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• W2005671749 abstract "Tumor necrosis factor-α (TNFα)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNFα induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNFα-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNFα signaling pathway.TNFα has been shown to activate at least two groups of caspases involved in the initiation and “execution” phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1β-converting enzyme-like proteases but is upstream of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in TNFα action and in connecting major components in the pathways leading to cell death. Tumor necrosis factor-α (TNFα)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNFα induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNFα-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNFα signaling pathway. TNFα has been shown to activate at least two groups of caspases involved in the initiation and “execution” phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1β-converting enzyme-like proteases but is upstream of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in TNFα action and in connecting major components in the pathways leading to cell death. Apoptosis, also known as programmed cell death, is an essential and closely regulated process important in the development and maintenance of multicellular organisms (1Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1992; 68: 251-306Crossref Scopus (6728) Google Scholar). Inducers of apoptosis include cytokines, chemotherapeutic agents, and stress conditions. Intracellular mediators of apoptosis include proteases of the interleukin-1β-converting enzyme/Ced-3 family (2Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2147) Google Scholar). The cytokine tumor necrosis factor-α (TNFα) 1The abbreviations used are: TNFα, tumor necrosis factor-α; CrmA, cytokine response modifier A; DTT, dithiothreitol; SM, sphingomyelin; SMase, sphingomyelinase; N-SMase, neutral magnesium-dependent sphingomyelinase; PARP, poly(ADP-ribose) polymerase; YVAD, Ac-Tyr-Val-Ala-Asp-chloromethylketone; GSH-ME, GSH-methylester; PBS, phosphate-buffered saline; NAC, N-acetylcysteine; BSO,l-buthionine-(S,R)-sulfoximine. induces apoptosis in target cells through binding to its cell surface receptor, TNFα receptor 1. Recently, it has been proposed that TNFα exerts its death signal through a series of protein domain-domain interactions; the cytoplasmic segment of TNFα receptor 1 binds to TRADD, which in turn associates with FADD, and the latter is thought to interact with MACH/FLICE (caspase-8) (3Hu S. Vincenz C. Buller M. Dixit V.M. J. Biol. Chem. 1997; 272: 9621-9624Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 4Boldin 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). Generation of oxidative stress has been proposed as a critical event for TNFα as well as other death-inducing agents in the process of initiating their cytotoxic activity (5Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2104) Google Scholar, 6Schwarz K.B. Free Radical Biol. Med. 1996; 21: 641-649Crossref PubMed Scopus (541) Google Scholar, 7Ciriolo M.R. Palamara A.T. Incerpi S. Lafavia E. Bue M.C. De Vito P. Rotilio G. J. Biol. Chem. 1997; 272: 2700-2708Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 8Um H.D. Orenstein J.M. Wahl S.M. J. Immunol. 1996; 156: 3469-3477PubMed Google Scholar, 9Ratan R.R. Murphy T.H. Baraban J.M. J. Neurochem. 1994; 62: 376-379Crossref PubMed Scopus (509) Google Scholar). Specifically, depletion of glutathione (GSH), the most abundant intracellular thiol-containing small molecule, has recently been found to either precede the onset of apoptotic cell death induced by various agents (10Macho A. Hirsch T. Marzo I. Marchetti P. Dallaporta B. Susin S. Zamzami N. Kroemer G. J. Immunol. 1997; 158: 4612-4619PubMed Google Scholar, 11Chiba T. Takahashi S. Sato N. Ishii S. Kikuchi K. Eur. J. Immunol. 1996; 26: 1164-1169Crossref PubMed Scopus (149) Google Scholar, 12Deas O. Dumont C. Mollereau B. Metivier D. Pasquier C. Bernard-Pomier G. Hirsch F. Charpentier B. Senik A. Int. Immunol. 1997; 9: 117-125Crossref PubMed Scopus (74) Google Scholar, 13Zhong L. Sarafian T. Kane D.J. Charles A.C. Mah S.P. Edwards R.H. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4533-4537Crossref PubMed Scopus (608) Google Scholar, 14van den Dobbelsteen D.J. Nobel C.S.I. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F.G. J. Biol. Chem. 1996; 271: 15420-15427Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 15Dhanbhoora C.M. Babson J.R. Arch. Biochem. Biophys. 1992; 293: 130-139Crossref PubMed Scopus (28) Google Scholar, 16Zucker B. Bauer G. Cell Death Differ. 1997; 4: 388-395Crossref PubMed Scopus (55) Google Scholar, 17Beaver J.P. Waring P. Eur. J. Cell Biol. 1995; 68: 47-54PubMed Google Scholar) or render the cells more sensitive to apoptotic agents (18Chiao C. Carothers A.M. Grunberger D. Solomon G. Preston G.A. Barrett J.C. Cancer Res. 1995; 55: 3576-3583PubMed Google Scholar, 19Christie N.A. Slutsky A.S. Freeman B.A. Tanswell A.K. Arch. Biochem. Biophys. 1994; 313: 131-138Crossref PubMed Scopus (29) Google Scholar, 20Kato S. Negishi K. Mawatari K. Kuo C.H. Neuroscience. 1992; 48: 903-914Crossref PubMed Scopus (122) Google Scholar), and it has been suggested that depletion of GSH is a relatively early event in the commitment to apoptosis (10Macho A. Hirsch T. Marzo I. Marchetti P. Dallaporta B. Susin S. Zamzami N. Kroemer G. J. Immunol. 1997; 158: 4612-4619PubMed Google Scholar). However, how this transmits cytotoxic signals remains to be answered. Ceramide, the product of cytokine-activated and sphingomyelinase (SMase)-catalyzed hydrolysis of sphingomyelin (SM), is an important regulator of apoptosis (21Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1618) Google Scholar). SM is one of the most abundant sphingolipid species in cell membrane with important structural and functional properties (22Hakomori S. Biochem. Soc. Trans. 1993; 21: 583-595Crossref PubMed Scopus (168) Google Scholar, 23Hakomori S. Annu. Rev. Biochem. 1981; 50: 733-764Crossref PubMed Scopus (1480) Google Scholar). Activation of SMases is believed to be involved in cell growth, differentiation, and apoptosis induced by cytokines, chemotherapeutic agents, and ionizing radiation (24Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar). To date, five types of SMase have been described, and they differ in subcellular location, pH optimum, cation dependence, and role in cell regulation (25Liu B. Obeid L.M. Hannun Y.A. Semin. Cell Dev. Biol. 1997; 8: 311-322Crossref PubMed Scopus (128) Google Scholar). The lysosomal acid SMase has been cloned, and this enzyme is activated in cells exposed to radiation (26Haimovitz-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 (855) Google Scholar), FAS (27Cifone 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), and TNFα (28Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Krönke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar, 29Jayadev S. Hayter H.L. Andrieu N. Gamard C.J. Liu B. Balu R. Hayakawa M. Ito F. Hannun Y.A. J. Biol. Chem. 1997; 272: 17196-17203Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The neutral and magnesium-dependent SMase, although not yet cloned, has been implicated in mediating apoptosis in cells exposed to serum starvation, FAS, TNFα, and cytosine arabinoside (29Jayadev S. Hayter H.L. Andrieu N. Gamard C.J. Liu B. Balu R. Hayakawa M. Ito F. Hannun Y.A. J. Biol. Chem. 1997; 272: 17196-17203Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 30Strum J.C. Small G.W. Pauig S.B. Daniel L.W. J. Biol. Chem. 1994; 269: 15493-15497Abstract Full Text PDF PubMed Google Scholar, 31Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Yu. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 32Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R.A. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 33Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar). Relatively little is known about the biological relevance of the other three SMases, namely the zinc-dependent and lysosomal acid SMase-derived acid enzyme (34Spence M.W. Byers D.M. Palmer F.B. Cook H.W. J. Biol. Chem. 1989; 264: 5358-5363Abstract Full Text PDF PubMed Google Scholar, 35Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), the magnesium-independent N-SMase (36Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar), and the alkaline SMase (37Nyberg L. Duan R.D. Axelson J. Nilsson A. Biochim. Biophys. Acta. 1996; 1300: 42-48Crossref PubMed Scopus (77) Google Scholar). We have observed that the N-SMase from the human acute lymphoblastic leukemic Molt-4 cells is inhibited in vitro by GSH (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). This suggested to us that N-SMase may be a direct target for transmitting at least some of the effects of GSH depletion. In the current study, we report that GSH inhibits the TNFα-induced activation of N-SMase in MCF7 cells, and depletion of GSH induced by TNFα involves activity of a cow pox virus cytokine modifier protein (cytokine response modifier A; CrmA)-sensitive interleukin-1β-converting enzyme-like protease but not Bcl-2. The implications of our results in bridging the fields of oxidative stress and sphingolipid signaling are discussed. MCF7 cells (39Dbaibo 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) were cultured in RPMI 1640 (Life Technologies, Inc.) containing 10% fetal bovine serum (Life Technologies, Inc.; complete medium) at 37 °C in 5% CO2. For cells transfected by Bcl-2 and its vector, hygromycin (150 μg/ml, Calbiochem) was included in the medium, and for cells transfected by CrmA and its vector, Geneticin (500 μg/ml, Life Technologies, Inc.) was used. For treatment, cells were normally seeded at 5 × 105 cells/10-cm culture dish (Falcon) in 10 ml of complete medium and grown for 48 h to 50–75% confluence. Prior to the initiation of treatment, cells were rested in fresh complete medium without the selection antibiotic but with 25 mm HEPES, pH 7.4. GSH was made as a 200 mm stock solution in RPMI and added to cells 2 h before the addition of TNFα. Unless otherwise indicated, the vector control cell line for the Bcl-2-transfected MCF7 cells was used and designated as MCF7 cells. TNFα was a gift from Dr. Phil Pekana (East Carolina University, Greenville, NC). [3H]choline chloride was purchased from NEN Life Science Products. GSH methyl ester and YVAD were from BACHEM (Torrance, CA). GSH and all other reagents were obtained from Sigma. MCF7 cells grown to near confluence in 175-cm2 flasks were harvested by trypsinization, washed with ice-cold phosphate-buffered saline (PBS), pelleted by centrifugation, frozen in a methanol-dry ice bath, and stored at −80 °C until use. To obtain N-SMase, detergent-solubilized membrane proteins were prepared from homogenate from pooled cell pellets (2 × 109 cells) and resolved on a DEAE-Sepharose column (1 × 10 cm) connected to an Amersham Pharmacia Biotech FPLC system essentially as described (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), except that both buffers A and B contained Triton X-100 (0.005%, w/v). N-SMase was efficiently resolved from A-SMase in MCF7 cells (25Liu B. Obeid L.M. Hannun Y.A. Semin. Cell Dev. Biol. 1997; 8: 311-322Crossref PubMed Scopus (128) Google Scholar) by the detergent extraction step and DEAE column, such that the final N-SMase preparation contained <1% of A-SMase activity under the assay conditions. The activity of N-SMase was determined using a mixed micelle assay system as described (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The reaction mixture contained enzyme preparation in 100 mmTris-HCl, pH 7.4, 10 nmol of [14C]sphingomyelin (100,000 dpm), 0.1% Triton X-100, and 5 mm magnesium chloride in a final volume of 100 μl. Cells grown in 10-cm Petri dishes were rinsed with ice-cold PBS and scraped into methanol. Cell lipids were extracted by the method of Bligh and Dyer (31Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Yu. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Ceramide content was determined using a modified diacylglycerol kinase assay as described previously (39Dbaibo 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). Cells were seeded at 2 × 105cells/10-cm culture dish and grown for 48 h. Then cells were switched to fresh complete medium containing [3H]choline (1 μCi/ml). After 48 h, cells were switched again to fresh medium and chased for 2 h before treatment with GSH and/or TNFα as described above. The level of SM was determined following a protocol essentially as described (40Andrieu N. Salvayre R. Levade T. Biochem. J. 1994; 303: 341-345Crossref PubMed Scopus (87) Google Scholar). Cells were seeded at 2 × 105 in 6-cm Petri dishes in 4 ml of complete medium. Two days later, cells were treated with the desired agents as described above. Treated cells were detached by trypsinization, washed (three times) with ice-cold PBS, and solubilized in 150 μl of water. 5-Sulfosalicylic acid was added to a final concentration of 2%, and the supernatant was separated from the acid-precipitated proteins by centrifugation. GSH content in the supernatant was determined by the Griffith (41Griffith O.W. Anal. Biochem. 1980; 106: 207-212Crossref PubMed Scopus (4060) Google Scholar) modification of the Tietze's enzymatic procedure (42Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5557) Google Scholar) as described (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Protein content was determined by the dye binding assay using bovine serum albumin as standard (43Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217546) Google Scholar). Cells were scraped into medium, pelleted by centrifugation, and washed (one time) with ice-cold PBS containing 1 mm phenylmethylsulfonyl fluoride. The cell pellet was resuspended in 50 μl of PBS-phenylmethylsulfonyl fluoride and solubilized in 2× SDS sample buffer. Western blot for PARP was performed as described (39Dbaibo 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, 44Zhang P. Liu B. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 9609-9612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The viability of cells was determined by their ability to exclude trypan blue. The survival of cells was determined with the WST-1 cell proliferation reagent from Boehringer Mannheim. Cells were seeded at 103 cells/well/200 μl of complete medium in a 96-well culture plate. Two days later, cells were treated in quadruplicate with TNFα as described above. At the end of treatment, WST-1 reagent was added, and after a 3-h incubation period the absorbance was measured at 450 nm with a multiwell plate reader as recommended by the manufacturer. Previously, we found that GSH inhibited in vitro the N-SMase from Molt-4 leukemic cells (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). To study the role of GSH in TNFα signaling, we chose the human mammary carcinoma cell line MCF7, which is very sensitive to TNFα (39Dbaibo 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). We partially purified N-SMase from MCF7 cells following the procedure described for rat brain (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) and tested the effects of GSH on N-SMase in vitro. When the enzyme was preincubated for 5 min at 37 °C with 1–20 mm GSH followed by incubation with substrate for 30 min, a dose-dependent inhibition of N-SMase by GSH was observed with a greater than 95% inhibition observed with 3 mm of GSH (Fig. 1 A). Preliminary experiments established that with the minimum preincubation time examined (1 min), GSH (3 mm) inhibited the enzyme activity by >80% (data not shown). The inhibition was specific for GSH, since two other small thiol-containing molecules, dithiothreitol (DTT) and β-mercaptoethanol, at concentrations up to 20 mm, were ineffective (Fig. 1 A), and co-incubation of GSH with 5 or 20 mm DTT did not alter the inhibitory profile for GSH (Fig. 1 B). When the N-SMase/GSH (4 mm) mixture was diluted by 3- or 5-fold, enzyme activity was recovered by 70 and 95%, respectively, suggesting that the inhibition was reversible (Fig. 1 C). GSH did not inhibit the acidic SMase (38Liu B. Hannun Y.A. J. Biol. Chem. 1997; 272: 16281-16287Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), although both the N-SMase and acidic SMase have been suggested to be activated in cells treated with TNFα (29Jayadev S. Hayter H.L. Andrieu N. Gamard C.J. Liu B. Balu R. Hayakawa M. Ito F. Hannun Y.A. J. Biol. Chem. 1997; 272: 17196-17203Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 33Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar). These results demonstrate that physiologic levels of GSH (3–10 mm) totally inhibit N-SMase. The results also suggest that the sharp drop in cellular levels of GSH may relieve this inhibition and cause activation of N-SMase. To investigate the effect of GSH on N-SMase in TNFα signaling, MCF7 cells were treated with TNFα (3 nm) for 2–24 h, and GSH levels were measured. As shown in Fig. 2 A, TNFα treatment resulted in an initial sharp drop in the level of GSH followed by a steady further decrease, with the first significant decrease observed at 8 h post-treatment. The most dramatic change in the level of GSH occurred between 8 and 10 h after TNFα treatment, where the GSH level plunged to nearly a third of that of control cells at the 10-h time point. GSH levels then steadily decreased to 7% of control cells by 24 h (Fig. 2 A). At the 10-h time point, cells treated with TNFα did not manifest any detectable sign of apoptosis. In particular, proteolysis of the “cell death substrate” poly(ADP-ribose) polymerase (PARP) (45Kaufmann S. Cancer Res. 1989; 49: 5870-5878PubMed Google Scholar) was only detected at 12–24 h (39Dbaibo 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). The TNFα-induced decrease in cellular GSH level was accompanied by an increase in the level of ceramide, with an initial significant rise observed at the 8–12-h time points. Ceramide levels at 12 h after TNFα treatment were twice control levels and reached more than 6-fold the control levels at 24 h (Fig. 2 B). When the effect of TNFα on cellular SM level was examined, significant SM hydrolysis was observed between 10 and 16 h, and a 30% hydrolysis of SM was detected at 14 h (Fig. 2 C). These kinetics raise the possibility that GSH may be involved in the regulation of SM hydrolysis and ceramide generation in cells treated with TNFα. Since GSH inhibited N-SMase in vitro at physiologically relevant concentrations and changes of GSH levels induced by TNFα preceded ceramide accumulation and SM hydrolysis, we next investigated the effects of manipulating intracellular GSH levels on TNFα-induced SM hydrolysis and ceramide generation. First, replenishment of intracellular GSH by the addition of 10 mm GSH to the culture medium prior to TNFα treatment (3 nm; 14 h) completely prevented the TNFα-induced SM hydrolysis (Fig. 3 A). Treatment of cells with GSH (10 mm) alone had no effect on SM levels. Second, GSH pretreatment significantly inhibited the accumulation of ceramide induced by TNFα (Fig. 3 B). A nearly complete inhibition of ceramide increase was observed at 12 h after TNFα treatment (Fig. 3 B). At 16 and 24 h, ceramide accumulation induced by TNFα was inhibited by about 50%. Third, in addition to GSH, significant inhibition of TNFα-induced ceramide accumulation was also achieved by treatment of cells with GSH methylester andN-acetylcysteine (NAC), which is converted intracellularly to cysteine, a precursor of GSH biosynthesis (Fig. 3 C). These results further support the notion that GSH levels regulate the activity of N-SMase in these cells. The partial protection observed with exogenous GSH may be explained by the fact that the addition of GSH, GSH-ME, or NAC could not completely restore the intracellular GSH levels in cells treated with TNFα. As shown in Fig. 3 D, pretreatment of cells with 20 mm GSH, GSH-ME, or NAC prior to a treatment with 3 nm TNFα for 16 h only brought the intracellular GSH levels from 14.5% to 52.8, 64.7, or 57.9% of that of control cells, respectively. Next, the biological consequences of changes in GSH levels in response to TNFα were studied by examination of cell death and survival. Whereas TNFα induced significant cell death, as determined by the ability of cells to exclude trypan blue dye, partial rescue of cells was achieved by replenishment of intracellular GSH with GSH added to the cell culture medium. Preincubation of cells with 10 and 15 mm GSH for 2 h prior to treatment with TNFα lowered TNFα-induced cell death from 58.5% to 35.5 and 26.5%, respectively (Fig. 4 A). The effect of TNFα on the viability of MCF7 cells was also determined using the WST-1 assay, which measures the activity of the mitochondrial respiratory chain in viable cells. TNFα significantly reduced the survival of MCF7 cells, and a 50% reduction in survival was observed when cells were treated with 1 nm TNFα for 24 h (Fig. 4 B). Inclusion of 10 mm GSH enhanced the survival of TNFα-treated cells to 75–95% of control level (Fig. 4 B). Mechanistically, the effects of TNFα on PARP cleavage, a close marker of the apoptotic response, were evaluated. TNFα-induced cleavage of PARP was partially inhibited by GSH and NAC in a dose-dependent manner (Fig. 4 C). Since PARP is a substrate for “execution” phase proteases such as CPP32/caspase-3 (46MacFarlane M. Cain K. Sun X.M. Alnemri E.S. Cohen G.M. J. Cell Biol. 1997; 137: 469-479Crossref PubMed Scopus (129) Google Scholar, 47Tewari 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: 1-20Abstract Full Text PDF PubMed Scopus (2279) Google Scholar), these results place GSH upstream of these proteases in the pathways leading to cell death. The hypothesis that GSH levels regulate the activity of N-SMase in response to TNFα suggests that the product ceramide should function downstream of GSH and that it probably would not alter the cellular level of GSH. Bacterial SMases are known to induce a fast and significant elevation of intracellular ceramide by hydrolysis of membrane sphingomyelin (44Zhang P. Liu B. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 9609-9612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 48Jarvis W.D. Kolesnick R.N. Fornari F.A. Traylor R.S. Gewirtz D.A. Grant S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 73-77Crossref PubMed Scopus (484) Google Scholar). However, when MCF7 cells were treated with 300 milliunits/ml of bacterial SMase from Staphylococcus aureus or Bacillus cereus for up to 24 h, no significant alteration in GSH levels was observed (Fig. 5 A). Similarly, treatment of cells with 2.5–10 μm of the cell-permeable short chain C6-ceramide for up to 48 h did not induce significant reduction in the GSH level (Fig. 5 B), although exogenous ceramide at these concentrations caused apoptosis in these cells (Fig. 5 C, Ref. 39Dbaibo 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). Conversely, pretreatment of cells with 10 mm GSH prior to ceramide treatment did not protect cells from death induced by ceramide (Fig. 5 C), in sharp contrast to the ability of GSH to inhibit the hydrolysis of SM and ceramide accumulation induced by TNFα. Finally, preincubation of cells with the inhibitor of GSH biosynthesis,l-buthionine-(S,R)-sulfoximine (BSO), for 24 h did not render the cells more sensitive to death induced by C6-ceramide (Fig. 5 D). The GSH level in the cells treated with 100 μm BSO for 24 h was 14.1 ± 1.3% of that of the control cells. These results place GSH upstream of N-SMase activation in the TNFα signaling pathway. TNFα-induced accumulation of ceramide has been shown to be inhibited by CrmA but not by Bcl-2 (39Dbaibo 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), placing activation of SMases downstream of CrmA-inhibitable proteases and upstream of Bcl-2 inhibitable proteases. To investigate the relationship between CrmA and GSH, MCF7 cells transfected with CrmA or empty vector were treated with TNFα, and GSH levels were determined. No significant changes in GSH levels were observed in CrmA-transfected cells treated with 3 nmTNFα over a time period ranging from 1 to 24 h, whereas a time-dependent depletion of GSH was observed in the vector-transfected cells (Fig. 6 A). The TNFα-induced depletion of GSH was also inhibited in a dose-dependent manner by the substrate-based tetrapeptide inhibitor of interleukin-1β-converting enzyme-like proteases, YVAD (49Margolin N. Raybuck S.A. Wilson K.P. Chen W. Fox T. Gu Y. Livingston D.J. J. Biol. Chem. 1997; 272: 7223-7228Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Pretreatment of MCF7 cells with 50 μm YVAD prior to TNFα (3 nm) brought the GSH level from 20% of control to 80% of control (Fig. 6 B). These results clearly demonstrate that the drop in GSH in response to TNFα is dependent upon activation of interleukin-1β-converting enzyme-like proteases (such as caspase-8/FLICE). Next, the effects of TNFα on the GSH levels in Bcl-2-transfected MCF7 cells were examined following treatment with 1–10 nmTNFα for 14 h. In both the Bcl-2- and vector-transfected cells, TNFα, at a dose as low as 1 nm, caused a drop in GSH levels to 45% of that of untreated control cells (Fig. 7). Concentrations of TNFα greater than 1 nm and up to 20 nm further decreased the GSH level to 20–30% of the control value. These results show that the drop in GSH levels is not downstream of the site of action of Bcl-2. Finally, the interrelation of GSH, Bcl-2, CrmA, and TNFα-induced hydrolysis of SM was investigated. When SM levels were analyzed in CrmA- and Bcl-2-transfected cells, Bcl-2 had no effect on TNFα-induced SM hydrolysis, and this SM hydrolysis in Bcl-2 cells was inhibited by pretreatment with GSH (Fig. 8). Cells with CrmA, however, were completely resistant to TNFα-induced SM hydrolysis (Fig. 8). Thus, similar to the drop in GSH, activation of N-SMase is downstream of the site of action of CrmA and most probably upstream of the site of action of Bcl-2. Our current study implicates GSH in regulation of a number of TNFα-induced processes. Specifically, we show that TNFα causes a dramatic depletion of GSH, which is closely related to regulation of neutral sphingomyelinase and activation of proteases. We show that GSH inhibits N-SMase in vitro and provide evidence for regulation of N-SMase in cells. Replenishment of GSH prevents hydrolysis of SM and offers partial protection against TNFα-induced generation of ceramide, PARP proteolysis, and cell death. Importantly, activation of interleukin-1β-converting enzyme-like proteases, those that are inhibited by CrmA or YVAD, is implicated in the depletion of GSH. On the other hand, Bcl-2 does not modulate GSH depletion. Since Bcl-2 inhibits activation of the CPP32 family of proteases (Group II; Ref. 68Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 30-38Abstract Full Text PDF Scopus (2187) Google Scholar) and since GSH plays a role in regulating proteolysis of PARP, the best studied substrate of this group, these results suggest that GSH depletion, SM hydrolysis, and ceramide accumulation (39Dbaibo 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, 50Zhang 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 (294) Google Scholar) occur at a point upstream of activation of this group of proteases and upstream of the site of action of Bcl-2. Depletion of GSH has been observed in response to many inducers of apoptosis, including TNFα (51Phelps D.T. Ferro T.J. Higgins P.J. Shankar R. Parker D.M. Johnson A. Am. J. Physiol. 1995; 269: L551-L559PubMed Google Scholar, 52Ishii Y. Partridge C.A. Del Vecchio P.J. Malik A.B. J. Clin. 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Pharmacol. 1994; 48: 361-370Crossref PubMed Scopus (18) Google Scholar), viral infections (7Ciriolo M.R. Palamara A.T. Incerpi S. Lafavia E. Bue M.C. De Vito P. Rotilio G. J. Biol. Chem. 1997; 272: 2700-2708Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and glutamate (20Kato S. Negishi K. Mawatari K. Kuo C.H. Neuroscience. 1992; 48: 903-914Crossref PubMed Scopus (122) Google Scholar, 57Froissard P. Monrocq H. Duval D. Eur. J. Pharmacol. 1997; 326: 93-99Crossref PubMed Scopus (79) Google Scholar). An important implication of this study is the prediction that those agents that cause GSH depletion will also cause activation of N-SMase as a direct consequence of this depletion, which would relieve the enzyme from inhibition by GSH. Ceramide generated through this mechanism would connect GSH depletion to a number of downstream targets known to be modulated by ceramide, such as caspase 3/CPP32 (58Perry D.P. Smyth M.J. Wang H-G. Reed J.C. Duriez P. Poirier G.G. Obeid L.M. Hannun Y.A. Cell Death Differ. 1997; 4: 29-33Crossref PubMed Scopus (37) Google Scholar), protein kinase Cα (59Lee J.Y. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1996; 271: 13169-13174Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), and phospholipase D (60Venable M.E. Bielawska A. Obeid L.M. J. Biol. Chem. 1996; 271: 24800-24805Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 61Gomez-Muñoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Abstract Full Text PDF PubMed Google Scholar, 62Suchard S.J. Hinkovska-Galcheva V. Mansfield P.J. Boxer L.A. Shayman J.A. Blood. 1997; 89: 2139-2147Crossref PubMed Google Scholar, 63Abousalham A. Liossis C. O'Brien L. Brindley D.N. J. Biol. Chem. 1997; 272: 1069-1075Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 64Nakamura Y. Nakashima S. Ojio K. Banno Y. Miyata H. Nozawa Y. J. Immunol. 1996; 156: 256-262PubMed Google Scholar). These observations also raise a number of important implications and questions. First, how does the activation of CrmA- and YVAD-inhibitable proteases result in depletion of GSH? Second, although this study identifies N-SMase as a direct target regulated by GSH, it is conceivable that the drop in GSH will affect other direct targets that may “communicate” additional messages in response to this drop. Third, although the results suggest an important role for GSH in regulating N-SMase with complete inhibition of SM hydrolysis by GSH, the effects on ceramide levels are incomplete. This raises the possibility that ceramide is regulated by additional pathways not involving N-SMase, such as the acidic SMase or ceramide synthase, each of which has been implicated in ceramide formation (33Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 65Kalén A. Borchardt R.A. Bell R.M. Biochim. Biophys. Acta. 1992; 1125: 90-96Crossref PubMed Scopus (48) Google Scholar, 66Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (786) Google Scholar, 67Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). Finally, GSH repletion did not reverse in full the effects of TNFα on PARP proteolysis or on cell death, suggesting that additional mechanisms are involved. In conclusion, these results seem to connect important regulators in TNFα-induced cell death. They provide for a direct target, N-SMase, that detects changes in the level of GSH, which may be of generalized significance in connecting oxidative damage with sphingolipid signaling." @default.
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• W2005671749 title "Glutathione Regulation of Neutral Sphingomyelinase in Tumor Necrosis Factor-α-induced Cell Death" @default.
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