©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1-induced Ceramide and Diacylglycerol Generation May Lead to Activation of the c-Jun NH-terminal Kinase and the Transcription Factor ATF2 in the Insulin-producing Cell Line RINm5F (*)

(Received for publication, October 16, 1995; and in revised form, January 18, 1996)

Nils Welsh (§)

From the Department of Medical Cell Biology, Uppsala University, Biomedicum, P. O. Box 571, S-751 23 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The aim of this investigation was to study the putative involvement of lipid second messengers, protein kinases, and transcription factors in interleukin-1beta (IL-1beta)-induced signal transduction in insulin-producing cells. For this purpose, insulin-producing RINm5F cells were exposed to IL-1beta (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-1beta 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-1beta. 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-1beta, 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-kappaB element was strongly induced by IL-1beta, but not by acetylsphingosine or PMA. Finally, acetylsphingosine and PMA did not mimic the nitric oxide-inducing effects of IL-1beta. It is concluded that IL-1beta-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 beta-cell nitric-oxide synthase induction.


INTRODUCTION

It has been demonstrated that interleukin-1beta (IL-1beta) (^1)exerts inhibitory and cytotoxic effects on rodent pancreatic beta cells in vitro(1, 2, 3) . 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, 5) . The IL-1beta effects are thought to be mediated by, at least in part, induction of nitric-oxide synthase (iNOS)(6, 7) . Nitric oxide production leads to inhibition of aconitase, glucose oxidation rates, ATP generation, and insulin production(2, 3, 7, 8, 9) . The intracellular signals generated in insulin-producing cells by the interaction between IL-1beta 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-alpha(10, 11) . 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) . Moreover, ceramide activation of this protein kinase has been reported to induce NF-kappaB, a stress response transcription factor(13) . In insulin-producing cells, indirect evidence has been presented suggesting a role of protein Ser-Thr and Tyr phosphorylation events (14, 15) leading to NF-kappaB activation (16) and induction of iNOS(17) . Therefore, it was investigated here whether IL-1beta stimulates sphingomyelin hydrolysis and ceramide generation and whether this putative event may participate in IL-1beta-induced transcription factor activation and the subsequent induction of iNOS.


EXPERIMENTAL PROCEDURES

Materials

Human recombinant IL-1beta 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-1beta preparation (National Institute for Biological Standards and Control, London)(18) . The chemicals were obtained from the following sources: Sigma, sphingosine, ceramide, 1,2-diacylglycerol, sphingomyelin, acetic anhydride, octyl beta-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 (C(2)-ceramide); Amersham International (Buckinghamshire, United Kingdom), [-P]ATP and [methyl-^3H]choline chloride; and Merck (Darmstadt, Germany), Silica Gel 60 F TLC plates.

Lipid Extraction

The clonal rat insulin secretory cell line RINm5F was cultured in RPMI 1640 medium + 10% fetal calf serum. Approximately 1.0 times 10^6 RINm5F cells were exposed to 25 units/ml IL-1beta 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 times 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-Diacylglycerol

Ceramide and 1,2-diacylglycerols were quantified essentially according to Preiss et al.(19) . Briefly, dried lipids were solubilized in 20 µl of an octyl beta-D-glucoside/cardiolipin solution (7.5% octyl beta-D-glucoside, 5 mM cardiolipin in 1 mM DTPA) by sonication in a sonicator bath. The reaction was then carried out in a final volume of 100 µl containing the 20-µl sample solution, 50 mM imidazole HCl, pH 6.6, 50 mM NaCl, 12.5 mM MgCl(2), 1 mM EGTA, 2 mM dithiothreitol, 6.6 µg of DAG kinase, and 1 mM [-P]ATP (specific activity of 1-5 times 10^5 cpm/nmol) for 30 min at room temperature. Lipids were extracted and evaporated as described above. Samples were then run on Kieselgel 60 plates activated by preheating at 120 °C. Plates were developed with chloroform/methanol/acetic acid (65:15:5, v/v) and subjected to autoradiography. Standard samples of D-1,2-dipalmitin and ceramide were phosphorylated and developed in parallel. The intensities of the spots corresponding to ceramide 1-phosphate (R(F) = 0.38) and phosphatidic acid (R(F) = 0.57) were quantified by autoradiography and densitometry and were expressed as arbitrary units (absorbance).

Assay for Sphingomyelin, Ceramide 1-Phosphate, and Phosphatidic Acid

For equilibrium-labeled studies, RINm5F cells were incubated for 48 h in RPMI 1640 medium + 10% fetal calf serum containing 50 µCi/ml P (phosphatidic acid and ceramide 1-phosphate) or 1 µCi/ml [^3H]choline (sphingomyelin)(20, 21) . The cells were then exposed to 25 units/ml IL-1beta for 0, 2, 5, and 20 min and rapidly washed with cold PBS. Lipids were extracted as described above, and glycerophospholipids were removed by incubation for 60 min at 37 °C in 0.1 M methanolic potassium hydroxide. Phosphatidic acid and ceramide 1-phosphate were resolved by TLC using chloroform/methanol/acetic acid (65:15:5, v/v) and sphingomyelin using chloroform/methanol/acetic acid/water (60:30:8:5, v/v) as solvents. Individual lipids were visualized by iodine vapor staining and autoradiography, scraped off the plates, and quantified by liquid scintillation counting.

N-Acetylsphingosine Synthesis

N-Acetylsphingosine was synthesized as described previously(22) . Briefly, 167 mg of sphingosine was allowed to react with 1 ml of acetic anhydride in 10 ml of methanol overnight. The reaction product was precipitated with 20 ml of water, washed, and filtered and then reprecipitated from a methanol solution by addition of water. The biological activity of synthesized N-acetylsphingosine was similar to that of a commercially available N-acetylsphingosine preparation (data not shown).

JNK1 Phosphorylation Assay

RINm5F cells (3 times 10^6) were labeled with P-labeled orthophosphate (0.5 mCi/ml) in Krebs-Ringer bicarbonate buffer containing 10 µM phosphate, 16.7 mM glucose, and 5% dialyzed fetal calf serum for 2 h. The cells were then exposed to 10 µM acetylsphingosine, 100 nM PMA, or 25 units/ml IL-1beta for 20 min. After washings with cold PBS, the cells were solubilized in PBS containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 100 µM sodium orthovanadate. The homogenates were briefly sonicated, and debris were removed by a 10-min centrifugation at 12,000 times g. To remaining supernatants was added 25 µl of anti-JNK1 antibody linked to agarose (Santa Cruz Biotechnology, Inc.). After 60 min, agarose conjugates were pelleted, washed in homogenization buffer, and boiled in SDS sample buffer (2% SDS, 100 mM Tris, pH 6.8, 10 mM beta-mercaptoethanol, 0.01% bromphenol blue, and 10% glycerol). Samples were run on 9% SDS-polyacrylamide gels.

JNK1 Kinase Assay

RINm5F cells that had been exposed to 10 µMN-acetylsphingosine, 100 nM PMA, and 25 units/ml IL-1beta for 20 min were solubilized in 500 µl of cold PBS containing 1% Triton X-100, 0.5% Nonidet P-40, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride. The homogenates were briefly sonicated and then centrifuged at 12,000 times g for 5 min. Aliquots of the supernatants were taken for SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining to verify that similar amounts of protein were using for immunoprecipitation. To the remaining supernatants was added 25 µl of agarose-conjugated anti-JNK1 antibodies. After 60 min on ice, the agarose conjugates were washed three times with homogenization buffer and once with kinase reaction buffer (12.5 mM MOPS, pH 7.5, 7.5 mM MgCl(2), 0.5 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM sodium orthovanadate)(23) . The immunoprecipitates were resuspended in 30 µl of kinase reaction buffer containing 1 µCi of [-P]ATP, 20 µM ATP, and 1 µg of GST-c-Jun(79) (GST-c-Jun(79) construct generously provided by Dr. J. Silvio Gutkind) fusion protein as substrate. GST was run in parallel as a negative control. Reactions were carried out at 30 °C for 20 min, after which 10 µl of 4 times SDS sample buffer was added. Samples were run on 12% SDS-polyacrylamide gels and analyzed using autoradiography and densitometry.

Immunoblot Analysis of ATF2

RINm5F cells that had been exposed to 0.1, 1.0, and 10 µMN-acetylsphingosine, 100 nM PMA, and 25 units/ml IL-1beta for 10, 20, and 60 min were solubilized in SDS/beta-mercaptoethanol sample buffer by boiling for 5 min. Samples were then run on 10% SDS-polyacrylamide gels and electroblotted to nitrocellulose filters, which were then incubated with anti-ATF2 antibodies (Santa Cruz Biotechnology, Inc.) diluted 1:1000 in PBS supplemented with 5% fat-free dry milk powder. Horseradish peroxidase-linked goat anti-rabbit Ig was used as a second layer. Immunodetection was performed as described for the ECL immunoblotting detection system (Amersham International). In a separate experiment, homogenates (100 µl) from control and IL-1beta-exposed RINm5F cells were treated with 1 unit of alkaline phosphatase (Boehringer, Mannheim, Germany) for 5 min at 37 °C prior to electrophoresis and immunoblot analysis.

Preparation of Nuclear Extracts

RINm5F cells (5 times 10^6) were incubated with N-acetylsphingosine (1 or 10 µM), PMA (100 nM), or IL-1beta (25 units/ml) for 20 min. Cells were then washed three times with cold PBS, harvested, and suspended in 100 µl of buffer A(24) . After 10 min at 4 °C, cells were pelleted and resuspended in 100 µl of the same buffer and mechanically homogenized. Nuclei were pelleted (20 s at 12,000 times g) and briefly sonicated in 100 µl of buffer C (24) . After a brief centrifugation, the remaining supernatants were frozen until further use.

Electrophoresis Mobility Shift Assay

For electrophoretic mobility shift assays of the transcription factors NF-kappaB, AP-1, C/EBP, and ATF/CREB, the following double-stranded oligonucleotides were used: 5`-AGCTTCAGAGGGGACTTCCGAGAGG(16) , 5`-CGCTTGATGACTCAGCCGGAA (Santa Cruz Biotechnology, Inc.), 5`-TGCAGATTGCGCAATGGCCTT (Santa Cruz Biotechnology, Inc.), and 5`-GATGAAGTGACGTCAGTGGGC (Santa Cruz Biotechnology, Inc.), respectively. The double-stranded oligonucleotides were labeled with [P]dCTP using a Megaprime labeling kit (Amersham International) and extracted once with an equal volume of phenol/chloroform/isoamyl alcohol (25:25:1, v/v). Binding reactions contained 10 mM Tris, pH 7.5, 0.2% deoxycholic acid, 40 mM NaCl, 1 mM EDTA, 1 mM beta-mercaptoethanol, 4% glycerol, 2 µg of polydeoxyinosinic-deoxycytidylic acid, 0.1 ng of DNA (14,000 cpm), and 4 µl of nuclear protein extract (20 µg). Samples supplemented with a 1000-fold excess of unlabeled double-stranded oligonucleotide were used as negative controls. Each 20-µl reaction was incubated at room temperature for 30 min. Samples were separated on 5% nondenaturing polyacrylamide gels in 0.5 times Tris borate/EDTA. Band intensities were quantified by densitometric scanning.

Nitrite Determination

To RINm5F cells (50-70% confluency) was added 10 µMN-acetylsphingosine, 5 µM sodium orthovanadate, 100 nM okadaic acid, or 25 units/ml IL-1beta as described in the figure legends. Six hours later, duplicate samples (2 times 80 µl) were taken for nitrite determination as described previously(7, 25) .

Statistics

Data are expressed as means ± S.E., and groups were compared using Student's paired t test.


RESULTS

Effects of IL-1beta on Lipid Content of RINm5F Cells

Since diacylglycerol kinase phosphorylates both diacylglycerol and ceramide using [-P]ATP as substrate, both lipids can be quantified in the same reaction. It was found that the DAG content of RINm5F cells was higher than the ceramide content (Fig. 1). IL-1beta induced a significant increase in the ceramide content after both 2 and 5 min of exposure (Fig. 1). After 20 min, this effect could no longer be observed (Fig. 1). Similarly, the DAG content was increased after 5 min, but not after 20 min (Fig. 1). The increase in ceramide was paralleled by an IL-1beta-induced decrease in sphingomyelin, which was 17% after 2 min, 35% after 5 min, and 28% after 20 min (Fig. 2). The ceramide 1-phosphate and phosphatidic acid contents of the RINm5F cells were not affected by IL-1beta (data not shown).


Figure 1: Effects of IL-1beta on DAG and ceramide contents of RINm5F cells. RINm5F cells were exposed to 25 units/ml IL-1beta for 0, 2, 5, and 20 min. Lipids were extracted, and DAG and ceramide contents were determined as described under ``Experimental Procedures.'' Results are means ± S.E. for 9-12 observations. * and **, p < 0.05 and p < 0.01, respectively, for a chance difference versus corresponding control using Student's paired t test.




Figure 2: Effects of IL-1beta on sphingomyelin content of RINm5F cells. RINm5F cells equilibrium-labeled with [^3H]choline were exposed to 25 units/ml IL-1beta for 0, 2, 5, and 20 min. Lipids were extracted and quantified as described under ``Experimental Procedures.'' Results are means ± S.E. for 9-11 observations. *, p < 0.05 for a chance difference versus corresponding control using Student's paired t test.



Effects of IL-1beta, Acetylsphingosine, and PMA on JNK1 Phosphorylation and JNK1 Kinase Activity

The Santa Cruz JNK1 (N-19) antibody is reactive mainly against the 46-kDa JNK1 protein, but cross-reacts also with the 54-kDa JNK2 protein. Immunoprecipitation of P-labeled RINm5F cell homogenates with this antibody demonstrated strong reactivity with both the 46- and 54-kDa phosphoproteins (Fig. 3A). Acetylsphingosine (10 µM) and PMA (100 nM) both increased the phosphorylation of the 46-kDa JNK1 protein when added during a 20-min exposure period (Fig. 3A). The phosphorylation of JNK1 was even more strongly enhanced by IL-1beta (25 units/ml) (Fig. 3A). The phosphorylation of the 54-kDa JNK2 protein was not affected by the different test substances (Fig. 3A).


Figure 3: A, effects of acetylsphingosine, PMA, and IL-1beta on JNK1 phosphorylation. RINm5F cells were P-labeled for 2 h and then exposed to 10 µM acetylsphingosine (lane 2), 100 nM PMA (lane 3), and 25 units/ml IL-1beta (lane 4) for 20 min. JNK1 was immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The position of JNK1 (46 kDa) is indicated by the lower arrow. The upper arrow indicates the position of the 54-kDa JNK2 phosphoprotein. This figure is representative of three separate experiments. B, effects of acetylsphingosine, PMA, and IL-1beta on JNK1 in vitro kinase activity. RINm5F cells were exposed for 20 min to 10 µM acetylsphingosine (lane 2), 100 nM PMA (lane 3), 10 µM acetylsphingosine + 10 nM PMA (lane 4), and 25 units/ml IL-1beta (lane 5). Lane 1 in A and B is the control. Homogenates were then immunoprecipitated for JNK1, and the in vitro kinase activity was determined using GST-c-Jun fusion protein as substrate. The position of phosphorylated GST-c-Jun is indicated by the arrow. C, Coomassie Brilliant Blue staining of cell homogenates separated by SDS-polyacrylamide gel electrophoresis. Lanes are the same as described for B.



The in vitro kinase activity of JNK1, measured as phosphorylation of the GST-c-Jun fusion protein, was weakly increased by 10 µM acetylsphingosine (37 ± 12%, n = 3) and by 100 nM PMA (95 ± 35%, n = 3). The combination of acetylsphingosine and PMA increased GST-c-Jun phosphorylation by 63 ± 12%, and IL-1beta (25 units/ml) increased GST-c-Jun phosphorylation by 275 ± 70% (n = 3) (Fig. 3B). There was no phosphorylation of a 38-kDa protein when only GST was used as a phosphorylation substrate (data not shown). Fig. 3C demonstrates that equal amounts of protein were used for immunoprecipitation and JNK1 activity determination.

Effects of IL-1beta, Acetylsphingosine, and PMA on Transcription Factor ATF2 Migration on SDS-Polyacrylamide Gel Electrophoresis

In control RINm5F cells, strong reactivity with a 69-kDa band was observed when performing immunoblot analysis for ATF2 (Fig. 4A). Addition of IL-1beta induced the appearance of an additional higher molecular mass band (72 kDa) immunoreactive with the anti-ATF2 antibody (Fig. 4A). The induction of the 72-kDa band by IL-1beta was mimicked by 100 nM PMA and, to a lesser extent, by 1 and 10 µM acetylsphingosine (Fig. 4A). There was no effect of 0.1 µM acetylsphingosine (Fig. 4A). Induction of the 72-kDa band was apparent after 10 min and persisted for at least 60 min (data not shown). The 72-kDa band, present in IL-1beta-treated RINm5F cells and not in control cells, could not be observed after treatment of the homogenate with alkaline phosphatase (Fig. 4B). This suggests that the 72-kDa band is the phosphorylated form of ATF2.


Figure 4: A, immunoblot analysis of ATF2 in RINm5F cells exposed to acetylsphingosine, PMA, and IL-1beta. RINm5F cells were exposed for 20 min to 0, 0.1, 1.0, and 10 µM acetylsphingosine (lanes 1-4, respectively), 100 nM PMA (lane 5), and 25 units/ml IL-1beta (lane 6). The lower arrow indicates the position of the 69-kDa ATF2 form, and upper arrow indicates the position of the phosphorylated 72-kDa ATF2 form. Positions of molecular mass markers (in kilodaltons) are given on the right. The figure is representative of three separate experiments. B, immunoblot analysis of ATF2. Homogenates from control RINm5F cells (lanes 1 and 2) or IL-1beta-exposed RINm5F cells (lanes 3 and 4) were either untreated (lanes 1 and 3) or incubated with alkaline phosphatase (lanes 2 and 4). The upper arrow shows the position of phosphorylated ATF2, and the lower arrow show that of nonphosphorylated ATF2.



Effects of IL-1beta, Ceramide, and DAG on Transcription Factor DNA Binding Activities

A 20-min exposure of RINm5F cells to IL-1beta (25 units/ml) induced a 15-fold increase in the DNA binding activity of NF-kappaB ( Fig. 5and Fig. 6). This effect was not mimicked by acetylsphingosine (1 and 10 µM) or PMA (100 nM); there was only a nonsignificant trend toward higher DNA binding activities with these agents (p > 0.05). The three agents, IL-1beta, acetylsphingosine, and PMA, all tended to moderately increase (50-70%) the DNA binding activities of AP-1 and C/EBP. Using Student's paired t test, 1 µM acetylsphingosine increased significantly the activities of AP-1 and C/EBP (p < 0.05), 100 nM PMA stimulated the activity of C/EBP (p < 0.05), and IL-1beta enhanced the activity of C/EBP (p < 0.01). Acetylsphingosine, PMA, and IL-1beta all stimulated similarly (4-5-fold; p < 0.05) the DNA binding activity of the ATF transcription factors ( Fig. 5and Fig. 6). The effect of acetylsphingosine was maximal already at 1 µM ( Fig. 5and Fig. 6).


Figure 5: Autoradiogram showing effects of acetylsphingosine, PMA, and IL-1beta on transcription factor gel retardation in an electrophoresis mobility shift assay experiment. Nuclear extracts from RINm5F cells exposed for 20 min to 1 and 10 µM acetylsphingosine (lanes 2 and 3), 100 nM PMA (lane 4), and 25 units/ml IL-1beta (lane 5) were incubated with P-labeled double-stranded oligonucleotides specific for NF-kappaB, AP-1, C/EBP, and ATF/CREB and run on 5% polyacrylamide gels. Lane 1 is the untreated control, and lane 6 is nuclear extracts from IL-1beta-treated RINm5F cells incubated with a 1000-fold excess of unlabeled oligonucleotide.




Figure 6: Effects of acetylsphingosine, PMA, and IL-1beta on transcription factor activation. Data are means ± S.E. from densitometric scannings of four to five separate experiments performed as described for Fig. 6. Bar 1, control; bars 2 and 3, 1 and 10 mM acetylsphingosine, respectively; bar 4, 100 nM PMA; bar 5, 25 units/ml IL-1beta.



Effects of IL-1beta, Okadaic Acid, Sodium Orthovanadate, and Acetylsphingosine on RINm5F Nitrite Production

The phosphatase inhibitors okadaic acid and sodium orthovanadate have previously been shown to enhance IL-1beta-induced nitrite production(14) . In this study, N-acetylsphingosine, okadaic acid, and vanadate were unable to induce nitrite formation, either alone or in combination (Fig. 7). IL-1beta induced by itself a marked response (Fig. 7), which was not further enhanced by N-acetylsphingosine (data not shown). N-Acetylsphingosine (10 µM) also did not induce nitrite production from isolated rat islets when added during a 24-h culture period (data not shown).


Figure 7: Effects of acetylsphingosine on RINm5F cell nitrite production. RINm5F cells were exposed to acetylsphingosine (ceramide (Cer.), okadaic acid (Ok. A), sodium orthovanadate, and IL-1beta as shown. After 6 h, medium samples were taken for nitrite determinations. Results are means ± S.E. for five to nine observations.




DISCUSSION

The results of this study suggest that IL-1 receptor activation in RINm5F cells leads to phospholipase C activation and DAG generation. This is in line with previous studies demonstrating IL-1 activation of a phosphatidylcholine-specific phospholipase C in Jurkat T cells, which generates DAG but not inositol trisphosphate, and showing that DAG was generated in isolated mouse pancreatic islets in response to IL-1beta without any concomitant increase in intracellular free Ca concentrations(26, 27) . The presently observed decrease in sphingomyelin and a concomitant increase in ceramide imply the activation of a sphingomyelinase by IL-1beta in RINm5F cells. It is unclear whether the sphingomyelinase is stimulated directly by the activated IL-1 receptor or whether sphingomyelinase activation is indirectly accomplished by the increased DAG level. Indeed, DAG is known to trigger sphingomyelinase activity in Jurkat T cells and in GH3 rat pituitary cells(28, 29) . The present finding that acetylsphingosine and PMA could not be clearly differentiated with respect to their activation profiles of JNK1 and the transcription factors may be explained by DAG- or PMA-induced sphingomyelinase activation and the concomitant generation of ceramide. Another possibility to be considered is that arachidonic acid can increase the activity of sphingomyelinase, a finding described in HL-60 cells(30) . Indeed, increased levels of prostaglandin E(2), a metabolite of arachidonic acid, have been observed in response to IL-1beta in isolated rat islets(31) .

Ceramide 1-phosphate has been suggested to act as a regulator of intracellular calcium stores and cell proliferation and is thought to be generated by ceramide kinase activation(20) . Although ceramide 1-phosphate contents appear to be high in RINm5F cells, they were not increased by IL-1beta, which speaks against cytokine-induced activation of ceramide kinase. The finding that phosphatidic acid was not increased suggests that IL-1beta does not stimulate the activity of phospholipase D in RINm5F cells.

Addition of ceramide analogues to dermal fibroblast and EL4 T helper cells mimics some IL-1 and tumor necrosis factor-alpha actions by inducing cyclooxygenase and IL-2 gene expression(21, 32) . These effects induced by the proinflammatory cytokines and ceramide analogues have been proposed to be mediated by the ceramide-activated protein kinase and downstream protein kinases belonging to the mitogen-activated protein kinase family(33) . The c-Jun NH(2)-terminal kinase (JNK1) (34) and its homologues, MAPKAP kinase-2 reactivating kinase(35) , stress-activated protein kinase-alpha (36) , HOG1(37) , p38(38) , and cytokine-suppressive anti-inflammatory drug-binding protein(39) , all belong to the mitogen-activated protein kinase family and are activated by cellular stress agonists such as proinflammatory cytokines, heat, arsenite, UV light, and osmotic and chemical stress. The present finding that IL-1beta and, to a lesser extent, acetylsphingosine and PMA induce the phosphorylation and activation of JNK1, an event possibly mediated by the extracellular signal-regulated kinase kinase kinase(40) , indicates that this pathway is active also in insulin-producing cells. In previous reports, it was demonstrated that JNK1 phosphorylates the stress protein hsp27(35) , c-Jun(41) , and a transcription factor that belongs to the ATF/CREB family, namely ATF2, leading to its transcriptional activation(42) . This is probably also the case in RINm5F cells since increased phosphorylation of ATF2 in response to acetylsphingosine, PMA, and IL-1beta was presently observed. Moreover, both PMA and acetylsphingosine induced enhanced transcription factor binding to the ATF/CREB element. Thus, IL-1beta-induced ceramide/DAG generation may contribute to the phosphorylation and activation of JNK1 and ATF2 in insulin-producing cells.

Even though relatively high concentrations of acetylsphingosine were presently used (1.0-10 µM), the effects of the ceramide analogue were generally less pronounced than those induced by IL-1beta. This may, on one hand, be due to a lower efficiency of the ceramide analogue in activating the ceramide-dependent protein kinase(s) compared with endogenously produced ceramide. On the other hand, it cannot be excluded that ceramide generation plays a less prominent role in this pathway and that other signals mediate or are necessary for IL-1beta-induced JNK1 and ATF2 activation. Indeed, it has recently been demonstrated that the GTP-binding proteins Cdc42 and Rac1 initiate a phosphorylation cascade that activates the stress-activated protein kinase/c-Jun NH(2)-terminal kinase signaling pathway (23) .

It has been proposed that cytokines and ceramide induce indirect phosphorylation events leading to the activation of the transcription factor NF-kappaB(13) . IL-1beta has been shown to induce NF-kappaB activation in RINm5F cells, an event that was necessary for iNOS gene expression(16) . However, the finding that acetylsphingosine and PMA did not activate NF-kappaB argues against a role for these lipids in this specific pathway. This is in line with other reports showing a dissociation between ceramide generation and NF-kappaB translocation(43, 44) . Interestingly, activated ATF2 has been demonstrated to interact not only with several viral proteins, but also with NF-kappaB and c-Jun (45, 46) . Thus, IL-1beta-induced iNOS expression may involve not only NF-kappaB activation, which seems to be ceramide- and PMA-independent, but also a parallel ceramide-dependent pathway leading to phosphorylation of c-Jun and ATF2, which both act synergistically in stimulating NF-kappaB and AP-1 activity.

It is unclear whether activated ATF2 interacts directly with the promoter region of the iNOS gene. Instead, ATF2 might induce the transcription of other genes necessary for iNOS induction. Indeed, the mRNA for iNOS appears as late as 3 h after IL-1beta addition in RINm5F cells, and iNOS mRNA expression requires protein synthesis since cycloheximide prevents the induction(47) . Thus, other transcription factors besides NF-kappaB and ATF2 are probably necessary for maximal iNOS gene expression. Since acetylsphingosine did not induce nitrite production in RINm5F and rat islet cells, either alone or together with the phosphatase inhibitors okadaic acid and vanadate, which both increase protein phosphorylation, ceramide generation alone does not appear to be sufficient for induction of iNOS.

No strong or specific effects were presently observed on AP-1 DNA binding activity. It should, however, be pointed out that phosphorylation and transcriptional activation of nuclear factors, such as c-Jun, do not necessarily lead to increased binding to specific promoter elements in gel shift experiments. Weak effects were also observed with the C/EBP element. To the C/EBP element binds, for example, C/EBPbeta, a transcriptional activator present in differentiated cells, such as hepatocytes, and involved in the acute-phase response and IL-1-induced gene expression(48) . This transcription factor becomes activated by the protein kinase C pathway, leading to enhancement of its transcriptional efficacy(49) .

In summary, IL-1beta stimulates the formation of ceramide and DAG in insulin-producing cells. This event may contribute to the phosphorylation of JNK1 and the transcription factors c-Jun and ATF2. A similar situation has recently been observed in human promyelocytic cells (HL-60 cells), in which sphingomyelinase activation and ceramide generation mediate tumor necrosis factor-alpha-induced activation of JNK1 (50) . Activation of this signaling pathway, however, does not lead to the activation of NF-kappaB and is not sufficient for induction of nitric oxide production in insulin-producing cells. Further studies are warranted for the identification of additional transcription factors that are activated or induced in response to IL-1beta.


FOOTNOTES

*
This work was supported in part by Swedish Medical Research Council Grants 12X-109, 12P-10151, 12X-11564, 12X-8273, and 12X-9886 and by the Swedish Diabetes Association, the Nordic Insulin Fund, the Juvenile Diabetes Foundation International, Barndiabetesfonden, and the Family Ernfors Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a European Association for the Study of Diabetes Mellitus/Eli Lilly research fellowship in diabetes research. To whom correspondence should be addressed. Tel.: 46-18-174212; Fax: 46-18-556401.

(^1)
The abbreviations used are: IL-1beta, interleukin-1beta; iNOS, inducible nitric-oxide synthase; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate-buffered saline; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; C/EBP, CCAAT/enhancer-binding protein; CREB, cAMP-responsive element-binding protein; ATF, activating transcription factor.


ACKNOWLEDGEMENTS

The excellent technical assistance of C. Göktürk and G. Kärf is gratefully acknowledged. Oligonucleotide synthesis was performed by J. Seibt (Department of Medical Immunology, Uppsala University).


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