiqi I. Chen D. Lotan R. Yang P. Newman R.A. Fischer S.M. Lippman S.M. Cancer Res. 2000; 60: 6846-6850PubMed Google Scholar), peroxisome proliferator-activated receptors (19He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar), extracellular signal-regulated kinase 1/2 signaling (20Rice P.L. Goldberg R.J. Ray E.C. Driggers L.J. Ahnen D.J. Cancer Res. 2001; 61: 1541-1547PubMed Google Scholar), NF-κB (21Kopp E. Ghosh S. Science. 1994; 265: 956-959Crossref PubMed Scopus (1637) Google Scholar), p70S6 kinase (22Law B.K. Waltner-Law M.E. Entingh A.J. Chytil A. Aakre M.E. Norgaard P. Moses H.L. J. Biol. Chem. 2000; 275: 38261-38267Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), p21 ras signaling (23Herrmann C. Block C. Geisen C. Haas K. Weber C. Winde G. Moroy T. Muller O. Oncogene. 1998; 17: 1769-1776Crossref PubMed Scopus (114) Google Scholar), and Akt/PKB kinase (5Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. J. Biol. Chem. 2000; 275: 11397-11403Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Recently, specific COX-2 inhibitors (coxib) have been generated for the treatment of rheumatoid arthritis and osteoarthritis (reviewed in Ref.24Everts B. Währborg P. Hedner T. Clin. Rheumatol. 2000; 19: 331-343Crossref PubMed Scopus (115) Google Scholar). Celecoxib reduces the formation of polyposis in familial adenomatous polyposis patients (25Steinbach G. Lynch P.M. Phillips R.K. Wallace M.H. Hawk E. Gordon G.B. Wakabayashi N. Saunders B. Shen Y. Fujimura T., Su, L.K. Levin B. N. Engl. J. Med. 2000; 342: 1946-1952Crossref PubMed Scopus (2332) Google Scholar), prevents colorectal tumor growth, and induces apoptosis in in vitro and in vivomodels (26Williams C.S. Watson A.J. Sheng H. Helou R. Shao J. DuBois R.N. Cancer Res. 2000; 60: 6045-6051PubMed Google Scholar, 27Oshima M. Murai N. Kargman S. Arguello M. Luk P. Kwong E. Taketo M.M. Evans J.F. Cancer Res. 2001; 61: 1733-1740PubMed Google Scholar, 28Jacoby R.F. Seibert K. Cole C.E. Kelloff G. Lubet R.A. Cancer Res. 2000; 60: 5040-5044PubMed Google Scholar). However, the proapoptotic effect of celecoxib was independent of inhibition of COX-2 in mouse embryo fibroblasts derived from COX-2 (+/−) and COX-2 (−/−) mice (26Williams C.S. Watson A.J. Sheng H. Helou R. Shao J. DuBois R.N. Cancer Res. 2000; 60: 6045-6051PubMed Google Scholar). Furthermore, Hsu et al. (5Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. J. Biol. Chem. 2000; 275: 11397-11403Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar) have recently demonstrated that celecoxib induced apoptosis in human prostate cancer cells by reducing phosphorylation of Akt/PKB kinase. Here, we report the first evidence that celecoxib potently induces apoptosis in the colon cancer cell line HT-29, which lacks COX-2 catalytic activity (29Hsi L.C. Baek S.J. Eling T.E. Exp. Cell Res. 2000; 256: 563-570Crossref PubMed Scopus (58) Google Scholar) and specifically inhibits the 3-phosphoinositide-dependent kinase PDK1 activity, the Akt/PKB upstream kinase. These data suggest that celecoxib induces apoptosis through a target other than COX-2 by controlling the major antiapoptotic PDK1/Akt/PKB pathway. Celecoxib was kindly provided by Pharmacia. Nitrocellulose membranes and BCA kit were purchased from Schleicher and Schuell and Pierce, respectively. Geneticin (G418) was purchased from Invitrogen. The SuperfectTMtransfection kit was from Qiagen (Courtaboeuf, France). Monoclonal antibody directed against polyhistidine tag (C terminus), ampicillin, and pcDNA3.0/myc-His A, B, C vectors were from Invitrogen. Monoclonal antibody against HA epitope was from Roche Molecular Biochemicals. Rabbit anti-COX-2 (PG27) was from Oxford Biochemical Research, and rabbit anti-Bcl-2 was from Calbiochem. PDK1 and Akt/PKB immunoprecipitation kinase assay® kits, rabbit anti-actin, rabbit anti-phospho-Akt/PKB (Thr308 and Ser473), sheep anti-Akt/PKB, sheep anti-phospho-GSK-3β, and sheep anti-GSK-3β were from Upstate Biotechnology, Inc. (Lake Placid, NY). [γ-32P]ATP (specific activity 3000 Ci/mmol) was from PerkinElmer Life Sciences. IL-13 and cDNA encoding for the myristoylated and dead forms of HA-tagged Akt/PKB were kindly provided by Dr. A. Minty (Sanofi Elf Biorecherche), Dr. T. F. Franke (Columbia University, New York), and Dr. P. N. Tsichlis (Fox Chase Cancer Center, Philadelphia, PA), respectively. cDNA encoding for wild-type PDK1, constitutively active form PDK1A280V, and kinase-defective PDK1K114G were a generous gift from Dr. F. Liu (University of Texas Health Science Center, San Antonio, TX). The enhanced chemiluminescence detection kit was from Amersham Biosciences. HT-29 cells were cultured as previously described (30Ogier-Denis E. Couvineau A. Maoret J.J. Houri J.J. Bauvy C., De Stefanis D. Isidoro C. Laburthe M. Codogno P. J. Biol. Chem. 1995; 270: 13-16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). 1 day after seeding, media were changed, and cells were treated with different concentrations of celecoxib (50–100 μm). An equivalent amount of the carrier (Me2SO) was added to untreated cells. In order to eliminate a putative interaction between G418 antibiotic and celecoxib, stable transfected cells were cultured in the absence of G418 during the drug treatment. When required, celecoxib at 50–100 μm and IL-13 (30 ng/ml) were added from 4 to 24 h. The expression constructs (pCMV6/HA-tagged MyrPKB, pcDNA3.0/His-tagged WT PDK1, pcDNA3.0/His-tagged PDK1A280V, and pcDNA3.0/His-tagged PDK1K114G) and control pCMV6 and pcDNA3.0 vectors were introduced into exponential growing HT-29 cells using the SuperfectTM transfection kit. Transfected cells were cultured in complete medium for 48 h and then selected for 3 weeks in a medium containing 800 μg/ml G418. Finally, G418-resistant cells were routinely maintained in a medium containing 250 μg of G418. Expression levels of each constructs were determined by Western blot analysis using anti-HA and anti-His antibodies. Parental and transfected HT-29 cells were washed twice with ice-cold phosphate-buffered NaCl solution and lysed in cold lysis buffer (20 mm Tris-HCl (pH 7.4); 150 mm NaCl; 1% Triton X-100; 0.5% Nonidet P-40; 1 mm EDTA; 1 mm EGTA; 5 μmphenylmethylsulfonyl fluoride; 5 μg/ml leupeptin, pepstatin A, and aprotinin; 1 mm Na3VO4; 2 mm NaF; and 2 mmNa4PO7) for 30 min on ice. After centrifugation, protein concentration of cell lysates was determined by BCA reagent. 100 μg of proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk in TBST (10 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 0.05% Tween 20) for 1 h at room temperature and then incubated with appropriate primary antibody for 1 h at room temperature or overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody at 1:3000 dilution for 1 h at room temperature. The polyclonal anti-COX-2, anti-actin, anti-phospho-Akt/PKB (Thr308 and Ser473), anti-Akt/PKB, anti-phospho-GSK-3β, and anti-GSK-3β were used at 1:1000 dilution. The polyclonal anti-Bcl-2 was used at 1:100 dilution. The monoclonal anti-His and anti-HA were used at 1:2500 and 1:3000 dilution, respectively. Cells were seeded for 24 h onto 24-well plates at 1 × 106 cells/plate and then were exposed to various concentrations of celecoxib for different times. When used, 100 μm caspase inhibitor zVAD-fmk (Cliniscience S.A.) was added 1 h before celecoxib. During the treatment, the percentage of cells floating in the medium increased over time. Adherent cells were rinsed three times with phosphate-buffered saline and were harvested by trypsinization. At each time of celecoxib treatment, floating cells were recovered by centrifugation of medium at 3200 × g for 5 min. Both adherent and floating cells were combined for the assessment of cell viability, which was determined by trypan blue exclusion. All data points shown are mean values ± S.E. of four independent experiments. The data points from triplicates of an individual experiment were averaged, and the data points shown are the mean of these averages from four experiments. Statistical calculation was done using Student's t test. HT-29 cells were cultured in the presence or absence of 100 μm celecoxib for various times. Floating and adherent cells were collected, rinsed twice with phosphate-buffered saline, and resuspended in lysis buffer containing 1% Nonidet P-40, 2 mm EDTA, and 50 mm Tris (pH 7.5) for 1 h. After centrifugation, the supernatants were treated with 5 μg/ml of ribonuclease A at 37 °C for 1 h, and then proteinase K was added at 2.5 μg/ml for 2 h at 37 °C. DNA was precipitated by 75% ethanol and 3 m sodium acetate at −80 °C for 2 h, and pellet was resuspended in TE buffer. Each sample was electrophoresed on a 1.6% agarose gel and visualized by ethidium bromide staining. In all experiments, floating and freshly trypsinized attached cells were pooled. Thereafter, apoptotic, necrotic, and damaged cells were separated by flow cytometry. Samples were then run on a FACSCalibur flow cytometer (San Jose, CA) equipped with an argon laser and filter configuration for fluorescein isothiocyanate (FITC)/propidium iodide (PI) dye combination. Light scatter and fluorescence signals were subjected to linear and logarithmic amplification, respectively. At least 10,000 events were acquired and analyzed with CellQuest software. The quantitative determination of the percentage of cells undergoing apoptosis was performed using an annexin V-FITC apoptosis detection kit (Cliniscience S.A.) according to the manufacturer's instructions. In brief, 5 × 105 washed cells resuspended in 100 μl of annexin V binding buffer were simultaneously incubated with 5 μl of FITC-conjugated annexin V and 5 μl of PI for 15 min at room temperature. Before cytometric analysis, the cell suspension was supplemented with 500 μl of annexin V binding buffer. Transmission electron microscopy was performed on cells treated or not with 100 μm celecoxib for 24 h. Samples were embedded in Epon as previously reported (31Lesuffleur T. Violette S. Vasile-Pandrea I. Dussaulx E. Barbat A. Muleris M. Zweibaum A. Int. J. Cancer. 1998; 76: 383-392Crossref PubMed Scopus (47) Google Scholar). Ultrathin sections were examined in a Jeol JEM-100 CX11 electron microscope operated at 100 kV. The assay was carried out in two stages. In the first stage, active PDK1 was immunoprecipitated from HT-29 cells transfected with the empty vector, WT PDK1, PDK1A280V, or PDK1K114G constructs. Cells were treated or not with 100 μm celecoxib for 24 h and lysed in buffer A containing 50 mm Tris-HCl (pH 7.5), 0.1% Triton X-100, 1 mm EDTA, 1 mmEGTA, 50 mm sodium fluoride, 10 mm sodium β-glycerophosphate, 1 mm activated sodium orthovanadate, 0.1% (v/v) 2-mercaptoethanol, and 1 μm microcystin at 4 °C for 1 h. Samples were centrifuged at 5000 ×g for 10 min, and supernatants were precleared with 50% protein G-agarose beads diluted in buffer A. In the same time, 4 μg of anti-PDK1 or normal sheep IgG were incubated with 100 μl of 50% protein G-agarose beads at 4 °C for 1 h. One mg of each precleared cell lysate was incubated for 2 h with protein G-agarose beads bearing anti-PDK1 on a rotator at 4 °C to immunoprecipitate active PDK1. In the second stage, inactive serum- and glucocorticoid-regulated kinase (SGK) was incubated with immunoprecipitated PDK1 and Mg2+/ATP to activate the SGK before the addition of [γ-32P]ATP and Akt/SGK-specific substrate peptide (RPRAATF). The activated SGK used Mg2+/[γ-32P]ATP to phosphorylate the Akt-specific substrate peptide. Immunoprecipitates were preincubated with inactive SGK enzyme (500 ng) for 30 min at 30 °C and incubated with 66 μm Akt/SGK substrate peptide in a shaking incubator for another 30 min, and 1 μCi/ml of [γ-32P]ATP was added to start the reaction. The phosphorylated peptide substrate was then separated from the residual [γ-32P]ATP using P81 phosphocellulose paper and quantitated by using a scintillation counter after three washes with 0.75% phosphoric acid and two washes with acetone. Values are from three separate experiments. HT-29 cells were treated or not with 100 μm celecoxib for different times in the presence or absence of 30 ng/ml IL-13 and then lysed at 4 °C for 1 h in buffer A containing 50 mmTris-HCl (pH 7.5); 1% Triton X-100; 1 mm EDTA; 1 mm EGTA; 50 mm sodium fluoride; 10 mm sodium β-glycerophosphate; 0.1% (v/v) 2-mercaptoethanol; 0.1 mm phenylmethylsulfonyl fluoride; and 1 μg/ml aprotinin, pepstatin, and leupeptin. Samples were centrifuged at 5000 × g for 10 min, and supernatants were precleared with 50% protein G-agarose beads diluted in buffer A. In the same time, 4 μg of anti-Akt/PKB or normal rabbit IgG were incubated with 100 μl of 50% protein G-agarose beads at 4 °C for 90 min. 1 mg of each precleared cell lysates was incubated for 2 h with protein G-agarose beads bearing anti-Akt/PKB on a rotator at 4 °C to immunoprecipitate Akt/PKB. This precipitate was next used to phosphorylate a specific substrate, the recombinant murine BAD protein expressed in Escherichia coli. Briefly, 3 μg of recombinant BAD were incubated with Akt/PKB-antibody-protein G-agarose complexes in the presence of magnesium/ATP mixture for 10 min at 37 °C. After three washes with buffer A, samples were boiled for 5 min, resolved by 10% SDS-PAGE, and transferred onto nitrocellulose membrane. The membranes were blocked with 5% nonfat dry milk in TBST and incubated with a 1:500 dilution of anti-phospho-BAD (Ser136) for 2 h at room temperature. Primary antibody was revealed using a goat anti-rabbit horseradish peroxidase-conjugated IgG for 1 h at room temperature. After revelation, the Akt/PKB activity was determined after gel scanning. Since evidence indicates that celecoxib induced apoptosis and inhibited cell cycle progression in colorectal carcinoma cells in culture by mechanisms independent of COX-2 inhibition (26Williams C.S. Watson A.J. Sheng H. Helou R. Shao J. DuBois R.N. Cancer Res. 2000; 60: 6045-6051PubMed Google Scholar), we have examined the dose- and time-dependent effects of celecoxib on the viability of HT-29 cells, a cell line that lacks COX-2 activity (29Hsi L.C. Baek S.J. Eling T.E. Exp. Cell Res. 2000; 256: 563-570Crossref PubMed Scopus (58) Google Scholar). As shown in Fig. 1 A, celecoxib significantly induced the cell death in a time- and dose-dependent manner. Treatment with 100 μmcelecoxib for 24 h caused a loss of viability up to 65%. This effect has been previously observed in many cell lines including prostate cancer PC3 cells (5Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. J. Biol. Chem. 2000; 275: 11397-11403Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar), lung carcinoma LLC cells (26Williams C.S. Watson A.J. Sheng H. Helou R. Shao J. DuBois R.N. Cancer Res. 2000; 60: 6045-6051PubMed Google Scholar), and colon cancer HCT-15 and HCT-116 cells (26Williams C.S. Watson A.J. Sheng H. Helou R. Shao J. DuBois R.N. Cancer Res. 2000; 60: 6045-6051PubMed Google Scholar). In order to determine whether the cytotoxic effect of celecoxib was due to apoptosis, HT-29 cells were treated with increasing concentrations of this drug in the presence of the large spectrum caspase inhibitor zVAD-fmk (Fig. 1 B). In the presence of zVAD-fmk, a reduction in cell death was observed, whatever the concentrations of celecoxib used. Celecoxib-dependent apoptosis was also confirmed by detection of oligonucleosomal cleavage of DNA in HT-29 cells after treatment with 100 μm celecoxib for 16 and 24 h (Fig. 2 A). However, as depicted in Fig. 1 B, a portion of HT-29 cells was always sensitive to celecoxib despite the presence of zVAD-fmk inhibitor, suggesting that a caspase-independent cell death may also occur in celecoxib-treated HT-29 cells. A better understanding of this caspase-independent cell death induced by celecoxib will require further investigations. Examination of the celecoxib-treated cells by electron microscopy revealed morphological alterations of apoptosis after being treated with 100 μm celecoxib for 24 h including chromatin condensation (Fig. 2 B). In addition, the externalization of phosphatidylserine, an early event of apoptotic process, was analyzed by flow cytometry with the annexin V-binding assay after treatment of HT-29 cells with 100 μmcelecoxib for the indicated times (Fig. 2 C). Apoptotic (annexin V+/PI−), necrotic (annexin V+/PI+), and viable cells (annexin V−/PI−) were separated on the basis of a double labeling for annexin V-FITC and PI, a membrane DNA stain. Treatment with 100 μm celecoxib caused a significant increase of apoptotic cells (annexin V+/PI−) with 12, 20, and 29% for 8, 16, and 24 h of treatment, respectively. In parallel, necrotic cells (annexin V+/PI+) were detected, whatever the time of treatment, to reach 37% after 24 h of treatment (Fig.2 C). This latter effect is consistent with the secondary necrosis process, which usually comes after apoptosis in cells growing in culture. In contrast to several reports that linked COX-2 inhibitor-induced apoptosis to Bcl-2 down-regulation (32Sheng H. Shao J. Morrow J.D. Beauchamp R.D. DuBois R.N. Cancer Res. 1998; 58: 362-366PubMed Google Scholar, 33Liu X.H. Yao S. Kirschenbaum A. Levine A.C. Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar), we demonstrated by Western blot analysis that celecoxib did not affect Bcl-2 expression in HT-29 cells after 24 h of treatment (Fig. 2 D). Furthermore, COX-2 expression level was unaffected throughout the time of celecoxib treatment (Fig. 2 D). Recent studies have reported that celecoxib induced apoptosis by inhibiting Akt/PKB activity in prostate cancer cells (5Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. J. Biol. Chem. 2000; 275: 11397-11403Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Since celecoxib caused programmed cell death in HT-29 cells that lack COX-2 activity (29Hsi L.C. Baek S.J. Eling T.E. Exp. Cell Res. 2000; 256: 563-570Crossref PubMed Scopus (58) Google Scholar), we checked whether the observed cytotoxic effect of celecoxib was mediated by a modulation of Akt/PKB activity. Akt/PKB, the major downstream effector of PI 3-kinase, is a Ser/Thr protein kinase that has been shown to exert a crucial role in the regulation of several cellular signaling pathways (reviewed in Ref. 34Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar). By stimulation of PI 3-kinase with growth factors and cytokines, Akt/PKB is recruited from the cytosol to the plasma membrane via its pleckstrin homology domain and is phosphorylated at two regulatory sites, Thr308 and Ser473, essential for its activation (34Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar). Activated Akt/PKB, that has been involved in protecting cells from apoptosis, can phosphorylate BAD (35Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (5003) Google Scholar), caspase 9 (36Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2751) Google Scholar), IkB kinase (37Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1927) Google Scholar), glycogen synthase kinase-3β (38Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (442) Google Scholar), and forkhead transcription factors (39Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P., Hu, L.S. Anderson M., J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5522) Google Scholar), leading to their inactivation and to cell survival. To evaluate the effect of celecoxib on Akt/PKB activity, HT-29 cells were treated with 100 μm celecoxib for 24 h, and phosphorylation at Thr308 and Ser473 was examined using specific phosphoantibodies (Fig.3 A). Despite the slight basal phosphorylation level of Akt/PKB in the control condition, celecoxib significantly reduced Akt/PKB phosphorylation at Thr308 and Ser473. The effect of celecoxib was confirmed in two independent ways: first, by measuring the Akt/PKB-mediated phosphorylation level of GSK-3β, a direct in vivodownstream substrate of Akt/PKB (40Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4474) Google Scholar) (Fig. 3 A), and second, by determining kinase activity of Akt/PKB after immunoprecipitation (Fig. 3 B). The loss of Akt/PKB phosphorylation mediated by celecoxib was correlated with an equivalent decrease of GSK-3β phosphorylation and an inhibition of its activity. In order to confirm the inhibitory effect of celecoxib, we increased Akt/PKB phosphorylation by using IL-13, a pleiotropic cytokine that is known to activate the PI 3-kinase/Akt/PKB pathway in HT-29 cells (41Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 2

Gα-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the α-subunit of the trimeric Gi3 protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Gαi3 protein (k = 0.187 ± 0.001 s−1, EC50 = 1.12 ± 0.10 μm) when compared with unphosphorylated recombinant GAIP (k = 0.145 ± 0.003 s−1, EC50 = 3.16 ± 0.12 μm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells. Gα-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the α-subunit of the trimeric Gi3 protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Gαi3 protein (k = 0.187 ± 0.001 s−1, EC50 = 1.12 ± 0.10 μm) when compared with unphosphorylated recombinant GAIP (k = 0.145 ± 0.003 s−1, EC50 = 3.16 ± 0.12 μm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells. regulators of G protein signaling bovine serum albumin 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole extracellular signal-regulated kinase Gα-interacting protein GTPase-activating protein Hanks' balanced salt solution lactate dehydrogenase mitogen-activated protein polymerase chain reaction wild-type polyacrylamide gel electrophoresis cAMP-dependent protein kinase protein kinase C Regulators of G protein signaling proteins (RGS)1 are a family of proteins that control the activity of trimeric G proteins (1Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 3Koelle M.R. Curr. Opin. Cell Biol. 1997; 9: 143-147Crossref PubMed Scopus (180) Google Scholar, 4De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (522) Google Scholar). More than 20 mammalian RGS proteins have been identified generally by reference to a conserved domain of about 115 amino acid residues known as the RGS box (5Zheng B. De Vries L. Farquhar M.G. Trends Biochem. Sci. 1999; 24: 411-414Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). RGS proteins are involved in modulating a variety of cell functions such as proliferation, differentiation, response to neurotransmitters, membrane trafficking, and embryonic development (4De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (522) Google Scholar, 6Arshavsky V.Y. Pugh Jr., E.N. Neuron. 1998; 20: 11-14Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). RGS act as negative regulators of several G proteins by accelerating the rate of GTP hydrolysis by the Gα proteins, thereby promoting their association with the βγ dimer (7Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (483) Google Scholar, 8Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (311) Google Scholar, 9Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). This GTPase-activating protein (GAP) activity is engaged in the desensitization of signaling by the trimeric G proteins, but it can also speed up the transmission of signals in some cases (10Doupnik C.A. Davidson N. Lester H.A. Kofuji P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (299) Google Scholar, 11Saitoh O. Kubo Y. Miyatani Y. Asano T. Nakata H. Nature. 1997; 390: 525-529Crossref PubMed Scopus (193) Google Scholar). Recently, the key role of RGS in the regulation of G protein-coupled receptor signaling has been demonstrated in vivo (12Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (409) Google Scholar, 13Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (338) Google Scholar). However, recent evidence supports the notion that RGS proteins may be engaged in functions distinct from the regulation of G protein-activity (14De Vries L. Gist Farquhar M. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). GAIP (Gα-interacting protein) is an RGS protein, which is known to interact with Gαi3 protein (15De Vries L. Mousli M. Wurmser A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11916-11920Crossref PubMed Scopus (267) Google Scholar). GAIP has been located to the Golgi apparatus membrane and newly budding Golgi vesicles (16Wylie F. Heimann K. Le T.L. Brown D. Rabnott G. Stow J.L. Am. J. Physiol. 1999; 276: C497-C506Crossref PubMed Google Scholar, 17Petiot A. Ogier-Denis E. Bauvy C. Cluzeaud F. Vandewalle A. Codogno P. Biochem. J. 1999; 337: 289-295Crossref PubMed Google Scholar) and associated with clathrin-coated vesicles (18De Vries L. Elenko E. McCaffery J.M. Fischer T. Hubler L. McQuistan T. Watson N. Farquhar M.G. Mol. Biol. Cell. 1998; 9: 1123-1134Crossref PubMed Scopus (89) Google Scholar), suggesting its potential role in vesicular transport. Recently, it has been demonstrated that posttranslational modifications of RGS can modulate their properties. Palmitoylation of conserved cysteines in RGS boxes has been shown to modify the GAP activity of RGS4 and RGS10 (19Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In addition, phosphorylation has been reported to influence the stability and the membrane association of the yeast RGS Sst2 and human GAIP, respectively (20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar). However, it is not known whether or not phosphorylation could modulate the GAP activity of RGS. In the present work, we show that the phosphorylation of GAIP is in part dependent upon the activation of the Erk1/2 MAP kinases in the human intestinal HT-29 cells, and both of these events were sensitive to PKC inhibitors and amino acids. Using a panel of Ser/Thr protein kinases in an in vitro assay, we demonstrate that the phosphorylation of GAIP by a recombinant Erk2 stimulated its GAP activity toward the Gαi3 protein when compared with the activity of unphosphorylated GAIP. This stimulation was abolished when serine at position 151 in the RGS domain was replaced by an alanine residue by site-directed mutagenesis. Previously, we have shown that GAIP and the Gαi3 protein are engaged in a signaling pathway that controls the lysosomal-autophagic route in HT-29 cells (22Ogier-Denis E. Couvineau A. Maoret J.J. Houri J.J. Bauvy C. De Stefanis D. Isidoro C. Laburthe M. Codogno P. J. Biol. Chem. 1995; 270: 13-16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A hallmark of autophagy in many mammalian cells is its sensitivity to extracellular amino acid levels, which reduce the formation of autophagic vacuoles containing cytoplasmic material destined to lysosomal degradation (25Blommaart E.F. Luiken J.J. Meijer A.J. Histochem. J. 1997; 29: 365-385Crossref PubMed Scopus (217) Google Scholar, 26Mortimore G.E. Miotto G. Venerando R. Kadowaki M. Lloyd J.B. Mason R.W. Subcellular Biochemistry. Plenium Publishing Corp., New York1996: 93-136Google Scholar). The inhibition of autophagy by the addition of amino acids was correlated with the inhibition of the Erk1/2 MAP kinases and a low level of GAIP phosphorylation in HT-29 cells. By contrast to cells expressing the wild-type GAIP, those expressing the S151A mutant were unable to increase their rate of autophagy in response to amino acid deprivation and Erk1/2 activation. In conclusion, these results demonstrate that an Erk1/2 MAP kinase-dependent phosphorylation stimulates the GAP activity of GAIP and they also identify GAIP as a target for amino acid-regulated catabolism in intestinal cells. cDNA were synthesized from mRNA isolated from HT-29 cells by reverse transcription and were used to amplify by PCR full-length cDNA encoding the wild-type GAIP (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Full-length GAIP mutant S151A was generated by oligonucleotide-directed mutagenesis using the following mismatched oligonucleotide: 5′-GTATCCATCCTGGCCCCCAAGGAGGTG-3′. Identification of the mutation was obtained by direct DNA sequencing. Inserts encoding wild-type GAIP and S151A mutant were subcloned into the eucaryote expression vector pcDNA3 (Invitrogen) at the BamHI/XbaI sites. Wild-type and S151A mutant cDNAs were amplified by PCR using the following primers: forward primer, 5′-CGCAAGCTTATGCCCACCCCGCATGAG-3′; and reverse primer, 5′-CGCGGATCCCAAGGCCTCGGAGGAGGA-3′. PCR products were subcloned into pcDNA3.1/c-Myc/His-tagged vector (Invitrogen) at the HindIII/BamHI sites. Recombinant proteins were obtained as described previously (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). HT-29 cells were cultured as described previously (22Ogier-Denis E. Couvineau A. Maoret J.J. Houri J.J. Bauvy C. De Stefanis D. Isidoro C. Laburthe M. Codogno P. J. Biol. Chem. 1995; 270: 13-16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). His-tagged wild-type GAIP and S151A mutant were introduced into exponentially growing HT-29 cells by the Effectene™ kit according to the supplier's conditions (Qiagen). Cells were used 72 h after cell transfection. All experiments were carried out in nutrient-free HBSS medium supplemented with 0.1% BSA, and when appropriate amino acids or drugs were added. The final concentrations of amino acids in the mixture were multiples (4×) of the normal plasma concentrations and were as follows (in μm): asparagine, 60; isoleucine, 100; leucine, 250; lysine, 300; methionine, 40; phenylalanine, 50; proline, 100; threonine, 180; tryptophan, 70; valine, 180; alanine, 400; aspartate, 30; glutamate, 100; glutamine, 350; glycine, 300; cysteine, 60; histidine, 60; serine, 200; tyrosine, 75; ornithine, 100. Metabolic labeling of HT-29 cells with 0.5 mCi of [32P]orthophosphoric acid (Amersham Pharmacia Biotech) was carried out for 3 h in nutrient-free medium (HBSS), in complete medium, or in the absence or in the presence of amino acids. When used, protein kinase inhibitors (100 μm H-89 (Calbiochem), 5 μm GF103209X (Calbiochem), 10 μm SB203580 (Calbiochem), 50 μm PD098059 (Calbiochem), 100 μm DRB (Alexis Biochemicals)) were added in the same time. Cells were then rinsed three times with phosphate-buffered saline and then scraped into buffer A (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.2% BSA containing a mixture of protease and phosphatase inhibitors). 32P-Labeled cell extracts were lysed for 1 h at 4 °C. The anti-GAIP antibody (1/200) (17Petiot A. Ogier-Denis E. Bauvy C. Cluzeaud F. Vandewalle A. Codogno P. Biochem. J. 1999; 337: 289-295Crossref PubMed Google Scholar) or the anti-His antibody (1/500; Invitrogen) was bound to protein A-Sepharose under agitation at 4 °C. 32P-Labeled lysates were incubated with the anti-GAIP/protein A-Sepharose (Amersham Pharmacia Biotech) complex for 16 h at 4 °C. Sepharose beads were sequentially washed with buffer A, three times with buffer B (buffer A containing 0.1% SDS), three times with buffer C (20 mm Tris-HCl, pH 8.0, 500 mm NaCl, 0.5% Triton X-100, 0.2% BSA), and once with buffer D (50 mm Tris-HCl, pH 8.0). Immunoprecipitates were analyzed by SDS-PAGE, transferred to nitrocellulose membranes, which were exposed to Kodak X-Omat film for 16 h at −80 °C. The same blots were used to perform immunoblotting experiments with anti-GAIP and developed using the ECL kit. Phosphoamino acid analysis of GAIP was performed using one-dimensional analysis by the method of Boyle et al. (27Boyle D.M. van der Walt L.A. J. Steroid Biochem. 1988; 30: 239-244Crossref PubMed Scopus (23) Google Scholar). 32P-Labeled GAIP band was located by autoradiography, excised, and then hydrolyzed in 0.25 ml of 5.7 n HCl for 1 h at 110 °C. The sample was dried using a Speed-Vac concentrator and then resuspended in 10 μl of 10/1/189 (v/v/v) acetic acid/pyridine/water. The sample was spotted on a cellulose-coated thin layer electrophoresis plate and subjected to electrophoresis at 1100 V for 45 min with water cooling. Phosphoserine, phosphothreonine, and phosphotyrosine (1 mg/ml) were used as markers. The plate was dried, sprayed with ninhydrin to localize the phosphoamino acid standards, and then subjected to autoradiography. In all cases, kinetics were done with each kinase to determine the optimum reaction time. Recombinant PKC, PKA, casein kinase II, p38 MAP kinase, and Erk2 were used for in vitro phosphorylation assays according the supplier's instructions. Erk2, p38 MAP kinase, and PKA were used in reaction buffer (50 mm Tris-HCl, 10 mmMgCl2, 1 mm EGTA, 2 mmdithiothreitol, 0.01% Brij 35). PKC was used in reaction buffer (20 mm HEPES, pH 7.4, 10 mm MgCl2, 2 mm MnCl2, 0.1 mm dithiothreitol, 0.5 mm CaCl2, 1 μg/ml diacylglycerol, and 18.6 μg/ml phosphatidylserine), and casein kinase II was used in 20 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2. PKC (0.03 units; Promega), PKA (215 picomolar units; Sigma), casein kinase II (500 units; New England Biolabs), p38 MAP kinase (0.2 units; Upstate Biotechnology), or Erk2 (100 units; New England Biolabs) was incubated with 5 μg of recombinant GAIP in the appropriate reaction buffer and 0.5 mm ATP containing 1 μCi of [γ-32P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech) at 30 °C for 1 h. Reactions were stopped by boiling in Laemmli buffer, and proteins were resolved by 13% SDS-PAGE and submitted to nitrocellulose transfer and autoradiography. The same membranes were used for immunoblotting with the GAIP antibody. The activity of each kinase was monitored using 10 μg of histones or 10 μg of MBP as control substrates (Sigma). The calculation of the stoichiometry of GAIP phosphorylation by Erk2 was based on the specific activity of the [γ-32P]ATP, the radioactivity incorporated in GAIP determined by Cerenkov counting of the GAIP band excised from a Coomassie Blue SDS-PAGE, and the amount of GAIP estimated by comparison to bovine serum albumin used as a standard. HT-29 cells were cultured for 4 h at 37 °C in HBSS with or without amino acids, 5 μmGF109203X, or 50 μm PD098059. Cells were scrapped in lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 0.25 m sucrose, 5 mm EDTA, 5 mmEGTA, 0.5% Triton X-100, 25 mm NaF, 5 mmNa3VO4, 5 mm β-glycerophosphate, 1 mm levamisole, 1 mm para-nitrophenylphosphate, 1.5 mmphenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml pepstatin, 10 mg/ml aprotinin, 1 mm diisopropylfluorophosphate, 1 mg/ml DNase I) and after sonication, the lysate was clarified by centrifugation at 50,000 × g for 15 min at 4 °C. Hundred-μg aliquots of proteins were submitted to 9% SDS-PAGE and transferred to nitrocellulose. The membrane was incubated for 1 h in blocking buffer (25 mm Tris, pH 7.5, 150 mmNaCl, 0.05% Tween 20) containing 5% milk. Antibodies phospho-Erk1/2 (1/4000; New England Biolabs) and phospho-p38 MAP kinase (1/2000; New England Biolabs) were incubated overnight at 4 °C in blocking buffer supplemented with 1% BSA. After washing in blocking buffer, membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. Bound antibodies were detected by enhanced chemoluminescence (ECL). The same membranes were then used with anti-Erk1 (1/1000; Santa Cruz) and anti-Erk2 (1/1000; Santa Cruz) or with anti-p38 MAP kinase (1/1000; Santa Cruz) to detect the complete pool of each MAP kinase. The secondary antibody (anti-rabbit) was linked to alkaline phosphatase. Single turnover GTPase activity measurements were carried out as follows; 250 nm recombinant Gαi3 were loaded with 1 μm[γ-32P]GTP (Amersham Pharmacia Biotech) for 30 min at 30 °C in 50 mm HEPES, pH 7.5, 5 mm EDTA, 1 mm dithiothreitol, 0.1% Lubrol PX, and reactions were next chilled at 4 °C. Free nucleotides were removed by size exclusion chromatography on microspin Sephadex G50. All hydrolysis experiments were done in solution at 4 °C under single turnover conditions. Reactions were started by addition of GAIP mix containing 15 mm MgSO4, 300 μm unlabeled GTP, and 30 nm to 30 μm recombinant proteins (WT GAIP or S151A mutant) when used. Aliquots (50 μl) were removed at different times, and reactions were stopped by addition of 750 μm 5% Norit activated charcoal in 50 mmNaH2PO4, pH 3.0. Charcoal was removed by centrifugation for 15 min at 12,000 × g, and 400 μl of free phosphate-containing supernatants were counted to determine the amount of Pi released per reaction. Zero time point was obtained by adding 30 μl of [γ -32P]GTP in Norit activated charcoal. No GAP activity could be detected by using boiled GAIP. The GTPase rate constants were calculated by fitting the experimental data to an exponential function: % GTP hydrolyzed = 100(1 − e −kt ), wherek is a rate constant for GTP hydrolysis. The results are expressed as the mean ± S.E. of triplicate measurements. Measurement of the degradation of [14C]valine-labeled long-lived proteins and LDH sequestration were monitored as reported previously (23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, degradation was measured by an extrapolation of the difference between soluble radioactivity and incorporated radioactivity in trichloroacetic acid-precipitable protein. To measure the sequestration of cytosolic LDH, we isolated autophagic vacuoles and determined the included LDH activity in sedimentable materials. Metabolic labeling of HT-29 cells with [32P]orthophosphoric acid followed by immunoprecipitation using an anti-GAIP antibody and SDS-PAGE showed that GAIP was a phosphoprotein (Fig. 1 a). Acid hydrolysis of32P-labeled GAIP and thin layer electrophoresis revealed only the presence of phosphoserine residues (Fig. 1 b). After a 3-h period of nutrient starvation in HBSS, the phosphorylation of GAIP was increased 1.5 times when compared with that observed in cells kept in complete medium. Addition of a mix amino acid to HBSS reduced by 60% the phosphorylation of GAIP, suggesting that an amino acid-dependent signaling pathway is involved in the control of the phosphorylation of GAIP.Figure 1GAIP is phosphorylated on serine residues. a, upper part, HT-29 cells were radiolabeled with 0.5 mCi of [32P]orthophosphoric acid for 3 h in complete medium, HBSS, or HBSS supplemented with amino acids (see “Experimental Procedures”). GAIP was immunoprecipitated using an anti-GAIP antibody and then submitted to SDS-PAGE. Middle part, Western blot (WB) of immunoprecipitates with an anti-GAIP antibody. -Lower part, the ratio [32P]GAIP/WB was determined after scanning.b, phosphoamino acid analysis after acid hydrolysis of immunoprecipitated [32P]GAIP by thin layer electrophoresis. Arrows indicate the mobility of the standards used: PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine. Results are representative of four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Phosphorylation of GAIP was investigated after metabolic labeling with [32P]orthophosphoric acid in the presence of a panel of inhibitors of Ser/Thr protein kinases (Fig. 2). The phosphorylation of GAIP was inhibited by 60% and 70% when bisindolylmaleimide (GF109203X), a broad spectrum PKC inhibitor, and PD098059, an inhibitor of the Erk1/2 pathway, were used, respectively. Inhibitors of casein kinase II (DRB) and p38 MAP kinase (SB203580) have only moderate inhibitory effects on the phosphorylation of GAIP (10% and 25%, respectively), whereas H-89, an inhibitor of PKA, has no effect. From the above results, we reasoned that the signaling pathway responsible for the phosphorylation of GAIP is controlled by Erk1/2 and/or PKC. Using antibodies directed against the activated forms of Erk1/2, we showed that amino acids and PD098059 were able to reduce the activation of Erk1/2 (Fig. 3 a). By contrast, these products have no effect on the activity of the closely related p38 MAP kinase (Fig. 3 b). These results are in agreement with the inhibition of GAIP phosphorylation stimulated by either amino acids or PD098059. It is well known that PKCs can stimulate the Erk1/2 pathway in different cell types (28Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar) including HT-29 cells (29Andre F. Rigot V. Remacle-Bonnet M. Luis J. Pommier G. Marvaldi J. Gastroenterology. 1999; 116: 64-77Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). According to these data, GF109203X greatly impaired the activation of Erk1/2 (Fig. 3 a) but has only a moderate effect on p38 MAP kinase (Fig. 3 b). This result would explain the inhibitory effect of GF109203X on the phosphorylation of GAIP without totally excluding the possibility that GAIP could be a PKC substrate (see below). In order to examine whether the GAP activity of GAIP is influenced by its phosphorylation, we performed an in vitro phosphorylation assay using recombinant GAIP and a panel of Ser/Thr protein kinases (corresponding to the inhibitors used in Fig. 2). We then tested the GAP activity of each recombinant phosphorylated GAIP toward the recombinant Gαi3 protein. Incubation of GAIP with purified kinases in the presence of [γ -32P]ATP revealed labeling by PKC, casein kinase II, and Erk2 but not by PKA and the p38 MAP kinase (Fig. 4 a). In parallel control experiments were conducted on histones and myelin basic protein whose substrate characteristics with respect to the different kinases used are well known (see Fig. 4 b and “Experimetnal Procedures”). The ability of recombinant GAIP incubated with PKC, casein kinase II, or Erk2 to accelerate the GTPase activity of Gαi3 protein was determined during a single round of [γ -32P]GTP hydrolysis experiment. At each concentration of GAIP used (from 30 nm to 30 μm), the rate of GTP hydrolysis (k), corresponding to the Pi liberation as a function of time, was calculated. These values were plotted as a function of GAIP concentration (Fig. 4 c). In all assays the Gαi3 protein was present at a concentration of 250 nm. The intrinsic rate of GTP hydrolysis by Gαi3 was 0.022 ± 0.001 s−1. Addition of 30 μm GAIP resulted in acceleration of the GTPase activity by more than 6.5 times (k = 0.145 ± 0.003 s−1) with an EC50 value of 3.16 ± 0.12 μm. This stimulating effect was abolished when boiled GAIP was used. The casein kinase II- and PKC-mediated phosphorylation of GAIP did not change the rate of GTP hydrolysis when compared with that of the recombinant GAIP (k = 0.146 ± 0.002 s−1, EC50 = 3.15 ± 0.11 μm andk = 0.158 ± 0.002 s−1, EC50 = 3.09 ± 0.12 μm, respectively). However, an increase in the rate of GTP hydrolysis and a 3-fold reduction of EC50 for GAIP were observed when was phosphorylation was effectuated by Erk2 (k = 0.187 ± 0.01 s−1, EC50 = 1.12 ± 0.1 μm) as compared with the unphosphorylated recombinant GAIP. The calculation of the estimated stoichiometry of in vitro Erk2 phosphorylation of GAIP indicates an average incorporation of 0.4 mol of phosphate/mol of protein, suggesting the presence of a single Erk1/2 phosphorylation site. The primary sequence of GAIP contains two consensus sites for PKC and seven consensus sites for casein kinase II (15De Vries L. Mousli M. Wurmser A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11916-11920Crossref PubMed Scopus (267) Google Scholar). However, several SP motifs that are potential phosphorylation sites for MAP kinases are also present. Among them, serine 151 in the RGS domain appears to be the most appropriate consensus site for Erk1/2 (ILSP) (30Kreegipuu A. Blom N. Brunak S. Jarv J. FEBS Lett. 1998; 430: 45-50Crossref PubMed Scopus (113) Google Scholar). This sequence is highly conserved among the GAIP subfamily (GAIP, Ret-RGS1, and RGSZ1) and absent among the five other RGS subfamilies (5Zheng B. De Vries L. Farquhar M.G. Trends Biochem. Sci. 1999; 24: 411-414Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) (Fig. 5 a). For this reason we performed site-directed mutagenesis on Ser151 in order to generate a recombinant S151A GAIP mutant. Both GAIP and the S151A GAIP mutant were then examined in an in vitrophosphorylation assay. The absence of phosphorylation of S151A GAIP mutant by Erk2, shown in Fig. 5 b, is not a consequence of gross modifications of the protein structure because: (i) the GAIP activity of the S151A GAIP protein is close to that of the wild-type GAIP (k = 0.119 ± 0.002 s−1, EC50 = 4.17 ± 0.09 μm versus k= 0.145 ± 0.003 s−1, EC50 = 3.16 ± 0.12 μm), (ii) PKC phosphorylates both the S151A GAIP mutant and wild-type GAIP in a similar manner (Fig. 5 b), and (iii) the heat-denatured S151A GAIP mutant is not a substrate for Erk2 (data not shown). After incubation with PKC or Erk2, the rate of GTP hydrolysis observed in the presence of S151A GAIP mutant was reduced compared with that observed with phosphorylated wild-type GAIP (Fig. 5 c). These data strongly suggest that phosphorylation of serine 151 residue by Erk2 is required for the increase of the GAP activity of GAIP. We have reported that the autophagic pathway is dependent upon the activity of the Gαi3protein and GAIP in HT-29 cells. To study the relationship between GAIP phosphorylation and macroautophagy, we have measured the autophagic sequestration of the cytosolic enzyme LDH in sedimentable material (Table I) and the degradation of long-lived [14C]valine-labeled proteins (Fig. 6) in HT-29 cells transfected with His-tagged expression vectors containing either the wild-type GAIP cDNA or the S151A GAIP mutant cDNA. According to our previous studies (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the overexpression of wild-type GAIP stimulates both autophagic parameters in HT-29 cells (Fig. 6 and Table I). These parameters were greatly reduced when amino acids or PD098059 were added (Fig. 6 and Table I). These results are in line with those shown in Figs. 1 and 2 on the effect of amino acids and PD098059 on both the phosphorylation of GAIP and Erk1/2 activation.Table IEffect of the S151A mutation of GAIP and Erk1/2 activity on autophagic sequestration of LDH in HT-29 cellsHis-tagged vectorGAIP expression 1-aThe ratio of overexpressed GAIP (WT or S151A mutant)/endogenous GAIP was calculated after scanning of a Western blot using an antibody directed against GAIP. The overexpression of His-tagged WT GAIP or His-tagged S151A GAIP was then detected by Western blot using an antibody directed against His tag.Autophagic sequestration of LDH 1-bThe values reported are the mean ± S.D. of four determinations.HBSSHBSS + amino acidsHBSS + PD098059%/hEmpty14.45 ± 0.672.15 ± 0.372.81 ± 0.42WT GAIP2.59.52 ± 1.352.75 ± 0.512.94 ± 0.58S151A GAIP2.44.25 ± 0.512.07 ± 0.352.31 ± 0.401-a The ratio of overexpressed GAIP (WT or S151A mutant)/endogenous GAIP was calculated after scanning of a Western blot using an antibody directed against GAIP. The overexpression of His-tagged WT GAIP or His-tagged S151A GAIP was then detected by Western blot using an antibody directed against His tag.1-b The values reported are the mean ± S.D. of four determinations. Open table in a new tab By contrast to the stimulatory effect of wild-type GAIP on autophagy, S151A GAIP failed to increase the rate of autophagy in the absence of amino acids under conditions where Erk1/2 MAP kinases were activated in S151 GAIP-expressing cells (data not shown) and the same level of transfected proteins were expressed (Table I). Several studies have shown that the activity of RGS is controlled at the transcriptional level (4De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (522) Google Scholar). Posttranslational modifications of RGS (including palmitoylation and phosphorylation) have been reported to be involved in their cellular localization and stability (19Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar, 31De Vries L. Elenko E. Hubler L. Jones T.L. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (158) Google Scholar). To our knowledge, our results provide the first evidence thatin vitro phosphorylation of GAIP by the Erk2 MAP kinase increases its GAP activity toward the Gαi3 protein. Substitution of Ser151 by Ala abrogates this stimulation. The GAP activity of S151A GAIP is comparable to that observed with unphosphorylated wild-type GAIP. This result strongly suggests that this GAIP mutant is still functional and able to interact with the Gαi3 protein. Ser151 is located in a loop connecting helices V and VI of GAIP in its RGS domain (32de Alba E. De Vries L. Farquhar M.G. Tjandra N. J. Mol. Biol. 1999; 291: 927-939Crossref PubMed Scopus (64) Google Scholar). Crystallographic data have shown that this loop is involved in the interaction of RGS4 and the Gαi1 protein (33Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). A critical residue in this loop is RGS4-Asn128. A serine residue (Ser156) in GAIP occupies this position. This characteristic defines a subfamily of RGS proteins, which includes GAIP, RET1-RGS, and RGSZ1 (5Zheng B. De Vries L. Farquhar M.G. Trends Biochem. Sci. 1999; 24: 411-414Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The sequence upstream of Ser156 is also conserved in this subfamily149Ileu-Leu-Ser-Pro152, whereas in other RGS subfamilies this tetrapeptide is not conserved. The determination of the soluble structure of GAIP by NMR techniques has suggested that, upon binding to Gαi, conformational rearrangements of the loop V-VI may facilitate the formation of electrostatic interactions that stabilize the RGS protein structure (32de Alba E. De Vries L. Farquhar M.G. Tjandra N. J. Mol. Biol. 1999; 291: 927-939Crossref PubMed Scopus (64) Google Scholar). Whether phosphorylation of Ser151 could directly interfere with the residues of Gαi actively involved in GTP hydrolysis or stabilize GAIP structure to optimize its GAP activity remains to be elucidated by structural studies. According to the presence of potential phosphorylation sites in its sequence, GAIP was in vitro phosphorylated by casein kinase II and PKC but not by PKA and p38 MAP kinase. These results are in good agreement with the inhibition profile of GAIP phosphorylation observedin vivo. Recently it has been reported that the casein kinase II-dependent phosphorylation of GAIP on Ser24 (outside of RGS domain) could regulate its membrane association (21Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar). Although Ser151 is a potential phosphorylation site for PKC, it is not likely to be a substrate for this kinase in vitro because the PKC-dependent phosphorylation of the S151A GAIP mutant was similar to that of the wild-type GAIP. However, it is interesting to note that a PKC site is located in the C-terminal part of GAIP (Thr201) in the vicinity of a GIPC, a PDZ domain-containing protein, interacting site (34De Vries L. Lou X. Zhao G. Zheng B. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12340-12345Crossref PubMed Scopus (190) Google Scholar). These data suggest that depending on the acting Ser/Thr protein kinase, the occupancy of different phosphorylation sites regulates different functional properties of GAIP. Following up the above reported results, we have shown that GAIP is phosphorylated in a Erk1/2-dependent manner in a cellular environment. This adds a novel mammalian non-nuclear substrate for Erk1/2 MAP kinases (35Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3254) Google Scholar). The list of non-nuclear MAP kinase substrates includes several proteins involved in the interruption of G protein signaling pathways (members of G protein-coupled receptor kinases and arrestins (20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 36Lin F.T. Miller W.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 15971-15974Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). This suggests that MAP kinases can act as feedback regulators of trimeric G protein signaling. The recent demonstration of this feedback control of MAP kinases on G protein signaling via RGS in yeast emphasizes the importance of this regulation loop, which has been conserved during evolution (20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Finally, our work concerns the signal control of the macroautophagic pathway. Previously, we have demonstrated that GAIP is a regulator of the Gi3 protein-dependent macroautophagic pathway in intestinal derived HT-29 cells (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A hallmark of macroautophagy in many mammalian cells including HT-29 cells is to be inhibited by amino acids (26Mortimore G.E. Miotto G. Venerando R. Kadowaki M. Lloyd J.B. Mason R.W. Subcellular Biochemistry. Plenium Publishing Corp., New York1996: 93-136Google Scholar). This inhibition has been demonstrated to be dependent upon the phosphorylation of the ribosomal S6 protein by the activation p70S6 kinase in rat hepatocytes (37Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). This signaling pathway is also operative in HT-29 cells. 2E. Ogier-Denis, S. Pattingre, J. El Benna, and P. Codogno, unpublished results. Here we report that amino acids can control autophagy by inhibiting Erk1/2-dependent GAIP phosphorylation. This control is dependent upon the presence of Ser151 in GAIP suggesting that the Erk1/2-dependent phosphorylation of Ser151 accelerates the GTP hydrolysis by the Gαi3 protein. This would be in line with our previous data showing that the GDP-bound form of the Gαi3 protein increases the rate of autophagy (23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In HT-29 cells amino acids have a coordinated inhibitory effect on autophagy by activating the p70S6 kinase and inhibiting the Erk1/2 pathway. The mechanism by which amino acids control the Erk1/2 pathway in this cell line remains to be investigated. The control of macroautophagy by the p38 MAP kinase has been reported in rat hepatocytes in response to change in cell volume (38Haussinger D. Schliess F. Dombrowski F. Vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Although care must be taken in extrapolating data from different experimental models, a role for the MAP kinase family in the control of the signaling of a major catabolic pathway could be a new function for these kinases. We thank Dr. S. E. H. Moore for critical reading of the manuscript.

Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Gα-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) <i>J. Biol. Chem.</i> 275, 39090–39095). Here we show that ERK1/2 activation by aurintricarboxylic acid (ATA) treatment induces the phosphorylation of GAIP in an amino acid-dependent manner. Accordingly, ATA challenge increased the rate of macroautophagy, whereas epidermal growth factor did not significantly affect macroautophagy and GAIP phosphorylation status. In fact, ATA activated the ERK1/2 signaling pathway, whereas epidermal growth factor stimulated both the ERK1/2 pathway and the class I phosphoinositide 3-kinase pathway, known to decrease the rate of macroautophagy. Amino acids interfered with the ATA-induced macroautophagy by inhibiting the activation of the kinase Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIP- and amino acid-dependent control of macroautophagy was confirmed in HT-29 cells expressing the Ras(G12V,T35S) mutant. Similar to the protein phosphatase 2A inhibitor okadaic acid, amino acids sustained the phosphorylation of Ser²⁵⁹, which is involved in the negative regulation of Raf-1. In conclusion, these results add a novel target to the amino acid signaling-dependent control of macroautophagy in intestinal cells.

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1wt-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation. The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1wt-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation. Macroautophagy (hereafter referred to as autophagy) is a lysosomal catabolic pathway for macromolecules and organelles (1Seglen P.O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (370) Google Scholar, 2Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 3Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (3014) Google Scholar). 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Type II is characterized by the accumulation of autophagic vacuoles. Recent studies have shown that autophagic machinery plays a role in cell death occurring in the absence of pro-apoptotic members of the Bcl-2 family (14Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1196) Google Scholar) and when caspases are inhibited (15Yu L. Alva A. Su H. Dutt P. Freundt E. Welsh S. Baehrecke E.H. Lenardo M.J. Science. 2004; 304: 1500-1502Crossref PubMed Scopus (1108) Google Scholar). However, in many situations apoptosis and autophagy may both contribute to cell death making difficult to clearly distinguish type I from type II cell death (16Edinger A.L. Thompson C.B. Curr. Opin. Cell Biol. 2004; 16: 663-669Crossref PubMed Scopus (1121) Google Scholar, 17Baehrecke E.H. Nat. Rev. Mol. Cell. Biol. 2005; 6: 505-510Crossref PubMed Scopus (852) Google Scholar, 18Golstein P. Kroemer G. 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Beclin 1 is a tumor suppressor gene product that plays an important role, together with the class III phosphatidylinositol 3-kinase (PI3K), during the formation of the autophagosome (22Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2771) Google Scholar, 23Kihara A. Kabeya Y. Ohsumi Y. Yoshimori T. EMBO Rep. 2001; 2: 330-335Crossref PubMed Scopus (728) Google Scholar). Beclin 1 haploinsufficiency induces a high level of tumor development in various mouse tissues (24Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.-L. Mizushima N. Ohsumi Y. Cattoretti G. Levine B. J. Clin. Invest. 2003; 112: 1809-1820Crossref PubMed Scopus (1889) Google Scholar, 25Yue Z. Jin S. Yang C. Levine A.J. Heintz N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15077-15082Crossref PubMed Scopus (1795) Google Scholar). 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However, the role of autophagy during tumor progression is probably more complex, because autophagic vacuoles have been observed in several types of human tumors (29Alva A.S. Gultekin S.H. Baehrecke E.H. Cell Death Differ. 2004; 11: 1046-1048Crossref PubMed Scopus (87) Google Scholar). Whether these autophagic vacuoles are associated with cell survival or cell death remains to be clearly demonstrated. Numerous drugs have been reported to trigger cell death with autophagic features in cancer cells (20Ogier-Denis E. Codogno P. Biochim. Biophys. Acta. 2003; 1603: 113-128PubMed Google Scholar, 21Gozuacik D. Kimchi A. Oncogene. 2004; 23: 2891-2906Crossref PubMed Scopus (1267) Google Scholar, 30Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1486) Google Scholar). However, the events that lead to the accumulation of autophagic vacuoles by cancer treatments have only begun to be identified (30Kondo Y. Kanzawa T. Sawaya R. Kondo S. Nat. Rev. Cancer. 2005; 5: 726-734Crossref PubMed Scopus (1486) Google Scholar). The antiestrogen drug tamoxifen triggers autophagic cell death in human breast cancer MCF-7 cells (31Bursch W. Ellinger A. Kienzl H. Torok L. Pandey S. Sikorska M. Walker R. Hermann R.S. Carcinogenesis. 1996; 17: 1595-1607Crossref PubMed Scopus (466) Google Scholar). We have recently shown that the sphingolipid ceramide is a downstream target of tamoxifen during autophagy in MCF-7 cells (32Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Ceramide stimulates autophagy by relieving the class I PI3K/Akt/PKB pathway and provoking the accumulation of the autophagy gene product Atg6/Beclin 1. In line with these findings, a cell-permeable analog of ceramide has recently been shown to induce autophagic cell death in glioma cells by increasing the expression of the mitochondrial bound BH3-only BNIP3 protein (33Daido S. Kanzawa T. Yamamoto A. Takeuchi H. Kondo Y. Kondo S. Cancer Res. 2004; 64: 4286-4293Crossref PubMed Scopus (356) Google Scholar). Sphingolipids are ubiquitous constituents of eukaryotic membranes. Their metabolism is a highly dynamic process generating second messengers, including ceramide, sphingosine, and sphingosine 1-phosphate (S1P) (34Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (621) Google Scholar, 35Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (443) Google Scholar, 36Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar, 37Levade T. Malagarie-Cazenave S. Gouazé V. Ségui B. Tardy C. Betito S. 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In the present work we show that SK1 modulates two major players of autophagy: the mTOR signaling pathway and the expression of Beclin 1. Stimulation of autophagy by SK1 is dependent upon the production of S1P. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy did not induce cell death but has a protective effect toward apoptosis during nutrient starvation. These findings, and others in the literature (32Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 33Daido S. Kanzawa T. Yamamoto A. Takeuchi H. Kondo Y. Kondo S. Cancer Res. 2004; 64: 4286-4293Crossref PubMed Scopus (356) Google Scholar), show that both the tumor-suppressor ceramide and the tumor-promoter S1P (38Ogretmen B. Hannun Y.A. Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1012) Google Scholar) are able to trigger autophagy but with different outcomes on cancer cell survival and death. C2-Cer and DMS were from Calbiochem (VWR International). Fumonisin B1 (FB1) was purchased from Sigma. 3MA was from Fluka. z-VAD-fmk was from Apotech. Cell culture medium and fetal bovine serum were from Invitrogen. Nitrocellulose membranes were from Schleicher & Schüll. Ceramide from porcine brain, used as an internal standard, was from Avanti Polar Lipids Inc. The radioisotopes l-[U-14C]valine (5.47 GBq/mmol) and [γ32-P]ATP (110 TBq/mmol) and [9,10-3H]myristic acid (1.69 TBq/mmol), the ECL™ Western blotting detection kit, and the donkey anti-rabbit antibody were purchased from BD Biosciences. The mouse monoclonal anti-Beclin1 was from Amersham Bioscience; the mouse monoclonal anti-PARP and the rabbit polyclonal anti-phospho-PKB Ser473 were from Cell Signaling. Goat anti-PKB was from Santa Cruz Biotechnology. The mouse monoclonal anti-actin was from Chemicon International Inc. The rabbit polyclonal anti-p70S6K, anti-phospho-p70S6K Thr389, anti-phospho-S6 Ser235, and anti-4E-BP1 were from Ozyme, and anti-FLAG M2 was from Sigma. The rabbit polyclonal anti-Atg7 was kindly provided by W. A. Dunn (University of Florida, Gainesville). Goat anti-mouse and swine anti-goat antibodies were obtained from Bio-Rad and Caltag, respectively. The plasmid expression vectors for human sphingosine kinase 1 and catalytically inactive mutant human sphingosine kinase 1 (pcDNA3-hSK1-FLAG and pcDNA3-hSK1G82D-FLAG) were a gift from S. Pitson and B. Wattenberg (Hanson Institute, Adelaide, Australia) (40Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The plasmid expression vector encoding for GFP-LC3 (pEGFP-LC3) was kindly provided by T. Yoshimori (National Institute of Genetics, Mishima, Japan). The plasmid expression vectors for PLD1 and catalytically inactive mutant PLD1 (pCGN-HA-hPLD1 and pCGN-HA-hPLD1K898R) were kindly given by M. Frohman (SUNY, New York). Human breast cancer MCF-7 cells were maintained at 37 °C in 10% CO2 in DMEM, supplemented with 10% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin as previously reported (32Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Beclin 1 MCF-7/tet-off (MCF-7.beclin 1) cells kindly provided by B. Levine (University of Texas Southwest Medical Center, Dallas) were used during starvation-induced autophagy. Briefly, cells were maintained in DMEM with 200 μg/ml hygromycin B and 2 μg/ml tetracycline (41Liang X.H. Yu J. Brown K. Levine B. Cancer Res. 2001; 61: 3443-3449PubMed Google Scholar). Expression of Beclin 1 was analyzed by Western blotting 5 days after tetracycline withdrawal. Starvation was induced by incubating cells in nutrient-free medium (Hanks' balanced salt solution plus 0.1% bovine serum albumin). Cells were transfected by using FuGENE 6 transfection reagent (Roche Applied Science), as recommended by the manufacturer. Briefly, cells (0.5 × 106 cells/well) were plated in 6-well plates. Two days later, 1 μg of plasmid and 3 μl of FuGENE 6 were suspended in 100 μl of DMEM and added to the culture medium. When cells were co-transfected with SK1, PLD1 (or empty vector), and GFP-LC3 vectors, the transfection efficiency was 40% as determined by counting GFP-positive cells. Cells were analyzed 24 or 48 h after co-transfection as detailed below. SK1 and Atg7 knockdowns were accomplished by transfecting MCF-7/GFP-LC3 cells with siRNAs. The RNAi target sequences were: sense, 5′-GGGCAAGGCCUUGCAGCUC-3′ for SK1 (42Bektas M. Jolly P.S. Muller C. Eberle J. Spiegel S. Geilen C.C. Oncogene. 2005; 24: 178-187Crossref PubMed Scopus (122) Google Scholar) and sense, 5′-CCAACACACUCGAGUCUUU-3′ for Atg7. As a control, siRNA targeting the unrelated protein phosphomannomutase-2 (sense, 5′-CUGGGAAAUGAUGUGGUUG) was used. siRNA were purchased from Eurogenetec (Seraing, Belgium). MCF-7 cells were seeded at 8 × 104/cm2. After 24 h, cells were transfected for 4 h with siRNA using Oligofectamine (Invitrogen) as recommended by the manufacturer. Cells were cultured for 24 h before analysis. Cells were disrupted in 4 m guanidine thiocyanate, and total RNA was isolated by sedimentation in cesium chloride. RNA was transcribed with superscript II (Invitrogen). SK1 was amplified by the following specific primers (42Bektas M. Jolly P.S. Muller C. Eberle J. Spiegel S. Geilen C.C. Oncogene. 2005; 24: 178-187Crossref PubMed Scopus (122) Google Scholar): forward, 5′-ATCCAGAAGCCCCTGTGTAGCCTCC-3′; reverse, 5′-GCAGCAAACATCTCACTGCCCAGGT-3′. Glyceraldehyde-3-phosphate dehydrogenase was amplified by following primers: forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′; reverse, 5′-AGCCTTCTCCATGGTCGTGAAGAC-3′. GFP-LC3 Staining—GFP-LC3 staining was carried out essentially as described previously (43Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5509) Google Scholar). At the indicated time after transfection GFP-LC3 staining was visualized using an Axioplan Zeiss microscope, and the number of GFP-LC3-positive cells with GFP-LC3 dots was determined. Proteolysis—Proteolysis was determined as described previously (44Pattingre S. Petiot A. Codogno P. Methods Enzymol. 2004; 390: 17-31Crossref PubMed Scopus (50) Google Scholar). Briefly, cells were incubated for 24 h at 37 °C in normal culture medium containing 7400 Bq/ml of [14C]valine. Cells were rinsed three times with PBS, pH 7.4, and then the cells were incubated for 1 h in complete medium supplemented with 10 mm valine. After incubating for 1 h, during which time short-lived proteins had been degraded, the medium was replaced by fresh chase medium for another 4 h. When required, C2-Cer (75 μm), FB1 (100 μm), DMS (1.5 μm), and 3MA (10 mm) were added to the chase medium. For the PLD1- or SK1-overexpressing cells, radiolabeling with [14C]valine was started 24 h after transfection. Cells and radiolabeled proteins from the 4-h chase medium were precipitated in trichloroacetic acid at a final concentration of 10% (v/v) at 4 °C. Radioactivity was determined by liquid scintillation counting. Protein degradation was calculated by dividing the acid-soluble radioactivity recovered from both cells and medium by the radioactivity contained in the precipitated proteins from both cells and medium. Cell viability was determined by the trypan blue exclusion test as previously described (32Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). When required, 40 μm z-VAD-fmk was added prior to incubation in nutrient-free medium (Hanks' balanced salt solution plus 0.1% of bovine serum albumin). Cells were collected by centrifuging (10 min, at 1200 × g), washing with PBS, and resuspending in lysis buffer (1% Triton X-100, 25 mm Tris-HCl, pH 7.4, 1 mm phenylmethylsulfonyl fluoride, 1% Protease Inhibitor Mixture (PIC), 1 mm Na3VO4, 1 mm NaF) for 1 h at 4°C. The cell lysate was centrifuged (20,000 × g for 30 min at 4 °C), and the supernatant was recovered. Protein concentrations were determined by the bicinchoninic acid (BCA) method as recommended by the manufacturer. Extracted proteins were first separated in SDS-polyacrylamide gels and then electrotransferred onto nitrocellulose membranes. After blocking overnight with fat-free milk, the membranes were incubated with appropriate primary antibodies: anti-Beclin 1 (1/2000), anti-PARP (1/1000), anti-p70S6K (1/1000), anti-phospho-p70S6K Thr389 (1/1000), anti-phospho-S6 Ser235(1/1000), anti-4E-BP1 (1/1000), anti-FLAG (1/1000), anti-Atg7 (1/1000), and anti-PKB and anti-phospho-PKB Ser473 (1/1000). Primary antibodies were detected by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies against rabbit, goat, or mouse immunoglobulins. Fluorographs were quantitatively scanned using the NIH image software. Ceramide levels were measured using the DGK assay as described previously (45Perry D.K. Hannun Y.A. Trends Biochem. Sci. 1999; 24: 226-227Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, cells were collected, and lipids were extracted according to a previous study (46Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43126) Google Scholar). The organic phase was divided into 1/2 and 1/6 aliquots, dried, and used for ceramide and total phospholipid measurements (47Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar), respectively. Briefly, 30 nmol of extracted lipids was incubated at room temperature for 45 min in the presence of β-octylglucoside/dioleoylphosphatidylglycerol micelles, 2 mm dithiothreitol, 5 μg of proteins of DGK containing membranes (Calbiochem), and 1 mm ATP mixed with [γ-32P]ATP (4.8 × 105 Bq/ml) in a final volume of 0.1 ml. At the end of the reaction, lipids were extracted, and [32P]ceramide 1-phosphate was determined by TLC separation in chloroform/acetone/methanol/acetic acid/water (10:4:3:3:1, v/v). The radioactivity associated with ceramide 1-phosphate spots was determined after scraping and counting in a scintillation counter. Ceramide levels were expressed in terms of the total phospholipid content. Sphingosine kinase activity was determined as described previously (48Granata R. Trovato L. Garbarino G. Taliano M. Ponti R. Sala G. Ghidoni R. Ghigo E. FASEB J. 2004; 18: 1456-1458Crossref PubMed Scopus (102) Google Scholar). Briefly, cells were collected and lysed by repeated freeze-thawing cycles in 200 μl of lysis buffer (20 mm Tris, pH 7.4, 20% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 0.01 mm MgCl2, 1 mm Na3VO4, 15 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% PIC, 0.1% Triton X-100, 0.5 mm 4-deoxypyridoxine). After centrifuging at 13,000 × g for 30 min, the protein concentration of supernatant was determined with a Coomassie Plus Protein Assay kit (Pierce). Proteins (100 μg) were then incubated with 25 μmd-erythrosphingosine dissolved in 0.1% Triton X-100, 2 mm ATP, and [γ-32P]ATP (3.7 × 105 Bq dissolved in 20 mm MgCl2) for 30 min at 37 °C in a final volume of 200 μl. The reaction was stopped by adding 20 μl of HCl, 1 n, followed by 800 μl of chloroform/methanol/HCl (100:200:1, v/v). After vigorous vortexing, 250 μl of chloroform and 250 μl of KCl 2 m were added, and phases were separated by centrifugation. The organic layer was dried and resuspended in chloroform/methanol/HCl 37% (100:100:0.2, v/v). Lipids were resolved on silica TLC plates (Whatman) in 1-butanol/acetic acid/water (3:1:1, v/v). Labeled S1P spots were visualized by autoradiography and quantified by scraping and counting in a scintillation counter. For quantification of S1P, MCF-7 cells were labeled for 7 h in serum-free medium with 0.3 μCi/ml d-erythro-[3-3H]sphingosine (PerkinElmer Life Sciences). After washing with PBS, cells were scraped and lysed in water. Radiolabeled S1P was extracted by phase partition as previously described (49Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar, 50Vessey D.A. Kelley M. Karliner J.S. Anal. Biochem. 2005; 337: 136-142Crossref PubMed Scopus (47) Google Scholar) and counted by liquid scintillation. Phospholipase D Activity—PLD activity was determined as described previously (51O'Luanaigh N. Pardo R. Fensome A. Allen-Baume V. Jones D. Holt M.R. Cockcroft S. Mol. Biol. Cell. 2002; 13: 3730-3746Crossref PubMed Scopus (85) Google Scholar) with some modifications. Briefly, 2 × 106 cells were plated on 25-cm2 flasks for 24 h; when required, cells were transfected with PLD1 vectors (wild-type and K898R mutant). After 24 h, cells were preincubated in 0.5% fetal bovine serum containing DMEM for 1 h. After removing the preincubation medium, cells were labeled with [3H]myristic acid (Amersham Biosciences) for 1 h at 1.11 × 105 Bq/ml in 0.5% fetal bovine serum containing DMEM. Cells were rinsed with PBS and incubated for 30 min in complete medium containing 0.5% butanol. Cells were rinsed with PBS and scraped in methanol/water (98:2, v/v). Cells were rinsed with methanol, and the lipids were extracted by adding equal amounts of chloroform and water. After centrifuging, the lower phase was collected and dried under vacuum before being redissolved in chloroform. Lipids were separated by TLC on silica-coated plastic sheets (Merck) in a solvent system of chloroform: methanol:acetic acid:water (75:45:3:0.4, v/v). Radioactive components were detected by autoradiography. PLD activity was determined by the [3H]phosphatidylbutanol/[3H]phosphatidylcholine ratio. Phosphatidylcholine (Avanti polar) and phosphatidylbutanol (Biomol) diluted in chloroform were loaded as standards and were visualized with iodine vapor. Statistical analysis of the differences between the groups was performed using Student's t test. p < 0.05 was considered statistically significant. Sphingosine Kinase 1 Overexpression Stimulates Autophagy—MCF-7 cells were transfected either with the cDNA encoding the wild-type SK1 (SK1wt) or that encoding a mutant inactive form (SK1G82D). The activity of SK was significantly increased in SK1wt-overexpressing cells when compared with that observed in control cells transfected with an empty vector or in SK1G82D-expressing cells (Fig. 1A). Accordingly, SK1wt-overexpressing cells displayed a higher level of S1P compared with control cells (Fig. 1A). In the presence of the SK inhibitor DMS, both S1P production and SK activity were dramatically reduced in these cells. In contrast, no change in the level of ceramide was detected in SK1wt-overexpressing cells at any time points after transfection (Fig. 1, A and B). Autophagy was first analyzed after transfection of GFP-LC3 (Fig. 2A). LC3 is a reliable marker of autophagosomes in mammalian cells, and its localization changes from a diffuse cytosolic pattern to a punctuate pattern representing its recruitment to the autophagosomal membrane

It has become clear in recent years that autophagy not only serves to produce amino acids for ongoing protein synthesis and to produce substrates for energy production when cells become starved but autophagy is also able to eliminate defective cell structures and for this reason the process may be implicated in several diseased states. Autophagy is controlled by complex signalling pathways, including that used by insulin. In these pathways, phosphatidylinositol 3-kinases and the protein kinase mTOR play important roles.

Autophagy is a highly regulated self-degradative mechanism required at a basal level for intracellular clearance and recycling of cytoplasmic contents. Upon intracellular pathogen invasion, autophagy can be induced as an innate immune mechanism to control infection. Nevertheless, pathogens have developed strategies to avoid or hijack autophagy for their own benefit. The molecular pathways inducing autophagy in response to infection remain poorly documented. We report here that the engagement of CD46, a ubiquitous human surface receptor able to bind several different pathogens, is sufficient to induce autophagy. CD46-Cyt-1, one of the two C-terminal splice variants of CD46, is linked to the autophagosome formation complex VPS34/Beclin1 via its interaction with the scaffold protein GOPC. Measles virus and group A Streptococcus, two CD46-binding pathogens, induce autophagy through a CD46-Cyt-1/GOPC pathway. Thus, upon microorganism recognition, a cell surface pathogen receptor can directly trigger autophagy, a critical step to control infection.

Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime self-destruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death.

Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity. The involvement of proteins with properties of tumor suppressor and oncogenic properties at different steps of the pathway implies that autophagy must be considered in tumor progression. Autophagy as a stress response mechanism protects cancer cells from low nutrient supply or therapeutic insults. Autophagy is also involved in the elimination of cancer cells by triggering a non-apoptotic cell death program, suggesting a negative role in tumor development. These two aspects of autophagy will be discussed in this review.

Autophagy, a lysosomal process involved in the maintenance of cellular homeostasis, is responsible for the turnover of long-lived proteins and organelles that are either damaged or functionally redundant. The process is tightly controlled by the insulin-amino acid-mammalian target of the rapamycin-dependent signal-transduction pathway. Research in the last decade has indicated not only that autophagy provides cells with oxidizable substrate when nutrients become scarce but also that it can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease, diabetes, and infections.

Macroautophagy is a vacuolar degradation pathway that terminates in the lysosomal compartment after formation of a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The identification of ATG (autophagy-related) genes that are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of macroautophagy, and its roles in cell function, which extend far beyond degradation and quality control of the cytoplasm. Macroautophagy, which plays a major role in tissue homeostasis, is now recognized as contributing to innate and adaptive immune responses. Recently, several mediators of apoptosis have been shown to control macroautophagy. Deciphering the cross talk between macroautophagy and apoptosis probably should help increase understanding of the role of macroautophagy in human disease and is likely to be of therapeutic importance.

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K+/H+ ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH+ population in HMLER cells than in the ALDH− population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH+ population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH+ population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.

A major side effect of the powerful immunosuppressive drug cyclosporine (CsA) is the development of a chronic nephrotoxicity whose mechanisms are not fully understood. Recent data suggest that tubular cells play a central role in the pathogenesis of chronic nephropathies. We have shown that CsA is responsible for endoplasmic reticulum (ER) stress in tubular cells. Autophagy has recently been described to be induced by ER stress and to alleviate its deleterious effects. In this study, we demonstrate that CsA induces autophagy in primary cultured human renal tubular cells through LC3II expression and autophagosomes visualization by electron microscopy. Autophagy is dependant of ER stress because various ER stress inducers activate autophagy and salubrinal, an inhibitor of eIF2α dephosphorylation that protects cells against ER stress, inhibited LC3II expression. Furthermore, autophagy inhibition during CsA treatment with beclin1 siRNA significantly increases tubular cell death. Finally, immunohistochemical analysis of rat kidneys demonstrates a positive LC3 staining on injured tubular cells, suggesting that CsA induces autophagy in vivo. Taken together, these results demonstrate that CsA, through ER stress induction, activates autophagy as a protection against cell death.

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy.

Article16 January 2015free access Unsaturated fatty acids induce non-canonical autophagy Mireia Niso-Santano Mireia Niso-Santano Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Shoaib Ahmad Malik Shoaib Ahmad Malik Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Government College University, Faisalabad, Pakistan Search for more papers by this author Federico Pietrocola Federico Pietrocola Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Guillermo Mariño Guillermo Mariño Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Valentina Cianfanelli Valentina Cianfanelli Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Search for more papers by this author Amena Ben-Younès Amena Ben-Younès Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Rodrigo Troncoso Rodrigo Troncoso Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Search for more papers by this author Maria Markaki Maria Markaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Search for more papers by this author Valentina Sica Valentina Sica Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author Valentina Izzo Valentina Izzo Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Kariman Chaba Kariman Chaba Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Chantal Bauvy Chantal Bauvy Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Oliver Kepp Oliver Kepp Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Search for more papers by this author Patrick Rockenfeller Patrick Rockenfeller Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Heimo Wolinski Heimo Wolinski Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Frank Madeo Frank Madeo Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Sergio Lavandero Sergio Lavandero Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Patrice Codogno Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Francis Harper Francis Harper Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis orcid.org/0000-0002-5253-1466 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Greece Search for more papers by this author Francesco Cecconi Francesco Cecconi Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Maria Chiara Maiuri Maria Chiara Maiuri Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Search for more papers by this author Mireia Niso-Santano Mireia Niso-Santano Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Shoaib Ahmad Malik Shoaib Ahmad Malik Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Government College University, Faisalabad, Pakistan Search for more papers by this author Federico Pietrocola Federico Pietrocola Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Guillermo Mariño Guillermo Mariño Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Valentina Cianfanelli Valentina Cianfanelli Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Search for more papers by this author Amena Ben-Younès Amena Ben-Younès Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Rodrigo Troncoso Rodrigo Troncoso Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Search for more papers by this author Maria Markaki Maria Markaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Search for more papers by this author Valentina Sica Valentina Sica Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author Valentina Izzo Valentina Izzo Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Kariman Chaba Kariman Chaba Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Chantal Bauvy Chantal Bauvy Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Oliver Kepp Oliver Kepp Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Search for more papers by this author Patrick Rockenfeller Patrick Rockenfeller Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Heimo Wolinski Heimo Wolinski Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Frank Madeo Frank Madeo Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Sergio Lavandero Sergio Lavandero Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Patrice Codogno Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Francis Harper Francis Harper Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis orcid.org/0000-0002-5253-1466 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Greece Search for more papers by this author Francesco Cecconi Francesco Cecconi Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Maria Chiara Maiuri Maria Chiara Maiuri Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Search for more papers by this author Author Information Mireia Niso-Santano1,2,3,‡, Shoaib Ahmad Malik1,2,3,4,‡, Federico Pietrocola1,2,3,5, José Manuel Bravo-San Pedro1,2,3, Guillermo Mariño1,2,3, Valentina Cianfanelli6,7, Amena Ben-Younès1,2,3, Rodrigo Troncoso8,9,10, Maria Markaki11, Valentina Sica1,2,3,5, Valentina Izzo1,2,3, Kariman Chaba1,12, Chantal Bauvy12,13,14, Nicolas Dupont12,13,14, Oliver Kepp1,3,15, Patrick Rockenfeller16,17, Heimo Wolinski16,17, Frank Madeo16,17, Sergio Lavandero8,9,10,18, Patrice Codogno12,13,14, Francis Harper2,19, Gérard Pierron2,19, Nektarios Tavernarakis11,20, Francesco Cecconi6,7,21, Maria Chiara Maiuri1,2,3, Lorenzo Galluzzi1,2,3,12,‡ and Guido Kroemer 1,3,12,15,22,‡ 1Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 2Gustave Roussy Comprehensive Cancer Center, Villejuif, France 3INSERM, U1138, Paris, France 4Government College University, Faisalabad, Pakistan 5Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France 6Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy 7Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark 8Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile 9Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile 10Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile 11Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece 12Université Paris Descartes, Sorbonne Paris Cité, Paris, France 13INSERM, U1151, Paris, France 14Institut Necker Enfants-Malades, Paris, France 15Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France 16Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria 17BioTechMed Graz, Graz, Austria 18Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA 19CNRS, UMR8122, Villejuif, France 20Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Greece 21Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy 22Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France ‡MN-S and SAM equally contributed to this paper ‡LG and GK share co-senior authorship *Corresponding author. Tel: +33 1 4211 6046; Fax: +33 1 4211 6047; E-mail: [email protected] The EMBO Journal (2015)34:1025-1041https://doi.org/10.15252/embj.201489363 See also: VA Bankaitis (April 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus. Synopsis A systematic screen in cancer cells reveals that unsaturated and saturated fatty acids induce autophagy via distinct pathways, with unsaturated fatty acids acting in a Golgi-dependent but Beclin-1-independent manner. Saturated and unsaturated fatty acids promote autophagy, in vitro and in vivo, via different molecular mechanisms. The saturated fatty acid palmitate stimulates canonical, BECN1- and PIK3C3-dependent autophagic responses that involve JNK1, PKR and AMPK. The unsaturated fatty acid oleate promotes a non-canonical BECN1-independent autophagic response that requires an intact Golgi apparatus. Oleate-induced non-canonical autophagy is conserved in human cells, mice, yeast and nematodes. Introduction Macroautophagy (here referred to as “autophagy”) relies on the sequestration of cytoplasmic structures in double-membraned vesicles that are commonly known as autophagosomes. Upon closure, autophagosomes fuse with lysosomes to generate autolysosomes, resulting in the degradation of the autophagosomal cargo and inner membrane by acidic hydrolases. The nucleation of autophagosomes is often initiated by unc-51-like autophagy-activating kinase 1 (ULK1) and generally requires phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, also known as VPS34). PIK3C3 encodes the catalytic subunit of a class III phosphatidylinositol-3 kinase that operates within a large macromolecular complex involving beclin 1, autophagy related (BECN1), ATG14 and phosphoinositide-3-kinase, regulatory subunit 4 (PIK3R4, also known as VPS15) (Weidberg et al, 2011). The elongation of autophagosomal membranes relies on two conjugation reactions, that is, the conjugation of ATG12 to ATG5 and that of mammalian orthologs of yeast Atg8 to phosphatidylethanolamine. In this setting, microtubule-associated protein 1 light chain 3 (MAP1LC3, best known as LC3) and GABA(A) receptor-associated protein (GABARAP) family members are conjugated to phosphatidylethanolamine in process that resembles ubiquitination. Such a cascade of reactions involves ATG7 (an E1-like enzyme) and ATG3 (an E2-like enzyme), eventually yielding phosphatidylethanolamine-conjugated and autophagosome-associated LC3 and GABARAP proteins (Rubinsztein et al, 2012). Thus, the formation of autophagosomes can be quantified by following the redistribution of LC3B (the most studied member of the LC3/Atg8 family) to cytoplasmic puncta or by assessing its lipidation (which increases its electrophoretic mobility). In addition, the so-called autophagic flux (i.e. the actual ability of autophagosomes to degrade intracellular components) can be monitored by measuring the turnover of long-lived proteins or by determining the degradation of specific autophagic substrates such as sequestosome 1 (SQSTM1, best known as p62) (Bjorkoy et al, 2009). Of note, autophagic responses are negatively regulated by mechanistic target of rapamycin (MTOR) complex I (MTORCI), a key hub for the control of cell survival, growth and proliferation (Laplante & Sabatini, 2012). Autophagy plays a major role in the differentiation and function of adipocytes (Singh et al, 2009b; Zhang et al, 2009), in the mobilization of lipid droplets within hepatocytes (a process that has been dubbed “lipophagy”) (Singh et al, 2009a), as well as in the oxidation of fatty acids (FAs) by cancer cells (Guo et al, 2013), underscoring its significant impact on lipid metabolism. Conversely, several saturated FAs (SFAs) and unsaturated FAs (UFAs) appear to modulate autophagy (Brenner et al, 2013). Moreover, neutral lipid droplets have recently been shown to contribute to autophagic responses by providing substrates for the formation of autophagosomes (Dupont et al, 2014a). The UFAs oleate (C18:1) and linoleate (C18:2) stimulate the autophagic flux in both mammary epithelial cells (Pauloin et al, 2010) and hepatocytes (Mei et al, 2011). Along similar lines, polyunsaturated FAs including di-homo-γ-linoleic acid (C20:3), arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) induce autophagy in multiple cell types (Fukui et al, 2013; O'Rourke et al, 2013). Conversely, the autophagy-modulatory potential of the SFA palmitate (C16:0) exhibits a significant degree of variation. Indeed, palmitate has been shown to inhibit (Las et al, 2011; Kim et al, 2013) as well as to activate (Khan et al, 2012; Martino et al, 2012) autophagy in pancreatic islet and vascular endothelial cells. Moreover, palmitate reportedly limits autophagy in hepatocytes (Mei et al, 2011), yet promotes it in mouse embryonic fibroblasts (Tan et al, 2012). At least in some cell types, the autophagic response to palmitate involves protein kinase C (PKC) (Tan et al, 2012), eukaryotic translation initiation factor 2, subunit 1α, 35 kDa (EIF2S1, best known as eIF2α) and its substrate eIF2α kinase 2 (EIF2AK2, best known as PKR), and mitogen-activated protein kinase 8 (MAPK8, best known as JNK1) (Shen et al, 2012). In addition, palmitate has been shown to stimulate or inhibit AMP-activated protein kinase (AMPK), an upstream regulator of autophagy impinging on MTORCI signaling, depending on experimental variables including cell type, concentration and exposure time (Fediuc et al, 2006; Sun et al, 2008). Although these discrepancies have not been investigated in detail, they may reflect the ability of palmitate to modulate autophagy in a manner that changes with time, initially promoting it and then inhibiting it. In support of this notion, feeding mice with a high-fat diet for a prolonged period has been shown to promote alterations in intracellular lipids that are accompanied by defects in the fusion between autophagosomes and lysosomes (Koga et al, 2010). Driven by the consideration that distinct FAs influence the composition of cellular membranes and affect long-term health in a different manner (Wijendran & Hayes, 2004; Cascio et al, 2012), we performed a systematic analysis of FAs for their capacity to induce autophagy in short-term experiments. Here, we report the unexpected finding that SFAs and UFAs promote autophagy by activating different molecular mechanisms. Thus, while SFAs stimulate canonical autophagy, UFAs trigger a BECN1- and PIK3C3-independent autophagic response that is accompanied by the redistribution of LC3 to Golgi-associated vesicles. Results Palmitate and oleate induce autophagy in vitro and in vivo To identify FAs that trigger autophagy, we exposed human osteosarcoma U2OS cells that stably express a green fluorescent protein (GFP)-LC3 chimera (Shen et al, 2011) to a panel of 26 FAs differing in the length of the carbon chain as well as in saturation status, followed by assessment of GFP-LC3+ puncta by automated fluorescence microscopy. SFAs with 15–18 carbon atoms such as pentadecanoic, hexadecanoic, heptadecanoic and octadecanoic acid (stearate), but neither smaller nor larger SFAs, promoted the formation of GFP-LC3+ dots in U2OS cells (Fig 1A). Similarly, several UFAs with 14–20 carbon atoms, including myristoleic, palmitoleic, oleic, linoleic and arachidonic acid, efficiently triggered the aggregation of GFP-LC3 in cytoplasmic dots (Fig 1A). Based on solubility considerations and natural abundance in human cells (as well as in commonly employed oils, i.e. palm and olive oil), we chose to focus our study on one UFA, that is, oleic acid (oleate) and one SFA, that is, hexadecanoic acid (palmitate). Figure 1. Induction of autophagy by palmitate and oleate A. Screening for autophagy-inducing fatty acids. U2OS cells expressing GFP-LC3 were cultured in control conditions (Co) or treated with the indicated saturated (in black) or unsaturated (in blue) fatty acids for 4 h, followed by the quantification of the number of cytoplasmic GFP-LC3+ dots per cell by automated fluorescence microscopy. Data are means ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01 versus untreated cells). B–D. Autophagic flux induced by palmitate, stearate and oleate. Wild-type (WT, C) and GFP-LC3-expressing (B) U2OS cells as well as HeLa cells (D) were maintained in control conditions, exposed to nutrient-free (NF) conditions or treated with 500 μM oleate (OL), 500 μM stearate (ST) or 500 μM palmitate (PA), alone or in combination with 10 μg/ml E-64d and 10 μg/ml pepstatin (Pep) for 4 (D) or 6 (B, C) h. Thereafter, the number of cytoplasmic GFP-LC3+ dots per cell (B), LC3 lipidation and p62 degradation (C) or the degradation of labeled long-lived proteins (D) was quantified. In (B) and (D), data are means ± SEM (B) or normalized means ± SD (D) of at least three independent experiments (B), or four replicate assessments from one representative (D) experiment(s) out of three performed (*P < 0.05, **P < 0.01 versus untreated cells; ##P < 0.01 versus cells treated with PA or OL only; n.s., not significant versus cells treated with E-64d plus Pep). In (C), β-actin levels were monitored to ensure equal loading of lanes, and densitometry was employed to quantify the abundance of lipidated LC3 (LC3-II) and p62 (both normalized to β-actin levels). E, F. Involvement of ATG5 and ATG7 in fatty acid-induced autophagy. WT (F) or GFP-LC3-expressing (E) U2OS cells were transfected with a control siRNA (siUNR) or with siRNAs targeting ATG5 (siATG5) or ATG7 (siATG7) for 48 h and either maintained in control conditions or treated with 500 μM PA or 500 μM OL. Six hours later, cells were processed for the quantification of the number of cytoplasmic GFP-LC3+ dots per cell by automated fluorescence microscopy (E) or for the assessment of LC3 lipidation and p62 degradation by immunoblotting (F). In (E), data are means ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01 versus untreated siUNR-transfected cells; #P < 0.05 versus siUNR-transfected cells treated with PA or OL only). In (F), β-actin levels were monitored to ensure equal loading of lanes. G. Induction of autophagy by fatty acids in mice. C57BL/6 mice were injected i.p. with vehicle only, 100 mg/kg PA or 100 mg/kg OL. One or two hours later, animals were euthanatized and LC3 lipidation, p62 degradation and AMPK phosphorylation were assessed by immunoblotting in the indicated tissues. GAPDH levels were monitored to ensure equal loading of lanes. Densitometry was employed to quantify the abundance of lipidated LC3 (LC3-II) and p62 (both normalized to GAPDH levels) and AMPK phosphorylation (normalized to total AMPK levels). Results are means ± SD of three mice (*P < 0.05, **P < 0.01, ***P < 0.001, versus vehicle-treated mice). Download figure Download PowerPoint The increase in GFP-LC3+ puncta, LC3 lipidation and p62 degradation induced by palmitate and oleate was exacerbated by the addition of E-64d and pepstatin (Fig 1B and C), two well-known inhibitors of lysosomal proteases. This indicates that palmitate and oleate do not stimulate the mere accumulation of LC3

Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1-8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.

Autophagy is a rapidly expanding field in the sense that our knowledge about the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. Similarly, the vocabulary associated with autophagy has grown concomitantly. This fact makes it difficult for readers, even those who work in the field, to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors or chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, or the role of accessory machinery or structures that are associated with autophagy.

Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

p53 and JNK are two apoptosis-regulatory factors frequently deregulated in cancer cells and also involved in the modulation of autophagy. We have recently investigated the links between these two signalling pathways in terms of the regulation of autophagy. We showed that 2-methoxyestradiol (2-ME), an antitumoral compound, enhances autophagy and apoptosis in Ewing sarcoma cells through the activation of both p53 and JNK pathways. In this context, p53 regulates, at least partially, JNK activation which in turn modulates autophagy through two distinct mechanisms: on the one hand it promotes Bcl-2 phosphorylation resulting in the dissociation of the Beclin 1-Bcl-2 complex and on the other hand it leads to the upregulation of DRAM (Damage-Regulated Autophagy Modulator), a p53 target gene. The critical role of DRAM in 2-ME-mediated autophagy and apoptosis is underlined by the fact that its silencing efficiently prevents the induction of both processes. These findings not only report the interplay between JNK and p53 in the regulation of autophagy but also uncover the role of JNK activation in the regulation of DRAM, a pro-autophagic and proapoptotic protein.

Macroautophagy (hereafter called autophagy) is a vacuolar, lysosomal pathway for catabolism of intracellular material that is conserved among eukaryotic cells. Autophagy plays a crucial role in tissue homeostasis, adaptation to stress situations, immune responses, and the regulation of the inflammatory response. Blockade or uncontrolled activation of autophagy is associated with cancer, diabetes, obesity, cardiovascular disease, neurodegenerative disease, autoimmune disease, infection, and chronic inflammatory disease. During the past decade, researchers have made major progress in understanding the three levels of regulation of autophagy in mammalian cells: signaling, autophagosome formation, and autophagosome maturation and lysosomal degradation. As we discuss in this review, each of these levels is potentially druggable, and, depending on the indication, may be able to stimulate or inhibit autophagy. We also summarize the different modulators of autophagy and their potential and limitations in the treatment of life-threatening diseases.

AbstractThe heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFκB transcription factor is activated during the recovery period after heat shock. In this study, we analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent (p65/RelA-depleted) cells. We demonstrate for the first time that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery and cell death is strongly increased in NFκB incompetent cells. Moreover, if autophagy is artificially induced in these cells, the cytotoxicity of heat shock is turned back to normal. We show that despite a post-heat shock increase of Beclin 1 level in NFκB competent cells, neither Beclin 1/class III PI3K complex, Bcl2/BclXL nor mTOR kinase are NFκB targets whose modulation of expression could be responsible for NFκB activation of autophagy during heat shock recovery. In contrast, we demonstrate that aberrantly folded/aggregated proteins are prime events in the signaling pathway leading to NFκB mediated autophagy after heat shock. Hence, our findings demonstrate that NFκB-induced autophagy during heat shock recovery is an additional cell response to HS-induced protein denaturation/aggregation; this mechanism increases cell survival, probably through clearance of irreversibly damaged proteins.

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.

<h2>Summary</h2> The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode <i>Caenorhabditis elegans</i> is lost when the expression of the EP300 ortholog <i>cpb-1</i> is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM.

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3(-/-) and atg5(-/-) MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress. While the signaling cascade upstream of p27 leading to its cytoplasmic localization and autophagy induction has been extensively studied, how p27 stimulates the autophagic process remains unclear. Here, we investigated the mechanism by which p27 promotes autophagy upon glucose deprivation. Mouse embryo fibroblasts (MEFs) lacking p27 exhibit a decreased autophagy flux compared to wild-type cells and this is correlated with an abnormal distribution of autophagosomes. Indeed, while autophagosomes are mainly located in the perinuclear area in wild-type cells, they are distributed throughout the cytoplasm in p27-null MEFs. Autophagosome trafficking towards the perinuclear area, where most lysosomes reside, is critical for autophagosome-lysosome fusion and cargo degradation. Vesicle trafficking is mediated by motor proteins, themselves recruited preferentially to acetylated microtubules, and autophagy flux is directly correlated to microtubule acetylation levels. p27-/- MEFs exhibit a marked reduction in microtubule acetylation levels and restoring microtubule acetylation in these cells, either by re-expressing p27 or with deacetylase inhibitors, restores perinuclear positioning of autophagosomes and autophagy flux. Finally, we find that p27 promotes microtubule acetylation by binding to and stabilizing α-tubulin acetyltransferase (ATAT1) in glucose-deprived cells. ATAT1 knockdown results in random distribution of autophagosomes in p27+/+ MEFs and impaired autophagy flux, similar to that observed in p27-/- cells. Overall, in response to glucose starvation, p27 promotes autophagy by facilitating autophagosome trafficking along microtubule tracks by maintaining elevated microtubule acetylation via an ATAT1-dependent mechanism.

Abstract Cells subjected to stress situations mobilize specific membranes and proteins to initiate autophagy. Phosphatidylinositol-3-phosphate (PI3P), a crucial lipid in membrane dynamics, is known to be essential in this context. In addition to nutriments deprivation, autophagy is also triggered by fluid-flow induced shear stress in epithelial cells, and this specific autophagic response depends on primary cilium (PC) signaling and leads to cell size regulation. Here we report that PI3KC2α, required for ciliogenesis and PC functions, promotes the synthesis of a local pool of PI3P upon shear stress. We show that PI3KC2α depletion in cells subjected to shear stress abolishes ciliogenesis as well as the autophagy and related cell size regulation. We finally show that PI3KC2α and VPS34, the two main enzymes responsible for PI3P synthesis, have different roles during autophagy, depending on the type of cellular stress: while VPS34 is clearly required for starvation-induced autophagy, PI3KC2α participates only in shear stress-dependent autophagy.

Journal Article hnRNP G: sequence and characterization of a glycosylated RNA-binding protein Get access Michel Soulard, Michel Soulard Search for other works by this author on: Oxford Academic PubMed Google Scholar Veèronique Della Valle, Veèronique Della Valle Search for other works by this author on: Oxford Academic PubMed Google Scholar Mikkiko C. Siomi, Mikkiko C. Siomi 1Howard Hughes Medical Institue, Department of Biochemistry and Biophysics, School of Medicine, University of PennsylvaniaPA 19104-6148 USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Serafin Pin`ol-Roma, Serafin Pin`ol-Roma 1Howard Hughes Medical Institue, Department of Biochemistry and Biophysics, School of Medicine, University of PennsylvaniaPA 19104-6148 USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Patrice codogno, Patrice codogno 2INSERM U-239 Faculteè de Meèdecine Xavier Bichat16 rue H. Huchard, 75018 Paris Search for other works by this author on: Oxford Academic PubMed Google Scholar Chantal Bauvy, Chantal Bauvy 2INSERM U-239 Faculteè de Meèdecine Xavier Bichat16 rue H. Huchard, 75018 Paris Search for other works by this author on: Oxford Academic PubMed Google Scholar Michel Bellini, Michel Bellini 3Laboratoire de Geèneètoque du Deèveloppement, Universite P.et M.curriePl.Jussieu, 75005 Paris, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Jean-Claude Lacroix, Jean-Claude Lacroix 3Laboratoire de Geèneètoque du Deèveloppement, Universite P.et M.curriePl.Jussieu, 75005 Paris, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Guillaume Monod, Guillaume Monod Search for other works by this author on: Oxford Academic PubMed Google Scholar Gidden Dreyfuss, Gidden Dreyfuss 1Howard Hughes Medical Institue, Department of Biochemistry and Biophysics, School of Medicine, University of PennsylvaniaPA 19104-6148 USA Search for other works by this author on: Oxford Academic PubMed Google Scholar ... Show more Christian-Jacques Larsen Christian-Jacques Larsen * * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 21, Issue 18, 11 September 1993, Pages 4210–4217, https://doi.org/10.1093/nar/21.18.4210 Published: 11 September 1993 Article history Received: 14 June 1993 Published: 11 September 1993 Revision received: 28 September 1993 Accepted: 28 September 1993

AbstractThe downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival, and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haplo-insufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that remains the major infectious cause of birth defects, as well as being an important opportunistic pathogen. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process responsible for the degradation of cytoplasmic macromolecules, and the elimination of damaged organelles via a lysosomal pathway. This process is also triggered in organisms by stressful conditions and by certain diseases. Previous observations have suggested that autophagy (also known as xenophagy in this case) may contribute to innate immunity against viral infections. Recent studies on HSV-1, another herpesvirus, have shown that HSV-1 is able to avoid this cellular defense by means of a viral protein, ICP34.5, which antagonizes the host autophagy response. However, it was not known whether HCMV was also able to counteract autophagy. Here, we show that HCMV infection drastically inhibits autophagosome formation in primary human fibroblasts. Autophagy was assessed by GFP-LC3 redistribution, LC3-II and p62 accumulation and electron microscopy. Inhibition of autophagy occurred early in the infection by a mechanism involving viral protein(s). Indeed, only infected cells expressing viral proteins displayed a striking decrease of autophagy; whereas bystander, non-infected cells displayed a level of autophagy similar to that of control cells. HCMV activated the mTOR signaling pathway, and rendered infected cells resistant to rapamycin-induced autophagy. Moreover, infected cells also became resistant to the stimulation of autophagy by lithium chloride, an mTOR-independent inducer of autophagy. These findings suggest that HCMV has developed efficient strategies for blocking the induction of autophagy during infection.

A paper by Scott et al.,1 suggested that p70S6 kinase (p70S6k) is a positive regulatory factor for autophagy. This finding is in contrast to previous data suggesting a negative role for this factor. The Scott et al. article was highlighted in Nature News & Views,2 which elicited a commentary by A.J. Meijer and P. Codogno. These authors present an alternate model for the role of p70S6k in autophagic induction, although still as a positive factor. Following the initial commentary is a response by T.P. Neufeld and R.C. Scott.

Abstract Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl1−/−) was compared with that of cells from heterozygous (Sgpl1+/−) or wild-type (Sgpl1+/+) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl1 gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl1−/− cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl1−/− cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA–mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S1P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant. [Cancer Res 2009;69(24):9346–53]

Dysregulation of autophagy contributes to aging and to diseases such as neurodegeneration, cardiomyopathy, and cancer. The paper by Yang et al., 2010Yang L. Li P. Fu S. Calay E.S. Hotamisligil G.S. Cell Metab. 2010; 11 (this issue): 467-478Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar in this issue of Cell Metabolism indicates that defective autophagy may also underlie impaired insulin sensitivity in obesity and that upregulating autophagy can combat insulin resistance. Dysregulation of autophagy contributes to aging and to diseases such as neurodegeneration, cardiomyopathy, and cancer. The paper by Yang et al., 2010Yang L. Li P. Fu S. Calay E.S. Hotamisligil G.S. Cell Metab. 2010; 11 (this issue): 467-478Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar in this issue of Cell Metabolism indicates that defective autophagy may also underlie impaired insulin sensitivity in obesity and that upregulating autophagy can combat insulin resistance. Autophagy is responsible for the turnover of long-lived proteins and of intracellular structures that are damaged or functionally redundant. The process is essential for the maintenance of cellular homeostasis and is activated by starvation (to supply ATP-producing substrates, e.g., amino acids) and other stress-inducing conditions. Its dysregulation is involved in many disorders and in aging (Meijer and Codogno, 2009Meijer A.J. Codogno P. Crit. Rev. Clin. Lab. Sci. 2009; 46: 210-240Crossref PubMed Scopus (151) Google Scholar). In this issue of Cell Metabolism, Yang et al., 2010Yang L. Li P. Fu S. Calay E.S. Hotamisligil G.S. Cell Metab. 2010; 11 (this issue): 467-478Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar now show that hepatic autophagy is defective in obesity and diabetes and that its upregulation improves insulin sensitivity. During autophagy, part of the cytoplasm is surrounded by a double membrane, presumably formed from the endoplasmic reticulum (ER), to form an autophagosome that then fuses with lysosomes, after which the sequestered material is degraded. This process requires the participation of autophagy-related (ATG) proteins (see Meijer and Codogno, 2009Meijer A.J. Codogno P. Crit. Rev. Clin. Lab. Sci. 2009; 46: 210-240Crossref PubMed Scopus (151) Google Scholar for review). Autophagy is inhibited by the insulin-amino acid-mTOR signaling pathway via both short-term and long-term regulation mechanisms. Short-term inhibition can be produced by the mammalian target of rapamycin (mTOR) complex 1, which causes phosphorylation and the inhibition of ULK1 (the human homolog of yeast ATG1). Long-term regulation occurs via the transcription factors FoxO1 and FoxO3 (Liu et al., 2009Liu H.Y. Han J. Cao S.Y. Hong T. Zhuo D. Shi J. Liu Z. Cao W. J. Biol. Chem. 2009; 284: 31484-31492Crossref PubMed Scopus (277) Google Scholar), which control the transcription of atg genes and become phosphorylated and inhibited by insulin-induced activation of protein kinase B (Figure 1). Recent evidence indicates that dysregulation of autophagy is implicated in obesity (characterized by ER stress, insulin resistance, and glucose intolerance; Hotamisligil, 2010Hotamisligil G.S. Cell. 2010; 140: 900-917Abstract Full Text Full Text PDF PubMed Scopus (1857) Google Scholar) and in diabetes. Mice fed a high-fat diet (HFD) have reduced hepatic autophagy (Liu et al., 2009Liu H.Y. Han J. Cao S.Y. Hong T. Zhuo D. Shi J. Liu Z. Cao W. J. Biol. Chem. 2009; 284: 31484-31492Crossref PubMed Scopus (277) Google Scholar). However, autophagy, which is essential for maintaining the structure and function of β cells, is increased in β cells during this period of HF feeding (Ebato et al., 2008Ebato C. Uchida T. Arakawa M. Komatsu M. Ueno T. Komiya K. Azuma K. Hirose T. Tanaka K. Kominami E. et al.Cell Metab. 2008; 8: 325-332Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). In contrast, the destruction of insulin production in β cells by streptozotocin (STZ) increases autophagy in the liver (Liu et al., 2009Liu H.Y. Han J. Cao S.Y. Hong T. Zhuo D. Shi J. Liu Z. Cao W. J. Biol. Chem. 2009; 284: 31484-31492Crossref PubMed Scopus (277) Google Scholar). Likewise, autophagy is also increased in β cells derived from human subjects with type-2 diabetes (Masini et al., 2009Masini M. Bugliani M. Lupi R. del Guerra S. Boggi U. Filipponi F. Marselli L. Masiello P. Marchetti P. Diabetologia. 2009; 52: 1083-1086Crossref PubMed Scopus (245) Google Scholar). Yang et al., 2010Yang L. Li P. Fu S. Calay E.S. Hotamisligil G.S. Cell Metab. 2010; 11 (this issue): 467-478Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar demonstrate that, not only is hepatic autophagy severely depressed in dietary (HFD for 16–22 weeks) and genetic (ob/ob, db/db) models of murine obesity and diabetes (as indicated by the severe downregulation [>90%] of the ATG proteins LC3, BECLIN1, ATG5, and ATG7), but also that this process may underlie the impaired insulin sensitivity in these models. Thus, the suppression of hepatic ATG7 using shRNAi (using an adenovirus-mediated approach) in lean control mice resulted in insulin resistance and ER stress. Similarly, suppression of ATG7 or ATG5 in in vitro cell models caused defective insulin signaling. Conversely, restoration of hepatic autophagy in HFD-fed or in ob/ob mice, by means of adenovirus-directed overexpression of ATG7, diminished ER stress, counteracted insulin resistance, improved hepatic fat metabolism, decreased gluconeogenesis, and increased peripheral glucose disposal, effects that could all be blunted by the expression of dominant-negative ATG5. The latter observation makes it unlikely that the effect of ATG7 overexpression on insulin sensitivity can be ascribed to some unknown function of ATG7 that is unrelated to autophagy. Assuming that food intake had remained unchanged under these conditions, these data clearly demonstrate that defective autophagy and insulin resistance are closely linked. At first sight, it is surprising that autophagy can be restored by increasing the expression of ATG7 alone, amidst many other severely depressed ATG proteins. Surprisingly, the expression of ATG7 alone also appeared to enhance the expression of BECLIN 1, ATG 5, ATG12, and LC3 protein. The inhibition of calpain 2 at least partially restored ATG7 levels, suggesting that this Ca2+-dependent protease is responsible for the decrease in ATG7 protein and other ATG proteins in the liver of ob/ob mice. The intimate mechanism of this effect remains to be explored. In order to rule out the possibility that the chronic high insulin levels in ob/ob mice were responsible for the reduced levels of ATG proteins, STZ was administered to the animals but did not restore the hepatic levels of ATGs (in contrast to what happened in lean mice, in which STZ increased hepatic autophagy (Liu et al., 2009Liu H.Y. Han J. Cao S.Y. Hong T. Zhuo D. Shi J. Liu Z. Cao W. J. Biol. Chem. 2009; 284: 31484-31492Crossref PubMed Scopus (277) Google Scholar; see above). Likewise, hepatic levels of ATGs in db/db mice, in which insulin levels were already very low, were also severely depressed. These findings indicate that it is the chronic obesity-related ER stress rather than the high insulin levels that is responsible for the low levels of ATGs in the livers of these mice. An additional contributing factor is that, in obesity, mTOR in the liver is overactivated, presumably as the result of increased amino acid concentrations (Newgard et al., 2009Newgard C.B. An J. Bain J.R. Muehlbauer M.J. Stevens R.D. Lien L.F. Haqq A.M. Shah S.H. Arlotto M. Slentz C.A. et al.Cell Metab. 2009; 9: 311-326Abstract Full Text Full Text PDF PubMed Scopus (1782) Google Scholar). This activates lipogenesis and decreases fatty acid oxidation (Li et al., 2010Li S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. USA. 2010; 107: 3441-3446Crossref PubMed Scopus (468) Google Scholar), which exacerbates the negative effects of the high fat content of the diet. Insulin inhibits autophagy, so one would expect insulin resistance to disinhibit autophagy in order to protect cells against oxidative stress (Meijer and Codogno, 2009Meijer A.J. Codogno P. Crit. Rev. Clin. Lab. Sci. 2009; 46: 210-240Crossref PubMed Scopus (151) Google Scholar). In the case of β cells, autophagy does indeed increase during the initial period of HFD feeding, presumably in order to protect the β cells and allow them to boost their insulin production in order to deal with the increased plasma glucose concentrations. In contrast, hepatic autophagy declines in obesity. However, it may be that during the first few weeks of HFD feeding, autophagy increases before it starts to decline as a result of continued stress. In the study by Yang et al., 2010Yang L. Li P. Fu S. Calay E.S. Hotamisligil G.S. Cell Metab. 2010; 11 (this issue): 467-478Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar, autophagy was unchanged after 7 weeks of HFD feeding but declined thereafter; earlier time points were not analyzed. One may conclude that simply increasing autophagy, e.g., by pharmacological means, would be sufficient to improve insulin sensitivity in obesity. Unfortunately, metabolism is more complicated because autophagy is also required for adipocyte differentiation; it is the inhibition, rather than the stimulation, of autophagy in adipocytes that gives them a brown-fat-cell-like appearance that favors fatty acid oxidation and increases insulin sensitivity (Zhang et al., 2009Zhang Y. Goldman S. Baerga R. Zhao Y. Komatsu M. Jin S. Proc. Natl. Acad. Sci. USA. 2009; 106: 19860-19865Crossref PubMed Scopus (441) Google Scholar, Singh et al., 2009Singh R. Xiang Y. Wang Y. Baikati K. Cuervo A.M. Luu Y.K. Tang Y. Pessin J.E. Schwartz G.J. Czaja M.J. J. Clin. Invest. 2009; 119: 3329-3339Crossref PubMed Scopus (82) Google Scholar). Yang et al., 2010Yang L. Li P. Fu S. Calay E.S. Hotamisligil G.S. Cell Metab. 2010; 11 (this issue): 467-478Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar stress the many parallels between pathologies associated with obesity and the age-related impairment of metabolism. During aging, autophagy declines, and insulin resistance can develop (Meijer and Codogno, 2009Meijer A.J. Codogno P. Crit. Rev. Clin. Lab. Sci. 2009; 46: 210-240Crossref PubMed Scopus (151) Google Scholar). The best way to increase autophagy in vivo is by restricting calorie intake. Though it has been known for decades that calorie restriction is an effective way to combat obesity-related insulin resistance (and also aging), the present study reveals a mechanism for these effects. Defective Hepatic Autophagy in Obesity Promotes ER Stress and Causes Insulin ResistanceYang et al.Cell MetabolismJune 09, 2010In BriefAutophagy is a homeostatic process involved in the bulk degradation of cytoplasmic components, including damaged organelles and proteins. In both genetic and dietary models of obesity, we observed a severe downregulation of autophagy, particularly in Atg7 expression levels in liver. Suppression of Atg7 both in vitro and in vivo resulted in defective insulin signaling and elevated ER stress. In contrast, restoration of the Atg7 expression in liver resulted in dampened ER stress, enhanced hepatic insulin action, and systemic glucose tolerance in obese mice. Full-Text PDF Open Archive

Prion protein (PrPc) was originally viewed solely as being involved in prion disease, but now several intriguing lines of evidence have emerged indicating that it plays a fundamental role not only in the nervous system, but also throughout the human body. PrPc is expressed most abundantly in the brain, but has also been detected in other non-neuronal tissues as diverse as lymphoid cells, lung, heart, kidney, gastrointestinal tract, muscle, and mammary glands. Recent data indicate that PrPc may be implicated in biology of glioblastoma, breast cancer, prostate and gastric cancer. Over expression of PrPc is correlated to the acquisition by tumor cells of a phenotype for resistance to cell death induced by TNF alpha and TRAIL or antitumor drugs such as paclitaxel and anthracyclines. PrPc may promote tumorigenesis, proliferation and G1/S transition in gastric cancer cells. This review revisits the physiological functions of PrPc, and its possible implications for cancer biology.

Ceramide is a sphingolipid bioactive molecule that induces apoptosis and other forms of cell death, and triggers macroautophagy (referred to below as autophagy). Like amino acid starvation, ceramide triggers autophagy by interfering with the mTOR-signaling pathway, and by dissociating the Beclin 1:Bcl-2 complex in a c-Jun N-terminal kinase 1 (JNK1)-mediated Bcl-2 phosphorylation-dependent manner. Dissociation of the Beclin 1:Bcl-2 complex, and the subsequent stimulation of autophagy have been observed in various contexts in which the cellular level of long-chain ceramides was increased. It is notable that the conversion of short-chain ceramides (C(2)-ceramide and C(6)-ceramide) into long-chain ceramide via the activity of ceramide synthase is required to trigger autophagy. The dissociation of the Beclin 1:Bcl-2 complex has also been observed in response to tamoxifen and PDMP (an inhibitor of the enzyme that converts ceramide to glucosylceramide), drugs that increase the intracellular level of long-chain ceramides. However, and in contrast to starvation, overexpression of Bcl-2 does not blunt ceramide-induced autophagy. Whether this autophagy that is unchecked by forced dissociation of the Beclin 1:Bcl-2 complex is related to the ability of ceramide to trigger cell death remains an open question. More generally, the question of whether ceramide-induced autophagy is a dedicated cell death mechanism deserves closer scrutiny.

Autophagy-related 16 like-1 (ATG16L-1), immunity-related GTPase-M (IRGM), and nucleotide-binding oligomerization domain-containing 2 (NOD2) regulate autophagy, and variants in these genes have been associated with predisposition to Crohn's disease (CD). However, little is known about the role of autophagy in CD. Intestinal biopsies from untreated pediatric patients with CD, celiac disease, or ulcerative colitis were analyzed by immunohistochemistry and electron microscopy. We observed that autophagy was specifically activated in Paneth cells from patients with CD, independently of mucosal inflammation or disease-associated variants of ATG16L1 or IRGM. In these cells, activation of autophagy was associated with a significant decrease in number of secretory granules and features of crinophagy. These observations might account for the disorganization of secretory granules previously reported in Paneth cells from patients with CD. Autophagy-related 16 like-1 (ATG16L-1), immunity-related GTPase-M (IRGM), and nucleotide-binding oligomerization domain-containing 2 (NOD2) regulate autophagy, and variants in these genes have been associated with predisposition to Crohn's disease (CD). However, little is known about the role of autophagy in CD. Intestinal biopsies from untreated pediatric patients with CD, celiac disease, or ulcerative colitis were analyzed by immunohistochemistry and electron microscopy. We observed that autophagy was specifically activated in Paneth cells from patients with CD, independently of mucosal inflammation or disease-associated variants of ATG16L1 or IRGM. In these cells, activation of autophagy was associated with a significant decrease in number of secretory granules and features of crinophagy. These observations might account for the disorganization of secretory granules previously reported in Paneth cells from patients with CD. Besides the well-known autophagy function in degradation and recycling of long-lived proteins,1Sumpter R. et al.Semin Cell Dev Biol. 2010; 21: 699-711Crossref PubMed Scopus (102) Google Scholar recent studies have highlighted the new roles of macroautophagy in the innate and adaptive immune responses. Among the CD susceptibility genes identified to date, several are involved in the autophagy process, including autophagy-related 16 like-1 (ATG16L1),2Hampe J. et al.Nat Genet. 2007; 39: 207-211Crossref PubMed Scopus (1555) Google Scholar immunity-related GTPase-M (IRGM),3McCarroll S.A. et al.Nat Genet. 2008; 40: 1107-1112Crossref PubMed Scopus (538) Google Scholar nucleotide-binding oligomerization domain-containing 2 (NOD2),4Hugot J.P. et al.Nature. 2001; 411: 599-603Crossref PubMed Scopus (4694) Google Scholar, 5Ogura Y. et al.Nature. 2001; 411: 603-606Crossref PubMed Scopus (4177) Google Scholar and X-box binding protein 1.6Kaser A. et al.Cell. 2008; 134: 743-756Abstract Full Text Full Text PDF PubMed Scopus (1083) Google Scholar Therefore, we studied the autophagy regulation in pediatric intestinal samples before any treatment. As microtubule-associated protein 1 light chain 3 (LC3) is commonly used as a hallmark of autophagosome formation,7Klionsky D.J. et al.Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1975) Google Scholar we first investigated this protein by immunohistochemistry (Supplementary Figure 1). The LC3-positive cells were identified as Paneth cells (Supplementary Figure 2), and the other mucosa cell types remained negative in CD and controls. Although LC3-positive Paneth cells were rare in controls, most CD Paneth cells highly expressed LC3 (Figure 1A). With a positive threshold fixed at 9% of LC3+ Paneth cells, this CD-associated finding had a sensibility and a specificity of 82.4 and 71.4 in duodenum and 93.3 and 90.9 in ileum, respectively (Figure 1B). As inflammation can induce autophagy,8Deretic V. et al.Cell Host Microbe. 2009; 5: 527-549Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar we looked at the density of LC3-positive Paneth cells in inflammatory and noninflammatory areas of the CD mucosa. No difference was observed (Figure 1C). In addition, no Paneth cells were LC3-positive in duodenal inflammatory samples from celiac patients (Figure 1C and Supplementary Figure 3), suggesting that the phenomenon is not a consequence of inflammation. The LC3 accumulation in Paneth cells can result from either an increased formation of autophagosomes or a deficiency in their degradation by lysosomes.7Klionsky D.J. et al.Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1975) Google Scholar Lysosomal activity was attested by the presence of numerous autophagic vacuoles, and multiple intermediary lysosomal structures in transmission electron microscopy (TEM) and by the accumulation of the lysosomal marker lysosomal-associated membrane protein 1 within the CD Paneth cells compared with controls (Figure 1D). Together with the absence of accumulation of P62 within Paneth cells (data not shown),7Klionsky D.J. et al.Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1975) Google Scholar these data demonstrated that LC3 staining is due to an increased active autophagic flux. Because CD susceptibility is linked to ATG16L1, IRGM, and NOD2 (all genes involved in autophagy), we genotyped the participants for the main risk alleles of these genes. The density of LC3-positive Paneth cells was not associated with ATG16L1 or IRGM polymorphisms (Supplementary Figure 4). No conclusion was reached regarding the possible effects of NOD2 mutations because only 2 of the CD patients carried NOD2 mutations. Patients with no risk allele for the 3 studied genes were also too rare to conclude. Considering that a dysfunction in autophagy was correlated with defects in the exocytic pathway of Paneth cells in Atg16l1 hypomorphic mice,9Cadwell K. et al.Nature. 2008; 456: 259-263Crossref PubMed Scopus (1170) Google Scholar, 10Cadwell K. et al.Cell. 2010; 141: 1135-1145Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar we looked for the repercussions of the abnormal induction of autophagy. In CD, transmission electron microscopy indicated that duodenal and ileal Paneth cells displayed a significant decrease in the number of secretory granules associated with images of fusions of secretory granules between them or with autophagic/lysosomal vacuoles (Figure 2, Supplementary Figure 5). These images are hallmarks of crinophagy, a degradation process that specifically targets secretory granules.11Glaumann H. et al.Exp Mol Pathol. 1989; 50: 167-182Crossref PubMed Scopus (7) Google Scholar, 12Triantafyllou A. et al.Arch Oral Biol. 2007; 52: 768-777Crossref PubMed Scopus (6) Google Scholar In conclusion, we identified a novel pathway of autophagy that specifically affects Paneth cells of CD patients. In this process, known as crinophagy, secretory granules are the target of autophagolysosomes. This observation is consistent with the previous observations of a reduced secretion of human defensin 5 in CD patients,13Wehkamp J. et al.Proc Natl Acad Sci U S A. 2005; 102: 18129-18134Crossref PubMed Scopus (808) Google Scholar, 14Simms L.A. et al.Gut. 2008; 57: 903-910Crossref PubMed Scopus (201) Google Scholar the defects of cryptdin 10 secretion in Nod2 knockout mice15Kobayashi K.S. et al.Science. 2005; 307: 731-734Crossref PubMed Scopus (1481) Google Scholar and the disorganization and decreased number of secretion granules in hypomorphic Atg16l1 mice.9Cadwell K. et al.Nature. 2008; 456: 259-263Crossref PubMed Scopus (1170) Google Scholar, 10Cadwell K. et al.Cell. 2010; 141: 1135-1145Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar The authors thank Maryline Roy for helpful technical assistance and Fanny Daniel for helpful discussions. The manuscript was corrected by Proof Reading Services (London, UK). Drs Berrebi and Viala contributed equally to this work and share equally the last-author role. Intestinal biopsies from a total number of 65 children followed in the Department of Pediatric Gastroenterology at Robert Debré Hospital (Paris, France) were retrospectively analyzed. Biopsies were obtained during routine upper and lower endoscopies performed for diagnostic procedures. All participants were free of medication. Diagnoses were as follows: 32 CD (10 female and 22 male; median age of 11 years), 4 ulcerative colitis (1 female and 3 male; median aged 10 years), 9 celiac disease (4 female and 5 male; median age, 1.3 years), and 20 noninflammatory controls with no inflammation of the digestive tract (10 female and 10 male; median age, 10 years). Controls included patients with familial adenomatous polyposis (n = 2), esophageal atresia (n = 1), and irritable bowel syndrome (n = 1). For each participant, one or more biopsies were taken from the duodenum, ileum, cecum, and rectosigmoid junction. The study protocol was validated by the local ethics committee and patients and families gave informed written consent. Single nucleotide polymorphisms genotyping was performed for the 3 main CD risk alleles of NOD2 (rs2066844, rs2066845, rs2066847), ATG16L1 (rs2241880), and IRGM (rs10065172) using Taqman single nucleotide polymorphisms genotyping Assays (Applied Biosystems, Villebon sur Yvette, France). The exonic rs10065172 of IRGM showed strong linkage disequilibrium with the CD-associated deletion polymorphism rs13361189.1Parkes M. Barrett J.C. Prescott N.J. et al.Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility.Nat Genet. 2007; 39: 830-832Crossref PubMed Scopus (971) Google Scholar Biopsies were fixed in 4% phosphate-buffered formalin, processed routinely, and stained with H&E for histological analysis. Intestinal sections were considered normal when none of the following lesions were observed: architectural changes, epithelial damage, infiltration of the lamina propria by mononuclear or polymorphonuclear cells, erosion and ulcers, and granulomas. The immunohistochemical analysis used rabbit polyclonal antibodies directed against LC3 (1:5000; MBL, Woburn, MA), lysosomal-associated membrane protein 1 (1:2500; Sigma, Lyon, France), P62 (1:5000; Abgent, Oxfordshire, UK), IRGM (1:5000; Promega, Charbonnieres, France), and anti-CD15 mouse monoclonal antibodies (1:50; Dako, Trappes, France). Immunostaining was based on the avidin-biotin-peroxidase complex method (Vectastain ABC Kit; Vectorlabs; Peterborough, UK). Briefly, 4-μm paraffin sections were deparaffinized with Xylene and dehydrated with alcohol. Endogenous peroxidase was blocked with a blocking solution (Dako Real, Trappes, France), and sections were then covered with 10% normal rabbit serum for 20 min at room temperature. Sections were then incubated with the primary antibodies and processed following the avidin-biotin method. Negative controls were prepared by omitting the primary antibody. The percentages of LC3 and IRGM positive cells were derived from the total number and the number of stained cells (counted on at least 15 crypts/section) present on serial sections stained with H&E or immunostained. Duodenal and ileal biopsies from 7 controls and 6 CD patients were fixed with 2.5% glutaraldehyde in phosphate-buffered saline, post-fixed with 1% osmium tetroxide in phosphate-buffered saline, dehydrated in a graduated series of ethanol dilutions, and embedded in Epoxy Resin 812. Blocks were cut using a Leica Ultracut ultramicrotome. Ultrathin sections were placed on 200 mesh copper and were stained with 1% uranyl acetate in 50% ethanol and Reynolds lead citrate and viewed on a Jeol 1010 electron microscope coupled with a MegaView III camera and Olympus SiS Analysis System version 3.2 software. Sections were examined under magnifications of ×2500 to ×100,000. The qualitative and quantitative analyses of Paneth cells were performed in a blinded manner. Statistics were calculated on a total number of 30 Paneth cells in CD patients and 48 Paneth cells in noninflammatory controls. Results are expressed as mean ± SEM. The unpaired 2-tailed Student t test was used to determine any statistical significance between 2 experimental groups and 1-way analysis of variance was used for multiple groups (GraphPad Prism 5 Software; GraphPad Software, Inc., La Jolla, CA). Differences were considered significant when P < .05 (*P < .0.05; **P < .0.01; ***P < .0.001).Supplementary Figure 2Mucosa LC3-positive cells are Paneth cells. In controls or CD intestinal samples, no LC3 staining was observed in enterocytes or mononuclear cells. The LC3-positive cells were identified as Paneth cells because of their localization at the bottom of the crypts (A), their progressive disappearance from the right to left colon, and their expression of the CD15 marker in double-labeling experiments using anti-CD15 (red staining) and anti-LC3 antibodies (dark-brown staining) (B). Although the number of LC3-positive Paneth cells was increased in CD patients, the total number of Paneth cells per crypt was not changed (C). In the left colon, some crypts expressed a high level of LC3 corresponding to the presence of metaplastic Paneth cells, which frequently occurs during CD, with a haphazard distribution (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 3The LC3 expression was not correlated to inflammation in ileum.Show full captionIn ileum biopsies, CD Paneth cells expressed LC3 in noninflamed (A) and inflamed samples (B), whereas the LC3 staining was negative in ulcerative colitis sections (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 4LC3 staining is not correlated to CD risk alleles of ATG16L1 or IRGM. (A, B) Quantitative analyses of LC3 expression in CD patients carrying ATG16L1 300A or 300T variants, and rs10065172 polymorphism localized in IRGM in duodenum and in terminal ileum. (C, D) IRGM duodenal staining within Paneth cells is similar from controls and CD patients, respectively. (E) Quantitative analyses of IRGM-positive Paneth cells per crypt in duodenum from CD patients and controls. (F) Quantitative analyse of IRGM-positive Paneth cells of CD patients carrying rs10065172 polymorphism of IRGM (n = 7). C allele and T allele are respectively the frequent and CD-associated alleles of IRGM. Values are mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 5Intermediary degradation steps of secretory granules during crinophagy in CD Paneth cells. High magnification visualization show partially degraded secretory granules occurring during crinophagy in different CD Paneth cells (A−D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In ileum biopsies, CD Paneth cells expressed LC3 in noninflamed (A) and inflamed samples (B), whereas the LC3 staining was negative in ulcerative colitis sections (C).

Liver fibrosis is a common wound healing response to chronic liver injury of all causes, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide. Fibrosis results from prolonged parenchymal cell apoptosis and necrosis associated with an inflammatory reaction that leads to recruitment of immune cells, activation and accumulation of fibrogenic cells, and extracellular matrix accumulation. The fibrogenic process is driven by hepatic myofibroblasts, that mainly derive from hepatic stellate cells undergoing a transdifferentiation from a quiescent, lipid-rich into a fibrogenic myofibroblastic phenotype, in response to paracrine/autocrine signals produced by neighbouring inflammatory and parenchymal cells. Autophagy is an important regulator of liver homeostasis under physiological and pathological conditions. This review focuses on recent findings showing that autophagy is a novel, but complex, regulatory pathway in liver fibrosis, with profibrogenic effects relying on its direct contribution to the process of hepatic stellate cell activation, but with antifibrogenic properties via indirect hepatoprotective and anti-inflammatory properties. Therefore, cell-specific delivery of drugs that exploit autophagic pathways is a prerequisite to further consider autophagy as a potential target for antifibrotic therapy.

Significance Autophagy allows the lysosomal degradation of intracellular material. It is a tightly regulated process controlled by the tumor-suppressor gene p53, among others. Here we report a unique regulator of autophagy, BAT3, which modulates the intracellular localization of the enzyme p300, an enzyme that adds some acetyl residues on targets proteins (acetylation) to modulate their activity. To stimulate autophagy, BAT3 allows the acetylation of p53 by p300 in the nucleus, but limits the p300-dependent acetylation of ATG7, a protein specific for autophagy, in the cytosol. Thus, BAT3 acts on both the cytosol and the nucleus to tightly modulate autophagy.

Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.

Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca²⁺-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5–50 μM), sorafenib (1–40 μM), staurosporine (20–500 nM), or thapsigargin (20–500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca²⁺ chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca²⁺/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7<i>H</i>-benzimidazo[2,1-<i>a</i>]benz[<i>de</i>]isoquinoline-3-carboxylic acid acetate (STO-609; 10–30 μg/ml) and 2-(<i>N</i>-[2-hydroxyethyl])-<i>N</i>-(4-methoxybenzenesulfonyl)amino-<i>N</i>-(4-chlorocinnamyl)-<i>N</i>-methylamine (KN-93; 1–10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca²⁺ mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α₁/α₂-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca²⁺-dependent signaling, including CaMKI independent of AMPKα₁/α₂. Our data also suggest that AMPKα₁/α₂ might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.

Autophagy is a cellular pathway crucial for development, differentiation, survival and homeostasis. Autophagy can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease and infection. Recent studies have reported new functions of autophagy in the regulation of cellular processes such as lipid metabolism and insulin sensitivity. Important links between the regulation of autophagy and obesity including food intake, adipose tissue development, β cell function, insulin sensitivity and hepatic steatosis exist. This review will provide insight into the current understanding of autophagy, its regulation, and its role in the complications associated with obesity.

Macroautophagy or autophagy is a self-digesting mechanism that the cellular contents are engulfed by autophagosomes and delivered to lysosomes for degradation. Although it has been well established that autophagy is an important protective mechanism for cells under stress such as starvation via provision of nutrients and removal of protein aggregates and damaged mitochondria, there is a very complex relation between autophagy and cell death. At present, the molecular cross-talk between autophagy and apoptosis has been well discussed, while the relationship between autophagy and programmed necrotic cell death is less understood. In this review we focus on the role of autophagy in necrotic cell death by detailed discussion on two important forms of necrotic cell death: (i) necroptosis and (ii) poly-(ADP-ribose) polymerase (PARP)-mediated cell death. It is believed that one important aspect of the pro-survival function of autophagy is achieved via its ability to block various forms of necrotic cell death.

Motile and primary cilia (PC) are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. Recent studies highlight the interplay between cilia and autophagy, a conserved cellular process responsible for intracellular degradation. Signaling from the PC recruits the autophagic machinery to trigger autophagosome formation. Conversely, autophagy regulates ciliogenesis by controlling the levels of ciliary proteins. The cross talk between autophagy and ciliated structures is a novel aspect of cell biology with major implications in development, physiology and human pathologies related to defects in cilium function.

Key Points miR-125b reduces mitochondrial respiration and promotes elongation of mitochondrial network through BIK and MTP18 silencing, respectively. The miR-125b/BIK/MTP18 axis promotes adaptation of monocytes to inflammation.

The epithelial to mesenchymal transition (EMT) is an essential process during development and during tumor progression. Here, we observed the accumulation of the selective autophagy receptor and signaling adaptor sequestosome-1 (SQSTM1/p62) during growth factor-induced EMT in immortalized and tumor-derived epithelial cell lines. Modulation of the p62 level regulated the expression of junctional proteins. This effect was dependent on the ubiquitin-associated domain of p62, which stabilized the TGFβ/Smad signaling co-activator Smad4 and the EMT transcription factor Twist. This study highlights a novel function of p62 in a major epithelial phenotypic alteration.

Nanoparticles (NPs) can be toxic, depending on their physico-chemical characteristics. Macroautophagy/autophagy could represent a potential underlying mechanism of this toxicity. We therefore set up a study aimed to characterize in depth the effects, on autophagy, of macrophage exposure to NPs, with a particular attention paid to the role of NP physico-chemical characteristics (specifically chemical composition, shape, size, length, crystal phase, and/or surface properties). We demonstrate that exposure to carbon nanotubes (CNT) but not to spherical NPs leads to the blockage of the autophagic flux. We further identified lysosomal dysfunction, in association with the downregulation of SNAPIN expression, as the underlying mechanism responsible for the CNT-induced autophagy blockade. These results identify for the first time the shape as a major determinant of the interaction of NPs with the autophagy pathway. Moreover, identifying the lysosomes and SNAPIN as primary targets of MWCNT toxicity opens new directions in the interpretation and understanding of nanomaterial toxicity.

LC3-associated phagocytosis is a protective mechanism against inflammation, with antifibrogenic properties in the liver.

Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric Gi3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the αi3-subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the αi2-subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the αi3-subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [14C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the βγ dimer from the GTP-bound αi3-subunit was impaired in cells transfected with the G203A mutant. In contrast, a high rate of pertussis toxin-sensitive autophagy was observed in cells transfected with an αi3-subunit mutant (S47N) which has an increased guanine nucleotide exchange rate and increased preference for GDP over GTP. Cells that express pertussis toxin-insensitive mutants of either wild-type αi3-subunit (C351S) or S47N αi3-subunit (S47N/C351S) exhibit a high rate of autophagy. Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric Gi3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the αi3-subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the αi2-subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the αi3-subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [14C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the βγ dimer from the GTP-bound αi3-subunit was impaired in cells transfected with the G203A mutant. In contrast, a high rate of pertussis toxin-sensitive autophagy was observed in cells transfected with an αi3-subunit mutant (S47N) which has an increased guanine nucleotide exchange rate and increased preference for GDP over GTP. Cells that express pertussis toxin-insensitive mutants of either wild-type αi3-subunit (C351S) or S47N αi3-subunit (S47N/C351S) exhibit a high rate of autophagy.

Human colon cancer HT-29 cells exhibit a differentiation-dependent autophagic-lysosomal pathway that is responsible for the degradation of a pool of newly synthesized N-linked glycoproteins in undifferentiated cells. In the present study, we have investigated the molecular control of this degradative pathway in undifferentiated HT-29 cells. For this purpose, we have modulated the function and expression of the heterotrimeric G-proteins (Gs and Gi) in these cells. After pertussis toxin treatment which ADP-ribosylates heterotrimeric Gi-proteins, we observed an inhibition of autophagic sequestration and the complete restoration of the passage of N-linked glycoproteins through the Golgi complex. In contrast, autophagic sequestration was not reduced by cholera toxin, which acts on heterotrimeric Gs-proteins. Further insights on the nature of the pertussis toxin-sensitive α subunit controlling autophagic sequestration were obtained by cDNA transfections of αi subunits. Overexpression of the αi3 subunit increased autophagic sequestration and degradation in undifferentiated cells, whereas overexpression of the αi3 subunit, the only other pertussis toxin-sensitive α subunit expressed in HT-29 cells, did not alter the rate of autophagy. Human colon cancer HT-29 cells exhibit a differentiation-dependent autophagic-lysosomal pathway that is responsible for the degradation of a pool of newly synthesized N-linked glycoproteins in undifferentiated cells. In the present study, we have investigated the molecular control of this degradative pathway in undifferentiated HT-29 cells. For this purpose, we have modulated the function and expression of the heterotrimeric G-proteins (Gs and Gi) in these cells. After pertussis toxin treatment which ADP-ribosylates heterotrimeric Gi-proteins, we observed an inhibition of autophagic sequestration and the complete restoration of the passage of N-linked glycoproteins through the Golgi complex. In contrast, autophagic sequestration was not reduced by cholera toxin, which acts on heterotrimeric Gs-proteins. Further insights on the nature of the pertussis toxin-sensitive α subunit controlling autophagic sequestration were obtained by cDNA transfections of αi subunits. Overexpression of the αi3 subunit increased autophagic sequestration and degradation in undifferentiated cells, whereas overexpression of the αi3 subunit, the only other pertussis toxin-sensitive α subunit expressed in HT-29 cells, did not alter the rate of autophagy. INTRODUCTIONAlthough recent reports indicate that the autophagic-lysosomal route could be modulated by intracellular signals including mobilization of calcium pools(1.Gordon P.B. Holen I. Fosse M. Rotnes J.S. Seglen P.O. J. Biol. Chem. 1993; 268: 26107-26112Abstract Full Text PDF PubMed Google Scholar), protein phosphorylation(2.Holen I. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1993; 215: 113-122Crossref PubMed Scopus (77) Google Scholar), and GTP hydrolysis(3.Kadowaki M. Venerando R. Miotto G. Mortimore G.E. J. Biol. Chem. 1994; 269: 3703-3710Abstract Full Text PDF PubMed Google Scholar), little information on the molecular control of this process has yet to emerge. We have previously shown that the N-glycan trimming, which reflects endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumCTXcholera toxindMM1-deoxymannojirimycin3-MA3-methyladeninePTXpertussis toxinPBSphosphate-buffered saline.to Golgi complex traffic(4.Schwaninger R. Beckers J.M. Balch W.E. J. Biol. Chem. 1991; 266: 13055-13063Abstract Full Text PDF PubMed Google Scholar), is dependent on the state of enterocytic differentiation of HT-29 cells (5.Ogier-Denis E. Codogno P. Chantret I. Trugnan G. J. Biol. Chem. 1988; 263: 6031-6037Abstract Full Text PDF PubMed Google Scholar). The conversion of high mannose oligosaccharides to their complex counterparts is observed in differentiated cells, whereas a partial blockade of high mannose oligosaccharide trimming is a characteristic of undifferentiated cells. This partial impairment of N-linked glycoprotein processing is the consequence of a bypass of the Golgi complex and delivery of high mannose type glycoproteins to the lysosomal compartment (6.Trugnan G. Ogier-Denis E. Sapin C. Darmoul D. Bauvy C. Aubery M. Codogno P. J. Biol. Chem. 1991; 266: 20849-20855Abstract Full Text PDF PubMed Google Scholar) via an autophagic pathway(7.Houri J.J. Ogier-Denis E. Trugnan G. Codogno P. Biochem. Biophys. Res. Commun. 1993; 197: 805-811Crossref PubMed Scopus (26) Google Scholar). We have taken advantage of our previous data to investigate the function of heterotrimeric G-proteins, pivotal players in membrane dynamics (reviewed in (8.Bomsel M. Mostov K. Mol. Biol. Cell. 1992; 3: 1317-1328Crossref PubMed Scopus (162) Google Scholar)), in the molecular control of autophagy.EXPERIMENTAL PROCEDURESReagentsCTX, PTX, leupeptin, dMM, asparagine, and 3-MA were from Sigma. Cell culture reagents and Geneticin (G418) were from Life Technologies, Inc. (Eragny, France). Nitrocellulose membrane was from Schleicher & Schuell (Dassel, Germany). BCA kit was from Pierce. Pronase grade CB and endo-β-N-acetylglucosaminidase H were from Calbiochem (Meudon, France) and Genzyme (Cambridge, MA), respectively. Rat cDNAs encoding the αi3 and αi3 subunits were kindly provided by Dr. R. Reed (John Hopkins University, Baltimore, MD). Polyclonal rabbit antibodies to the α subunits of G i3-, G i3-proteins were from Euromedex (Souffelweyersheim, France). Each antibody was raised to decapeptides from the C termini of the respective α subunit(9.Simonds W.F. Goldsmith P.K. Codina J. Unson C.G. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7809-7813Crossref PubMed Scopus (309) Google Scholar). The radioisotopes [32P]NAD (1000 Ci/mmol), [14C]leucine (312 mCi/mmol), and 125I-labeled sheep anti-rabbit IgG (13 mCi/mg) were from Amersham (Les Ulis, France). D-[2-3H]mannose (20-30 Ci/mmol) was from ICN Biomedicals (Orsay, France). [3H]Raffinose (5-15 Ci/mmol) was from NEN Dupont de Nemours (Les Ulis, France).Cell Labeling and Glycoprotein AnalysisHT-29 cells were cultured as described previously(5.Ogier-Denis E. Codogno P. Chantret I. Trugnan G. J. Biol. Chem. 1988; 263: 6031-6037Abstract Full Text PDF PubMed Google Scholar, 10.Wice B.M. Trugnan G. Pinto M. Rousset M. Chevalier G. Dussaulx E. Lacroix B. Zweibaum A. J. Biol. Chem. 1985; 260: 139-146Abstract Full Text PDF PubMed Google Scholar). Cells were radiolabeled with 400 μCi/ml D-[2-3H]mannose for 10 min and then chased for the indicated times(5.Ogier-Denis E. Codogno P. Chantret I. Trugnan G. J. Biol. Chem. 1988; 263: 6031-6037Abstract Full Text PDF PubMed Google Scholar). PTX (200 ng/ml) was added 18 h before the labeling period and was present throughout the pulse-chase experiment. When used, 2 mM dMM was added 6 h before the labeling period and was present throughout the pulse-chase experiment (6.Trugnan G. Ogier-Denis E. Sapin C. Darmoul D. Bauvy C. Aubery M. Codogno P. J. Biol. Chem. 1991; 266: 20849-20855Abstract Full Text PDF PubMed Google Scholar). N-Linked glycoproteins were isolated from delipidated cell homogenates, and N-glycans were analyzed after Pronase digestion as described(5.Ogier-Denis E. Codogno P. Chantret I. Trugnan G. J. Biol. Chem. 1988; 263: 6031-6037Abstract Full Text PDF PubMed Google Scholar, 6.Trugnan G. Ogier-Denis E. Sapin C. Darmoul D. Bauvy C. Aubery M. Codogno P. J. Biol. Chem. 1991; 266: 20849-20855Abstract Full Text PDF PubMed Google Scholar). PTX does not affect either the synthesis of lipid-linked oligosaccharides or their transfer en bloc to polypeptides.Autophagic Sequestration of [3H]Raffinose[3H]Raffinose sequestration was monitored using a modification of the method of Seglen et al.(11.Seglen P.O. Gordon P.B. Tolleshaug H. H⊘yvik H. Exp. Cell Res. 1986; 162: 273-277Crossref PubMed Scopus (34) Google Scholar). Briefly, the cells were resuspended at a density of 5 × 106/500 μl with 2 μCi of [3H]raffinose, after which they were incubated for 15 min at 37°C and submitted to electroinjection by a single voltage pulse (330 V, 1000 millifarads). After electroinjection, the suspension was maintained at 4°C for 30 min and incubated at 37°C for 15 min. At the end of the incubation period, cells were washed twice with PBS and resuspended in complete medium with or without 5 mM 3-methyladenine. When used toxins were added 18 h before the electroinjection and present during the incubation period. Subsequently, at different times (see “Results”), the cells were washed twice with 10% sucrose at 4°C, resuspended in 0.5 ml of 10% sucrose and homogenized by 5 strokes in a glass/Teflon homogenizer on ice. Immediately after homogenization, 0.5 ml ice-cold phosphate buffer (100 mM potassium phosphate, 2 mM EDTA and 2 mM dithiothreitol, 100 μg/ml bovine serum albumin, 0.01% Tween 20, pH 7.5) was added, and 1 ml of cell homogenate was layered on the top of a 4 ml density cushion of buffered metrizamide/sucrose (10% sucrose, 8% metrizamide, 1 mM EDTA, 100 μg/ml bovine serum albumin, 0.01% Tween 20, pH 7.5) and centrifuged at 7000 × g for 60 min. The radioactivity associated with the pellet and total homogenate was measured by liquid scintillation counting.DNA TransfectionsRat cDNAs encoding either the αi3 or αi3 subunits were subcloned into the expression vector pBK/CMV (Stratagene). Plasmids were introduced into undifferentiated HT-29 cells by the calcium phosphate precipitation method(12.Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6475) Google Scholar). Twenty-four hours after transfection, cells were grown in selective medium containing 400 μg/ml G418 for at least 3 weeks. Resistant cells were cloned by serial dilution. Eight and 12 clones were selected and screened for their level of αi3 and αi3 expression, respectively.Immunoblotting of G-protein α SubunitsCell homogenates, crude membranes, and cytosolic fractions were prepared exactly according to Wilson et al.(13.Wilson B.S. Komuro M. Farquhar M.G. Endocrinology. 1994; 134: 233-244Crossref PubMed Scopus (76) Google Scholar). One hundred micrograms of protein were resolved by SDS-polyacrylamide gel electrophoresis (10% gel) and were transferred onto a nitrocellulose membrane. Thereafter the membrane was blocked in blotting buffer (5% nonfat dry milk in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 2 mM CaCl2, 1% Nonidet P-40), incubated with either anti-αi3 (1/500) or anti-αi3 (1/1000). After washing, bound IgG was labeled with 125I-labeled sheep anti-rabbit IgG.ADP-ribosylation of HT-29 MembranesCell-free ADP-ribosylation was conducted as described by van den Berg et al.(14.van den Berghe N. Nieuwkoop N.J. Vaandrager A.B. de Jonge H.R. Biochem. J. 1991; 278: 565-571Crossref PubMed Scopus (25) Google Scholar). Briefly, cells were incubated in either the absence or presence of 200 ng/ml PTX for 18 h and ADP-ribosylation was performed by incubating 150 μg of membrane proteins for 60 min at 32°C in the substrate mix(14.van den Berghe N. Nieuwkoop N.J. Vaandrager A.B. de Jonge H.R. Biochem. J. 1991; 278: 565-571Crossref PubMed Scopus (25) Google Scholar). Control incubations in the absence of PTX were also included. Reactions were terminated by addition of 400 μl of 20% trichloroacetic acid.Protein DegradationCells were labeled for 6 h with 0.2 μCi of [14C]leucine and chased for different times in medium containing 5 mM leucine. PTX (200 ng/ml) was added 18 h before the labeling period and was present throughout the chase period; 3-MA (5 mM), asparagine (10 mM) and NH4Cl (10 mM) were added at the beginning of the chase period. Degradation of [14C]leucine-labeled proteins was measured after trichloroacetic acid-phosphotungstic acid precipitation as described previously(7.Houri J.J. Ogier-Denis E. Trugnan G. Codogno P. Biochem. Biophys. Res. Commun. 1993; 197: 805-811Crossref PubMed Scopus (26) Google Scholar).Immunofluorescence MicroscopyCells grown on 12-mm glass coverslips were fixed at room temperature with 2% paraformaldehyde in PBS for 15 min, washed with PBS, quenched in 50 mM NH4Cl in PBS, and then blocked and permeabilized in 0.2% gelatin and 0.075% saponin in PBS for 20 min. The coverslips were then incubated with anti-αi3 (diluted 1/50) or anti-αi3 (diluted 1/50) for 45 min. Antibodies were diluted in gelatin-saponin-PBS. After washing, the coverslips were incubated for 45 min with a fluorescein isothiocyanate goat anti-rabbit antibody (diluted 1/500). The coverslips were mounted in Glycergel.RESULTS AND DISCUSSIONWhen undifferentiated HT-29 cells (hereafter referred to as HT-29 cells) are treated with PTX, which ADP-ribosylates the αi subunits of heterotrimeric G-proteins, resulting in the inhibition of GDP/GTP exchange(15.Moss J. Vaughan M. Meister A. Advances in Enzymology. 61. Interscience, New York1988: 303-379Google Scholar), a quantitative trimming of high mannose to complex-type oligosaccharides was observed (Fig. 1, a and b). This PTX-induced processing is not a consequence of either an increase in protein synthesis, as determined by [14C]leucine incorporation, or modification of Golgi complex-associated glycosyltransferase activities (evaluated by the activity of β1,4 galactosyltransferase activity: 1.58 and 1.37 nmol/h/mg protein in control and PTX-treated cells, respectively). Moreover, PTX treatment was also associated with the cessation of N-linked glycoprotein degradation. This was demonstrated after treatment of cells with dMM (Fig. 1a, inset), an inhibitor of ER α-mannosidase and Golgi complex α-mannosidase I(16.Elbein A.D. Annu. Rev. Biochem. 1987; 56: 497-534Crossref PubMed Google Scholar). Under these conditions we have shown previously that high mannose glycoproteins were unstable in HT-29 cells ((10.Wice B.M. Trugnan G. Pinto M. Rousset M. Chevalier G. Dussaulx E. Lacroix B. Zweibaum A. J. Biol. Chem. 1985; 260: 139-146Abstract Full Text PDF PubMed Google Scholar) and Fig. 1a, inset). In contrast, in the presence of PTX high mannose glycoproteins were stable during a pulse-chase experiment (Fig. 1a, inset). The above described effect of PTX suggests that the restoration of the trimming of N-glycan chains in HT-29 cells could be due to either a cessation of the autophagic sequestration that impairs the Golgi delivery of N-linked glycoproteins (6.Trugnan G. Ogier-Denis E. Sapin C. Darmoul D. Bauvy C. Aubery M. Codogno P. J. Biol. Chem. 1991; 266: 20849-20855Abstract Full Text PDF PubMed Google Scholar, 7.Houri J.J. Ogier-Denis E. Trugnan G. Codogno P. Biochem. Biophys. Res. Commun. 1993; 197: 805-811Crossref PubMed Scopus (26) Google Scholar) or to an accelerated rate of ER to Golgi transport that prevents glycoproteins from entering the autophagic pathway. Indeed the presence of PTX-sensitive heterotrimeric Gi-proteins in ER and Golgi membranes is compatible with a function in protein transport from ER to Golgi(17.Audigier Y. Nigam S.N. Blobel G. J. Biol. Chem. 1988; 263: 16352-16357Abstract Full Text PDF PubMed Google Scholar, 18.Ercolani L. Stow J.L.F.B. J. Holtzman E.J. Lin H. Grove J.R. Ausiello D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4635-4639Crossref PubMed Scopus (92) Google Scholar, 19.Hermouet S. de Mazancourt P. Spiegel A.M. Farquhar M.G. Wilson B.S. FEBS Lett. 1992; 312: 223-228Crossref PubMed Scopus (24) Google Scholar). We therefore examined the rate of maturation of the N-linked glycans of either Lamp 1, a glycoprotein associated with lysosomal membranes (20.Carlsson S.R. Roth J. Piller F. Fukuda M. J. Biol. Chem. 1988; 263: 18911-18928Abstract Full Text PDF PubMed Google Scholar) or dipeptidylpeptidase IV, a plasma membrane glycoprotein(21.Darmoul D. Lacasa M. Baricault L. Marguet D. Sapin C. Trotot P. Barbat A. Trugnan G. J. Biol. Chem. 1992; 267: 4824-4833Abstract Full Text PDF PubMed Google Scholar). In both cases the kinetics of high mannose oligosaccharide maturation to complex oligosaccharides was not changed by PTX treatment (data not shown), in agreement with other results(22.Schwaninger R. Plutner H. Bokoch G.M. Balch W.E. J. Cell Biol. 1992; 119: 1077-1096Crossref PubMed Scopus (81) Google Scholar, 23.Pimplikar S.W. Simons K. Nature. 1993; 362: 456-458Crossref PubMed Scopus (144) Google Scholar), indicating that ER to Golgi complex transport is unaffected by PTX. In contrast, PTX dramatically inhibits the autophagic sequestration of [3H]raffinose electroloaded into HT-29 cells (Fig. 2a), the level of PTX inhibition is similar to that observed in the presence of 3-MA (an inhibitor of autophagic sequestration, (24.Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1889-1892Crossref PubMed Scopus (1164) Google Scholar)). This sequestration of electroloaded [3H]raffinose was insensitive to CTX, which ADP-ribosylates the αs subunit of G-proteins(25.Stryer L. Bourne H.R. Annu. Rev. Cell Biol. 1986; 2: 391-419Crossref PubMed Scopus (624) Google Scholar), although this toxin is able to activate Gs-proteins in HT-29 cells as determined by the increase of cAMP production(26.Lencer W.I. Reinhart F.D. Neutra M. Am. J. Physiol. 1990; 258: G96-G102Crossref PubMed Google Scholar, 27.Jarry A. Merlin D. Velcich A. Hopfer U. Augenlicht L.H. Laboisse C.L. Eur. J. Pharmacol. 1994; 267: 95-103Crossref PubMed Scopus (30) Google Scholar). In addition, the absence of in vitro PTX-dependent ADP-ribosylation of αi subunits in homogenates from PTX-treated cells confirmed that PTX could modify the Gi-proteins in vivo (Fig. 2b). In order to determine the identity of the PTX-sensitive G-protein αi subunit that controls the autophagic sequestration we have transfected HT-29 cells with cDNA encoding either the αi3 or the αi3 subunits. We focused our study on these two subunits since as in normal intestinal cells(14.van den Berghe N. Nieuwkoop N.J. Vaandrager A.B. de Jonge H.R. Biochem. J. 1991; 278: 565-571Crossref PubMed Scopus (25) Google Scholar, 28.Couvineau A. Darmoul D. Blais A. Rouyer-Fessard C. Daviaud D. Voisin T. Paris H. Rouot B. Laburthe M. Am. J. Physiol. 1992; 262: C1478-C1484Crossref PubMed Google Scholar), they are the only PTX-sensitive subunits expressed in HT-29 cells (data not shown). Several stably transfected clones expressing different levels of αi subunits were selected. Whatever the αi subunit considered, Western blot analysis showed that these clones could be classified into two groups: the first one has a 1.5-fold increase in the level of the expression and the second a 3-fold increase. All the screened clones have phenotypic characteristics similar to that of parental untransfected cells. The rate of autophagy was measured in representative clones by assaying the sequestration of electroloaded [3H]raffinose (Fig. 3a) and the degradation of [14C]leucine-labeled proteins (Fig. 3b). As shown in Fig. 3, the overexpression of αi3 (3-fold) does not change autophagic sequestration/degradation in HT-29 cells. Similar results were obtained using other clones with lower (1.5-fold) overexpression of αi3 (data not shown). In contrast we observed a relationship between the overexpression of αi3 and the rate of autophagy. Clones 1 (1.5-fold increase) and 2 (3-fold increase) have 2.2- and 3.8-fold increases, respectively, in the sequestration of [3H]raffinose when compared to that observed in untransfected cells. In both clones the autophagic sequestration was inhibited by PTX and 3-MA treatment (Fig. 3a). This increase in the [3H]raffinose sequestration was correlated to an increase in protein degradation in overexpressing αi3 cells: by 2.6- and 4.0-fold in clones 1 and 2, respectively (Fig. 3b). The degradation of proteins in overexpressing αi3 cells was inhibited by PTX and drugs that impair the autophagic-lysosomal pathway at different steps (Fig. 3c), i.e. sequestration (3-MA), fusion of autophagic vacuoles with lysosomes (asparagine), lysosomal degradation (NH4Cl). These results underscore the involvement of αi3 in the control of the autophagic pathway. As shown in Fig. 4a, the overexpression of αi3 (3-fold) does not change the cellular localization of both αi3 and αi3 subunits as observed previously in other cell lines(19.Hermouet S. de Mazancourt P. Spiegel A.M. Farquhar M.G. Wilson B.S. FEBS Lett. 1992; 312: 223-228Crossref PubMed Scopus (24) Google Scholar, 29.Stow J.L. de Almeida J.B. Narula N. Holtzman E.J. Ercolani L. Ausiello D.A. J. Cell Biol. 1991; 114: 1113-1124Crossref PubMed Scopus (239) Google Scholar). In contrast to most of the cell lines studied so far(13.Wilson B.S. Komuro M. Farquhar M.G. Endocrinology. 1994; 134: 233-244Crossref PubMed Scopus (76) Google Scholar, 18.Ercolani L. Stow J.L.F.B. J. Holtzman E.J. Lin H. Grove J.R. Ausiello D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4635-4639Crossref PubMed Scopus (92) Google Scholar, 19.Hermouet S. de Mazancourt P. Spiegel A.M. Farquhar M.G. Wilson B.S. FEBS Lett. 1992; 312: 223-228Crossref PubMed Scopus (24) Google Scholar), a high amount of αi subunits was found in the cytosolic fraction, which was more evident for αi3 subunit (Fig. 4b). Whether this distribution of αi3 is related to the autophagic capacity of HT-29 cells remains to be explored. Nevertheless the absolute amount of membrane-bound αi3 was increased in overexpressing αi3 cells by 1.5-3.0-fold (Fig. 4b). This increase was correlated with changes in autophagic degradation and autophagic sequestration (see above), which is compatible with an amplified response for heterotrimeric G-protein pathways (see (29.Stow J.L. de Almeida J.B. Narula N. Holtzman E.J. Ercolani L. Ausiello D.A. J. Cell Biol. 1991; 114: 1113-1124Crossref PubMed Scopus (239) Google Scholar) and (30.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar), and references therein). The PTX-dependent inhibition of autophagic sequestration/degradation would suggest that the stabilization of membrane-bound G i3-protein in association with GDP is a key regulatory step in autophagy. As autophagic sequestration is constitutively expressed in HT-29 cells, our results suggest, by analogy with the functioning of plasma membrane-bound G-proteins(31.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar), that an endomembraneassociated effector is under a conformational state that permanently activates G i3-protein. This putative effector could be in an inactive state in differentiated HT-29 cells where autophagic sequestration is down-regulated (6.Trugnan G. Ogier-Denis E. Sapin C. Darmoul D. Bauvy C. Aubery M. Codogno P. J. Biol. Chem. 1991; 266: 20849-20855Abstract Full Text PDF PubMed Google Scholar, 7.Houri J.J. Ogier-Denis E. Trugnan G. Codogno P. Biochem. Biophys. Res. Commun. 1993; 197: 805-811Crossref PubMed Scopus (26) Google Scholar) despite the fact that the αi3 subunit is expressed at the same level as in undifferentiated HT-29 cells (data not shown). Such a change in the conformation of a membrane-bound effector would fit with the rapidity of G-protein mediated signal transduction (31.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar) and induction of autophagic sequestration in stimulated cells, e.g. hepatocytes(32.Hirsim ä ki P. Arstila A.U. Trump B.F. Marzella L. Arstila A.U. Trump B.F. Pathology of Cell Membranes. 3. Academic Press, Orlando, FL1983: 201-235Google Scholar, 33.Mortimore G.E. Pösö A.R. Lardeux B.R. Diabetes/Metabol. 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Recently, Kadowaki et al.(3.Kadowaki M. Venerando R. Miotto G. Mortimore G.E. J. Biol. Chem. 1994; 269: 3703-3710Abstract Full Text PDF PubMed Google Scholar) reported that GTP γS inhibits the stimulated autophagic sequestration in rat hepatocytes, suggesting a role for GTP-binding proteins in the regulation of autophagy. Since GTP γS acts as well on heterotrimeric and monomeric G-proteins(22.Schwaninger R. Plutner H. Bokoch G.M. Balch W.E. J. Cell Biol. 1992; 119: 1077-1096Crossref PubMed Scopus (81) Google Scholar), it cannot be concluded from this study on the identity of G-proteins involved in the autophagic sequestration. However, from the results reported in the present work and the possible localization of Rab24 (37.Olkkonen V.M. Dupree P. Killish I. Lütcke A. Zerial M. Simons K. J. Cell Sci. 1993; 106: 1249-1261Crossref PubMed Google Scholar), a monomeric GTP-binding protein, along the autophagic pathway it could be suggested that autophagy is under the control of multiple GTP-binding proteins as already observed for membrane dynamics along the exocytic and endocytic routes(26.Lencer W.I. Reinhart F.D. Neutra M. Am. J. Physiol. 1990; 258: G96-G102Crossref PubMed Google Scholar, 38.Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (591) Google Scholar, 39.Carter L.L. Redelmeier T.E. Woollenweber L.A. Schmid S.L. J. Cell Biol. 1993; 120: 37-45Crossref PubMed Scopus (139) Google Scholar, 40.Ostermann J. Orci L. Tani K. Amherdt M. Ravazzola M. Elazar Z. Rothman J.E. Cell. 1993; 75: 1015-1025Abstract Full Text PDF PubMed Scopus (232) Google Scholar).Figure 2The effect of toxins on the autophagic sequestration of electroloaded [3H]raffinose in undifferentiated HT-29 cells. a, autophagic sequestration was determined either in the absence or presence of 200 ng/ml PTX, 500 ng/ml CTX, or 5 mM 3-MA. After homogenization and centrifugation in a sucrose/metrizamide gradient, the radioactivity was measured in the sedimentable material. Values are the mean ± S.D. (n = 5). b, cell-free PTX-catalyzed ADP-ribosylation were performed with [32P]NAD on cell homogenates prepared from PTX-treated (+) or control(−) cells. Proteins were subsequently analyzed by SDS-polyacrylamide gel electrophoresis (10% gel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Overexpression of αi3 stimulates autophagic sequestration and degradation. a, autophagic sequestration of [3H]raffinose was determined as detailed in the legend to Fig. 2 in overexpressing αi3 cells (3-fold overexpression), overexpressing αi3 cells (clone 1: 1.5-fold overexpression, clone 2: 3-fold overexpression), and control cells. b, protein degradation in untransfected cells (control) and αi3 and αi3 overexpressing cells (clone 1 and clone 2). c, inhibition of degradation by drugs in overexpressing αi3 cells (clone 2). For protein degradation studies, cells were labeled for 6 h with 0.2 μCi of [14C]leucine and chased for different times in medium containing 5 mM leucine. PTX (200 ng/ml) was added 18 h before the labeling period and was present throughout the chase period; 3-MA (5 mM), asparagine (10 mM), and NH4Cl (10 mM) were added at the beginning of the chase period. Degradation of [14C]leucine-labeled proteins was measured as described previously(11.Seglen P.O. Gordon P.B. Tolleshaug H. H⊘yvik H. Exp. Cell Res. 1986; 162: 273-277Crossref PubMed Scopus (34) Google Scholar). In panels a-c, values are the mean ± S.D. (n = 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Localization and distribution of αi subunits. a, immunofluorescent localization of αi3 and αi3 in untransfected cells and in αi3 overexpressing cells (clone 2); bar represents 15 μm. b, Western blot of cytosolic (C) and membrane-bound (M) αi3 and αi3 subunits in HT-29 cells (untransfected) and overexpressing αi3 cells (clone 2). A 3.0-fold increase (measured by densitometry) in the amount of αi3 subunit was found in the membrane-bound fraction of clone 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) INTRODUCTIONAlthough recent reports indicate that the autophagic-lysosomal route could be modulated by intracellular signals including mobilization of calcium pools(1.Gordon P.B. Holen I. Fosse M. Rotnes J.S. Seglen P.O. J. Biol. Chem. 1993; 268: 26107-26112Abstract Full Text PDF PubMed Google Scholar), protein phosphorylation(2.Holen I. Gordon P.B. Seglen P.O. Eur. J. Biochem. 1993; 215: 113-122Crossref PubMed Scopus (77) Google Scholar), and GTP hydrolysis(3.Kadowaki M. Venerando R. Miotto G. Mortimore G.E. J. Biol. Chem. 1994; 269: 3703-3710Abstract Full Text PDF PubMed Google Scholar), little information on the molecular control of this process has yet to emerge. We have previously shown that the N-glycan trimming, which reflects endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumCTXcholera toxindMM1-deoxymannojirimycin3-MA3-methyladeninePTXpertussis toxinPBSphosphate-buffered saline.to Golgi complex traffic(4.Schwaninger R. Beckers J.M. Balch W.E. J. Biol. Chem. 1991; 266: 13055-13063Abstract Full Text PDF PubMed Google Scholar), is dependent on the state of enterocytic differentiation of HT-29 cells (5.Ogier-Denis E. Codogno P. Chantret I. Trugnan G. J. Biol. Chem. 1988; 263: 6031-6037Abstract Full Text PDF PubMed Google Scholar). The conversion of high mannose oligosaccharides to their complex counterparts is observed in differentiated cells, whereas a partial blockade of high mannose oligosaccharide trimming is a characteristic of undifferentiated cells. This partial impairment of N-linked glycoprotein processing is the consequence of a bypass of the Golgi complex and delivery of high mannose type glycoproteins to the lysosomal compartment (6.Trugnan G. Ogier-Denis E. Sapin C. Darmoul D. Bauvy C. Aubery M. Codogno P. J. Biol. Chem. 1991; 266: 20849-20855Abstract Full Text PDF PubMed Google Scholar) via an autophagic pathway(7.Houri J.J. Ogier-Denis E. Trugnan G. Codogno P. Biochem. Biophys. Res. Commun. 1993; 197: 805-811Crossref PubMed Scopus (26) Google Scholar). We have taken advantage of our previous data to investigate the function of heterotrimeric G-proteins, pivotal players in membrane dynamics (reviewed in (8.Bomsel M. Mostov K. Mol. Biol. Cell. 1992; 3: 1317-1328Crossref PubMed Scopus (162) Google Scholar)), in the molecular control of autophagy.

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for proteinN-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast geneALG12 that encodes the dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl α6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man7GlcNAc2 from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum. Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for proteinN-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast geneALG12 that encodes the dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl α6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man7GlcNAc2 from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum. endoplasmic reticulum congenital disorders of glycosylation lipid-linked oligosaccharide expressed sequence tag open reading frame endo-β-d-N-acetylglucosaminidase H human immunodeficiency virus, type 1 enhanced green fluorescent protein concanavalin A castanospermine Epstein-Barr virus self-inactivating human phosphoglycerate kinase promoter internal ribosome entry site Oligosaccharides N-linked to glycoproteins are initially synthesized on a lipid carrier in the endoplasmic reticulum (ER)1 membrane by the stepwise addition of monosaccharides onto dolichol pyrophosphate to form Glc3Man9GlcNAc2-PP-dolichol (1Abeijon C. Hirschberg C.B. J. Biol. Chem. 1990; 265: 14691-14695Abstract Full Text PDF PubMed Google Scholar, 2Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3752) Google Scholar). The oligosaccharide moiety is then transferred from lipid onto nascent proteins in the lumen of the ER. The resulting oligomannose-type oligosaccharides N-linked to glycoproteins are involved in the folding (3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1956) Google Scholar), degradation (5Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar), and efficient transport (7Hauri H. Appenzeller C. Kuhn F. Nufer O. FEBS Lett. 2000; 476: 32-37Crossref PubMed Scopus (131) Google Scholar) of glycoproteins in the secretory pathway. During their passage through the Golgi apparatus, the oligomannose-type sugar units attached to glycoproteins are often partially demannosylated and subsequently decorated with GlcNAc, galactose, sialic acid, and fucose residues (2Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3752) Google Scholar). The resulting glycoproteins, bearing complex-type oligosaccharide, are subsequently secreted into the extracellular space or exported to the cell surface where they are involved in diverse regulatory processes (8Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4963) Google Scholar). The importance of this widespread cotranslational modification is attested to by the identification of a rapidly growing number of diseases whose molecular origins have been linked to abnormalities in glycoprotein biosynthesis (9Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Freeze H.H. Glycobiology. 2001; 11: R129-R143Crossref PubMed Scopus (145) Google Scholar, 11Jaeken J. Carchon H. J. Inherited Metab. Dis. 1993; 16: 813-820Crossref PubMed Scopus (113) Google Scholar, 12Kornfeld S. J. Clin. Invest. 1998; 101: 1293-1295Crossref PubMed Scopus (46) Google Scholar, 13Schachter H. Cell. Mol. Life Sci. 2001; 58: 1085-1104Crossref PubMed Scopus (74) Google Scholar, 14Schachter H. J. Clin. Invest. 2001; 108: 1579-1582Crossref PubMed Scopus (27) Google Scholar).Type I congenital disorders of glycosylation (CDG I) are newly described rare metabolic diseases presenting a severe clinical picture in which multisystemic lesions including central and peripheral nervous system deficits are apparent (9Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 11Jaeken J. Carchon H. J. Inherited Metab. Dis. 1993; 16: 813-820Crossref PubMed Scopus (113) Google Scholar, 15Freeze H.H. J. Pediatr. 1998; 133: 593-600Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Although the molecular events leading to this complicated clinical picture are multifactorial and not well understood, the disease is characterized at the biochemical level by under-glycosylated serum glycoproteins of hepatic origin (16Seta N. Barnier A. Hochedez F. Besnard M.A. Durand G. Clin. Chim. Acta. 1996; 254: 131-140Crossref PubMed Scopus (54) Google Scholar). Disruption of any one of the enzymic steps involved in the biosynthesis of lipid-linked oligosaccharides (LLO) and the transfer of the mature oligosaccharide onto protein could potentially lead to the glycoprotein hypoglycosylation that is considered to be the hallmark of CDG I. Work on the underlying genetic bases of CDG I is still at an early stage because genes encoding the proteins involved in glycoprotein biosynthesis are numerous and, at least in humans, have for the most part yet to be identified (9Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Notwithstanding these difficulties, mutations in six of the genes encoding proteins of the glycosylation pathway have been shown to underlie CDG I, and these disease types have been classified as CDG I subtypes a–f as follows: (Ia, phosphomannomutase 2 (17Matthijs G. Schollen E. Pardon E. Veiga-Da-Cunha M. Jaeken J. Cassiman J.-J. van Schaftingen E. Nat. Genet. 1997; 16: 88-92Crossref PubMed Scopus (304) Google Scholar, 18Körner C. Lehle L. von Figura K. Glycobiology. 1998; 8: 165-171Crossref PubMed Scopus (40) Google Scholar); Ib, phosphomannose isomerase (19Niehues R. Hasilik M. Alton G. Körner C. Schiebe-Sukumar M. Koch H.G. Zimmer K.-P., Wu, R. Harms E. Reiter K. von Figura K. Freeze H.H. Harms H.K. Marquardt T. J. Clin. Invest. 1998; 101: 1414-1420Crossref PubMed Google Scholar, 20de Koning T.J. Nikkels P.G. Dorland L. Bekhof J., De Schrijver J.E. van Hattum J. van Diggelen O.P. Duran M. Berger R. Poll-The B.T. Virchows Arch. 2000; 437: 101-105Crossref PubMed Scopus (27) Google Scholar); Ic, dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl α3-glucosyltransferase: (21Grünewald S. Imbach T. Huijben K. Rubio-Gozalbo M.E. Verrips A. de Klerk J.B.C. Stroink H. de Rijk-van Andel J.F. Van Hove J.L.K. Wendel U. Matthijs G. Hennet T. Jaeken J. Wevers R.A. Ann. Neurol. 2000; 47: 776-781Crossref PubMed Scopus (68) Google Scholar, 22Körner C. Knauer R. Holzbach U. Hanefeld F. Lehle L. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13200-13205Crossref PubMed Scopus (103) Google Scholar, 23Imbach T. Burda P. Kuhnert P. Wevers R.A. Aebi M. Berger E.G. Hennet T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6982-6987Crossref PubMed Scopus (99) Google Scholar); Id, dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl α3-mannosyltransferase (24Körner C. Knauer R. Stephani U. Marquardt T. Lehle L. von Figura K. EMBO J. 1999; 18: 6816-6822Crossref PubMed Scopus (121) Google Scholar); Ie, dolichol-P-Man synthase I (25Kim S. Westphal V. Srikrishna G. Mehta D.P. Peterson S. Filiano J. Karnes P.S. Patterson M.C. Freeze H.H. J. Clin. Invest. 2000; 105: 191-198Crossref PubMed Scopus (148) Google Scholar,26Imbach T. Schenk B. Schollen E. Burda P. Stutz A. Grünewald S. Bailie N.M. King M.D. Jaeken J. Matthijs G. Berger E.G. Aebi M. Hennet T. J. Clin. Invest. 2000; 105: 233-239Crossref PubMed Scopus (148) Google Scholar); and If, the MPDU1 gene product of unknown function, (27Schenk B. Imbach T. Frank C.G. Grubenmann C.E. Raymond G.V. Hurvitz H. Raas-Rotschild A. Luder A.S. Jaeken J. Berger E.G. Matthijs G. Hennet T. Aebi M. J. Clin. Invest. 2001; 108: 1687-1695Crossref PubMed Scopus (117) Google Scholar, 28Kranz C. Denecke J. Lehrman M.A. Ray S. Kienz P. Kreissel G. Sagi D. Peter-Katalinic J. Freeze H.H. Schmid T. Jackowski-Dohrmann S. Harms E. Marquardt T. J. Clin. Invest. 2001; 108: 1613-1619Crossref PubMed Scopus (108) Google Scholar). CDG I cases for which the genetic origins remain unknown are defined as CDG subtype Ix. Identification of the molecular bases of type I CDG is crucial from the clinical standpoint in that CDG Ib is treatable by administering oral mannose to the patient (29Freeze H.H. Biochem. Biophys. Res. Commun. 1999; 255: 189-193Crossref PubMed Scopus (25) Google Scholar). In addition, identification of the underlying genetic defects will ultimately facilitate the design of antenatal diagnostic tests for different disease subtypes.Here we report on the genetic deficit underlying a new subtype of CDG I. We show that skin biopsy fibroblasts from a CDG I patient have a reduced capacity to add the eighth mannose residue onto the dolichol-PP-oligosaccharide precursor required for protein glycosylation. Sequencing of cDNA derived from the patient and control cells indicated that the patient was homozygous for a point (F142V) mutation in the human homolog of the yeast ALG12gene that encodes dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl α6-mannosyltransferase.DISCUSSIONHere we report on a CDG I patient with a deficiency in the ability to add the eighth mannose onto LLO. Structural analysis of the lipid-linked Man7GlcNAc2 that was found to accumulate in fibroblasts from this patient revealed the same isomeric configuration of mannose residues to that which has been shown to occur in the normal LLO intermediate generated during glycoprotein biosynthesis (2Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3752) Google Scholar, 46Romero P. Herscovics A. J. Biol. Chem. 1986; 261: 15936-15940Abstract Full Text PDF PubMed Google Scholar). This result allowed us to eliminate the hypothesis that the block was due to a rare “change of function” mutation potentially causing the seventh mannose residue to be added to an inappropriate position of the LLO acceptor. Additionally, it is noteworthy that in a yeast strain deficient in ALG11p, the enzyme that adds the fifth mannose to the growing oligosaccharide-lipid (50Cipollo J.F. Trimble R.B. Chi J.H. Yan Q. Dean N. J. Biol. Chem. 2001; 276: 21828-21840Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), an abnormal Man7GlcNAc2 structure linked to dolichol has been observed (50Cipollo J.F. Trimble R.B. Chi J.H. Yan Q. Dean N. J. Biol. Chem. 2001; 276: 21828-21840Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) further emphasizing the necessity to carry out detailed structural analyses on the oligosaccharide species that occur in these unusual circumstances. The most likely hypothesis to explain the block in LLO biosynthesis was a deficiency in dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl α6-mannosyltransferase. In yeast, this enzyme is thought to be encoded by the ALG12 gene (48Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (300) Google Scholar, 51Burda P. Aebi M. Biochim. Biophys. Acta. 1999; 1426: 239-257Crossref PubMed Scopus (523) Google Scholar). The proteins encoded by the ALG12 genes of different species are all hydrophobic, and like some membrane transporters may possess up to 10–12 transmembrane domains. Nevertheless, the peptide sequences of theALG12 gene products have features in common to those of the ALG3p which has been shown to have dolichyl-P-mannose:Man5GlcNAc2-PP-dolichyl mannosyltransferase activity (52Sharma C.B. Knauer R. Lehle L. Biol. Chem. Hoppe-Seyler. 2001; 382: 321-328Crossref PubMed Scopus (41) Google Scholar), indicating that ALG12p is also likely to have mannosyltransferase activity. In the work presented here we characterized the human homolog of this gene, and the accuracy of the sequence we deposited in the data banks was attested to by the subsequent appearance of an identical “anonymous” cDNA sequence (GenBankTM accession number BC001729). Interestingly, we noted the presence of a consensus glycosylation site (N(K/R)S, see Fig.3) which was conserved in all species except yeast. Theoretical predictions of hALG12p transmembrane regions indicate that this glycosylation site is situated in an extramembrane loop sufficiently large to support glycosylation. The question of whether or not hALG12p is glycosylated will have to await experimental evidence.When the human wild type ALG12 gene was introduced into the fibroblasts of the patient, we noted a remarkable normalization of LLO biosynthesis (Fig. 5 B), and a concomitant increase in the fraction of fully mannosylated oligosaccharides was transferred from dolichol onto polypeptide (results not shown). These observations strongly suggest that the ALG12 gene is defective in the patient described here.We found the hALG12 cDNA of the patient to be homozygous for a single point mutation that leads to replacement of a Phe residue by Val in the encoded protein. Several lines of argument suggest that this mutation is the underlying cause of the Man7GlcNAc2-PP-dolichyl accumulation observed in fibroblasts obtained from patient ME. First, examination of the size of the hALG12 mRNA transcript from the fibroblasts of the patient revealed it to be identical to that observed for the transcript present in other cell lines, indicating normal processing and the absence of an important deletion. Second, this mRNA transcript appeared to be as abundant as that observed in control fibroblasts, signifying on the one hand that both alleles are expressed and on the other hand an absence of mRNA instability. Third, analysis of the human EST banks allowed us to identify 28 separate human ESTs containing the region of the mutation, but we were unable to detect the T571G base change in any of these sequences, suggesting that the mutation is not simply a common polymorphism. Fourth, the F142V replacement occurs in one of the small highly conserved patches toward the NH2 terminus of ALG12p. In fact, in all species for which sequence data are available, we noted that the position of the replaced Phe residue in the ALG12p is invariably occupied by the aromatic amino acids Phe or Tyr (Schizosaccharomyces pombe) and that this position is next to a completely conserved Tyr residue. At present the role of this pair of highly conserved aromatic amino acids in ALG12p function is not understood, but our observations suggest that they are situated in, or near, a transmembrane domain. Indeed, several point mutations in glycosyltransferases that lead to CDG type I have been noted to occur in the transmembrane regions of these proteins.2 To conclude, the above arguments strongly favor our assertion that the point mutation identified in thehALG12 cDNA of patient ME is responsible for the accumulation of Man7GlcNAc2-PP-dolichyl observed in skin biopsy fibroblasts obtained from this subject.As observed for other types of CDG I, the observed mutation is leaky (18Körner C. Lehle L. von Figura K. Glycobiology. 1998; 8: 165-171Crossref PubMed Scopus (40) Google Scholar, 22Körner C. Knauer R. Holzbach U. Hanefeld F. Lehle L. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13200-13205Crossref PubMed Scopus (103) Google Scholar, 24Körner C. Knauer R. Stephani U. Marquardt T. Lehle L. von Figura K. EMBO J. 1999; 18: 6816-6822Crossref PubMed Scopus (121) Google Scholar) because small amounts of fully mannosylated LLO can be detected in cells from the patient. This leakiness allows a substantial transfer of Glc3Man9GlcNAc2 from dolichol onto nascent polypeptides in the fibroblasts of the patient. We also observed that N-linked Glc3Man7GlcNAc2 and Man7GlcNAc2 account for the remaining two-thirds of the total oligosaccharide transferred onto protein from oligosaccharide lipid. The relative abundance of these latter two species linked on the one hand to lipid and on the other handN-linked to polypeptide suggests the following: first, the lipid-linked Man7GlcNAc2 is poorly glucosylated; second, the small amount of lipid-linked Glc3Man7GlcNAc2 that is formed can be more efficiently transferred to protein than its non-glucosylated counterpart. The observation that substantial amounts of non-glucosylated Man7GlcNAc2 can be transferred from lipid onto protein was surprising considering that human skin biopsy fibroblasts deficient in dolichol-P-Glc:Man9GlcNAc2-PP-dolichol transferase do not appear to be able to transfer Man9GlcNAc2 directly onto protein (22Körner C. Knauer R. Holzbach U. Hanefeld F. Lehle L. von Figura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13200-13205Crossref PubMed Scopus (103) Google Scholar). However, this phenomenon has been observed in mouse F9 teratocarcinoma cells (46Romero P. Herscovics A. J. Biol. Chem. 1986; 261: 15936-15940Abstract Full Text PDF PubMed Google Scholar). It would be interesting to look for mutations in the mouseALG12 gene in the F9 cell line. To summarize, it appears that in addition to leading to only a partial block in hALG12p function, the truncated Man7GlcNAc2intermediate that accumulates can be transferred directly onto protein. Despite these relieving factors, it is apparent the clinical picture for patient ME is severe, comprising both central nervous system and peripheral deficits. At present it is unclear whether or not a deficiency in hALG12p will invariably lead to a severe type of CDG I; only characterization of other patients with the same enzymic deficit will answer this question. However, one compounding factor may contribute to the severity of the symptoms observed in this case. In fact, observations made with a yeast strain deficient inALG12 led investigators to suspect that misfolded glycoproteins bearing N-linked Man7GlcNAc2 are poor substrates for ER-associated glycoprotein degradation (48Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (300) Google Scholar). Although these observations have yet to be extended to mammalian cells, it is possible that in fibroblasts from ME such a phenomenon could exacerbate the ER accumulation of glycoproteins that may result from the misfolding of hypoglycosylated polypeptides (53Marquardt T. Ullrich K. Zimmer K.-P. Hasilik A. Deufel T. Harms E. Eur. J. Cell Biol. 1995; 66: 268-273PubMed Google Scholar). Under these conditions, the resulting stress generated in the ER could have profound effects on the cellular homeostasis of such cells. Furthermore, it has been shownin vitro that the “processing” isomers of Man7GlcNAc (generated by ER mannosidases) that are substrates for UDP-glucose:glycoprotein glucosyltransferase have only 15% of the acceptor activity of Man9GlcNAc (54Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (267) Google Scholar). However, we show here that the “biosynthetic” Man7GlcNAc structure that is transferred directly from dolichol onto protein in cells from patient ME can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase, presumably as part of the glycoprotein “quality control” system.To conclude, our results indicate that a F142V replacement in hALG12p is the cause of inefficient addition of the eighth mannose residue onto Man7GlcNAc2-PP-dolichol during glycoprotein biosynthesis in a patient with type I CDG. The insufficiency in this step of the pathway for lipid-linked oligosaccharide biosynthesis defines a new subtype of the disease that we suggest should be called type Ig.Note Added in ProofThe GenBank™ AL671710 genomic sequence located in chromosome 22q13.3 contains the ALG12 gene. Oligosaccharides N-linked to glycoproteins are initially synthesized on a lipid carrier in the endoplasmic reticulum (ER)1 membrane by the stepwise addition of monosaccharides onto dolichol pyrophosphate to form Glc3Man9GlcNAc2-PP-dolichol (1Abeijon C. Hirschberg C.B. J. Biol. Chem. 1990; 265: 14691-14695Abstract Full Text PDF PubMed Google Scholar, 2Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3752) Google Scholar). The oligosaccharide moiety is then transferred from lipid onto nascent proteins in the lumen of the ER. The resulting oligomannose-type oligosaccharides N-linked to glycoproteins are involved in the folding (3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1956) Google Scholar), degradation (5Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar), and efficient transport (7Hauri H. Appenzeller C. Kuhn F. Nufer O. FEBS Lett. 2000; 476: 32-37Crossref PubMed Scopus (131) Google Scholar) of glycoproteins in the secretory pathway. During their passage through the Golgi apparatus, the oligomannose-type sugar units attached to glycoproteins are often partially demannosylated and subsequently decorated with GlcNAc, galactose, sialic acid, and fucose residues (2Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3752) Google Scholar). The resulting glycoproteins, bearing complex-type oligosaccharide, are subsequently secreted into the extracellular space or exported to the cell surface where they are involved in diverse regulatory processes (8Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4963) Google Scholar). The importance of this widespread cotranslational modification is attested to by the identification of a rapidly growing number of diseases whose molecular origins have been linked to abnormalities in glycoprotein biosynthesis (9Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Freeze H.H. Glycobiology. 2001; 11: R129-R143Crossref PubMed Scopus (145) Google Scholar, 11Jaeken J. Carchon H. J. Inherited Metab. Dis. 1993; 16: 813-820Crossref PubMed Scopus (113) Google Scholar, 12Kornfeld S. J. Clin. 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Although the molecular events leading to this complicated clinical picture are multifactorial and not well understood, the disease is characterized at the biochemical level by under-glycosylated serum glycoproteins of hepatic origin (16Seta N. Barnier A. Hochedez F. Besnard M.A. Durand G. Clin. Chim. Acta. 1996; 254: 131-140Crossref PubMed Scopus (54) Google Scholar). Disruption of any one of the enzymic steps involved in the biosynthesis of lipid-linked oligosaccharides (LLO) and the transfer of the mature oligosaccharide onto protein could potentially lead to the glycoprotein hypoglycosylation that is considered to be the hallmark of CDG I. Work on the underlying genetic bases of CDG I is still at an early stage because genes encoding the proteins involved in glycoprotein biosynthesis are numerous and, at least in humans, have for the most part yet to be identified (9Aebi M. Hennet T. Trends Cell Biol. 2001; 11: 136-141Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Notwithstanding these difficulties, mutations in six of the genes encoding proteins of the glycosylation pathway have been shown to underlie CDG I, and these disease types have been classified as CDG I subtypes a–f as follows: (Ia, phosphomannomutase 2 (17Matthijs G. Schollen E. Pardon E. Veiga-Da-Cunha M. Jaeken J. Cassiman J.-J. van Schaftingen E. Nat. Genet. 1997; 16: 88-92Crossref PubMed Scopus (304) Google Scholar, 18Körner C. Lehle L. von Figura K. Glycobiology. 1998; 8: 165-171Crossref PubMed Scopus (40) Google Scholar); Ib, phosphomannose isomerase (19Niehues R. Hasilik M. Alton G. Körner C. Schiebe-Sukumar M. Koch H.G. Zimmer K.-P., Wu, R. Harms E. Reiter K. von Figura K. Freeze H.H. Harms H.K. Marquardt T. J. Clin. Invest. 1998; 101: 1414-1420Crossref PubMed Google Scholar, 20de Koning T.J. Nikkels P.G. Dorland L. 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The NF-κappaB transcription factor is an important anti-apoptotic factor, which is frequently deregulated in cancer cells. We have recently demonstrated that NF-kappaB activation mediates the repression of autophagy in response to TNFα in three models of cancer cell lines. In contrast, in the absence of NF-kappaB activation, TNFα induces autophagy, which requires reactive oxygen species (ROS) production and participates in the TNFα-induced apoptotic signaling pathway. Autophagy-dependent apoptosis was also observed following direct addition of ROS to cells. Moreover, addition of rapamycin to TNFα renders these cells susceptible to the cytotoxic effect of this cytokine. These findings highlight the regulation of autophagy by oxidative stress and support the idea that repression of autophagy by NF-kappaB may constitute a novel anti-apoptotic function of this transcription factor. We also bring evidence that direct stimulation of autophagy may represent a new therapeutic strategy for overcoming the NF-κappaB-dependent chemoresistance of cancer cells.Addendum to:NF-κappaB Activation Represses TNF-alpha-Induced AutophagyM. Djavaheri-Mergny, M. Amelotti, J. Mathieu, F. Besançon, C. Bauvy, S. Souquère, G. Pierron and P. CodognoJ Biol Chem 2006; 281:30373-82