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TNF Receptor Death Domain-associated Proteins TRADD and FADD Signal Activation of Acid Sphingomyelinase

1998; Elsevier BV; Volume: 273; Issue: 10 Linguagem: Inglês

10.1074/jbc.273.10.5916

1083-351X

Ralf Schwandner, Katja Wiegmann, Katussevani Bernardo, Dirk Kreder, Martin Krönke,

Lipid Membrane Structure and Behavior

Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. Here we describe the TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells did not change basal activity of A-SMase but enhanced TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which were reported to mediate TNF R55-mediated activation of nuclear factor κB, did not affect activation of A-SMase. Caspase inhibitors markedly reduced A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) did not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b. Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. Here we describe the TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells did not change basal activity of A-SMase but enhanced TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which were reported to mediate TNF R55-mediated activation of nuclear factor κB, did not affect activation of A-SMase. Caspase inhibitors markedly reduced A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) did not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b. Ceramide produced by sphingomyelinases (SMases) 1The abbreviations used are: SMase, sphingomyelinase; TNF, tumor necrosis factor; A-SMase, acidic sphingomyelinase; N-SMase, neutral sphingomyelinase; TRADD, TNF receptor-associated protein with death domain; TRAF2, TNF receptor-associated factor 2; FADD, Fas-associated protein with death domain; RIP, receptor interacting kinase; ICE, interleukin-1β-converting enzyme; Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde; Ac-YVAD.cmk, acetyl-Try-Val-Ala-Asp-chloromethylketone; NFκB, nuclear factor-κB; NSD, N-SMase activation domain; crmA, cytokine response modifier A; PBS, phosphate-buffered saline; NFκB, nuclear transcription factor κ B. has been recognized as an important second messenger in membrane receptor signaling. Through binding to the p55 TNF receptor (TNF-R55), TNF rapidly activates two distinct forms of SMases, a membrane-associated neutral (N-) SMase and an acid (A-) SMase (1Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar), which reside in caveolae (2Liu P. Anderson R.G. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar) and the endosomal-lysosomal compartment. Each type of SMase hydrolyzes the phosphodiester bond of sphingomyelin to yield the neutral lipid second messenger ceramide and phosphorylcholine. Ceramide generated by N-SMase at the plasma membrane directs the activation of ceramide-activated protein kinase (CAPK) (3Mathias S. Dressler K.A. Kolesnick R.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10009-10013Crossref PubMed Scopus (349) Google Scholar) that phosphorylates and activates Raf-1 kinase (4Yao B. Zhang Y. Delikat S. Mathias S. Basu S. Kolesnick R. Nature. 1995; 378: 307-310Crossref PubMed Scopus (303) Google Scholar, 5Zhang Y. Yao B. Delikat S. Bayoumy S. Lin X.H. Basu S. McGinley M. Chan-Hui P.Y. Lichenstein H. Kolesnick R. Cell. 1997; 89: 63-72Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Activated Raf-1 in turn phosphorylates and stimulates a dual specificity mitogen-activated kinase (MEK-1), which eventually phosphorylates and activates extracellular signal-regulated kinases (ERKs). The downstream targets of endosomal-derived ceramide produced by A-SMase are not yet defined but may include protein kinase C (PKCζ), JNK, and caspases (6Müller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (471) Google Scholar, 7Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar, 8Westwick J.K. Bielawska A.E. Dbaibo G. Hannun Y.A. Brenner D.A. J. Biol. Chem. 1995; 270: 22689-22692Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). N-SMase and A-SMase are activated independently by distinct cytoplasmic domains of TNF-R55 (1Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar). N-SMase activation is mediated by neutral sphingomyelinase domain (NSD), spanning an 11-amino acid motif N-terminally adjacent to the death domain of TNF-R55 (9Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Krönke M. Cell. 1996; 86: 937-947Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 10Adam D. Wiegmann K. Adam-Klages S. Ruff A. Krönke M. J. Biol. Chem. 1996; 271: 14617-14622Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The NSD binds a WD-repeat protein, FAN, that mediates activation of N-SMase. The domain of TNF-R55 activating the A-SMase pathway strikingly corresponds to the death domain signaling the cytotoxic effects of TNF (1Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 11Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1169) Google Scholar). The cytoplasmic protein TRADD was recently identified to associate with the death domain of TNF-R55 in a TNF-dependent process (12Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar, 13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar). TRADD serves as an adapter protein that recruits other proteins to the cytoplasmic TNF receptor complex (14Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 15Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (367) Google Scholar). Overexpression of TRADD potently activates distinct signaling cascades leading to activation of NF-κB and induction of cell death (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar). FADD, which directly binds to TRADD, has been shown to mediate activation of a pro-apoptotic protease, caspase-8/a (FLICE/MACH), eventually leading to apoptosis (16Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 17Muzio 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 (2743) Google Scholar). Here we show that TNF-induced activation of A-SMase is mediated through TRADD and FADD. Neither caspase-8/a nor a dominant negative mutant of caspase-8/a alter the TNF-induced activation of A-SMase. However, several tetrapeptide caspase inhibitors as well as cytokine response modifier A (crmA) attenuated TNF-induced activation of A-SMase activity, suggesting that FADD mediates enhancement of A-SMase activity through activation of a protease distinct from FLICE. The human embryonic kidney cell line HEK 293 was kindly provided by Dr. M. Schmidt, Essen, FRG. Hela and COS7 cells were obtained from ATCC. Cells were maintained in high glucose Dulbecco's modified Eagle's medium (ICN) supplemented with 10% fetal calf serum, 10 mm glutamine, and 50 μg/ml each of streptomycin and penicillin in a humidified incubator at 5% CO2. Highly purified recombinant human TNF (3 × 107 units/mg) was kindly provided by Dr. G. Adolf, Boehringer Research Institute, Vienna, Austria. Tetrapeptide caspase inhibitors were obtained from Bachem, Heidelberg, FRG. Mammalian cell expression vectors encoding TRADD, TRAF2, RIP, crmA, caspase-8/a and dominant negative deletion mutants of this protein, as well as the control expression plasmids pRK5 and pCMV-β-gal were kindly provided by Drs. D. V. Goeddel and M. Rothe, Tularik Inc., S. San Francisco, and have been described previously (12Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar, 13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar, 18Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (933) Google Scholar). FADD and caspase-10/b cDNA were kindly provided by Dr. V. Dixit, Genentech, San Francisco. The expression vector pRK-FADD was generated by inserting aSalI-HindIII fragment of FADD cDNA into the pRK5 vector. Deletion mutants of FADD were generated by inserting corresponding sequences in-frame into the expression vector pRK5. For transient expression experiments, 1.5 × 106 HEK 293 cells or 1.0 × 106 COS7 or Hela cells were seeded on 100-mm dishes (Falcon, 3803). Cells were transfected the following day by the calcium phosphate precipitation method (19Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A.K. Current Protocols in Molecular Biology. John Wiley and Sons Inc., New York1994Google Scholar). After 9 h of incubation, cells were harvested, and enzymatic SMase assays were performed. HEK 293 or COS7 cells were transfected with different TRADD expression constructs, or with identical amounts of expression vectors, either alone or in combination with other expression constructs for TNF-R55-associated proteins. After 9 h, cells were harvested either for SMase assays or Western blot analysis. The micellar SMase assay using exogenous radiolabeled sphingomyelin was performed as described previously (1Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar). Briefly, at 9 h post-transfection, cells were treated in triplicate in 0.5 ml of medium with 100 ng/ml human recombinant TNF for the indicated periods of time. To measure acid SMase, cells were homogenized in 0.2% Triton X-100 lysis buffer. Radioactive phosphorylcholine produced from [N-methyl-14C]sphingomyelin (labeled in the choline moiety, 47 mCi/mmol, NEN Life Science Products) was determined in the aqueous phase by liquid scintillation counting. Cells were washed once with phosphate-buffered saline (PBS) and lysed for 20 min at 4 °C in 0.4 ml of lysis buffer (50 mm Hepes, pH 7.4, 100 mmNaCl, 10% glycerol, 5 mm EDTA, and protease inhibitor mix Complete® (Boehringer Mannheim, FRG). Cell lysates were fractionated by 10% SDS-PAGE and transferred on Porablot NCL filters (0.45 μm, Macherey-Nagel, Düren, FRG) Filters were blocked overnight in Tris-bufferd saline containing 0.1% (v/v) Tween-20, 5% milk powder, and 1% bovine serum albumin. After incubation at room temperature with either mAb FLAG M2 (Kodak Inc.), mAb myc (Life Technologies, Inc.), rabbit anti-TRADD, rabbit anti-TRAF2 (12Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar), (M. Rothe), or mAb FADD (Transduction Laboratories), filters were washed in Tris-bufferd saline containing 0.1% (v/v) Tween-20 and incubated with a 1:2400 dilution of goat anti-mouse/goat anti-rabbit horseradish peroxidase conjugate (Coulter-Immunotech) in blocking buffer. Filters were washed and developed with ECL detection reagent (Amersham Corp.). Cells were washed twice with cold PBS/EDTA, 5 mm, and 1 × 106 cells were resuspended in 0.5 ml of PBS/EDTA and fixed by adding 0.5 ml of ethanol. After 30 min of incubation at room temperature, cells were centrifuged and resuspended in 0.5 ml of PBS/EDTA. 20 μl of RNase A (1 μg/ml) was added, and after incubation for 30 min at room temperature, 0.5 ml staining solution (500 μg/ml propidium iodide in PBS/EDTA, 5 mm) was added. After 1 h of incubation at 4 °C, cell cycle analyses were performed by flow cytometry using a Becton Dickinson Calibur FACS Analyzer. 293 cells were transfected with 1.5 μg of expression constructs and vector control. Cells were harvested the next day and left untreated or treated with 10 ng/ml TNF and incubated for 20 min at 37 °C. Cells were washed two times with ice-cold PBS, and nuclear extracts were prepared as described previously (20Kruppa G. Thoma B. Machleidt T. Wiegmann K. Krönke M. J. Immunol. 1992; 148: 3152-3157PubMed Google Scholar). Electrophoretic mobility shift assays were performed as described (21Adam D. Kessler U. Krönke M. J. Biol. Chem. 1995; 270: 17482-17487Abstract Full Text Full Text PDF PubMed Google Scholar). Recent studies have identified a C-terminal domain, corresponding to the death domain of TNF-R55, that is responsible for activation of A-SMase and generation of the lipid second messenger ceramide (1Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 22Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Krönke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar). To examine whether one of the so far identified TNF-R55 death domain-associated proteins mediates the activation of A-SMase, 293 cells were first transfected with an expression vector coding for FLAG-tagged TRADD. Expression of TRADD protein was monitored by Western blotting using a specific FLAG antibody (Fig. 1 B). However, at 9 h after transfection, cells did not show any signs of apoptosis, which could be readily observed 24–36 h post-transfection (data not shown). Overexpression of TRADD enhances TNF-induced A-SMase activity in a dose-dependent manner (Fig. 1 A). Notably, basal levels of A-SMase activity remained unaffected, suggesting that the effects of TRADD on A-SMase were dependent on oligomerization of TNF receptors. Similar results were obtained in different cell lines. For example, TRADD also enhanced TNF-induced activation of A-SMase in COS7 cells (Fig. 1 C). Basal activity of N-SMase as well as TNF-induced activation of N-SMase remained unaltered in 293 cells transfected with TRADD expression constructs (data not shown). We next evaluated the effects of individual TRADD interacting proteins TRAF2, RIP, and FADD on the activation of A-SMase. TRAF2 belongs to a family of signal transducers sharing a highly conserved C-terminal TRAF domain, which binds to a N-terminal region of TRADD distinct from the death domain (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar, 23Takeuchi M. Rothe M. Goeddel D.V. J. Biol. Chem. 1996; 271: 19935-19942Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). TRAF2 was shown to mediate TNF-R55-induced activation of nuclear transcription factor κ B (NFκB) (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar, 15Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (367) Google Scholar). As shown in Fig. 2 A, TRAF2 had no effect on A-SMase activity. Similarly, overexpression of a dominant negative mutant of TRAF2 (amino acids 87–501), lacking the N-terminal RING finger motif essential for NFκB activation, had no inhibitory effect on A-SMase activity (Fig. 2 A). Finally, co-transfection with TRADD cDNA did not reveal TRAF2 action on A-SMase (Fig. 2 D). The expression and biological function of TRAF2 cDNA were ensured by Western blot analysis and by NFκB-activation in 293 cells, respectively (Fig. 2, B and C). These findings strongly suggest that TRAF2 is not involved in TNF-induced activation of A-SMase. The receptor interacting protein kinase RIP has been shown to directly interact with TRAF proteins and the death domain of TRADD (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar) and to be capable of inducing both the activation of NFκB and apoptosis (24Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (869) Google Scholar). As shown in Fig. 3 A, neither overexpression of RIP nor a dominant negative deletion mutant RIP-DN, encoding only the death domain of RIP, altered TNF-induced activation of A-SMase. Notably, both expression constructs induced apoptosis when transfected into 293 cells (Fig. 3 B). The lack of the ability of RIP-DN to signal for the activation of NFκB may contribute to the greater extent of hypodiploid nuclei in HeLa cells when compared with the full-length RIP expression construct. Co-transfection of TRADD and RIP did not result in enhanced activity of A-SMase (Fig. 3 C). These results suggest that RIP, like TRAF2, is not involved in TNF-induced activation of A-SMase. It has been recently reported that overexpression of FADD causes the activation of caspase-8/a (FLICE/MACH) and eventually apoptosis (16Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar,17Muzio 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 (2743) Google Scholar). As shown in Fig. 4 A, expression of FADD cDNA in 293 cells led to enhanced A-SMase activity in a dose- and TNF-dependent manner. Basal activity of A-SMase remained unchanged. A dominant negative mutant of FADD, lacking the N-terminal death effector domain (DED), FADD-DN, was found to decrease TNF-induced activation of A-SMase in a dose-dependent manner (Fig. 4 B). Co-expression of FADD along with a constant amount of TRADD cDNA (Fig. 4 C) enhanced the A-SMase activity slightly over the values obtained with FADD expression constructs alone. These findings indicate, that FADD, like TRADD, can enhance TNF-induced A-SMase activation. To obtain a more detailed estimation for TRADD and FADD-mediated enhancement of A-SMase activity, we performed enzyme kinetic analysis from cells transfected with expression constructs for TRADD and FADD. Apparent Vmax and Km values were determined for the substrate [N-methyl-14C]sphingomyelin by fitting initial rates of SM hydrolysis at various substrate concentrations to the Michaelis-Menten equation. As shown in Fig. 5, A-SMase has a basal activity of Vmax = 21 nmol/h/mg and a Km of 11 μm for sphingomyelin in 293 cells. The maximal velocity Vmax is enhanced upon TNF treatment toVmax = 33 nmol/h/mg, whereasKm remained unchanged. Overexpression of TRADD further enhanced the activity of A-SMase, yielding a maximal velocityVmax = 45 nmol/h/mg. The highest maximal velocity was observed with TNF-treated cells previously co-transfected with expression vectors for TRADD and FADD (Vmax= 83 nmol/h/mg). In all instances, Km values remained unchanged. The kinetic profile of A-SMase activation after TNF treatment is shown in Fig. 6. After stimulation of 293 cells with TNF, the activity of A-SMase is transiently increased, peaking at 3 min, and declined over a period of 10 min to reach basal levels again. Cells transfected with expression vectors for TRADD and FADD showed enhanced A-SMase activity at any time after TNF treatment, while basal levels of A-SMase activity remained unaffected. Caspase-8/a (FLICE/MACH) is a downstream signaling molecule of FADD that transduces the TNF and FAS-induced death signal. When 293 cells were transfected with various doses of expression constructs for caspase-8/a the basal activity of A-SMase remained unchanged (Fig. 7 A). Furthermore, overexpression of caspase-8/a (FLICE/MACH) did not alter the magnitude of TNF-induced A-SMase activation. However, caspase-8/a strongly induced apoptosis in these cells, within 15–24 h post-transfection, which was blocked by the coexpression of a 2-fold excess of crmA expression construct (Fig. 7 B). These findings ensured the expression of a functional caspase-8/a zymogen. A dominant negative mutant of caspase-8/a lacking 43 amino acids of the C terminus was shown to prevent TNF-induced cell death (25Hu S. Vincenz C. Ni J. Gentz R. Dixit V.M. J. Biol. Chem. 1997; 272: 17255-17257Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). However, overexpression of FLICE-DN did not alter activity of A-SMase (data not shown). Similarly, overexpression of caspase-10/b (FLICE2) did not alter basal or TNF-induced A-SMase activation in 293 cells (data not shown). These findings suggest that caspase-8/a or caspase-10/b are not involved in TNF-induced A-SMase activation in 293 cells. To investigate a possible involvement of caspases other than FLICE/MACH in A-SMase activation, we finally employed a set of potent caspase inhibitors. When cow pox virus crmA was overexpressed in 293 cells, TNF-induced activation of A-SMase was markedly reduced (Fig. 8 A). As shown in Fig. 8 D, crmA also inhibited enhancing effects of TRADD and FADD on TNF-induced A-SMase activity. Again, the basal A-SMase activity in these cells remained unaltered. Similar results were obtained with tetrapeptide inhibitors of caspases, z-VAD.fmk, Ac-DEVD.CHO, and Ac-YVAD.cmk. Treatment of cells with Ac-DEVD.CHO, which preferentially inhibits caspase-3-like death proteases, resulted in reduced TNF activation of A-SMase (Fig. 8 B). A more pronounced inhibitory effect was obtained with Ac-YVAD.cmk, an inhibitor of caspase-1 (Fig. 8 C). In contrast to Ac-DEVD.CHO, Ac-YVAD.cmk also slightly reduced basal activity of A-SMase. The broad range caspase inhibitor z-VAD.fmk also reduced the enhanced activation of A-SMase in TRADD- and FADD-transfected 293 cells. These results suggest that FADD-mediated activation of A-SMase involves an ICE-like caspase activity distinct from caspase-8/a (FLICE/MACH) and caspase-10/b (FLICE2). Here we show that acid sphingomyelinase is activated through p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells enhanced the TNF-induced activity of A-SMase. Activation of A-SMase could be blocked in cells expressing a dominant negative mutant of FADD. Basal velocity of A-SMase in cells transfected with TRADD/FADD expression plasmids was not altered, suggesting that cross-linking of the TNF-R55·TRADD·FADD complex is essential for the activation of A-SMase. Enzyme kinetic analysis revealed that the maximal velocity Vmax of A-SMase is enhanced in TNF-treated 293 cells overexpressing TRADD or FADD. The highest maximal velocity of A-SMase was obtained in 293 cells transfected with both TRADD and FADD expression plasmids. Notably, the Km remained unchanged, indicating that the enzyme activity of A-SMase was enhanced rather than the accessibility of the substrate. In contrast, overexpression of neither TRAF2 nor a dominant negative deletion mutant TRAF2-DN altered TNF-induced activity of A-SMase in 293 cells, suggesting that TRAF2 is not required for the activation of A-SMase by TNF. TRAF2 has been shown to mediate TNF-induced activation of SAP/JNK (26Natoli G. Costanzo A. Ianni A. Templeton D.J. Woodgett J.R. Balsano C. Levrero M. Science. 1997; 275: 200-203Crossref PubMed Scopus (415) Google Scholar) and NFκB (15Shu H.B. Takeuchi M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13973-13978Crossref PubMed Scopus (367) Google Scholar), which involves a MAP3 kinase-related protein kinase, NIK, and the IκB-kinase IKKα (27Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1166) Google Scholar, 28Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 29DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar). In 293 cells, TRAF2 is apparently not involved in activation of a A-SMase, suggesting that the A-SMase activation pathway is distinct from that causing induction of NFκB and SAPK/JNK. These data confirm a recent report by Hannun et al. (30Gamard C.J. Dbaibo G.S. Liu B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1997; 272: 16474-16481Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), who showed that ceramide is not a direct member of the NFκB activation pathway and rather may antagonize NFκB activation in some systems. The previous notion, that A-SMase and ceramide activate NFκB may be explained by tissue-specific activation and function of A-SMase or as a result of a nonspecific stress response caused by exogenous ceramide or SMase. Overexpression of the receptor interacting protein RIP has been shown to be capable of inducing both activation of NFκB and apoptosis. (26Natoli G. Costanzo A. Ianni A. Templeton D.J. Woodgett J.R. Balsano C. Levrero M. Science. 1997; 275: 200-203Crossref PubMed Scopus (415) Google Scholar,31Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1784) Google Scholar). Notably, RIP was found to be important for TNF-R55-mediated activation of NFκB but not for Fas/Apo-1-initiated induction of apoptosis (32Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar). The results of our study indicate that RIP does not alter TNF-induced activation of A-SMase, suggesting that RIP is not a component of TNF R55-triggered A-SMase activation pathway. It is well established that FADD, through its death effector domain (DED), recruits caspase-8 (FLICE/MACH) to the DISC where it is activated (16Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 17Muzio 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 (2743) Google Scholar, 33Medema 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 (1043) Google Scholar, 34Scaffidi C. Medema J.P. Krammer P.H. Peter M.E. J. Biol. Chem. 1997; 272: 26953-26958Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). However, overexpression of caspase-8/a or of its dominant negative, did not affect TNF-induced activation of A-SMase. Thus, the apoptotic signaling of caspase-8 pathway to segregate from the A-SMase activation pathway at the level of FADD. While caspase-8/a did not seem to be involved in activation of A-SMase, tetrapeptide caspase inhibitors markedly inhibited TNF-induced A-SMase activation. In addition, overexpression of crmA, a potent inhibitor of ICE-like proteases, was able to block the TRADD and/or FADD-mediated enhancement of TNF-induced activation of A-SMase. Together, these results suggest that ICE-like protease distinct from caspase-8/a are involved in A-SMase activation. Hannun and co-workers (36Smyth 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) reported that ceramide signaled apoptosis in vincristine-treated ALL-697 leukemia cells via a caspase-3 (CPP32)-like protease and that this effect was inhibitable by Bcl-2 (35Tsai E.Y. Yie J. Thanos D. Goldfeld A.E. Mol. Cell. Biol. 1996; 16: 5232-5244Crossref PubMed Scopus (178) Google Scholar, 36Smyth 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). Cells overexpressing Bcl-2, like wild-type cells, still responded to vincristine treatment with ceramide generation but failed to show PARP cleavage and apoptosis in response to a C6-ceramide analog. Further support for the notion that ceramide may be downstream of caspases is derived from experiments which showed that REAPER-induced ceramide generation was blocked by caspase inhibitor z-VAD.fmk (37Pronk G.J. Ramer K. Amiri P. Williams L.T. Science. 1996; 271: 808-810Crossref PubMed Scopus (260) Google Scholar). Furthermore, crmA, a pox virus serpin that is an inhibitor of ICE-like proteases but does not block caspase-3 (CPP32), abolished ceramide generation in TNF-treated cells (38Dbaibo 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). These studies suggested that ceramide generation occurs downstream of caspases. However, the source of ceramide was not addressed in these studies. The data of this report indicate that overexpression of TRADD and FADD enhances TNF-induced activation of A-SMase, which favors A-SMase to be downstream of the death domain adapter complex rather than ceramide synthase and other pathways of ceramide generation. The question arises whether A-SMase is able to confer apoptosis in some instance of a specific cell type/stimulus combination. This idea is consistent with previous reports demonstrating that deletions of the death domain region of the TNF-R55 and CD95 (1Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 39Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (598) Google Scholar, 40Cifone 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, 41Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar), as well as overexpression of dominant negative FADD-blocked ligand-induced ceramide generation and apoptosis (42Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar). Further, Dixit and co-workers (42Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar) reported that treatment with ceramide analogs can bypass the anti-apoptotic effect of dominant negative FADD and restore apoptosis, indicating that apoptotic elements downstream of ceramide generation are intact in these cells. The results of the present study clearly indicate that A-SMase activation can be dissociated from caspase-8-induced apoptosis. Overexpression of caspase-8 readily induced apoptosis of 293 cells in the absence of changes of A-SMase activity levels. Thus, A-SMase does not seem to be necessary for caspase-8-driven apoptosis. A-SMase seems neither sufficient for apoptosis of 293 cells because overexpression of A-SMase did not cause cell death. 2R. Schwandner, K. Wiegmann, K. Bernardo, D. Kreder, and M. Krönke, unpublished observation. The data do not rule out, though, that A-SMase constitutes an accessory pathway that can enhance the apoptotic response of 293 cells. The role of A-SMase in apoptosis induced by various stimuli in diverse cell types remains an open issue (for review see Ref. 43Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60 (in press)Crossref PubMed Scopus (731) Google Scholar), and its precise delineation will require further extensive investigation. We thank Drs. S. Adam-Klages and D. Adam for assistance with fluorescence-activated cell sorter analysis and helpful discussions. We thank Drs. M. Rothe and D. V. Goeddel, Tularik Inc., S. San Francisco, for providing reagents and fruitful discussions and Dr. V. Dixit for FADD and FLICE2 expression vectors.