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CD95/Fas-induced Ceramide Formation Proceeds with Slow Kinetics and Is Not Blocked by Caspase-3/CPP32 Inhibition

1997; Elsevier BV; Volume: 272; Issue: 39 Linguagem: Inglês

10.1074/jbc.272.39.24308

ISSN

1083-351X

Autores

Annemiek D. Tepper, Jeanine G.R. Boesen-de Cock, Evert de Vries, Jannie Borst, Wim J. van Blitterswijk,

Tópico(s)

Axial and Atropisomeric Chirality Synthesis

Resumo

The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (hours) often parallels apoptosis onset. We investigated CD95-induced ceramide formation in Jurkat cells, using two methods (radiolabeling/thin layer chromatography and benzoylation/high performance liquid chromatography), which, unlike the commonly used diglyceride kinase assay, discriminate between ceramide species and de novoformed dihydroceramide. We demonstrate that ceramide accumulates after several hours, reaching a 7-fold increase after 8 h, kinetics closely paralleling apoptosis induction. No fast response was observed, not even in the presence of inhibitors of ceramide metabolism. The majority (∼70%) of the ceramide response remained unaffected when apoptosis was completely inhibited at the level of caspase-3/CPP32 processing by the inhibitor peptide DEVD-CHO. Exogenous cell-permeable C2-ceramide induced the proteolytic processing of caspase-3, albeit with somewhat slower kinetics than with CD95. DEVD-CHO dose-dependently inhibited C2-ceramide- or exogenous sphingomyelinase-induced apoptosis. The results support the idea that ceramide acts in conjunction with the caspase cascade in CD95-induced apoptosis. The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (hours) often parallels apoptosis onset. We investigated CD95-induced ceramide formation in Jurkat cells, using two methods (radiolabeling/thin layer chromatography and benzoylation/high performance liquid chromatography), which, unlike the commonly used diglyceride kinase assay, discriminate between ceramide species and de novoformed dihydroceramide. We demonstrate that ceramide accumulates after several hours, reaching a 7-fold increase after 8 h, kinetics closely paralleling apoptosis induction. No fast response was observed, not even in the presence of inhibitors of ceramide metabolism. The majority (∼70%) of the ceramide response remained unaffected when apoptosis was completely inhibited at the level of caspase-3/CPP32 processing by the inhibitor peptide DEVD-CHO. Exogenous cell-permeable C2-ceramide induced the proteolytic processing of caspase-3, albeit with somewhat slower kinetics than with CD95. DEVD-CHO dose-dependently inhibited C2-ceramide- or exogenous sphingomyelinase-induced apoptosis. The results support the idea that ceramide acts in conjunction with the caspase cascade in CD95-induced apoptosis. Important physiological triggers for apoptosis include ligation of the CD95 (Apo-1/Fas) cell surface receptor, which belongs to the tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; Cer, ceramide; C2-Cer,N-acetyl-d-sphingosine; SM, sphingomyelin; SMase, sphingomyelinase; FACS, fluorescence-activated cell sorting; HPLC, high performance liquid chromatography; TLC, thin layer chromatography; d-e-MAPP,d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; PPMP,d-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol; mAb, monoclonal antibody; DEVD-CHO, Ac-Asp-Glu-Val-Asp aldehyde.1The abbreviations used are: TNF, tumor necrosis factor; Cer, ceramide; C2-Cer,N-acetyl-d-sphingosine; SM, sphingomyelin; SMase, sphingomyelinase; FACS, fluorescence-activated cell sorting; HPLC, high performance liquid chromatography; TLC, thin layer chromatography; d-e-MAPP,d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; PPMP,d-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol; mAb, monoclonal antibody; DEVD-CHO, Ac-Asp-Glu-Val-Asp aldehyde.receptor superfamily. A cascade consisting of several cysteine proteases of the ICE/CED-3 family (caspases; Ref. 1Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2123) Google Scholar) has been implicated in apoptosis in different cell types (2Chinnaiyan A.M. Dixit V.M. Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar, 3Nicholson D. Nat. Biotechnol. 1996; 14: 297-301Crossref PubMed Scopus (239) Google Scholar). Upon stimulation, the CD95 receptor trimerizes and recruits caspase-8/FLICE/Mch5 (4Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2714) Google Scholar, 5Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2096) Google Scholar, 6Fernandes-Alnemri T. Litwack G. Alnemri E.S. J. Biol. Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar) to the CD95 death-inducing signaling complex, where it is activated (7Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1757) Google Scholar, 8Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1032) Google Scholar). Subsequently, the cytosolic caspase-3/CPP32/Yama becomes activated (9Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2259) Google Scholar, 10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (479) Google Scholar). There is little insight into the mechanism by which the cascade is regulated. Although FLICE can cleave CPP32 in vitro (10Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (479) Google Scholar), it is still unclear whether these caspases directly interact in vivo. A second pathway leading to apoptosis involves activation of sphingomyelinase (SMase), which hydrolyzes sphingomyelin (SM) to ceramide (Cer). SM hydrolysis is evoked not only by triggering of the TNF or CD95 receptor (11Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (672) Google Scholar, 12Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier l. L. Santoni A. Testi R. J. Exp. Med. 1993; 177: 1547-1552Google Scholar, 13Cifone M.G. Roncaioli P. De Maria R. Camarda G. Santoni A. Ruberti G. Testi R. EMBO J. 1995; 14: 5859-5868Crossref PubMed Scopus (277) Google Scholar, 14Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (431) Google Scholar) but also by other apoptotic inputs such as oxidative stress, UV, and γ-irradiation (15Haimovitz-Friedman A. Kan C.-C. Ehleiter D. Persaud R.S. McLoughlin M. Fuks Z. Kolesnick R.N. J. Exp. Med. 1994; 180: 525-535Crossref PubMed Scopus (842) Google Scholar, 16Verheij M. Bose R. Lin X.-H. Yao B. Grant S. Birrer M.J. Szabo E. Zon I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1707) Google Scholar, 17Santana P. Peña L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R.N. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). A specific role for Cer in mediating apoptotic signals is suggested by the apoptotic effect of exogenous short-chain Cer, the structurally closely related compound dihydroCer being inactive. Likewise, treatment with bacterial SMase or drugs that inhibit metabolic conversion of Cer causes apoptosis, supposedly via elevated endogenous Cer levels (18Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1483) Google Scholar). There is currently much confusion about the role of endogenous Cer in apoptosis, inasmuch as kinetics and magnitude of the Cer response differ widely among various studies (18Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1483) Google Scholar). Some investigators observe a response within minutes after stimulation, which never exceeds 200% (12Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier l. L. Santoni A. Testi R. J. Exp. Med. 1993; 177: 1547-1552Google Scholar, 14Gulbins E. Bissonette R. Mahboubi A. Martin S. Nishioka W. Brunner T. Baier G. Baier-Bitterlich G. Byrd C. Lang F. Kolesnick R. Altman A. Green D. Immunity. 1995; 2: 341-351Abstract Full Text PDF PubMed Scopus (431) Google Scholar), whereas others, using the same stimulus, only measure significant elevation after several hours up to 5–7-fold above basal (19Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 20Sawai H. Okazaki T. Takeda M. Tashima M. Sawada H Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 21Sillence D.J. Allan D. Biochem. J. 1997; 324: 29-32Crossref PubMed Scopus (54) Google Scholar). One may question whether late ceramide formation is a consequence rather than a cause of apoptosis, inasmuch as kinetics often parallels the onset of apoptosis (19Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 21Sillence D.J. Allan D. Biochem. J. 1997; 324: 29-32Crossref PubMed Scopus (54) Google Scholar). On the other hand, overexpression of Bcl-2 in ALL-697 cells did not affect vincristine- or TNFα-induced delayed ceramide formation whereas it completely prevented apoptosis (22Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (293) Google Scholar, 23Dbaibo G.S. Perry D.K. Gamard C.J. Platt R. Poirier G.G. Obeid L.M. Hannun Y.A. J. Exp. Med. 1997; 185: 481-490Crossref PubMed Scopus (208) Google Scholar), demonstrating that late ceramide is not necessarily a result of cell death. Activation of both SMase and caspases has been implicated in apoptosis, especially when induced by members of the TNF receptor family, but the connection between both pathways has not been elucidated. In the present study, we have thoroughly investigated CD95-induced Cer formation by two different quantitation methods, other than the commonly used Escherichia coli diglyceride kinase assay. Unlike the latter assay, the new assays based on metabolic radiolabeling/TLC and benzoylation/HPLC, respectively, allow the resolution of Cer with different chain lengths as well as separation from de novo synthesized dihydroCer. We demonstrate that, in Jurkat T cells, CD95 only induces late Cer formation paralleling caspase-3 activation. Direct inhibition of caspase-3 processing prevents apoptosis but does not block the Cer response. Because exogenous Cer can activate caspase-3, our findings suggest that Cer acts upstream of caspase-3 in the CD95-induced apoptotic signaling pathway. [3-14C]Serine and [methyl-14C]choline chloride were from Amersham; C2-Cer was from Biomol; Bacillus cereus SMase, Type III Cer,N-stearoyl-d-sphingosine,N-nervonoyl-d-sphingosine,N-nervonoyl-d-sphinganine, dimethylaminopyridine, benzoic anhydride, and tamoxifen (free base) were from Sigma; Silica G60 TLC plates and cyclohexane Lichrosolv® were obtained from Merck; Silica HPLC column (250 × 4.6 mm) and HPLC sample filters were from Chrompack (The Netherlands); anti-CD95 monoclonal antibody (CH-11) was from Immunotech (Marseille, France). Polyclonal antiserum raised against a glutathioneS-transferase fusion protein of human caspase-3/CPP32 was prepared by Dr. G. Gil-Gomez in our institute. DEVD-CHO was from Calbiochem and PPMP from Matreya. d-e-MAPP was kindly provided by Dr. A. H. Merrill Jr. (Emory University School of Medicine, Atlanta, GA). The J16 wild type clone was derived by limiting dilution from the human T-cell line Jurkat and selected for high sensitivity to CD95-induced apoptosis. It was cultured in Iscove's modified Dulbecco's medium supplemented with 10% (v/v) fetal calf serum, 2 mm glutamine, and 100 IU/ml penicillin/streptomycin, at 37 °C, 5% CO2. Before cell stimulation, cells were incubated overnight in synthetic Yssel's medium (24Yssel H. de Vries J.E. Koken M. van Blitterswijk W.J. Spits H. J. Immunol. Methods. 1984; 72: 219-227Crossref PubMed Scopus (339) Google Scholar), resuspended at 5–10 × 106 cells/ml in Yssel's medium in a 24-well culture plate, and stimulated with CH-11 mAb, C2-Cer (prepared as 10 mm stock in Me2SO), or SMase for various time periods at 37 °C, 5% CO2. For apoptosis measurements, cells were seeded at 1 × 106 cells/ml, 200 μl/well in round-bottomed, 96-well microtiter plates in Yssel's medium (24Yssel H. de Vries J.E. Koken M. van Blitterswijk W.J. Spits H. J. Immunol. Methods. 1984; 72: 219-227Crossref PubMed Scopus (339) Google Scholar). Cells were lysed in 0.1% sodium citrate, 0.1% Triton X-100, and 50 μg/ml propidium iodide (25Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4398) Google Scholar). Fluorescence intensity of propidium iodide-stained DNA was determined in 5000 cells on a FACScan (Becton Dickinson, San Jose, CA), and data were analyzed using Lysys software. Fragmented, apoptotic nuclei are recognized by their subdiploid DNA content. Cells (1 × 107) were collected by brief centrifugation and washed twice with phosphate-buffered saline. Lipids were extracted with chloroform/methanol (1:2,v/v) and phases separated (26Bligh E.G. Dyer W.J. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41817) Google Scholar). Benzoate-derivatives were prepared essentially as has been reported for mono- and diglycerides (27Louie K. Wiegand R.D. Anderson R.E. Biochemistry. 1988; 27: 9014-9020Crossref PubMed Scopus (41) Google Scholar). Dried lipid extracts of 1 × 107 cells were dissolved in 0.25 ml of toluene containing 8 mg of benzoic anhydride and 3 mg of 4-(dimethylamino)pyridine and allowed to stand at room temperature for 2 h. Under these conditions, no amide acylation occurs (28Gross S.K. McCluer R.H. Anal. Biochem. 1980; 102: 429-433Crossref PubMed Scopus (39) Google Scholar). Samples were cooled on ice, and 2 ml of 25% NH4OH was added dropwise. Benzoate-derivatives were extracted three times with 2 ml ofn-hexane. The samples were dried, reconstituted in 1 ml of chloroform, passed through a 0.2-μm HPLC sample filter, dried under N2, and dissolved in a small volume (usually 50 μl) of carbon tetrachloride. HPLC was performed using a Waters 600E System Controller. Separation of benzoylated lipids was accomplished using isocratic HPLC with a ChromSpher silica analytical column (4.6 × 250 mm, 5-μm pore size) based on published methods (29Iwamori M. Costello C. Moser H.W. J. Lipid Res. 1974; 20: 86-96Abstract Full Text PDF Google Scholar, 30Previati M. Bertolaso L. Tramarin M. Bertagnolo V. Capitani S. Anal. Biochem. 1996; 233: 108-114Crossref PubMed Scopus (65) Google Scholar). Samples of 15 μl, corresponding to lipids from 3 × 106 cells, were injected, and eluates were monitored at 230 nm using a Waters 486 Tunable Absorbance Detector. The attenuation was usually set at a full scale of 0.075 absorbance unit. Data were collected with a Waters 741 Data Module integrator. The mobile phase consisted of cyclohexane, 0.45% (v/v) isopropanol at a flow rate of 1.5 ml/min. This system not only allows the separation of benzoyl-Cer from other benzoylated lipid molecules, it also resolves dihydroCer and Cer molecular species differing in fatty acid chain length. Cells (1 × 106/ml) in Yssel's medium (24Yssel H. de Vries J.E. Koken M. van Blitterswijk W.J. Spits H. J. Immunol. Methods. 1984; 72: 219-227Crossref PubMed Scopus (339) Google Scholar) were labeled with [14C]serine (0.2 μCi/ml) for 16–20 h. When inhibitors of Cer metabolism were used, these were added at this point. Cells were washed twice, stimulated, and extracted as described above. Lipid extracts were spotted on TLC silica plates and developed to 70% of the total length in CHCl3/MeOH/H2O/25% NH4OH (50/50/2/1, v/v). Plates were dried under nitrogen and rechromatographed to the top of the plate in CHCl3/MeOH/H2O/25% NH4OH (90/10/0.5/0.5, v/v). Under these conditions, Cer, glucosylceramide, sphingosine, SM, and glycerophospholipids are well separated. Radioactive lipids were visualized and quantitated using a Fujix BAS 2000 TR phosphorimager (exposure time 1–2 days). For chromatographic reference, radiolabeled Cer was prepared from endogenous [14C]SM isolated by TLC and sonicated in 1.5 ml of 0.1m Tris-HCl, pH 7.4, 0.1% Triton X-100, 6 mmMgCl2, by incubation overnight with B. cereusSMase (1 units/ml) at 37 °C. [14C]Cer, extracted with diethylether, yielded a doublet on TLC corresponding to Cer species containing either C22–24 (upper spot) or C16–18 (lower spot) fatty acid chains, as was confirmed with bovine brain Cer (mainly C24:1 and C18:0 species), C24:1-Cer, and C18:0-Cer as commercial standards (data not shown). Cells (2 × 106) were lysed for 30 min at 4 °C in 30 μl of 10 mm triethanolamine HCl, pH 7.8, 0.15 m NaCl, 5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, containing 1% Nonidet P-40, 0.02 mg/ml trypsin inhibitor, and 0.02 mg/ml leupeptin. Lysates were centrifuged, and supernatants were taken up in reducing SDS sample buffer and separated on 12% SDS-polyacrylamide gels (equivalents of 0.8–1 × 106 cells/lane). Proteins were transferred to nitrocellulose (Schleicher & Schüll, Dassel, Germany). Blots were blocked with 5% (w/v) nonfat dry milk in phosphate-buffered saline, 0.1% Tween 20; probed with purified anti-Ig (10 μg/ml) in phosphate-buffered saline, 0.1% Tween 20, 1% nonfat dry milk, followed by a 1:7500 dilution of horseradish peroxidase-conjugated swine anti-rabbit Ig (DAKO A/S, Glostrup, Denmark); and developed by enhanced chemiluminescence (Amersham). Previous studies suggested the involvement of acidic SMase, generating Cer, in transmitting signals from the CD95 receptor to the apoptotic machinery (12Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier l. L. Santoni A. Testi R. J. Exp. Med. 1993; 177: 1547-1552Google Scholar, 13Cifone M.G. Roncaioli P. De Maria R. Camarda G. Santoni A. Ruberti G. Testi R. EMBO J. 1995; 14: 5859-5868Crossref PubMed Scopus (277) Google Scholar, 19Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar). However, widely conflicting data regarding kinetics and magnitude of Cer formation have caused much confusion concerning the role of Cer as a mediator of apoptosis as induced by CD95 or other apoptotic stimuli (18Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1483) Google Scholar). Therefore, we decided to analyze Cer formation by methods alternative to the commonly used procedure for Cer mass measurement involving E. colidiglyceride kinase (31Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar). The major drawback of this assay is that it does not discriminate between Cer and dihydroCer (32Smith E.R. Merrill Jr., A.H. J. Biol. Chem. 1995; 270: 18749-18758Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), which lacks the C4–C5 trans double bond in the sphingoid backbone and is, therefore, biologically inactive (33Bielawska A. Crane H.M. Liotta D. Obeid L.M. Hannun Y.A J. Biol. Chem. 1993; 268: 26226-26232Abstract Full Text PDF PubMed Google Scholar). Considering the outstanding question whether Cer plays a role in apoptosis induction, we investigated the kinetics and magnitude of CD95-induced Cer formation in apoptosis-sensitive Jurkat T cells, using two different quantitation methods that discriminate between Cer, released from existing sphingolipids, and dihydroCer, produced via stimulated de novo synthesis. The first method involves metabolic sphingolipid labeling in the sphingoid backbone using [14C]serine and analysis of Cer formation by TLC. Besides a clear distinction between dihydroCer and Cer, this method allows separation of Cer species containing either long (C22–24) or intermediate (C16 -18) fatty acids represented by a doublet (Fig. 1 A). Exposure of Jurkat T cells to monoclonal anti-human CD95/Fas IgM (CH-11) caused accumulation of Cer, which was most prominent for the intermediate species (lower spot). Analysis of Cer species generated by bacterial SMase treatment of [14C]serine-labeled cells (data not shown) or total isolated endogenous [14C]SM (Fig.1 A) also yielded a doublet with the majority of the radioactivity also residing in the lower spot. Because the Cer species distribution produced upon CD95 stimulation reflects the fatty acid composition of total SM, there is no evidence for the selective hydrolysis of SM with intermediate acyl chains. Quantification of total Cer showed a first significant increase between 3 and 4 h after CD95 stimulation, which further increased to approximately 7-fold above basal at 8 h (Fig. 1 B). Although the accumulation of C16–18 species (referred to as Cer lower spot) may somewhat precede, there is a clear temporal correlation between total Cer formation and the onset of apoptosis measured by nuclear segmentation. Others have shown a similar correlation in SKW6.4 cells, when the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide viability assay (19Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar) or bisbenzamide staining (21Sillence D.J. Allan D. Biochem. J. 1997; 324: 29-32Crossref PubMed Scopus (54) Google Scholar) were used to monitor kinetics of CD95-induced cell death. In separate experiments, using several assays (diglyceride kinase, [14C]serine labeling, or HPLC; see below), we have extensively looked for rapid (within 30 min) Cer responses, but these were never detected (data not shown). Alternatively, we metabolically labeled SM with [14C]choline (48 h). Subsequent CD95 triggering did not reveal an acute decrease in [14C]SM, which would be indicative of SMase activity (results not shown). We next considered the possibility that detection of an acute Cer signal could have been masked by its rapid attenuation through the action of ceramidase or glucosyltransferase. Cer degradation to sphingosine by neutral ceramidase is inhibited byd-e-MAPP (34Bielawska A. Greenberg M.S. Perry D. Jayadev S. Shayman J.A. McKay C. Hannun Y.A. J. Biol. Chem. 1996; 271: 12646-12654Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), whereas PPMP (35Abe A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; 271: 14383-14389Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and tamoxifen (36Cabot M.C. Giuliano A.E. Volner A. Han T. FEBS Lett. 1996; 394: 129-131Crossref PubMed Scopus (73) Google Scholar) block conversion of Cer to glucosylceramide. Interestingly, these agents have been reported to be inducers of apoptosis, presumably via increasing endogenous Cer levels (18Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1483) Google Scholar). After [14C]serine labeling of sphingolipids, cells were pretreated overnight with 500 nm PPMP or 10 μm tamoxifen, which indeed increased the steady-state ratio Cer/glucosylceramide (data not shown), or with 10 μmd-e-MAPP. Subsequent CD95 triggering in the presence of these agents, however, did not reveal any acute Cer formation, whereas all agents induced apoptosis at higher concentrations (results not shown). To exclude the possibility that [14C]serine might have selectively labeled a subpopulation of SM molecules inaccessible to possible rapid CD95-induced hydrolysis, we also developed an HPLC method to quantify Cer mass. In this assay, total cellular lipids were subjected to a derivatization procedure to yield chromophoric, nonpolar benzoate-derivatives. Exposure of Jurkat cells to anti-CD95 mAb caused Cer accumulation, which, again, was most prominent for the second peak of a doublet, corresponding to C16–18 Cer species (Fig.2). Also with this assay, kinetics of Cer formation closely paralleled apoptosis and no acute Cer formation was detected, in agreement with the radiolabeling assay. From these extensive analyses, we have to conclude that CD95 triggering does not evoke rapid activation of SMase, but only causes a late and sustained elevation of Cer species that predominantly contain intermediate chain fatty acids. The relevance of late Cer formation in mediating CD95-induced apoptosis is a topic of debate. In a number of studies, cell lines resistant to apoptosis induced by CD95 (19Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 20Sawai H. Okazaki T. Takeda M. Tashima M. Sawada H Okuma M. Kishi S. Umehara H. Domae N. J. Biol. Chem. 1997; 272: 2452-2458Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 37Wright S.C. Zheng H. Zhong J. FASEB J. 1996; 10: 325-332Crossref PubMed Scopus (63) Google Scholar) and anti-IgM (38Gottschalk A.R. McShan C.L. Kilkus J. Dawson G. Quintas J. Eur. J. Immunol. 1995; 25: 1032-1038Crossref PubMed Scopus (41) Google Scholar) were reported to lack Cer formation. However, whether this is really the cause, as was suggested, rather than a consequence of their resistance remains a question. In the case of TNFα-induced apoptosis in MCF-7 cells, ceramide accumulation is also slow but clearly precedes apoptosis (23Dbaibo G.S. Perry D.K. Gamard C.J. Platt R. Poirier G.G. Obeid L.M. Hannun Y.A. J. Exp. Med. 1997; 185: 481-490Crossref PubMed Scopus (208) Google Scholar). Activation of the ICE/Ced-3-like cysteine protease caspase-3/CPP32 was previously shown to be required for CD95-induced apoptosis (39Schlegel J. Peters I. Orrenius S. Miller D.K. Thornberry N.A. Yamin T.-T. Nicholson D.W. J. Biol. Chem. 1996; 271: 1841-1844Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Fig.3 shows the kinetics of proteolytic activation of caspase-3 as triggered by CD95 in Jurkat cells. A first significant decline in the 32-kDa proform with concomitant appearance of a proteolytic fragment is observed after 4 h. The caspase inhibitor DEVD-CHO (40Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T.T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3766) Google Scholar) efficiently blocked caspase-3 processing (Fig.3) and apoptosis (Fig. 6). However, this inhibitor did not prevent CD95-induced Cer formation (Fig.4 A). Interestingly, Fig.4 B shows that inhibition of caspase-3 and apoptosis hardly affected the kinetics of Cer formation, although its magnitude is reduced by approximately 30%. These data suggest that CD95-induced ceramide formation occurs upstream of caspase-3 activation. In a different system, i.e. TNFα-stimulated MCF-7 cells, Dbaiboet al. (23Dbaibo G.S. Perry D.K. Gamard C.J. Platt R. Poirier G.G. Obeid L.M. Hannun Y.A. J. Exp. Med. 1997; 185: 481-490Crossref PubMed Scopus (208) Google Scholar) also concluded that Cer was generated upstream of caspases involved in the "execution stage" of apoptosis, inasmuch as Bcl-2 overexpression did not interfere with TNFα-induced ceramide generation, while it provided protection from exogenous Cer-induced poly(ADP-ribose) polymerase cleavage and apoptosis (22Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (293) Google Scholar, 23Dbaibo G.S. Perry D.K. Gamard C.J. Platt R. Poirier G.G. Obeid L.M. Hannun Y.A. J. Exp. Med. 1997; 185: 481-490Crossref PubMed Scopus (208) Google Scholar).Figure 6Caspase-3 activation is required for Cer- and SMase-induced apoptosis. After a 2-h preincubation with DEVD-CHO at varying concentrations, Jurkat cells were exposed to anti-CD95 mAb (CH-11; 500 ng/ml; 8 h), C2-Cer (50 μm; 18 h), B. cereus SMase (300 milliunits/ml; 18 h), or control medium (18 h). Apoptosis was determined by FACS analysis (25Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4398) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4DEVD-CHO does not prevent CD95-induced Cer formation. Jurkat cells prelabeled with [14C]serine were preincubated for 2 h in the absence or presence of 100 μm DEVD-CHO, as indicated. Next, they were left untreated or treated with anti-CD95 mAb (CH-11; 1 μg/ml). At indicated times, lipids were extracted and separated by TLC as described under "Experimental Procedures." A, lipid analysis of cells that were left untreated (control) or treated with CH-11 (antiCD95) for 6 and 8 h. Apoptosis (percentage indicated) was quantified in the same set of samples. B, time course of CD95-induced [14C]Cer generation in the absence (open circles) or presence (closed circles) of DEVD-CHO. Data (means of three experiments ± S.D.) are expressed as -fold increase in total Cer relative to control cells that received no anti-CD95.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our data are at variance with the work by Gamen et al. (41Gamen S. Marzo I. Anel A. Piñeiro A. Naval J. FEBS Lett. 1996; 390: 233-237Crossref Scopus (80) Google Scholar), wherein DEVD-CHO completely prevented CD95-induced Cer generation. However, they measured only [1-14C]stearic acid-labeled Cer at 16 h and showed no kinetics of Cer formation or apoptosis. Their 16-h timepoint might well reflect an end point of the apoptotic process (dead cells). Others, using the caspase inhibitor zVAD.fmk to inhibit CD95-induced cell death, observed complete inhibition of ceramide production in SKW 6.4 cells (21Sillence D.J. Allan D. Biochem. J. 1997; 324: 29-32Crossref PubMed Scopus (54) Google Scholar) and concluded that Cer is formed as a consequence of processes downstream of the caspase cascade. However, because zVAD.fmk is a broad spectrum caspase inhibitor and can inhibit FLICE activation in vivo (8Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1032) Google Scholar), their results do not exclude that Cer is formed in between steps of sequential caspase activation. As the majority of Cer accumulation is not downstream of caspase-3 and because kinetics parallel caspase-3 proteolytic processing, we investigated whether caspase-3 could be a Cer target. Indeed, exogenous C2-Cer induced the proteolytic cleavage of the 32-kDa proform of the protease after 6–8 h (Fig.5), which is somewhat later than by CD95 stimulation. Notably, in a different system, i.e.C6-Cer-treated Molt-4 cells, and using a different read-out, i.e. proteolytic cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase, Smyth et al. (42Smyth M.J. Perry D.K. Zhang J. Poirier G.G. Hannun Y.A. Obeid L.M. Biochem. J. 1996; 316: 25-28Crossref PubMed Scopus (198) Google Scholar) have also found activation of caspase-3 by exogenous Cer. Fig.6 shows that nuclear fragmentation in response to exogenous Cer and bacterial SMase, like that induced by CD95, was inhibited by DEVD-CHO in a dose-dependent fashion. These results provide evidence for the specific requirement of caspase-3 in mediating the apoptotic effect of both short-chain Cer as well as of naturally occurring Cer species. Taken together, we find that CD95 evokes no rapid (<1 h) Cer response but only induces late Cer formation paralleling caspase-3 activation and the appearance of nuclear fragmentation. Direct inhibition of caspase-3 processing by the caspase inhibitor DEVD-CHO completely prevents the apoptotic phenotype but does not block the majority (∼70%) of the Cer response. These results indicate that late Cer is not a mere result of CD95-induced apoptosis but rather could be instrumental in the execution of apoptosis. Exogenous Cer was shown to induce caspase-3 processing, and, moreover, caspase-3 activation appeared a requirement for apoptosis induced either by cell-permeable Cer or bacterial SMase. Thus, our results support the model in which the SMase pathway acts upstream of the caspase-3 member of the family of caspases in CD95-induced apoptosis and pose intriguing questions concerning the role of Cer acting in concert with members of the proteolytic cascade. We thank E. Noteboom for his contribution to the FACS analyses and Dr. G. Gil-Gomez for kindly providing the polyclonal antiserum against human caspase-3/CPP32.

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