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• W2024149904 abstract "Among its diverse biologic effects, the cytokine tumor necrosis factor α causes the rapid nuclear translocation of the transcription factor, nuclear factor κB (NF-κB). The p55 tumor necrosis factor (TNF) receptor shares with the related APO-1/Fas antigen the ability to initiate apoptosis. We investigated the role of the sphingolipid mediator ceramide in the cytokine-induced signaling mechanisms leading to NF-κB activation and cell death. Several lines of evidence presented here suggest that ceramide generated in response to TNFα or Fas activation is not involved in NF-κB activation. (i) Cell-permeable ceramides and exogenous sphingomyelinase failed to induce either nuclear translocation of NF-κB or degradation of its cytosolic inhibitor, I-κB, in Jurkat T cells. (ii) Ceramide treatment of cells inhibited phorbol ester-induced activation of NF-κB. (iii) TNFα potently activated NF-κB in a cell line deficient in acid sphingomyelinase. (iv) TNFα activated NF-κB within minutes without altering ceramide levels. (v) Treatment of Jurkat cells with cross-linking antibodies to APO-1/Fas induced large scale increases in ceramide and apoptosis without affecting NF-κB. (vi) Ceramide generation in response to Fas activation was inhibited byN-acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone, a peptide inhibitor of interleukin-1β-converting enzyme-like proteases, whereas TNFα-induced NF-κB activation was unaffected by the inhibitor. These results show that ceramide accumulation belongs selectively to the apoptotic pathway(s) induced by cytokines, and, if anything, ceramide may participate in negative feedback regulation of NF-κB. Among its diverse biologic effects, the cytokine tumor necrosis factor α causes the rapid nuclear translocation of the transcription factor, nuclear factor κB (NF-κB). The p55 tumor necrosis factor (TNF) receptor shares with the related APO-1/Fas antigen the ability to initiate apoptosis. We investigated the role of the sphingolipid mediator ceramide in the cytokine-induced signaling mechanisms leading to NF-κB activation and cell death. Several lines of evidence presented here suggest that ceramide generated in response to TNFα or Fas activation is not involved in NF-κB activation. (i) Cell-permeable ceramides and exogenous sphingomyelinase failed to induce either nuclear translocation of NF-κB or degradation of its cytosolic inhibitor, I-κB, in Jurkat T cells. (ii) Ceramide treatment of cells inhibited phorbol ester-induced activation of NF-κB. (iii) TNFα potently activated NF-κB in a cell line deficient in acid sphingomyelinase. (iv) TNFα activated NF-κB within minutes without altering ceramide levels. (v) Treatment of Jurkat cells with cross-linking antibodies to APO-1/Fas induced large scale increases in ceramide and apoptosis without affecting NF-κB. (vi) Ceramide generation in response to Fas activation was inhibited byN-acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone, a peptide inhibitor of interleukin-1β-converting enzyme-like proteases, whereas TNFα-induced NF-κB activation was unaffected by the inhibitor. These results show that ceramide accumulation belongs selectively to the apoptotic pathway(s) induced by cytokines, and, if anything, ceramide may participate in negative feedback regulation of NF-κB. Membrane glycerophospholipids, once thought to serve only as structural components of the cell, are now known to play central roles in a host of signal transduction pathways. Another class of lipids, the sphingolipids, have emerged recently as regulators of such diverse processes as cell growth and differentiation (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 2Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar, 3Kim M.-Y. Linardic C.M. Obeid L. Hannun Y. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar), cell cycle arrest (4Rani C.S. Abe A. Chang Y. Rosenzweig N. Saltiel A.R. Radin N.S. Shayman J.A. J. Biol. Chem. 1995; 270: 2859-2867Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 5Jayadev S. Liu B. Bielawska A. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar), cellular senescence (6Venable M.E. Lee J.Y. Smyth M.J. Bielawska A. Obeid L.M. J. Biol. Chem. 1995; 270: 30701-30708Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), and programmed cell death (7Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1604) Google Scholar, 8Ji L. Zhang G. Uematsu S. Akahori Y. Hirabayashi Y. FEBS Lett. 1995; 358: 211-214Crossref PubMed Scopus (164) Google Scholar, 9Hannun Y.A. Obeid L.M. Trends Biochem. Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (573) Google Scholar). In particular, the sphingolipid ceramide, produced by hydrolysis of membrane sphingomyelin (for review, see Ref. 10Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar), has received attention as an important bioeffector molecule, which may participate in mediating some of the actions of extracellular agents such as tumor necrosis factor α (TNFα) 1The abbreviations used are: TNF, tumor necrosis factor; TNFR, TNF receptor; NF-κB, nuclear factor κB; PMA, phorbol 12-myristate 13-acetate; C6-ceramide,N-hexanoylsphingosine; C2-ceramide,N-acetylsphingosine; SMase, sphingomyelinase; NPA, Niemann-Pick type A; YVAD.CMK,N-acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; IL, interleukin; ICE, interleukin-1β-converting enzyme. 1The abbreviations used are: TNF, tumor necrosis factor; TNFR, TNF receptor; NF-κB, nuclear factor κB; PMA, phorbol 12-myristate 13-acetate; C6-ceramide,N-hexanoylsphingosine; C2-ceramide,N-acetylsphingosine; SMase, sphingomyelinase; NPA, Niemann-Pick type A; YVAD.CMK,N-acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; IL, interleukin; ICE, interleukin-1β-converting enzyme. (11Dressler K.A. Mathias S. Kolesnick R.N. Science. 1992; 255: 1715-1718Crossref PubMed Scopus (367) Google Scholar, 12Dbaibo G.S. Obeid L.M Hannun Y.A. J. Biol. Chem. 1993; 268: 17762-17766Abstract Full Text PDF PubMed Google Scholar, 13Tepper 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), 1α,25-dihydroxyvitamin D3 (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 2Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar), γ-interferon (3Kim M.-Y. Linardic C.M. Obeid L. Hannun Y. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar), and APO-1/Fas (13Tepper 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, 14Cifone 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 (597) Google Scholar). TNFα is a pleiotropic cytokine, which has a central role in mediating immune regulation and inflammatory response via binding to its 55- and 75-kDa membrane receptors, termed TNFR-1 and TNFR-2, respectively (for review, see Refs. 15Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1835) Google Scholar and 16Beutler B. Van Huffel C. Science. 1994; 264: 667-668Crossref PubMed Scopus (420) Google Scholar). The APO-1/Fas antigen is a related member of the TNF receptor superfamily, which shares the ability to induce apoptosis in a number of hematopoietic cell lines (for review, see Ref. 17Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3972) Google Scholar). Recent studies have shed some light on the upstream events that may mediate a common death signaling pathway for both TNF and Fas involving recruitment of the death domain-associated protein FADD (18Chinnaiyan 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 (706) Google Scholar) and the sequential activation of members of the interleukin-1β-converting enzyme (ICE)-like protease family (19Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2104) Google Scholar, 20Muzio 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 (2730) Google Scholar,45Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar). TNFα and Fas also both induce sphingomyelinase activation and the generation of ceramide, which can induce apoptosis and may play a role in apoptotic signaling by these cytokines (7Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1604) Google Scholar, 8Ji L. Zhang G. Uematsu S. Akahori Y. Hirabayashi Y. FEBS Lett. 1995; 358: 211-214Crossref PubMed Scopus (164) Google Scholar, 9Hannun Y.A. Obeid L.M. Trends Biochem. Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (573) Google Scholar). TNFα is additionally known to activate the transcription factor NF-κB (21Osborn L. Kunkel W. Nabel G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1366) Google Scholar, 22Lowenthal J.W. Ballard D.W. Bogerd H. Böhnlein E. Greene W.C. J. Immunol. 1989; 142: 3121-3128PubMed Google Scholar) which is thought to mediate the TNFα-induced expression of a variety of genes including the IL-2 receptor. NF-κB belongs to the Rel family of transcription factors and in its inactive state exists in the cytosol as a heterodimer bound to the inhibitory complex I-κB (for review, see Refs. 23Grilli M. Chiu J.J. Lenardo M.J. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (881) Google Scholar and 24Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4584) Google Scholar). Stimulation at the cell surface by cytokines such as TNFα and interleukin-1β (IL-1β) or by lipopolysaccharide initiates a poorly understood set of signaling events, which result in the phosphorylation and degradation of I-κB, thus allowing the free NF-κB dimer to translocate to the nucleus and initiate transcription of κB-responsive elements (25Beg A.A. Finco T.S. Nantermet P.V. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar, 26Henkel T. Machleidt T. Alkalay I. Krönke M. Ben-Neriah Y. Baeuerle P. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1034) Google Scholar). The TNF receptor-associated proteins TRADD and TRAF-2 have been implicated in signaling to NF-κB by TNFR-1 (27Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1739) Google Scholar, 28Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (975) Google Scholar). It is unclear whether ceramide generated in response to TNFα is involved in NF-κB activation. Some studies have suggested an essential role for this lipid second messenger in NF-κB activation and a dependence on ceramide generated by acid sphingomyelinase activity in particular (29Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 30Yang Z. Costanzo M. Golde D.W. Kolesnick R.N. J. Biol. Chem. 1993; 268: 20520-20523Abstract Full Text PDF PubMed Google Scholar, 31Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Krönke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). However, other studies have provided evidence against a role for ceramide in this signaling pathway (12Dbaibo G.S. Obeid L.M Hannun Y.A. J. Biol. Chem. 1993; 268: 17762-17766Abstract Full Text PDF PubMed Google Scholar, 32Betts J.C. Agranoff A.B. Nabel G.J. Shayman J.A. J. Biol. Chem. 1994; 269: 8455-8458Abstract Full Text PDF PubMed Google Scholar, 33Kuno K. Sukegawa K. Ishikawa Y. Orii T. Matsushima K. Int. Immunol. 1994; 6: 1269-1272Crossref PubMed Scopus (58) Google Scholar, 34Johns L.D. Sarr T. Ranges G.E. J. Immunol. 1994; 152: 5877-5882PubMed Google Scholar, 35Higuchi M. Singh S. Jaffrezou J.P. Aggarwal B. J. Immunol. 1996; 157: 297-304PubMed Google Scholar). Therefore, in the current study we sought to clarify the potential role of ceramide in the TNFα and Fas mechanisms of NF-κB activation. In this study, we demonstrate that cell-permeable analogs of ceramide were unable to induce either I-κB degradation or nuclear translocation of NF-κB in intact Jurkat T cells. Likewise, treatment of cells with bacterial sphingomyelinase, which has been shown to increase intracellular ceramide levels via cleavage of membrane sphingomyelin (36Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 23530-23537Abstract Full Text PDF PubMed Google Scholar), failed to activate NF-κB. TNFα remained a potent activator of NF-κB in cells from a patient with Niemann-Pick disease type A (NPA), which lack acid sphingomyelinase activity (37Ferlinz K. Hurwitz R. Sandhoff K. Biochem. Biophys. Res. Commun. 1991; 179: 1187-1191Crossref PubMed Scopus (39) Google Scholar,38Schuchman E.H. Desnick R.J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2601-2624Google Scholar). Treatment of Jurkat T cells with cross-linking antibodies to APO-1/Fas caused both a marked increase in intracellular ceramide levels and apoptosis. However, Fas was unable to signal nuclear translocation of NF-κB at early or late time points. Pretreatment of Jurkat cells with YVAD.CMK, a site-specific inhibitor of ICE-like proteases (39Cain K. Inayat-Hussein S.H. Couet C. Cohen G.M. Biochem. J. 1996; 314: 27-32Crossref PubMed Scopus (71) Google Scholar), inhibited both Fas-induced ceramide generation and apoptosis, but not TNFα-induced NF-κB activation. Furthermore, in Jurkat cells treated with TNFα, we observed no increase in intracellular ceramide formation in the time course needed for activation of NF-κB (1–10 min). Thus, Fas induced intracellular ceramide increases in an ICE-like protease-dependent manner without activating NF-κB, whereas TNFα activated NF-κB within minutes independent of ceramide and ICE-like proteases. Finally, we show that ceramide inhibits PMA-induced activation of NF-κB. Taken together, these lines of evidence strongly suggest that ceramide is a specific component of apoptotic signaling pathways and not of the pathways leading to NF-κB activation. Jurkat T cells were obtained from ATCC, Rockville, MD. Niemann-Pick A and normal skin fibroblasts were obtained from the Coriell Institute (National Institute on Aging). TNFα was a kind gift from Dr. Phil Pekala (East Carolina University). Anti-Fas monoclonal antibody was purchased from Upstate Biotechnology, Inc.Staphylococcus aureus sphingomyelinase was purchased from Sigma. C2- and C6-ceramide were synthesized as described (2Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar). [γ-32P]ATP was from NEN Life Science Products. Poly(dI·dC) and poly(dN6) were from Pharmacia Biotech Inc. Anti-NF-κB monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-I-κB monoclonal antibody was purchased from Rockland, Inc. YVAD.CMK (Bachem Bioscience, King of Prussia, PA) was dissolved in Me2SO before addition to medium (final Me2SO concentration 0.2%, v/v), and appropriate solvent controls were used. Jurkat (acute lymphocytic T cell leukemia) cells were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum. Niemann-Pick type A skin fibroblasts and normal skin fibroblasts were grown in minimal essential medium (Life Technologies, Inc.) supplemented with 20% (v/v) fetal bovine serum. Cells were maintained at densities between 2 × 105 and 1.2 × 106 cells/ml under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C). Treatment with bacterial sphingomyelinase was carried out as described (35Higuchi M. Singh S. Jaffrezou J.P. Aggarwal B. J. Immunol. 1996; 157: 297-304PubMed Google Scholar). The nuclear extraction procedure was modified from Dignam (40Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar) and Osborn (21Osborn L. Kunkel W. Nabel G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1366) Google Scholar). After treatments, medium was removed and approximately 107 cells were washed once in ice-cold PBS. The cell pellet was rapidly frozen in dry ice and ethanol and then thawed by resuspending in 100 μl of ice-cold Buffer A (10 mm Hepes (pH 7.9), 10 mm KCl, 1.5 mm MgCl2, 1 mm dithiothreitol) resulting in approximately 100% lysis. The nuclei were pelleted by microcentrifugation at 3500 rpm for 10 min at 4 °C. The supernatant was discarded, and the nuclei were suspended in 15 μl of Buffer C (20 mm Hepes (pH 7.9), 0.4 m NaCl, 1.5 mm MgCl2, 25% (v/v) glycerol, 0.2 mm EDTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). The suspension was mixed gently for 20 min at 4 °C, and then microcentrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was diluted with 40–70 μl of Buffer D (20 mm Hepes (pH 7.9), 50 mm KCl, 25% (v/v) glycerol, 0.2 mm EDTA, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride), and aliquots were stored at −80 °C. Protein concentrations were determined using the Bio-Rad assay. Reactions were performed in a 20-μl volume, using 8–10 μg of nuclear extract in the presence of 1 μg of poly(dI·dC), 1 μg of poly(dN)6, and 10 μg of bovine serum albumin. Incubations were in the presence of HDKE buffer with the following final concentrations: 20 mm Hepes, pH 7.9, 50 mm KCl, 1 mm EDTA, and 5 mm dithiothreitol. 1 μl of radiolabeled oligonucleotide probe (20,000–50,000 cpm) was added to each reaction. After incubation for 20 min, the reaction was terminated by adding 6 μl of 15% Ficoll solution containing indicator dyes. For supershift experiments, 1 μl of antibody was added to appropriate samples, which were then incubated for 1 h on ice prior to addition of Ficoll solution. Equal amounts of the reaction mixture were loaded on a 5% nondenaturing polyacrylamide gel in 1 × TBE and were run at 200 V. Gels were transferred to Whatman filter paper, dried at 80 °C for 2 h, and exposed to film at −80 °C for 4–12 h. The probe utilized was a synthetic NF-κB consensus oligonucleotide with the following sequence: 5′-AGTTGAGGGGACTTTCCCAGGC-3′. It was end-labeled using T4 kinase and [γ-32P]ATP. The mutant oligonucleotide used in competition experiments had the following sequence: 5′-AGTTGAGGCGACTTTCCCAGGC-3′. After treatments were carried out, cytosolic extracts from Jurkat cells were prepared by washing 107 cells in ice-cold PBS and resuspending pellet in ice-cold homogenizing buffer (20 mm Tris-HCl (pH 7.5), 250 mm sucrose, 10 mm EGTA (pH 7.4), 2 mm EDTA (pH 7.4), 1 mm phenylmethylsulfonyl fluoride, 0.02% leupeptin, and 0.1% Triton X-100). The cells were then lysed by sonication and ultracentrifuged at 40,000 rpm for 40 min at 4 °C to separate cytosolic from nuclear and membrane components. An aliquot of the supernatant was removed for protein determination, and the remainder of the supernatant was mixed (1:1) with 2 × sodium dodecyl sulfate sample buffer and boiled for 5 min. Samples containing equivalent amounts of protein were then analyzed by Western blot analysis using enhanced chemiluminescence (ECL) by Amersham. Jurkat T cells were seeded at 5 × 105 cells/ml and treated for the indicated times as described. Cells were harvested, and lipids were extracted by the Bligh and Dyer method (41Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42389) Google Scholar); lipids were dried and resuspended in 1 ml of chloroform. Duplicate aliquots of 100 μl were set aside for phosphate measurements (42Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1314) Google Scholar), and 100 μl was utilized in the Escherichia coli diacylglycerol kinase assay as modified for ceramide (43Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar, 44Van Veldhoven P.P. Bishop W.R. Bell R.M. Anal. Biochem. 1989; 183: 177-189Crossref PubMed Scopus (74) Google Scholar). Ceramide was quantitated by using external standards and was normalized to phosphate. Niemann-Pick fibroblasts were washed with PBS and resuspended in cold lysis buffer (25 mmTris-HCl, pH 7.4, 5 mm EDTA, 1 mm ATP, 20 μg/ml chymostatin, 20 μg/ml leupeptin, 20 μg/ml antipain, 20 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride) to attain a final concentration of 5 × 107 cells/ml. Cells were lysed via three cycles of freezing and thawing. Homogenate was obtained by centrifuging the total cell lysate for 10 min at 1000 × g at 4 °C. Sphingomyelinase activity was assayed in all fractions using 14C-labeled sphingomyelin as described (45Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar). To investigate whether TNFα-induced ceramide generation is a sufficient signal for NF-κB activation, Jurkat T cells were treated with varying concentrations of synthetic cell-permeable ceramide analogs and were then assayed for nuclear translocation of NF-κB (Fig. 1, A and B). These cell-permeable analogs have been shown to mimic the cytotoxic (apoptotic) effects of TNFα at micromolar concentrations (7Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1604) Google Scholar). However, neither C2- nor C6-ceramide was able to induce nuclear translocation and activation of NF-κB as compared with untreated and TNFα-treated controls over both short and extended time courses. To evaluate the possibility that endogenously generated ceramide may provide a signal that the synthetic analogs lack, cells were treated with bacterial sphingomyelinase (Fig. 1 C). Incubation of leukemic cell lines with bacterial sphingomyelinase has been shown to result in the hydrolysis of membrane sphingomyelin and the generation of intracellular ceramide in a dose- and time-dependent fashion (36Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 23530-23537Abstract Full Text PDF PubMed Google Scholar). However, this treatment likewise failed to signal nuclear translocation of NF-κB in Jurkat T cells. Activation of NF-κB was also studied by using Western blot analysis of its cytosolic inhibitor, I-κBα. Upon treatment of cells with a variety of inducers of NF-κB, I-κBα is phosphorylated and proteolyzed, thereby releasing NF-κB and allowing the free heterodimer to enter the nucleus and bind to target gene promoter regions (23Grilli M. Chiu J.J. Lenardo M.J. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (881) Google Scholar, 24Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4584) Google Scholar). I-κBα proteolysis has been shown to be a necessary regulated step in the activation of NF-κB by TNFα (25Beg A.A. Finco T.S. Nantermet P.V. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar,26Henkel T. Machleidt T. Alkalay I. Krönke M. Ben-Neriah Y. Baeuerle P. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1034) Google Scholar). Treatment of Jurkat T cells with TNFα resulted in proteolysis of I-κBα within 10 min (data not shown), and after 30 min near-complete proteolysis of the band was observed (Fig.2). Treatment with varying concentrations of cell-permeable ceramide and bacterial sphingomyelinase did not induce I-κBα proteolysis as compared with untreated control (Fig. 2), further suggesting the divergence of ceramide-mediated pathways from the signaling events leading to NF-κB activation. TNFα signaling through the 55-kDa receptor has been shown to result in activation of sphingomyelinase with both neutral and acidic pH optima, and there has been evidence that a ceramide signal generated by the acid sphingomyelinase in particular is a necessary and sufficient signal for NF-κB activation (29Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (674) Google Scholar). To more closely evaluate the possible role of acid sphingomyelinase in this pathway, we studied skin fibroblasts from a patient with Niemann-Pick disease type A, a lysosomal storage disease characterized at the cellular level by complete lack of acid sphingomyelinase activity and clinically by pathological sphingomyelin accumulation (37Ferlinz K. Hurwitz R. Sandhoff K. Biochem. Biophys. Res. Commun. 1991; 179: 1187-1191Crossref PubMed Scopus (39) Google Scholar, 38Schuchman E.H. Desnick R.J. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2601-2624Google Scholar). To confirm the phenotype of the cell line, post-nuclear extracts from the Niemann-Pick fibroblasts and from age-matched normal skin fibroblasts were assayed for sphingomyelinase activity in neutral and acidic pH ranges (Table I). The Niemann-Pick fibroblast line completely lacked acid sphingomyelinase activity and displayed only a small amount of neutral sphingomyelinase activity, while the normal skin fibroblast line displayed acid and neutral sphingomyelinase activities of 49.25 and 2.50 nmol/mg protein/h, respectively. Despite a complete lack of acid sphingomyelinase activity in the Niemann-Pick fibroblasts, the TNFα-induced nuclear translocation of NF-κB remained unimpaired as compared with controls (Fig. 3 A). Nuclear translocation was evident within 15 min of treatment with TNFα. Therefore, the kinetics of NF-κB activation in the enzymatically deficient fibroblast line are identical to those seen in the skin fibroblast controls and in Jurkat and other hematopoietic cell lines. The specificity of NF-κB activation by TNFα in the Niemann-Pick fibroblasts is shown in Fig. 3 B. A monoclonal antibody specific for the p65 subunit of the NF-κB dimer caused retardation of the TNFα-induced band, resulting in a characteristic “supershift,” whereas an anti-c-Rel monoclonal antibody did not cause a supershift (lanes 3 and4). Specificity was further demonstrated by competitive washout of the band by addition of excess cold κB consensus oligonucleotide and the inability of mutant κB oligonucleotide to compete with the band (Fig. 3 B, lanes 5 and6). This evidence suggests that activation of acid sphingomyelinase is not a required step in the signaling pathway linking TNFα binding at the cell surface to the activation of NF-κB.Table IAcid and neutral sphingomyelinase activities in Niemann-Pick disease type A (NPA) and normal human skin fibroblastsNPANormal skin fibroblastsAcid sphingomyelinase1-anmol/mg protein/h.049.27Neutral magnesium-dependent sphingomyelinase1-anmol/mg protein/h.0.252.50Cells were harvested and enzyme activities measured as described under “Experimental Procedures.”1-a nmol/mg protein/h. Open table in a new tab Cells were harvested and enzyme activities measured as described under “Experimental Procedures.” Binding of the APO1/Fas cell surface antigen by either its ligand or cross-linking antibodies initiates a poorly understood set of signaling events resulting in programmed cell death in a number of hematopoietic cell lines (17Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3972) Google Scholar). Recent work has begun to define the upstream mediators of Fas signaling, including the death domain-associated protein FADD (18Chinnaiyan 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 (706) Google Scholar). Other evidence has implicated ceramide as a downstream effector of the apoptotic pathway; treatment of SKW6.4 cells with a monoclonal cross-linking antibody has been shown to result in activation of membrane bound neutral sphingomyelinase and a subsequent 2–3-fold increase in intracellular ceramide levels within 16 h of treatment (13Tepper 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). In the present study, we treated Jurkat T cells with anti-Fas antibodies and observed a greater than 5-fold increase in ceramide levels over control within 12 h and a greater than 7-fold increase over control within 20 h as assessed by diacylglycerol kinase assay (Fig. 4 A). At the concentrations studied, anti-Fas antibody induced 80–90% cell death after 24 h (data not shown). TNFα induces a well characterized activation of NF-κB in Jurkat and other hematopoietic cell lines (21Osborn L. Kunkel W. Nabel G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1366) Google Scholar, 22Lowenthal J.W. B" @default.
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