Calcium Has a Permissive Role in Interleukin-1?-Induced c-Jun N-Terminal Kinase Activation in Insulin-Secreting Cells
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内分泌学杂志 2005年第7期
Laboratory for ?-Cell Biology (J.S., A.E.K, N.B., T.M.-P.), Steno Diabetes Center, DK-2820 Gentofte, Denmark; The Rolf Luft Center for Diabetes Research (S.V.Z., I.L.K., P.-O.B., T.M.-P.), Department of Molecular Medicine, Karolinska Institutet, S-171 76 Stockholm, Sweden; and Belozersky Institute of Physico-Chemical Biology (S.V.Z.), Moscow State University, Moscow 119899, Russia
Address all correspondence and requests for reprints to: Thomas Mandrup-Poulsen, M.D., D.M.Sc., Steno Diabetes Center, Niels Steensensvej 2, DK-2820 Gentofte, Denmark. E-mail: tmpo@steno.dk; or Joachim St?rling, M.Sc., Steno Diabetes Center, Niels Steensensvej 8, NSPP, DK-2820 Gentofte, Denmark. E-mail: jstq@steno.dk.
Abstract
The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1?-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic ?-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1? activation of JNK in ?-cells are largely unknown. In this study, we investigated whether Ca2+ plays a role for IL-1?-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mM glucose, combined pharmacological blockade of L- and T-type Ca2+ channels suppressed IL-1?-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1?-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1?-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca2+ channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse ?TC3 cells. Ca2+ influx, specifically via L-type but not T-type channels, contributed to IL-1? activation of JNK. Activation of p38 and ERK in response to IL-1? was also dependent on L-type Ca2+ influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca2+ ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase, all caused an amplification of IL-1?-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca2+ [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca2+/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1?-stimulated c-jun phosphorylation in INS-1 or ?TC3 cells. Our data suggest that Ca2+ plays a permissive role in IL-1? activation of the JNK signaling pathway in insulin-secreting cells.
Introduction
THE LOSS OF ?-cell mass in the pancreatic islets of Langerhans leading to insufficient insulin release and hyperglycemia characteristic of type 1 diabetes is believed to be an immune-mediated process in which proinflammatory cytokines, in particular IL-1?, play an essential role (1). Furthermore, recent findings have established a role for IL-1? in ?-cell failure in type 2 diabetes (2). Under in vitro conditions, exposure of insulin-secreting cells or pancreatic islets to IL-1? alone or in combination with interferon (IFN) and/or TNF leads to impairment of ?-cell function and induction of apoptotic cell death (1).
IL-1? activates the c-jun N-terminal kinase (JNK) pathway in ?-cells (3, 4, 5). JNK constitutes a stress-activated member of the MAPK family of threonine/serine kinases and is involved in the transmission of stress and apoptotic signaling in many cells. Activation of JNK is achieved by the dual phosphorylation on Thr183 and Tyr185 in the activation loop of the kinase by the dual specificity MAPK kinases MKK4 and MKK7, which are activated by phosphorylation by a MAPK kinase kinase (6, 7). JNK plays a critical role in cytokine-mediated apoptosis of ?-cells, as suggested by the finding that apoptosis induced by IL-1? in insulin-secreting cells can be prevented by inhibition of the JNK pathway (8, 9, 10). Furthermore, in primary ?-cells, oxidative stress-induced suppression of insulin gene transcription is mediated by JNK (11). Thus, strong evidence points toward JNK as a key regulator of ?-cell function and apoptosis. However, the regulatory events in the activation of JNK by IL-1? in insulin-secreting cells remain largely unknown. Insight into these mechanisms may identify targets for pharmacological intervention in diabetes and islet graft failure.
Pancreatic ?-cells are highly active in Ca2+ handling. Thus, ?-cell uptake and metabolism of glucose lead to closure of ATP-sensitive K+ channels, depolarization of the plasma membrane, and, subsequently, influx of Ca2+ through voltage-gated Ca2+ channels (VGCCs). The Ca2+ signal provided by this influx of Ca2+ is accompanied by release of Ca2+ from the endoplasmic reticulum (ER) (12, 13, 14, 15). The subsequent increase in the cytosolic free Ca2+ concentration ([Ca2+]i) is an important determinant for insulin granule exocytosis. Ca2+ entry in response to glucose in insulin-secreting cells also leads to activation of ERK MAPK (16, 17, 18), but not of JNK or p38 MAPK to any significant degree (5, 17). VGCCs also regulate ?-cell apoptosis. Hence, influx of Ca2+ via L-type VGCCs is required for induction of ?-cell apoptosis resulting from glucotoxicity (19) and in response to serum from type 1 diabetes patients (20). Furthermore, cytokine-mediated ?-cell death seems to be dependent on VGCC-mediated Ca2+ entry as suggested by the findings that blockers of L-type VGCCs can suppress ?-cell apoptosis induced by IL-1? (21, 22) or IFN plus TNF (23) and blockers of T-type VGCCs can repress ?-cell apoptosis induced by a mixture of cytokines (24).
The aim of the present study was to test the hypothesis that Ca2+ contributes to IL-1? activation of proapoptotic JNK in ?-cells. Our findings provide evidence for a permissive and potentiating role of Ca2+ in IL-1?-induced signaling via the JNK pathway in insulin-secreting cells.
Materials and Methods
Materials
Recombinant mouse or human IL-1? was obtained from BD PharMingen (San Diego, CA), Novo Nordisk (Bagsv?rd, Denmark), or Calbiochem (San Diego, CA). All reagents for SDS-PAGE were from Invitrogen (Carlsbad, CA). The JNK substrate glutathione S-transferase (GST)-c-jun (1–79) was from Calbiochem. Recombinant murine heat shock protein-25 (Hsp25) was from Stressgen (Victoria, Canada). GST-Elk-1 was a gift from Klaus Seedorf, then at Hagedorn Research Institute (Gentofte, Denmark). [-32P]ATP (3000 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). Antibodies to Thr183/Tyr185-phosphorylated JNK1/2, JNK1/2, Thr202/Tyr204-phosphorylated ERK1/2, ERK1/2, Thr180/Tyr182-phosphorylated p38, and p38 were obtained from Cell Signaling (Beverly, MA). Inhibitory B (IB) antibody was obtained from Acive Motif (Carlsbad, CA). Antibody to ?-tubulin was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and antibody to inducible nitric oxide synthase (iNOS) was from BD PharMingen. Mibefradil was kindly provided by Roche (Basel, Switzerland). Nimodipine, Ca2+ ionophore A23187, thapsigargin, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM), W7, KN62, and KN93 were obtained from Calbiochem. FPL64176was obtained from Sigma (St. Louis, MO). Luciferase reporter plasmids pNFB Luc and pRL-TK were from Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI), respectively. iNOS promoter construct piNOS-1002 Luc (25) was a gift from Decio Eizirik (Université Libre de Bruxelles, Brussels, Belgium). All other reagents were from Sigma or Merck (Darmstadt, Germany). Nimodipine was dissolved in polyethylene glycol or dimethylsulfoxide (DMSO). Mibefradil and KN93 were dissolved in water. FPL64176was dissolved in ethanol. All other inhibitors were dissolved in DMSO.
Cell culture
INS-1 or ?TC3 cells were maintained in RPMI 1640 medium (11 mM glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies, Inc., Grand Island, NY) in 80-cm2 tissue culture flasks (Nunc, Roskilde, Denmark). In addition, the media for INS-1 cells contained 50 μM ?-mercaptoethanol (Sigma). Cells were trypsinized and passaged weekly. For experimentation, cells were seeded in 12-well dishes (Nunc) (0.5 x 106 cells per well) or in 25-cm2 tissue culture flasks (Nunc) (2 x 106 cells per flask).
Islet isolation and culture
Pancreatic islets of Langerhans from 3- to 4-d-old Wistar Furth rats (Charles River, Sulzfeldt, Germany) were isolated and cultured as previously described (4). On the day of experimentation, 150 rat islets were placed in four-well dishes (Nunc) in RPMI 1640 medium (Life Technologies, Inc.) containing 11 mM glucose and supplemented with 0.5% human serum. Islets from adult ob/ob mice were obtained from a local noninbred colony. The islets were isolated by collagenase digestion technique and cultured overnight at 37 C in RPMI 1640 medium supplemented with 10% fetal calf serum (Flow Laboratories, Erwing, UK) and 11 mM glucose. Islets from ob/ob mice contain more than 90% ?-cells (26). All animal work was carried out according to national and international law and ethical standards.
Cell and islet stimulation and lysis
At the time of experimentation (1 or 2 d after seeding of cells), the culture medium was removed and fresh medium (11 mM glucose) with or without drugs was added. Cells or islets were incubated in the presence of drugs for 30 min to 1 h before exposure to IL-1?. When necessary, solvents (DMSO, polyethylene glycol, or ethanol) used for drugs were added to all conditions (0.1–1% final concentration). After IL-1? exposure, cells or islets were washed in cold PBS, and lysed for 30 min on ice in lysis buffer containing 20 mM Tris-acetate (pH 7.0), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% vol/vol Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1 mM benzamidine, 1 mM dithiothreitol, 1 mM Na3VO4, and 4 μg/ml leupeptin. Detergent-insoluble material was pelleted by centrifugation at 15,000 x g for 5 min at 2–4 C. The supernatants (whole-cell lysates) were stored at –80 C until assayed.
In vitro kinase assay
Phosphotransferase activities toward GST-c-jun, GST-Elk-1 and Hsp25 were measured by a whole-cell lysate in vitro kinase assay essentially as described in Ref. 4 except that 2 μg of GST-c-jun (1–79) was used instead of activating transcription factor 2. Phosphorylated substrates were visualized by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Immunoblotting
Whole cell lysate was mixed with 4x SDS sample buffer (Invitrogen), boiled for 10 min, and subjected to 10% SDS-PAGE. Proteins were electrotransferred to nitrocellulose filter membranes. Blocking of nonspecific protein binding was done by incubating the filter membrane in blocking buffer (1x TBS, pH 7.6; 0.1% Tween 20; 5% nonfat dry milk) for 1 h. After washing in TBST (1x TBS, pH 7.6; 0.1% Tween 20), filters were incubated with the respective antibodies diluted in either TBST containing 5% BSA or in blocking buffer. Filter membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Immune complexes were detected by chemiluminescence using LumiGLO (Cell Signaling), and light emission was captured on x-ray film or digitally using FUJI LAS3000.
Transient transfection and luciferase reporter assay
INS-1 cells (0.2 x 106 cells) in duplicates were transiently transfected with luciferase reporter plasmids [for nuclear factor-B (NFB) construct: 0.4 μg pNFB Luc and 0.15 μg pRL-TK; for iNOS promoter construct: 0.7 μg piNOS-1002 Luc and 0.05 μg pRL-TK] using Lipofectamine2000 (Invitrogen) as described by the manufacturer. The day after transfection, fresh medium with or without nimodipine was added to cells. After 30 min preincubation, cells were exposed for 6 h to IL-1?, washed in PBS and lysed in passive lysis buffer (Promega). Luciferase assays were carried out using the dual luciferase reporter system kit according to the specifications of the manufacturer (Promega). NFB and iNOS promoter activities were normalized to the coexpressed Renilla luciferase.
Measurements of intracellular calcium concentration ([Ca2+]i)
INS-1 cells were cultured on glass coverslips in plastic Petri dishes under the same conditions as those used for the evaluation of the effect of A23187 on IL-1?-induced JNK phosphorylation. Ca2+ fluorescent indicator fura 2/AM (2 μM) was added to the culture medium 1 h before the measurements of [Ca2+]i were performed. The coverslips with fura 2 loaded cells formed the bottom of an open chamber and in the course of [Ca2+]i. Measurements were perfused with the buffer containing 125 mM NaCl; 5.9 mM KCl; 1.2 mM MgCl2; 1.28 mM CaCl2; 25 mM HEPES; 11 mM glucose; and 0.1% BSA, pH 7.4, at 37 C. Measurements of [Ca2+]i were made as previously described (14). The fluorescence ratio at 340:380 nm was determined using a Spex Fluorolog spectrophotometer coupled to a Zeiss Axiovert 35 microscope with a Zeiss Fluar 40x/1.30 oil objective (Carl Zeiss, G?ttingen, Germany).
Methylthiazolyl Tetrazolium (MTT) viability analysis
Cell viability was assessed by the MTT assay (Promega) measuring mitochondrial activity by the conversion of a tetrazolium salt to a colored formazan product by the mitochondrial enzyme succinate dehydrogenase. Thus, it should be noted that the assay is a marker of mitochondrial activity and not per se cell viability. Briefly, cells in triplicates in 96-well plates were left untreated or treated with IL-1? in the presence or absence of inhibitors for 2 d. Twenty-five microliters of MTT dye reagent were added to each well followed by incubation at 37 C for 1–3 h. Then 90 μl per well of MTT Stop solution was added followed by overnight incubation. Absorbance was measured at 578 nm.
Statistical analysis
Statistical differences between groups were calculated by paired t test using Excel (Microsoft).
Results
IL-1? activates JNK in insulin-secreting INS-1 cells
We first characterized the dose-response relationship between IL-1? exposure and JNK activation in insulin-secreting rat INS-1 cells. Measuring in vitro kinase activity toward the specific JNK substrate GST-c-jun (amino acids 1–79) in whole-cell lysates prepared from INS-1 cells exposed to increasing concentrations of IL-1? for 1 h revealed that IL-1?, in a dose-dependent manner, induced an increase in the phosphotransferase activity toward GST-c-jun (Fig. 1A, upper panel). The effect of IL-1? on in vitro GST-c-jun phosphorylation correlated well with an increase in the amount of the endogenous, phosphorylated and active form of JNK as determined by immunoblot analysis using antibodies against Thr183/Tyr185-phosphorylated JNK1/2 (Fig. 1A, middle panel). Under these conditions, the level of total JNK1/2 protein was unchanged (Fig. 1A, lower panel). From this experiment, it was found that an IL-1? concentration of 80–160 pg/ml, giving roughly a half-maximal effect on JNK, would be appropriate for the remainder of the study. A time-course study of IL-1?-induced phosphorylation of JNK showed that JNK1/2 phosphorylation in response to IL-1? peaked at 1 h and persisted for at least 9 h (Fig. 1B).
FIG. 1. IL-1? activates JNK in INS-1 cells. A, INS-1 cells were exposed to the indicated concentrations of IL-1? for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP. Phosphorylated GST-c-jun was visualized by autoradiography. The activation state of JNK was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Results shown are representative of two experiments. B, INS-1 cells were exposed to IL-1? (160 pg/ml) for the indicated time periods. After lysis of cells, the activation state of JNK was examined by immunoblot analysis. Blots shown are representative of three experiments. KA, Kinase assay; IB, immunoblot.
Nimodipine plus mibefradil reduce IL-1?-induced JNK activation
To determine whether Ca2+ plays a role in JNK activation by IL-1?, we first examined whether combined blockade of L- and T-type plasma membrane VGCCs, the two types of Ca2+ channels that previously were shown to be implicated in cytokine-induced ?-cell apoptosis (21, 22, 23, 24), affects IL-1?-stimulated JNK activity in INS-1 cells. Nimodipine was used to block L-type channels and mibefradil to block T-type channels. The increase in kinase activity toward GST-c-jun as well as the phosphorylation of JNK1/2 induced by a 1-h exposure to IL-1? in standard culture medium containing 11 mM glucose were abolished when cells were coincubated with 10 μM nimodipine plus 1 μM mibefradil (Fig. 2A, upper panels). Quantitative analysis of GST-c-jun revealed that IL-1?-induced GST-c-jun phosphorylation was reduced from 3.1 ± 0.2-fold above baseline to 0.9 ± 0.1 (P < 0.005) by the presence of nimodipine and mibefradil. In similar experiments conducted on isolated intact rat or ob/ob mouse islets or on insulin-secreting mouse ?TC3 cells, the IL-1?-induced increase in in vitro phosphorylation of GST-c-jun was also significantly inhibited by combined Ca2+ channel blockade (Fig. 2A, lower panels). When each channel blocker was used separately on INS-1 cells, it was found that nimodipine was effective in inhibiting IL-1?-stimulated GST-c-jun phosphorylation and activation of JNK1/2, whereas mibefradil was ineffective (Fig. 2B). This suggests that Ca2+ entering preferentially through L-type channels contribute to IL-1? activation of JNK. IL-1? dose experiments further revealed that nimodipine suppressed IL-1?-induced JNK activity as measured by GST-c-jun phosphorylation (Fig. 2C). Because it was previously shown in mouse ?-cells that exposure for 6 or 24 h to a combination of cytokines (IL-1? plus IFN with or without TNF) specifically led to an increase in the activity of T-type Ca2+ channels (24), we stimulated ?TC3 cells with a combination of cytokines for 6 h in the presence or absence of T-type channel blockade and examined the cells for JNK activity. As seen in Fig. 2D, cytokine-exposed cells had activated JNK1/2, which was not affected by T-type Ca2+ channel blockade by mibefradil or NiCl2 (10 μM). Collectively, these results suggest that Ca2+ influx specifically via L-type VGCC plays a role in the activation of JNK by IL-1? in ?-cells.
FIG. 2. Calcium channel blockade reduces IL-1?-induced JNK activation. A, INS-1 cells, rat islets, ob/ob mouse islets, or ?TC3 cells were incubated with or without IL-1? (INS-1 and rat islets, 80 pg/ml; ?TC3 and ob/ob mouse islets, 2 ng/ml) in the presence or absence of 10 μM nimodipine (Nimo) plus 1 μM mibefradil (Mibe) for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP. Phosphorylated GST-c-jun was visualized by autoradiography. Autoradiograms shown are representative of four (INS-1), three (rat islets), or five (ob/ob and ?TC3) experiments. In addition, the activation state of JNK in INS-1 cells was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Blots shown are representative of two experiments. B, INS-1 cells were incubated with or without IL-1? (80 pg/ml) in the presence or absence of 10 μM nimodipine or 1 μM mibefradil for 1 h. After lysis of cells, whole-cell lysates were assayed as in A. Autoradiogram and blots shown are representative of two to four experiments. C, INS-1 cells were incubated with increasing concentrations of IL-1? (160–800 pg/ml) in the presence or absence of 10 μM nimodipine for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun. Autoradiogram shown is representative of four experiments. D, ?TC3 cells were incubated with or without IL-1? (63 pg/ml) + IFN (10 pg/ml) + TNF (5 pg/ml) (Cyt. mix) in the presence or absence of 10 μM NiCl2 or 1 μM mibefradil for 6 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun and the activation state of JNK was examined by immunoblot analysis. Autoradiogram and blots shown are representative of three experiments. KA, Kinase assay; IB, immunoblot.
Nimodipine inhibits IL-1?-induced activation of p38 and ERK
We next examined whether Ca2+ influx also regulates IL-1? activation of p38 and ERK MAPKs. As demonstrated by immunoblotting of the activated kinases as well as by in vitro kinase activity toward Hsp25, a substrate of the p38-activated kinase MAPK-activated protein kinase 2, and GST-Elk-1, a substrate of ERK in ?-cells (4), IL-1? activation of both p38 and ERK1/2 in INS-1 cells cultured at 11 mM glucose was inhibited by nimodipine (Fig. 3, A and B). These findings suggest that L-type Ca2+ entry also contributes to IL-1? activation of p38 and ERK MAPKs.
FIG. 3. The L-type channel blocker nimodipine reduces IL-1?-induced activation of p38 and ERK, but only modestly affects IL-1? signaling via NFB. A and B, INS-1 cells were incubated with or without IL-1? (A, 80 pg/ml; B, 0.8 ng/ml) in the presence or absence of 10 μM nimodipine for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward Hsp25 and GST-Elk-1 in the presence of [32P]ATP. Phosphorylated substrates were visualized by autoradiography. The activation states of p38 and ERK were examined by immunoblot analysis using antibodies to Thr180/Tyr182-phosphorylated p38, p38, and Thr202/Tyr204-phosphorylated ERK1/2. Immunoblot analysis of tubulin in B confirmed equal protein loading. Autoradiograms and blots shown are representative of two to three experiments. C, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 10 μM nimodipine for 30 min. After lysis of cells, the lysates were analyzed for content of IB by immunoblot analysis. Results shown are representative of three experiments. D and F, INS-1 cells were transfected with either NFB-driven or iNOS promoter luciferase reporter gene constructs. Cells were then stimulated with or without IL-1? (160 pg/ml) in the presence or absence of 10 μM nimodipine for 6 h. Luciferase activity was normalized to coexpressed Renilla luciferase. Data are expressed as means ± SEM of four experiments. E, INS-1 cells were treated with or without IL-1? in the presence or absence of 10 μM nimodipine for 6 h. Cell lysates were analyzed for content of iNOS by immunoblot analysis. Results are representative of two experiments. G, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 10 μM nimodipine for 2 d. Cell viability was determined by MTT analysis. Data are means ± SEM of five experiments. *, P < 0.005 vs. control; #, P < 0.02 vs. IL-1?. KA, Kinase assay; IB, immunoblot.
Nimodipine only modestly affects IL-1?-induced signaling via NFB
IL-1? is a potent activator of the transcription factor NFB in ?-cells (27, 28), and this transcription factor contributes to apoptosis in cytokine-exposed ?-cells (29, 30). To investigate whether IL-1? signaling via the NFB pathway also involves Ca2+, the effect of nimodipine on IL-1?-stimulated degradation of IB was determined. Figure 3C shows that stimulation of INS-1 cells with IL-1? led to a pronounced degradation of IB within 30 min. This degradation of IB was not affected by coincubation with nimodipine. Because other factors down-stream to the degradation of IB are involved in the transcriptional capacity of NFB, we investigated by luciferase reporter gene assay the effect of nimodipine on IL-1?-stimulated NFB transactivation in INS-1 cells. Luciferase activity in cells transiently transfected with a NFB-driven luciferase reporter plasmid was decreased from 1.79 ± 0.08-fold to 1.52 ± 0.03 (P < 0.005) corresponding to a 34% inhibition by nimodipine (Fig. 3D). To see whether this partial inhibition of NFB transactivation affects the expression of a gene whose transcription requires NFB, we tested the effect of L-type Ca2+ channel blockade on IL-1?-mediated expression of iNOS, an enzyme whose expression depends on NFB (25, 27, 28, 31). Figure 3E shows that a 6-h exposure to IL-1? induced the expression of iNOS, which was not affected by nimodipine. INS-1 cells were then transiently transfected with an iNOS promoter luciferase reporter gene construct to see whether Ca2+ channel blockade affects IL-1?-induced iNOS promoter activity. This experiment revealed that the IL-1?-stimulated induction of iNOS promoter activity was not modulated by nimodipine (Fig. 3F). In accordance with these observations, analysis of accumulated nitrite in the culture medium after a 24-h exposure to IL-1?, showed no effect of nimodipine on IL-1?-induced NO production (13.2 vs. 12.1 μM for IL-1? alone and IL-1? + nimo, respectively, means of n = 2). Together, these findings indicate that opposed to MAPKs, IL-1? signaling via NFB is not regulated by L-type Ca2+ influx. Consistent with a role of L-type Ca2+ influx for IL-1? signaling via proapoptotic JNK/MAPKs, nimodipine afforded a significant partial protection against the suppressive effect of IL-1? on INS-1 cell viability as determined by MTT analysis measuring mitochondrial activity (Fig. 3G), whereas 10 μM nimodipine alone did not affect INS-1 cell mitochondrial activity (104 ± 3.5% of control, n = 5). To exclude that T-type VGCCs might be involved in IL-1? signaling to NFB, the effect of mibefradil on NFB activation was also determined. However, mibefradil did not affect IL-1?-mediated IB degradation (Fig. 4A) and had no effect on IL-1?-induced NFB and iNOS promoter activities in reporter gene assays (Fig. 4, C and D). Furthermore, mibefradil neither affected INS-1 cell viability alone (101 ± 3.8% of control, n = 3) nor protected against IL-1?-mediated suppression of cell viability (Fig. 4B).
FIG. 4. The T-type channel blocker mibefradil does not affect IL-1?-induced IB degradation or toxicity. A, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 1 μM mibefradil for 30 min. After lysis of cells, the lysates were analyzed for content of IB by immunoblot analysis. Results shown are representative of three experiments. B, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 1 μM mibefradil for 2 d. Cell viability was determined by MTT analysis. Data are means ± SEM of three experiments. C and D, INS-1 cells were transfected with either NFB-driven or iNOS promoter luciferase reporter gene constructs. Cells were then stimulated with or without IL-1? (160 pg/ml) in the presence or absence of 1 μM mibefradil (mibe) for 6 h. Luciferase activity was normalized to coexpressed Renilla luciferase. Data are expressed as means ± SEM of three experiments.
Agents that increase [Ca2+]i augment IL-1?-induced JNK activation
Because blocking Ca2+ entry via VGCCs inhibited IL-1?-induced JNK activity, the hypothesis that an increase in Ca2+ influx may lead to higher JNK activity in IL-1?-exposed cells was tested. To increase Ca2+ entry via L-type channels, we used a membrane depolarizing stimulus (KCl) in the presence of the L-type channel agonist FPL64176 As a positive control for L-type Ca2+ influx during this treatment, we first measured the effect on ERK, which is known to be activated by Ca2+ entry through L-type channels in response to membrane depolarization in insulin-secreting cells. Figure 5A shows that exposure of INS-1 cells to 50 mM KCl plus FPL64176(1 μM) stimulated ERK activation, which was abolished by nimodipine. In contrast to that observed for ERK, KCl plus FPL64176in itself did not stimulate activation of JNK1/2 to any significant degree, whereas IL-1?-induced JNK1/2 activation was augmented by KCl and FPL64176(Fig. 5B). We then examined whether glucose would also modulate JNK activation. As seen in Fig. 5C, IL-1? activation of JNK1/2 was higher in the presence of 27 mM glucose compared with 5 mM glucose. Nimodipine decreased the potentiating effect of glucose on JNK verifying that the effect of glucose involved L-type Ca2+ influx. To address whether another physiological Ca2+-mobilizing stimulus would affect IL-1?-induced JNK, we exposed cells to IL-1? for 1 h in the presence of increasing concentrations of glucagon-like peptide (GLP)-1, which is known to promote Ca2+ signaling (32, 33, 34). However, in contrast to glucose, GLP-1 failed to modulate IL-1?-induced phosphorylation of JNK1/2 (Fig. 5D), suggesting that not all physiological stimuli that induce Ca2+ signaling have the capability to augment IL-1?-induced JNK activation. To more directly examine the effect of [Ca2+]i on IL-1? activation of JNK, the effect of Ca2+ ionophore A23187 was determined. Alone, A23187 (1 μM) did not affect basal JNK1/2 phosphorylation, but caused a dose-dependent increase in IL-1?-induced JNK1/2 activation as well as IL-1?-stimulated in vitro GST-c-jun phosphorylation (Fig. 6A) To determine the correlation between A23187-induced increases in [Ca2+]i and IL-1?-induced JNK activation, we measured [Ca2+]i after treatment with IL-1? in the presence or absence of A23187. Although exposure to IL-1? alone for 1 h did not cause any significant change in [Ca2+]i compared with nontreated cells (157.7 ± 12.2 vs. 198.5 ± 17.9 nM, n = 47–59), A23187 dose-dependently caused a rise in [Ca2+]i in the presence of IL-1? (Fig. 6B). Therefore, the A23187-induced increases in [Ca2+]i directly correlated with the augmenting effect of A23187 on IL-1?-induced JNK phosphorylation. Finally, because entry of Ca2+ via VGCCs into ?-cells is accompanied by Ca2+ release from the ER (14), we next looked at whether Ca2+ release from the ER modulates JNK activation. Thapsigargin (1 μM), an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase leading to impairment of ER Ca2+ uptake and thus an increase in [Ca2+]i, in itself stimulated kinase activity toward GST-c-jun (Fig. 6C, upper panel). However, the combination of IL-1? and thapsigargin resulted in much higher GST-c-jun phosphorylation as compared with each treatment alone. The increase in phosphorylation of GST-c-jun was quantified to be 313% (P < 0.005) above the level induced by IL-1? alone. In line with this, thapsigargin augmented IL-1?-induced phosphorylation of JNK1/2 (Fig. 6C, lower panels). Taken together, these observations indicate that most stimuli that increase [Ca2+]i cause higher JNK activation in response to IL-1?.
FIG. 5. KCl and glucose, but not GLP-1, potentiate IL-1?-induced JNK activation. A, INS-1 cells were incubated with or without KCl (50 mM) plus FPL64176(1 μM) in the presence or absence of 10 μM nimodipine for 1 h. After lysis of cells, the activation state of ERK in whole-cell lysates was examined by immunoblot analysis using antibodies to Thr202/Tyr204-phosphorylated ERK1/2 and ERK1/2. B, INS-1 cells were incubated with or without KCl plus FPL64176in the presence or absence of IL-1? (160 pg/ml) for 1 h. The activation state of JNK in whole-cell lysates was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Blots shown are representative of six experiments. C, INS-1 cells were incubated in the presence of 5 mM glucose overnight. Medium was then changed to new medium containing either 5 or 27 mM glucose in the presence or absence of IL-1? with or without 10 μM nimodipine. After a 1-h stimulation, the activation state of JNK in whole-cell lysates was examined by immunoblot analysis. Blots shown are representative of three experiments. D, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of increasing concentrations (1, 5, 10, 100 nM) of GLP-1. After a 1-h stimulation, the activation state of JNK in whole-cell lysates was examined by immunoblot analysis. Blots shown are representative of three experiments. IB, Immunoblot.
FIG. 6. Ca2+ ionophore and thapsigargin potentiate IL-1?-induced JNK activation. A, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of increasing concentrations (0.1, 0.25, 0.5, 1 μM) of Ca2+ ionophore A23187 for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP, and the activation state of JNK was examined by immunoblot analysis. Autoradiogram and blots shown are representative of three experiments. B, INS-1 cells were incubated with IL-1? (160 pg/ml) in the presence or absence of increasing concentrations (0.25, 0.5, 1 μM) of A23187 for 1 h. [Ca2+]i was measured by fura 2 method. Data are expressed as means ± SEM of n = 42–59 for each condition. C, INS-1 cells were incubated with IL-1? (160 pg/ml) in the presence or absence of thapsigargin (Tg) (1 μM) for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP and the activation state of JNK was examined by immunoblot analysis. Autoradiogram and blots shown are representative of four experiments. *, P < 0.01 vs. IL-1? alone. KA, Kinase assay; IB, immunoblot.
BAPTA-AM, W7, and KN62 or KN93 reduce IL-1?-induced JNK activation
We next applied various inhibitors of Ca2+ and Ca2+-activated proteins to assess the involvement of these in IL-1? activation of JNK in INS-1 cells. As shown in Fig. 7A, BAPTA-AM (100 nM) reduced IL-1?-stimulated in vitro GST-c-jun phosphorylation. Consistent with this, both W7 (5 and 25 μM), a calmodulin antagonist, and KN62 (10 μM), an inhibitor of Ca2+/calmodulin-dependent kinases (CaMKs), diminished IL-1?-stimulated GST-c-jun phosphorylation (Fig. 7, B and C). Quantitative analysis revealed that BAPTA-AM, W7, and KN62 decreased IL-1?-stimulated GST-c-jun phosphorylation by 32% (P = 0.01), 33% (P < 0.05), and 39% (P < 0.02), respectively. In ?TC3 cells, another CaMK inhibitor (KN93) in a dose-dependent manner decreased IL-1?-stimuled phosphorylation of GST-c-jun, which correlated with reduced IL-1?-induced phosphorylation of JNK1/2 (Fig. 7D).
FIG. 7. BAPTA-AM, W7, KN62, and KN93 reduce IL-1?-induced JNK activation. INS-1 cells were incubated with or without IL-1? (80 or 160 pg/ml) in the presence or absence of BAPTA-AM (100 nM) (A), W7 (5 or 25 μM) (B), or KN62 (10 μM) (C) for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP. Phosphorylated GST-c-jun was visualized by autoradiography. Autoradiograms shown are representative of three to four experiments. D, ?TC3 cells were incubated with or without IL-1? (320 pg/ml) in the presence or absence of the indicated concentrations of KN93 for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP, and the activation state of JNK was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Autoradiograms and blots shown are representative of four and two experiments, respectively. KA, Kinase assay; IB, immunoblot.
Discussion
IL-1? may be involved in the pathogenesis of type 1 and 2 diabetes by causing cellular dysfunction and apoptosis of pancreatic ?-cells. IL-1? activates JNK in ?-cells and signaling via this pathway is critically involved in mediating IL-1?-induced apoptosis in insulin-secreting cells (8, 9, 10). However, the cellular regulatory mechanisms contributing to IL-1?-induced JNK activation in ?-cells are not well understood.
In this study, we investigated the role of Ca2+ in IL-1? activation of JNK. By using Ca2+ channel blockers, agents that increase or decrease [Ca2+]i and inhibitors of Ca2+-activated proteins, we provide evidence to suggest a role for Ca2+ in controlling IL-1? activation of JNK in insulin-secreting cells and rodent islets. The molecular mechanism may involve activation of CaMKs by increases in [Ca2+]i. In analogy, CAMK IV mediates Ca2+-dependent JNK activation in PC12 cells (35).
Although our data indicate a role for Ca2+ entry via L-type VGCCs in regulating IL-1? ?-cell JNK signaling, previous studies in ?-cells showed no apparent alterations in VGCC activity or steady-state [Ca2+]i after exposure to IL-1? (21, 24, 36, 37, 38), except in one study, where a 2-h exposure to IL-1? stimulated a cellular net-uptake of Ca2+ via L-type channels in rat islets (39). The results of our study also do not show an increase in [Ca2+]i after a 1-h exposure to IL-1?. This favors the idea that Ca2+ has an important but strictly permissive role in IL-1?-induced JNK activation.
In the presence of in vitro mid-range (typically 11 mM) concentrations of glucose, glucose-induced Ca2+ influx is critical for the stimulus-secretion coupling. However, if the ?-cell is under concurrent challenge with IL-1?, an increased [Ca2+]i produced by glucose may augment the capability of IL-1? to evoke intracellular signaling and, thereby, the capability of IL-1? to signal ?-cell destruction. Thus, although IL-1? does not induce detectable net increases in [Ca2+]i, Ca2+ plays a role in IL-1? signal transduction in that glucose-stimulated Ca2+ signaling indeed exerts a permissive and potentiating role to propagate IL-1? signaling. This concept is supported by the observations that the cytotoxic effects of cytokines are more pronounced in high vs. low glucose (40) and that activation of Ca2+ influx by membrane depolarization by KCl was insufficient to induce JNK activity in the absence of IL-1?. We have recently found that even in the presence of a low, but physiologically relevant glucose concentration (5.5 mM), IL-1?-induced human islet ?-cell apoptosis is dependent on Ca2+ influx as determined by the use of nimodipine or the K+ channel openers diazoxide and NN414 (22). This suggests that IL-1?-induced ?-cell signaling and death is dependent upon Ca2+ entry also under conditions where glucose-induced Ca2+ signaling is modest. We acknowledge the limitations in the use of pharmacological [Ca2+]i-modulating agents. On the other hand, KCl, A23187, and thapsigargin provided uniform results that were consistent with the physiological stimulus glucose, i.e. all these stimuli that lead to elevation in [Ca2+]i also augmented IL-1? activation of JNK. Congruently, Ca2+ chelator BAPTA reduced IL-1?-stimulated in vitro GST-c-jun phosphorylation. Taken together, the results obtained by the use of both glucose and nonphysiological [Ca2+]i modulators indicate that [Ca2+]i plays a modulatory role in IL-1? signal transduction in ?-cells. This may explain the extraordinary sensitivity of ?-cells to IL-1?-mediated toxicity compared with other islet cells (41, 42, 43). Although we did not address this issue experimentally, the subcellular localization and kinetics of the Ca2+ signal are of importance for how increases in [Ca2+]i induced by different physiological stimuli are translated into a specific cellular response, and therefore, it is likely that the spatio-temporal nature of ?-cell Ca2+ signaling plays an important role in Ca2+-dependent IL-1? signaling.
The requirement of Ca2+ entry through L-type VGCCs was not limited to IL-1? activation of the JNK pathway, because IL-1? stimulation of p38 and ERK MAPKs was also inhibited by nimodipine. Therefore, [Ca2+]i seems to be involved in IL-1? signaling leading to activation of all three MAPK signaling pathways. Consistent with this, we have recently found that IL-1?-induced ERK activation in human islets is dependent on L-type Ca2+ influx (22). Furthermore, it has previously been observed that Ca2+ influx is required for ERK activation in response to IL-1? in human fibroblasts (44). However, in that study, IL-1?-induced activation of p38 and JNK did not require Ca2+ influx. This probably reflects cell-specific requirements and regulatory mechanisms in IL-1? signal propagation to MAPKs.
In previous studies, the functional role of Ca2+ entry via L-type VGCCs in IL-1?-induced cytotoxicity was addressed. Thus, in both ob/ob mouse islets and human islets, IL-1?-induced apoptosis could be abrogated by L-type Ca2+ channel blockade by D600 or nimodipine, respectively (21, 22). Our data obtained with INS-1 cells showing that nimodipine affords a partial, but significant protection against the suppressive effect of IL-1? on cell viability further supports that Ca2+ plays a role in IL-1?-induced ?-cell apoptosis. Although an earlier study showed dependence on T-type Ca2+ influx for ?-cell apoptosis induced by a combination of cytokines (24), the present findings using mibefradil suggest that Ca2+ entry through T-type VGCCs does not contribute to IL-1? activation of JNK or IL-1?-mediated suppression of cell viability. Even if cells were incubated for 6 h with a mixture of cytokines previously shown to cause activation of T-type VGCCs, blockade of T-type channels had no effect on activation of JNK induced by IL-1? + IFN + TNF. This may suggest that the increase in influx of Ca2+ through T-type VGCCs observed after a 6-h exposure to a combination of cytokines (24) is an event distal to JNK/MAPK activation.
We examined the role of Ca2+ influx via VGCC in IL-1?-signaling via the NFB pathway. The data obtained demonstrated that neither T- or L-type channel blockade affected IL-1?-mediated degradation of IB. However, L-type blockade by nimodipine, but not T-type blockade by mibefradil caused a partial reduction of IL-1?-induced NFB-driven reporter gene activity. The discrepancy between the lack of effect of nimodipine on IB degradation and the inhibitory effect on NFB transactivation could be explained by the fact that nimodipine inhibited p38 and ERK. These MAPKs have previously been required for phosphorylation of NFB, a step that is obligatory for NFB-dependent transactivation (45, 46). ERK-dependent NFB transactivation in response to membrane depolarization was recently demonstrated in insulin-producing mouse MIN6 cells (47). However, despite a partial inhibitory effect of nimodipine on NFB transactivation, IL-1?-stimulated iNOS expression and iNOS promoter activity were not modulated by nimodipine. Therefore, we suggest that IL-1? signaling via the NFB pathway in insulin-secreting cells is mainly independent of Ca2+ influx.
What is the molecular mechanism(s) behind IL-1?-induced, Ca2+-dependent JNK/MAPK activation? By the use of KN62 and KN93, inhibitors of CaMKs, we found evidence for involvement of CaMKs in JNK activation. Indirect evidence for a role of CaMKs was also provided by the use of the calmodulin inhibitor W7. Previously, CaMK IV was shown to mediate Ca2+-dependent JNK activation in PC12 cells (35). Because expression of CaMK IV has been verified in ?-cells (48), CaMK IV may be the CaMK involved in mediating Ca2+-dependent JNK activation in insulin-secreting cells.
The findings obtained using the sarco(endo)plasmic reticulum Ca2+ ATPase blocker thapsigargin suggest that Ca2+ release from internal stores posses the capability to activate JNK and to augment IL-1? activation of JNK. Thapsigargin is a known activator of ER stress and the latter is known to activate the JNK signaling pathway (49, 50). We have found recently that ER stress induced by thapsigargin leads to a late and prolonged JNK activation in insulin-producing cells (51). However, the results of that study showed that cytokines induced an early and more transitory JNK phosphorylation that preceded the subsequent ER stress. Hence it is reasonable to suggest that under our experimental time frame (1 h) the effect of thapsigargin on IL-1?-stimulated JNK activity in INS-1 cells is probably related to an increase in [Ca2+]i. However, it cannot be excluded that modulations in [Ca2+]i by different stimuli eventually will have an impact on ER stress status by affecting ER Ca2+ homeostasis, e.g. Ca2+-induced Ca2+-release from the ER.
One interesting observation of this study is the absence of effect of GLP-1 on IL-1?-induced JNK activation. GLP-1 is known to activate the GLP-1 receptor with subsequent activation of adenylyl cyclase and elevation in cAMP giving rise to activation of protein kinase A and exchange protein activated by cAMP, which in turn leads to an increase in [Ca2+]i by mobilization of Ca2+ from intracellular stores and also from cAMP-dependent activation of VGCCs (52, 53, 54). The lack of a Ca2+-mediated potentiating effect of GLP-1 on IL-1?-induced JNK activation can be explained by parallel signaling mechanisms that dampen JNK activity. For example, in addition to activation cAMP-dependent pathways, GLP-1 is known to induce activation of PI3-kinase and Akt (55), which has been shown to suppress JNK activation (56, 57).
In conclusion, this study provides evidence for a permissive role of Ca2+ in the regulation of ?-cell IL-1?-induced proapoptotic JNK signaling. Even though the present findings were mainly obtained by a pharmacological approach and these findings should be confirmed by molecular approaches, e.g. transfection with Ca2+-binding proteins to lower [Ca2+]i and/or overexpression of kinase mutant CaMK, the results of the present study may help design future rational intervention strategies for the preservation of residual ?-cell mass in type 1 and 2 diabetes patients and to protect transplanted islets from immune-mediated damage.
Acknowledgments
We thank Ann-Sofie Hilles?, Anna Hlin Schram, and Hanne Foght for excellent technical assistance.
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Address all correspondence and requests for reprints to: Thomas Mandrup-Poulsen, M.D., D.M.Sc., Steno Diabetes Center, Niels Steensensvej 2, DK-2820 Gentofte, Denmark. E-mail: tmpo@steno.dk; or Joachim St?rling, M.Sc., Steno Diabetes Center, Niels Steensensvej 8, NSPP, DK-2820 Gentofte, Denmark. E-mail: jstq@steno.dk.
Abstract
The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1?-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic ?-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1? activation of JNK in ?-cells are largely unknown. In this study, we investigated whether Ca2+ plays a role for IL-1?-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mM glucose, combined pharmacological blockade of L- and T-type Ca2+ channels suppressed IL-1?-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1?-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1?-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca2+ channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse ?TC3 cells. Ca2+ influx, specifically via L-type but not T-type channels, contributed to IL-1? activation of JNK. Activation of p38 and ERK in response to IL-1? was also dependent on L-type Ca2+ influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca2+ ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase, all caused an amplification of IL-1?-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca2+ [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca2+/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1?-stimulated c-jun phosphorylation in INS-1 or ?TC3 cells. Our data suggest that Ca2+ plays a permissive role in IL-1? activation of the JNK signaling pathway in insulin-secreting cells.
Introduction
THE LOSS OF ?-cell mass in the pancreatic islets of Langerhans leading to insufficient insulin release and hyperglycemia characteristic of type 1 diabetes is believed to be an immune-mediated process in which proinflammatory cytokines, in particular IL-1?, play an essential role (1). Furthermore, recent findings have established a role for IL-1? in ?-cell failure in type 2 diabetes (2). Under in vitro conditions, exposure of insulin-secreting cells or pancreatic islets to IL-1? alone or in combination with interferon (IFN) and/or TNF leads to impairment of ?-cell function and induction of apoptotic cell death (1).
IL-1? activates the c-jun N-terminal kinase (JNK) pathway in ?-cells (3, 4, 5). JNK constitutes a stress-activated member of the MAPK family of threonine/serine kinases and is involved in the transmission of stress and apoptotic signaling in many cells. Activation of JNK is achieved by the dual phosphorylation on Thr183 and Tyr185 in the activation loop of the kinase by the dual specificity MAPK kinases MKK4 and MKK7, which are activated by phosphorylation by a MAPK kinase kinase (6, 7). JNK plays a critical role in cytokine-mediated apoptosis of ?-cells, as suggested by the finding that apoptosis induced by IL-1? in insulin-secreting cells can be prevented by inhibition of the JNK pathway (8, 9, 10). Furthermore, in primary ?-cells, oxidative stress-induced suppression of insulin gene transcription is mediated by JNK (11). Thus, strong evidence points toward JNK as a key regulator of ?-cell function and apoptosis. However, the regulatory events in the activation of JNK by IL-1? in insulin-secreting cells remain largely unknown. Insight into these mechanisms may identify targets for pharmacological intervention in diabetes and islet graft failure.
Pancreatic ?-cells are highly active in Ca2+ handling. Thus, ?-cell uptake and metabolism of glucose lead to closure of ATP-sensitive K+ channels, depolarization of the plasma membrane, and, subsequently, influx of Ca2+ through voltage-gated Ca2+ channels (VGCCs). The Ca2+ signal provided by this influx of Ca2+ is accompanied by release of Ca2+ from the endoplasmic reticulum (ER) (12, 13, 14, 15). The subsequent increase in the cytosolic free Ca2+ concentration ([Ca2+]i) is an important determinant for insulin granule exocytosis. Ca2+ entry in response to glucose in insulin-secreting cells also leads to activation of ERK MAPK (16, 17, 18), but not of JNK or p38 MAPK to any significant degree (5, 17). VGCCs also regulate ?-cell apoptosis. Hence, influx of Ca2+ via L-type VGCCs is required for induction of ?-cell apoptosis resulting from glucotoxicity (19) and in response to serum from type 1 diabetes patients (20). Furthermore, cytokine-mediated ?-cell death seems to be dependent on VGCC-mediated Ca2+ entry as suggested by the findings that blockers of L-type VGCCs can suppress ?-cell apoptosis induced by IL-1? (21, 22) or IFN plus TNF (23) and blockers of T-type VGCCs can repress ?-cell apoptosis induced by a mixture of cytokines (24).
The aim of the present study was to test the hypothesis that Ca2+ contributes to IL-1? activation of proapoptotic JNK in ?-cells. Our findings provide evidence for a permissive and potentiating role of Ca2+ in IL-1?-induced signaling via the JNK pathway in insulin-secreting cells.
Materials and Methods
Materials
Recombinant mouse or human IL-1? was obtained from BD PharMingen (San Diego, CA), Novo Nordisk (Bagsv?rd, Denmark), or Calbiochem (San Diego, CA). All reagents for SDS-PAGE were from Invitrogen (Carlsbad, CA). The JNK substrate glutathione S-transferase (GST)-c-jun (1–79) was from Calbiochem. Recombinant murine heat shock protein-25 (Hsp25) was from Stressgen (Victoria, Canada). GST-Elk-1 was a gift from Klaus Seedorf, then at Hagedorn Research Institute (Gentofte, Denmark). [-32P]ATP (3000 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). Antibodies to Thr183/Tyr185-phosphorylated JNK1/2, JNK1/2, Thr202/Tyr204-phosphorylated ERK1/2, ERK1/2, Thr180/Tyr182-phosphorylated p38, and p38 were obtained from Cell Signaling (Beverly, MA). Inhibitory B (IB) antibody was obtained from Acive Motif (Carlsbad, CA). Antibody to ?-tubulin was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and antibody to inducible nitric oxide synthase (iNOS) was from BD PharMingen. Mibefradil was kindly provided by Roche (Basel, Switzerland). Nimodipine, Ca2+ ionophore A23187, thapsigargin, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM), W7, KN62, and KN93 were obtained from Calbiochem. FPL64176was obtained from Sigma (St. Louis, MO). Luciferase reporter plasmids pNFB Luc and pRL-TK were from Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI), respectively. iNOS promoter construct piNOS-1002 Luc (25) was a gift from Decio Eizirik (Université Libre de Bruxelles, Brussels, Belgium). All other reagents were from Sigma or Merck (Darmstadt, Germany). Nimodipine was dissolved in polyethylene glycol or dimethylsulfoxide (DMSO). Mibefradil and KN93 were dissolved in water. FPL64176was dissolved in ethanol. All other inhibitors were dissolved in DMSO.
Cell culture
INS-1 or ?TC3 cells were maintained in RPMI 1640 medium (11 mM glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies, Inc., Grand Island, NY) in 80-cm2 tissue culture flasks (Nunc, Roskilde, Denmark). In addition, the media for INS-1 cells contained 50 μM ?-mercaptoethanol (Sigma). Cells were trypsinized and passaged weekly. For experimentation, cells were seeded in 12-well dishes (Nunc) (0.5 x 106 cells per well) or in 25-cm2 tissue culture flasks (Nunc) (2 x 106 cells per flask).
Islet isolation and culture
Pancreatic islets of Langerhans from 3- to 4-d-old Wistar Furth rats (Charles River, Sulzfeldt, Germany) were isolated and cultured as previously described (4). On the day of experimentation, 150 rat islets were placed in four-well dishes (Nunc) in RPMI 1640 medium (Life Technologies, Inc.) containing 11 mM glucose and supplemented with 0.5% human serum. Islets from adult ob/ob mice were obtained from a local noninbred colony. The islets were isolated by collagenase digestion technique and cultured overnight at 37 C in RPMI 1640 medium supplemented with 10% fetal calf serum (Flow Laboratories, Erwing, UK) and 11 mM glucose. Islets from ob/ob mice contain more than 90% ?-cells (26). All animal work was carried out according to national and international law and ethical standards.
Cell and islet stimulation and lysis
At the time of experimentation (1 or 2 d after seeding of cells), the culture medium was removed and fresh medium (11 mM glucose) with or without drugs was added. Cells or islets were incubated in the presence of drugs for 30 min to 1 h before exposure to IL-1?. When necessary, solvents (DMSO, polyethylene glycol, or ethanol) used for drugs were added to all conditions (0.1–1% final concentration). After IL-1? exposure, cells or islets were washed in cold PBS, and lysed for 30 min on ice in lysis buffer containing 20 mM Tris-acetate (pH 7.0), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% vol/vol Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1 mM benzamidine, 1 mM dithiothreitol, 1 mM Na3VO4, and 4 μg/ml leupeptin. Detergent-insoluble material was pelleted by centrifugation at 15,000 x g for 5 min at 2–4 C. The supernatants (whole-cell lysates) were stored at –80 C until assayed.
In vitro kinase assay
Phosphotransferase activities toward GST-c-jun, GST-Elk-1 and Hsp25 were measured by a whole-cell lysate in vitro kinase assay essentially as described in Ref. 4 except that 2 μg of GST-c-jun (1–79) was used instead of activating transcription factor 2. Phosphorylated substrates were visualized by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Immunoblotting
Whole cell lysate was mixed with 4x SDS sample buffer (Invitrogen), boiled for 10 min, and subjected to 10% SDS-PAGE. Proteins were electrotransferred to nitrocellulose filter membranes. Blocking of nonspecific protein binding was done by incubating the filter membrane in blocking buffer (1x TBS, pH 7.6; 0.1% Tween 20; 5% nonfat dry milk) for 1 h. After washing in TBST (1x TBS, pH 7.6; 0.1% Tween 20), filters were incubated with the respective antibodies diluted in either TBST containing 5% BSA or in blocking buffer. Filter membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Immune complexes were detected by chemiluminescence using LumiGLO (Cell Signaling), and light emission was captured on x-ray film or digitally using FUJI LAS3000.
Transient transfection and luciferase reporter assay
INS-1 cells (0.2 x 106 cells) in duplicates were transiently transfected with luciferase reporter plasmids [for nuclear factor-B (NFB) construct: 0.4 μg pNFB Luc and 0.15 μg pRL-TK; for iNOS promoter construct: 0.7 μg piNOS-1002 Luc and 0.05 μg pRL-TK] using Lipofectamine2000 (Invitrogen) as described by the manufacturer. The day after transfection, fresh medium with or without nimodipine was added to cells. After 30 min preincubation, cells were exposed for 6 h to IL-1?, washed in PBS and lysed in passive lysis buffer (Promega). Luciferase assays were carried out using the dual luciferase reporter system kit according to the specifications of the manufacturer (Promega). NFB and iNOS promoter activities were normalized to the coexpressed Renilla luciferase.
Measurements of intracellular calcium concentration ([Ca2+]i)
INS-1 cells were cultured on glass coverslips in plastic Petri dishes under the same conditions as those used for the evaluation of the effect of A23187 on IL-1?-induced JNK phosphorylation. Ca2+ fluorescent indicator fura 2/AM (2 μM) was added to the culture medium 1 h before the measurements of [Ca2+]i were performed. The coverslips with fura 2 loaded cells formed the bottom of an open chamber and in the course of [Ca2+]i. Measurements were perfused with the buffer containing 125 mM NaCl; 5.9 mM KCl; 1.2 mM MgCl2; 1.28 mM CaCl2; 25 mM HEPES; 11 mM glucose; and 0.1% BSA, pH 7.4, at 37 C. Measurements of [Ca2+]i were made as previously described (14). The fluorescence ratio at 340:380 nm was determined using a Spex Fluorolog spectrophotometer coupled to a Zeiss Axiovert 35 microscope with a Zeiss Fluar 40x/1.30 oil objective (Carl Zeiss, G?ttingen, Germany).
Methylthiazolyl Tetrazolium (MTT) viability analysis
Cell viability was assessed by the MTT assay (Promega) measuring mitochondrial activity by the conversion of a tetrazolium salt to a colored formazan product by the mitochondrial enzyme succinate dehydrogenase. Thus, it should be noted that the assay is a marker of mitochondrial activity and not per se cell viability. Briefly, cells in triplicates in 96-well plates were left untreated or treated with IL-1? in the presence or absence of inhibitors for 2 d. Twenty-five microliters of MTT dye reagent were added to each well followed by incubation at 37 C for 1–3 h. Then 90 μl per well of MTT Stop solution was added followed by overnight incubation. Absorbance was measured at 578 nm.
Statistical analysis
Statistical differences between groups were calculated by paired t test using Excel (Microsoft).
Results
IL-1? activates JNK in insulin-secreting INS-1 cells
We first characterized the dose-response relationship between IL-1? exposure and JNK activation in insulin-secreting rat INS-1 cells. Measuring in vitro kinase activity toward the specific JNK substrate GST-c-jun (amino acids 1–79) in whole-cell lysates prepared from INS-1 cells exposed to increasing concentrations of IL-1? for 1 h revealed that IL-1?, in a dose-dependent manner, induced an increase in the phosphotransferase activity toward GST-c-jun (Fig. 1A, upper panel). The effect of IL-1? on in vitro GST-c-jun phosphorylation correlated well with an increase in the amount of the endogenous, phosphorylated and active form of JNK as determined by immunoblot analysis using antibodies against Thr183/Tyr185-phosphorylated JNK1/2 (Fig. 1A, middle panel). Under these conditions, the level of total JNK1/2 protein was unchanged (Fig. 1A, lower panel). From this experiment, it was found that an IL-1? concentration of 80–160 pg/ml, giving roughly a half-maximal effect on JNK, would be appropriate for the remainder of the study. A time-course study of IL-1?-induced phosphorylation of JNK showed that JNK1/2 phosphorylation in response to IL-1? peaked at 1 h and persisted for at least 9 h (Fig. 1B).
FIG. 1. IL-1? activates JNK in INS-1 cells. A, INS-1 cells were exposed to the indicated concentrations of IL-1? for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP. Phosphorylated GST-c-jun was visualized by autoradiography. The activation state of JNK was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Results shown are representative of two experiments. B, INS-1 cells were exposed to IL-1? (160 pg/ml) for the indicated time periods. After lysis of cells, the activation state of JNK was examined by immunoblot analysis. Blots shown are representative of three experiments. KA, Kinase assay; IB, immunoblot.
Nimodipine plus mibefradil reduce IL-1?-induced JNK activation
To determine whether Ca2+ plays a role in JNK activation by IL-1?, we first examined whether combined blockade of L- and T-type plasma membrane VGCCs, the two types of Ca2+ channels that previously were shown to be implicated in cytokine-induced ?-cell apoptosis (21, 22, 23, 24), affects IL-1?-stimulated JNK activity in INS-1 cells. Nimodipine was used to block L-type channels and mibefradil to block T-type channels. The increase in kinase activity toward GST-c-jun as well as the phosphorylation of JNK1/2 induced by a 1-h exposure to IL-1? in standard culture medium containing 11 mM glucose were abolished when cells were coincubated with 10 μM nimodipine plus 1 μM mibefradil (Fig. 2A, upper panels). Quantitative analysis of GST-c-jun revealed that IL-1?-induced GST-c-jun phosphorylation was reduced from 3.1 ± 0.2-fold above baseline to 0.9 ± 0.1 (P < 0.005) by the presence of nimodipine and mibefradil. In similar experiments conducted on isolated intact rat or ob/ob mouse islets or on insulin-secreting mouse ?TC3 cells, the IL-1?-induced increase in in vitro phosphorylation of GST-c-jun was also significantly inhibited by combined Ca2+ channel blockade (Fig. 2A, lower panels). When each channel blocker was used separately on INS-1 cells, it was found that nimodipine was effective in inhibiting IL-1?-stimulated GST-c-jun phosphorylation and activation of JNK1/2, whereas mibefradil was ineffective (Fig. 2B). This suggests that Ca2+ entering preferentially through L-type channels contribute to IL-1? activation of JNK. IL-1? dose experiments further revealed that nimodipine suppressed IL-1?-induced JNK activity as measured by GST-c-jun phosphorylation (Fig. 2C). Because it was previously shown in mouse ?-cells that exposure for 6 or 24 h to a combination of cytokines (IL-1? plus IFN with or without TNF) specifically led to an increase in the activity of T-type Ca2+ channels (24), we stimulated ?TC3 cells with a combination of cytokines for 6 h in the presence or absence of T-type channel blockade and examined the cells for JNK activity. As seen in Fig. 2D, cytokine-exposed cells had activated JNK1/2, which was not affected by T-type Ca2+ channel blockade by mibefradil or NiCl2 (10 μM). Collectively, these results suggest that Ca2+ influx specifically via L-type VGCC plays a role in the activation of JNK by IL-1? in ?-cells.
FIG. 2. Calcium channel blockade reduces IL-1?-induced JNK activation. A, INS-1 cells, rat islets, ob/ob mouse islets, or ?TC3 cells were incubated with or without IL-1? (INS-1 and rat islets, 80 pg/ml; ?TC3 and ob/ob mouse islets, 2 ng/ml) in the presence or absence of 10 μM nimodipine (Nimo) plus 1 μM mibefradil (Mibe) for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP. Phosphorylated GST-c-jun was visualized by autoradiography. Autoradiograms shown are representative of four (INS-1), three (rat islets), or five (ob/ob and ?TC3) experiments. In addition, the activation state of JNK in INS-1 cells was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Blots shown are representative of two experiments. B, INS-1 cells were incubated with or without IL-1? (80 pg/ml) in the presence or absence of 10 μM nimodipine or 1 μM mibefradil for 1 h. After lysis of cells, whole-cell lysates were assayed as in A. Autoradiogram and blots shown are representative of two to four experiments. C, INS-1 cells were incubated with increasing concentrations of IL-1? (160–800 pg/ml) in the presence or absence of 10 μM nimodipine for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun. Autoradiogram shown is representative of four experiments. D, ?TC3 cells were incubated with or without IL-1? (63 pg/ml) + IFN (10 pg/ml) + TNF (5 pg/ml) (Cyt. mix) in the presence or absence of 10 μM NiCl2 or 1 μM mibefradil for 6 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun and the activation state of JNK was examined by immunoblot analysis. Autoradiogram and blots shown are representative of three experiments. KA, Kinase assay; IB, immunoblot.
Nimodipine inhibits IL-1?-induced activation of p38 and ERK
We next examined whether Ca2+ influx also regulates IL-1? activation of p38 and ERK MAPKs. As demonstrated by immunoblotting of the activated kinases as well as by in vitro kinase activity toward Hsp25, a substrate of the p38-activated kinase MAPK-activated protein kinase 2, and GST-Elk-1, a substrate of ERK in ?-cells (4), IL-1? activation of both p38 and ERK1/2 in INS-1 cells cultured at 11 mM glucose was inhibited by nimodipine (Fig. 3, A and B). These findings suggest that L-type Ca2+ entry also contributes to IL-1? activation of p38 and ERK MAPKs.
FIG. 3. The L-type channel blocker nimodipine reduces IL-1?-induced activation of p38 and ERK, but only modestly affects IL-1? signaling via NFB. A and B, INS-1 cells were incubated with or without IL-1? (A, 80 pg/ml; B, 0.8 ng/ml) in the presence or absence of 10 μM nimodipine for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward Hsp25 and GST-Elk-1 in the presence of [32P]ATP. Phosphorylated substrates were visualized by autoradiography. The activation states of p38 and ERK were examined by immunoblot analysis using antibodies to Thr180/Tyr182-phosphorylated p38, p38, and Thr202/Tyr204-phosphorylated ERK1/2. Immunoblot analysis of tubulin in B confirmed equal protein loading. Autoradiograms and blots shown are representative of two to three experiments. C, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 10 μM nimodipine for 30 min. After lysis of cells, the lysates were analyzed for content of IB by immunoblot analysis. Results shown are representative of three experiments. D and F, INS-1 cells were transfected with either NFB-driven or iNOS promoter luciferase reporter gene constructs. Cells were then stimulated with or without IL-1? (160 pg/ml) in the presence or absence of 10 μM nimodipine for 6 h. Luciferase activity was normalized to coexpressed Renilla luciferase. Data are expressed as means ± SEM of four experiments. E, INS-1 cells were treated with or without IL-1? in the presence or absence of 10 μM nimodipine for 6 h. Cell lysates were analyzed for content of iNOS by immunoblot analysis. Results are representative of two experiments. G, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 10 μM nimodipine for 2 d. Cell viability was determined by MTT analysis. Data are means ± SEM of five experiments. *, P < 0.005 vs. control; #, P < 0.02 vs. IL-1?. KA, Kinase assay; IB, immunoblot.
Nimodipine only modestly affects IL-1?-induced signaling via NFB
IL-1? is a potent activator of the transcription factor NFB in ?-cells (27, 28), and this transcription factor contributes to apoptosis in cytokine-exposed ?-cells (29, 30). To investigate whether IL-1? signaling via the NFB pathway also involves Ca2+, the effect of nimodipine on IL-1?-stimulated degradation of IB was determined. Figure 3C shows that stimulation of INS-1 cells with IL-1? led to a pronounced degradation of IB within 30 min. This degradation of IB was not affected by coincubation with nimodipine. Because other factors down-stream to the degradation of IB are involved in the transcriptional capacity of NFB, we investigated by luciferase reporter gene assay the effect of nimodipine on IL-1?-stimulated NFB transactivation in INS-1 cells. Luciferase activity in cells transiently transfected with a NFB-driven luciferase reporter plasmid was decreased from 1.79 ± 0.08-fold to 1.52 ± 0.03 (P < 0.005) corresponding to a 34% inhibition by nimodipine (Fig. 3D). To see whether this partial inhibition of NFB transactivation affects the expression of a gene whose transcription requires NFB, we tested the effect of L-type Ca2+ channel blockade on IL-1?-mediated expression of iNOS, an enzyme whose expression depends on NFB (25, 27, 28, 31). Figure 3E shows that a 6-h exposure to IL-1? induced the expression of iNOS, which was not affected by nimodipine. INS-1 cells were then transiently transfected with an iNOS promoter luciferase reporter gene construct to see whether Ca2+ channel blockade affects IL-1?-induced iNOS promoter activity. This experiment revealed that the IL-1?-stimulated induction of iNOS promoter activity was not modulated by nimodipine (Fig. 3F). In accordance with these observations, analysis of accumulated nitrite in the culture medium after a 24-h exposure to IL-1?, showed no effect of nimodipine on IL-1?-induced NO production (13.2 vs. 12.1 μM for IL-1? alone and IL-1? + nimo, respectively, means of n = 2). Together, these findings indicate that opposed to MAPKs, IL-1? signaling via NFB is not regulated by L-type Ca2+ influx. Consistent with a role of L-type Ca2+ influx for IL-1? signaling via proapoptotic JNK/MAPKs, nimodipine afforded a significant partial protection against the suppressive effect of IL-1? on INS-1 cell viability as determined by MTT analysis measuring mitochondrial activity (Fig. 3G), whereas 10 μM nimodipine alone did not affect INS-1 cell mitochondrial activity (104 ± 3.5% of control, n = 5). To exclude that T-type VGCCs might be involved in IL-1? signaling to NFB, the effect of mibefradil on NFB activation was also determined. However, mibefradil did not affect IL-1?-mediated IB degradation (Fig. 4A) and had no effect on IL-1?-induced NFB and iNOS promoter activities in reporter gene assays (Fig. 4, C and D). Furthermore, mibefradil neither affected INS-1 cell viability alone (101 ± 3.8% of control, n = 3) nor protected against IL-1?-mediated suppression of cell viability (Fig. 4B).
FIG. 4. The T-type channel blocker mibefradil does not affect IL-1?-induced IB degradation or toxicity. A, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 1 μM mibefradil for 30 min. After lysis of cells, the lysates were analyzed for content of IB by immunoblot analysis. Results shown are representative of three experiments. B, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of 1 μM mibefradil for 2 d. Cell viability was determined by MTT analysis. Data are means ± SEM of three experiments. C and D, INS-1 cells were transfected with either NFB-driven or iNOS promoter luciferase reporter gene constructs. Cells were then stimulated with or without IL-1? (160 pg/ml) in the presence or absence of 1 μM mibefradil (mibe) for 6 h. Luciferase activity was normalized to coexpressed Renilla luciferase. Data are expressed as means ± SEM of three experiments.
Agents that increase [Ca2+]i augment IL-1?-induced JNK activation
Because blocking Ca2+ entry via VGCCs inhibited IL-1?-induced JNK activity, the hypothesis that an increase in Ca2+ influx may lead to higher JNK activity in IL-1?-exposed cells was tested. To increase Ca2+ entry via L-type channels, we used a membrane depolarizing stimulus (KCl) in the presence of the L-type channel agonist FPL64176 As a positive control for L-type Ca2+ influx during this treatment, we first measured the effect on ERK, which is known to be activated by Ca2+ entry through L-type channels in response to membrane depolarization in insulin-secreting cells. Figure 5A shows that exposure of INS-1 cells to 50 mM KCl plus FPL64176(1 μM) stimulated ERK activation, which was abolished by nimodipine. In contrast to that observed for ERK, KCl plus FPL64176in itself did not stimulate activation of JNK1/2 to any significant degree, whereas IL-1?-induced JNK1/2 activation was augmented by KCl and FPL64176(Fig. 5B). We then examined whether glucose would also modulate JNK activation. As seen in Fig. 5C, IL-1? activation of JNK1/2 was higher in the presence of 27 mM glucose compared with 5 mM glucose. Nimodipine decreased the potentiating effect of glucose on JNK verifying that the effect of glucose involved L-type Ca2+ influx. To address whether another physiological Ca2+-mobilizing stimulus would affect IL-1?-induced JNK, we exposed cells to IL-1? for 1 h in the presence of increasing concentrations of glucagon-like peptide (GLP)-1, which is known to promote Ca2+ signaling (32, 33, 34). However, in contrast to glucose, GLP-1 failed to modulate IL-1?-induced phosphorylation of JNK1/2 (Fig. 5D), suggesting that not all physiological stimuli that induce Ca2+ signaling have the capability to augment IL-1?-induced JNK activation. To more directly examine the effect of [Ca2+]i on IL-1? activation of JNK, the effect of Ca2+ ionophore A23187 was determined. Alone, A23187 (1 μM) did not affect basal JNK1/2 phosphorylation, but caused a dose-dependent increase in IL-1?-induced JNK1/2 activation as well as IL-1?-stimulated in vitro GST-c-jun phosphorylation (Fig. 6A) To determine the correlation between A23187-induced increases in [Ca2+]i and IL-1?-induced JNK activation, we measured [Ca2+]i after treatment with IL-1? in the presence or absence of A23187. Although exposure to IL-1? alone for 1 h did not cause any significant change in [Ca2+]i compared with nontreated cells (157.7 ± 12.2 vs. 198.5 ± 17.9 nM, n = 47–59), A23187 dose-dependently caused a rise in [Ca2+]i in the presence of IL-1? (Fig. 6B). Therefore, the A23187-induced increases in [Ca2+]i directly correlated with the augmenting effect of A23187 on IL-1?-induced JNK phosphorylation. Finally, because entry of Ca2+ via VGCCs into ?-cells is accompanied by Ca2+ release from the ER (14), we next looked at whether Ca2+ release from the ER modulates JNK activation. Thapsigargin (1 μM), an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase leading to impairment of ER Ca2+ uptake and thus an increase in [Ca2+]i, in itself stimulated kinase activity toward GST-c-jun (Fig. 6C, upper panel). However, the combination of IL-1? and thapsigargin resulted in much higher GST-c-jun phosphorylation as compared with each treatment alone. The increase in phosphorylation of GST-c-jun was quantified to be 313% (P < 0.005) above the level induced by IL-1? alone. In line with this, thapsigargin augmented IL-1?-induced phosphorylation of JNK1/2 (Fig. 6C, lower panels). Taken together, these observations indicate that most stimuli that increase [Ca2+]i cause higher JNK activation in response to IL-1?.
FIG. 5. KCl and glucose, but not GLP-1, potentiate IL-1?-induced JNK activation. A, INS-1 cells were incubated with or without KCl (50 mM) plus FPL64176(1 μM) in the presence or absence of 10 μM nimodipine for 1 h. After lysis of cells, the activation state of ERK in whole-cell lysates was examined by immunoblot analysis using antibodies to Thr202/Tyr204-phosphorylated ERK1/2 and ERK1/2. B, INS-1 cells were incubated with or without KCl plus FPL64176in the presence or absence of IL-1? (160 pg/ml) for 1 h. The activation state of JNK in whole-cell lysates was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Blots shown are representative of six experiments. C, INS-1 cells were incubated in the presence of 5 mM glucose overnight. Medium was then changed to new medium containing either 5 or 27 mM glucose in the presence or absence of IL-1? with or without 10 μM nimodipine. After a 1-h stimulation, the activation state of JNK in whole-cell lysates was examined by immunoblot analysis. Blots shown are representative of three experiments. D, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of increasing concentrations (1, 5, 10, 100 nM) of GLP-1. After a 1-h stimulation, the activation state of JNK in whole-cell lysates was examined by immunoblot analysis. Blots shown are representative of three experiments. IB, Immunoblot.
FIG. 6. Ca2+ ionophore and thapsigargin potentiate IL-1?-induced JNK activation. A, INS-1 cells were incubated with or without IL-1? (160 pg/ml) in the presence or absence of increasing concentrations (0.1, 0.25, 0.5, 1 μM) of Ca2+ ionophore A23187 for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP, and the activation state of JNK was examined by immunoblot analysis. Autoradiogram and blots shown are representative of three experiments. B, INS-1 cells were incubated with IL-1? (160 pg/ml) in the presence or absence of increasing concentrations (0.25, 0.5, 1 μM) of A23187 for 1 h. [Ca2+]i was measured by fura 2 method. Data are expressed as means ± SEM of n = 42–59 for each condition. C, INS-1 cells were incubated with IL-1? (160 pg/ml) in the presence or absence of thapsigargin (Tg) (1 μM) for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP and the activation state of JNK was examined by immunoblot analysis. Autoradiogram and blots shown are representative of four experiments. *, P < 0.01 vs. IL-1? alone. KA, Kinase assay; IB, immunoblot.
BAPTA-AM, W7, and KN62 or KN93 reduce IL-1?-induced JNK activation
We next applied various inhibitors of Ca2+ and Ca2+-activated proteins to assess the involvement of these in IL-1? activation of JNK in INS-1 cells. As shown in Fig. 7A, BAPTA-AM (100 nM) reduced IL-1?-stimulated in vitro GST-c-jun phosphorylation. Consistent with this, both W7 (5 and 25 μM), a calmodulin antagonist, and KN62 (10 μM), an inhibitor of Ca2+/calmodulin-dependent kinases (CaMKs), diminished IL-1?-stimulated GST-c-jun phosphorylation (Fig. 7, B and C). Quantitative analysis revealed that BAPTA-AM, W7, and KN62 decreased IL-1?-stimulated GST-c-jun phosphorylation by 32% (P = 0.01), 33% (P < 0.05), and 39% (P < 0.02), respectively. In ?TC3 cells, another CaMK inhibitor (KN93) in a dose-dependent manner decreased IL-1?-stimuled phosphorylation of GST-c-jun, which correlated with reduced IL-1?-induced phosphorylation of JNK1/2 (Fig. 7D).
FIG. 7. BAPTA-AM, W7, KN62, and KN93 reduce IL-1?-induced JNK activation. INS-1 cells were incubated with or without IL-1? (80 or 160 pg/ml) in the presence or absence of BAPTA-AM (100 nM) (A), W7 (5 or 25 μM) (B), or KN62 (10 μM) (C) for 1 h. After lysis of cells, whole-cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP. Phosphorylated GST-c-jun was visualized by autoradiography. Autoradiograms shown are representative of three to four experiments. D, ?TC3 cells were incubated with or without IL-1? (320 pg/ml) in the presence or absence of the indicated concentrations of KN93 for 1 h. Whole cell lysates were assayed for in vitro kinase activity toward GST-c-jun in the presence of [32P]ATP, and the activation state of JNK was examined by immunoblot analysis using antibodies to Thr183/Tyr185-phosphorylated JNK1/2 and JNK1/2. Autoradiograms and blots shown are representative of four and two experiments, respectively. KA, Kinase assay; IB, immunoblot.
Discussion
IL-1? may be involved in the pathogenesis of type 1 and 2 diabetes by causing cellular dysfunction and apoptosis of pancreatic ?-cells. IL-1? activates JNK in ?-cells and signaling via this pathway is critically involved in mediating IL-1?-induced apoptosis in insulin-secreting cells (8, 9, 10). However, the cellular regulatory mechanisms contributing to IL-1?-induced JNK activation in ?-cells are not well understood.
In this study, we investigated the role of Ca2+ in IL-1? activation of JNK. By using Ca2+ channel blockers, agents that increase or decrease [Ca2+]i and inhibitors of Ca2+-activated proteins, we provide evidence to suggest a role for Ca2+ in controlling IL-1? activation of JNK in insulin-secreting cells and rodent islets. The molecular mechanism may involve activation of CaMKs by increases in [Ca2+]i. In analogy, CAMK IV mediates Ca2+-dependent JNK activation in PC12 cells (35).
Although our data indicate a role for Ca2+ entry via L-type VGCCs in regulating IL-1? ?-cell JNK signaling, previous studies in ?-cells showed no apparent alterations in VGCC activity or steady-state [Ca2+]i after exposure to IL-1? (21, 24, 36, 37, 38), except in one study, where a 2-h exposure to IL-1? stimulated a cellular net-uptake of Ca2+ via L-type channels in rat islets (39). The results of our study also do not show an increase in [Ca2+]i after a 1-h exposure to IL-1?. This favors the idea that Ca2+ has an important but strictly permissive role in IL-1?-induced JNK activation.
In the presence of in vitro mid-range (typically 11 mM) concentrations of glucose, glucose-induced Ca2+ influx is critical for the stimulus-secretion coupling. However, if the ?-cell is under concurrent challenge with IL-1?, an increased [Ca2+]i produced by glucose may augment the capability of IL-1? to evoke intracellular signaling and, thereby, the capability of IL-1? to signal ?-cell destruction. Thus, although IL-1? does not induce detectable net increases in [Ca2+]i, Ca2+ plays a role in IL-1? signal transduction in that glucose-stimulated Ca2+ signaling indeed exerts a permissive and potentiating role to propagate IL-1? signaling. This concept is supported by the observations that the cytotoxic effects of cytokines are more pronounced in high vs. low glucose (40) and that activation of Ca2+ influx by membrane depolarization by KCl was insufficient to induce JNK activity in the absence of IL-1?. We have recently found that even in the presence of a low, but physiologically relevant glucose concentration (5.5 mM), IL-1?-induced human islet ?-cell apoptosis is dependent on Ca2+ influx as determined by the use of nimodipine or the K+ channel openers diazoxide and NN414 (22). This suggests that IL-1?-induced ?-cell signaling and death is dependent upon Ca2+ entry also under conditions where glucose-induced Ca2+ signaling is modest. We acknowledge the limitations in the use of pharmacological [Ca2+]i-modulating agents. On the other hand, KCl, A23187, and thapsigargin provided uniform results that were consistent with the physiological stimulus glucose, i.e. all these stimuli that lead to elevation in [Ca2+]i also augmented IL-1? activation of JNK. Congruently, Ca2+ chelator BAPTA reduced IL-1?-stimulated in vitro GST-c-jun phosphorylation. Taken together, the results obtained by the use of both glucose and nonphysiological [Ca2+]i modulators indicate that [Ca2+]i plays a modulatory role in IL-1? signal transduction in ?-cells. This may explain the extraordinary sensitivity of ?-cells to IL-1?-mediated toxicity compared with other islet cells (41, 42, 43). Although we did not address this issue experimentally, the subcellular localization and kinetics of the Ca2+ signal are of importance for how increases in [Ca2+]i induced by different physiological stimuli are translated into a specific cellular response, and therefore, it is likely that the spatio-temporal nature of ?-cell Ca2+ signaling plays an important role in Ca2+-dependent IL-1? signaling.
The requirement of Ca2+ entry through L-type VGCCs was not limited to IL-1? activation of the JNK pathway, because IL-1? stimulation of p38 and ERK MAPKs was also inhibited by nimodipine. Therefore, [Ca2+]i seems to be involved in IL-1? signaling leading to activation of all three MAPK signaling pathways. Consistent with this, we have recently found that IL-1?-induced ERK activation in human islets is dependent on L-type Ca2+ influx (22). Furthermore, it has previously been observed that Ca2+ influx is required for ERK activation in response to IL-1? in human fibroblasts (44). However, in that study, IL-1?-induced activation of p38 and JNK did not require Ca2+ influx. This probably reflects cell-specific requirements and regulatory mechanisms in IL-1? signal propagation to MAPKs.
In previous studies, the functional role of Ca2+ entry via L-type VGCCs in IL-1?-induced cytotoxicity was addressed. Thus, in both ob/ob mouse islets and human islets, IL-1?-induced apoptosis could be abrogated by L-type Ca2+ channel blockade by D600 or nimodipine, respectively (21, 22). Our data obtained with INS-1 cells showing that nimodipine affords a partial, but significant protection against the suppressive effect of IL-1? on cell viability further supports that Ca2+ plays a role in IL-1?-induced ?-cell apoptosis. Although an earlier study showed dependence on T-type Ca2+ influx for ?-cell apoptosis induced by a combination of cytokines (24), the present findings using mibefradil suggest that Ca2+ entry through T-type VGCCs does not contribute to IL-1? activation of JNK or IL-1?-mediated suppression of cell viability. Even if cells were incubated for 6 h with a mixture of cytokines previously shown to cause activation of T-type VGCCs, blockade of T-type channels had no effect on activation of JNK induced by IL-1? + IFN + TNF. This may suggest that the increase in influx of Ca2+ through T-type VGCCs observed after a 6-h exposure to a combination of cytokines (24) is an event distal to JNK/MAPK activation.
We examined the role of Ca2+ influx via VGCC in IL-1?-signaling via the NFB pathway. The data obtained demonstrated that neither T- or L-type channel blockade affected IL-1?-mediated degradation of IB. However, L-type blockade by nimodipine, but not T-type blockade by mibefradil caused a partial reduction of IL-1?-induced NFB-driven reporter gene activity. The discrepancy between the lack of effect of nimodipine on IB degradation and the inhibitory effect on NFB transactivation could be explained by the fact that nimodipine inhibited p38 and ERK. These MAPKs have previously been required for phosphorylation of NFB, a step that is obligatory for NFB-dependent transactivation (45, 46). ERK-dependent NFB transactivation in response to membrane depolarization was recently demonstrated in insulin-producing mouse MIN6 cells (47). However, despite a partial inhibitory effect of nimodipine on NFB transactivation, IL-1?-stimulated iNOS expression and iNOS promoter activity were not modulated by nimodipine. Therefore, we suggest that IL-1? signaling via the NFB pathway in insulin-secreting cells is mainly independent of Ca2+ influx.
What is the molecular mechanism(s) behind IL-1?-induced, Ca2+-dependent JNK/MAPK activation? By the use of KN62 and KN93, inhibitors of CaMKs, we found evidence for involvement of CaMKs in JNK activation. Indirect evidence for a role of CaMKs was also provided by the use of the calmodulin inhibitor W7. Previously, CaMK IV was shown to mediate Ca2+-dependent JNK activation in PC12 cells (35). Because expression of CaMK IV has been verified in ?-cells (48), CaMK IV may be the CaMK involved in mediating Ca2+-dependent JNK activation in insulin-secreting cells.
The findings obtained using the sarco(endo)plasmic reticulum Ca2+ ATPase blocker thapsigargin suggest that Ca2+ release from internal stores posses the capability to activate JNK and to augment IL-1? activation of JNK. Thapsigargin is a known activator of ER stress and the latter is known to activate the JNK signaling pathway (49, 50). We have found recently that ER stress induced by thapsigargin leads to a late and prolonged JNK activation in insulin-producing cells (51). However, the results of that study showed that cytokines induced an early and more transitory JNK phosphorylation that preceded the subsequent ER stress. Hence it is reasonable to suggest that under our experimental time frame (1 h) the effect of thapsigargin on IL-1?-stimulated JNK activity in INS-1 cells is probably related to an increase in [Ca2+]i. However, it cannot be excluded that modulations in [Ca2+]i by different stimuli eventually will have an impact on ER stress status by affecting ER Ca2+ homeostasis, e.g. Ca2+-induced Ca2+-release from the ER.
One interesting observation of this study is the absence of effect of GLP-1 on IL-1?-induced JNK activation. GLP-1 is known to activate the GLP-1 receptor with subsequent activation of adenylyl cyclase and elevation in cAMP giving rise to activation of protein kinase A and exchange protein activated by cAMP, which in turn leads to an increase in [Ca2+]i by mobilization of Ca2+ from intracellular stores and also from cAMP-dependent activation of VGCCs (52, 53, 54). The lack of a Ca2+-mediated potentiating effect of GLP-1 on IL-1?-induced JNK activation can be explained by parallel signaling mechanisms that dampen JNK activity. For example, in addition to activation cAMP-dependent pathways, GLP-1 is known to induce activation of PI3-kinase and Akt (55), which has been shown to suppress JNK activation (56, 57).
In conclusion, this study provides evidence for a permissive role of Ca2+ in the regulation of ?-cell IL-1?-induced proapoptotic JNK signaling. Even though the present findings were mainly obtained by a pharmacological approach and these findings should be confirmed by molecular approaches, e.g. transfection with Ca2+-binding proteins to lower [Ca2+]i and/or overexpression of kinase mutant CaMK, the results of the present study may help design future rational intervention strategies for the preservation of residual ?-cell mass in type 1 and 2 diabetes patients and to protect transplanted islets from immune-mediated damage.
Acknowledgments
We thank Ann-Sofie Hilles?, Anna Hlin Schram, and Hanne Foght for excellent technical assistance.
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