Alfredo Criollo

Administration of spermidine, a polyamine whose concentration declines during ageing, extends lifespan in yeast, flies, worms and in human immune cells. Spermidine prevents early oxidative stress and necrotic cell death and increases the expression of autophagy genes by inhibiting histone acetyltransferases action on histone H3. Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

Article19 April 2007free access Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1 M Chiara Maiuri M Chiara Maiuri INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Università degli studi di Napoli ‘Federico II’, Facoltà di Scienze Biotecnologiche, Napoli, Italy Search for more papers by this author Gaëtane Le Toumelin Gaëtane Le Toumelin Institut de Recherche Servier, Croissy sur Seine, France Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Search for more papers by this author Jean-Christophe Rain Jean-Christophe Rain Hybrigenics, Paris, France Search for more papers by this author Fabien Gautier Fabien Gautier INSERM, U601-Equipe 4, University of Nantes, Faculty of MedicineM, Nantes, France Search for more papers by this author Philippe Juin Philippe Juin INSERM, U601-Equipe 4, University of Nantes, Faculty of MedicineM, Nantes, France Search for more papers by this author Ezgi Tasdemir Ezgi Tasdemir INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron CNRS, FRE 2937, Institut André Lwoff, Villejuif, France Search for more papers by this author Kostoula Troulinaki Kostoula Troulinaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Crete, Greece Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Crete, Greece Search for more papers by this author John A Hickman John A Hickman Institut de Recherche Servier, Croissy sur Seine, France Search for more papers by this author Olivier Geneste Corresponding Author Olivier Geneste Institut de Recherche Servier, Croissy sur Seine, France Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Search for more papers by this author M Chiara Maiuri M Chiara Maiuri INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Università degli studi di Napoli ‘Federico II’, Facoltà di Scienze Biotecnologiche, Napoli, Italy Search for more papers by this author Gaëtane Le Toumelin Gaëtane Le Toumelin Institut de Recherche Servier, Croissy sur Seine, France Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Search for more papers by this author Jean-Christophe Rain Jean-Christophe Rain Hybrigenics, Paris, France Search for more papers by this author Fabien Gautier Fabien Gautier INSERM, U601-Equipe 4, University of Nantes, Faculty of MedicineM, Nantes, France Search for more papers by this author Philippe Juin Philippe Juin INSERM, U601-Equipe 4, University of Nantes, Faculty of MedicineM, Nantes, France Search for more papers by this author Ezgi Tasdemir Ezgi Tasdemir INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron CNRS, FRE 2937, Institut André Lwoff, Villejuif, France Search for more papers by this author Kostoula Troulinaki Kostoula Troulinaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Crete, Greece Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Crete, Greece Search for more papers by this author John A Hickman John A Hickman Institut de Recherche Servier, Croissy sur Seine, France Search for more papers by this author Olivier Geneste Corresponding Author Olivier Geneste Institut de Recherche Servier, Croissy sur Seine, France Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud—Paris 11, Villejuif, France Search for more papers by this author Author Information M Chiara Maiuri1,2,3,4, Gaëtane Le Toumelin5, Alfredo Criollo1,2,3, Jean-Christophe Rain6, Fabien Gautier7, Philippe Juin7, Ezgi Tasdemir1,2,3, Gérard Pierron8, Kostoula Troulinaki9, Nektarios Tavernarakis9, John A Hickman5, Olivier Geneste 5 and Guido Kroemer 1,2,3 1INSERM U848, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Université Paris Sud—Paris 11, Villejuif, France 4Università degli studi di Napoli ‘Federico II’, Facoltà di Scienze Biotecnologiche, Napoli, Italy 5Institut de Recherche Servier, Croissy sur Seine, France 6Hybrigenics, Paris, France 7INSERM, U601-Equipe 4, University of Nantes, Faculty of MedicineM, Nantes, France 8CNRS, FRE 2937, Institut André Lwoff, Villejuif, France 9Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology—Hellas, Crete, Greece ‡These authors contributed equally to this work *Corresponding authors: INSERM U848, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, Villejuif 94805, France. Tel.: +33 1 42 11 60 46; Fax: +33 1 42 11 60 47; E-mail: [email protected] Institut de Recherche Servier, 125 chemin de ronde, Croissy sur Seine 78290, France. Tel.: +33 1 55 72 21 68; Fax: +33 1 55 72 21 80; E-mail: [email protected] The EMBO Journal (2007)26:2527-2539https://doi.org/10.1038/sj.emboj.7601689 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The anti-apoptotic proteins Bcl-2 and Bcl-XL bind and inhibit Beclin-1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin-1 (residues 114–123). The physical interaction between Beclin-1 and Bcl-XL is lost when the BH3 domain of Beclin-1 or the BH3 receptor domain of Bcl-XL is mutated. Mutation of the BH3 domain of Beclin-1 or of the BH3 receptor domain of Bcl-XL abolishes the Bcl-XL-mediated inhibition of autophagy triggered by Beclin-1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin-1 and Bcl-2/Bcl-XL, antagonizes autophagy inhibition by Bcl-2/Bcl-XL and hence stimulates autophagy. Knockout or knockdown of the BH3-only protein Bad reduces starvation-induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain-of-function mutation of the sole BH3-only protein from Caenorhabditis elegans, EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy. These results reveal a novel autophagy-stimulatory function of BH3-only proteins beyond their established role as apoptosis inducers. BH3-only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin-1 and Bcl-2 or Bcl-XL. Introduction Two self-destructive processes, apoptosis (self-killing) and autophagy (self-eating), have captured the attention of cell biologists over the last decades. While apoptosis involves the activation of catabolic enzymes leading to the demolition of cellular structures and organelles, autophagy is a slow, localized phenomenon in which parts of the cytoplasm are sequestered within double-membraned autophagic vacuoles and finally digested by lysosomal hydrolases (Gozuacik and Kimchi, 2004; Kroemer and Jaattela, 2005). The relationship between apoptosis and autophagy is complex and autophagy may either contribute to cell death (Shimizu et al, 2004; Yu et al, 2004) or constitute a cellular defense against acute stress, in particular stress induced by starvation from nutrients or obligate growth factors (Boya et al, 2005; Lum et al, 2005). The crosstalk between autophagy and apoptosis is mediated at least in part by the functional and physical interaction between Beclin-1, an essential autophagy gene, and Bcl-2, one of the paradigmatic apoptosis-inhibitory proteins (Liang et al, 1999; Pattingre et al, 2005; Takacs-Vellai et al, 2005). Bcl-2 is the prototype of a family of proteins containing at least one Bcl-2 homology (BH) region. The family is split into anti-apoptotic multidomain proteins (such as Bcl-2 and Bcl-XL), which contain four BH domains (BH1234), pro-apoptotic multidomain proteins (prototypes Bax and Bak), which contain three BH domains (BH123), and the pro-apoptotic BH3-only protein family (Letai et al, 2002). As a rule, BH1234 proteins (Bcl-2, Bcl-XL, Mcl-1) mainly reside in mitochondria, protecting these organelles against mitochondrial outer membrane permeabilization (MOMP), one of the rate-limiting events of apoptosis induction. Either of the two BH123 proteins (Bax and Bak) are required for MOMP, in a series of different models of apoptosis induction (Wei et al, 2001). BH3-only proteins are suggested to kill cells by interacting with the BH3 receptor, which is a hydrophobic groove formed by apposition of the BH1, BH2 and BH3 domains, hence activating BH123 proteins and/or by neutralizing BH1234 proteins. The so-called ‘BH3 mimetics’, pharmacological compounds that bind to BH3 receptors, can induce apoptosis or facilitate apoptosis induction in cancer cells (Letai et al, 2002; Oltersdorf et al, 2005). Beclin-1 (also called ATG6) is a phylogenetically conserved protein that is essential for the initiation of autophagy, perhaps via its interaction with the class III phosphatidylinositol-3-kinase Vps34 (Zeng et al, 2006). Originally, human Beclin-1 has been identified as an interactor of Bcl-2 (Liang et al, 1999). Caenorhabditis elegans Beclin-1 (BEC-1) interacts with the Bcl-2 homolog CED-9, and inactivation of the C. elegans bec-1 gene causes apoptosis (Takacs-Vellai et al, 2005). In mammalian cells, knockdown of beclin-1 sensitizes to apoptosis induction by starvation (Boya et al, 2005; Lum et al, 2005). However, Beclin-1 downregulation can also inhibit cell death induction by conditions in which essential pro-apoptotic MOMP or caspase activation are blocked (Shimizu et al, 2004; Yu et al, 2004), and restoration of normal Beclin-1 levels in Beclin-1 deficient tumor cells can facilitate the induction of cell death by vitamin D analogues (Hoyer-Hansen et al, 2005). Importantly, beclin-1 is a haploinsufficient tumor suppressor gene (Qu et al, 2003; Yue et al, 2003). Transfection-enforced overexpression of Beclin-1 stimulates autophagy, and this autophagy-stimulatory effect is enhanced by depletion of Bcl-2 and reduced by Bcl-2 overexpression (Pattingre et al, 2005). Based on these premises and incognita, we explored the fine mechanisms which govern the interaction between Beclin-1 and Bcl-2/Bcl-XL. As shown here, Beclin-1 possesses a BH3-like domain, thus elucidating the structural basis for the functional Beclin-1-Bcl-2/Bcl-XL interaction. Disrupting this interaction by BH3-only proteins or BH3 mimetics increases the autophagic activity of Beclin-1, thus revealing a novel physiological role for BH3 domains. Results Identification of a BH3-like domain in Beclin-1 In multiple yeast two-hybrid screens, using complex human DNA libraries, a number of Beclin-1 fragments interacted with Bcl-2 as well as with Bcl-XL, allowing us to narrow down the precise interaction domain of Beclin-1 to amino acids (aa) 112 to 159 (Figure 1A). A stretch of 10 amino acids (aa 114–123) within this domain showed significant similarity with BH3 domains from the Bcl-2 family (Figure 1B). An eicosahexapeptide (aa 107–132) comprising this BH3-like domain in an α-helical conformation displaced a Bak-derived BH3-containing peptide binding to the hydrophobic groove of purified recombinant Bcl-XL measured in a fluorescence anisotropy assay. Mutation of the most conserved residue in the BH3-like domain of Beclin-1 (L116A) as well as that of another amino acid (F123A) (Pattingre et al, 2005) abolished the competition with Bak-BH3 for Bcl-XL binding (Figure 1C). The affinity of the wild-type BH3 peptide from Beclin-1 for Bcl-XL was high (Kd 203±6 nM, n=3) (Figure 1D) and of an order of magnitude similar to Bax-BH3 (145 nM) or Bak-BH3 (40 nM), although lower than Bad-BH3 (10 nM) (data not shown). In contrast Bcl-XL carrying a single mutation (G138A) in the BH3 binding groove (Ottilie et al, 1997) was unable to interact with the Beclin-derived BH3-like peptide. Figure 1.Physical interaction between Bcl-XL/Bcl-2 and the BH3-like domain of Beclin-1. (A) Selection of Beclin-1 fragments that interact with Bcl-XL/Bcl-2 in the yeast-two-hybrid system. Black lines indicate the fragments of Beclin-1 (grey) that interact with Bcl-XL (upper part of the graph) or with Bcl-2 (lower part). ▪, CEMC7 library; •, human thymocyte library; ▴, placenta library and ★, human brain library. The minimal interacting domain required for interaction with Bcl-XL/Bcl-2 is situated between aa 112 and 159. (B) Alignment of the BH3-like domain of Beclin-1 with established BH3 domains. L116A Beclin-1 and F123A Beclin-1 describe two Beclin-1 mutant peptides. (C) Fluorescence polarization assays demonstrating that a peptide containing the BH3-like domain of Beclin-1 interacts with Bcl-XL. Recombinant Bcl-XL ΔTM was incubated with carboxyfluorescein-labeled Bak BH3 peptide, in the absence or presence of wild-type (WT) Beclin-1 BH3 or either of the two mutant peptides described in B. (D) Affinity determination of the interaction between recombinant Bcl-XL ΔTM and fluorescent Beclin-1 BH3, by fluorescence polarization. A comparison between WT Bcl-XL(squares) and G138A Bcl-XL (triangles) is shown (means±s.d., n=3 separate experiments). (E) Co-immunoprecipitation of Bcl-XL and WT or mutant Beclin-1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag-tagged Bcl-XL and immunochemical detection of Beclin-1. (F) Co-immunoprecipitation of Beclin-1 with-WT and mutant (G138A) Bcl-XL. Results are representative of at least three experiments yielding similar results. Download figure Download PowerPoint To corroborate the role of the BH3-like domain for the Beclin-1 interaction with Bcl-XL, cells were transfected with wild-type or mutant Beclin-1 and/or with Bcl-XL, followed by co-immunoprecipitation assays. The BH3-disrupting mutation L116A almost abolished the interaction of Beclin-1 with Bcl-XL (Figure 1E), as well as that of Beclin-1 with Bcl-2 and Mcl-1 (not shown). Moreover, F123A mutation reduced the Beclin-1-Bcl-XL interaction (Figure 1E). The G138A mutation within the BH3-binding cleft of Bcl-XL abrogated the binding of Beclin-1 in the cellular context (Figure 1F). These results suggest that a novel BH3-like domain in Beclin-1 is critical for the interaction of Beclin-1 with anti-apoptotic members of the Bcl-2 family. The BH3-like domain of Beclin-1 is phylogenetically conserved, because the Beclin-1 orthologs from fugu (Takifugu rubripes), latipes (Oryzia latipes) and zebrafish (Danio rerio) exhibit sequence homology within their BH3-like domain with human Beclin-1 (Supplementary Figure 1A). Peptides corresponding to these BH3-like domains induced apoptosis in a Bax-dependent manner when they were microinjected into human HCT116 cells (Supplementary Figure 1B and C), and the peptides from fugu and latipes (but not the ones from zebrafish) are able to displace the Bak BH3 peptide from recombinant Bcl-XL or Bcl-2 protein in vitro (Supplementary Figure 1D and E). The fact that the zebrafish-derived Beclin-1 peptide did not bind to human Bcl-XL yet induced Bax-dependent apoptosis, suggests that this particular peptide induces cell death by acting on a multidomain Bcl-2 family protein other than Bcl-XL. Inhibition of the Beclin-1-Bcl-XL/Bcl-2 interaction by a BH3 mimetic The BH3-mimetic compound ABT737 (Oltersdorf et al, 2005), inhibits the binding of Beclin-1 BH3 peptide to Bcl-XL in a competitive manner, with an IC50 in the micromolar range, as determined by fluorescence polarization of synthetic peptide binding to purified recombinant Bcl-XL in vitro (Figure 2A). Pretreatment of cells with ABT737 inhibited the co-immunoprecipitation of Flag-tagged Bcl-XL and His-tagged (Figure 2B) or endogenous Beclin-1 (Figure 2C). ABT737 also reduced the interaction between Bcl-2 and Beclin-1 (Figure 2D), yet had no effect on the interaction between Mcl-1 and Beclin-1 (Figure 2E). This can be explained by the selectivity of ABT737, which binds to Bcl-2 and Bcl-XL but not to Mcl-1, and hence has a Bad-like profile (Oltersdorf et al, 2005, Van Delft et al, 2006). It is important to note that ABT737 also abolished the interaction between endogenous Bcl-2 and Beclin-1 (see below), meaning that these effects cannot be attributed to overexpression-associated artifacts. Altogether, these data indicate that the BH3-like domain of Beclin-1 binds to the BH3 receptor region of Bcl-XL/Bcl-2 and that ABT737 competitively disrupts this interaction. Figure 2.Inhibition of the interaction between Beclin-1 and anti-apoptotic Bcl-2 proteins by ABT737. (A) Competition between Beclin-1 BH3 and ABT737 for Bcl-XL binding. A fluorescent 25-mer peptide containing the BH3-like domain of Beclin-1 (Figure 1D) was docked to recombinant Bcl-XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl-XL ΔTM, was 1.7 μM. (B, C) Abolition of the interaction between Bcl-XL and Beclin-1 by ABT737 in intact cells. Co-immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co-transfected Bcl-XL and Beclin-1 (B) and for endogenous Beclin-1 interacting with Flag-tagged Bcl-XL (C). (D, E) Differential effect of ABT737 on the interaction between Bcl-2 (D) or Mcl-1 (E) and Beclin-1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results. Download figure Download PowerPoint The BH3 mimetic ABT737 stimulates Beclin-1-dependent autophagy If the physiological function of the physical Beclin-1-Bcl-XL/Bcl-2 interaction were to control Beclin-1-initiated autophagy (Pattingre et al, 2005), then inhibition of this interaction would be expected to stimulate autophagy. Accordingly, ABT737 increased the frequency of cells that manifested cytoplasmic (non-nuclear) aggregation of the marker of autophagic vacuoles, LC3-GFP. This aggregation of LC3-GFP (which, in non-autophagic cells, is diffuse in the cytosol as well as in the nucleus) is an established sign of autophagy (Mizushima et al, 2004), and was induced both in complete medium as well as in conditions of nutrient depletion (Figure 3A). Knockdown of Beclin-1 by two distinct small interfering RNA (siRNA) heteroduplexes (insert in Figure 3B) inhibited the ABT737-stimulated LC3-GFP aggregation (Figure 3A and B). Similarly, knockdown of other essential ATG proteins (ATG5, 10, 12) reduced ABT737-induced LC3-GFP aggregation, confirming that ABT737 engages the classical autophagic pathway (Supplementary Figure 2S). Since LC3-GFP might interfere with the normal regulation of autophagy, cytoplasmic vacuoles (which are bona fide autophagic vacuoles) were detected by staining with CMFDA, without prior transfection with LC3-GFP. Again, ABT737 induced signs of autophagy that could be completely suppressed by the knockdown of Beclin-1 (Figure 3C and D). These results could be confirmed by transmission electron microscopy showing double-membraned autophagic vacuoles that were elicited by ABT737 and suppressed by Beclin-1-specific siRNAs (Figures 3E and 4). The ABT737-stimulated autophagy was organelle-specific in the sense that LC3-GFP colocalized with the mitochondrial marker HSP60 (Supplementary Figure 3A and C) but not with the endoplasmic reticulum (ER) marker calreticulin (Supplementary Figure 3B and C). It is noteworthy that these results were obtained by observing viable, adherent cells (Figure 3A–E) and that the cytotoxic pro-apoptotic effects of ABT737 were minor. Thus, ABT737 failed to induce a major loss of the mitochondrial transmembrane potential (which may be associated with apoptosis or necrosis) (Figure 3F) or phosphatidylserine exposure (which is associated with apoptosis) (Supplementary Figure 4), unless Beclin-1 was depleted simultaneously. This is in accord with the notion that ABT737 as a single agent only kills a limited set of transformed cell lines (Oltersdorf et al, 2005). ABT737 can stimulate autophagy without inducing cell death. Figure 3.Beclin-1-dependent autophagy stimulated by ABT737. (A, B) Detection of autophagic vacuoles by LC3-GFP and their modulation by ABT737 and by Beclin-1-specific siRNAs. HeLa cells were transfected with control or Beclin-1-specific siRNAs, 24 h later re-transfected with LC3-GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient-free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3-GFP-transfected cells bearing LC3-GFP aggregates in the cytoplasm (LC3-GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin-1-specific siRNAs, as quantified by immunoblot. (C, D) Detection of cytoplasmic vacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin-1-specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmic vacuole (arrow head) (means±s.d., n=3 separate experiments). Download figure Download PowerPoint Figure 4.(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown. (F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm-sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3)low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low PI− (dying) population. Results are means±s.d. of three independent experiments. Download figure Download PowerPoint Figure 5.Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin-1 at 0 h) were re-transfected with LC3-GFP finally cultured in nutrient-free (NF) conditions (60–72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopic detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A). The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B). Download figure Download PowerPoint BH3 dependency of the functional interaction between Beclin-1 and Bcl-XL/Bcl-2 Overexpression of Beclin-1 stimulates autophagy (Liang et al, 1999; Hoyer-Hansen et al, 2005; Pattingre et al, 2005), and this effect was increased by ABT737 (Figure 5A and B). The Beclin-1 mutants L116A and F123A were more efficient than wild-type Beclin-1 in stimulating autophagy, in line with the fact that these mutations disrupt the interaction with the Beclin-1 inhibitors Bcl-2/Bcl-XL. This result was obtained when using two different read-outs, namely LC3-GFP aggregation (Figure 5A and B) and CMFDA-quantifiable vacuolization (Figure 5C and D). In the presence of ABT737, the differential capacity of wild-type Beclin-1 and of its mutants L116A and F123A to induce autophagy was matched, suggesting that it was indeed the interaction between the BH3-like domain of Beclin-1 and a BH3 receptor (inhibitable by ABT737) that regulated the pro-autophagy activity of Beclin-1. These data also suggests that endogenous Mcl-1 (which is not inhibitable by ABT737) (van Delft et al, 2006) does not play a crucial role in controlling autophagy induced by overexpressed Beclin-1. Moreover, Bcl-XL (but not Bcl-XL G138A), Bcl-2 and Mcl-1 all inhibited the induction of autophagy by Beclin-1 (Figure 5E). The autophagy-inhibitory effect of Bcl-XL and Bcl-2 was abrogated by ABT737. However, ABT737 did not affect the suppression of autophagy by Mcl-1 (Figure 5E), in accord with its incapacity to block the Beclin-1–Mcl-1 interaction (Figure 2E). Altogether, these data suggest that ABT737 stimulates (or de-inhibits) autophagy by competitively inhibiting the interaction between the BH3 domain of Beclin-1 and the BH3 receptor regions of Bcl-2/Bcl-XL. Figure 6.Impact of the BH3-like domain on Beclin-1-induced autophagy. (A–D) Modulation of Beclin-1-induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP-LC3 together with an empty control vector or Beclin-1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3-GFP-positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3-GFP method. (E) BH3-dependent modulation of Beclin-1-induced autophagy by anti-apoptotic Bcl-2 proteins. Cells were transfected simultaneously with Beclin-1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl-XL, Bcl-XL G138A, Bcl-2 or Mcl-1 (or control vector), as well as GFP-LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h-long experiment. The percentage of cells exhibiting the accumulation of LC3-GFP in vacuoles (LC3-GFPvac) is quantified as means±s.d. (n=3 separate experiments). Download figure Download PowerPoint Spatially restricted, regulated interactions between Bcl-2 and Beclin-1 are inhibited by ABT737 ABT737 as well as nutrient depletion reduced the amount of endogenous Beclin-1 that immunoprecipitated with endogenous Bcl-2, both in HeLa and in MV4.11 cells (Figure 6A), confirming that physiological levels of these proteins can interact in a fashion that is inhibited by nutrient depletion or ABT737. Bcl-2 associates both with ER and mitochondrial membranes (Germain and Shore, 2003). Wild-type and ER-targeted Bcl-2 (Bcl-2-Cb5) but not mitochondrion-targeted Bcl-2 (Bcl-2-Acta) inhibits starvation-induced autophagy (Pattingre et al, 2005), suggesting that the autophagy-regulatory pool of Bcl-2 is localized on ER. To investigate this, we determined the inhibitory effect of ABT737 and nutrient depletion on the Bcl-2-Beclin-1 interaction in cell lines that stably express wild-type Bcl-2, Bcl-2-Cb5, or Bcl-2-Acta. The amount of Beclin-1 that co-immunoprecipitated with wild type and Bcl-2-Cb5 diminished after treatment with ABT737 or starvation, whereas the amount of Beclin-1 that co-immunoprecipiated with Bcl-2-Acta remained constant (Figure 6B). These results could be further substantiated by subcellular fractionation. The interaction between wild-type Bcl-2 and Beclin-1 measurable in microsomes (ER) was reduced by ABT737 or starvation, but remained constant within the heavy membrane fraction (mitochondria) (Figure 6C and D). Hence, only the ER-targeted pool of Bcl-2 is relevant to the inhibition of autophagy. Figure 7.Interaction between endogenous and spatially restricted Bcl-2 and Beclin-1 in starvation and after addition of ABT737. (A) Immunoprecipitation of endogenous Bcl-2 and Beclin-1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient-free EBSS or with ABT737 (1 μM). (B) Immunoprecipitation of wild-type and ER-targeted Bcl-2 with Beclin-1. Cells stably transfected with wild type, ER- and mitochondrion-targeted Bcl-2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl-2 and immunodetection of Beclin-1. (C, D) Immunoprecipitation of Bcl-2 and Beclin-1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl-2 and immunodetection of Beclin-1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations. Download figure Download PowerPoint Starvation-induced induction of autophagy via the BH3-only protein Bad The results above imply that, to the very least in certain instances, induction of Beclin-1-mediated autophagy should correlate with its release from inhibitory complexes. Upon starvation, the amount of endogenous Beclin-1 that co-immunoprecipitated with Bcl-XL declined within the first hour of serum and nutrient withdrawal, whereas the amount of the BH3 protein Bad (whose activation is known to be triggered by serum withdrawal) (Danial and Korsmeyer, 2004) that co-immunoprecipitated with Bcl-XL increased (Figure 7A). In contrast, addition of rapamycin (which induces auto

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.

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.

For the last four decades, the treatment of cancer has relied on four treatment modalities, namely surgery, radiotherapy, cytotoxic chemotherapy, and hormonotherapy. Most of these therapies are believed to directly attack and eradicate tumor cells. The emerging concept that cancer is not just a disease of a tissue or an organ but also a host disease relies on evidence of tumor-induced immunosuppression and polymorphisms in genes involved in host protection against tumors. This theory is now gaining new impetus, based on our recent data showing that optimal therapeutic effects require the immunoadjuvant effect of tumor cell death induced by cytotoxic anticancer agents. Here, we show that the release of the high mobility group box 1 protein (HMGB1) by dying tumor cells is mandatory to license host dendritic cells (DCs) to process and present tumor antigens. HMGB1 interacts with Toll-like receptor 4 (TLR4) on DCs, which are selectively involved in the cross-priming of anti-tumor T lymphocytes in vivo. A TLR4 polymorphism that affects the binding of HMGB1 to TLR4 predicts early relapse after anthracycline-based chemotherapy in breast cancer patients. This knowledge may be clinically exploited to predict the immunogenicity and hence the efficacy of chemotherapeutic regimens.

Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-XL) by virtue of its BH3 domain, an amphipathic α-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-XL. The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-XL. This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (ER-phagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-XL can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-XL complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-XL complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-XL at the level of the endoplasmic reticulum.Addendum to:Functional and Physical Interaction Between Bcl-XL and a BH3-Like Domain in Beclin-1 M.C. Maiuri, G. Le Toumelin, A. Criollo, J.-C. Rain, F. Gautier, P. Juin, E. Tasdemir, G. Pierron, K. Troulinaki, N. Tavernarakis, J.A. Hickman, O. Geneste and G. KroemerEMBO J 2007; In press

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

Multiple oncogenes (in particular phosphatidylinositol 3-kinase, PI3K; activated Akt1; antiapoptotic proteins from the Bcl-2 family) inhibit autophagy. Similarly, several tumor suppressor proteins (such as BH3-only proteins; death-associated protein kinase-1, DAPK1; the phosphatase that antagonizes PI3K, PTEN; tuberous sclerosic complex 1 and 2, TSC1 and TSC2; as well as LKB1/STK11) induce autophagy, meaning that their loss reduces autophagy. Beclin-1, which is required for autophagy induction acts as a haploinsufficient tumor suppressor protein, and other essential autophagy mediators (such as Atg4c, UVRAG and Bif-1) are bona fide oncosuppressors. One of the central tumor suppressor proteins, p53 exerts an ambiguous function in the regulation of autophagy. Within the nucleus, p53 can act as an autophagy-inducing transcription factor. Within the cytoplasm, p53 exerts a tonic autophagy-inhibitory function, and its degradation is actually required for the induction of autophagy. The role of autophagy in oncogenesis and anticancer therapy is contradictory. Chronic suppression of autophagy may stimulate oncogenesis. However, once a tumor is formed, autophagy inhibition may be a therapeutic goal for radiosensitization and chemosensitization. Altogether, the current state-of-the art suggests a complex relationship between cancer and deregulated autophagy that must be disentangled by further in-depth investigation.

Keeping Cancer Cells At Bay Cancer cells are often aneuploid; that is, they have an abnormal number of chromosomes. But to what extent this contributes to the tumorigenic phenotype is not clear. Senovilla et al. (p. 1678 ; see the Perspective by Zanetti and Mahadevan ) found that tetraploidization of cancer cells can cause them to become immunogenic and thus aid in their clearance from the body by the immune system. Cells with excess chromosomes put stress on the endoplasmic reticulum, which leads to movement of the protein calreticulin to the cell surface. Calreticulin exposure in turn caused recognition of cancer cells in mice by the host immune system. Thus, the immune system appears to serve a protective role in eliminating hyperploid cells that must be overcome to allow unrestricted growth of cancer cells.

Autophagy constitutes one of the major responses to stress in eukaryotic cells, and is regulated by a complex network of signaling cascades. Not surprisingly, autophagy is implicated in multiple pathological processes, including infection by pathogens, inflammatory bowel disease, neurodegeneration and cancer. Both oncogenesis and tumor survival are influenced by perturbations of the molecular machinery that controls autophagy. Numerous oncoproteins, including phosphatidylinositol 3-kinase, Akt1 and anti-apoptotic members of the Bcl-2 family suppress autophagy. Conversely, several tumor suppressor proteins (e.g., Atg4c; beclin 1; Bif-1; BH3-only proteins; death-associated protein kinase 1; LKB1/STK11; PTEN; UVRAG) promote the autophagic pathway. This does not entirely apply to p53, one of the most important tumor suppressor proteins, which regulates autophagy in an ambiguous fashion, depending on its subcellular localization. Irrespective of the controversial role of p53, basal levels of autophagy appear to inhibit tumor development. On the contrary, chemotherapy- and metabolic stress-induced activation of the autophagic pathway reportedly contribute to the survival of formed tumors, thereby favoring resistance. In this context, autophagy inhibition would represent a major therapeutic target for chemosensitization. Here, we will review the current knowledge on the dual role of autophagy as an anti- and pro-tumor mechanism.

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Background— The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Methods and Results— Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1 . Beclin 1 +/− mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Conclusions— Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity.

Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53-/- cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g. starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy. Addendum to: Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, D'Amelio M, Criollo A, Morselli E, Zhu C, Harper F, Nannmark U, Samara C, Pinton P, Vicencio JM, Carnuccio R, Moll UM, Madeo F, Paterlini-Brechot P, Rizzuto R, Szabadkai G, Pierron G, Blomgren K, Tavernarakis N, Codogno P, Cecconi F, Kroemer G. Regulation of autophagy by cytoplasmic p53. Nat Cell Biol 2008; 10:676-87.

In response to stress, cells start transcriptional and transcription‐independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IκB kinase) complex that is best known for its essential role in the activation of the transcription factor NF‐κB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation‐induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF‐κB or ablation of the p65/RelA NF‐κB subunit failed to suppress IKK‐induced autophagy, indicating that IKK can promote the autophagic pathway in an NF‐κB‐independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK‐β suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non-small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27(Kip1) as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC.

The inositol 1,4,5-trisphosphate receptor (IP3R) is a major regulator of apoptotic signaling. Through interactions with members of the Bcl-2 family of proteins, it drives calcium (Ca2+) transients from the endoplasmic reticulum (ER) to mitochondria, thereby establishing a functional and physical link between these organelles. Importantly, the IP3R also regulates autophagy, and in particular, its inhibition/depletion strongly induces macroautophagy. Here, we show that the IP3R antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by the IP3R and Beclin 1, an interaction that is increased or inhibited by overexpression or knockdown of Bcl-2, respectively. An effect of Beclin 1 on Ca2+ homeostasis was discarded as siRNA-mediated knockdown of Beclin 1 did not affect cytosolic or luminal ER Ca2+ levels. Xestospongin B- or starvation-induced autophagy was inhibited by overexpression of the IP3R ligand-binding domain, which coimmunoprecipitated with Beclin 1. These results identify IP3R as a new regulator of the Beclin 1 complex that may bridge signals converging on the ER and initial phagophore formation.

The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

Macrophage activation is intimately linked to metabolic reprogramming. Inflammatory (M1) macrophages are able to sustain inflammatory responses and to kill pathogens, mostly by relying on glycolysis and fatty acid biosynthesis. Glycolysis is a swift way of producing ATP, and fatty acids serve as precursors for the synthesis of inflammatory mediators. On the opposite side, anti-inflammatory (M2) macrophages mediate the resolution of inflammation and tissue repair, switching their metabolism to fatty acid oxidation and oxidative phosphorylation. Over the years, this classical view has been challenged by recent discoveries pointing to a more complex metabolic network during macrophage activation. Lipid metabolism plays critical roles in the activation of both M1 and M2 macrophages. Recent evidence shows that fatty acid oxidation is also essential for inflammasome activation in M1 macrophages, and glycolysis is now known to fuel fatty acid oxidation in M2 macrophages. Ultimately, targeting lipid metabolism in macrophages can improve the outcome of metabolic diseases. Here, we review the main aspects of macrophage immunometabolism form the perspective of the metabolism of lipids. Building a reliable metabolic network during macrophage activation will bring us closer to targeting macrophages for improving human health.

Although the essential genes for autophagy (Atg) have been identified, the molecular mechanisms through which Atg proteins control 'self eating' in mammalian cells remain elusive. Beclin 1 (Bec1), the mammalian orthologue of yeast Atg6, is part of the class III phosphatidylinositol 3-kinase (PI3K) complex that induces autophagy. The first among an increasing number of Bec1-interacting proteins that has been identified is the anti-apoptotic protein Bcl-2. The dissociation of Bec1 from Bcl-2 is essential for its autophagic activity, and Bcl-2 only inhibits autophagy when it is present in the endoplasmic reticulum (ER). A paper in this issue of the EMBO Journal has identified a novel protein, NAF-1 (nutrient-deprivation autophagy factor-1), that binds Bcl-2 at the ER. NAF-1 is a component of the inositol-1,4,5 trisphosphate (IP3) receptor complex, which contributes to the interaction of Bcl-2 with Bec1 and is required for Bcl-2 to functionally antagonize Bec1-mediated autophagy. This work provides mechanistic insights into how autophagy- and apoptosis-regulatory molecules crosstalk at the ER.

Many features of aging result from the incapacity of cells to adapt to stress conditions. When damage accumulates irreversibly, mitotic cells from renewable tissues rely on either of two mechanisms to avoid replication. They can permanently arrest the cell cycle (cellular senescence) or trigger cell death programs. Apoptosis (self-killing) is the best-described form of programmed cell death, but autophagy (self-eating), which is a lysosomal degradation pathway essential for homeostasis, reportedly contributes to cell death as well. Unlike mitotic cells, postmitotic cells like neurons or cardiomyocytes cannot become senescent since they are already terminally differentiated. The fate of these cells entirely depends on their ability to cope with stress. Autophagy then operates as a major homeostatic mechanism to eliminate damaged organelles, long-lived or aberrant proteins and superfluous portions of the cytoplasm. In this mini-review, we briefly summarize the molecular networks that allow damaged cells either to adapt to stress or to engage in programmed-cell-death pathways.

In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes.Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation.Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

Although autophagy has widely been conceived as a self-destructive mechanism that causes cell death, accumulating evidence suggests that autophagy usually mediates cytoprotection, thereby avoiding the apoptotic or necrotic demise of stressed cells. Recent evidence produced by our groups demonstrates that autophagy is also involved in pharmacological manipulations that increase longevity. Exogenous supply of the polyamine spermidine can prolong the lifespan of (while inducing autophagy in) yeast, nematodes and flies. Similarly, resveratrol can trigger autophagy in cells from different organisms, extend lifespan in nematodes, and ameliorate the fitness of human cells undergoing metabolic stress. These beneficial effects are lost when essential autophagy modulators are genetically or pharmacologically inactivated, indicating that autophagy is required for the cytoprotective and/or anti-aging effects of spermidine and resveratrol. Genetic and functional studies indicate that spermidine inhibits histone acetylases, while resveratrol activates the histone deacetylase Sirtuin 1 to confer cytoprotection/longevity. Although it remains elusive whether the same histones (or perhaps other nuclear or cytoplasmic proteins) act as the downstream targets of spermidine and resveratrol, these results point to an essential role of protein hypoacetylation in autophagy control and in the regulation of longevity.

Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

Autophagy is a highly regulated catabolic process that involves lysosomal degradation of proteins and organelles, mostly mitochondria, for the maintenance of cellular homeostasis and reduction of metabolic stress. Problems in the execution of this process are linked to different pathological conditions, such as neurodegeneration, aging, and cancer. Many of the proteins that regulate autophagy are either oncogenes or tumor suppressor proteins. Specifically, tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, Rheb, and AKT, inhibit autophagy, suggesting that autophagy is a tumor suppressor mechanism. Consistent with this hypothesis, the inhibition of autophagy promotes oxidative stress, genomic instability, and tumorigenesis. Nevertheless, autophagy also functions as a cytoprotective mechanism under stress conditions, including hypoxia and nutrient starvation, that promotes tumor growth and resistance to chemotherapy in established tumors. Here, in this brief review, we will focus the discussion on this ambiguous role of autophagy in the development and progression of cancer.

Article25 February 2010free access Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos Ilio Vitale Ilio Vitale INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Laura Senovilla Laura Senovilla INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mohamed Jemaà Mohamed Jemaà INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mickaël Michaud Mickaël Michaud INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Oliver Kepp Oliver Kepp INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lisa Nanty Lisa Nanty INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Santiago Rello-Varona Santiago Rello-Varona INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Gwenola Manic Gwenola Manic INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Didier Métivier Didier Métivier INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Sonia Vivet Sonia Vivet INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicolas Tajeddine Nicolas Tajeddine INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicholas Joza Nicholas Joza INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alexander Valent Alexander Valent Unité de Recherche Translationnelle, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Maria Castedo Maria Castedo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Ilio Vitale Ilio Vitale INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Laura Senovilla Laura Senovilla INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mohamed Jemaà Mohamed Jemaà INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Mickaël Michaud Mickaël Michaud INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Oliver Kepp Oliver Kepp INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Lisa Nanty Lisa Nanty INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Santiago Rello-Varona Santiago Rello-Varona INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Gwenola Manic Gwenola Manic INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Didier Métivier Didier Métivier INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Sonia Vivet Sonia Vivet INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicolas Tajeddine Nicolas Tajeddine INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Nicholas Joza Nicholas Joza INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, France Search for more papers by this author Alexander Valent Alexander Valent Unité de Recherche Translationnelle, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Maria Castedo Maria Castedo INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM, Villejuif, France Institut Gustave Roussy, Villejuif, France Faculté de Médecine, Université Paris-Sud XI, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Author Information Ilio Vitale1,2,3, Laura Senovilla1,2,3, Mohamed Jemaà1,2,3, Mickaël Michaud1,2,3, Lorenzo Galluzzi1,2,3, Oliver Kepp1,2,3, Lisa Nanty1,2,3, Alfredo Criollo1,2,3, Santiago Rello-Varona1,2,3, Gwenola Manic1,2,3, Didier Métivier1,2,3, Sonia Vivet1,2,3, Nicolas Tajeddine1,2,3, Nicholas Joza1,2,3, Alexander Valent4, Maria Castedo1,2,3 and Guido Kroemer 1,2,3 1INSERM, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Faculté de Médecine, Université Paris-Sud XI, Villejuif, France 4Unité de Recherche Translationnelle, Institut Gustave Roussy, Villejuif, France *Corresponding author. INSERM, Institut Gustave Roussy, Pavillon de Recherche 1, rue Camille Desmoulins, Villejuif F-94805, France. Tel.: +33 1 4211 6046; Fax: +33 1 4211 6047; E-mail: [email protected] The EMBO Journal (2010)29:1272-1284https://doi.org/10.1038/emboj.2010.11 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 Tetraploidy can constitute a metastable intermediate between normal diploidy and oncogenic aneuploidy. Here, we show that the absence of p53 is not only permissive for the survival but also for multipolar asymmetric divisions of tetraploid cells, which lead to the generation of aneuploid cells with a near-to-diploid chromosome content. Multipolar mitoses (which reduce the tetraploid genome to a sub-tetraploid state) are more frequent when p53 is downregulated and the product of the Mos oncogene is upregulated. Mos inhibits the coalescence of supernumerary centrosomes that allow for normal bipolar mitoses of tetraploid cells. In the absence of p53, Mos knockdown prevents multipolar mitoses and exerts genome-stabilizing effects. These results elucidate the mechanisms through which asymmetric cell division drives chromosomal instability in tetraploid cells. Introduction Tetraploid cells are detected in some precancerous lesions such as Barrett's oesophagus and cervical dysplasia, where their presence coexists with the loss of functional p53 (Heselmeyer et al, 1996; Maley et al, 2004). Owing to the increase in the number of chromosomes, perhaps coupled to changes in the geometry of the mitotic machinery (Storchova et al, 2006; Storchova and Kuffer, 2008), tetraploid cells frequently activate the DNA damage response and become genomically unstable. Thus, tetraploidy may be considered as a metastable state that links normal diploidy to cancer-associated aneuploidy (Storchova and Pellman, 2004; Fujiwara et al, 2005; Margolis, 2005). Numerous tumour suppressor genes including p53 (Margolis, 2005), BRCA1 (Schlegel et al, 2003), LATS2 (Aylon et al, 2006) and APC (Tighe et al, 2004) actively repress tetraploidy, meaning that their removal can either stimulate the spontaneous tetraploidization of cells or facilitate the survival of tetraploid cells generated upon cytokinesis or karyokinesis inhibition. The former has been shown for the knockdown of APC in cultured cells and for the conditional knockout of APC in small intestine epithelia in vivo (Caldwell et al, 2007). The latter has been demonstrated in cultured cancer cell lines that were depleted from p53 by gene knockout or RNA interference (Cross et al, 1995; Andreassen et al, 2001; Castedo et al, 2006a, 2006b), as well as in p53−/− primary mouse mammary epithelial cells (Fujiwara et al, 2005; Senovilla et al, 2009). Moreover, the expression of some oncogenes including Myc (Yin et al, 1999), Aurora-A (Wang et al, 2006) and human papillomavirus (HPV)-encoded E6 (Incassati et al, 2006) can stimulate tetraploidization. The mechanisms through which tetraploidy favours oncogenesis are complex and have not yet been entirely elucidated. One single tetraploid cell can undergo multipolar mitosis, which often leads to the generation of three or more daughter cells (Storchova and Pellman, 2004). This process causes the near-to-stochastic distribution of chromosomes and hence is lethal for most daughter cells. Nullisomy (the total absence of one particular chromosome) and polysomy (the presence of extra copies of one chromosome), indeed, result in major genetic defects involving the incorrect assembly of multiprotein complexes and fatal linkage disequilibria, which are rarely compatible with cell survival (Zhivotovsky and Kroemer, 2004; Roumier et al, 2005; Ganem et al, 2007). Moreover, during mitosis, the presence of more than two centrosomes in tetraploid cells can also favour merotelic chromosome attachments and hence chromosomal lagging, which may favour chromosome loss or asymmetric distribution among daughter cells, even when the division is bipolar (Ganem et al, 2009). Multipolar and asymmetric cell division, as they can result from tetraploidy (Levine et al, 1991; Ganem et al, 2007), are commonly observed in malignant lesions and have been suspected to contribute to oncogenesis for over a century (Boveri, 2008). Supernumerary centrosomes, as they are detected in malignant cells (Levine et al, 1991; D'Assoro et al, 2002), can be induced experimentally, and this reportedly suffices to trigger oncogenesis (Basto et al, 2008; Gergely and Basto, 2008). Moreover, ‘anisocytosis’ and ‘anisokaryosis’ (heterogeneity in cell size and nuclear size, respectively), which presumably result from asymmetric divisions, are well-established histological hallmarks of malignancy (Boveri, 2008; Holland and Cleveland, 2009). It is interesting to note that in some cancer types (e.g., non-small cell lung cancer), these morphological traits of malignancy correlate with the expression of one particular oncogene, Mos (Gorgoulis et al, 2001). Mos (also called c-Mos) is the first human oncogene cloned, and has been identified as the cellular homologue of the viral oncogene v-Mos, which is encoded by the Moloney murine sarcoma virus (Oskarsson et al, 1980; Watson et al, 1982). The p39Mos protein (hereafter referred to as Mos) has been shown to stimulate the transformation of murine fibroblasts in vitro (Okazaki and Sagata, 1995; Fukasawa and Vande Woude, 1997). Moreover, transfection-enforced overexpression of Mos reportedly inhibits mitotic progression (Wang et al, 1994) and causes the generation of binucleated cells due to the inhibition of cytokinesis (Okazaki et al, 1992; Fukasawa and Vande Woude, 1995). Apparently, Mos can stabilize another oncogene product, c-Fos, (Okazaki and Sagata, 1995) and enhance the expression of cyclins (Rhodes et al, 1997), thereby stimulating cell proliferation. The genetic invalidation of Mos has no obvious phenotypic consequences in mice (Colledge et al, 1994; Hashimoto et al, 1994). However, although Mos−/− male mice exhibit normal reproduction rates, Mos−/− female are nearly infertile (Colledge et al, 1994; Hashimoto et al, 1994), in line with the fact that Mos is strictly necessary for the first meiotic division of oocytes (Sagata et al, 1989a), and then exerts a critical checkpoint function during metaphase II (Sagata et al, 1989b). Both the meiosis-regulatory and the transforming effects of Mos require its serine–threonine kinase activity (Haccard et al, 1993; Okazaki and Sagata, 1995). Known Mos substrates include cyclin B2, tubulin and MEK1 (Roy et al, 1990; Zhou et al, 1991; Sagata, 1997), and the meiotic checkpoint function of Mos depends on its capacity to activate the mitogen-activated protein kinase (MAPK) pathway (Haccard et al, 1993). Thus, very little is known about the role of Mos in somatic cells and on the mechanisms by which Mos can act as an oncogene. Here, we developed a cellular model of aneuploidization in which p53−/− cells were driven into tetraploidy, which was followed by multipolar mitosis and re-acquisition of a near-to-diploid chromosome content. We found that Mos was upregulated in p53-deficient tetraploid cells and that it was strictly required for the occurrence of multipolar divisions, presumably because Mos acts as an inhibitor of centrosome coalescence. Results and discussion Generation of sub-tetraploid derivatives from p53-deficient tetraploid cells Shortly (2 days) after a 48 h-long treatment with the microtubule poison nocodazole or the cytokinesis inhibitor cytochalasin D, p53−/− human colon carcinoma HCT 116 cell cultures contained a higher fraction of polyploid cells (with a ⩾4n DNA content), yet a lower amount of dying and dead cells (exhibiting the dissipation of the mitochondrial transmembrane potential (ΔΨm) and the breakdown of plasma membranes, respectively) (Kroemer et al, 2007; Galluzzi et al, 2009) than their p53-proficient counterparts (Figure 1A and Supplementary Figure S1). Moreover, fluorescence-activated cell sorter (FACS)-purified cells with an ∼8n DNA content formed colonies more efficiently when they did not express p53 than when they did so (Figure 1B), in line with the notion that p53 deficiency is permissive for the generation and survival of tetraploid cells (Castedo et al, 2006b). Cultures derived from FACS-purified cells with an ∼8n DNA content were characterized by a 4n DNA content in the G1 phase of the cell cycle and by an 8n DNA content in the G2 and M phases, thereby exhibiting bona fide traits of tetraploidy. However, after several passages, p53−/− (but neither p21−/− nor Bax−/−) cultures progressively accumulated a population of cells with a ∼2n DNA content (Figure 1C and D). This was observed in six independent experiments in which the initial contamination with ∼2n cells (measured immediately after FACS purification) was undetectable. To understand the origin of such ∼2n population, we followed the fate of tetraploid cells 1 week after their generation by videomicroscopy, and found that p53−/− cells underwent multipolar (mostly tri- or tetrapolar) divisions—which are associated with Y- and X-shaped metaphases (Figure 2A)—much more frequently than p53+/+ control cells (Figure 2B and Supplementary Videos 1 and 2). These results suggest that cells with a ∼2n DNA content (hereafter referred to as ‘sub-tetraploid’) appearing at significant frequencies (⩾10%), as early as 15 days after tetraploidization, might result from a peculiar process of multipolar division. A significant percentage of daughter cells that originated from p53−/− tetraploid cells by multipolar mitosis could enter and terminate normal bipolar divisions (Figure 2C and D, and Supplementary Video 3), suggesting that such sub-tetraploid cells can give rise to a new lineage. The reduction of the chromosomal content was significantly more frequent among p53−/− tetraploid cells than among control ones (Figures 1 and 2), and, in another cell line, was exacerbated by pharmacological inhibition of p53 by cyclic pifithrin-α (Komarov et al, 1999) (Supplementary Figure S2). Thus, the tumour suppressor p53 reduces the probability of sub-tetraploidy. Figure 1.Effect of p53 on the survival and on the genomic stability of tetraploid HCT 116 cells. (A, B) The absence of p53 increases the clonogenic survival of freshly generated polyploid cells. Wild type (WT), p53−/−, Bax−/− and p21−/− diploid human colon carcinoma HCT 116 cells and HCT 116 cells stably transfected with a plasmid encoding the baculoviral inhibitor of caspases p35 (p35) were left untreated or treated with 100 nM nocodazole (Noco) for 48 h. After washing, cells were cultured for additional 48 h in drug-free culture medium, then stained with Hoechst 33342, followed by fluorescence-activated cell sorter (FACS) purification of diploid (2n, white and grey symbols for untreated and nocodazole-treated cells, respectively) or polyploid (>4n, black symbols) cell populations. In A, representative cell cycle distributions of WT and p53−/− cells from one out of three independent experiments are shown. X-axis = Hoechst 33342 fluorescence (DNA content); Y-axis = cell number per channel (counts). In B, the results of clonogenic survival assays carried out on such FACS-purified cells are reported. The upper part shows representative pictures of colonies formed by WT and p53−/− cells as observed upon crystal violet staining 10 days after FACS purification. Columns depict the survival fraction (mean±s.e.m., n=3 parallel wells, normalized for plating efficiency and to control diploid cells) of the diploid and polyploid cell populations with the indicated genotype and corresponding to the FACS-purified populations represented in A. Asterisks indicate statistically significant differences as compared to WT tetraploid cells (Student's t-test, P<0.05). (C, D) Genomic instability of viable polyploid cells. Diploid HCT 116 cells with the indicated genotype were treated for 48 h with nocodazole and tetraploid populations were FACS-purified as in A (>4n, black symbols). Cells then were cultured for the indicated number of days and analyzed for DNA content. Representative cell cycle profiles of WT and p53−/− tetraploid cells are shown in C, whereas quantitative data are reported in D (mean±s.e.m., n=6 independent determinations). In C, the percentages of cells with a sub-tetraploid DNA content (indicating chromosomal loss) are indicated. Statistical significance resulting from the comparison to WT cells is highlighted by asterisks (Student's t-test, P<0.05). Download figure Download PowerPoint Figure 2.Multipolar divisions of p53-deficient tetraploid cells. Isogenic wild type (WT) and p53−/− diploid HCT 116 cells expressing a histone 2B–green fluorescent protein (H2B–GFP) chimera were treated for 48 h with nocodazole and then cultured in drug-free medium for additional 48 h. Polyploid cells were fluorescence-activated cell sorter (FACS)-sorted as in Figure 1A (>4n, black symbols) and monitored by fluorescence videomicroscopy for 48 h. In A snapshots taken at the indicated time points exemplify the appearance of tripolar and tetrapolar mitosis, thus exhibiting the representative Y- and X-shaped metaphase, respectively. Full-length movies proving the completion of cell division are available as Supplementary Videos S1 and S2. B reports the percentage of cell cycle arrest, apoptosis induction and various mitotic aberrations as quantified among 150 to 200 mitoses for each genotype (mean ± s.e.m., n=3 independent experiments). In C representative images captured at the indicated time points show that some daughter cells that originated from p53−/− tetraploid HCT 116 cells by multipolar mitosis can enter and terminate normal bipolar divisions (red arrows), whereas others undergo apoptosis (yellow arrows). A full-length movie that demonstrates the completion of a normal cell division by such a daughter cell is available as Supplementary Video S3. D reports the percentage of these cells (arisen from p53−/− tetraploid HCT 116 cells by multipolar mitosis) that underwent the indicated fate by the end of the experiment (as quantified among 150-200 daughter cells, mean±s.e.m., n=3 independent experiments). Note that only cells entering multipolar mitosis within the first 18 h of the assays were tracked for the subsequent 30 h. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Sub-tetraploidy is linked to centrosome defects and aneuploidy Next, we generated multiple tetraploid clones from isogenic HCT 116 cells. As compared with wild type (WT), Bax−/−, p21−/− or apoptosis-resistant (owing to the expression of the caspase inhibitor p35 from Baculovirus) tetraploid cells, p53−/− tetraploid clones displayed some difficulties in maintaining a stable tetraploid genome. Four weeks after cloning, indeed, only one-third of p53−/− tetraploid clones still exhibited a clean tetraploid DNA content profile, whereas the other two-thirds accumulated viable sub-tetraploid populations (Figure 3A and B), first as rather broad shoulders (‘phase 1’) and later as sharper peaks (‘phase 2’) of sub-tetraploid cells. Such sharper peaks presumably arise in cultures that are dominated by one or few viable sub-clones with a comparable sub-tetraploid DNA content. Phase 1 and phase 2 unstable sub-tetraploid populations also arose after repeated transfection (approximately once every 5 days) with a small interfering RNA (siRNA) targeting p53 (Supplementary Figure S2). Chromosome counting confirmed that phase 2 unstable p53−/− tetraploid cells frequently contained a roughly diploid number of chromosomes (Figure 3C and D). Phase 1 unstable p53−/− tetraploid cells were characterized by a high frequency of aberrant mitoses that were either monopolar, bipolar characterized by lagging chromosomes or multipolar linked to supernumerary centrosomes. Multipolar mitoses were also more frequent among phase 2 unstable tetraploids as compared to WT or stable p53−/− tetraploids (Figure 3E). As compared to phase 1 cells, phase 2 cells proliferated more quickly, and among them the sub-tetraploid population had shorter duplication times and shorter mitoses than the tetraploid one (Supplementary Figure S3). This explains the outgrowth of sub-tetraploid cells over their tetraploid counterparts. When such sub-tetraploid cells were exposed to nocodazole, they could again tetraploidize and then revert once more to sub-tetraploidy, but this process of reversion was not accelerated (Supplementary Figure S4). Figure 3.Chromosome instability and centrosome amplification in p53−/− tetraploid HCT 116 clones. (A, B) p53 deficiency increases the percentage of unstable tetraploid clones. Tetraploid HCT 116 clones were generated from wild type (WT), p53−/−, Bax−/−, p21−/− and p35-expressing parental cells as described in Supplementary Data. Cell cycle distribution and apoptosis-related parameters were evaluated 4 weeks after cloning by multiparametric cytofluorometry upon staining with Hoechst 33342 (which measures DNA content), the mitochondrial transmembrane potential (ΔΨm)-sensitive dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) and propidium iodide (PI, an exclusion dye that only stains dead cells). Representative plots are shown in A, the inserts therein showing positive controls for ΔΨm dissipation (as obtained by treating the cells for 30 min with 100 μM protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP) and plasma membrane permeabilization (as resulting from a 2 min-long incubation in 0.5% (w/v) saponin (Sapo)). Tetraploid clones were classified according to genomic instability (as evaluated by the quantification of viable sub-tetraploid populations) in stable (sub-tetraploid cells <10%) and unstable (sub-tetraploid cells >10%). Unstable clones were subdivided into ‘phase 1’ and ‘phase 2’ clones, characterized by broad and sharp sub-tetraploid peaks, respectively. B reports the frequency of unstable clones generated from parental tetraploid cells of the indicated genotype. Frequency was calculated among 50–100 clones for each genotype (mean±s.e.m., n=3 independent series of clones). (C, D) Chromosome count in tetraploid clones. Representative examples of 4′,6 diamidino-2-phenylindole (DAPI)-stained metaphase spreads with the corresponding number of chromosomes are shown in C. For WT and p53−/− clones of the indicated type, D reports the percentage of cells containing 40–60, 61–80, 81–100 or >101 chromosomes, as quantified among 100 metaphases per condition (mean±s.e.m., n=4 different clones in independent assessments) (E) Centrosome amplification correlates with genomic instability. WT and p53−/− clones of the indicated class were cultured on glass coverslips and subjected to immunofluorescence detection of mitotic spindles (β-tubulin staining, green fluorescence) and centrosomes (γ-tubulin staining, red fluorescence). Nuclei were counterstained with Hoechst 33342 (emitting in blue). According to the number of centrosomes, interphase cells were divided in normal (1 or 2 centrosomes) and abnormal (more than 2 centrosomes, either congressed in a single pole or not), whereas metaphases were classified as monopolar (2 congressed centrosomes), normal and abnormal bipolar (2 centrosomes, the latter exhibiting the misalignment of one or more chromosomes), and multipolar (tripolar, tetrapolar or of higher-order polarity, characterized by 3, 4 or more centrosomes, respectively). Representative immunofluorescence microscopy images of each category are shown. The percentage of occurrence of each category is reported, as quantified among 150 to 200 cells for WT and p53−/− clones of the indicated type (mean±s.e.m., n=4 distinct clones). A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Fluorescence in situ hybridization (FISH) carried out during interphase proved that a large portion of sub-tetraploid cells (which were FACS-purified from unstable p53−/− tetraploid clones) was aneuploid (Figure 4A-C). Thus, especially in phase 1 cultures, nullisomies (which are always lethal) were frequently detected (Figure 4C). Accordingly, most (>99%) of such phase 1 sub-tetraploid cells failed to form stable offspring in clonogenic assays and died (Figure 4D). In phase 2 cultures, the frequency of aneuploid cells was lower, and nullisomies were infrequent (Figure 4C), presumably because viable cells (which efficiently form clones, Figure 4D) had been positively selected. To further explore the behaviour of sub-tetraploid cells, we generated phase 1 and phase 2 clones from p53−/− tetraploid cells expressing histone H2B fused to the N-terminus of green fluorescent protein (H2B–GFP, which labels chromatin in green and hence allows for monitoring chromosome movement), and followed the fate of their sub-tetraploid derivatives after FACS purification (Figure 4E, F and Supplementary Figure S5, and Supplementary Videos 4, 5 and 6). Although phase 1 sub-tetraploid cells rarely (∼3%) engaged in subsequent cycles of division, phase 2 sub-tetraploid cells did so much more frequently (∼60%). Nonetheless, cell cycle blockade, mitosis without cytokinesis and cell death occurred more frequently among sub-tetraploid cells than among their diploid progenitors (Figure 4E, F and Supplementary Figure S5, and Supplementary Videos 4, 5 and 6). Figure 4.Characterization and fate of the sub-tetraploid offspring of p53−/− tetraploid cells. (A–C) An important fraction of sub-tetraploid cells are aneuploid. Sub-tetraploid cells derived from phase 1 and 2 unstable p53−/− tetraploid HCT 116 clones or the 2n population of the parental p53−/− diploid cell line were fluorescence-activated cell sorter (FACS)-purified as indicated in A. Thereafter, nuclei from the sorted po

The life span of various model organisms can be extended by caloric restriction as well as by autophagy-inducing pharmacological agents. Life span-prolonging effects have also been observed in yeast cells, nematodes and flies upon the overexpression of the deacetylase Sirtuin-1. Intrigued by these observations and by the established link between caloric restriction and Sirtuin-1 activation, we decided to investigate the putative implication of Sirtuin-1 in the response of human cancer cells and Caenorhabditis elegans to multiple triggers of autophagy. Our data indicate that the activation of Sirtuin-1 (by the pharmacological agent resveratrol and/or genetic means) per se ignites autophagy, and that Sirtuin-1 is required for the autophagic response to nutrient deprivation, in both human and nematode cells, but not for autophagy triggered by downstream signals such as the inhibition of mTOR or p53. Since the life spanextending effects of Sirtuin-1 activators are lost in autophagy-deficient C. elegans, our results suggest that caloric restriction and resveratrol extend longevity, at least in experimental settings, by activating autophagy.

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53 (K382R) failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

Tetraploidy and the depolyploidization of tetraploid cells may contribute to oncogenesis. Several mechanisms have evolved to avoid the generation, survival, proliferation and depolyploidization of tetraploids. Cells that illicitly survive these checkpoints are prone to chromosomal instability and aneuploidization. Along with their replication, tetraploids constantly undergo chromosomal rearrangements that eventually lead to pseudodiploidy by two non-exclusive mechanisms: (i) multipolar divisions and (ii) illicit bipolar divisions in the presence of improper microtubule-kinetochore attachments. Here, we describe the regulation and the molecular mechanisms that underlie such a 'polyploidization-depolyploidization' cascade, while focusing on the role of oncogenes and tumor suppressor genes in tetraploidy-driven tumorigenesis. We speculate that the identification of signaling/metabolic cascades that are required for the survival of tetraploid or aneuploid (but not diploid) cancer cells may pave the way for the development of novel broad-spectrum anticancer agents.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-XL, thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-κB (IκB) kinase (IKK) and the hyperphosphorylation of the IKK substrate IκB, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways.

Background: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. Methods: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. Results: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor β-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. Conclusions: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor β-1–SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

AbstractIt is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.View correction statement:Erratum to Criollo A, et al. Cell Cycle Volume 11, Issue 1; pp. 194-9

With increased life expectancy, women will spend over three decades of life postmenopause. The menopausal transition increases susceptibility to metabolic diseases such as obesity, diabetes, cardiovascular disease, and cancer. Thus, it is more important than ever to develop effective hormonal treatment strategies to protect aging women. Understanding the role of estrogens, and their biological actions mediated by estrogen receptors (ERs), in the regulation of cardiometabolic health is of paramount importance to discover novel targeted therapeutics. In this brief review, we provide a detailed overview of the literature, from basic science findings to human clinical trial evidence, supporting a protective role of estrogens and their receptors, specifically ERα, in maintenance of cardiometabolic health. In so doing, we provide a concise mechanistic discussion of some of the major tissue-specific roles of estrogens signaling through ERα. Taken together, evidence suggests that targeted, perhaps receptor-specific, hormonal therapies can and should be used to optimize the health of women as they transition through menopause, while reducing the undesired complications that have limited the efficacy and use of traditional hormone replacement interventions.

Chaperone Mediated Autophagy (CMA) is a lysosomal-dependent protein degradation pathway. At least 30% of cytosolic proteins can be degraded by this process. The two major protein players of CMA are LAMP-2A and HSC70. While LAMP-2A works as a receptor for protein substrates at the lysosomal membrane, HSC70 specifically binds protein targets and takes them for CMA degradation. Because of the broad spectrum of proteins able to be degraded by CMA, this pathway has been involved in physiological and pathological processes such as lipid and carbohydrate metabolism, and neurodegenerative diseases, respectively. Both, CMA, and the mentioned processes, are affected by aging and by inadequate nutritional habits such as a high fat diet or a high carbohydrate diet. Little is known regarding about CMA, which is considered a common regulation factor that links metabolism with neurodegenerative disorders. This review summarizes what is known about CMA, focusing on its molecular mechanism, its role in protein, lipid and carbohydrate metabolism. In addition, the review will discuss how CMA could be linked to protein, lipids and carbohydrate metabolism within neurodegenerative diseases. Furthermore, it will be discussed how aging and inadequate nutritional habits can have an impact on both CMA activity and neurodegenerative disorders.

When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-alpha), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-X(L) with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G(1) phase, less so in the S phase and spares the G(2)/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.

Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

Background— L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress–induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein–coupled receptor–like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results— We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown–dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction–induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction–elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusion— PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein.

Non-communicable diseases (NCDs), also known as chronic diseases, are long-lasting conditions that affect millions of people around the world. Different factors contribute to their genesis and progression; however they share common features, which are critical for the development of novel therapeutic strategies. A persistently altered inflammatory response is typically observed in many NCDs together with redox imbalance. Additionally, dysregulated proteostasis, mainly derived as a consequence of compromised autophagy, is a common feature of several chronic diseases. In this review, we discuss the crosstalk among inflammation, autophagy and oxidative stress, and how they participate in the progression of chronic diseases such as cancer, cardiovascular diseases, obesity and type II diabetes mellitus.

Autophagy is a catabolic mechanism where intracellular material is degraded by vesicular structures called autophagolysosomes. Autophagy is necessary to maintain the normal function of the central nervous system (CNS), avoiding the accumulation of misfolded and aggregated proteins. Consistently, impaired autophagy has been associated with the pathogenesis of various neurodegenerative diseases. The proteins TAR DNA-binding protein-43 (TDP-43), which regulates RNA processing at different levels, and chromosome 9 open reading frame 72 (C9orf72), probably involved in membrane trafficking, are crucial in the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Additionally, recent studies have identified a role for these proteins in the control of autophagy. In this manuscript, we review what is known regarding the autophagic mechanism and discuss the involvement of TDP-43 and C9orf72 in autophagy and their impact on neurodegenerative diseases.

In this review, we discuss the observations that, following chronic high-fat diet (HFD) exposure, male mice have higher levels of saturated fatty acids (FAs) and total sphingolipids, whereas lower amounts of polyunsaturated FAs in the central nervous system (CNS) than females. Furthermore, males, when compared with female mice, have higher levels of inflammatory markers in the hypothalamus following exposure to HFD. The increase in markers of inflammation in male mice is possibly due to the reductions in proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and estrogen receptor alpha (ERα), which is not recapitulated in female mice. Consistently, hypothalamic inflammation is induced both in male and female ERα total-body knockout mice when exposed to a HFD, thus confirming the key role of ERα in the regulation of HFD-induced hypothalamic inflammation. Finally, the HFD-induced depletion of hypothalamic ERα is associated with dysregulation in metabolic homeostasis, as evidenced by reductions in glucose tolerance and decrements in myocardial function.

Chronic consumption of high fat diets (HFDs), rich in saturated fatty acids (SatFAs) like palmitic acid (PA), is associated with the development of obesity and obesity-related metabolic diseases such as type II diabetes mellitus (T2DM). Previous studies indicate that PA accumulates in the hypothalamus following consumption of HFDs; in addition, HFDs consumption inhibits autophagy and reduces insulin sensitivity. Whether malfunction of autophagy specifically in hypothalamic neurons decreases insulin sensitivity remains unknown. PA does activate the Free Fatty Acid Receptor 1 (FFAR1), also known as G protein-coupled receptor 40 (GPR40); however, whether FFAR1 mediates the effects of PA on hypothalamic autophagy and insulin sensitivity has not been shown. Here, we demonstrate that exposure to PA inhibits the autophagic flux and reduces insulin sensitivity in a cellular model of hypothalamic neurons (N43/5 cells). Furthermore, we show that inhibition of autophagy and the autophagic flux reduces insulin sensitivity in hypothalamic neuronal cells. Interestingly, the inhibition of the autophagic flux, and the reduction in insulin sensitivity are prevented by pharmacological inhibition of FFAR1. Our findings show that dysregulation of autophagy reduces insulin sensitivity in hypothalamic neuronal cells. In addition, our data suggest FFAR1 mediates the ability of PA to inhibit autophagic flux and reduce insulin sensitivity in hypothalamic neuronal cells. These results reveal a novel cellular mechanism linking PA-rich diets to decreased insulin sensitivity in the hypothalamus and suggest that hypothalamic autophagy might represent a target for future T2DM therapies.

Proper execution of cellular function, maintenance of cellular homeostasis and cell survival depend on functional integration of cellular processes and correct orchestration of cellular responses to stresses. Cancer transformation is a common negative consequence of mismanagement of coordinated response by the cell. In this scenario, by maintaining the balance among synthesis, degradation, and recycling of cytosolic components including proteins, lipids, and organelles the process of autophagy plays a central role. Several environmental stresses activate autophagy, among those hypoxia, DNA damage, inflammation, and metabolic challenges such as starvation. In addition to these chemical challenges, there is a requirement for cells to cope with mechanical stresses stemming from their microenvironment. Cells accomplish this task by activating an intrinsic mechanical response mediated by cytoskeleton active processes and through mechanosensitive protein complexes which interface the cells with their mechano-environment. Despite autophagy and cell mechanics being known to play crucial transforming roles during oncogenesis and malignant progression their interplay is largely overlooked. In this review, we highlight the role of physical forces in autophagy regulation and their potential implications in both physiological as well as pathological conditions. By taking a mechanical perspective, we wish to stimulate novel questions to further the investigation of the mechanical requirements of autophagy and appreciate the extent to which mechanical signals affect this process.

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with <i>N</i>-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.

HeLa and HCT116 cells respond differentially to sorbitol, an osmolyte able to induce hypertonic stress. In these models, sorbitol promoted the phenotypic manifestations of early apoptosis followed by complete loss of viability in a time-, dose-, and cell type-specific fashion, by eliciting distinct yet partially overlapping molecular pathways. In HCT116 but not in HeLa cells, sorbitol caused the mitochondrial release of the caspase-independent death effector AIF, whereas in both cell lines cytochrome c was retained in mitochondria. Despite cytochrome c retention, HeLa cells exhibited the progressive activation of caspase-3, presumably due to the prior activation of caspase-8. Accordingly, caspase inhibition prevented sorbitol-induced killing in HeLa, but only partially in HCT116 cells. Both the knock-out of Bax in HCT116 cells and the knock-down of Bax in A549 cells by RNA interference reduced the AIF release and/or the mitochondrial alterations. While the knock-down of Bcl-2/Bcl-XL sensitized to sorbitol-induced killing, overexpression of a Bcl-2 variant that specifically localizes to mitochondria (but not of the wild-type nor of a endoplasmic reticulum-targeted form) strongly inhibited sorbitol effects. Thus, hyperosmotic stress kills cells by triggering different molecular pathways, which converge at mitochondria where pro- and anti-apoptotic members of the Bcl-2 family exert their control.

The second messenger myo-inositol-1,4,5-trisphosphate (IP3) acts on the IP3 receptor (IP3R), an IP3-activated Ca2+ channel of the endoplasmic reticulum (ER). The IP3R agonist IP3 inhibits starvation-induced autophagy. The IP3R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-XL, two proteins that physically interact with IP3R. Autophagy can also be induced by depletion of the IP3R by small interfering RNAs. Autophagy induction by IP3R blockade cannot be explained by changes in steady state levels of Ca2+ in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP3R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.Addendum to:Regulation of Autophagy by the Inositol Trisphosphate ReceptorA. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgo, J. Diaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai and G. KroemerCell Death Differ 2007; In press

Cellular organelles do not function as isolated or static units, but rather form dynamic contacts between one another that can be modulated according to cellular needs. The physical interfaces between organelles are important for Ca2+ and lipid homeostasis, and serve as platforms for the control of many essential functions including metabolism, signaling, organelle integrity and execution of the apoptotic program. Emerging evidence also highlights the importance of organelle communication in disorders such as Alzheimer's disease, pulmonary arterial hypertension, cancer, skeletal and cardiac muscle dysfunction. Here, we provide an overview of the current literature on organelle communication and the link to human pathologies.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver's CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain's CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains.

Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.

Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy.Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types.Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

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Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

We show that chronic high fat diet (HFD) feeding affects the hypothalamus of male but not female mice. In our study we demonstrate that palmitic acid and sphingolipids accumulate in the central nervous system of HFD-fed males. Additionally, we show that HFD-feeding reduces proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) thus reducing estrogen receptor α (ERα) and driving hypothalamic inflammation in male but not female mice. Hypothalamic inflammation correlates with markers of metabolic dysregulation as indicated by dysregulation in glucose intolerance and myocardial function. Lastly, we demonstrate that there are blockages in mitophagy and lipophagy in hypothalamic tissues in males. Our data suggest there is a sexually dimorphic response to chronic HDF exposure, females; despite gaining the same amount of body weight following HFD-feeding, appear to be protected from the adverse metabolic effects of the HFD.

Estrogens and their receptors play key roles in regulating body weight, energy expenditure, and metabolic homeostasis. It is known that lack of estrogens promotes increased food intake and induces the expansion of adipose tissues, for which much is known. An area of estrogenic research that has received less attention is the role of estrogens and their receptors in influencing intermediary lipid metabolism in organs such as the brain. In this review, we highlight the actions of estrogens and their receptors in regulating their impact on modulating fatty acid content, utilization, and oxidation through their direct impact on intracellular signaling cascades within the central nervous system.

High-fat diet (HFD)-induced obesity is associated with increased cancer risk. Long-term feeding with HFD increases the concentration of the saturated fatty acid palmitic acid (PA) in the hypothalamus. We previously showed that, in hypothalamic neuronal cells, exposure to PA inhibits the autophagic flux, which is the whole autophagic process from the synthesis of the autophagosomes, up to their lysosomal fusion and degradation. However, the mechanism by which PA impairs autophagy in hypothalamic neurons remains unknown. Here, we show that PA-mediated reduction of the autophagic flux is not caused by lysosomal dysfunction, as PA treatment does not impair lysosomal pH or the activity of cathepsin B.Instead, PA dysregulates autophagy by reducing autophagosome-lysosome fusion, which correlates with the swelling of endolysosomal compartments that show areduction in their dynamics. Finally, because lysosomes undergo constant dynamic regulation by the small Rab7 GTPase, we investigated the effect of PA treatment on its activity. Interestingly, we found PA treatment altered the activity of Rab7. Altogether, these results unveil the cellular process by which PA exposure impairs the autophagic flux. As impaired autophagy in hypothalamic neurons promotes obesity, and balanced autophagy is required to inhibit malignant transformation, this could affect tumor initiation, progression, and/or response to therapy of obesity-related cancers.

Oral squamous cell carcinoma, the most common type of oral cancer, affects more than 275,000 people per year worldwide. Oral squamous cell carcinoma is very aggressive, as most patients die after 3 to 5 years post-diagnosis. The initiation and progression of oral squamous cell carcinoma are multifactorial: smoking, alcohol consumption, and human papilloma virus infection are among the causes that promote its development. Although oral squamous cell carcinoma involves abnormal growth and migration of oral epithelial cells, other cell types such as fibroblasts and immune cells form the carcinoma niche. An underlying inflammatory state within the oral tissue promotes differential stress-related responses that favor oral squamous cell carcinoma. Autophagy is an intracellular degradation process that allows cancer cells to survive under stress conditions. Autophagy degrades cellular components by sequestering them in vesicles called autophagosomes, which ultimately fuse with lysosomes. Although several autophagy markers have been associated with oral squamous cell carcinoma, it remains unclear whether up- or down-regulation of autophagy favors its progression. Autophagy levels during oral squamous cell carcinoma are both timing- and cell-specific. Here we discuss how autophagy is required to establish a new cellular microenvironment in oral squamous cell carcinoma and how autophagy drives the phenotypic change of oral squamous cell carcinoma cells by promoting crosstalk between carcinoma cells, fibroblasts, and immune cells.

The acetylase inhibitor, spermidine and the deacetylase activator, resveratrol, both induce autophagy and prolong life span of the model organism Caenorhabditis elegans in an autophagydependent fashion. Based on these premises, we investigated the differences and similarities in spermidine and resveratrol-induced autophagy. The deacetylase sirtuin 1 (SIRT1) and its orthologs are required for the autophagy induction by resveratrol but dispensable for autophagy stimulation by spermidine in human cells, Saccharomyces cerevisiae and C. elegans. SIRT1 is also dispensable for life-span extension by spermidine. Mass spectrometry analysis of the human acetylproteome revealed that resveratrol and/or spermidine induce changes in the acetylation of 560 peptides corresponding to 375 different proteins. Among these, 170 proteins are part of the recently elucidated human autophagy protein network. Importantly, spermidine and resveratrol frequently affect the acetylation pattern in a similar fashion. In the cytoplasm, spermidine and resveratrol induce convergent protein de-acetylation more frequently than convergent acetylation, while in the nucleus, acetylation is dominantly triggered by both agents. We surmise that subtle and concerted alterations in the acetylproteome regulate autophagy at multiple levels.

General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.

Organelles are membrane-lined structures that compartmentalize subcellular biochemical functions. Therefore, interorganelle communication is crucial for cellular responses that require the coordination of such functions. Multiple principles govern interorganelle interactions, which arise from the complex nature of organelles: position, multilingualism, continuity, heterogeneity, proximity, and bidirectionality, among others. Given their importance, alterations in organelle communication have been linked to many diseases. Among the different types of contacts, endoplasmic reticulum mitochondria interactions are the best known; however, mounting evidence indicates that other organelles also have something to say in the pathophysiological conversation.

We have recently shown that hyperosmotic stress activates p65/RelB NFκB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death. It remains unexplored how NFκB is regulated in cultured rat cardiomyocytes exposed to hyperosmotic stress. We study here: (a) if hyperosmotic stress triggers reactive oxygen species (ROS) generation and in turn whether they regulate NFκB and (b) if insulin‐like growth factor‐1 (IGF‐1) modulates ROS production and NFκB activation in hyperosmotically‐stressed cardiomyocytes. The results showed that hyperosmotic stress generated ROS in cultured cardiac myocytes, in particular the hydroxyl and superoxide species, which were inhibited by N ‐acetylcysteine (NAC). Hyperosmotic stress‐induced NFκB activation as determined by IκBα degradation and NFκB DNA binding. NFκB activation and procaspase‐3 and ‐9 fragmentation were prevented by NAC and IGF‐1. However, this growth factor did not decrease ROS generation induced by hyperosmotic stress, suggesting that its actions over NFκB and caspase activation may be due to modulation of events downstream of ROS generation. We conclude that hyperosmotic stress induces ROS, which in turn activates NFκB and caspases. IGF‐1 prevents NFκB activation by a ROS‐independent mechanism.

The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies.

Recurrence and resistance of Candida spp. infections is associated with the ability of these microorganisms to present several virulence patterns such as morphogenesis, adhesion, and biofilm formation. In the search for agents with antivirulence activity, essential oils could represent a strategy to act against biofilms and to potentiate antifungal drugs.To evaluate the antivirulence effect of Origanum vulgare L. essential oil (O-EO) against Candida spp. and to potentiate the effect of fluconazole and nystatin.The effect of O-EO was evaluated on ATCC reference strains of C. albicans and non-albicans Candida species. Minimum inhibitory concentration (MIC) was determined through broth microdilution assay. Adhesion to microplates was determined by crystal violet (CV) assay. An adapted scratch assay in 24-well was used to determine the effect of essential oil on biofilms proliferation. Viability of biofilms was evaluated by MTT reduction assay and through a checkerboard assay we determined if O-EO could act synergistically with fluconazole and nystatin.MIC for C. albicans ATCC-90029 and ATCC-10231 was 0.01 mg/L and 0.97 mg/L, respectively. For non-albicans Candida strains MIC values were 2.6 mg/L for C. dubliniensis ATCC-CD36 and 5.3 mg/L for C. krusei ATCC-6258. By using these concentrations, O-EO inhibited morphogenesis, adhesion, and proliferation at least by 50% for the strains assayed. In formed biofilms O-EO decreased viability in ATCC 90029 and ATCC 10231 strains (IC50 7.4 and 2.8 mg/L respectively). Finally, we show that O-EO interacted synergistically with fluconazole and nystatin.This study demonstrate that O-EO could be considered to improve the antifungal treatment against Candida spp.

We have previously shown that tetraploid cancer cells succumb through a p53-dependent apoptotic pathway when checkpoint kinase 1 (Chk1) is depleted by small interfering RNAs (siRNAs) or inhibited with 7-hydroxystaurosporine (UCN-01). Here, we demonstrate that Chk1 inhibition results in the activating phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Depletion of p38 MAPK by transfection with a siRNA targeting the alpha isoform of p38 MAPK (p38alpha MAPK) abolishes the phosphorylation of p53 on serines 15 and 46 that is induced by Chk1 knockdown. The siRNA-mediated downregulation and pharmacological inhibition of p38alpha MAPK (with SB 203580) also reduces cell death induced by Chk1 knockdown or UCN-01. These results underscore the role of p38 MAPK as a pro-apoptotic kinase in the p53-dependant pathway for the therapeutic elimination of polyploidy cells.

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of β-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.

Abstract Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

&lt;p&gt;Original Western Blot images&lt;/p&gt;

&lt;p&gt;Original Western Blot images&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis.&lt;/p&gt;Experimental Design:&lt;p&gt;We performed a model of tobacco-induced oral cancer &lt;i&gt;in vitro&lt;/i&gt;, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an &lt;i&gt;in vivo&lt;/i&gt; model of oral carcinogenesis and &lt;i&gt;ex vivo&lt;/i&gt; samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59.&lt;/p&gt;Results:&lt;p&gt;Both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;ex vivo&lt;/i&gt; oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 &lt;i&gt;in vitro&lt;/i&gt; resulted in diminished cell migration, viability, and invasion. Finally, by using an &lt;i&gt;in vivo&lt;/i&gt; model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions.&lt;/p&gt;Conclusions:&lt;p&gt;The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Characterization of the in vitro human oral carcinogenesis model&lt;/p&gt;

&lt;p&gt;Effect of C59 on cell invasion in CAL-27 cells&lt;/p&gt;

&lt;p&gt;Characterization of the in vitro human oral carcinogenesis model&lt;/p&gt;

&lt;p&gt;Effect of C59 on cell invasion in CAL-27 cells&lt;/p&gt;

&lt;p&gt;Clinical presentation of human oral lesions used as oral carcinogenesis ex vivo modeL&lt;/p&gt;

&lt;p&gt;Specificity of C59 over endogenous Wnt3a signaling in DOK-TC cells&lt;/p&gt;

&lt;p&gt;Clinical presentation of human oral lesions used as oral carcinogenesis ex vivo modeL&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis.&lt;/p&gt;Experimental Design:&lt;p&gt;We performed a model of tobacco-induced oral cancer &lt;i&gt;in vitro&lt;/i&gt;, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an &lt;i&gt;in vivo&lt;/i&gt; model of oral carcinogenesis and &lt;i&gt;ex vivo&lt;/i&gt; samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59.&lt;/p&gt;Results:&lt;p&gt;Both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;ex vivo&lt;/i&gt; oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 &lt;i&gt;in vitro&lt;/i&gt; resulted in diminished cell migration, viability, and invasion. Finally, by using an &lt;i&gt;in vivo&lt;/i&gt; model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions.&lt;/p&gt;Conclusions:&lt;p&gt;The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Specificity of C59 over endogenous Wnt3a signaling in DOK-TC cells&lt;/p&gt;

The primary cilium, hereafter cilium, is an antenna-like organelle that modulates intracellular responses, including autophagy, a lysosomal degradation process essential for cell homeostasis. Dysfunction of the cilium is associated with impairment of autophagy and diseases known as "ciliopathies". The discovery of autophagy-related proteins at the base of the cilium suggests its potential role in coordinating autophagy initiation in response to physiopathological stimuli. One of these proteins, beclin-1 (BECN1), it which is necessary for autophagosome biogenesis. Additionally, polycystin-2 (PKD2), a calcium channel enriched at the cilium, is required and sufficient to induce autophagy in renal and cancer cells. We previously demonstrated that PKD2 and BECN1 form a protein complex at the endoplasmic reticulum in non-ciliated cells, where it initiates autophagy, but whether this protein complex is present at the cilium remains unknown. Anorexigenic pro-opiomelanocortin (POMC) neurons are ciliated cells that require autophagy to maintain intracellular homeostasis. POMC neurons are sensitive to metabolic changes, modulating signaling pathways crucial for controlling food intake. Exposure to the saturated fatty acid palmitic acid (PA) reduces ciliogenesis and inhibits autophagy in these cells. Here, we show that PKD2 and BECN1 form a protein complex in N43/5 cells, an in vitro model of POMC neurons, and that both PKD2 and BECN1 locate at the cilium. In addition, our data show that the cilium is required for PKD2-BECN1 protein complex formation and that PA disrupts the PKD2-BECN1 complex, suppressing autophagy. Our findings provide new insights into the mechanisms by which the cilium controls autophagy in hypothalamic neuronal cells.

Comment on: Analysis of DRAM-related proteins reveals evolutionarily conserved and divergent roles in the control of autophagy.Jim O’Prey, Joanna Skommer, Simon Wilkinson and Kevin M. Ryan.

In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca2+ levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca2+ signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca2+ level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca2+-dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca2+/PKCα/ERK pathway without the participation of calcineurin. J. Cell. Biochem. 115: 712–720, 2014. © 2013 Wiley Periodicals, Inc.

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy.

NFkappaB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFkappaB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKalpha/beta as well as degradation of IkappaBalpha. All five members of the NFkappaB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFkappaB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFkappaB to the consensus DNA kappaB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFkappaB to DNA. Hyperosmotic stress also resulted in activation of the NFkappaB-lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFkappaB with AdIkappaBalpha, an IkappaBalpha dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFkappaB isoforms and NFkappaB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFkappaB-independent.

Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of β-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of β-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the β-catenin destruction complex allows nuclear accumulation of β-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of β-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3β, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3β, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the β-catenin destruction complex leads to stabilization and nuclear accumulation of β-catenin in oral dysplasia.

The brain is, after the adipose tissue, the organ with the greatest amount of lipids and diversity in their composition in the human body. In neurons, lipids are involved in signaling pathways controlling autophagy, a lysosome-dependent catabolic process essential for the maintenance of neuronal homeostasis and the function of the primary cilium, a cellular antenna that acts as a communication hub that transfers extracellular signals into intracellular responses required for neurogenesis and brain development. A crosstalk between primary cilia and autophagy has been established; however, its role in the control of neuronal activity and homeostasis is barely known. In this review, we briefly discuss the current knowledge regarding the role of autophagy and the primary cilium in neurons. Then we review the recent literature about specific lipid subclasses in the regulation of autophagy, in the control of primary cilium structure and its dependent cellular signaling in physiological and pathological conditions, specifically focusing on neurons, an area of research that could have major implications in neurodevelopment, energy homeostasis, and neurodegeneration.

Abstract Purpose: Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. Experimental Design: We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. Results: Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. Conclusions: The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

Polycystin-2 (PC2) is a calcium channel that can be found in the endoplasmic reticulum, the plasmatic membrane, and the primary cilium. The structure of PC2 is characterized by a highly ordered C-terminal tail with an EF-motif (calcium-binding domain) and a canonical coiled-coil domain (CCD; interaction domain), and its activity is regulated by interacting partners and post-translational modifications. Calcium mobilization into the cytosol by PC2 has been mainly associated with cell growth and differentiation, and therefore mutations or dysfunction of PC2 lead to renal and cardiac consequences. Interestingly, PC2-related pathologies are usually treated with rapamycin, an autophagy stimulator. Autophagy is an intracellular degradation process where recycling material is sequestered into autophagosomes and then hydrolyzed by fusion with a lysosome. Interestingly, several studies have provided evidence that PC2 may be required for autophagy, suggesting that PC2 maintains a physiologic catabolic state.

Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.

Autophagy is an intracellular degradation mechanism that allows recycling of organelles and macromolecules. Autophagic function increases metabolite availability modulating metabolic pathways, differentiation and cell survival. The oral environment is composed of several structures, including mineralized and soft tissues, which are formed by complex interactions between epithelial and mesenchymal cells. With aging, increased prevalence of oral diseases such as periodontitis, oral cancer and periapical lesions are observed in humans. These aging-related oral diseases are chronic conditions that alter the epithelial-mesenchymal homeostasis, disrupting the oral tissue architecture affecting the quality of life of the patients. Given that autophagy levels are reduced with age, the purpose of this review is to discuss the link between autophagy and age-related oral diseases.

Muscle atrophy involves a massive catabolism of intracellular components leading to a significant reduction in cellular and tissue volume. In this regard, autophagy, an intracellular mechanism that degrades proteins and organelles, has been implicated with muscle breakdown. Recently, it has shown that polycystin-2 (PC2), a membrane protein that belongs to the transient receptor potential (TRP) family, is required for the maintenance of cellular proteostasis, by regulating autophagy in several cell types. The role of PC2 in the control of atrophy and autophagy in skeletal muscle remains unknown. Here, we show that PC2 is required for the induction of atrophy in C2C12 myotubes caused by nutrient deprivation or rapamycin exposure. Consistently, overexpression of PC2 induces atrophy in C2C12 myotubes as indicated by decreasing of the myogenic proteins myogenin and caveolin-3. In addition, we show that inhibition of mTORC1, by starvation or rapamycin is inhibited in cells when PC2 is silenced. Importantly, even if PC2 regulates mTORC1, our results show that the regulation of atrophy by PC2 is independent of autophagy. This study provides novel evidence regarding the role of PC2 in skeletal muscle cell atrophy.

The intake of food with high levels of saturated fatty acids (SatFAs) is associated with the development of obesity and insulin resistance. SatFAs, such as palmitic (PA) and stearic (SA) acids, have been shown to accumulate in the hypothalamus, causing several pathological consequences. Autophagy is a lysosomal-degrading pathway that can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Previous studies showed that PA impairs macroautophagy function and insulin response in hypothalamic proopiomelanocortin (POMC) neurons. Here, we show in vitro that the exposure of POMC neurons to PA or SA also inhibits CMA, possibly by decreasing the total and lysosomal LAMP2A protein levels. Proteomics of lysosomes from PA- and SA-treated cells showed that the inhibition of CMA could impact vesicle formation and trafficking, mitochondrial components, and insulin response, among others. Finally, we show that CMA activity is important for regulating the insulin response in POMC hypothalamic neurons. These in vitro results demonstrate that CMA is inhibited by PA and SA in POMC-like neurons, giving an overview of the CMA-dependent cellular pathways that could be affected by such inhibition and opening a door for in vivo studies of CMA in the context of the hypothalamus and obesity.