Regulation of Autophagy by Sphingosine Kinase 1 and Its Role in Cell Survival during Nutrient Starvation
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m506182200
ISSN1083-351X
AutoresGrégory Lavieu, Francesca Scarlatti, G Sala, Stéphane Carpentier, Thierry Levade, Riccardo Ghidoni, Joëlle Botti, Patrice Codogno,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThe sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1wt-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation. The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1wt-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation. Macroautophagy (hereafter referred to as autophagy) is a lysosomal catabolic pathway for macromolecules and organelles (1Seglen P.O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (370) Google Scholar, 2Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 3Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (3014) Google Scholar). The discovery of ATG 2The abbreviations used are: ATG, autophagy-related genes; C2-Cer, C2-ceramide; DGK, diacylglycerol kinase; DMEM, Dulbecco's modified Eagle's medium; 4E-BP1, eukaryotic translational initiation factor 4E-binding protein-1; FB1, fumonisin B1; GFP, green fluorescent protein; LC3, light chain 3; 3MA, 3-methyladenine; PBS, phosphate-buffered saline; Akt/PKB, protein kinase B; p70S6K, p70S6 kinase; PI3K, phosphatidylinositol 3-kinase; PLD, phospholipase D; S1P, sphingosine 1-phosphate; SK, sphingosine kinase; mTOR, mammalian target of rapamycin; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone; DMS, dimethylsphingosine; HA, hemagglutinin; siRNA, small interference RNA; PIC, Protease Inhibitor Mixture; wt, wild type; PARP, poly(ADP-ribose) polymerase. in yeast has revealed the evolutionarily conserved mechanism for the formation of autophagosomes that sequester cytoplasmic material before they fuse with the endo/lysosomal compartment (4Klionsky D.J. 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Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1012) Google Scholar) are able to trigger autophagy but with different outcomes on cancer cell survival and death. C2-Cer and DMS were from Calbiochem (VWR International). Fumonisin B1 (FB1) was purchased from Sigma. 3MA was from Fluka. z-VAD-fmk was from Apotech. Cell culture medium and fetal bovine serum were from Invitrogen. Nitrocellulose membranes were from Schleicher & Schüll. Ceramide from porcine brain, used as an internal standard, was from Avanti Polar Lipids Inc. The radioisotopes l-[U-14C]valine (5.47 GBq/mmol) and [γ32-P]ATP (110 TBq/mmol) and [9,10-3H]myristic acid (1.69 TBq/mmol), the ECL™ Western blotting detection kit, and the donkey anti-rabbit antibody were purchased from BD Biosciences. The mouse monoclonal anti-Beclin1 was from Amersham Bioscience; the mouse monoclonal anti-PARP and the rabbit polyclonal anti-phospho-PKB Ser473 were from Cell Signaling. Goat anti-PKB was from Santa Cruz Biotechnology. The mouse monoclonal anti-actin was from Chemicon International Inc. The rabbit polyclonal anti-p70S6K, anti-phospho-p70S6K Thr389, anti-phospho-S6 Ser235, and anti-4E-BP1 were from Ozyme, and anti-FLAG M2 was from Sigma. The rabbit polyclonal anti-Atg7 was kindly provided by W. A. Dunn (University of Florida, Gainesville). Goat anti-mouse and swine anti-goat antibodies were obtained from Bio-Rad and Caltag, respectively. The plasmid expression vectors for human sphingosine kinase 1 and catalytically inactive mutant human sphingosine kinase 1 (pcDNA3-hSK1-FLAG and pcDNA3-hSK1G82D-FLAG) were a gift from S. Pitson and B. Wattenberg (Hanson Institute, Adelaide, Australia) (40Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The plasmid expression vector encoding for GFP-LC3 (pEGFP-LC3) was kindly provided by T. Yoshimori (National Institute of Genetics, Mishima, Japan). The plasmid expression vectors for PLD1 and catalytically inactive mutant PLD1 (pCGN-HA-hPLD1 and pCGN-HA-hPLD1K898R) were kindly given by M. Frohman (SUNY, New York). Human breast cancer MCF-7 cells were maintained at 37 °C in 10% CO2 in DMEM, supplemented with 10% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin as previously reported (32Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Beclin 1 MCF-7/tet-off (MCF-7.beclin 1) cells kindly provided by B. Levine (University of Texas Southwest Medical Center, Dallas) were used during starvation-induced autophagy. Briefly, cells were maintained in DMEM with 200 μg/ml hygromycin B and 2 μg/ml tetracycline (41Liang X.H. Yu J. Brown K. Levine B. Cancer Res. 2001; 61: 3443-3449PubMed Google Scholar). Expression of Beclin 1 was analyzed by Western blotting 5 days after tetracycline withdrawal. Starvation was induced by incubating cells in nutrient-free medium (Hanks' balanced salt solution plus 0.1% bovine serum albumin). Cells were transfected by using FuGENE 6 transfection reagent (Roche Applied Science), as recommended by the manufacturer. Briefly, cells (0.5 × 106 cells/well) were plated in 6-well plates. Two days later, 1 μg of plasmid and 3 μl of FuGENE 6 were suspended in 100 μl of DMEM and added to the culture medium. When cells were co-transfected with SK1, PLD1 (or empty vector), and GFP-LC3 vectors, the transfection efficiency was 40% as determined by counting GFP-positive cells. Cells were analyzed 24 or 48 h after co-transfection as detailed below. SK1 and Atg7 knockdowns were accomplished by transfecting MCF-7/GFP-LC3 cells with siRNAs. The RNAi target sequences were: sense, 5′-GGGCAAGGCCUUGCAGCUC-3′ for SK1 (42Bektas M. Jolly P.S. Muller C. Eberle J. Spiegel S. Geilen C.C. Oncogene. 2005; 24: 178-187Crossref PubMed Scopus (122) Google Scholar) and sense, 5′-CCAACACACUCGAGUCUUU-3′ for Atg7. As a control, siRNA targeting the unrelated protein phosphomannomutase-2 (sense, 5′-CUGGGAAAUGAUGUGGUUG) was used. siRNA were purchased from Eurogenetec (Seraing, Belgium). MCF-7 cells were seeded at 8 × 104/cm2. After 24 h, cells were transfected for 4 h with siRNA using Oligofectamine (Invitrogen) as recommended by the manufacturer. Cells were cultured for 24 h before analysis. Cells were disrupted in 4 m guanidine thiocyanate, and total RNA was isolated by sedimentation in cesium chloride. RNA was transcribed with superscript II (Invitrogen). SK1 was amplified by the following specific primers (42Bektas M. Jolly P.S. Muller C. Eberle J. Spiegel S. Geilen C.C. Oncogene. 2005; 24: 178-187Crossref PubMed Scopus (122) Google Scholar): forward, 5′-ATCCAGAAGCCCCTGTGTAGCCTCC-3′; reverse, 5′-GCAGCAAACATCTCACTGCCCAGGT-3′. Glyceraldehyde-3-phosphate dehydrogenase was amplified by following primers: forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′; reverse, 5′-AGCCTTCTCCATGGTCGTGAAGAC-3′. GFP-LC3 Staining—GFP-LC3 staining was carried out essentially as described previously (43Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5509) Google Scholar). At the indicated time after transfection GFP-LC3 staining was visualized using an Axioplan Zeiss microscope, and the number of GFP-LC3-positive cells with GFP-LC3 dots was determined. Proteolysis—Proteolysis was determined as described previously (44Pattingre S. Petiot A. Codogno P. Methods Enzymol. 2004; 390: 17-31Crossref PubMed Scopus (50) Google Scholar). Briefly, cells were incubated for 24 h at 37 °C in normal culture medium containing 7400 Bq/ml of [14C]valine. Cells were rinsed three times with PBS, pH 7.4, and then the cells were incubated for 1 h in complete medium supplemented with 10 mm valine. After incubating for 1 h, during which time short-lived proteins had been degraded, the medium was replaced by fresh chase medium for another 4 h. When required, C2-Cer (75 μm), FB1 (100 μm), DMS (1.5 μm), and 3MA (10 mm) were added to the chase medium. For the PLD1- or SK1-overexpressing cells, radiolabeling with [14C]valine was started 24 h after transfection. Cells and radiolabeled proteins from the 4-h chase medium were precipitated in trichloroacetic acid at a final concentration of 10% (v/v) at 4 °C. Radioactivity was determined by liquid scintillation counting. Protein degradation was calculated by dividing the acid-soluble radioactivity recovered from both cells and medium by the radioactivity contained in the precipitated proteins from both cells and medium. Cell viability was determined by the trypan blue exclusion test as previously described (32Scarlatti F. Bauvy C. Ventruti A. Sala G. Cluzeaud F. Vandewalle A. Ghidoni R. Codogno P. J. Biol. Chem. 2004; 279: 18384-18391Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). When required, 40 μm z-VAD-fmk was added prior to incubation in nutrient-free medium (Hanks' balanced salt solution plus 0.1% of bovine serum albumin). Cells were collected by centrifuging (10 min, at 1200 × g), washing with PBS, and resuspending in lysis buffer (1% Triton X-100, 25 mm Tris-HCl, pH 7.4, 1 mm phenylmethylsulfonyl fluoride, 1% Protease Inhibitor Mixture (PIC), 1 mm Na3VO4, 1 mm NaF) for 1 h at 4°C. The cell lysate was centrifuged (20,000 × g for 30 min at 4 °C), and the supernatant was recovered. Protein concentrations were determined by the bicinchoninic acid (BCA) method as recommended by the manufacturer. Extracted proteins were first separated in SDS-polyacrylamide gels and then electrotransferred onto nitrocellulose membranes. After blocking overnight with fat-free milk, the membranes were incubated with appropriate primary antibodies: anti-Beclin 1 (1/2000), anti-PARP (1/1000), anti-p70S6K (1/1000), anti-phospho-p70S6K Thr389 (1/1000), anti-phospho-S6 Ser235(1/1000), anti-4E-BP1 (1/1000), anti-FLAG (1/1000), anti-Atg7 (1/1000), and anti-PKB and anti-phospho-PKB Ser473 (1/1000). Primary antibodies were detected by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies against rabbit, goat, or mouse immunoglobulins. Fluorographs were quantitatively scanned using the NIH image software. Ceramide levels were measured using the DGK assay as described previously (45Perry D.K. Hannun Y.A. Trends Biochem. Sci. 1999; 24: 226-227Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, cells were collected, and lipids were extracted according to a previous study (46Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43126) Google Scholar). The organic phase was divided into 1/2 and 1/6 aliquots, dried, and used for ceramide and total phospholipid measurements (47Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar), respectively. Briefly, 30 nmol of extracted lipids was incubated at room temperature for 45 min in the presence of β-octylglucoside/dioleoylphosphatidylglycerol micelles, 2 mm dithiothreitol, 5 μg of proteins of DGK containing membranes (Calbiochem), and 1 mm ATP mixed with [γ-32P]ATP (4.8 × 105 Bq/ml) in a final volume of 0.1 ml. At the end of the reaction, lipids were extracted, and [32P]ceramide 1-phosphate was determined by TLC separation in chloroform/acetone/methanol/acetic acid/water (10:4:3:3:1, v/v). The radioactivity associated with ceramide 1-phosphate spots was determined after scraping and counting in a scintillation counter. Ceramide levels were expressed in terms of the total phospholipid content. Sphingosine kinase activity was determined as described previously (48Granata R. Trovato L. Garbarino G. Taliano M. Ponti R. Sala G. Ghidoni R. Ghigo E. FASEB J. 2004; 18: 1456-1458Crossref PubMed Scopus (102) Google Scholar). Briefly, cells were collected and lysed by repeated freeze-thawing cycles in 200 μl of lysis buffer (20 mm Tris, pH 7.4, 20% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 0.01 mm MgCl2, 1 mm Na3VO4, 15 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1% PIC, 0.1% Triton X-100, 0.5 mm 4-deoxypyridoxine). After centrifuging at 13,000 × g for 30 min, the protein concentration of supernatant was determined with a Coomassie Plus Protein Assay kit (Pierce). Proteins (100 μg) were then incubated with 25 μmd-erythrosphingosine dissolved in 0.1% Triton X-100, 2 mm ATP, and [γ-32P]ATP (3.7 × 105 Bq dissolved in 20 mm MgCl2) for 30 min at 37 °C in a final volume of 200 μl. The reaction was stopped by adding 20 μl of HCl, 1 n, followed by 800 μl of chloroform/methanol/HCl (100:200:1, v/v). After vigorous vortexing, 250 μl of chloroform and 250 μl of KCl 2 m were added, and phases were separated by centrifugation. The organic layer was dried and resuspended in chloroform/methanol/HCl 37% (100:100:0.2, v/v). Lipids were resolved on silica TLC plates (Whatman) in 1-butanol/acetic acid/water (3:1:1, v/v). Labeled S1P spots were visualized by autoradiography and quantified by scraping and counting in a scintillation counter. For quantification of S1P, MCF-7 cells were labeled for 7 h in serum-free medium with 0.3 μCi/ml d-erythro-[3-3H]sphingosine (PerkinElmer Life Sciences). After washing with PBS, cells were scraped and lysed in water. Radiolabeled S1P was extracted by phase partition as previously described (49Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar, 50Vessey D.A. Kelley M. Karliner J.S. Anal. Biochem. 2005; 337: 136-142Crossref PubMed Scopus (47) Google Scholar) and counted by liquid scintillation. Phospholipase D Activity—PLD activity was determined as described previously (51O'Luanaigh N. Pardo R. Fensome A. Allen-Baume V. Jones D. Holt M.R. Cockcroft S. Mol. Biol. Cell. 2002; 13: 3730-3746Crossref PubMed Scopus (85) Google Scholar) with some modifications. Briefly, 2 × 106 cells were plated on 25-cm2 flasks for 24 h; when required, cells were transfected with PLD1 vectors (wild-type and K898R mutant). After 24 h, cells were preincubated in 0.5% fetal bovine serum containing DMEM for 1 h. After removing the preincubation medium, cells were labeled with [3H]myristic acid (Amersham Biosciences) for 1 h at 1.11 × 105 Bq/ml in 0.5% fetal bovine serum containing DMEM. Cells were rinsed with PBS and incubated for 30 min in complete medium containing 0.5% butanol. Cells were rinsed with PBS and scraped in methanol/water (98:2, v/v). Cells were rinsed with methanol, and the lipids were extracted by adding equal amounts of chloroform and water. After centrifuging, the lower phase was collected and dried under vacuum before being redissolved in chloroform. Lipids were separated by TLC on silica-coated plastic sheets (Merck) in a solvent system of chloroform: methanol:acetic acid:water (75:45:3:0.4, v/v). Radioactive components were detected by autoradiography. PLD activity was determined by the [3H]phosphatidylbutanol/[3H]phosphatidylcholine ratio. Phosphatidylcholine (Avanti polar) and phosphatidylbutanol (Biomol) diluted in chloroform were loaded as standards and were visualized with iodine vapor. Statistical analysis of the differences between the groups was performed using Student's t test. p < 0.05 was considered statistically significant. Sphingosine Kinase 1 Overexpression Stimulates Autophagy—MCF-7 cells were transfected either with the cDNA encoding the wild-type SK1 (SK1wt) or that encoding a mutant inactive form (SK1G82D). The activity of SK was significantly increased in SK1wt-overexpressing cells when compared with that observed in control cells transfected with an empty vector or in SK1G82D-expressing cells (Fig. 1A). Accordingly, SK1wt-overexpressing cells displayed a higher level of S1P compared with control cells (Fig. 1A). In the presence of the SK inhibitor DMS, both S1P production and SK activity were dramatically reduced in these cells. In contrast, no change in the level of ceramide was detected in SK1wt-overexpressing cells at any time points after transfection (Fig. 1, A and B). Autophagy was first analyzed after transfection of GFP-LC3 (Fig. 2A). LC3 is a reliable marker of autophagosomes in mammalian cells, and its localization changes from a diffuse cytosolic pattern to a punctuate pattern representing its recruitment to the autophagosomal membrane duri
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