Interleukin-1β-induced Rat Pancreatic Islet Nitric Oxide Synthesis Requires Both the p38 and Extracellular Signal-regulated Kinase 1/2 Mitogen-activated Protein Kinases
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.15294
ISSN1083-351X
AutoresClaus M. Larsen, Karin Wadt, Lone Juhl, Henrik U. Andersen, Allan E. Karlsen, Michael Su, Klaus Seedorf, Leland Shapiro, Charles A. Dinarello, Thomas Mandrup‐Poulsen,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoInterleukin-1β (IL-1β) is cytotoxic to rat pancreatic β-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1β induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1β was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1β-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1β-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1β but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1β-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1β-mediated β-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1β in β-cells. Interleukin-1β (IL-1β) is cytotoxic to rat pancreatic β-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1β induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1β was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1β-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1β-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1β but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1β-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1β-mediated β-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1β in β-cells. Interleukin-1β (IL-1β) 1The abbreviations used are: IL, interleukin; ATF2, activating transcription factor 2; ERK, extracellular signal-regulated kinase; GST, glutathione S -transferase; Hsp25, 25-kDa heat shock protein; NO, nitric oxide; iNOS, inducible NO synthase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAP-K2, MAPK-activated protein kinase-2; MEKi, MAPK/ERK kinase inhibitor; NF-κB, nuclear factor-κB; p38i, p38 inhibitor; RIN, rat insulinoma; PAGE, polyacrylamide gel electrophoresis; CM, complete medium; PCR, polymerase chain reaction. 1The abbreviations used are: IL, interleukin; ATF2, activating transcription factor 2; ERK, extracellular signal-regulated kinase; GST, glutathione S -transferase; Hsp25, 25-kDa heat shock protein; NO, nitric oxide; iNOS, inducible NO synthase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAP-K2, MAPK-activated protein kinase-2; MEKi, MAPK/ERK kinase inhibitor; NF-κB, nuclear factor-κB; p38i, p38 inhibitor; RIN, rat insulinoma; PAGE, polyacrylamide gel electrophoresis; CM, complete medium; PCR, polymerase chain reaction. is cytotoxic to rat pancreatic β-cells (1Mandrup-Poulsen T. 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Nature. 1994; 372: 739-746Crossref PubMed Scopus (3133) Google Scholar) signaling pathways have been identified, providing effective tools for investigating the role of ERK1/2 and p38 in cellular signaling. The contribution of p38 and ERK1/2 to IL-1β signal transduction in insulin-producing β-cells has not been investigated. Therefore, we examined the involvement of ERK1/2 and p38 in IL-1β signaling in intact rat islets of Langerhans and in a RIN β-cell line. We report that both ERK1/2 and p38 are activated by IL-1β in intact islets and in a RIN β-cell line and that ERK1/2 and p38 are both important steps in signaling leading to NO synthesis, but p38 is also involved in signaling of NO-independent effects of IL-1β in intact islets. All reagents were from Sigma unless otherwise specified. Recombinant human IL-1β (400 units/ng) was from Novo Nordisk (Bagsværd, Denmark). Reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were all from Bio-Rad. [γ-32P]ATP (3000 Ci/mmol) was from Amersham Pharmacia Biotech, glutathione S -transferase (GST)-Elk-1 was a gift from Peter Shaw (Max-Planck-Institut für Immunbiologie, Freiburg, Germany), GST-ATF2 (1–109) was a gift form Roger J. Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA), GST-c-Jun was a gift from Peter Angel (Deutsh Krebsforschungszentrum, Heidelberg, Germany), and recombinant murine 25-kDa heat shock protein (Hsp25) was from Stressgen (Victoria, Canada). Antibodies against p38, ERK1/2, phospho-p38, or phospho-ERK1/2 were from New England Biolabs (Hitchin Hertfordshire, United Kingdom), and MAPK-activated protein kinase-2 (MAPKAP-K2) antibody was from Upstate Biotechnology (Lake Placid, NY). The highly specific inhibitor of p38 (compound VK-19.577, identical to SB203580 and p38i) (26Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemans I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3133) Google Scholar, 27Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google Scholar) was from Vertex Pharmaceuticals Inc. (Cambridge, MA). Compound PD 098059 from New England Biolabs is the specific inhibitor of MAPK/ERK kinase activation (hereafter termed MEK inhibitor (MEKi)) (25Dudley D.T. Pang L. Decker T. Bridges A. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2589) Google Scholar, 28Alessi D.R. Cuenda A. Cohen P. Dudly D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar). p38i and MEKi were both dissolved in DMSO to stock concentrations of 25 and 100 mm, respectively. Inhibitors were added 1 h before islet stimulation, and a final 0.14% (v/v) DMSO was added to all conditions as control for the DMSO used to dissolve MEKi and p38i. Islets of Langerhans from 5–7 day old Wistar Furth rats (Charles River, Sulzfeldt, Germany) were isolated by handpicking after collagenase digestion (Collagenase A from Boehringer Mannheim) of the pancreata (29Brunstedt J. Nielsen J.H. Lernmark Å. the Hagedorn Study GroupLarner J. Pohl S.L. Methods in Diabetes Research. John Wiley & Sons, Inc., New York1984: 254-288Google Scholar). Islets were precultured in 5-ml dishes (Nunc, Roskilde, Denmark) for 7 days at 37 °C in atmospheric humidified air in complete RPMI medium (CM)) + 10% FCS (Life Technologies, Inc.): RPMI 1640 medium (11 mm glucose) supplemented with 100,000 IU/liter penicillin, 100 mg/liter streptomycin, 20 mm HEPES buffer, 2 mml-glutamine, and 0.038% NaHCO3 (all from Life Technologies, Inc.). 150 randomly picked islets/300 μl of CM + 0.5% human serum (final osmolarity, 300 mosm) were placed in 4-well dishes (Nunc) treated with IL-1β, with or without inhibitors, or exposed to hyperosmolarity (addition of hyperosmolar saline to CM as described previously (17Shapiro L. Dinarello C.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12230-12234Crossref PubMed Scopus (233) Google Scholar)), as indicated in the figure legends. For immunoprecipitation and Western blotting experiments, each condition was made in duplicate, and islets were pooled prior to lysis. RIN-5AH-T2B cells of low passage number (10Welsh N. Biosci. Rep. 1994; 14: 43-50Crossref PubMed Scopus (17) Google Scholar, 11Corbett J.A. Sweetland M.A. Lancaster Jr., J.R. McDaniel M.L. FASEB J. 1993; 7: 369-374Crossref PubMed Scopus (73) Google Scholar, 12Welsh N. J. Biol. Chem. 1996; 271: 8307-8312Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 13Cano E. Mahadevan L.C. TIBS. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (997) Google Scholar, 14Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar, 15Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2040) Google Scholar, 16Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 17Shapiro L. Dinarello C.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12230-12234Crossref PubMed Scopus (233) Google Scholar) were maintained in 80-cm2 tissue culture flasks (Nunc) in CM supplied with 10% heat-inactivated FCS (HyClone, Logan, UT) at 37 °C in a 5% CO2/95% air mixture. When confluent RIN cells were trypsinized, 150,000 cells seeded in 96-well dishes (Costar, Cambridge, UK) containing 200 μl of CM + 10% heat-inactivated FCS and precultured for 24 h before experimentation, by which time the cell number had doubled. The RIN cells were then exposed to IL-1β as described in the Fig. 2legend. Following stimulation, islets and RIN cells were lysed for 30 min on ice in 25, 50, and 100 μl of lysis buffer (20 mm Tris acetate, pH 7.0, 0.27 msucrose, 1 mm EDTA, 1 mm EGTA, 1 mmNa3VO4, 50 mm NaF, 1% Triton X-100, 5 mm sodium pyrophosphate, 10 mmβ-glycerophosphate, 1 mm dithiothreitol, 1 mmbenzamidine, and 4 μg/ml leupeptin) for whole cell lysate kinase assay, Western blotting, and immunoprecipitation, respectively. The detergent-insoluble material was pelleted by centrifugation at 15,000 rpm for 5 min at 4 °C. The supernatants containing whole cell lysate were either immediately used for whole cell lysate kinase assay, immunoprecipitation, or Western blotting or stored at −80 °C. The GST-Elk-1, GST-ATF2, and Hsp25 phosphotransferase reactions were carried out in a final volume of 25 μl at 30 °C for 30 min after addition of 5 μl of whole cell lysate, 17 μl of reaction buffer (2 μg of GST-Elk-1, 2 μg of GST-ATF2, 1 μg of Hsp25, 25 mm Tris-HCl, pH 7.5, 0.1 mm EGTA, 0.1 mm Na3VO4, 1 μm cAMP-dependent protein kinase inhibitor peptide, and 10 mm Mg-acetate), and 3 μl of ATP mixture (1 mm ATP and 3 μCi [γ-32P]ATP). Reactions were terminated by addition of 25 μl of SDS sample buffer (125 mm Tris-HCl, pH 6.8, 4% SDS, 0.1 mdithiothreitol, 10% glycerol, and 0.02% bromphenol blue) and boiling for 5 min. The samples were then subjected to SDS-PAGE as described by Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar), using a 4% stacking gel and a 12% separating gel. After electrophoresis, the separating gel was washed for 15 min in a mixture of 10% acetate and 40% methanol. The gels were dried, and the proteins were visualized by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). To asses the possible contamination of the used GST-ATF2, we performed Coomassie Blue staining and kinase assay with GST-ATF2 and ATF2 (1–96 and 1–505) from Santa Cruz Biotechnology (Santa Cruz, CA), which confirmed the substrate specificity and purity of the GST-ATF2. 100 μl of whole cell lysates from RIN cells or 300 islets, diluted 1:4 in washing buffer (lysis buffer with 0.1% Triton X-100), were immunoprecipitated by incubation overnight with anti-MAPKAP-K2 or anti-ERK1/2 antibodies and then with protein A-Sepharose beads (Amersham Pharmacia Biotech) for 3 h at 4 °C (MAPKAP-K2 and ERK1/2) or incubated with GST-c-Jun (5 μg) coupled to glutathione-Sepharose beads for 3 h at 4 °C (JNK kinase assay). The beads were washed three times in washing buffer and twice in kinase buffer (20 mm HEPES (pH 7.5), 20 mmβ-glycerophosphate, 10 mm MgCl2, 1 mm dithiothreitol, 50 μmNa3VO4). Kinase reactions were carried out for 30 min at 30 °C in 30 μl of kinase buffer containing 10 μCi [γ-32P]ATP and 1 μg of Hsp25 (MAPKAP-K2) or 5 μg of GST-Elk-1 (ERK1/2). Reactions were terminated with 30 μl of SDS sample buffer, and the samples were analyzed as in the whole cell lysate kinase assay. Pooled duplicate experiments (a total of 300 islets/condition) were lysed in 50 μl of lysis buffer. Twenty μl (∼20 μg) of whole cell lysates were added to 20 μl of SDS sample buffer and boiled for 5 min. SDS-PAGE (12%) was performed, and Western blotting was carried out according to standard protocols (31Seedorf K. Kostka G. Lammers R. Bashkin P. Daly R. Burgess W.H. van der Bliek A.M. Schlessinger J. Ullrich A. J. Biol. Chem. 1994; 269: 16009-16014Abstract Full Text PDF PubMed Google Scholar). Anti-total p38, anti-total ERK1/2, anti-phosphospecific p38, or anti-phosphospecific ERK1/2 antibodies were used. Enhanced chemiluminescence was used for detection. Lysate from the same experiment was separated on two gels and probed with either phosphospecific or total antibody as control for sample variation in protein content. Islet NO production was measured as nitrite accumulation in conditioned media determined by the Griess reaction (32Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10793) Google Scholar). In brief, 150 μl of medium were mixed with an equal volume of the Griess reagent (one part 0.1% naphtylethylene diamine dihydrochloride and one part 1% sulfanilamide in 5% H3PO4 (Merck, Darmstadt, Germany)) and incubated for 10 min at room temperature. The absorbance at 550 nm was measured on an immunoreader (Nippon Inter Med., Tokyo, Japan). The detection limit was 1 μm, equal to 2 pmol/islet in our conditions. Values below the detection limit were assigned the value of 2 pmol/islet. Intra- and interassay coefficients of variation calculated from three points on the standard curve were as follows: 1 μm, 2.3 and 16.8%; 10 μm, 1.8 and 8.1%; and 25 μm, 1.9 and 11.2%. Accumulated insulin release in the conditioned media was measured by radioimmunoassay (33Heding L.G. Diabetologia. 1972; 8: 260-266Crossref PubMed Scopus (730) Google Scholar). The detection limit was 35 fmol/ml. Intra- and interassay coefficients of variation between three known controls were as follows: A, 6.1 and 12.2%; B, 4.3 and 11.4%; and C, 3.1 and 8.2%. Total RNA from snap frozen islets was extracted and cDNA was prepared with cDNA Cycle® kit (Invitrogen, Leek, The Netherlands) as described previously (34Reimers J.I. Andersen H.U. Mauricio D. Pociot F. Karlsen A.E. Petersen J.S. Mandrup-Poulsen T. Nerup J. Diabetes. 1996; 45: 771-778Crossref PubMed Google Scholar). Reverse transcription-PCR was performed using [α-32P]dCTP (Amersham Pharmacia Biotech) and a fixed volume (5 μl) of cDNA dilution. Each analysis was performed with a set of iNOS primers in combination with a set of primers for TATA-binding protein (35Jensen J. Serup P. Karlsen C. Nielsen T.F. Madsen O.D. J. Biol. Chem. 1996; 271: 18749-18758Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) as an internal standard. The PCR products were separated on a 6% polyarylamide gel (Life Technologies, Inc.), visualized by autoradiography, and quantitated by PhosphorImager. Expression data are given as ratios to the co-amplified internal standard (TATA-binding protein). Results are presented as mean ± S.E. (n > 2) or as mean ± range (n = 2). Wilcoxon's matched-pair test was used, andp < 0.05 was chosen as the level of significance. To investigate whether p38 and ERK1/2 were activated by IL-1β in rat islets, we first assayed the kinase activities toward Elk-1, ATF2, and Hsp25 in whole cell lysates of IL-1β stimulated islets. Detectable kinase activity using Elk-1, ATF2, and Hsp25 as substrates was present in control islets (0′, baseline) (Fig.1 A ). IL-1β-enhanced phosphorylation of Elk-1 was evident within 20 min, peaked at 90 min with a 4.1-fold increase over baseline, and was sustained at 12 h. Enhanced ATF2 phosphorylation was found within 1 min, with a maximum 3.8-fold activation of phosphotransferase activity at 20 min, and was sustained at 12 h. Increased Hsp25 phosphorylation was apparent within 1 min, with a 3.1-fold peak activity at 20 min, and was sustained at 12 h. To demonstrate that the IL-1β-stimulated kinase activities toward Elk-1, ATF2, and Hsp25 in islets were associated with the presence and increased phosphorylation of ERK1/2 and p38, we performed Western blotting with phosphospecific antibodies recognizing only Tyr204- and Tyr182-phosphorylated ERK1/2 and p38, respectively. As seen in Fig. 1 B , a time-dependent IL-1β-mediated enhancement of the phosphorylation of ERK1/2 (pERK1/2) and p38 (pp38) was detected. The time courses of IL-1β-mediated phosphorylation of ERK1 and 2 were similar: a weak phosphorylation over baseline (0′) at 2.5–5 min, followed by a peak phosphorylation at 20 min, with only a slight decrease until 3 h, after which a marked decline was observed. However, a weak phosphorylation over baseline level was seen even at 24 h. Enhanced p38 phosphorylation by IL-1β was visible at 1 min, peaking and reaching a plateau between 20 min and 12 h and not detectable after 24 h. Control experiments using antibodies to total ERK1/2 and p38 (ERK1/2 and p38) showed no effects of IL-1β. These findings were substantiated by directly measuring the activity of ERK1/2 and MAPKAP-K2 (a specific substrate of p38 (27Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google Scholar)) (Fig.1 C ) in an immunocomplex kinase assay, showing a 3.1- and 3.6-fold activation of ERK1/2 and MAPKAP-K2, respectively, after a 20-min IL-1β exposure. Following 24 h IL-1β stimulation, kinase activities were below baseline. To investigate whether the ERK1/2 and p38 activities found in intact islets were due to the presence of these kinases in β-cells, the RIN β-cell line was assayed for ERK1/2 and p38 activities. The RIN β-cell line is comparable to primary β-cells in terms of IL-1β-mediated NO production, iNOS expression, and cytotoxicity, albeit at a higher concentration than is needed in islets (36Kwon G. Corbett J.A. Rodi C. Sullivan P. McDaniel M.L. Endocrinology. 1995; 136: 4790-4795Crossref PubMed Google Scholar, 37Ankarcrona M. Dypbukt J.M. Brüne B. Nicotera P. Exp. Cell Res. 1994; 213: 172-177Crossref PubMed Scopus (219) Google Scholar). Based on the IL-1β time course from intact islets (Fig.1 A ), an IL-1β exposure period of 20 min was used. IL-1β stimulated ATF2 and Hsp25 kinase activities in a dose-dependent manner, whereas IL-1β-induced Elk-1 kinase was not further activated by an increased concentration of IL-1β above 150 pg/ml (Fig. 2 A ). The binding of phosphospecific antibodies (pERK1/2 and pp38) showed that both ERK1/2 and p38 were phosphorylated in RIN cells after a 20-min exposure to 1500 pg/ml IL-1β (Fig. 2 B ). p38 and ERK1/2 activation by IL-1β was further demonstrated by the immunocomplex kinase assay that showed a 3.9- and 8.1-fold increase in ERK1/2 and MAPKAP-K2 activity in IL-1β-exposed RIN cells, respectively (Fig.2 C ). To dissect the relative contributions of ERK1/2 and p38 signaling pathways in the IL-1β-induced Elk-1, ATF2, and Hsp25 kinase activities, islets were incubated with p38i and MEKi 1 h prior to IL-1β exposure (Fig. 3 A ). The MEKi inhibited both basal and IL-1β stimulated Elk-1 kinase activity without affecting the ATF2 and Hsp25 kinase activities. A maximal inhibition was seen at 100 μm of MEKi. The p38i inhibited both basal and IL-1β-induced ATF2 and Hsp25 kinase activities; it was more effective in Hsp25 kinase inhibition, which was completely blocked at 10 μm, whereas this concentration of p38i inhibited IL-1β-induced ATF2 kinase activity by 71%. When the inhibitors were combined, the kinase activities toward the three substrates were completely blocked. The specificity of the inhibitors was further evaluated in an immunocomplex kinase assay, where the activities of immunoprecipitated ERK1/2 and MAPKAP-K2 in lysates of islets that had been preincubated with MEKi and/or p38i prior to IL-1β exposure were determined. MEKi inhibited IL-1β-induced ERK1/2 activity and did not affect the activity of MAPKAP-K2, whereas p38i inhibited IL-1β-stimulated MAPKAP-K2 activity but not that of ERK1/2 (Fig. 3 B ,top and middle panels ). Because JNK has both Elk-1 and ATF2 kinase activities (20Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (880) Google Scholar, 38Gupta S. Campbell D. Dérijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar), the complete inhibition of ATF2 and Elk-1 kinase activities in whole cell lysates of IL-1β stimulated islets preincubated with MEKi and p38i questioned the involvement of JNK in IL-1β signaling in islets. However, we found substantial IL-1β-stimulated JNK activity determined by c-Jun phosphorylation in an in vitro solid-phase kinase assay in islets, and as expected, JNK activity was unaffected by either of the inhibitors (Fig. 3 B , bottom panel ). This indicates that JNK is neither an IL-1β-activated Elk-1 nor ATF2 kinase in islets. MEKi and p38i were then used to evaluate the impact of ERK1/2 and p38 on IL-1β-mediated β-cell dysfunction. NO production was not detectable from untreated islets, and neither MEKi nor p38i added alone or in combination resulted in NO production (TableI). At a low (25 pg/ml) IL-1β concentration, p38i caused a 35% decrease in the IL-1β-induced islet NO production, whereas MEKi blocked NO production at that concentration of IL-1β. At a high (150 pg/ml) IL-1β concentration, p38i and MEKi caused a 25 and 33% reduction, respectively of IL-1β-induced islet NO production. However, in the presence of the two inhibitors, a synergistic effect was found, and IL-1β-induced NO synthesis was completely blocked.Table IEffects of p38i and MEKi on IL-1β-stimulated islet NO productionInhibitorNitrite0 pg/ml IL-1β25 pg/ml IL-1β150 pg/ml IL-1βpmol/islet/24 hNone29.8 ± 1.41-aP < 0.05 versus 0 IL-1 ÷ inhibitors (i).13.5 ± 1.01-aP < 0.05 versus 0 IL-1 ÷ inhibitors (i).,1-bP < 0.05 versus 25 IL-1 ÷ i.10 μmp38i26.4 ± 1.21-bP < 0.05 versus 25 IL-1 ÷ i.,1-cP < 0.05 versus 0 IL-1 + p38i.10.1 ± 1.11-dP < 0.05 versus 150 IL-1 ÷ i.,1-eP < 0.05 versus either 0 IL-1 + p38i or 25 IL-1 + p38i.100 μm MEKi22.7 ± 0.31-bP < 0.05 versus 25 IL-1 ÷ i.,1-fP < 0.05 versus 25 IL-1 + p38i.9.1 ± 1.01-dP < 0.05 versus 150 IL-1 ÷ i.,1-gP < 0.05 versus either 0 IL-1 + MEKi or 25 IL-1 + MEKi.10 μm p38i + 100 μmMEKi22.1 ± 0.11-bP < 0.05 versus 25 IL-1 ÷ i.,1-fP < 0.05 versus 25 IL-1 + p38i.2.8 ± 0.31-dP < 0.05 versus 150 IL-1 ÷ i.,1-hP < 0.05 versus either 150 IL-1 + p38i or 150 IL-1 + MEKi.Groups of 150 islets were incubated for 24 h in 0, 25, or 150 pg/ml IL-1 ± 10 μm p38i and/or 100 μmMEKi added 1 h prior to IL-1. Medium was sampled for nitrite determination. Values are mean ± S.E. of n = 6.1-a P < 0.05 versus 0 IL-1 ÷ inhibitors (i).1-b P < 0.05 versus 25 IL-1 ÷ i.1-c P < 0.05 versus 0 IL-1 + p38i.1-d P < 0.05 versus 150 IL-1 ÷ i.1-e P < 0.05 versus either 0 IL-1 + p38i or 25 IL-1 + p38i.1-f P < 0.05 versus 25 IL-1 + p38i.1-g P < 0.05 versus either 0 IL-1 + MEKi or 25 IL-1 + MEKi.1-h P < 0.05 versus either 150 IL-1 + p38i or 150 IL-1 + MEKi. Open table in a new tab Groups of 150 islets were incubat
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