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• W2025787534 abstract "The role of cytosolic phospholipase A2 (cPLA2) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor α (TNFα). In L929 cells, the addition of TNFα resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5–12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg2+-dependent, neutral Mg2+-independent, and acidic enzymes). The variant cell line C12, which lacks cPLA2, is resistant to the cytotoxic action of TNFα. TNFα was able to activate nuclear factor κB in both the wild-type L929 cells and the C12 cells. However, TNFα was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C6-ceramide overcame the resistance to TNFα and caused cell death in C12 cells to a level similar to that in L929 cells. The introduction of the cPLA2 gene into C12 cells resulted in partial restoration of TNFα-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA2 is a necessary component in the pathways leading to ceramide accumulation and cell death. The role of cytosolic phospholipase A2 (cPLA2) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor α (TNFα). In L929 cells, the addition of TNFα resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5–12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg2+-dependent, neutral Mg2+-independent, and acidic enzymes). The variant cell line C12, which lacks cPLA2, is resistant to the cytotoxic action of TNFα. TNFα was able to activate nuclear factor κB in both the wild-type L929 cells and the C12 cells. However, TNFα was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C6-ceramide overcame the resistance to TNFα and caused cell death in C12 cells to a level similar to that in L929 cells. The introduction of the cPLA2 gene into C12 cells resulted in partial restoration of TNFα-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA2 is a necessary component in the pathways leading to ceramide accumulation and cell death. The sphingomyelin (SM) 1The abbreviations used are: SM, sphingomyelin; SMase, sphingomyelinase; TNFα, tumor necrosis factor α AA, arachidonic acid; PLA2, phopholipase A2; cPLA2, cytosolic PLA2; PBS, phosphate-buffered saline; NF-κB, nuclear factor κB. 1The abbreviations used are: SM, sphingomyelin; SMase, sphingomyelinase; TNFα, tumor necrosis factor α AA, arachidonic acid; PLA2, phopholipase A2; cPLA2, cytosolic PLA2; PBS, phosphate-buffered saline; NF-κB, nuclear factor κB. cycle, first described by Okazaki et al. (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar), has gained recognition over the past few years as a key mechanism for regulating anti-mitogenic signals. Activation of this cycle through the regulation of a signal-induced sphingomyelinase (SMase) results in generation of the lipid second messenger ceramide. Ceramide then modulates a number of biological fates, including growth inhibition (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 2Bielawska A. Linardic C.M. Hannun Y.A. FEBS Lett. 1992; 307: 211-214Crossref PubMed Scopus (89) Google Scholar, 3Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (549) Google Scholar), differentiation (2Bielawska A. Linardic C.M. Hannun Y.A. FEBS Lett. 1992; 307: 211-214Crossref PubMed Scopus (89) Google Scholar), apoptosis (4Pushkareva M. Obeid L.M. Hannun Y.A. Immunol. Today. 1995; 16: 294-297Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 5Obeid L.M. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Crossref PubMed Scopus (235) Google Scholar, 6Kolesnick R.N. Haimovitz-Friedman A. Fuks Z. Biochem. Cell Biol. 1994; 72: 471-474Crossref PubMed Scopus (141) Google Scholar), and cell cycle arrest (7Jayadev S. Liu B. Bielawska A.E. 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). Although recent studies have begun to catalogue inducers such as TNFα, interleukin-1β, nerve growth factor, and Fas that are capable of signaling through the SM cycle (see Refs. 5Obeid L.M. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Crossref PubMed Scopus (235) Google Scholar, 6Kolesnick R.N. Haimovitz-Friedman A. Fuks Z. Biochem. Cell Biol. 1994; 72: 471-474Crossref PubMed Scopus (141) Google Scholar, and 8Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar for reviews), the mechanisms by which these inducers stimulate SMase activity remain poorly understood.TNFα, through interaction with either a 55- or 75-kDa TNF receptor (9Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (999) Google Scholar, 10Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1831) Google Scholar), impacts upon a myriad of intracellular signaling cascades, including protein phosphorylation cascades, transcription factors, and lipid messengers (11Heller R.A. Kronke M. J. Cell Biol. 1994; 126: 5-9Crossref PubMed Scopus (401) Google Scholar). Two classes of lipid mediators have been implicated in TNFα signaling, glycerophospholipid metabolites and sphingolipid metabolites (11Heller R.A. Kronke M. J. Cell Biol. 1994; 126: 5-9Crossref PubMed Scopus (401) Google Scholar, 12Kronke M. Schutze S. Scheurich P. Pfizenmaier K. Aggarwal B.B. Vilcek J. Tumor Necrosis Factors: Structure, Function, and Mechanism of Action. Marcel Dekker, Inc., New York1992: 189-216Google Scholar), and recent evidence suggests that these two classes of lipids may interact (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar). In HL-60 cells, a linear correlation was established among TNFα stimulation, AA generation, and SM cycle activation: TNFα-stimulated AA liberation preceded ceramide generation, and AA reproduced the effects of TNFα on the SM cycle (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar). Although these studies suggested that AA and/or its metabolites may be involved in activation of SMase, the physiologic role of the PLA2/AA pathway in regulating SMase activity has not been determined.In this study, we examined the role of PLA2 in SMase activation in the L929 murine fibroblast cell line. In L929 cells, TNFα treatment is known to produce potent cytotoxic effects (14Hayakawa M. Oku N. Takagi T. Hori T. Shibamoto S. Yamanaka Y. Takeuchi K. Tsujimoto M. Ito F. Cell Struct. Funct. 1991; 16: 333-340Crossref PubMed Scopus (16) Google Scholar). TNFα-resistant L929 cells have also been generated via clonal selection from resistant populations of L929 mouse fibroblasts grown in the presence of TNFα (14Hayakawa M. Oku N. Takagi T. Hori T. Shibamoto S. Yamanaka Y. Takeuchi K. Tsujimoto M. Ito F. Cell Struct. Funct. 1991; 16: 333-340Crossref PubMed Scopus (16) Google Scholar, 15Hayakawa M. Ishida N. Takeuchi K. Shibamoto S. Hori T. Oku N. Ito F. Tsujimoto M. J. Biol. Chem. 1993; 268: 11290-11295Abstract Full Text PDF PubMed Google Scholar). One of these cell lines, C12, differed from the original L929 cells by the absence of cPLA2 and by the lack of inducibility of AA in response to TNFα. However, in all parameters of TNFα receptor binding and internalization, this resistant cell line was found to be analogous to the parental L929 line.Using these two cell lines, L929 and C12, we investigated the necessity of cPLA2 activation and AA generation for TNFα-induced ceramide generation. In the L929 model system, we found that the kinetics of cytokine-induced lipid mobilization occurred much later than previously documented in HL-60 (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar, 16Kim M.-Y. Linardic C. Obeid L. Hannun Y. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar) and U937 (17Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1598) Google Scholar) cells. Although the kinetics of activation were protracted in the L929 system, we found that, similar to HL-60 and U937 cells, the mechanism of ceramide generation was still through the activation of SMase and the subsequent hydrolysis of SM. Furthermore, we found that the generation of ceramide in response to TNFα did not occur in the resistant line, which was incapable of liberating AA following cytokine treatment. Finally, we found that TNFα-induced AA generation, ceramide generation, and cytotoxicity could be partly re-established in a C12 variant containing a cPLA2 expression vector. This study implicates cPLA2 activation and AA generation as necessary precursors to TNFα-induced activation of the SM cycle. The implications of these findings are discussed.EXPERIMENTAL PROCEDURESMaterialsThe L929 cell line and its C12 variant have been previously described (14Hayakawa M. Oku N. Takagi T. Hori T. Shibamoto S. Yamanaka Y. Takeuchi K. Tsujimoto M. Ito F. Cell Struct. Funct. 1991; 16: 333-340Crossref PubMed Scopus (16) Google Scholar, 15Hayakawa M. Ishida N. Takeuchi K. Shibamoto S. Hori T. Oku N. Ito F. Tsujimoto M. J. Biol. Chem. 1993; 268: 11290-11295Abstract Full Text PDF PubMed Google Scholar). Dulbecco's modified Eagle's medium and kanamycin sulfate were purchased from Life Technologies, Inc. Heat-inactivated fetal calf serum was purchased from Summit Biotechnologies (Fort Collins, CO). Arachidonic acid was purchased from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). [3H]Choline chloride and [3H]arachidonic acid were purchased from DuPont NEN. TNFα was a gift from Hoffmann-La Roche (Basel, Switzerland). All other reagents were obtained from Sigma.MethodsCell CultureAll cells were maintained for up to 20 passages at 37 °C in a 5% CO2 incubator. For general maintenance, L929 and C12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 60 mg/liter kanamycin sulfate. L929/neo and CPL4 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 0.8 mg/ml G418. For studies, cells were plated at 2 × 104/well in 12-well plates, at 5 × 104/well in 6-well plates, or at 1 × 105/well in 10-cm Petri plates. Cells were allowed to grow to 50–70% confluence and then washed, refed, rested for 4 h, and treated as indicated. Time-matched controls were always run concurrently.Arachidonic Acid ReleaseCells seeded in 6- or 12-well plates were grown for 2 days and then labeled with 1 μCi/ml [3H]arachidonic acid for 24 h. Post-labeling, cells were washed, refed, and rested for 4 h. Cells were then treated as indicated, and 1.0–1.2 ml of culture medium was harvested from each treatment well. Non-adherent cells were pelleted out of the harvested medium, and 400-μl aliquots were counted to determine the levels of released label. The total counts associated with cells were ∼150,000 dpm, and 7% of the total label (10,500 dpm) was released in response to TNFα. The medium was also subjected to TLC to assess whether metabolites such as phospholipids or acylglycerols accounted for the observed counts. We found that minimal counts, if any, were associated with such metabolites.Ceramide and Diacylglycerol QuantitationCells grown in 10-cm Petri plates were grown for 2–4 days, refed, rested, and treated. Following the indicated times of treatment, the media were removed from the plates, and adherent cells were washed with PBS. Cells were then scraped, and the lipids were extracted via the method of Bligh and Dyer (18Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42153) Google Scholar). In instances where cell floaters were analyzed, the treatment media and the PBS wash were pooled and spun down. The resultant pellet was then considered the “floater” population, and lipids were harvested from these cells. Extracted lipids were dried, resuspended in chloroform, and aliquoted for phosphate (19Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar) and diacylglycerol kinase (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar) analyses as described previously. Ceramide and diacylglycerol levels were quantitated using external standards, and the resultant values were normalized against total lipid phosphate.SM QuantitationCells grown in 10-cm Petri plates for 2–4 days were treated as described for the ceramide measurements. Lipids were extracted via the method of Bligh and Dyer (18Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42153) Google Scholar), and SM was quantitated by the bacterial SMase method described previously (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar).SMase Isolation and AssayCells were seeded at 3 × 105/30-cm Petri plate in 30 ml of regular growth medium. Cells were allowed to grow for 3–4 days and then washed, refed, and treated. Following the indicated treatment times, cells were scraped into a minimal volume of serum-free medium and pelleted. Retrieved cells were resuspended in cold lysis buffer (20Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (673) Google Scholar) and lysed via three cycles of freeze-thawing (one cycle = 3 min in a methanol/dry ice bath, 3 min at room temperature, and vortexing). By this protocol, ≥95% of the cells were lysed. Cells were spun at 2100 rpm (1000 × g) for 10 min to remove nuclei and the few unlysed cells. The resulting homogenate was assayed for SMase activity as described previously (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar). Assay conditions for the three different sphingomyelinases were as follows: 1) neutral Mg2+-dependent: 10 nmol of SM (2 × 105 cpm), 0.1% Triton X-100, 0.1 m Tris-HCl, pH 7.4, and 5 mm MgCl2; 2) neutral Mg2+-independent: 10 nmol of SM (2 × 105cpm), 0.1% Triton X-100, and 0.1 m Tris-HCl, pH 7.4; and 3) acidic: 10 nmol of SM (2 × 105 cpm), 0.1% Triton X-100, and 0.1 m sodium acetate, pH 5.0.Thymidine IncorporationCells were grown in 6-well plates for 2 days and then washed, refed, rested, and treated. Four hours prior to harvest, 1 μCi/ml [3H]thymidine was added to each well. Following the indicated treatment times, cells were harvested via a modification of a previously described method (21Smyth M.J. Runnels B. Wharton W. J. Cell. Biochem. 1992; 50: 210-218Crossref PubMed Scopus (4) Google Scholar). Briefly, the medium was removed, and cells were washed twice with cold PBS. Cells remaining in wells were washed twice with 5% trichloroacetic acid and solubilized in 0.5 ml of 0.25 nNaOH, and 0.3 ml was collected and counted.Crystal Violet AssayCells were grown in 6- or 12-well plates for 2 days and then washed, refed, rested, and treated. Following treatment, the medium was removed, and cells were washed with PBS. The amount of cells remaining adhered to the plate was assessed via crystal violet staining as described previously (22Suffys P. Beyaert R. Van Roy F. Fiers W. Biochem. Biophys. Res. Commun. 1987; 149: 735-743Crossref PubMed Scopus (112) Google Scholar).NF-κB Gel ShiftCells were grown to 70–80% confluence and then treated in the presence of regular culture medium. Following the indicated treatment times, cells were harvested via trypsinization, and the pellets were washed one time with cold PBS. Nuclear extractions and electrophoretic mobility shift assays were run via a modification of methods previously described (23Dbaibo G.S. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1993; 268: 17762-17766Abstract Full Text PDF PubMed Google Scholar). Briefly, the cell pellets were quick-frozen using an ethanol/dry ice bath, and the pellets were then resuspended in 50–100 μl of hypotonic buffer (10 mmHEPES, pH 7.9, 10 mm KCl, 1.5 mmMgCl2, and 1 mm dithiothreitol). This hypotonic lysis yields ∼100% lysis of cells. The lysed cells were then spun, and the nuclear pellet was recovered and resuspended in 15 μl of hypertonic buffer (20 mm HEPES, pH 7.9, 0.4 mNaCl, 1.5 mm MgCl2, 25% glycerol, 0.2 mm EDTA, 1 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride). Extraction of the nuclear protein was achieved by gentle mixing of this mixture for 30 min at 4 °C. The debris was then spun down, and the resultant supernatant was diluted with 20–70 μl of dilution buffer (20 mm HEPES, pH 7.9, 50 mm KCl, 20% glycerol, 0.2 mm EDTA, 1 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride). Approximately 2-μl aliquots were used for the Bio-Rad protein assay, and the remaining portion was quick-frozen and stored at −80 °C until gel shift assays were run. Protein-DNA reactions were performed in a 20-μl volume and contained 8–10 μg of nuclear extract, 1 μg of poly[d(I·C)], 1 μg of poly[d(N)6], 10 μg of bovine serum albumin, 20 mm HEPES, pH 7.9, 50 mm KCl, 1 mm EDTA, 5 mmdithiothreitol, and 10,000–50,000 cpm radiolabeled oligonucleotide probe (see Ref. 23Dbaibo G.S. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1993; 268: 17762-17766Abstract Full Text PDF PubMed Google Scholar for sequences used). Reactions were allowed to proceed for 20 min and then terminated by the addition of 6 μl of 15% Ficoll. Nondenaturing polyacrylamide gels (5%) that had been prerun for 1–1.5 h at 200 V were loaded with equal volumes of reaction mixture and run at 200 V for 1.5–2 h. Gels were then dried and exposed to film. Shown (Fig. 10) is a representation of an autoradiogram obtained in this manner.DISCUSSIONThis study demonstrates the importance of PLA2 to ceramide generation and cytotoxicity. Previous studies in the HL-60 cell system suggested a link between AA and the SM cycle in TNFα signaling (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar); however, the L929 system, used here, has allowed the further development of these initial studies. L929 cells vary markedly from the HL-60 model in two critical respects. First, the temporal correlation between receptor activation and lipid mobilization (both AA release and ceramide release) is greatly attenuated in L929 cells, taking hours as opposed to minutes. Second, the magnitude of change in ceramide elicited by TNFα stimulation of L929 cells (2–4-fold changes) exceeds the levels attainable upon cytokine stimulation of HL-60 cells (at best, 1.5–1.8-fold changes) (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar, 16Kim M.-Y. Linardic C. Obeid L. Hannun Y. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar). Despite these differences, both systems show TNFα-induced PLA2activation/AA generation to occur prior to SM hydrolysis and ceramide generation. Thus, the L929 model demonstrates that AA-mediated signaling to ceramide is not restricted to one cell system, but is indeed a cascade that may have greater implications.Furthermore, the L929 model has been used here to extend our studies from the correlative level to establish the necessity of AA liberation for ceramide generation. These studies were possible because of the availability of L929 clones resistant to the cytotoxic effects of TNFα that are defective in cPLA2. We found that, whereas L929 cells responded to TNFα stimulation by elevating AA levels within 2–4 h, C12 cells showed little if any elevation of AA release. Likewise, we found that, unlike the parental L929 line, this resistant line was incapable of responding to TNFα through the production of ceramide. In contrast, TNFα-stimulated NF-κB activation appeared to be intact, suggesting that only AA-dependent cascades were affected in the resistant cells. Thus, initial observations with the resistant lines illustrated the specificity of the interplay between AA and ceramide. We further established the necessity for AA generation through utilization of a variant strain of C12, the CPL4 line, which differed from C12 only with respect to the presence of an expression plasmid containing the murine cPLA2 gene. We found that, through recapitulating PLA2 activity in the CPL4 lines, we were able to re-establish 1) cytokine-induced AA generation, 2) TNFα-induced ceramide generation, and 3) TNFα-induced growth inhibition. Although the CPL4 system did not fully restore cytokine responsiveness, it did serve to demonstrate the interconnection among AA, ceramide, and growth.A number of key points emerge from this study. First, it becomes apparent that the kinetics of ceramide generation are dependent upon the type of SMase activated. Whereas rapid ceramide mobilization has been attributed to the activation of one enzyme (depending on the system, a neutral, cytosolic, or acidic SMase), more prolonged ceramide generation appears to be the consequence of multiple activities perhaps acting together: a neutral Mg2+-dependent SMase, a neutral Mg2+-independent SMase, and an acidic SMase. Whether all three of these enzyme activities can be regulated by AA remains to be determined. Also, since the long-term accumulation of ceramide involves multiple enzymes, multiple pools of SM could be implicated. At a first level of examination, however, the ceramide generated in both the L929 and HL-60 systems appears to be similar (as determined by TLC) (data not shown). Whether all three SMase activities found to be stimulated by TNFα in L929 cells can act on the same pool of SM, however, remains unknown.Second, these results raise the question of how PLA2/AA couples to SMase. The exact links between PLA2activation/AA generation and ceramide generation remain elusive. Furthermore, other components that may be involved in regulating the signaling cascade between TNF receptor stimulation and SMase activation remain to be determined. It is important to note in this context that Fas ligand-induced death, in contrast to TNF, may not require cPLA2 (27Enari M. Hug H. Hayakawa M. Ito F. Nishimura Y. Nagata S. Eur. J. Biochem. 1996; 236: 533-538Crossref PubMed Scopus (52) Google Scholar).Finally, the prolonged kinetics of ceramide generation raise a possibility that ceramide may function as a long-term regulator of cell growth/viability. Indeed, most studies examining the changes in ceramide associated with cell death or growth suppression disclose similar long-term changes in ceramide. These include serum withdrawal,fas stimulation, and dexamethasone-induced apoptosis (7Jayadev S. Liu B. Bielawska A.E. 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,24Tepper C.G. Jayadev S. 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, 25Gottschalk A.R. Quintans J. Immunol. Cell Biol. 1995; 73: 8-16Crossref PubMed Google Scholar). These studies have raised the question whether ceramide generation is a precedent to cell death and differentiation or whether ceramide elevations are a consequence of the biological fate and serve more as a marker of phenotype. The kinetics established in this study clearly show that both SMase activation and ceramide elevation precede the appearance of a dead/floater population of cells. The observed increase in ceramide in the adherent population of cells demonstrates cytokine-responsive SM hydrolysis prior to the onset of death, suggesting that ceramide precedes cell death. Ongoing studies also support a role for ceramide prior to the onset of actual cell death. In cells overexpressing the anti-apoptotic protein Bcl2, it appears that ceramide generation in response to chemotherapeutic agents is not perturbed, although cell death is greatly reduced (26Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (293) Google Scholar). Such studies continue to expand our understanding of where AA and ceramide fit in the overall scheme between cell stimulation and onset of response, whether it be death, differentiation, or cell cycle arrest. The sphingomyelin (SM) 1The abbreviations used are: SM, sphingomyelin; SMase, sphingomyelinase; TNFα, tumor necrosis factor α AA, arachidonic acid; PLA2, phopholipase A2; cPLA2, cytosolic PLA2; PBS, phosphate-buffered saline; NF-κB, nuclear factor κB. 1The abbreviations used are: SM, sphingomyelin; SMase, sphingomyelinase; TNFα, tumor necrosis factor α AA, arachidonic acid; PLA2, phopholipase A2; cPLA2, cytosolic PLA2; PBS, phosphate-buffered saline; NF-κB, nuclear factor κB. cycle, first described by Okazaki et al. (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar), has gained recognition over the past few years as a key mechanism for regulating anti-mitogenic signals. Activation of this cycle through the regulation of a signal-induced sphingomyelinase (SMase) results in generation of the lipid second messenger ceramide. Ceramide then modulates a number of biological fates, including growth inhibition (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 2Bielawska A. Linardic C.M. Hannun Y.A. FEBS Lett. 1992; 307: 211-214Crossref PubMed Scopus (89) Google Scholar, 3Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (549) Google Scholar), differentiation (2Bielawska A. Linardic C.M. Hannun Y.A. FEBS Lett. 1992; 307: 211-214Crossref PubMed Scopus (89) Google Scholar), apoptosis (4Pushkareva M. Obeid L.M. Hannun Y.A. Immunol. Today. 1995; 16: 294-297Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 5Obeid L.M. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Crossref PubMed Scopus (235) Google Scholar, 6Kolesnick R.N. Haimovitz-Friedman A. Fuks Z. Biochem. Cell Biol. 1994; 72: 471-474Crossref PubMed Scopus (141) Google Scholar), and cell cycle arrest (7Jayadev S. Liu B. Bielawska A.E. 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). Although recent studies have begun to catalogue inducers such as TNFα, interleukin-1β, nerve growth factor, and Fas that are capable of signaling through the SM cycle (see Refs. 5Obeid L.M. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Crossref PubMed Scopus (235) Google Scholar, 6Kolesnick R.N. Haimovitz-Friedman A. Fuks Z. Biochem. Cell Biol. 1994; 72: 471-474Crossref PubMed Scopus (141) Google Scholar, and 8Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar for reviews), the mechanisms by which these inducers stimulate SMase activity remain poorly understood. TNFα, through interaction with either a 55- or 75-kDa TNF receptor (9Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (999) Google Scholar, 10Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1831) Google Scholar), impacts upon a myriad of intracellular signaling cascades, including protein phosphorylation cascades, transcription factors, and lipid messengers (11Heller R.A. Kronke M. J. Cell Biol. 1994; 126: 5-9Crossref PubMed Scopus (401) Google Scholar). Two classes of lipid mediators have been implicated in TNFα signaling, glycerophospholipid metabolites and sphingolipid metabolites (11Heller R.A. Kronke M. J. Cell Biol. 1994; 126: 5-9Crossref PubMed Scopus (401) Google Scholar, 12Kronke M. Schutze S. Scheurich P. Pfizenmaier K. Aggarwal B.B. Vilcek J. Tumor Necrosis Factors: Structure, Function, and Mechanism of Action. Marcel Dekker, Inc., New York1992: 189-216Google Scholar), and recent evidence suggests that these two classes of lipids may interact (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar). In HL-60 cells, a linear correlation was established among TNFα stimulation, AA generation, and SM cycle activation: TNFα-stimulated AA liberation preceded ceramide generation, and AA reproduced the effects of TNFα on the SM cycle (13Jayadev S. Linardic C.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 5757-5763Abstract Full Text PDF PubMed Google Scholar). Although these studies suggested that AA and/or its metabolites may be involved in activation of SMase, the physiologic role of the PLA2/AA pathway in regulating SMase activity has not been determined. In this study, we examined the role of PLA2 in SMase activation in the L929 murine fibroblast cell line. In L929 cells, TNFα treatment is known to produce potent cytotoxic effects (14Hayakawa M. Oku N. Takagi T. Hori T. Shibamoto S. Yamanaka Y. Takeuchi K. Tsujimoto M. Ito F. Cell Struct. Funct. 1991; 16: 333-340Crossref PubMed Scopus (16) Google Scholar). TNFα-resistant L929" @default.
• W2025787534 created "2016-06-24" @default.
• W2025787534 creator A5009713793 @default.
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