Aimee L. Edinger

Apoptosis is the principal mechanism by which cells are physiologically eliminated in metazoan organisms. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response. Autophagy has also been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy in times of stress. Recent advances have helped to define the function of and mechanism for programmed necrosis and the role of autophagy in cell survival and suicide.

In multicellular organisms, constituent cells depend on extracellular signals for growth, proliferation, and survival. When cells are withdrawn from growth factors, they undergo apoptosis. Expression of constitutively active forms of the serine/threonine kinase Akt/PKB can prevent apoptosis upon growth factor withdrawal. Akt-mediated survival depends in part on the maintenance of glucose metabolism, suggesting that reduced glucose utilization contributes to growth factor withdrawal-induced death. However, it is unclear how restricting access to extracellular glucose alone would lead to the metabolic collapse observed after growth factor withdrawal. We report herein that growth factor withdrawal results in the loss of surface transporters for not only glucose but also amino acids, low-density lipoprotein, and iron. This coordinated decline in transporters and receptors for extracellular molecules creates a catabolic state characterized by atrophy and a decline in the mitochondrial membrane potential. Activated forms of Akt maintained these transporters on the cell surface in the absence of growth factor through an mTOR-dependent mechanism. The mTOR inhibitor rapamycin diminished Akt-mediated increases in cell size, mitochondrial membrane potential, and cell survival. These results suggest that growth factors control cellular growth and survival by regulating cellular access to extracellular nutrients in part by modulating the activity of Akt and mTOR.

Safaiyan et al. demonstrate that myelin fragments progressively pinch off from aged myelin sheaths and are taken up and cleared by microglia. Age-associated myelin breakdown is substantial and saturates the degradative capacities of microglia, leading to lysosomal storage and an immune activation in microglia with time. Myelin is synthesized as a multilamellar membrane, but the mechanisms of membrane turnover are unknown. We found that myelin pieces were gradually released from aging myelin sheaths and were subsequently cleared by microglia. Myelin fragmentation increased with age and led to the formation of insoluble, lipofuscin-like lysosomal inclusions in microglia. Thus, age-related myelin fragmentation is substantial, leading to lysosomal storage and contributing to microglial senescence and immune dysfunction in aging.

While cancer cell proliferation depends on access to extracellular nutrients, inadequate tumour perfusion means that glucose, amino acids and lipids are often in short supply. To overcome this obstacle to growth, cancer cells utilize multiple scavenging strategies, obtaining macromolecules from the microenvironment and breaking them down in the lysosome to produce substrates for ATP generation and anabolism. Recent studies have revealed four scavenging pathways that support cancer cell proliferation in low-nutrient environments: scavenging of extracellular matrix proteins via integrins, receptor-mediated albumin uptake and catabolism, macropinocytic consumption of multiple components of the tumour microenvironment and the engulfment and degradation of entire live cells via entosis. New evidence suggests that blocking these pathways alone or in combination could provide substantial benefits to patients with incurable solid tumours. Both US Food and Drug Administration (FDA)-approved drugs and several agents in preclinical or clinical development shut down individual or multiple scavenging pathways. These therapies may increase the extent and durability of tumour growth inhibition and/or prevent the development of resistance when used in combination with existing treatments. This Review summarizes the evidence suggesting that scavenging pathways drive tumour growth, highlights recent advances that define the oncogenic signal transduction pathways that regulate scavenging and considers the benefits and detriments of therapeutic strategies targeting scavenging that are currently under development.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.Significance: As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. Cancer Discov; 8(7); 866-83. ©2018 AACR.See related commentary by Commisso and Debnath, p. 800This article is highlighted in the In This Issue feature, p. 781.

Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis) offers additional anabolic benefits. A click chemistry-based flux assay reveals that necrocytosis provides not only amino acids, but sugars, fatty acids and nucleotides for biosynthesis, conferring resistance to therapies targeting anabolic pathways. Indeed, necrotic cell debris allow macropinocytic breast and prostate cancer cells to proliferate, despite fatty acid synthase inhibition. Standard therapies such as gemcitabine, 5-fluorouracil (5-FU), doxorubicin and gamma-irradiation directly or indirectly target nucleotide biosynthesis, creating stress that is relieved by scavenged nucleotides. Strikingly, necrotic debris also render macropinocytic, but not non-macropinocytic, pancreas and breast cancer cells resistant to these treatments. Selective, genetic inhibition of macropinocytosis confirms that necrocytosis both supports tumor growth and limits the effectiveness of 5-FU in vivo. Therefore, this study establishes necrocytosis as a mechanism for drug resistance.

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.

A comparison of Akt- and Bcl-xL-dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic geneBcl-xL. Although both genes prevent cell death, Akt-protected cells can be distinguished fromBcl-xL-protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-xL-expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-xL suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-xL maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells. A comparison of Akt- and Bcl-xL-dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic geneBcl-xL. Although both genes prevent cell death, Akt-protected cells can be distinguished fromBcl-xL-protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-xL-expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-xL suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-xL maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells. There is increasing evidence that tissue homeostasis in multicellular organisms is controlled by the availability of growth factors (1Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2486) Google Scholar). Within a given tissue, high levels of relevant growth factors promote increased cellular mass, metabolism, and proliferation. In contrast, when the availability of growth factor becomes limiting, cellular atrophy and an increased rate of apoptosis are observed. Many growth factors affect cellular responses through receptor-mediated recruitment and activation of the phosphoinositide 3-kinase (PI3K) 1The abbreviations used are:PI3Kphosphoinositide 3-kinasemAktmyristoylated AktTMREtetramethylrhodamine ethyl esterHAhemagglutininILinterleukinBrdUrdbromodeoxyuridine and the serine/threonine kinase Akt (2Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2212) Google Scholar, 3Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1284) Google Scholar). Once activated, Akt can phosphorylate substrates involved in controlling a variety of cellular processes, including cellular metabolism and survival (4Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (785) Google Scholar). The PI3K/Akt pathway is an important regulator of cellular homeostasis in vivo, as activating mutations of this pathway are correlated with multiple types of cancer. Akt itself was first identified as a viral oncogene, and deficiency in PTEN, an inositol phosphatase that opposes the activity of PI3K, is frequently found in numerous types of advanced cancers (5Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (780) Google Scholar, 6Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2487) Google Scholar, 7Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4201) Google Scholar). phosphoinositide 3-kinase myristoylated Akt tetramethylrhodamine ethyl ester hemagglutinin interleukin bromodeoxyuridine Akt activation by growth factor receptors prevents apoptosis by blocking the release of cytochrome c (8Kennedy S.G. Kandel E.S. Cross T.K. Hay N. Mol. Cell. Biol. 1999; 19: 5800-5810Crossref PubMed Scopus (584) Google Scholar). A number of molecular targets for the inhibition of apoptosis by Akt have been proposed. Akt phosphorylates and inactivates pro-apoptotic proteins, including Bad and Forkhead family transcription factors (9Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4895) Google Scholar, 10Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (252) Google Scholar). In addition, Akt has been reported to stimulate the expression of anti-apoptotic Bcl-2 proteins, such as Bcl-xL and Mcl-1, through the activation of NF-κB (11Jones R.G. Parsons M. Bonnard M. Chan V.S. Yeh W.C. Woodgett J.R. Ohashi P.S. J. Exp. Med. 2000; 191: 1721-1734Crossref PubMed Scopus (285) Google Scholar, 12Zong W.X. Edelstein L.C. Chen C. Bash J. Gelinas C. Genes Dev. 1999; 13: 382-387Crossref PubMed Scopus (638) Google Scholar). However, it is not clear if these targets entirely account for the effects of Akt on cell survival. Members of the Bcl-2 family are attractive Akt targets, as they have been shown to be potent regulators of apoptosis following growth factor withdrawal. Transgenic overexpression of anti-apoptotic family members prevents the induction of programmed cell death and leads to an accumulation of cells, whereas transgenic overexpression of pro-apoptotic family members can result in decreased cell numbers within an organ (13Chao D.T. Linette G.P. Boise L.H. White L.S. Thompson C.B. Korsmeyer S.J. J. Exp. Med. 1995; 182: 821-828Crossref PubMed Scopus (379) Google Scholar, 14Grillot D.A. Merino R. Pena J.C. Fanslow W.C. Finkelman F.D. Thompson C.B. Nunez G. J. Exp. Med. 1996; 183: 381-391Crossref PubMed Scopus (167) Google Scholar). Anti-apoptotic Bcl-2 family proteins, such as Bcl-xL, are localized to the outer mitochondrial membrane and function to maintain mitochondrial homeostasis upon growth factor withdrawal. Bcl-xL and Bcl-2 have been reported to promote mitochondrial homeostasis by promoting continued transport of metabolites across the outer membrane, despite decreases in cellular metabolism (15Vander Heiden M.G. Chandel N.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 16Vander Heiden M.G. Chandel N.S. Li X.X. Schumacker P.T. Colombini M. Thompson C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4666-4671Crossref PubMed Scopus (373) Google Scholar). In the absence of growth factor, Bcl-xL facilitates cell survival by preserving cellular ATP production following the decrease in glycolysis that accompanies growth factor withdrawal. Since both Akt and Bcl-xL can regulate cell survival and cellular metabolism, we have compared the bioenergetic properties of growth factor-deprived FL5.12 cells expressing either Akt or Bcl-xL. FL5.12 cells are nontransformed pro-B cells that depend on IL-3 for survival and proliferation. The results demonstrate that cells expressing Akt or Bcl-xL maintain distinct metabolic states in the absence of growth factor. Akt expression sustains sufficient glucose uptake and glycolysis to maintain a physiologic mitochondrial membrane potential in growth factor withdrawn cells. In contrast, Bcl-xL promotes the survival of growth factor-deprived cells by maintaining mitochondrial integrity and function, despite a decrease in mitochondrial potential that results from a decline in glucose-derived substrates. These differences in cellular metabolism suggest that there are fundamental differences in the mechanisms by which Akt and Bcl-xL promote cell survival. FL5.12 cells were cultured as described previously (17Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). Myristoylated Akt (mAkt) gene expression was induced for 18 h with 1 μg/ml of doxycycline (Sigma) treatment. Cell volume measurements were made using a Coulter Z2 instrument (Beckman Coulter). Where indicated, wortmannin (Calbiochem) and LY294002 (Sigma) were added to cultures at final concentrations of 100 nm and 10 μm, respectively. Glucose- and glutamine-free medium was made using the RPMI 1640 Select Amine Kit (Life Technologies, Inc.) and 10% dialyzed fetal bovine serum (Life Technologies, Inc.). Glucose and glutamine were supplemented to final concentrations as indicated. pUHD172–1Neo expressing rtTA was generously provided by J. Leiden, Abbott. Akt constructs were generously provided by N. Hay, University of Illinois, Chicago. mAkt was hemagglutinin (HA)-tagged at the C terminus, and both mAkt-HA and K179M Akt were cloned into pRevTRE (pRT, CLONTECH). Bcl-xL was ligated into pBabeMN-IRESGFP (Bcl-xL1, generously provided by G. Nolan, Stanford University) or pRT (Bcl-xL2). Retroviral expression vectors were virally transduced using the Phoenix packaging cell line (generously provided by G. Nolan) as described previously (18Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2275) Google Scholar). Briefly, target cells were combined with the retrovirus and 4 μg/ml hexadimethrine bromide (Sigma), spun at 2500 rpm for 1.5 h, and selected with 3 mg/ml hygromycin (CLONTECH). Cell populations were cloned by limiting dilution. Three independently isolated clones expressing the epitope-tagged mAkt in an inducible manner (mAkt1, mAkt2, and mAkt3) were obtained and subjected to further study. Lysates from cell lines were standardized for protein content and separated by SDS-polyacrylamide gel electrophoresis (Invitrogen). Blots were probed with either rabbit anti-Bcl-xL (13.6) (19Chang B.S. Kelekar A. Harris M.H. Harlan J.E. Fesik S.W. Thompson C.B. Mol. Cell. Biol. 1999; 19: 6673-6681Crossref PubMed Google Scholar), rabbit anti-Akt (New England Biolabs), or mouse anti-tubulin (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). Total RNA was prepared using TRIZOL (Life Technologies, Inc.) from cells cultured with or without IL-3 for 14 h. 10 μg of RNA were separated on a 1% formaldehyde agarose gel and probed with rat Glut1 cDNA (generously provided by M. Birnbaum, University of Pennsylvania). Loading was assessed by visualizing gels stained with ethidium bromide. Cell viability assays were performed using propidium iodide as described (17Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). For mitochondrial potential determination, live cells were enriched by centrifugation over Ficoll, rested for 1 h, and incubated for 30 min at 37 °C with 200 nm tetramethylrhodamine ethyl ester (Molecular Probes). Analysis was performed in a FacsCalibur flow cytometer (Becton Dickinson). After 6 days without IL-3, 1 × 106 Ficoll-enriched live cells were resuspended in pre-warmed CO2-buffered Krebs solution (115 mmNaCl, 2 mm KCl, 25 mm NaHCO3, 1 mm MgCl2, 2 mm CaCl2, 0.25% bovine serum albumin, pH 7.4) lacking glucose for 30 min at 37 °C. Cells were then washed and resuspended in 500 μl of Krebs buffer containing 10 mm glucose supplemented with 20 μCi/ml [5-3H]glucose (PerkinElmer Life Sciences) for 1 h at 37 °C, and the reaction was stopped by adding equal volume 0.2 n HCl. 3H2O was separated from [3H]glucose by evaporative diffusion of3H2O in a closed chamber, as described (20Ashcroft S.J. Weerasinghe L.C. Bassett J.M. Randle P.J. Biochem. J. 1972; 126: 525-532Crossref PubMed Scopus (246) Google Scholar). Total ATP levels were determined from 1 × 106 live cells, enriched by Ficoll, using the ATP bioluminescence assay kit HS II (Roche Molecular Biochemicals) (15Vander Heiden M.G. Chandel N.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Akt is involved in transmitting intracellular signals from growth factor receptors (21Conlon I. Raff M. Cell. 1999; 96: 235-244Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). We sought to determine whether Akt is involved in IL-3-dependent signal transduction in FL5.12 cells. FL5.12 cells were deprived of growth factor for 12 h, and recombinant IL-3 was reconstituted for the indicated time intervals. Addition of IL-3 resulted in rapid and transient phosphorylation of Akt, which was maximal 15 min after IL-3 addition (Fig.1A). Activation of Akt was dependent on PI3K, since the PI3K inhibitors wortmannin or LY294002 prevented the induction of Akt phosphorylation (Fig. 1A and data not shown). Thus, Akt is a component of the proximal signal transduction machinery of the IL-3 receptor. When FL5.12 cells are withdrawn from growth factor, they undergo progressive atrophy until they initiate programmed cell death. Bcl-xL protects from cell death in response to growth factor withdrawal, but it does not prevent cellular atrophy (22Rathmell J.C. Vander Heiden M.G. Harris M.H. Frauwirth K.A. Thompson C.B. Mol. Cell. 2000; 6: 683-692Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Since the PI3K/Akt pathway is an important regulator of cell size inDrosophila (23Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (304) Google Scholar), we investigated whether the PI3K/Akt pathway is required for cell growth following IL-3 readdition. Reconstitution of IL-3 in cultures that had been withdrawn from growth factor resulted in rapid recovery of cell size (Fig. 1B). Addition of the PI3K inhibitor LY294002 simultaneously with IL-3 resulted in a delay in the recovery of cell size, indicating that the PI3K pathway regulated cell growth in response to stimulation with IL-3. These data indicate that the PI3K/Akt pathway is important in transmitting cell growth signals in mammalian cells, as has been observed in Drosophila cells (23Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (304) Google Scholar, 24Weinkove D. Neufeld T.P. Twardzik T. Waterfield M.D. Leevers S.J. Curr. Biol. 1999; 9: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In addition to promoting cell growth, the PI3K/Akt pathway also transmits cell survival signals from growth factor receptors (2Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2212) Google Scholar, 25Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (962) Google Scholar). To assess the role of Akt in promoting cell survival in FL5.12 cells, we established cell lines expressing constitutively active, mAkt (26Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsichlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (977) Google Scholar). Addition of the Src myristoylation sequence to the N terminus of Akt targets it to the plasma membrane, conferring constitutive activity to the kinase (27Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). FL5.12 cells were infected with a retrovirus encoding an epitope-tagged mAkt under the control of a tetracycline response element. Three independent clones were isolated which expressed doxycycline-induced mAkt at a level comparable to the level of total Akt expressed in parental FL5.12 cells (Fig.2). In the absence of induction, all three clones expressed low levels of mAkt. Withdrawal of IL-3 from wild-type FL5.12 cells resulted in loss of viability within 48 h, as determined by the ability of cells to exclude propidium iodide (Fig.2, A and B). All three mAkt clones maintained viability over 6 days, whereas cells expressing kinase-deficient Akt died with kinetics similar to wild-type cells (Fig. 2A). As has been shown previously, cells expressing high and low levels of Bcl-xL (Bcl-xL1 and 2, respectively) maintained viability in the absence of growth factor in a dose-dependent manner (28Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2908) Google Scholar). After 6 days of growth factor withdrawal, the remaining viable cells could be recovered quantitatively from mAkt- and Bcl-xL-expressing populations as assayed by IL-3 readdition and cloning by limiting dilution (data not shown). Thus, increased viability over 6 days represented true cell survival over the entire time course. Constitutively active Akt promotes cell survival in a dose-dependent manner, as doxycycline addition increased mAkt expression and cell survival concomitantly (Fig. 2, B and C). Akt signal transduction has been implicated in the control of cell cycle progression by stimulating increased translation of cyclin D (29Muise-Helmericks R.C. Grimes H.L. Bellacosa A. Malstrom S.E. Tsichlis P.N. Rosen N. J. Biol. Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). To determine whether Akt-dependent survival following growth factor withdrawal involved continued cell proliferation, we assessed the rates of DNA synthesis in cells growing in IL-3 and in cells that had been withdrawn from IL-3. Incorporation of the nucleotide analog bromodeoxyuridine (BrdUrd) was similar for all clones growing in IL-3 (Fig. 3). Following growth factor withdrawal, cells expressing either mAkt or Bcl-xL lacked significant levels of DNA synthesis, while maintaining significant viability (Fig. 3). In contrast, vector control cells also lack the ability to incorporate BrdUrd, but this reflects the fact that all these cells have died as indicated by their sub-diploid DNA content and inability to exclude propidium iodide (Fig.2 and data not shown). These data suggest that Akt-dependent survival in FL5.12 cells does not require cell cycle progression. FL5.12 cells undergo progressive atrophy when deprived of growth factor (22Rathmell J.C. Vander Heiden M.G. Harris M.H. Frauwirth K.A. Thompson C.B. Mol. Cell. 2000; 6: 683-692Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Since Akt transmits cell growth signals from the IL-3 receptor, expression of constitutively active Akt could prevent cellular atrophy upon growth factor withdrawal. Cell size was assessed in cells that had been withdrawn from IL-3 by measuring forward scatter of equal numbers of live cells in a flow cytometer. Cells expressing mAkt maintained a larger cell size in the absence of growth factor, compared with cells expressing Bcl-xL (Fig. 4). Cell size correlated with mAkt expression, as cells induced with doxycycline were larger than uninduced cells. Although cells expressing mAkt remained larger than Bcl-xL cells, they still atrophied after growth factor withdrawal (data not shown). This indicates that mAkt can transmit a cell growth signal in the absence of IL-3, but it does not completely substitute for the cell growth and proliferative signals transmitted from the IL-3 receptor. Thus, activated Akt both promotes cell survival and prevents cellular atrophy in the absence of growth factor. Cellular atrophy is correlated with decreases in cellular metabolism and macromolecular synthesis (30Franklin J.L. Johnson E.M. J. Cell Biol. 1998; 142: 1313-1324Crossref PubMed Scopus (43) Google Scholar, 31Deshmukh M. Vasilakos J. Deckwerth T.L. Lampe P.A. Shivers B.D. Johnson Jr., E.M. J. Cell Biol. 1996; 135: 1341-1354Crossref PubMed Scopus (205) Google Scholar, 32Deckwerth T.L. Johnson Jr., E.M. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (513) Google Scholar). Since Akt signaling attenuates cellular atrophy, the metabolic status of mAkt and Bcl-xLcells was compared in the absence of growth factor. Total cellular ATP levels were assessed in cells that had been withdrawn from IL-3. Cells expressing mAkt contained more ATP than cells expressing Bcl-xL (Fig. 5), suggesting that cells expressing mAkt remained metabolically more active than cells expressing Bcl-xL. Furthermore, the prevention of cellular atrophy by Akt is correlated with increased metabolism in FL5.12 cells. In insulin receptor signal transduction, Akt regulates several aspects of glucose uptake, metabolism, and storage (33Deprez J. Vertommen D. Alessi D.R. Hue L. Rider M.H. J. Biol. Chem. 1997; 272: 17269-17275Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 34Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar, 35Barthel A. Okino S.T. Liao J. Nakatani K. Li J. Whitlock Jr., J.P. Roth R.A. J. Biol. Chem. 1999; 274: 20281-20286Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The elevated amounts of ATP in mAkt cells may be due in part to the ability of mAkt to maintain glucose metabolism in the absence of growth factor. Akt can increase glucose metabolism in response to insulin by inducing the expression of the glucose transporters in insulin-responsive cell types (35Barthel A. Okino S.T. Liao J. Nakatani K. Li J. Whitlock Jr., J.P. Roth R.A. J. Biol. Chem. 1999; 274: 20281-20286Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). In contrast to insulin-responsive tissues, which primarily express Glut4, the major glucose transporter in lymphocytes is Glut1. 2J. C. Rathmell, unpublished observations. Analysis of Glut1 mRNA in cells growing in the presence of IL-3 revealed no difference in mRNA levels between vector control cells and cells expressing mAkt or Bcl-xL (Fig.6A). Following IL-3 withdrawal, Glut1 mRNA was undetectable in vector control and Bcl-xL cells but was still detectable in cells expressing mAkt (Fig. 6A). In addition to promoting increased Glut1 expression, mAkt sustained an overall increase in glycolysis in the absence of IL-3 (Fig. 6B). Following growth factor withdrawal, cells expressing mAkt mediated significantly increased glycolytic rates compared with cells expressing Bcl-xL(Fig. 6B). However, constitutively active Akt did not completely substitute for growth factor receptor signaling, since the rates of glycolysis in Akt-protected cells were lower than in cells growing in IL-3. Mitochondrial potential is indicative of mitochondrial activity, and perturbations in inner membrane potential following growth factor withdrawal have been associated with the commitment to cell death (15Vander Heiden M.G. Chandel N.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Therefore, mitochondrial membrane potential was assessed using the potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Following IL-3 withdrawal, mitochondrial membrane potential was significantly reduced in cells expressing Bcl-xL (Fig.7A). In contrast, there was no reduction in the mitochondrial potential of cells expressing mAkt following IL-3 withdrawal. TMRE fluorescence was reflective of mitochondrial potentials in these cells, since addition of agents that collapse the mitochondrial potential resulted in a decrease in TMRE staining (Fig. 7B). Thus, the difference in staining with TMRE alone was due to the difference in mitochondrial potential in cells expressing mAkt or Bcl-xL. These data indicate that cells expressing mAkt contain sufficient electron transport substrates to maintain mitochondrial potential, despite the absence of growth factors. To survive growth factor withdrawal, cells must maintain cellular metabolism at a level sufficient to sustain viability. The data indicate that Akt-expressing cells are metabolically more active than Bcl-xL-expressing cells in the absence of growth factor, as determined by total cellular ATP, glycolytic rates, and mitochondrial potentials. One difference between Akt and Bcl-xL is the ability of Akt to regulate glucose metabolism. Glucose and glutamine are the major carbon sources for lymphocytes in culture medium (36Brand K. Leibold W. Luppa P. Schoerner C. Schulz A. Immunobiology. 1986; 173: 23-34Crossref PubMed Scopus (63) Google Scholar,37Brand K. Fekl W. von Hintzenstern J. Langer K. Luppa P. Schoerner C. Metabolism. 1989; 38: 29-33Abstract Full Text PDF PubMed Scopus (72) Google Scholar), and limiting concentrations of these nutrients resulted in decreased cellular glycolysis (data not shown). To determine whether the increased rate of glycolysis was necessary for Akt-dependent survival, we tested cell survival under conditions of nutrient limitation. As the concentrations of glucose and glutamine were decreased in the culture medium, a progressive impairment in the ability of mAkt to promote growth factor-independent survival was evident (Fig. 8). In contrast, Bcl-xL cells were unaffected by decreasing concentrations of carbon sources. Cells expressing mAkt also decreased in cell size prior to undergoing apoptosis as nutrients became more limiting (data not shown). These data suggest that Bcl-xLpromotes cell survival by regulating a cell intrinsic effect on bioenergetics, rather than by regulating glucose uptake or utilization. In contrast, Akt is reliant on the availability of exogenous nutrients to promote cell survival. The data indicate that Akt can function to promote both cell growth and cell survival downstream of growth factor receptors. Akt is rapidly phosphorylated following stimulation with IL-3, identifying the kinase as a component of the proximal IL-3 receptor signaling pathway. Inhibition of the PI3K/Akt pathway prevents the rapid induction of cell growth upon re-stimulation of FL5.12 cells with IL-3. Constitutively active Akt both promotes cell survival and attenuates cellular atrophy in the absence of growth factor. Thus, Akt regulates both cell survival and cell size in mammalian cells. Increases in Akt activity have been shown to result in increases in cyclin D expression, which results in cell cycle progression (29Muise-Helmericks R.C. Grimes H.L. Bellacosa A. Malstrom S.E. Tsichlis P.N. Rosen N. J. Biol. Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). However, constitutively active Akt does not support IL-3-independent proliferation in FL5.12 cells. Cells expressing Akt are slower to exit the cell cycle than cells expressing Bcl-xL following growth factor withdrawal (data not shown). Nonetheless, cells expressing constitutively active Akt do exit from the cell cycle while maintaining cellular viability. This indicates that mAkt is not sufficient to transform FL5.12 cells and that cellular proliferation is not necessary for Akt-dependent cell survival. The dual role of Akt in controlling both cell growth and cell survival has been suggested by genetic studies of Akt inDrosophila. Ablation of Akt in embryos results in increases in apoptosis throughout the embryo (38Staveley B.E. Ruel L. Jin J. Stambolic V. Mastronardi F.G. Heitzler P. Woodgett J.R. Manoukian A.S. Curr. Biol. 1998; 8: 599-602Abstract Full Text Full Text PDF PubMed Google Scholar). Overexpression of Akt in eye and wing imaginal discs results in increases in cell size, without increases in cell number (23Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (304) Google Scholar). Importantly, overexpression of Akt does not increase cellular proliferation and does not overcome cell cycle arrest in the zone of nonproliferating cells in the wing imaginal disc (23Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (304) Google Scholar). This indicates that Akt can promote increases in cell size without altering cell proliferation in Drosophila, in agreement with the findings in FL5.12 cells. How can Akt simultaneously control both cell growth and cell survival? The answer may be linked to the control of cellular metabolism. Cellular growth requires the uptake and utilization of energy-rich metabolites. Similarly, to survive growth factor withdrawal, cells must establish a mechanism to sustain their metabolism. It is possible that Akt influences both cell growth and cell survival by sustaining increased cellular bioenergetics. In the absence of growth factor, constitutively active Akt promotes increases in cellular ATP levels, glycolytic rates, and mitochondrial potential, indicating that Akt mediates a global increase in cellular metabolism. This global increase may attenuate the alterations in cellular metabolism that are associated with growth factor withdrawal-induced programmed cell death. Akt can control cellular metabolism on a number of levels. Here we report that IL-3 signal transduction is required to maintain Glut1 expression in FL5.12 cells. However, Akt activation is sufficient to induce Glut1 expression in these cells, even in the absence of IL-3. In addition to stimulating glucose uptake, Akt also controls glucose utilization within cells. Constitutively active Akt is sufficient to increase the overall rate of glycolysis in cells surviving growth factor withdrawal. Akt may increase glucose utilization by phosphorylating GSK-3β or PFK-2 (33Deprez J. Vertommen D. Alessi D.R. Hue L. Rider M.H. J. Biol. Chem. 1997; 272: 17269-17275Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 34Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar). The finding that GSK-3β overexpression results in apoptosis lends support to the possibility that Akt control of glucose metabolism contributes to its ability to promote cell survival (39Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar). The ability of Akt to maintain the glycolytic rate of a cell is sufficient to explain how Akt overexpression maintains the mitochondrial membrane potential. A higher glycolytic rate will result in greater substrate availability for mitochondrial electron transport. Consistent with this hypothesis, the ability of Akt to maintain the mitochondrial potential was found to be dependent on glucose. In contrast, Bcl-2 family proteins have been reported to maintain mitochondrial integrity following growth factor withdrawal by facilitating mitochondrial exchange of metabolites and ATP/ADP (15Vander Heiden M.G. Chandel N.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). This allows mitochondria to sustain coupled respiration in the face of a fall in membrane potential that occurs as a result of the decrease in glycolytic substrates. Biochemical analysis of cells surviving growth factor withdrawal in an Akt-dependent manner indicates that these cells are metabolically more active than their Bcl-xL counterparts. To sustain high levels of metabolism and prevent atrophy, cells expressing Akt are reliant on external sources of energy. Akt cannot promote cell survival in the context of insufficient external energy sources. In contrast, cells that express Bcl-xL can maintain viability even in the context of limited external nutrient sources. Since Akt is a component of growth factor receptor signal transduction, constitutively active Akt may promote survival by transmitting partial cell growth signals that sustain cellular metabolism, whereas Bcl-xL promotes survival by allowing cells to adapt to a reduced metabolic state. The data suggest that Akt does not promote cell survival solely by inactivating pro-apoptotic factors. If Akt functioned exclusively as an anti-apoptotic protein, cells surviving growth factor withdrawal would adopt a low energy phenotype, as do cells expressing Bcl-xL. The difference in the metabolic requirements of cells surviving due to Akt or Bcl-xLindicates cellular contexts in which each gene might be functional. Bcl-xL can mediate survival under limiting concentrations of nutrients. Thus, newly transformed cells that have not yet established an effective blood supply would be better served by expressing Bcl-xL than by expressing Akt. Vascularized tumors, in contrast, can survive the absence of specific growth factors by expressing Akt. These cells would maintain greater levels of metabolism, which might contribute to their oncogenic potential. The requirement for a rich source of nutrients may partially explain why mutations in PTEN, which result in elevations in Akt activity, are correlated with late stage, aggressive tumors (6Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2487) Google Scholar, 7Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4201) Google Scholar). In contrast, overexpression of Bcl-2 proteins correlates with low grade tumors with low mitotic indices (40Zhang G.J. Kimijima I. Abe R. Watanabe T. Kanno M. Hara K. Tsuchiya A. Anticancer Res. 1998; 18: 1989-1998PubMed Google Scholar). We thank N. Hay (University of Illinois, Chicago) for Akt constructs and F. Matschinsky (University of Pennsylvania) for help with the glycolysis assay. In addition, we thank members of the Thompson laboratory for technical help and reviewing the manuscript, especially Matthew Vander Heiden.

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo . Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.

Leukocyte chemoattractants act through a rapidly growing subfamily of G protein-coupled receptors. We report the cloning of a novel human gene encoding an orphan receptor (ChemR23) related to the C3a, C5a and formyl Met-Leu-Phe receptors, and more distantly to the subfamilies of chemokine receptors. ChemR23 transcripts were found to be abundant in monocyte-derived dendritic cells and macrophages, treated or not with LPS. Low expression could also be detected by reverse transcription-PCR in CD4+ T lymphocytes. The gene encoding ChemR23 was assigned by radiation hybrid mapping to the q21.2-21.3 region of human chromosome 12, outside the gene clusters identified so far for chemoattractant receptors. Given the increasing number of chemoattractant receptors used by HIV-1, HIV-2 and SIV as coreceptors, ChemR23 was tested in fusion assays for potential coreceptor activity by a range of viral strains. None of the tested HIV-2 strains made use of ChemR23 as a coreceptor, but several SIV strains (SIVmac316, SIVmac239, SIVmacl7E-Fr and SIVsm62A), as well as a primary HIV-1 strain (92UG024-2) used it efficiently. ChemR23 therefore appears as a coreceptor for immunodeficiency viruses that does not belong to the chemokine receptor family. It is also a putative chemoattractant receptor relatively specific for antigen-presenting cells, and it could play an important role in the recruitment or trafficking of these cell populations. Future work will be required to identify the ligand(s) of this new G protein-coupled receptor and to define its precise role in the physiology of dendritic cells and macrophages.

Certain chemokine receptors serve as cofactors for HIV type 1 envelope (env)-mediated cell-cell fusion and virus infection of CD4-positive cells. Macrophage tropic (M-tropic) HIV-1 isolates use CCR5, and T cell tropic (T-tropic) strains use CXCR4. To investigate the cofactors used by simian immunodeficiency viruses (SIV), we tested four T-tropic and two M-tropic SIV env proteins for their ability to mediate cell-cell fusion with cells expressing CD4 and either human or nonhuman primate chemokine receptors. Unlike HIV-1, both M- and T-tropic SIV envs used CCR5 but not CXCR4 or the other chemokine receptors tested. However, by testing a panel of CCR5/CCR2b chimeras, we found that the structural requirements for CCR5 utilization by M-tropic and T-tropic SIV strains were different. T-tropic SIV strains required the second extracellular loop of CCR5 whereas a closely related M-tropic SIV strain could, like M-tropic HIV-1 strains, use the amino-terminal domain of CCR5. As few as two amino acid changes in the SIV env V3 domain affected the regions of CCR5 that were critical for fusogenic activity. Receptor signaling was not required for either fusion or infection. Our results suggest that viral tropism may be influenced not only by the coreceptors used by a given virus strain but also by how a given coreceptor is used.

Cellular proteins are degraded within two distinct compartments: the proteasome and the lysosome. Alterations in proteasomal degradation can contribute to carcinogenesis. In contrast, alterations in autophagic protein degradation through the lysosome have not been linked to cancer. Now two reports demonstrate that the autophagic gene, Beclin 1, is a haploinsufficient tumor suppressor gene. These new data suggest that autophagic degradation provides an important mechanism to prevent cellular transformation.

ABSTRACT Both CD4 and an appropriate coreceptor are necessary for infection of cells by human immunodeficiency virus type 1 (HIV-1) and most strains of HIV-2. The chemokine receptors CCR5 and CXCR4 are the major HIV-1 coreceptors, although some virus strains can also utilize alternative coreceptors such as CCR3 to infect cells. In contrast, most if not all simian immunodeficiency virus (SIV) strains use CCR5 as a coreceptor, and many SIV strains can use CCR5 independently of CD4. In addition, several orphan seven-transmembrane receptors which can serve as HIV-1 and SIV coreceptors have been identified. Here we report that APJ, an orphan seven-transmembrane domain receptor with homology to the angiotensin receptor family, functions as a coreceptor for a number of HIV-1 and SIV strains. APJ was expressed widely in the human brain and in NT2N neurons. APJ transcripts were also detected by reverse transcription-PCR in the CD4-positive T-cell line C8166, but not in peripheral blood leukocytes, microglia, phytohemagglutinin (PHA)- or PHA/interleukin-2-stimulated peripheral blood mononuclear cells, monocytes, or monocyte-derived macrophages. The widespread distribution of APJ in the central nervous system coupled with its use as a coreceptor by some HIV-1 strains indicates that it may play a role in neuropathogenesis.

Ceramide induces cell death in response to many stimuli. Its mechanism of action, however, is not completely understood. Ceramide induces autophagy in mammalian cells maintained in rich media and nutrient permease downregulation in yeast. These observations suggested to us that ceramide might kill mammalian cells by limiting cellular access to extracellular nutrients. Consistent with this proposal, physiologically relevant concentrations of ceramide produced a profound and specific downregulation of nutrient transporter proteins in mammalian cells. Blocking ceramide-induced nutrient transporter loss or supplementation with the cell-permeable nutrient, methyl pyruvate, reversed ceramide-dependent toxicity. Conversely, cells became more sensitive to ceramide when nutrient stress was increased by acutely limiting extracellular nutrients, inhibiting autophagy, or deleting AMP-activated protein kinase (AMPK). Observations that ceramide can trigger either apoptosis or caspase-independent cell death may be explained by this model. We found that methyl pyruvate (MP) also protected cells from ceramide-induced, nonapoptotic death consistent with the idea that severe bioenergetic stress was responsible. Taken together, these studies suggest that the cellular metabolic state is an important arbiter of the cellular response to ceramide. In fact, increasing nutrient demand by incubating cells in high levels of growth factor sensitized cells to ceramide. On the other hand, gradually adapting cells to tolerate low levels of extracellular nutrients completely blocked ceramide-induced death. In sum, these results support a model where ceramide kills cells by inducing intracellular nutrient limitation subsequent to nutrient transporter downregulation.

The small GTPase Rab7 promotes fusion events between late endosomes and lysosomes. Rab7 activity is regulated by extrinsic signals, most likely via effects on its guanine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP). Based on their homology to the yeast proteins that regulate the Ypt7 GTP binding state, TBC1D15, and mammalian Vps39 (mVps39) have been suggested to function as the Rab7 GAP and GEF, respectively. We developed an effector pull-down assay to test this model. TBC1D15 functioned as a Rab7 GAP in cells, reducing Rab7 binding to its effector protein RILP, fragmenting the lysosome, and conferring resistance to growth factor withdrawal-induced cell death. In a cellular context, TBC1D15 GAP activity was selective for Rab7. TBC1D15 overexpression did not inhibit transferrin internalization or recycling, Rab7-independent processes that require Rab4, Rab5, and Rab11 activation. TBC1D15 was thus renamed Rab7-GAP. Contrary to expectations for a Rab7 GEF, mVps39 induced lysosomal clustering without increasing Rab7 GTP binding. Moreover, a dominant-negative mVps39 mutant fragmented the lysosome and promoted growth factor independence without decreasing Rab7-GTP levels. These findings suggest that a protein other than mVps39 serves as the Rab7 GEF. In summary, although only TBC1D15/Rab7-GAP altered Rab7-GTP levels, both Rab7-GAP and mVps39 regulate lysosomal morphology and play a role in maintaining growth factor dependence.

The BCL-2 family members BAK and BAX are required for apoptosis and trigger mitochondrial outer membrane permeabilization (MOMP). Here we identify a MOMP-independent function of BAK as a required factor for long-chain ceramide production in response to pro-apoptotic stress. UV-C irradiation of wild-type (WT) cells increased long-chain ceramides; blocking ceramide generation prevented caspase activation and cell death, demonstrating that long-chain ceramides play a key role in UV-C-induced apoptosis. In contrast, UV-C irradiation did not increase long-chain ceramides in BAK and BAX double knock-out cells. Notably, this was not specific to the cell type (baby mouse kidney cells, hematopoietic) nor the apoptotic stimulus employed (UV-C, cisplatin, and growth factor withdrawal). Importantly, long-chain ceramide generation was dependent on the presence of BAK, but not BAX. However, ceramide generation was independent of the known downstream actions of BAK in apoptosis (MOMP or caspase activation), suggesting a novel role for BAK in apoptosis. Finally, enzymatic assays identified ceramide synthase as the mechanism by which BAK regulates ceramide metabolism. There was no change in CerS expression at the message or protein level, indicating regulation at the post-translational level. Moreover, CerS activity in BAK KO microsomes can be reactivated upon addition of BAK-containing microsomes. The data presented indicate that ceramide-induced apoptosis is dependent upon BAK and identify a novel role for BAK during apoptosis. By establishing a unique role for BAK in long-chain ceramide metabolism, these studies further demonstrate that the seemingly redundant proteins BAK and BAX have distinct mechanisms of action during apoptosis induction.

Highly proliferative cells, including cancer cells, require a constant supply of molecular building blocks to support their growth. To acquire substrates such as glucose and amino acids from the extracellular space, dividing cells rely on transporter proteins in the plasma membrane. Numerous studies link transcriptional and post-translational control of nutrient transporter expression with proliferation, highlighting the importance of nutrient transporters in both physiologic and pathologic growth. Here we review recent work that spotlights the crucial role of nutrient transporters in cell growth and proliferation, discuss post-translational mechanisms for coordinating expression of different transporters, and consider the therapeutic potential of targeting these proteins in cancer and other diseases characterized by inappropriate cell division. Highly proliferative cells, including cancer cells, require a constant supply of molecular building blocks to support their growth. To acquire substrates such as glucose and amino acids from the extracellular space, dividing cells rely on transporter proteins in the plasma membrane. Numerous studies link transcriptional and post-translational control of nutrient transporter expression with proliferation, highlighting the importance of nutrient transporters in both physiologic and pathologic growth. Here we review recent work that spotlights the crucial role of nutrient transporters in cell growth and proliferation, discuss post-translational mechanisms for coordinating expression of different transporters, and consider the therapeutic potential of targeting these proteins in cancer and other diseases characterized by inappropriate cell division. also known as cluster of differentiation 98 heavy chain (CD98hc)/solute carrier family 3 member 2 (SLC3A2), 4F2hc is a ubiquitous cell surface transmembrane protein that associates with both amino acid transporters and integrins. also known as CD147/extracellular matrix metalloproteinase inducer (EMMPRIN), basigin is an ancillary protein required for proper targeting of monocarboxylate transporters 1, 3, and 4. a transporter that forms the amino acid transport system xc– when coupled with 4F2hc. It exchanges cystine for glutamate in the pancreas and brain, but is frequently upregulated in cancer and cultured cells. a lysosomal transmembrane protein that transports cystine and cysteine. a protein that transports glutamate and aspartate across the plasma membrane in an Na+, H+, and K+-dependent manner. Its primary function is in neurotransmitter reuptake, but it is expressed in some cancers as a fusion protein. the first protein to be characterized that facilitates the transport of glucose across the plasma membrane. It is ubiquitously expressed and highly conserved. a high-affinity glucose transporter that is highly expressed in cell types with elevated glucose demand, including neurons, cells in the testes, and tumor cell lines. an insulin-sensitive glucose transporter expressed in cardiac and skeletal muscle and adipose tissue. a ubiquitously expressed (with the exception of liver) cationic amino acid transporter. a transporter that associates with 4F2hc to exchange bulky and aromatic amino acids across the plasma membrane. Overexpression of the LAT1/4F2hc dimer is linked to proliferation in a number of cancers. members of the SLC16 family of proteins that transport monocarboxylates such as lactate, pyruvate, and ketone bodies bi-directionally across the membrane in a proton-dependent manner. MCT1, 3, and 4 require the chaperone basignin for proper localization. also known as lysosomal amino acid transporter 1 (LYAAT1)/SLC36A1, PAT1 is a broadly expressed, low-affinity, pH-dependent transporter of glycine, proline, and alanine, localized to the lysosomal membrane. a transporter with specificity for all essential amino acids. Cotransport with H+, Na+, and Cl– allows net amino acid import. Expression is low in normal tissues but increased in some cancers. also known as SLC38A1 and SLC28A2, SNAT1/2 are transporter proteins that use the Na+ gradient to concentrate neutral amino acids within the cell. SNAT2 expression is nearly ubiquitous, whereas SNAT1 expression is more limited. the first active transporter of glucose to be identified. Its primary function is glucose absorption in the intestine. a neutral amino acid exchanger frequently upregulated in cancers.

Human and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell–cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell–cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism.

Growth factor withdrawal results in the endocytosis and degradation of transporter proteins for glucose and amino acids. Here, we show that this process is under the active control of the small GTPase Rab7. In the presence of growth factor, Rab7 inhibition had no effect on nutrient transporter expression. In growth factor-deprived cells, however, blocking Rab7 function prevented the clearance of glucose and amino acid transporter proteins from the cell surface. When Rab7 was inhibited, growth factor deprived cells maintained their mitochondrial membrane potential and displayed prolonged, growth factor-independent, nutrient-dependent cell survival. Thus, Rab7 functions as a proapoptotic protein by limiting cell-autonomous nutrient uptake. Consistent with this, dominant-negative Rab7 cooperated with E1A to promote the transformation of p53−/− mouse embryonic fibroblasts (MEFs). These results suggest that proteins that limit nutrient transporter expression function to prevent cell-autonomous growth and survival.

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.

Summary: Intense research efforts over the last two decades have focused on establishing the significance of apoptotic signaling in adaptive immunity. Without doubt, caspase‐dependent apoptosis plays vital roles in many immune processes, including lymphocyte development, positive and negative selection, homeostasis, and self‐tolerance. Cell biologists have developed new insights into cell death, establishing that other modes of cell death exist, such as programmed necrosis and type II/autophagic cell death. Additionally, immunologists have identified a number of immunological processes that are highly dependent upon cellular autophagy, including antigen presentation, lymphocyte development and function, pathogen recognition and destruction, and inflammatory regulation. In this review, we provide detailed mechanistic descriptions of cellular autophagy and programmed necrosis induced in response to death receptor ligation, including methods to identify them, and compare and contrast these processes with apoptosis. The crosstalk between these three processes is emphasized as newly formulated evidence suggests that this interplay is vital for efficient T‐cell clonal expansion. This new evidence indicates that in addition to apoptosis, autophagy and programmed necrosis play significant roles in the termination of T‐cell‐dependent immune responses.

Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34-/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.

The constitutive anabolism of cancer cells not only supports proliferation but also addicts tumor cells to a steady influx of exogenous nutrients. Limiting access to metabolic substrates could be an effective and selective means to block cancer growth. In this review, we define the pathways by which cancer cells acquire the raw materials for anabolism, highlight the actionable proteins in each pathway, and discuss the status of therapeutic interventions that disrupt nutrient acquisition. Critical open questions to be answered before apical metabolic inhibitors can be successfully and safely deployed in the clinic are also outlined. In summary, recent studies provide strong support that substrate limitation is a powerful therapeutic strategy to effectively, and safely, starve cancer cells to death.

Rapamycin and its analogues have shown promising anticancer activities in preclinical and clinical studies. However, the mechanism whereby rapamycin inhibits signaling through the mammalian target of rapamycin (mTOR) remains poorly understood. Here, we show that the FKBP12/rapamycin complex is an essentially irreversible inhibitor of mTOR kinase activity in vitro. However, we observe no suppression of mTOR catalytic activity after immunoprecipitation from rapamycin-treated cells. These results suggest either that rapamycin acts as a reversible kinase inhibitor in intact cells or that the cellular effects of rapamycin are not mediated through global suppression in mTOR kinase activity. To better understand the cellular pharmacology of rapamycin, we compared the individual and combined effects of rapamycin and kinase-inactive mTOR expression on a panel of mTOR-dependent cellular responses. These studies identified glycolytic activity, amino acid transporter trafficking, and Akt kinase activity as novel, mTOR-modulated functions in mammalian cells. Whereas kinase-inactive mTOR did not enhance the decreases in cell size and glycolysis induced by rapamycin, expression of this mTOR mutant significantly enhanced the inhibitory effects of rapamycin on cell proliferation, 4EBP1 phosphorylation, and Akt activity. Unexpectedly, amino acid transporter trafficking was perturbed by kinase-inactive mTOR but not by rapamycin, indicating that this process is rapamycin insensitive. These results indicate that rapamycin exerts variable inhibitory actions on mTOR signaling functions and suggest that direct inhibitors of the mTOR kinase domain will display substantially broader anticancer activities than rapamycin.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

ABSTRACT With rare exceptions, all simian immunodeficiency virus (SIV) strains can use CCR5 as a coreceptor along with CD4 for viral infection. In addition, many SIV strains are capable of using CCR5 as a primary receptor to infect CD4-negative cells such as rhesus brain capillary endothelial cells. By using coupled fluorescence-activated cell sorter (FACS) and infection assays, we found that even very low levels of CCR5 expression could support CD4-independent virus infection. CD4-independent viruses represent valuable tools for finely dissecting interactions between Env and CCR5 which may otherwise be masked due to the stabilization of these contacts by Env-CD4 binding. Based on the ability of SIV Env to bind to and mediate infection of cells expressing CCR5 chimeras and mutants, we identified the N terminus of CCR5 as a critical domain for direct Env binding and for supporting CD4-independent virus infection. However, the activity of N-terminal domain CCR5 mutants could be rescued by the presence of CD4, indicating that other regions of CCR5 are important for post-binding events that lead to viral entry. Rhesus CCR5 supported CD4-independent infection and direct Env binding more efficiently than did human CCR5 due to a single amino acid difference in the N terminus. Interestingly, uncleaved, oligomeric SIV Env protein bound to both CD4 and CCR5 less efficiently than did monomeric gp120. Finally, several mutations present in chronically infected monkey populations are shown to decrease the ability of CCR5 to serve as a primary viral receptor for the SIV isolates examined.

Although all cells depend upon nutrients they acquire from the extracellular space, surprisingly little is known about how nutrient uptake is regulated in mammalian cells. Most nutrients are brought into cells by means of specific transporter proteins. In yeast, the expression and trafficking of a wide variety of nutrient transporters is controlled by the TOR (target of rapamycin) kinase. Consistent with this, recent studies in mammalian cells have shown that mTOR (mammalian TOR) and the related protein, PI3K (phosphoinositide 3-kinase), play central roles in coupling nutrient transporter expression to the availability of extrinsic trophic and survival signals. In the case of lymphocytes, it has been particularly well established that these extrinsic signals stimulate cell growth and proliferation in part by regulating nutrient transporter expression. The ability of growth factors to control nutrient access may also play an important role in tumour suppression: the non-homoeostatic growth of tumour cells requires that nutrient transporter expression is uncoupled from trophic factor availability. Also supporting a link between nutrient transporter expression levels and oncogenesis, several recent studies demonstrate that nutrient transporter expression drives, rather than simply parallels, cellular metabolism. This review summarizes the evidence that regulated nutrient transporter expression plays a central role in cellular growth control and highlights the implications of these findings for human disease.

The ribosome is a macromolecular machine that undergoes global conformational rearrangements during translation, that involve coupled movements of ribosomal subunits, tRNAs, and the L1 stalk. However, there is a disagreement between different ...The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal subunits with respect to each other. Upon peptidyl transfer, the small ribosomal subunit ...

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7. Here, we show that growth factor deprivation increased both the fraction of Rab7 that was associated with cellular membranes and the percentage of Rab7 bound to guanosine triphosphate (GTP). Moreover, expressing a constitutively GTP-bound mutant of Rab7, Rab7-Q67L, was sufficient to trigger cell death even in the presence of growth factors. This activated Rab7 mutant was also able to reverse the growth factor-independent cell survival conferred by protein kinase C (PKC) delta inhibition. PKCdelta is one of the most highly induced proteins after growth factor withdrawal and contributes to the induction of apoptosis. To evaluate whether PKCdelta regulates Rab7, we first examined lysosomal morphology in cells with reduced PKCdelta activity. Consistent with a potential role as a Rab7 activator, blocking PKCdelta function caused profound lysosomal fragmentation comparable to that observed when Rab7 was directly inhibited. Interestingly, PKCdelta inhibition fragmented the lysosome without decreasing Rab7-GTP levels. Taken together, these results suggest that Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis and that Rab7-dependent fusion reactions may be targeted by signaling pathways that limit growth factor-independent cell survival.

Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cell's ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720's dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.

ABSTRACT In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmΔB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmΔB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.

Mouse models lacking proteins essential for autophagosome formation have demonstrated that autophagy plays a critical role in T cell development and activation. To better understand the function of autophagy in quiescent and activated lymphocytes, we have generated a mouse deficient in rab7 selectively in T cells and compared the effects of blocking autophagy at an early (atg5(-/-)) or late (rab7(-/-)) stage on T cell biology. rab7(-/-) murine embryonic fibroblasts (MEFs) and T cells generated from these mice exhibit a profound block in autophagosome degradation and are as sensitive as atg5(-/-) cells to extracellular nutrient limitation. Rab7(flox/flox)CD4-Cre(+) mice lacking the RAB7 protein in both CD4 and CD8 T cells had reduced numbers of peripheral T cells, but this defect was not as severe as in Atg5(flox/flox)CD4-Cre(+) mice despite efficient rab7 deletion and inhibition of autophagic flux. This difference may stem from the reduced ROS generation and enhanced survival of rab7(-/-) T cells compared with wild-type and atg5(-/-) T cells in the absence of cytokine stimulation. rab7(-/-) and atg5(-/-) T cells exhibited similar defects in proliferation both following antibody-mediated T cell receptor (TCR) cross-linking and using a more physiologic activation protocol, allogeneic stimulation. Interestingly, autophagy was not required to provide building blocks for the upregulation of nutrient transporter proteins immediately following activation. Together, these studies suggest that autophagosome degradation is required for the survival of activated T cells, but that loss of rab7 is better tolerated in naïve T cells than the loss of atg5.

Ceramide-induced mitochondrial fission drives high-fat diet (HFD)-induced obesity. However, molecules targeting mitochondrial dynamics have shown limited benefits in murine obesity models. Here, we reveal that these compounds are either unable to block ceramide-induced mitochondrial fission or require extended incubation periods to be effective. In contrast, targeting endolysosomal trafficking events important for mitochondrial fission rapidly and robustly prevented ceramide-induced disruptions in mitochondrial form and function. By simultaneously inhibiting ARF6- and PIKfyve-dependent trafficking events, the synthetic sphingolipid SH-BC-893 blocked palmitate- and ceramide-induced mitochondrial fission, preserved mitochondrial function, and prevented ER stress in vitro. Similar benefits were observed in the tissues of HFD-fed mice. Within 4 h of oral administration, SH-BC-893 normalized mitochondrial morphology in the livers and brains of HFD-fed mice, improved mitochondrial function in white adipose tissue, and corrected aberrant plasma leptin and adiponectin levels. As an interventional agent, SH-BC-893 restored normal body weight, glucose disposal, and hepatic lipid levels in mice consuming a HFD. In sum, the sphingolipid analog SH-BC-893 robustly and acutely blocks ceramide-induced mitochondrial dysfunction, correcting diet-induced obesity and its metabolic sequelae.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has antineoplastic actions. Our published work suggests that at least some of FTY720’s anticancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720’s S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective antitumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for antileukemic activity.

Ceramide generation is increased by a broad array of signals. In general, ceramide limits cell survival and proliferation and promotes differentiation and senescence. Despite its role in the pathogenesis of multiple human diseases, ceramide’s mechanism of action remains poorly defined. Understanding how this sphingolipid modulates cell physiology is therefore an important goal. Building on prior observations that ceramide induces autophagy, we demonstrate that ceramide kills cells by inducing severe bioenergetic stress secondary to nutrient transporter down-regulation. In support of this model, maintaining nutrient access blocks ceramide-induced autophagy and cell death. This bioenergetic mechanism of action may explain the increased sensitivity of cancer cells to ceramide. Starvation induces quiescence in normal cells. Tumor cells, in contrast, carry oncogenic mutations that block the switch to catabolism and prevent a reduction in metabolic demand leading to a bioenergetic crisis when nutrients become scarce. We propose that the non-lethal effects of ceramide might also stem from ceramide-induced starvation. While severe nutrient stress kills cells, mild nutrient limitation slows proliferation and may contribute to the induction of senescence. In sum, our new model for ceramide action suggests that regulated nutrient transporter expression may play a previously unappreciated role in cancer and other diseases where ceramide metabolism is altered.

Ceramide induces differentiation, proliferative arrest, senescence and death in mammalian cells. The mechanism by which ceramide produces these outcomes has proved difficult to define. Building on observations that ceramide stimulates autophagy, we have identified a novel mechanism of action for this sphingolipid: ceramide starves cells to death subsequent to profound nutrient transporter down-regulation. In yeast, ceramide generated in response to heat stress adaptively slows cell growth by down-regulating nutrient permeases. In mammalian cells, a lethal dose of ceramide triggers a bioenergetic crisis by so severely limiting cellular access to extracellular nutrients that autophagy is insufficient to meet the metabolic demands of the cell. In keeping with this bioenergetic explanation for ceramide toxicity, methyl pyruvate, a membrane-permeable nutrient, protects cells from ceramide-induced starvation. Also consistent with this model, we have found that the metabolic state of the cell determines its sensitivity to ceramide. Thus the increased sensitivity of cancer cells to ceramide may relate to their inflexible biosynthetic metabolic programme. These studies highlight the value of assessing nutrient transporter expression in autophagic cells and the important role that culture conditions play in determining the cellular response to ceramide.

Nutrient stress that produces quiescence and catabolism in normal cells is lethal to cancer cells, because oncogenic mutations constitutively drive anabolism. One driver of biosynthesis in cancer cells is the mammalian target of rapamycin complex 1 (mTORC1) signaling complex. Activating mTORC1 by deleting its negative regulator tuberous sclerosis complex 2 (TSC2) leads to hypersensitivity to glucose deprivation. We have previously shown that ceramide kills cells in part by triggering nutrient transporter loss and restricting access to extracellular amino acids and glucose, suggesting that TSC2-deficient cells would be hypersensitive to ceramide. However, murine embryonic fibroblasts (MEFs) lacking TSC2 were highly resistant to ceramide-induced death. Consistent with the observation that ceramide limits access to both amino acids and glucose, TSC2−/− MEFs also had a survival advantage when extracellular amino acids and glucose were both reduced. As TSC2−/− MEFs were resistant to nutrient stress despite sustained mTORC1 activity, we assessed whether mTORC1 signaling might be beneficial under these conditions. In low amino acid and glucose medium, and following ceramide-induced nutrient transporter loss, elevated mTORC1 activity significantly enhanced the adaptive upregulation of new transporter proteins for amino acids and glucose. Strikingly, the introduction of oncogenic Ras abrogated the survival advantage of TSC2−/− MEFs upon ceramide treatment most likely by increasing nutrient demand. These results suggest that, in the absence of oncogene-driven biosynthetic demand, mTORC1-dependent translation facilitates the adaptive cellular response to nutrient stress.

Rapidly proliferating cancer cells increase flux through anabolic pathways to build the mass necessary to support cell division. Imported amino acids and glucose lie at the apex of the anabolic pyramid. Consistent with this, elevated expression of nutrient transporter proteins is characteristic of aggressive and highly malignant cancers. Because tumour cells are more dependent than their normal neighbours on accelerated nutrient import, these up-regulated transporters could be excellent targets for selective anti-cancer therapies. A study by Babu et al. in a recent issue of the Biochemical Journal definitively shows that SLC6A14 (where SLC is solute carrier) is one such cancer-specific amino acid transporter. Although mice completely lacking SLC6A14 are viable and exhibit normal mammary gland development, these animals are highly resistant to mammary tumour initiation and progression driven by potent oncogenes. Because SLC6A14 is essential for tumour growth yet dispensable for normal development and tissue maintenance, small molecules that block amino acid import through this transporter could be effective and selective anti-cancer agents, particularly as components of rational drug combinations.

Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.

The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720’s anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.

SUMMARY The stability of tight junctions (TJs) between endothelial cells (ECs) is essential to maintain blood-brain barrier (BBB) function in the healthy brain. Following ischemic stroke, TJ strand dismantlement due to protein degradation leads to BBB dysfunction, yet the mechanisms driving this process are poorly understood. Here, we show that endothelial-specific ablation of Rab7a, a small GTPase that regulates endolysosomal protein degradation, reduces stroke-induced TJ strand disassembly resulting in decreased paracellular BBB permeability and improved neuronal outcomes. Two pro-inflammatory cytokines, TNFα and IL1β, but not glucose and oxygen deprivation, induce Rab7a activation via Ccz1 in brain ECs in vitro , leading to increased TJ protein degradation and impaired paracellular barrier function. Silencing Rab7a in brain ECs in vitro reduces cytokine-driven endothelial barrier dysfunction by suppressing degradation of a key BBB TJ protein, Claudin-5. Thus, Rab7a activation by inflammatory cytokines promotes degradation of select TJ proteins leading to BBB dysfunction after ischemic stroke.

Abstract Macropinocytosis is a bulk endocytic process that can fuel tumor growth and drug resistance by providing access to extracellular protein and necrotic cell debris. This process is primarily studied by utilizing the chemical inhibitor, 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA), but this inhibitor is nonselective can hinder proliferation even in non-macropinocytic breast cancer cells. Hence, a more selective, genetic approach is needed. Formation of macropinosomes is dependent on the actin cytoskeleton, which in turn is regulated by CARMIL1, a large multimeric protein that regulates the actin capping protein. CARMIL1 is essential for macropinocytosis, and mutation of two residues in the Capping-Protein Interaction (CPI) region of CARMIL1 to alanine (CARMIL1-AA) limits the CARMIL1-capping protein interaction and more importantly, inhibits macropinocytosis upon introduction of exogenous CARMIL1-AA in tumor cells. Therefore, CARMIL1-AA is a genetic tool to study the effects of macropinocytosis on other cellular processes. However, autophagy, a lysosome-dependent recycling system that enables cell survival, additionally utilizes actin to form autophagosomes. The overall goal of this project is to determine whether CARMIL1-AA specifically inhibits macropinocytosis while still supporting autophagy. CARMIL1-WT and -AA mutant cells were generated using the MMTV-PyMT tumor model and HeLa cell lines and validated for CARMIL1 expression by western blot and quantitative PCR and for macropinocytosis by dextran analysis. These cells were evaluated for autophagic flux by western blot for LC3 I/II and by utilizing a GFP-LC3-RFP-LC3ΔG reporter. Our observation that CARMIL1-AA does not impact autophagy enables further investigation of the importance of macropinocytosis in therapeutic resistance and in shaping the tumor microenvironment. Citation Format: Rebecca M Lim, Brandon Chu, Aimee Lara Edinger. A genetic approach to selectively inhibit macropinocytosis while sparing autophagy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B071.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE, VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

The mouse leukotriene B4receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4(LTB4, K d = 0.24 ± 0.03 nm). In competition binding experiments, LTB4was the most potent competitor (K i = 0.23 ± 0.05 nm) followed by 20-hydroxy-LTB4(K i = 1.1 ± 0.2 nm) and by 6-trans-12-epi-LTB4 and LTD4(K i > 1 μm). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation. The mouse leukotriene B4receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4(LTB4, K d = 0.24 ± 0.03 nm). In competition binding experiments, LTB4was the most potent competitor (K i = 0.23 ± 0.05 nm) followed by 20-hydroxy-LTB4(K i = 1.1 ± 0.2 nm) and by 6-trans-12-epi-LTB4 and LTD4(K i > 1 μm). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation. Leukotriene B4(LTB4) 1The abbreviations used are:LTB4, leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; BLTR, leukotriene B4 receptor; h-BLTR, human leukotriene B4 receptor; HIV, human immunodeficiency virus; m-BLTR, mouse leukotriene B4 receptor; 6-trans-12-epi-LTB4, 5(S),12(S)-dihydroxy-14-cis-6,8,10-trans-eicosatetraenoic acid; kb, kilobase(s); PCR, polymerase chain reaction; HEK, human embryonic kidney; CHO, Chinese hamster ovary; HEK-m-BLTR, stable transfected HEK cells with pCR3.1-m-BLTR; CHO-m-BLTR, stable transfected CHO cells with pCR3.1-m-BLTR; SIV, simian immunodeficiency virus.1The abbreviations used are:LTB4, leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; BLTR, leukotriene B4 receptor; h-BLTR, human leukotriene B4 receptor; HIV, human immunodeficiency virus; m-BLTR, mouse leukotriene B4 receptor; 6-trans-12-epi-LTB4, 5(S),12(S)-dihydroxy-14-cis-6,8,10-trans-eicosatetraenoic acid; kb, kilobase(s); PCR, polymerase chain reaction; HEK, human embryonic kidney; CHO, Chinese hamster ovary; HEK-m-BLTR, stable transfected HEK cells with pCR3.1-m-BLTR; CHO-m-BLTR, stable transfected CHO cells with pCR3.1-m-BLTR; SIV, simian immunodeficiency virus. and the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4), derived from arachidonic acid metabolism, are synthesized sequentially by 5-lipoxygenase and then by either LTA4 hydrolase or LTC4 synthase, respectively (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2320) Google Scholar). The biological actions of the cysteinyl-leukotrienes are mediated through at least two G protein-coupled receptors referred to as CysLT1 and CysLT2 whose molecular identities remain uncharacterized (2Metters K.M. J. Lipid Mediators Cell Signal. 1995; 12: 413-427Crossref PubMed Scopus (69) Google Scholar). LTB4 mediates its effects through a membrane G-protein-coupled receptor termed BLTR (3Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar). Additionally, LTB4 was shown to bind to the intracellular transcription factor peroxisome proliferator-activated receptor (4Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1205) Google Scholar). This facet of binding has been proposed to be part of a negative feedback mechanism to limit the inflammatory actions of LTB4. However the binding to peroxisome proliferator-activated receptor has been questioned in recent experiments (5Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1861) Google Scholar). LTB4, a dihydroxy fatty acid, is one of the most potent known chemoattractant mediators, acting mainly on neutrophils but also on related myeloid cells, mast cells, and endothelial cells (6Ford-Hutchinson A.W. Bray M.A. Doig M.V. Shipley M.E. Smith M.J. Nature. 1980; 286: 264-265Crossref PubMed Scopus (1584) Google Scholar, 7Nohgawa M. Sasada M. Maeda A. Asagoe K. Harakawa N. Takano K. Yamamoto K. Okuma M. J. Leukocyte Biol. 1997; 62: 203-209Crossref PubMed Scopus (42) Google Scholar). LTB4 induces chemotaxis, chemokinesis, and aggregation, causing the migration of neutrophils to sites of inflammation where the cells degranulate, resulting in the release of lysosomal enzymes in addition to other antibacterial and anti-microbicidal agents (8Henderson Jr., W.R. Ann. Intern. Med. 1994; 121: 684-697Crossref PubMed Scopus (588) Google Scholar). LTB4 also promotes the adherence of neutrophils to vascular endothelial cells and their transmigration, which amplifies the inflammatory response. LTB4 has been implicated in the pathophysiology of various diseases like arthritis, inflammatory bowel disease, and psoriasis. The exact role of LTB4 in the etiology of these disorders has been debated vigorously. Inhibitors of 5-lipoxygenase and the 5-lipoxygenase-activating protein have been used efficiently in models of ulcerative colitis (9Kjeldsen J. Laursen L.S. Hillingso J. Mertz-Nielsen A. Bukhave K. Rask-Madsen J. Lauritsen K. Pharmacol. Toxicol. 1995; 77: 371-376Crossref PubMed Scopus (7) Google Scholar, 10Hillingso J. Kjeldsen J. Laursen L.S. Lauritsen K. von Spreckelsen S. Depre M. Friedman B.S. Malmstrom K. Shingo S. Bukhave K. Raskmadsen J. Clin. Pharmacol. Ther. 1995; 57: 335-341Crossref PubMed Scopus (19) Google Scholar), endotoxic shock (11Yoshikawa D. Goto F. Circul. Shock. 1992; 38: 29-33PubMed Google Scholar), and induced asthma (12Shindo K. Koide K. Fukumura M. Thorax. 1997; 52: 1024-1029Crossref PubMed Scopus (27) Google Scholar, 13Diamant Z. Timmers M.C. van der Veen H. Friedman B.S. De Smet M. Depre M. Hilliard D. Bel E.H. Sterk P.J. J. Allergy Clin. Immunol. 1995; 95: 42-51Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Fisher A.R. Rosenberg M.A. Roth M. Loper M. Jungerwirth S. Israel E. Thorax. 1997; 52: 1074-1077Crossref PubMed Scopus (19) Google Scholar). The development of specific and highly potent BLTR antagonists has lagged behind cysteinyl receptor antagonists, which are currently available in the clinic for treatment of asthmatic inflammatory symptoms. One BLTR antagonist has shown encouraging results in a murine model of collagen-induced arthritis (15Griffiths R.J. Pettipher E.R. Koch K. Farrell C.A. Breslow R. Conklyn M.J. Smith M.A. Hackman B.C. Wimberly D.J. Milici A.J. Scampoli D.N. Cheng J.B. Pillor J.S. Pazoles C.J. Doherty N.S. Melvin L.S. Reiter L.A. Biggars M.S. Falkner F.C. Mitchell D.Y. Liston T.E. Showell H.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 517-521Crossref PubMed Scopus (197) Google Scholar). There has been considerable controversy about the molecular identification of the BLTR. In 1996 two independent research groups (16Owman C. Nilsson C. Lolait S.J. Genomics. 1996; 37: 187-194Crossref PubMed Scopus (50) Google Scholar, 17Raport C.J. Schweickart V.L. Chantry D. Eddy Jr., R.L. Shows T.B. Godiska R. Gray P.W. J. Leukocyte Biol. 1996; 59: 18-23Crossref PubMed Scopus (82) Google Scholar) cloned identical orphan receptor genes believed to encode members of the chemotactic factor subfamily of G protein-coupled seven transmembrane receptors. Initially, one group indicated that the receptor was unable to bind LTB4 (16Owman C. Nilsson C. Lolait S.J. Genomics. 1996; 37: 187-194Crossref PubMed Scopus (50) Google Scholar) but later retracted this finding to indicate specific binding (18Owman C. Lolait S.J. Santen S. Olde B. Biochem. Biophys. Res. Commun. 1997; 241: 390-394Crossref PubMed Scopus (7) Google Scholar). A third group cloned the identical receptor sequence, which was classified as a purinergic P2Y7 receptor on the basis of its affinity for binding ATP (19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). However, Yokomizo et al. (3Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar) challenged this identification and provided convincing proof that the human BLTR (h-BLTR) had been cloned. Intense interest in the role of chemokine receptors for facilitation of HIV entry into CD4-positive cells is evident from recent surveys of the literature (20Doms R.W. Peiper S.C. Virology. 1997; 235: 179-190Crossref PubMed Scopus (236) Google Scholar, 21Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1984) Google Scholar). The h-BLTR is structurally related to chemokine receptors (e.g. CCR5) and is expressed in various immune cells. Recent evidence has suggested that the h-BLTR may function as a coreceptor for entry of primary HIV-1 isolates into CD4-positive cells (22Owman C. Garzino-Demo A. Cocchi F. Popovic F. Sabirsh A. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9530-9534Crossref PubMed Scopus (53) Google Scholar). If true, this finding would add a significant new dimension to the interplay of leukotrienes, inflammation, and AIDS pathogenesis. We report here the cloning and characterization of the m-BLTR, the signaling pathways for this G protein-coupled receptor, and a detailed analysis as to whether the h-BLTR can function as an HIV coreceptor. [α-32P]dCTP was purchased from NEN Life Science Products and [3H]LTB4 from Amersham Pharmacia Biotech. The mouse strain 129 Sv genomic library in Lambda Fix II and the cloning vector pBluescript II KS were from Stratagene (La Jolla, CA), and the mammalian expression vector pCR3.1 Uni was from Invitrogen (Carlsbad, CA). LTB4, 20-hydroxy-LTB4, 6-trans-12-epi LTB4, and LTD4 were purchased from Cayman Chemical Co. (Ann Arbor, MI). Dulbecco's modified Eagle's medium, Ham's F-12, Opti-MEM, L-15, conditioned frog medium, phosphate-buffered saline, fetal bovine serum, and LipofectAMINE were from Life Technologies, Inc., and restriction enzymes were from New England Biolabs (Beverly, MA). Ampli-Taq DNA polymerase was obtained from Perkin-Elmer. A 1.1-kb fragment (NheI-PstI) from the cDNA, described in Ref.19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar and labeled with [α-32P]dCTP, was used as probe to screen the genomic mouse library by standard procedures. Putative positive clones were taken through two additional rounds of screening until plaque-purified. Phage DNA was purified and subjected to restriction enzyme digestion and Southern blot analysis. A 2.2- kb XbaI fragment that hybridized to the probe was gel extracted and inserted into theXbaI site of pBluescript II KS. The insert was sequenced entirely on both strands using automated sequencing (Applied Biosystems Big Dye Terminator, Ready Reaction Kit Reagents; ABI 373 sequencer) at the Department of Genetics, University of Pennsylvania. The open reading frame was amplified by PCR using primers CDF367 (5′-GCCATGGCTGCAAACACTACATCTC-3′) and CDF370 (5′-AGTTCACTTCGAAGACTCAGG-3′). The sequence of the amplified product was verified and cloned into pCR3.1 Uni. Human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells from the American Type Culture Collection (ATCC) were maintained in Dulbecco's modified Eagle's medium and Ham's F-12, respectively, containing 100 units/ml of penicillin and 100 μg/ml streptomycin, supplemented with 10% (v/v) fetal bovine serum in a 5% CO2 incubator at 37 °C. Cells were seeded at a density of 1–1.5 × 106cells/100-mm dish. Plasmid DNA and LipofectAMINE were mixed with Opti-MEM and added for 12 h on cells, at 70–80% confluence for transient transfection or at 40–50% confluence to select stable transfected cell lines. Cells were selected 24 h after transfection with G418. Isolation of stable clones was achieved by serial dilution or pipette lifting colonies followed by trypsinization and replating colonies in medium containing 2 mg/ml G418. When single clones where isolated and passed several times, the G418 concentration was reduced gradually to 0.5 mg/ml. Experiments were performed on two cell lines transfected with pCR3.1-m-BLTR (HEK-m-BLTR and CHO-m-BLTR). Total RNA was prepared from different murine tissues using TRIzol reagent (Life Technologies, Inc.). RNA blot analysis was carried out with 15 μg of total RNA from different tissues. The full-length m-BLTR DNA was used as probe, labeled with [α-32P]dCTP. Blots were prehybridized 20 min at 68 °C in QuickHyb solution (Stratagene) and hybridized at 68 °C for 1 h. The final washing conditions were 0.1 × SSC, 0.1% SDS at 60 °C. cDNA was synthesized from 5 μg of total RNA with random primers (for reverse transcriptase-PCR). PCR primers CDF370 and CDF367 were used to amplify m-BLTR cDNA. Cells were washed twice with cold phosphate-buffered saline without calcium and magnesium, harvested, and homogenized with a hand-held Polytron on ice in 5 ml of buffer A (10 mm HEPES, 2 mm EDTA, pH 7.4, and a mixture of protease inhibitors (Boehringer Mannheim)). The homogenate was centrifuged 5 min at 140 × g, and the supernatant was then centrifuged at 100,000 g for 1 h. The pellet was resuspended in buffer A to 2 mg/ml of protein for [3H]LTB4 binding experiments performed within several hours. To determine the specific binding on membrane fractions isolated from transiently transfected cells, cells were isolated 36–40 h after transfection. For cold competition experiments, HEK-m-BLTR cell membrane fractions (200 μg/ml of protein) were incubated with 0.1 nm[3H]LTB4 in 0.5 ml of 10 mmHEPES, pH 7.4, containing 20 mm MgCl2 and various concentrations of either LTB4 (0.01–100 nm), 20-hydroxy-LTB4, 6-trans-12-epi LTB4, or LTD4 (0.1 nm-1 μm). For saturation experiments, stable transfected cell membranes (200 μg/ml of protein) were incubated with various concentrations of [3H]LTB4 (0.01–2.5 nm) in 0.25 ml of 10 mm HEPES, pH 7.4, containing 20 mm MgCl2 in the presence or absence of 2.5 μm LTB4. All samples, in duplicate, were incubated at room temperature for 1 h. The total and nonspecific binding were determined as the amount of [3H]LTB4 bound to the membrane fractions in the presence or absence of 2.5 μm LTB4. Bound and free radioligand were separated by filtration through Whatman GF/C filters presoaked with 0.1% bovine serum albumin in 10 mmHEPES. Confluent CHO-m-BLTR cells were harvested and washed with HEPES-buffered saline. Cells were then loaded with the fluorescent dye FURA-2/AM (Molecular Probes, Eugene, OR) at 10 μm, washed and resuspended in HEPES-buffered saline containing: 142 mm NaCl, 2.4 mm KCl, 1.2 mm K2HPO4, 1.3 mm Ca2+, 10 mm d-glucose, 10 mm HEPES, pH 7.4, and 250 μm sulfinpyrazone, the latter being added in order to reduce excretion of the dye (23Di Virgilio F. Fasolato C. Steinberg T.H. Biochem. J. 1988; 256: 959-963Crossref PubMed Scopus (123) Google Scholar). Measurements of change in Ca2+ levels in stirred cell suspensions were made using a Perkin-Elmer model LS50B luminescence spectrometer and were expressed as ratios of fluorescence emitted at 510 nm in response to excitation at 340 and 380 nm (data sampling interval, 0.5 s). Calcium concentrations were calculated from these ratios after determining the maximum and minimum ratios of fluorescence in the presence and absence of saturating levels of Ca2+, respectively, according to the ratiometric method described previously (24Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Xenopus laevis melanophores were maintained in culture and used as described previously (25Graminski G.F. Jayawickreme C.K. Potenza M.N. Lerner M.R. J. Biol. Chem. 1993; 268: 5957-5964Abstract Full Text PDF PubMed Google Scholar, 26Lerner M.R. Trends Neurosci. 1994; 17: 142-146Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 27Potenza M.N. Graminski G.F. Schmauss C. Lerner M.R. J. Neurosci. 1994; 14: 1463-1476Crossref PubMed Google Scholar, 28McClintock T.S. Graminski G.F. Potenza M.N. Jayawickreme C.K. Roby-Shemkovitz A. Lerner M.R. Anal. Biochem. 1993; 209: 298-305Crossref PubMed Scopus (40) Google Scholar). Briefly, transient expression of pCR3.1-m-BLTR in melanophores was achieved by electroporation. Melanophores were plated (15,000/well) in 96-well tissue culture plates and cultured for 2 days. Before the addition of agonist, cells were washed, incubated with 0.7 × L-15, 0.1% bovine serum albumin as described (25Graminski G.F. Jayawickreme C.K. Potenza M.N. Lerner M.R. J. Biol. Chem. 1993; 268: 5957-5964Abstract Full Text PDF PubMed Google Scholar), and then exposed to room light for 1 h. This exposure causes the cells to disperse their pigment granules and darken. The plates were incubated for 1 h in room light and base-line absorbance (A 0) reading obtained at 620 nm using a Molecular DevicesV max kinetic microtiter plate reader. The agonist LTB4 was added to the microtiter wells in 20-μl aliquots at 10 × their final concentration. Dose-response data were obtained 1 h later (A f). Data were plotted with y = 1 − (A f/A 0). Data are presented as mean ± S.E. CHO-m-BLTR were plated in 12-well plates at a density of 200,000 cells/well. 2 days later, the cultured medium was removed and replaced with 0.5 ml of culture medium with 25 μm forskolin. After 15 min, different concentrations of LTB4 were added. The medium was removed 10 min after the addition of agonist, and the cAMP produced was extracted by adding 0.5 ml of ethanol to each well. The supernatant was evaporated to dryness, and the pellet was dissolved in Tris (0.05 m) EDTA (4 mm) buffer, pH = 7.5. The cAMP content was measured using a [3H]cAMP radioimmunoassay kit from Amersham Pharmacia Biotech. 5-Lipoxygenase-deficient mice (29Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (354) Google Scholar) and wild-type controls (five mice each) from our colony were injected intraperitoneally with glycogen and the neutrophils harvested 5 h later as described previously (29Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (354) Google Scholar). Binding was carried out on membranes as mentioned above. For cell-cell fusion assays, quail QT6 target cells were transfected with plasmids expressing CD4, the desired coreceptor, and luciferase under control of the T7 promoter. The h-BLTR, previously characterized as the P2Y7 receptor (gift from S. Kunapuli; Ref. 19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), was used for these studies (see “Results”). The next day, QT6 effector cells expressing the desired Env protein by transfection and T7 polymerase as a consequence of infection by recombinant vaccinia viruses were added. In this assay, cell-cell fusion results in cytoplasmic mixing and luciferase production, which can be easily quantified. Additional details can be found in previous papers (30Nussbaum O. Broder C.C. Berger E.A. J. Virol. 1994; 68: 5411-5422Crossref PubMed Google Scholar, 31Rucker J. Doranz B.J. Edinger A.E. Long D. Berson J.F. Doms R.W. Methods Enzymol. 1997; 288: 118-133PubMed Google Scholar). For infection assays, we used luciferase reporter viruses. Human 293T cells were transfected with plasmids expressing the desired Env (in a pcDNA3 or psv7d background) and with the NL4–3 luciferase virus backbone (pNL-Luc-E−R−) (32Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar, 33Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1088) Google Scholar). Virus was harvested from the media the next day and used to infect feline CCCS cells (for HXBch) or 293T cells (for all other Evs) expressing the indicated CD4/coreceptor combinations. Infection was quantified by measuring luciferase activity 3-days postinfection. Multiple analysis of variance tests followed by Bonferroni analysis were performed on cAMP data. *p< 0.05; ***p < 0.001. To clarify the confusion surrounding the molecular identity of the h-BLTR and to advance the study of the role of LTB4 in murine inflammation models, we sought to clone and characterize the m-BLTR. A mouse genomic library was screened using a 1.1-kb fragment from the cDNA identified previously as encoding the purinergic P2Y7 receptor (19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Among the positive clones that hybridized to the probe, one genomic clone with a 2.2-kb XbaI fragment was found to display an open reading frame of 1.056 kb, encoding a 351-amino acid receptor with seven putative transmembrane domains, two potential glycosylation sites, and several phosphorylation sites (GenBank accession numberAF077673). This open reading frame revealed a deduced amino acid sequence with 77% identity to the h-BLTR (3Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar). The third intracellular loop of the m-BLTR showed 100% identity to the human receptor, with two protein kinase C phosphorylation sites. TheN-glycosylation sites and several of the phosphorylation sites are conserved between both species. The putative promoter region (−1 to −618) contained a CAAT-like box, a 36-nucleotide poly(A) tract, and several other conserved sequences for the putative binding of GATA-1, PEA-3, c-Myc, c-Myb, Sp-1, and NF-IL6 transcription factors. While this manuscript was under review, Huang et al. (34Huang W.-W. Garcia-Zepeda E.A. Sauty A. Oettgen H.C. Rothenberg M.E. Luster A.D. J. Exp. Med. 1998; 188: 1063-1074Crossref PubMed Scopus (133) Google Scholar) published a m-BLTR sequence cloned from a murine eosinophil cDNA library that was identical to the m-BLTR gene cloned here. Because the genomic clone appeared to be intronless, we proceeded directly to expression studies. m-BLTR was subcloned in the expression vector pCR3.1 and used to transfect HEK 293 cells. Transient transfected HEK cells displayed specific binding for LTB4 (Fig.1 A), as did cells transfected with the original human P2Y7 receptor clone (not shown), whereas nontransfected (Ct) and mock transfected cells did not. Membrane fractions of HEK-m-BLTR cells showed a reversible, saturable and high affinity binding for LTB4 with aK d = 0.24 ± 0.03 nm andB max = 743 ± 168 fmol/mg of protein (Fig.1 B). Displacement curves of [3H]LTB4 binding indicated that the binding site was specific for LTB4 with a K i of 0.23 ± 0.05 nm (n = 4), followed by 20-hydroxy-LTB4, a metabolite of LTB4(K i = 1.1 ± 0.2 nm,n = 4), and by the nonenzymatic breakdown product of LTA4, 6-trans-12-epi-LTB4, and LTD4 (K i > 1 μm) (Fig.1 C). The tissue distribution of the m-BLTR was investigated by Northern blot analysis of total RNA using two different probes. No signal was detected in the tissues tested (spleen, lung, kidney, liver, pancreas, uterus, testis, heart, aorta, brain). However, using reverse transcriptase-PCR, the m-BLTR mRNA was detected in all tissues, but not aorta (data not shown). The mouse heart and lung cDNAs were sequenced and found to be identical with the positive clone identified by mouse genomic library screening. X. laevis melanophores provide a rapid, functional and visual readout of receptor activation. These cells disperse their pigment granules upon stimulation of receptors that are coupled to Gs, Go, and Gq and lead to accumulation of second messengers and thus appear dark. In contrast, stimulation of receptors that are coupled to Gi cause a decrease in second messenger levels and result in pigment granule aggregation, and the cells appear light (27Potenza M.N. Graminski G.F. Schmauss C. Lerner M.R. J. Neurosci. 1994; 14: 1463-1476Crossref PubMed Google Scholar). A long term culture of melanophores was used to evaluate the functional activation of the m-BLTR. Stimulation of the receptor caused pigment aggregation in a concentration-dependent fashion with an EC50 = 0.13 nm (Fig. 2) consistent with coupling to Gi. In CHO-m-BLTR cells, LTB4 inhibited in a concentration-dependent manner the cAMP production induced by 25 μm forskolin (Fig.3 A). Maximum inhibition (58%) was obtained at 100 nm. With the same cell line, LTB4 induced a concentration-dependent increase in the intracellular calcium levels (EC50 = 4.4 nm; Fig.3 B). With 1 μm thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase pump, the intracellular increase of calcium induced by 100 nm LTB4 was inhibited by 89.5 ± 1.4% (n = 4). Previously, we (29Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (354) Google Scholar) and another group (35Goulet J.L. Snouwaert J.N. Latour A.M. Coffman T.M. Koller B.H. Proc. Natl. Acad. Sci U. S. A. 1994; 91: 12852-12856Crossref PubMed Scopus (188) Google Scholar) developed mice with disruptions in the 5-lipoxygenase gene. These mice were unable to synthesize cysteinyl-leukotrienes or LTB4 in various inflammatory cell types. To test whether the absence of ligand influences receptor expression, we tested 5-lipoxygenase-deficient and control mice for alterations in LTB4 binding using membranes from glycogen-elicited neutrophils. Although we obtained specific and competitive binding in these membranes, the lack of ability to synthesize LTB4 in 5-lipoxygenase deficient mice did not substantially alter BLTR binding (Fig.4). Primate lentiviruses utilize CD4 and a coreceptor (most often the chemokine receptors CCR5 and CXCR4) to enter target cells (20Doms R.W. Peiper S.C. Virology. 1997; 235: 179-190Crossref PubMed Scopus (236) Google Scholar). The importance of CCR5 as a HIV-1 coreceptor was demonstrated by the finding that individuals who lack CCR5 because of a naturally occurring polymorphism are highly resistant to HIV infection (36Samson M. Libert F. Doranz B.J. Rucker J. Liesnard C. Farber C.M. Saragosti S. Lapoumeroulie C. Cognaux J. Forceille C. Muyldermans G. Verhofstede C. Burtonboy G. Georges M. Imai T. Rana S. Yi Y. Smyth R.J. Collman R.G. Doms R.W. Vassart G. Parmentier M. Nature. 1996; 382: 722-725Crossref PubMed Scopus (2447) Google Scholar). In addition to CCR5 and CXCR4, approximately one dozen other chemokine and related orphan receptors have been shown to function as coreceptors for more limited numbers of virus strains in vitro (37Hoffman T.L. Doms R.W. AIDS. 1998; 12: S17-S26PubMed Google Scholar). However, the in vivo relevance of these alternative coreceptors is uncertain. Recently, using a PCR-based entry assay, h-BLTR was shown to serve as a coreceptor for some X4 HIV-1 stains (22Owman C. Garzino-Demo A. Cocchi F. Popovic F. Sabirsh A. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9530-9534Crossref PubMed Scopus (53) Google Scholar). To investigate the ability of the BLTR to serve as a coreceptor for SIV and additional HIV-1 strains using a virus infection assay, we expressed CD4 and h-BLTR in human 293T or CCCS cells. The cells were then infected with luciferase reporter viruses bearing various HIV-1 and SIV Env proteins (Fig.5). None of the viral Env proteins tested could mediate infection of cells expressing CD4 and h-BLTR, although infection was readily observed when cells expressed either CCR5 or CXCR4 (depending on the virus strain). In addition to the results depicted in Fig. 5, SIVmac1A11, smPBj6.6, macB670

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.

AbstractAutophagy is triggered by ceramide, a sphingolipid that regulates diverse cellular processes including survival, differentiation, and senescence. Both ceramide and autophagy play important, but incompletely understood, roles in type 2 diabetes and cancer. We reasoned that defining the connection between ceramide and autophagy might provide important insight into these highly prevalent diseases. Our recently published work demonstrates that ceramide-induced autophagy is a homeostatic response to starvation caused by nutrient transporter down-regulation. Preventing nutrient transporter loss or supplementation with transporter-independent nutrients protects cells from ceramide-induced death and delays the onset of autophagy. Thus, we propose a model where ceramide kills cells by inducing acute and severe intracellular nutrient limitation. Consistent with this idea, AMPK-deficient cells that are less able to deal with bioenergetic stress are also more sensitive to ceramide than wild-type cells. Our observation that gradually adapting cells to tolerate low levels of extracellular nutrients confers striking resistance to ceramide toxicity further supports this model. These results highlight the value of measuring nutrient transporter expression in cells undergoing protective autophagy. In addition, this novel mechanism for ceramide-induced cell death suggests new approaches to studying and treating multiple human diseases.This article refers to:

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.

Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture.To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change.Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation.This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.

Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. These effects are mediated by the activation of protein phosphatase 2A (PP2A). To identify putative PP2A substrates, we used quantitative phosphoproteomics to profile the temporal changes in protein phosphorylation in FL5.12 cells following incubation with SH-BC-893 or the specific PP2A inhibitor LB-100. These analyses enabled the profiling of more than 15,000 phosphorylation sites, of which 958 sites on 644 proteins were dynamically regulated. We identified 114 putative PP2A substrates including several nutrient transporter proteins, GTPase regulators (e.g. Agap2, Git1), and proteins associated with actin cytoskeletal remodeling (e.g. Vim, Pxn). To identify SH-BC-893-induced cell signaling events that disrupt lysosomal trafficking, we compared phosphorylation profiles in cells treated with SH-BC-893 or C2-ceramide, a non-vacuolating sphingolipid that does not impair lysosomal fusion. These analyses combined with functional assays uncovered the differential regulation of Akt and Gsk3b by SH-BC-893 (vacuolating) and C2-ceramide (non-vacuolating). Dynamic phosphoproteomics of cells treated with compounds affecting PP2A activity thus enabled the correlation of cell signaling with phenotypes to rationalize their mode of action.

Growth factors provide permission signals that allow mammalian cells to grow, proliferate and survive. One mechanism by which growth factors maintain this control is through the regulation of cell surface nutrient transporter expression. Following growth factor withdrawal, nutrient transporters are endocytosed and degraded in the lysosome, effectively terminating the cell's ability to obtain nutrients. This results in a state of pseudostarvation in which cells atrophy and initiate a catabolic metabolic programme in the midst of abundant extracellular nutrients. Oncogenic forms of Akt can support growth factor-independent nutrient transporter expression through a mechanism that depends upon mTOR (mammalian target of rapamycin). The ability of activated Akt to support nutrient transporter expression is an essential component of its prosurvival function. When the destruction of nutrient transporters is inhibited, cells are capable of long-term growth-factor-independent cell survival in the absence of receptor-dependent signal transduction. These results imply that proteins involved in nutrient transporter turnover in response to growth factor withdrawal are components of a novel tumour suppressor pathway. Preliminary data suggest that Rab7, a GTPase required for transporter degradation, functions as a tumour suppressor protein, as inhibiting Rab7 activity promotes colony formation in soft agar. These studies indicate that drugs affecting this pathway might have utility as anti-cancer chemotherapeutic agents.

Does the LKB1–AMPK (AMP-activated protein kinase) pathway act to suppress tumorigenesis or to rescue cancer cells from metabolic collapse? New work from the Alessi laboratory in this issue of the Biochemical Journal shows conclusively that AMPK activators delay the growth of tumours that occur spontaneously in PTEN (phosphatase and tensin homologue deleted on chromosome 10) heterozygous mice.

Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound.

A synthetic sphingolipid related to a ring-constrained hydroxymethyl pyrrolidine analog of FTY720 that was known to starve cancer cells to death was chemically modified to include a series of alkoxy-tethered 3,6-substituted 1,2-pyridazines. These derivatives exhibited excellent antiproliferative activity against eight human cancer cell lines from four different cancer types. A 2.5- to 9-fold reduction in IC50 in these cell lines was observed relative to the lead compound, which lacked the appended heterocycle.

Abstract Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.

Because non-homeostatic proliferation increases anabolic demand, tumor cells reprogram metabolism in ways that support growth. Although tumor cells retain some metabolic flexibility, the constitutive activation of oncogenes and mutation or loss of tumor suppressors limits their metabolic choices and creates nutrient dependencies not present in their normal counterparts (1-8). Identifying and targeting these differences in metabolic wiring will likely be an effective means to limit tumor growth while sparing normal cells. Clearly, different oncogenic mutations activate distinct downstream gene expression programs that drive metabolic reprogramming in ways that favor certain biosynthetic routes. At the same time, oncogenic events occur in the divergent epigenetic landscapes associated with different tissues of origin. As normal lung and pancreatic cells from the same individual contain identical genomes yet exhibit markedly different gene expression patterns, it stands to reason that tumor cells with shared oncogenic drivers but different tissue origins would do the same.

Significance Statement Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. Background Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. Methods To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. Results Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. Conclusions Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

A series of compounds containing pyrrolidine and pyrrolizidine cores with appended hydrophobic substituents were prepared as constrained analogs of FTY720 and phytosphingosine. The effect of these compounds on the viability of cancer cells, on downregulation of the nutrient transport systems, and on their ability to cause vacuolation was studied. An attempt to inhibit HDACs with some phosphate esters of our analogs was thwarted by our failure to reproduce the reported inhibitory action of FTY720-phosphate.

A method is presented for the measurement of ceramide species in biological fluids using flow injection tandem mass spectrometry. Ceramides are important signaling compounds in a number of cell:cell interactions including apoptosis and neurodegeneration. Because of the large number of potential fatty acid constituent moieties on ceramide molecules, a method which accurately distinguishes different chain-length species was required. The present method does not require HPLC separation and is designed to be applicable to high throughput analysis required for clinical studies. We provide a reference range for all measurable ceramide species in normal human plasma and an example of the utility of the assay in providing biomarkers in an in vitro apoptotic cell death study using murine hematopoietic cells treated with daunorubicin.

Inspired by the cytotoxicity of perphenazine toward cancer cells and its ability to activate the serine/threonine protein phosphatase 2A (PP2A), we prepared series of ether-carbon linked analogs of a constrained synthetic sphingolipid analog 3, known for its cytotoxicity, nutrient transporter down-regulation and vacuolation properties, incorporating the tricyclic neuroleptics phenoxazine and phenothiazine to represent hybrid structures with possible synergistic cytotoxic activity. While the original activity of the lead compound 3 was diminished by fusion with the phenoxazine or phenothiazine tethered moieties, the corresponding 3-pyridyltetryl ether analog 10 showed cytotoxicity and nutrient transporter down-regulation similar to the lead compound 3, although it separated these PP2A-dependent phenotypes from that of vacuolation.

Targeting the inability of cancerous cells to adapt to metabolic stress is a promising alternative to conventional cancer chemotherapy. FTY720 (Gilenya), an FDA-approved drug for the treatment of multiple sclerosis, has recently been shown to inhibit cancer progression through the down-regulation of essential nutrient transport proteins, selectively starving cancer cells to death. However, the clinical use of FTY720 for cancer therapy is prohibited because of its capability of inducing immunosuppression (lymphopenia) and bradycardia when phosphorylated upon administration. A prodrug to specifically prevent phosphorylation during circulation, hence avoiding bradycardia and lymphopenia, was synthesized by capping its hydroxyl groups with polyethylene glycol (PEG) via an acid-cleavable ketal linkage. Improved aqueous solubility was also accomplished by PEGylation. The prodrug reduces to fully potent FTY720 upon cellular uptake and induces metabolic stress in cancer cells. Enhanced release of FTY720 at a mildly acidic endosomal pH and the ability to substantially down-regulate cell-surface nutrient transporter proteins in leukemia cells only by an acid-cleaved drug were confirmed. Importantly, the prodrug demonstrated nearly identical efficacy to FTY720 in an animal model of BCR-Abl-driven leukemia without inducing bradycardia or lymphopenia in vivo, highlighting its potential clinical value. The prodrug formulation of FTY720 demonstrates the utility of precisely engineering a drug to avoid undesirable effects by tackling specific molecular mechanisms as well as a financially favorable alternative to new drug development. A multitude of existing cancer therapeutics may be explored for prodrug formulation to avoid specific side effects and preserve or enhance therapeutic efficacy.

Biological membrane fusion is an important part of many cellular processes and is a critical step in the entry of enveloped viruses, such as HIV-1, into cells. For HIV-1 to infect cells, the virus must bind to the cell surface, after which the viral Env protein must be triggered to undergo a conformational change that mediates membrane fusion. Cell-surface binding has long been known to occur via a high-affinity interaction between Env and CD4. However, the cell-surface molecules responsible for triggering the fusion-inducing conformational change in the Env protein have been only recently identified, permitting the study of HIV-1 Env-mediated membrane fusion in much greater detail (for review, see 1). These molecules, termed coreceptors, have been shown to be members of the nine-transmembrane domain receptor family. The most important HIV-1 coreceptors are the chemokine receptors CCR5 and CXCR4 (2–7), although at least nine other chemokine receptors or orphan receptors have been shown to support cellular entry for subsets of HIV-1 or SIV strains (3,5,8–11). The ability of a given virus strain to utilize particular chemokine receptors is a major determinant of cellular tropism. Thus, it is desirable to identify the receptors used by virus strains in a rapid, quantitative, and reproducible manner.

Our recent work demonstrates that inactivating mutations in phosphatase and tensin homolog (PTEN) are sufficient to drive macropinocytosis in the context of AMP-activated protein kinase (AMPK) activation. Given that blocking macropinocytosis limits PTEN-deficient prostate tumor growth, AMPK or phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitors could have therapeutic value in castration-resistant prostate cancer patients, particularly when used in combination with standard of care therapies. Abbreviations: ATG5: autophagy related 5; NHE: Na(+)/H(+) exchanger; PAK1: p21-activated kinase 1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; PIP2: phosphatidylinositol (4,5)-bisphosphate; RAC1: Rac family small GTPase 1.

Abstract Tumor cells have devised unique metabolic strategies to garner enough nutrients to sustain continuous growth and cell division. Oncogenic mutations may alter metabolic pathways to unlock new sources of energy, and cells take the advantage of various scavenging pathways to ingest material from their environment. These changes in metabolism result in a metabolic profile that, in addition to providing the building blocks for macromolecules, can also influence cell signaling pathways to promote tumor initiation and progression. Understanding what pathways tumor cells use to synthesize the materials necessary to support metabolic growth can pave the way for new cancer therapeutics. Potential strategies include depriving tumors of the materials needed to grow or targeting pathways involved in dependencies that arise by virtue of their altered metabolis.

Abstract A wealth of data has consistently demonstrated that a diverse faculty maximizes productivity and innovation in the research enterprise and increases the persistence and success of groups that are underrepresented in STEM. While the diversity of students in graduate programs has steadily increased, faculty diversity, particularly in the biomedical sciences, continues to remain relatively flat. Several issues contribute to this mismatch between the pipeline and the professoriate including biases in search and hiring practices, lack of equity and equal opportunities for individuals from underrepresented backgrounds, and unwelcoming campus climates that lead to marginalization and isolation in academic life. A comprehensive approach that addresses these challenges is necessary for institutions of higher education to achieve their faculty diversity goals and create a climate where individuals from all groups feel welcomed and succeed. This article focuses on the first step in this approach–diversifying faculty recruitment through adopting search practices that generate an applicant pool that matches national availability, ensures equity in evaluation and hiring practices, and promotes inclusion and belonging in the hiring experience. These strategies have been recently used at the University of California, Irvine's School of Biological Sciences and while the long‐term impact remains unknown, short‐term outcomes in recruitment and hiring have demonstrated significant improvement over previous years.

Commentary to: Identification of Akt-selective cytotoxic compounds that enhance cytotoxic responses to rapamycin Jennifer F. Barger, Catherine A. Gallo, Kyle A. Torni, Lisa Merk, William L. Seibel, Sandra Nelson and David R. Plas

Abstract Rab7 is a small GTPase that mediates endosomal functions and regulates T cell homeostasis. Here, we have addressed the B cell-inherent role of Rab7 in antibody responses and underlying mechanisms by constructing Igh+/Cγ1-cre Rab7fl/fl mice. These mice displayed normal B cell and T cell development and survival, and were knocked-out of the Rab7 gene specifically in B cells activated to undergo IgH germline Iγ1-Sγ1-Cγ1 transcription. This transcription process is induced by IL-4 plus a primary CSR-inducing stimulus, and is required for antibody switching from IgM to IgG1, but not to other Ig isotypes. Igh+/Cγ1-cre Rab7fl/fl mice had virtually abrogated levels of IgG1, but normal levels of IgM and other switched Ig isotypes, and failed to mount specific IgG1 responses to T-dependent or T-independent antigens. They showed markedly reduced IgG1+ B cells and antibody-forming cells, with unaltered B cell proliferation, survival and differentiation into germinal center B cells and plasma cells. Purified naïve B cells from these mice were defective in CSR to IgG1, despite normal proliferation and plasma cell differentiation. These together with the unaltered germline transcription but significantly decreased expression of AID (essential for CSR and SHM) demonstrate that B cell Rab7 mediates CSR and, therefore, the antibody response by upregulating AID, likely by regulating intracellular membranes that facilitate signal transduction for activation of specific transcription factors.

Abstract IgH class switch DNA recombination (CSR) is central to the maturation of the antibody response, as it diversifies antibody biologic effector functions. By constructing conditional KO Igh+/Cγ1-creRab7fl/fl mice, we have previously identified a B cell-intrinsic role for Rab7, a small GTPase that regulates endosome maturation, in CSR and antibody responses. In Igh+/Cγ1-creRab7fl/fl B cells, Rab7 gene is ablated within 48 h of stimulation by IL-4 plus CD154 or LPS, resulting in reduced CSR to IgG1 and IgE. Here, we have addressed the mechanisms underlying the role of Rab7 in CSR. Once Rab7 was ablated, B cells were also defective in CSR to IgA, indicating that Rab7 regulates the central CSR machinery in an Ig isotype-independent manner. Indeed, expression of AID, as essential to CSR, was reduced in Rab7 KO B cells and enforced AID expression rescued CSR. Finally, super-resolution imaging of CD154-stimulated B cells revealed that Rab7 formed foci, within which CD40 was enriched. These findings, together with our demonstration that Rab7 mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in specific signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Wiley's Corporate Citizenship initiative seeks to address the environmental, social, economic, and ethical challenges faced in our business and which are important to our diverse stakeholder groups.Since launching the initiative, we have focused on sharing our content with those in need, enhancing community philanthropy, reducing our carbon impact, creating global guidelines and best practices for paper use, establishing a vendor code of ethics, and engaging our colleagues and other stakeholders in our efforts.Follow our progress at www.