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The aim of this investigation was to study the putative involvement of lipid second messengers, protein kinases, and transcription factors in interleukin-1β (IL-1β)-induced signal transduction in insulin-producing cells. For this purpose, insulin-producing RINm5F cells were exposed to IL-1β (25 units/ml), and the ceramide, ceramide 1-phosphate, sphingomyelin, diacylglycerol, and phosphatidic acid contents of the cells were subsequently determined. It was found that IL-1β induced a transient increase (2-5 min) in ceramide and diacylglycerol, which was not paralleled by an increase in ceramide 1-phosphate and phosphatidic acid. A rapid decrease in the sphingomyelin content of the cells was, however, observed. The cell-permeable ceramide analogue N-acetylsphingosine and the phorbol ester phorbol 12-myristate 13-acetate (PMA) both induced the phosphorylation and increased the activities of the protein kinase JNK1 and the transcription factor ATF2. These effects were, however, not as pronounced as those induced by IL-1β. The DNA binding activity of transcription factors in nuclear extracts was determined using the electrophoretic mobility shift assay method. Transcription factor binding to the ATF/cAMP-responsive element consensus sequence was increased 4-5-fold by acetylsphingosine, PMA, or IL-1β, whereas binding to the CCAAT/enhancer-binding protein and AP-1 elements was found to be only slightly stimulated by these three agents. Binding to the NF-κB element was strongly induced by IL-1β, but not by acetylsphingosine or PMA. Finally, acetylsphingosine and PMA did not mimic the nitric oxide-inducing effects of IL-1β. It is concluded that IL-1β-stimulated formation of ceramide and diacylglycerol may contribute to JNK1 and ATF2 transcription factor activation, which may be a necessary (but not sufficient) step in β-cell nitric-oxide synthase induction. The aim of this investigation was to study the putative involvement of lipid second messengers, protein kinases, and transcription factors in interleukin-1β (IL-1β)-induced signal transduction in insulin-producing cells. For this purpose, insulin-producing RINm5F cells were exposed to IL-1β (25 units/ml), and the ceramide, ceramide 1-phosphate, sphingomyelin, diacylglycerol, and phosphatidic acid contents of the cells were subsequently determined. It was found that IL-1β induced a transient increase (2-5 min) in ceramide and diacylglycerol, which was not paralleled by an increase in ceramide 1-phosphate and phosphatidic acid. A rapid decrease in the sphingomyelin content of the cells was, however, observed. The cell-permeable ceramide analogue N-acetylsphingosine and the phorbol ester phorbol 12-myristate 13-acetate (PMA) both induced the phosphorylation and increased the activities of the protein kinase JNK1 and the transcription factor ATF2. These effects were, however, not as pronounced as those induced by IL-1β. The DNA binding activity of transcription factors in nuclear extracts was determined using the electrophoretic mobility shift assay method. Transcription factor binding to the ATF/cAMP-responsive element consensus sequence was increased 4-5-fold by acetylsphingosine, PMA, or IL-1β, whereas binding to the CCAAT/enhancer-binding protein and AP-1 elements was found to be only slightly stimulated by these three agents. Binding to the NF-κB element was strongly induced by IL-1β, but not by acetylsphingosine or PMA. Finally, acetylsphingosine and PMA did not mimic the nitric oxide-inducing effects of IL-1β. It is concluded that IL-1β-stimulated formation of ceramide and diacylglycerol may contribute to JNK1 and ATF2 transcription factor activation, which may be a necessary (but not sufficient) step in β-cell nitric-oxide synthase induction. INTRODUCTIONIt has been demonstrated that interleukin-1β (IL-1β) ( 1The abbreviations used are: IL-1βinterleukin-1βiNOSinducible nitric-oxide synthaseDTPAdiethylenetriaminepentaacetic acidPBSphosphate-buffered salineDAGdiacylglycerolPMAphorbol 12-myristate 13-acetateMOPS4-morpholinepropanesulfonic acidGSTglutathione S-transferaseC/EBPCCAAT/enhancer-binding proteinCREBcAMP-responsive element-binding proteinATFactivating transcription factor.) exerts inhibitory and cytotoxic effects on rodent pancreatic beta cells in vitro(1.Bendtzen K. Mandrup-Poulsen T. Nerup J. Dinarello C.A. Svenson M. Nielsen J.H. Science. 1986; 232: 1545-1547Google Scholar, 2.Sandler S. Andersson A. Hellerström C. Endocrinology. 1987; 121: 1424-1431Google Scholar, 3.Sandler S. Bendtzen K. Borg L.A.H. Eizirik D.L. Strandell E. Welsh N. Endocrinology. 1989; 124: 1492-1501Google Scholar). This has led to the suggestion that this cytokine, alone or in combination with other cytokines, may be an important mediator of the autoimmune destruction of beta cells during the course of insulin-dependent diabetes mellitus(4.Nerup J. Mandrup-Poulsen T. M⊘lvig J. Helqvist S. Wogensen L. Egeberg J. Diabetes Care. 1988; 11: 16-23Google Scholar, 5.Bendtzen K. Autoimmunity. 1989; 2: 177-189Google Scholar). The IL-1β effects are thought to be mediated by, at least in part, induction of nitric-oxide synthase (iNOS)(6.Southern C. Schulster D. Green I.C. FEBS Lett. 1990; 276: 42-44Google Scholar, 7.Welsh N. Eizirik D.L. Bendtzen K. Sandler S. Endocrinology. 1991; 129: 3167-3173Google Scholar). Nitric oxide production leads to inhibition of aconitase, glucose oxidation rates, ATP generation, and insulin production(2.Sandler S. Andersson A. Hellerström C. Endocrinology. 1987; 121: 1424-1431Google Scholar, 3.Sandler S. Bendtzen K. Borg L.A.H. Eizirik D.L. Strandell E. Welsh N. Endocrinology. 1989; 124: 1492-1501Google Scholar, 7.Welsh N. Eizirik D.L. Bendtzen K. Sandler S. Endocrinology. 1991; 129: 3167-3173Google Scholar, 8.Corbett J.A. Wang J.L. Hughes J.L. Wolf B.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. Biochem. J. 1992; 287: 229-235Google Scholar, 9.Welsh N. Sandler S. Biochem. Biophys. Res. Commun. 1992; 182: 333-340Google Scholar). The intracellular signals generated in insulin-producing cells by the interaction between IL-1β and its receptor have, however, not yet been elucidated.Sphingomyelin hydrolysis and ceramide generation constitute a signal transduction pathway that mediates some of the effects of the cytokines IL-1 and tumor necrosis factor-α(10.Kolesnick R.N. Trends Cell Biol. 1992; 2: 232-237Google Scholar, 11.Kolesnick R.N. Golde D.W. Cell. 1994; 77: 325-328Google Scholar). Sphingomyelin consists of sphingosine, a fatty acid, and a phosphocholine head group. Upon hydrolysis, the phosphocholine head group is released, and ceramide is formed. The ceramide generated by a cytokine-stimulated sphingomyelinase is thought to activate a 97-kDa proline-directed serine/threonine kinase(12.Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Google Scholar). Moreover, ceramide activation of this protein kinase has been reported to induce NF-κB, a stress response transcription factor(13.Machleidt T. Wiegmann K. Henkel T. Schütze S. Baeuerle P. Krönke M. J. Biol. Chem. 1994; 269: 13760-13765Google Scholar). In insulin-producing cells, indirect evidence has been presented suggesting a role of protein Ser-Thr and Tyr phosphorylation events (14.Welsh N. Biosci. Rep. 1994; 14: 43-50Google Scholar, 15.Corbett J.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. FASEB J. 1993; 7: 369-374Google Scholar) leading to NF-κB activation (16.Saldeen J. Welsh N. Biochem. Biophys. Res. Commun. 1994; 203: 149-155Google Scholar) and induction of iNOS(17.Eizirik D.L. Cagliero E. Björklund A. Welsh N. FEBS Lett. 1992; 308: 249-252Google Scholar). Therefore, it was investigated here whether IL-1β stimulates sphingomyelin hydrolysis and ceramide generation and whether this putative event may participate in IL-1β-induced transcription factor activation and the subsequent induction of iNOS.EXPERIMENTAL PROCEDURESMaterialsHuman recombinant IL-1β was kindly provided by Dr. K. Bendtzen (Laboratory of Medical Immunology, Rigshospitalet, Copenhagen). The cytokine was produced by Immunex (Seattle, WA) and had a biological activity of 50 units/ng as compared with an interim international standard recombinant IL-1β preparation (National Institute for Biological Standards and Control, London)(18.Svenson M. Bendtzen K. Scand. J. Immunol. 1988; 27: 593-599Google Scholar). The chemicals were obtained from the following sources: Sigma, sphingosine, ceramide, 1,2-diacylglycerol, sphingomyelin, acetic anhydride, octyl β-D-glucoside, cardiolipin, DTPA, sodium orthovanadate, okadaic acid, naphthylethylenediamine dihydrochloride, and sulfanilamide; Calbiochem, sn-1,2-diacylglycerol kinase; Matreya Inc. (Pleasant Gap, PA), N-acetylsphingosine (C2-ceramide); Amersham International (Buckinghamshire, United Kingdom), γ-32PATP and methyl-3Hcholine chloride; and Merck (Darmstadt, Germany), Silica Gel 60 F254 TLC plates.Lipid ExtractionThe clonal rat insulin secretory cell line RINm5F was cultured in RPMI 1640 medium + 10% fetal calf serum. Approximately 1.0 × 106 RINm5F cells were exposed to 25 units/ml IL-1β for 0, 2, 5, and 20 min. The cells were then rapidly washed with cold PBS and sonicated in 400 μl of a chloroform solution consisting of chloroform/methanol/HCl (100:100:1, v/v) and 100 μl of PBS containing 10 mM EDTA. After centrifugation for 5 min at 12,000 × g, the aqueous phase was removed and re-extracted with 100 μl of chloroform, which was subsequently added to the chloroform phase. The combined chloroform phases were evaporated under a stream of nitrogen and resolubilized in 40 μl of the chloroform solution. This solution was re-extracted with 10 μl of PBS with 10 mM EDTA and then re-evaporated. The samples were then stored at −70°C under nitrogen until analyzed for ceramide and 1,2-diacylglycerols.Assay for Ceramide and 1,2-DiacylglycerolCeramide and 1,2-diacylglycerols were quantified essentially according to Preiss et al.(19.Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Google Scholar). Briefly, dried lipids were solubilized in 20 μl of an octyl β-D-glucoside/cardiolipin solution (7.5% octyl β-D-glucoside, 5 mM cardiolipin in 1 mM DTPA) by in a The was then in a of 100 μl containing the 50 mM 50 mM mM 1 mM mM of and 1 mM γ-32PATP activity of × for min at were and evaporated as were then on 60 by at were with acid v/v) and to samples of and ceramide were and in The of the to ceramide 1-phosphate and phosphatidic acid were quantified by and and were as for Ceramide and RINm5F cells were for in RPMI 1640 medium + 10% fetal calf containing 50 acid and ceramide or 1 R.N. J. Biol. Chem. 1990; Scholar, S. A. C. R.N. Science. 1993; Scholar). The cells were then exposed to 25 units/ml IL-1β for 0, 2, 5, and 20 min and rapidly washed with cold were as and were removed by for 60 min at in acid and ceramide 1-phosphate were by TLC using acid v/v) and sphingomyelin using v/v) as lipids were by and the and quantified by was as J. Chem. Scholar). Briefly, of was to with 1 of acetic in 10 of The was with 20 of and and then from a solution by of The biological activity of N-acetylsphingosine was to that of a N-acetylsphingosine preparation not cells × were with in containing 10 mM and fetal calf for The cells were then exposed to 10 acetylsphingosine, 100 PMA, or 25 units/ml IL-1β for 20 min. After with cold the cells were solubilized in PBS containing sodium 1 mM and 100 sodium The were and were removed by a centrifugation at 12,000 × was added 25 μl of to After 60 were washed in and in 100 mM 10 mM and 10% were on cells that had been exposed to 10 100 PMA, and 25 units/ml IL-1β for 20 min were solubilized in μl of cold PBS containing and 1 mM The were sonicated and then at 12,000 × for 5 min. of the were for and to that of protein were using for the was added 25 μl of After 60 min on the were washed three with and with kinase mM mM mM mM sodium and mM sodium M. P. N. T. Cell. Scholar). The were in μl of kinase containing 1 of 20 and 1 of provided by Dr. J. protein as was in as a were at for 20 which 10 μl of × was were on and analyzed using and of cells that had been exposed to and 10 100 PMA, and 25 units/ml IL-1β for and 60 min were solubilized in by for 5 min. were then on 10% and to which were then with in PBS with was used as a second was as for the In a from and RINm5F cells were with 1 of for 5 min at to and of cells × were with N-acetylsphingosine or 10 PMA or IL-1β (25 for 20 min. were then washed three with cold and in 100 μl of R.M. Res. 11: Scholar). After 10 min at cells were and in 100 μl of the and were at 12,000 × and sonicated in 100 μl of R.M. Res. 11: Scholar). After a the were until electrophoretic mobility shift of the transcription factors NF-κB, and the following were J. Welsh N. Biochem. Biophys. Res. Commun. 1994; 203: 149-155Google and The were with using a and with an of Binding 10 mM acid, 40 mM 1 mM 1 mM of acid, of DNA and μl of nuclear protein with a of were used as was at for min. were on in × were quantified by RINm5F cells was added 10 5 sodium orthovanadate, 100 okadaic acid, or 25 units/ml IL-1β as in the samples × were for as N. Eizirik D.L. Bendtzen K. Sandler S. Endocrinology. 1991; 129: 3167-3173Google Scholar, J. Biochem. are as and were compared using of IL-1β on of RINm5F diacylglycerol kinase both diacylglycerol and ceramide using γ-32PATP as both lipids be quantified in the It was found that the content of RINm5F cells was the ceramide content IL-1β induced a increase in the ceramide content both and 5 min of After 20 this be the content was increased 5 but not 20 min The increase in ceramide was paralleled by an IL-1β-induced decrease in sphingomyelin, which was 5 and 20 min The ceramide 1-phosphate and phosphatidic acid contents of the RINm5F cells were not by IL-1β not of IL-1β on sphingomyelin content of RINm5F cells. RINm5F cells with were exposed to 25 units/ml IL-1β for 0, 2, 5, and 20 min. were and quantified as under are for for a using of IL-1β, and PMA on JNK1 and JNK1 JNK1 is the JNK1 but with the of RINm5F cell with this demonstrated with both the and and PMA both increased the phosphorylation of the JNK1 protein added during a The phosphorylation of JNK1 was strongly by IL-1β (25 The phosphorylation of the protein was not by the effects of acetylsphingosine, PMA, and IL-1β on JNK1 RINm5F cells were for and then exposed to 10 acetylsphingosine 100 PMA and 25 units/ml IL-1β for 20 min. JNK1 was and analyzed by The of JNK1 is by the The the of the This is of three effects of acetylsphingosine, PMA, and IL-1β on JNK1 in kinase RINm5F cells were exposed for 20 min to 10 acetylsphingosine 100 PMA 10 acetylsphingosine + 10 PMA and 25 units/ml IL-1β 1 in A and is the were then for and the in kinase activity was determined using protein as The of is by the of cell by are the as for in kinase activity of as phosphorylation of the was increased by 10 acetylsphingosine and by 100 PMA The combination of acetylsphingosine and PMA increased phosphorylation by and IL-1β (25 increased phosphorylation by was phosphorylation of a protein only was used as a phosphorylation not that of protein were used for and JNK1 activity of IL-1β, and PMA on Transcription ATF2 on Gel RINm5F cells, with a was for ATF2 of IL-1β induced the of an with the The induction of the by IL-1β was by 100 PMA to a by 1 and 10 acetylsphingosine was of acetylsphingosine of the was 10 min and for at least 60 min not The in RINm5F cells and not in cells, not be of the with This that the is the of of ATF2 in RINm5F cells exposed to acetylsphingosine, PMA, and IL-1β. RINm5F cells were exposed for 20 min to 0, and 10 acetylsphingosine 100 PMA and 25 units/ml IL-1β The the of the ATF2 and the of the ATF2 of are on the The is of three of ATF2. from RINm5F cells 1 and or RINm5F cells and were 1 and or with and The the of and the that of of IL-1β, and on Transcription DNA Binding of RINm5F cells to IL-1β (25 induced a increase in the DNA binding activity of NF-κB This was not by acetylsphingosine and 10 or PMA was only a DNA binding activities with these The three IL-1β, acetylsphingosine, and PMA, to increase the DNA binding activities of AP-1 and 1 acetylsphingosine increased the activities of AP-1 and 100 PMA stimulated the activity of and IL-1β the activity of PMA, and IL-1β stimulated the DNA binding activity of the transcription factors The of acetylsphingosine was at 1 of IL-1β, and on RINm5F okadaic acid and sodium been to IL-1β-induced N. Biosci. Rep. 1994; 14: 43-50Google Scholar). In this okadaic acid, and were to induce alone or in combination IL-1β induced by a response which was not by N-acetylsphingosine not did not induce production from rat added during a not of acetylsphingosine, PMA, and IL-1β on transcription factor are from of to as for and 1 and 10 mM acetylsphingosine, 100 5, 25 units/ml effects of acetylsphingosine, PMA, and IL-1β on transcription factor in an mobility shift assay extracts from RINm5F cells exposed for 20 min to 1 and 10 acetylsphingosine and 100 PMA and 25 units/ml IL-1β were with for NF-κB, and and on 1 is the and is nuclear extracts from RINm5F cells with a of of acetylsphingosine on RINm5F cell RINm5F cells were exposed to acetylsphingosine okadaic acid sodium orthovanadate, and IL-1β as After medium samples were for are for to of this study that IL-1 receptor activation in RINm5F cells leads to activation and This is in line with IL-1 activation of a in cells, which but not and that was generated in pancreatic in response to IL-1β increase in intracellular N. Dinarello C.A. Cell. 1988; Scholar, D.L. Sandler S. Welsh N. L. Cell. Scholar). The decrease in sphingomyelin and a increase in ceramide the activation of a sphingomyelinase by IL-1β in RINm5F cells. It is whether the sphingomyelinase is stimulated by the IL-1 receptor or whether sphingomyelinase activation is by the increased is to sphingomyelinase activity in cells and in rat S. K. T. D. Wiegmann K. Krönke M. Cell. 1992; Scholar, R.N. J. Biol. Chem. 1987; Scholar). The that acetylsphingosine and PMA not be with to activation of JNK1 and the transcription factors may be by or sphingomyelinase activation and the generation of to be is that acid increase the activity of a in S. Hannun Y.A. J. Biol. Chem. 1994; 269: Scholar). increased of a of acid, been in response to IL-1β in rat J.A. J. McDaniel M.L. 1993; 1-phosphate has been to as a of intracellular and cell and is thought to be generated by ceramide kinase R.N. J. Biol. Chem. 1990; Scholar). ceramide 1-phosphate contents to be in RINm5F cells, were not increased by IL-1β, which activation of ceramide The that phosphatidic acid was not increased that IL-1β not the activity of in RINm5F of ceramide to and cells some IL-1 and tumor necrosis by and S. A. C. R.N. Science. 1993; Scholar, M.A. R. J. Biol. Chem. 1992; Scholar). These effects induced by the cytokines and ceramide been to be mediated by the protein kinase and protein to the protein kinase M.A. R.N. Golde D.W. J. Biol. Chem. 1993; Scholar). The kinase M. T. T. M. Cell. 1994; Scholar) and its J. P. S. M. A. D. T. Cell. 1994; protein J. P. E. T. J. J.R. 1994; Science. 1994; J. L. Science. 1994; and S. Green D. D. J.R. J.E. J.R. J.L. 1994; to the protein kinase and are by stress as cytokines, and and The that IL-1β to a acetylsphingosine and PMA induce the phosphorylation and activation of an event mediated by the kinase kinase A. A. M. C. M. Science. that this pathway is in insulin-producing cells. In it was demonstrated that JNK1 the stress protein J. P. S. M. A. D. T. Cell. 1994; Hughes K. J.R. J. 1994; and a transcription factor that to the leading to its S. D. Science. Scholar). This is the in RINm5F cells increased phosphorylation of ATF2 in response to acetylsphingosine, PMA, and IL-1β was observed. Moreover, both PMA and acetylsphingosine induced transcription factor binding to the IL-1β-induced generation may contribute to the phosphorylation and activation of JNK1 and ATF2 in insulin-producing of acetylsphingosine were used the effects of the ceramide analogue were pronounced those induced by IL-1β. This on be to a of the ceramide analogue in the protein compared with produced the other it be that ceramide generation a role in this pathway and that other signals or are necessary for IL-1β-induced JNK1 and ATF2 it has been demonstrated that the and a phosphorylation that the protein kinase pathway M. P. N. T. Cell. has been that cytokines and ceramide induce indirect phosphorylation events leading to the activation of the transcription factor T. Wiegmann K. Henkel T. Schütze S. Baeuerle P. Krönke M. J. Biol. Chem. 1994; 269: 13760-13765Google Scholar). IL-1β has been to induce NF-κB activation in RINm5F cells, an event that was necessary for J. Welsh N. Biochem. Biophys. Res. Commun. 1994; 203: 149-155Google Scholar). the that acetylsphingosine and PMA did not activate NF-κB a role for these lipids in this This is in line with other a between ceramide generation and NF-κB J.A. J. Biol. Chem. 1994; 269: Scholar, T. J. Immunol. 1994; Scholar). ATF2 has been demonstrated to not only with but with NF-κB and 1990; Scholar, D. T. Cell. 1993; Scholar). IL-1β-induced may not only NF-κB activation, which to be and but a pathway leading to phosphorylation of and which both in NF-κB and AP-1 is whether ATF2 with the of the ATF2 induce the transcription of other necessary for induction. the for as as IL-1β in RINm5F cells, and protein the N. A. Sandler S. Eizirik D.L. Biochem. Biophys. Res. Commun. 1994; 203: Scholar). other transcription factors NF-κB and ATF2 are necessary for acetylsphingosine did not induce production in RINm5F and rat cells, alone or with the okadaic acid and which both increase protein ceramide generation alone not to be for induction of or effects were on AP-1 DNA binding It however, be that phosphorylation and activation of nuclear as not to increased binding to elements in shift effects were with the the element for a in cells, as and in the response and R. Cell. 1990; Scholar). This transcription factor by the protein kinase leading to of its C. C. P. T. M. M. 1993; IL-1β stimulates the formation of ceramide and in insulin-producing cells. This event may contribute to the phosphorylation of JNK1 and the transcription factors and ATF2. A has been in cells in which sphingomyelinase activation and ceramide generation tumor necrosis activation of JNK1 Bielawska Hannun Y.A. J. Biol. Chem. Scholar). of this however, not to the activation of NF-κB and is not for induction of nitric oxide production in insulin-producing cells. are for the of transcription factors that are or induced in response to IL-1β. INTRODUCTIONIt has been demonstrated that interleukin-1β (IL-1β) ( 1The abbreviations used are: IL-1βinterleukin-1βiNOSinducible nitric-oxide synthaseDTPAdiethylenetriaminepentaacetic acidPBSphosphate-buffered salineDAGdiacylglycerolPMAphorbol 12-myristate 13-acetateMOPS4-morpholinepropanesulfonic acidGSTglutathione S-transferaseC/EBPCCAAT/enhancer-binding proteinCREBcAMP-responsive element-binding proteinATFactivating transcription factor.) exerts inhibitory and cytotoxic effects on rodent pancreatic beta cells in vitro(1.Bendtzen K. Mandrup-Poulsen T. Nerup J. Dinarello C.A. Svenson M. Nielsen J.H. Science. 1986; 232: 1545-1547Google Scholar, 2.Sandler S. Andersson A. Hellerström C. Endocrinology. 1987; 121: 1424-1431Google Scholar, 3.Sandler S. Bendtzen K. Borg L.A.H. Eizirik D.L. Strandell E. Welsh N. Endocrinology. 1989; 124: 1492-1501Google Scholar). This has led to the suggestion that this cytokine, alone or in combination with other cytokines, may be an important mediator of the autoimmune destruction of beta cells during the course of insulin-dependent diabetes mellitus(4.Nerup J. Mandrup-Poulsen T. M⊘lvig J. Helqvist S. Wogensen L. Egeberg J. Diabetes Care. 1988; 11: 16-23Google Scholar, 5.Bendtzen K. Autoimmunity. 1989; 2: 177-189Google Scholar). The IL-1β effects are thought to be mediated by, at least in part, induction of nitric-oxide synthase (iNOS)(6.Southern C. Schulster D. Green I.C. FEBS Lett. 1990; 276: 42-44Google Scholar, 7.Welsh N. Eizirik D.L. Bendtzen K. Sandler S. Endocrinology. 1991; 129: 3167-3173Google Scholar). Nitric oxide production leads to inhibition of aconitase, glucose oxidation rates, ATP generation, and insulin production(2.Sandler S. Andersson A. Hellerström C. Endocrinology. 1987; 121: 1424-1431Google Scholar, 3.Sandler S. Bendtzen K. Borg L.A.H. Eizirik D.L. Strandell E. Welsh N. Endocrinology. 1989; 124: 1492-1501Google Scholar, 7.Welsh N. Eizirik D.L. Bendtzen K. Sandler S. Endocrinology. 1991; 129: 3167-3173Google Scholar, 8.Corbett J.A. Wang J.L. Hughes J.L. Wolf B.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. Biochem. J. 1992; 287: 229-235Google Scholar, 9.Welsh N. Sandler S. Biochem. Biophys. Res. Commun. 1992; 182: 333-340Google Scholar). The intracellular signals generated in insulin-producing cells by the interaction between IL-1β and its receptor have, however, not yet been elucidated.Sphingomyelin hydrolysis and ceramide generation constitute a signal transduction pathway that mediates some of the effects of the cytokines IL-1 and tumor necrosis factor-α(10.Kolesnick R.N. Trends Cell Biol. 1992; 2: 232-237Google Scholar, 11.Kolesnick R.N. Golde D.W. Cell. 1994; 77: 325-328Google Scholar). Sphingomyelin consists of sphingosine, a fatty acid, and a phosphocholine head group. Upon hydrolysis, the phosphocholine head group is released, and ceramide is formed. The ceramide generated by a cytokine-stimulated sphingomyelinase is thought to activate a 97-kDa proline-directed serine/threonine kinase(12.Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Google Scholar). Moreover, ceramide activation of this protein kinase has been reported to induce NF-κB, a stress response transcription factor(13.Machleidt T. Wiegmann K. Henkel T. Schütze S. Baeuerle P. Krönke M. J. Biol. Chem. 1994; 269: 13760-13765Google Scholar). In insulin-producing cells, indirect evidence has been presented suggesting a role of protein Ser-Thr and Tyr phosphorylation events (14.Welsh N. Biosci. Rep. 1994; 14: 43-50Google Scholar, 15.Corbett J.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. FASEB J. 1993; 7: 369-374Google Scholar) leading to NF-κB activation (16.Saldeen J. Welsh N. Biochem. Biophys. Res. Commun. 1994; 203: 149-155Google Scholar) and induction of iNOS(17.Eizirik D.L. Cagliero E. Björklund A. Welsh N. FEBS Lett. 1992; 308: 249-252Google Scholar). Therefore, it was investigated here whether IL-1β stimulates sphingomyelin hydrolysis and ceramide generation and whether this putative event may participate in IL-1β-induced transcription factor activation and the subsequent induction of
Nils Welsh (Mon,) studied this question.