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Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy

2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês

10.1074/jbc.m805920200

1083-351X

Sophie Pattingre, Chantal Bauvy, Stéphane Carpentier, Thierry Levade, Beth Levine, Patrice Codogno,

Erythrocyte Function and Pathophysiology

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C2-ceramide and C6-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr69, Ser70, and Ser87, located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress. Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C2-ceramide and C6-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr69, Ser70, and Ser87, located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress. Macroautophagy (referred to below as “autophagy”) is a vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is conserved in eukaryotic cells (1Levine B. Klionsky D.J. Dev. Cell.. 2004; 6: 463-477Google Scholar, 2Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Nature.. 2008; 451: 1069-1075Google Scholar, 3Xie Z. Klionsky D.J. Nat. Cell Biol... 2007; 9: 1102-1109Google Scholar). Autophagy is initiated by the formation of a multimembrane-bound autophagosome that engulfs cytoplasmic proteins and organelles. The last stage in the process results in fusion with the lysosomal compartments, where the autophagic cargo undergoes degradation. Basal autophagy is important in controlling the quality of the cytoplasm by removing damaged organelles and protein aggregates. Inhibition of basal autophagy in the brain is deleterious, and leads to neurodegeneration in mouse models (4Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Nature.. 2006; 441: 885-889Google Scholar, 5Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Nature.. 2006; 441: 880-884Google Scholar). Stimulation of autophagy during periods of nutrient starvation is a physiological response present at birth and has been shown to provide energy in various tissues of newborn pups (6Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature.. 2004; 432: 1032-1036Google Scholar). In cultured cells, starvation-induced autophagy is an autonomous cell survival mechanism, which provides nutrients to maintain a metabolic rate and level of ATP compatible with cell survival (7Lum J.J. Bauer D.E. Kong M. Harris M.H. Li C. Lindsten T. Thompson C.B. Cell.. 2005; 120: 237-248Google Scholar). In addition, starvation-induced autophagy blocks the induction of apoptosis (8Boya P. Gonzalez-Polo R.A. Casares N. Perfettini J.L. Dessen P. Larochette N. Metivier D. Meley D. Souquere S. Yoshimori T. Pierron G. Codogno P. Kroemer G. Mol. Cell. Biol... 2005; 25: 1025-1040Google Scholar). In other contexts, such as drug treatment and a hypoxic environment, autophagy has also been shown to be cytoprotective in cancer cells (9Levine B. Nature.. 2007; 446: 745-747Google Scholar, 10Mathew R. Karantza-Wadsworth V. White E. Nat. Rev. Cancer.. 2007; 7: 961-967Google Scholar). However, autophagy is also part of cell death pathways in certain situations (11Maiuri M.C. Zalckvar E. Kimchi A. Kroemer G. Nat. Rev. Mol. Cell. Biol... 2007; 8: 741-752Google Scholar). Autophagy can be a player in apoptosis-independent type-2 cell death (type-1 cell death is apoptosis), also known as autophagic cell death. This situation has been shown to occur when the apoptotic machinery is crippled in mammalian cells (12Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol... 2004; 6: 1221-1228Google Scholar, 13Yu L. Alva A. Su H. Dutt P. Freundt E. Welsh S. Baehrecke E.H. Lenardo M.J. Science.. 2004; 304: 1500-1502Google Scholar). Autophagy can also be part of the apoptotic program, for instance in tumor necrosis factor-α-induced cell death when NF-κB is inhibited (14Djavaheri-Mergny M. Amelotti M. Mathieu J. Besancon F. Bauvy C. Souquere S. Pierron G. Codogno P. J. Biol. Chem... 2006; 281: 30373-30382Google Scholar), or in human immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T cells (15Espert L. Denizot M. Grimaldi M. Robert-Hebmann V. Gay B. Varbanov M. Codogno P. Biard-Piechaczyk M. J. Clin. Invest... 2006; 116: 2161-2172Google Scholar). Moreover autophagy has recently been shown to be required for the externalization of phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of apoptotic cells (16Qu X. Zou Z. Sun Q. Luby-Phelps K. Cheng P. Hogan R.N. Gilpin C. Levine B. Cell.. 2007; 128: 931-946Google Scholar). The complex relationship between autophagy and apoptosis reflects the intertwined regulation of these processes (17Codogno P. Meijer A.J. Cell Death Differ.. 2005; 12: 1509-1518Google Scholar, 18Gozuacik D. Kimchi A. Curr. Top. Dev. Biol... 2007; 78: 217-245Google Scholar). Many signaling pathways involved in the regulation of autophagy also regulate apoptosis. This intertwining has recently been shown to occur at the level of the molecular machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been shown to inhibit starvation-induced autophagy by interacting with the autophagy protein Beclin 1 (19Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell.. 2005; 122: 927-939Google Scholar). Beclin 1 is one of the Atg proteins conserved from yeast to humans (it is the mammalian orthologue of yeast Atg6) and is involved in autophagosome formation (20Levine B. Yuan J. J. Clin. Invest... 2005; 115: 2679-2688Google Scholar). Beclin 1 is a platform protein that interacts with several different partners, including hVps34 (class III phosphatidylinositol 3-kinase), which is responsible for the synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is important for events associated with the nucleation of the isolation membrane before it elongates and closes to form autophagosomes in response to other Atg proteins, including the Atg12 and LC3 2The abbreviations used are: LC3, Light Chain 3; C2-Cer, C2-ceramide; C2-DHCer, C2-dihydroceramide; C6-Cer, C6-ceramide; C6-DHCer, C6-dihydroceramide; DAG, diacylglycerol; dnJNK1, dominant-negative JNK1; FB1, fumonisin B1; EBSS, Earle's balanced salt solution; Tam, tamoxifen; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ER, endoplasmic reticulum; PBS, phosphate-buffered saline; CM, complete medium; mTOR, mammalian target of rapamycin. (microtubule-associated protein light chain 3 is the mammalian orthologue of the yeast Atg8) ubiquitin-like conjugation systems (3Xie Z. Klionsky D.J. Nat. Cell Biol... 2007; 9: 1102-1109Google Scholar, 21Ohsumi Y. Nat. Rev. Mol. Cell. Biol... 2001; 2: 211-216Google Scholar). Various partners associated with the Beclin 1 complex modulate the activity of hVps34. For instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1, and Bif-1 all up-regulate it (22Cao Y. Klionsky D.J. Cell Res... 2007; 17: 839-849Google Scholar, 23Pattingre S. Espert L. Biard-Piechaczyk M. Codogno P. Biochimie (Paris).. 2008; 90: 313-323Google Scholar). In view of the intertwining between autophagy and apoptosis, it is noteworthy that Beclin 1 belongs to the BH3-only family of proteins (24Erlich S. Mizrachy L. Segev O. Lindenboim L. Zmira O. Adi-Harel S. Hirsch J.A. Stein R. Pinkas-Kramarski R. Autophagy.. 2007; 3: 561-568Google Scholar, 25Maiuri M.C. Le Toumelin G. Criollo A. Rain J.C. Gautier F. Juin P. Tasdemir E. Pierron G. Troulinaki K. Tavernarakis N. Hickman J.A. Geneste O. Kroemer G. EMBO J.. 2007; 26: 2527-2539Google Scholar, 26Oberstein A. Jeffrey P.D. Shi Y. J. Biol. Chem... 2007; 282: 13123-13132Google Scholar). However, and unlike most of the proteins in this family, Beclin 1 is not able to trigger apoptosis when its expression is forced in cells (27Levine B. Sinha S. Kroemer G. Autophagy.. 2008; 4: 600-606Google Scholar). A BH3-mimetic drug, ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger autophagy by mirroring the effect of starvation (25Maiuri M.C. Le Toumelin G. Criollo A. Rain J.C. Gautier F. Juin P. Tasdemir E. Pierron G. Troulinaki K. Tavernarakis N. Hickman J.A. Geneste O. Kroemer G. EMBO J.. 2007; 26: 2527-2539Google Scholar). The sphingolipids constitute a family of bioactive lipids (28Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell. Biol... 2008; 9: 139-150Google Scholar, 29Hla T. Semin. Cell Dev. Biol... 2004; 15: 513-520Google Scholar, 30Kolesnick R. J. Clin. Invest... 2002; 110: 3-8Google Scholar, 31Ogretmen B. Hannun Y.A. Nat. Rev. Cancer.. 2004; 4: 604-616Google Scholar, 32Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol... 2003; 4: 397-407Google Scholar) of which several members, such as ceramide and sphingosine 1-phosphate, are signaling molecules. These molecules constitute a “sphingolipid rheostat” that determines the fate of the cell, because in many settings ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic effect (31Ogretmen B. Hannun Y.A. Nat. Rev. Cancer.. 2004; 4: 604-616Google Scholar, 32Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol... 2003; 4: 397-407Google Scholar). However, ceramide is also engaged in a wide variety of other cell processes, such as the formation of exosomes (33Trajkovic K. Hsu C. Chiantia S. Rajendran L. Wenzel D. Wieland F. Schwille P. Brügger B. Simons M. Science.. 2008; 319: 1244-1247Google Scholar), differentiation, cell proliferation, and senescence (34Hannun Y.A. Science.. 1996; 274: 1855-1859Google Scholar). Recently we showed that both ceramide and sphingosine 1-phosphate are able to stimulate autophagy (35Lavieu G. Scarlatti F. Sala G. Carpentier S. Levade T. Ghidoni R. Botti J. Codogno P. J. Biol. Chem... 2006; 281: 8518-8527Google Scholar, 36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar). It has also been shown that ceramide triggers autophagy in a large panel of mammalian cells (37Daido S. Kanzawa T. Yamamoto A. Takeuchi H. Kondo Y. Kondo S. Cancer Res... 2004; 64: 4286-4293Google Scholar, 38Zeng X. Overmeyer J.H. Maltese W.A. J. Cell Sci... 2006; 119: 259-270Google Scholar, 39Zheng W. Kollmeyer J. Symolon H. Momin A. Munter E. Wang E. Kelly S. Allegood J.C. Liu Y. Peng Q. Ramaraju H. Sullards M.C. Cabot M. Merrill Jr., A.H. Biochim. Biophys. Acta.. 2006; 1758: 1864-1884Google Scholar). However, elucidation of the mechanism by which ceramide stimulates autophagy is still in its infancy. We have previously demonstrated that ceramide induces autophagy in breast and colon cancer cells by inhibiting the Class I phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central role in inhibiting autophagy (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar). Inhibition of mTOR is another hallmark of starvation-induced autophagy (17Codogno P. Meijer A.J. Cell Death Differ.. 2005; 12: 1509-1518Google Scholar). This finding led us to investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results presented here show that ceramide is more potent than starvation in dissociating the Beclin 1-Bcl-2 complex (see Ref. 40Wei Y. Pattingre S. Sinha S. Bassik M. Levine B. Mol. Cell.. 2008; 30: 678-688Google Scholar). This dissociation is dependent on three phosphorylation sites (Thr69, Ser70, and Ser87) located in a non-structural loop of Bcl-2. Ceramide induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2. Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation, and thus the induction of autophagy by ceramide. These findings help to explain how autophagy is regulated by a major lipid second messenger. Reagents—C2-Cer, C6-Cer, C2-DHCer, and C6-DHCer were from Sigma and were dissolved in ethanol before use. FB1, Myriocin, and TAM were purchased from Biomol. Cell culture medium, Lipofectamine 2000, and fetal bovine serum were from Invitrogen. The radioisotope l-[U-14C]valine (256 mCi/mmol), the ECL™ Western blotting detection kit, and the donkey anti-rabbit antibody were purchased from Amersham Biosciences. Goat anti-mouse and swine anti-goat antibodies were obtained from Bio-Rad and Caltag (Burlingame, CA), respectively. Mouse monoclonal anti-p62 antibody was obtained from BD Biosciences. Mouse monoclonal anti-Bcl-2 and goat polyclonal anti-Beclin 1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit monoclonal antibodies against p-Bcl-2, p-JNK, and total JNK were obtained from Cell Signaling. Rabbit polyclonal anti-Beclin 1 antibody was obtained from Novus Biologicals. Rabbit polyclonal anti-LC3 antibody was obtained as previously described (41Chaumorcel M. Souquere S. Pierron G. Codogno P. Esclatine A. Autophagy.. 2008; 4: 46-53Google Scholar). Cell Culture—Human breast cancer cell line MCF-7 cells stably transfected with beclin 1 (MCF7.beclin 1) were cultured as previously described (42Furuya N. Yu J. Byfield M. Pattingre S. Levine B. Autophagy.. 2005; 1: 46-52Google Scholar). HeLa cells were obtained from ATCC, and maintained at 37 °C in 10% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin. HeLa GFP-LC3 cells, kindly provided by A. M. Tolkovsky (University of Cambridge, UK), were cultured in the presence of 200 μg/ml G418. The human colon cancer cell line HT-29 transfected either with empty vector or vector encoding Bcl-2 were kindly provided by M. T. Dimanche-Boitrel (INSERM U620, France), and cultured at 37 °C in 10% CO2 in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin plus 200 μg/ml G418. MEF WT and Atg5–/– were kindly given by N. Mizushima (Tokyo Medical and Dental University, Japan), and cultured as previously described (6Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature.. 2004; 432: 1032-1036Google Scholar). The trypan blue exclusion test showed that cell viability was greater than 90% under all the experimental conditions used. Quantification of Endogenous Ceramide—Ceramide was determined using Escherichia coli DAG kinase as previously reported (43Bielawska A. Perry D.K. Hannun Y.A. Anal. Biochem... 2001; 298: 141-150Google Scholar). The E. coli strain was kindly provided by Drs. D. K. Perry and Y. A. Hannun (Medical University of South Carolina, Charleston, SC). Beclin 1 and Bcl-2 Co-immunoprecipitation—To immunoprecipitate endogenous Beclin 1 and endogenous Bcl-2 in HeLa cells or stably transfected Beclin 1 in MCF.7 cells, the cells were lysed in CHAPS lysis buffer (20 mm Tris, pH 7.4, 137 mm NaCl, 2 mm EDTA, 10% glycerol, and 2% CHAPS) for 3 h at 4 °C, and immunoprecipitation was performed overnight at 4 °C with a goat polyclonal antibody (1:80 dilution, Santa Cruz Biotechnology). Protein A-Sepharose beads (Amersham Biosciences) were added for 2 h at 4 °C, washed twice with 137 mm NaCl CHAPS wash buffer (20 mm Tris, pH 7.4, 137 mm NaCl, 2 mm EDTA, 10% glycerol, and 0.5% CHAPS), and twice with 274 mm NaCl CHAPS wash buffer. Anti-Beclin 1 immunoprecipitates were subjected to SDS-PAGE, and Bcl-2 was detected by immunoblot analysis (19Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell.. 2005; 122: 927-939Google Scholar). GFP-LC3 Assay—The assay was performed in HeLa cells either stably transfected with rat GFP-LC3 (kindly provided by T. Yoshimori, Osaka University, Japan) or in transiently transfected with MCF-7. beclin 1, HT-29, and HeLa cells using Lipofectamine 2000 (19Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell.. 2005; 122: 927-939Google Scholar, 42Furuya N. Yu J. Byfield M. Pattingre S. Levine B. Autophagy.. 2005; 1: 46-52Google Scholar). HeLa cells were also transfected either with human GFP-LC3B or the mutant GFP-LC3BΔG (kindly provided by I. Tanida, National Institute of Infectious Disease, Tokyo, Japan). The vector, pcDNA3-FLAG-MKK7-JNK1(APF) (dominant-negative JNK1, dnJNK1), was provided by R. J. Davis (Howard Hughes Medical Institute) (44Lei K. Nimnual A. Zong W.X. Kennedy N.J. Flavell R.A. Thompson C.B. Bar-Sagi D. Davis R.J. Mol. Cell. Biol... 2002; 22: 4929-4942Google Scholar). When required, co-transfection with a plasmid encoding GFP-LC3 and a vector encoding for dnJNK1, or the empty vector pcDNA3, was performed in MCF-7.beclin 1 cells (ratio GFP-LC3:vector, 1:3). Prior to analysis, the cells were starved for 4 h in Earle's balanced salt solution (EBSS, starvation medium), maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum (control medium), or treated as described in the text. Autophagy was then measured by light microscopic counting of cells with GFP-LC3 puncta as described previously (42Furuya N. Yu J. Byfield M. Pattingre S. Levine B. Autophagy.. 2005; 1: 46-52Google Scholar). A minimum of 50–100 cells per sample was counted in triplicate samples per condition per experiment. Analysis of Protein Degradation—HT-29 cells were incubated for 24 h at 37 °C with 0.2 μCi/ml l-[14C]valine. Three hours before the end of the radiolabeling period, cells were exposed to increasing concentrations of C2-Cer or C2-DHCer, and when required 100 nm FB1 was added, also 3 h before the end of radiolabeling. At the end of radiolabeling period, the cells were washed three times with PBS, pH 7.4. Cells were then incubated in complete medium supplemented with 10 mm cold valine. After incubating for 1 h, by which time short-lived proteins are degraded, the medium was replaced with fresh nutrient-free medium (EBSS plus 0.1% of bovine serum albumin and 10 mm cold valine), and the incubation was continued for an additional 4 h. Cells and radiolabeled proteins from the 4-h chase medium were precipitated in trichloroacetic acid at a final concentration of 10% (v/v) at 4 °C. The precipitated proteins were separated from the soluble radioactivity by centrifugation at 600 × g for 10 min, and then dissolved in 0.5 ml of 0.2 n NaOH. Radioactivity was determined by liquid scintillation counting. Protein degradation was calculated by dividing the acid-soluble radioactivity recovered from both cells and medium by the radioactivity contained in precipitated proteins from both cells and medium (45Bauvy C. Meijer A.J. Codogno P. Methods Enzymol.2008Google Scholar). Immunoblotting—After being resolved by SDS-PAGE, proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non fat dry milk in PBST (PBS and 0.1% Tween 20) for 1 h at room temperature, and then incubated with the appropriate primary antibody overnight at 4 °C in PBST. The antibody dilutions were as follows: anti-LC3 1:10,000; anti-JNK1-P(Thr183/Tyr185), anti-JNK1, anti-Bcl-2-P(Ser70), anti-Bcl-2 1:1000; anti-p62 1:2000; anti-Beclin 1 1:2000; anti-actin 1:5000). After three washes in PBST, the membrane was incubated for 1 h at room temperature with the appropriate horseradish peroxidase-labeled secondary antibody. Bound antibodies were detected using ECL. Statistical Analysis—Statistical analysis of the differences between the groups was performed using Student's t test. p < 0.05 was considered statistically significant. Ceramide Induces Autophagy in Several Cell Lines—We have previously reported that the permeant short-chain C2-ceramide (C2-Cer) induces autophagy both in MCF-7 breast cancer cells and in HT-29 colon carcinoma cells via the production of endogenous long-chain ceramides (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar). Autophagy was monitored by electron microscopy and by measuring the autophagic flux by analyzing the rate of long-lived protein degradationsensitive to 3-methyladenine, an inhibitor of the formation of autophagosomes (46Seglen P.O. Gordon P.B. Proc. Natl. Acad. Sci. U. S. A... 1982; 79: 1889-1892Google Scholar). In a first series of experiments, we extended these findings to HeLa GFP-LC3 cells by analyzing the formation of GFP-LC3 puncta. During autophagy, the LC3 protein is relocated to the autophagosomal membranes as a result of C-terminal conjugation to phosphatidylethanolamine. Thus, the accumulation of GFP-LC3 puncta provides an effective way of detecting autophagosomes (47Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J.. 2000; 19: 5720-5728Google Scholar). C2-Cer treatment induced the accumulation of GFP-LC3 puncta in HeLa GFP-LC3 cells, whereas treatment with C2-dihydroceramide (C2-DHCer), a C2-Cer analogue that does not induce autophagy in MCF-7 and HT-29 cells (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar), failed to do so (Fig. 1A). To check that the formation of GFP-LC3 puncta in response to C2-Cer was not simply due to clumping of the chimeric protein induced by C2-Cer treatment, we repeated the experiment in cells transfected with the mutant GFP-LC3ΔG, which is unable to support the formation of autophagosomes (48Tanida I. Yamaji T. Ueno T. Ishiura S. Kominami E. Hanada K. Autophagy.. 2008; 4: 131-134Google Scholar). Under these conditions, the mutant chimeric protein did not form puncta in response to C2-Cer treatment (Fig. 1B). Furthermore, C2-Cer was also unable to induce GFP-LC3 puncta in Atg5–/– MEF cells (supplemental Fig. S1A) that lack the essential autophagy protein Atg5 (49Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol... 2001; 152: 657-668Google Scholar). Unlike C2-Cer, C2-DHCer did not induce autophagy in wt MEF (supplemental Fig. S1A). The accumulation of GFP-LC3 puncta was also observed in MCF-7.beclin 1 cells. This cell line was engineered from a MCF-7 cell population with a low level of Beclin 1 expression and provides a convenient tool to for investigating autophagy, because the expression of Beclin 1 is under the control of a Tet-OFF system (50Liang X.H. Yu J. Brown K. Levine B. Cancer Res... 2001; 61: 3443-3449Google Scholar). In the absence of tetracycline we observed that C2-Cer, but not C2-DHCer, induced the formation of GFP-LC3 puncta (supplemental Fig. S2A). To confirm that the formation of GFP-LC3 puncta induced by C2-Cer was indeed attributable to stimulation of the autophagic pathway, i.e. to increases in both the formation of autophagosomes and their consumption by the lysosomal compartment, we analyzed the effect of C2-Cer on two independent assays of autophagic flux, the degradation of p62 and that of [14C]valine-labeled long-lived protein. In HeLa GFP-LC3 cells, we observed that protein p62, a substrate for starvation-induced autophagy (51Mizushima N. Yoshimori T. Autophagy.. 2007; 3: 542-545Google Scholar), was just as well degraded in nutrient-free medium (EBSS) as in C2-Cer-treated cells (Fig. 1C). In contrast, cells treated with either complete media (CM) or C2-DHCer were unable to stimulate p62 degradation (Fig. 1C). In MCF-7.beclin 1 cells, C2-Cer treatment increased the degradation of long-lived proteins sensitive to 3-methyladenine (supplemental Fig. S2B). Our previous results had shown that the induction of autophagy depends on the elongation of C2-Cer to form long chain ceramides (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar). In HeLa GFP-LC3 (Fig. 1D), MEF (supplemental Fig. S1B) and MCF-7.beclin 1 cells (supplemental Fig. S2C) an accumulation of long-chain ceramide was observed after C2-Cer treatment. The elongation step depends on the activity of ceramide synthase is sensitive to FB1 (52Merrill Jr., A.H. J. Biol. Chem... 2002; 277: 25843-25846Google Scholar). In all the cell lines used, FB1 treatment (100 nm) blocked both the elongation of C2-Cer and C2-Cer-induced autophagy (Fig. 1 and supplemental Figs. S1 and S2). In line with our previous findings (35Lavieu G. Scarlatti F. Sala G. Carpentier S. Levade T. Ghidoni R. Botti J. Codogno P. J. Biol. Chem... 2006; 281: 8518-8527Google Scholar, 36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar), these results strongly suggest that endogenous long-chain ceramides are potent stimulators of autophagy. To further substantiate the role of long-chain ceramides in autophagy, we repeated the above experiments using the short chain C6-Cer, which is a good substrate for generating the sphingosine backbone for long-chain ceramides (53Kitatani K. Idkowiak-Baldys J. Hannun Y.A. Cell Signal.. 2008; 20: 1010-1018Google Scholar). We found that exposing HeLa GFP-LC3 cells to 100 μm of C6-Cer (for 4 h) significantly increased autophagy assessed by counting the number of puncta per cell (Fig. 2A). In contrast, its inactive counterpart, C6-DHCer was unable to regulate autophagy (Fig. 2A). During the course of these experiments we observed an accumulation of long-chain ceramides in C6-Cer-treated cells. Once again FB1 treatment blocked both the elongation of C6-Cer (Fig. 2C) and C6-Cer-induced autophagy (data not shown). Stimulation of autophagy and accumulation of long-chain ceramides were also observed when C6-Cer was used at a lower concentration (40 μm) for different periods of time (supplemental Fig. S3). C6-Cer also increased the autophagic flux as determined by analyzing the rate of long-lived protein degradation (data not shown). We have previously shown that Tamoxifen (Tam) and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol stimulate autophagy in MCF-7 by increasing the level of endogenous long-chain ceramides (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar). These treatments both increased the formation of long-chain ceramides presumably either by stimulating the de novo ceramide synthesis in the ER, or by inhibiting the conversion of ceramide to glucosylceramide (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar, 54Bleicher R.J. Cabot M. Biochim. Biophys. Acta.. 2002; 1585: 172-178Google Scholar). Here we show that Tam and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol stimulated autophagy (Fig. 2B) and increased the formation of long-chain ceramides (Fig. 2C) in HeLa-GFP-LC3 cells. Moreover, Myriocin, a potent inhibitor of serine palmitoyltransferase the key rate-limiting enzyme of the de novo synthesis of ceramide (52Merrill Jr., A.H. J. Biol. Chem... 2002; 277: 25843-25846Google Scholar), blocked both autophagy and the accumulation of long-chain ceramides in Tam-treated HeLa GFP-LC3 cells (Fig. 2, B and C). In a previous study, we demonstrated that ceramide stimulates autophagy by interfering with the activation of Akt/PKB upstream of mTOR, a key regulator of autophagy signaling (36Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem... 2004; 279: 18384-18391Google Scholar). However, we wonder whether ceramide could also stimulate autophagy by directly modulating the activity of the Atg machinery involved in autophagosome formation. Ceramide Induces Dissociation of the Beclin 1-Bcl-2 Complex—Autophagy is tightly regulated by the activity of the Beclin 1 complex in initiating the formation of autophagosomes (22Cao Y. Klionsky D.J. Cell Res... 2007; 17: 839-849Google Scholar, 23Pattingre S. Espert L. Biard-Piechaczyk M. Codogno P. Biochimie (Paris).. 2008; 90: 313-323Google Scholar). In this complex, the anti-apoptotic protein Bcl-2 represses autophagy. Dissociation of the Beclin 1-Bcl-2 complex stimulates autophagy whether induced by starvation or in response to BH3 mimetic molecules (19Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell.. 2005; 122: 927-939Google Scholar, 25Maiuri M.C. Le Toumelin G. Criollo A. Rain J.C. Gautier F. Juin P. Tasdemir E. Pierron G. Troulinaki K. Tavernarakis N. Hickman J.A. Geneste O. Kroemer