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Reactive Oxygen Species-Mediated Pancreatic β-Cell Death Is Regulated by Interactions between Stress-Activated Protein Kinases, p38 and c-Jun N-Terminal Kinase, and Mitogen-Activated Protein Kinase Phosphatases

Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan

Address all correspondence and requests for reprints to: Toshiyuki Takeuchi, Secretion Biology Lab, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan. E-mail: tstake{at}showa.gunma-u.ac.jp.

	Abstract

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Pancreatic β-cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose (LG), hypoxic, or cytokine-producing conditions. When we cultured mouse β-cell-derived MIN6 cells in a LG condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the LG culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and manganese(III)tetrakis(4-benzoic acid) porphyrin. We next investigated the mechanism of stress-activated protein kinases (SAPKs), c-jun N-terminal kinase (JNK) and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the LG culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death, whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased β-cell death, whereas JNK inhibitors decreased it. We then examined the role of MAPK phosphatases (MKPs) specific for stress-activated protein kinases in β-cell death. We found that MKP-1 presented an increase in its oxidized product after the LG culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to β-cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the LG culture, and decreased MIN6 β-cell death. We suggest that β-cell death is regulated by interactions between JNK and its specific MKPs.

	Introduction

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PANCREATIC β-CELLS are reportedly vulnerable to oxidative stress, which may induce β-cell apoptosis and a decrease in β-cell mass (1). This decrease results in the dysfunction of insulin secretion, and leads to the onset of type 1 and 2 diabetes (2, 3, 4). The susceptibility of β-cells to oxidative stress may be related to their possessing relatively low levels of antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD), Mn SOD, catalase, and glutathione peroxide dismutase (1). Furthermore, β-cells are reported to lack hyperglycemia/hypoxia-inducible mitochondrial protein, which is important for cell survival against oxidative stress-induced apoptosis (5). However, there is evidence from transgene-expressing nonobese diabetic mice studies that antioxidant enzymes, such as metallothionein and catalase, accelerate the progress of diabetes (6). Whether oxidative stress is prodiabetic or antidiabetic, it does result from increased levels of reactive oxygen species (ROS), which include free radicals such as superoxide and hydroxyl radical, and nonradical species such as hydrogen peroxide (7). These radicals are generated when β-cells are under conditions of low glucose (LG) (8), high glucose (HG) (9, 10), or hypoxia (11, 12).

Oxidative stresses trigger cellular responses by activating several protein phosphorylation pathways, including the MAPK pathways, namely, the stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK) and p38 MAPK (13, 14, 15). When isolated rat islets were exposed to oxidative stress agents such as hydrogen peroxide, the JNK, p38, and protein kinase C pathways were activated preceding a decrease in insulin gene expression (16). Research into the molecular mechanisms of oxidative stress-mediated JNK activation has focused on redox-sensitive proteins such as thioredoxin and glutaredoxin (17). The reduced form of thioredoxin reportedly binds to one of the MAPK kinase kinases, an apoptosis signal-regulating kinase 1 (ASK1), and blocks its kinase activity (18). Conversely, ROS oxidizes thioredoxin to dissociate from ASK1 for its activation, resulting in the JNK-mediated apoptosis. Alternatively, thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated JNK activation and apoptosis (19). Recently, it was proposed that ROS promote TNF-induced death and sustain JNK activation by inhibiting MAPK phosphatases (MKPs) in IB kinase β-deficient mouse fibroblast cells (20). Like JNK, p38 is reportedly activated by oxidative stresses via ASK1 activation (15), and changes the β-cell survival-promoting effect of glucose-dependent insulinotropic polypeptide (21). However, it is unclear whether ROS-induced β-cell death is mediated by JNK and p38 additively, simultaneously, or successively.

Phosphorylation of both Thr and Tyr is required for JNK or p38 kinase activation, but inactivation of the kinase activity is sufficient by dephosphorylation of either residue by dual specific MKP family phosphatases (22) or by serine/threonine-specific phosphatases such as Wip1 (23). The dual specificity MKP family includes 10 phosphatases (22), among which MKP-1 was initially discovered as a stress-responsive protein phosphatase that dephosphorylates ERK MAPK after oxidative stress (24). Subsequent studies revealed that MKP-1 preferentially dephosphorylates activated JNK and p38 relative to ERK (25). Previous studies have shown that JNK and p38 are highly activated in MKP-1-deficient mouse embryonic fibroblasts (26), and in the skeletal muscle of mkp^–/– mice (27). In general, sustained activation of SAPKs appears to be regulated by a balance between the activation of the upstream kinases and the protein phosphatases, including MKPs (22, 28).

We previously identified at least five MKPs [MKP-1, MKP-2, MKP-3, MKP-5, and vaccinia H1-related phosphatase (VHR)] in mouse pancreatic β-cell-derived MIN6 cells and found that MKP-1 is inducible by parathyroid hormone-related protein via a cAMP pathway (29). Because MKP-1 and the two SAPKs, JNK and p38, are known to be oxidative stress-activated proteins and play a role in cell survival and apoptosis (30, 31), we investigated the interaction between MKP-1 and SAPKs for β-cell survival and death during oxidative stress. We also investigated whether MKP-1 causes dephosphorylation of JNK and p38 simultaneously or successively. Most studies of oxidative stress exposed β-cells to H₂O₂, but the actual involvement of endogenous H₂O₂ and ROS in β-cell death has not been fully investigated (32, 33, 34, 35). Several previous studies have reported increased production of H₂O₂ from HG-exposed β-cells, whereas Rösen et al. (7) and Martens et al. (8) reported high H₂O₂ production from LG-exposed β-cells. We compared H₂O₂ production between HG and LG-exposed β-cells and found that high levels of H₂O₂ are produced from β-cells in the low-glucose culture. Furthermore, a relatively HG concentration of over 10 mM is essential for β-cell survival, whereas physiological glucose concentrations of less than or equal to 5.5 mM are cytocidal (36, 37, 38). In the present study, we investigated the effect of oxidative stress induced by a LG culture on β-cell survival and death based on the interaction between two SAPKs, p38 and JNK, and one MKP, MKP-1, using mouse β-cell-derived MIN6 cells.

	Materials and Methods

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Cell culture and LG culture
MIN6 cells derived from mouse pancreatic β-cells were cultured in DMEM (Invitrogen Corp., Carlsbad, CA) containing 25 mmol/liter glucose with 15% fetal bovine serum (FBS) and 100 µM of β-mercaptoethanol (β-ME) at 37 C in a humidified atmosphere of 95% O₂:5% CO₂ with medium changes every 2 d. MIN6 cells before passage 25 were used because later-passage β-cells are known to lose certain functions and have a decreased insulin content (39). For the LG culture, MIN6 cells were passaged 2 d before measurement and were changed to a serum-free DMEM culture containing 2 mM glucose for 8–48 h. Rat insulinoma INS-1 cells were cultured in RPMI 1640 (Invitrogen) containing 10% FBS, 100 U/ml sodium pyruvate, 10 mM HEPES (pH 7.4), and 100 µM of β-ME with 11 mM glucose (40). Experiments were done under culture conditions of serum-free RPMI 1640 containing 16, 11, 2.8, and 2 mM glucose separately for 24 h.

Construction of recombinant adenoviruses
Full-length cDNAs of p38 MAPK and MKP-7 were generated by PCR using the MIN6 cDNA library as a template. The pEF-Flag-Wip1 was kindly provided by Dr. Ogata (Mie University, Tsu, Japan). p38 cDNA with a nonphosphorylatable mutated site, Ala-Gly-Phe from Thr-Gly-Tyr (p38-AGF), was generated by PCR using a pair of primers: 5'-GAGATGGCCGGCTTCGTGGCTACCAGGTGG-3' and 5'-AGCCACGAAGCCGGCCATCTCATCATCAGT-3'. All the subcloned PCR products were directly sequenced. Each cDNA with Flag-tag was transferred to the cassette cosmid pAdex vector (pAxCAwt). Adenoviruses were propagated in HEK 293 cells and purified by CsCl density gradient centrifugation. The virus titer was expressed as the multiplicity of infection (MOI). Recombinant adenoviruses of MKP1-Flag, JNK-Flag, and JNK-Ala-Pro-Phe (APF)-Flag were described and characterized previously (29). Overexpression of MKP3 was performed by a double-adenoviral infection method described previously (41). Briefly, two kinds of adenoviruses were generated: one with a regulatory vector expressing Cre recombinase, and the other consisting of MKP3 or β-galactosidase (LacZ) interrupted by loxP. We performed recombinant adenovirus infection by MOI 520 to obtain a similar expression level for each expression unit. We monitored adenoviral expression of each protein by immunofluorescence analysis (results can be found in supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) and confirmed most of the cells infected.

View larger version (20K): [in this window] [in a new window]   FIG. 1. LG culture induces hydrogen peroxide production in MIN6 cells. Aa and Ab, Mouse β-cell-derived MIN6 cells were incubated in DMEM containing 25 mM glucose (Aa, HG) or 2 mM glucose (Ab, LG) for 24 h. Ac and Ad, MIN6 cells were exposed to 5 or 20% O2 for 24 h (Ac, normoxia; Ad, hypoxia). Ae–Ag, MIN6 cells were treated with none (Ae), 10 nM TNF (Af), or 300 pg/ml IL-1β (Ag) for 12 h. The cells were incubated in the dark with 5 µM CM-H2DCFDA in PBS for 5 min, and generated a DCF fluorescent signal visualized by microscopy with constant fluorescent parameters. B, MIN6 cells were cultured in DMEM containing 2 mM glucose for an indicated time. Intracellular ROS generation was monitored by fluorescence of CM-H2DCFDA as in A. C, MIN6 cells were cultured in LG or HG for an indicated time. Intracellular H2O2 was determined for each cell extract, as described in Materials and Methods. Results are the mean ± SEM of three independent experiments. All experiments were repeated several times with reproducible results. Bar, 20 µm.

Measurement of intracellular ROS and H₂O₂
Intracellular ROS generation was assessed using 5-(and-6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) (Invitrogen). MIN6 cells were washed and then incubated in the dark with 5 µM CM-H₂DCFDA for 5 min. The fluorescence of dichlorofluorescein (DCF) was detected with an epifluorescence microscope (BX-50) at an excitation wavelength of 488 nM. To avoid photooxidation of the indicator dye, the fluorescence images were collected by a single rapid scan with identical parameters, such as contrast and brightness, for all samples. The concentration of cellular H₂O₂ was determined using a quantitative H₂O₂ assay kit (Sigma-Aldrich). Cells were lysed by sonication in a phosphate buffer [50 mM (pH 6.0)] and then incubated at room temperature with a xylenol orange solution. Absorbance was measured at 550 nm using a MTP-500F microplate reader (Corona Electric, Hitachinaka-shi, Japan).

Assessment of cell viability
Cell viability was determined by Trypan blue dye-exclusion assay. MIN6 or INS-1 cells were collected at an indicated time and incubated with 0.4% Trypan blue, observed under a microscope, and the stained and unstained cells were then counted on a hemocytometer separately. Cell viability was calculated according to the following formula: cell viability (%) = (unstained cells number/total cells number) x 100. Cell death was calculated by the following formula: cell death (%) = (stained cells number/total cells number) x 100.

Western blot analysis
MIN6 cells were harvested from culture dishes, and were lysed in a lysis buffer [50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% Nonidet P-40 (Sigma-Aldrich), 100 mM NaF, 0.2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin]. Cell lysates (15 µg protein/lane) were separated on a SDS-PAGE for nitrocellulose membrane blotting. The blotted membranes were blocked with 5% skim milk for 30 min, and were incubated with primary antibodies (1:1000 dilution for ERK1/2, phospho-ERK1/2, p38, phospho-p38, JNK, and phospho-JNK; 1:10000 dilution for FLAG; 1:1000 dilution for MKP3; 1:3000 dilution for MKP7; 1:1000 dilution for MKP1; 1:1000 dilution for phospho-MKK4, phospho-ATF-2, and phospho-c-Jun; and 1:5000 dilution for -tubulin). The antibodies for phospho-ERK1/2, p38, phospho-p38, JNK, phospho-JNK, phospho-MKK4, phospho-ATF-2, and phospho-c-Jun were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti-ERK1/2 and anti-FLAG (M2) antibodies were purchased from BD Biosciences (San Jose, CA) and Sigma-Aldrich, respectively. Anti-MKP1 and anti-MKP3 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-MKP7 antibody was a kind gift from Dr. Mary Collins (Royal Free and University College Medical School, London, UK). The immunoreactive bands were visualized by enhanced chemiluminescence using horseradish peroxidase-conjugated IgG secondary antibodies. Band density was measured by densitometry, quantified using Gel plotting macros of the National Institutes of Health image 1.62 program (National Institutes of Health, Bethesda, MD), and normalized to an indicated sample in the identical membrane.

Analysis of MKP-1 oxidation
The MKP-1 redox state was detected by EMSA on SDS-PAGE under a nonreducing condition, as described previously (20). MIN6 cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin. The lysates (30 µg protein/lane) were separated by SDS-PAGE in the presence or absence of β-ME, and were immunoblotted with the MKP-1 antibody.

RT-PCR
RT-PCR was used to detect MKP family RNAs in the MIN6 cells. MIN6 cells were cultured under HG or LG conditions for 24 h. Total RNAs were extracted and reverse-transcribed with an oligo(dT) primer. This single-strand cDNA was used as a template to amplify Wip-1, VHR, MKP-1, MKP-3, MKP-7, and MKP-5 by PCR using the following specific primers: VHR, 5'-CGTTCGAACTCTCGGTGCAAGA-3' and 5'-CATTTTTATGGGCCAGCGCCT-3' (342 bp); MKP-1, 5'-TCAACGTCTCAGCCAATTGTCCT-3' and 5'-CGTCCAGCTTTACCCGGTTAGTC-3' (237 bp); MKP-3, 5'-GTGTTCTCATTCCAGTCGCTG-3' and 5'-TAGATACGCTCAGACCCGTG-3' (322 bp); MKP-7, 5'-GAGGATGGACATGTCTCTAG-3' and 5'-ATGTCGGCAGTGAGAATCTC-3' (322 bp); MKP-5, 5'-GCAGGATGCTCAGGACCTAGACA-3' and 5'-CATCCGTGTGTGCTTCATCAAGT-3' (285 bp); and Wip-1, 5'-CTACTTACAACAGCCAGGAG-3' and 5'-GTGAGACAGTTTGACTGGGA-3' (520 bp). The primers for VHR, MKP-1, MKP-3, and MKP-5 were kindly provided by Dr. Ogata.

Primary islet cell culture and apoptosis analysis
Mouse pancreatic islets were isolated from C57BL/6J mouse (CLEA Japan, Inc., Tokyo, Japan) by pancreatic ductal injection of 500 U/ml collagenase (type XI; Sigma-Aldrich). Islets were cultured in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C. After 48 h, they were dispersed into cells by 0.05% trypsin/0.02% EDTA and cultured on eight-well laboratory-Tek chamber slides (Nalge Nunc International Corp., Naperville, IL) precoated with poly-(L)-lysine (Sigma-Aldrich). Experiments were done under culture conditions of RPMI 1640 with 0.5% (wt/vol) BSA (fatty acid free; Sigma-Aldrich) containing 11.1 or 2.0 mM glucose for 2–4 d.

Apoptosis assay in dispersed islet β-cells was performed as described previously (42). Briefly, islet cells on chamber slide were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100. Cells were then incubated with anti-insulin (Sigma-Aldrich), followed by Rhodamine Red-X-conjugated, anti-genuine pig secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) to detect β-cells. Staining with 0.1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) was performed for identifying nucleus. The condensed or fragmented nucleus is the morphological characteristic of apoptosis. The number of insulin-positive islet β-cells with normal or apoptotic nucleus was counted, and the apoptotic index was calculated. At least four different fields from each well were selected to count at least 300 cells to calculate the rate of apoptosis.

Data analysis
Data are expressed as the mean ± SE values of the SEM with the number of individual experiments described in the figure legends. Differences between groups were analyzed using the Student’s t test. P values less than 0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LG culture induces ROS-mediated cell death in MIN6 cells
Because ROS are generated when pancreatic β-cells are kept under conditions of LG (8), HG (9, 10, 43), or hypoxia (11, 12), we placed MIN6 cells in the culture environment of LG (2 mM, 24 h), hypoxia (5% O₂, 24 h), or cytokine treatments (20 nM TNF, 12 h; 300 pg/ml IL-1β, 12 h), and examined the increase in the fluorescence of an oxidant indicator dye, CM-H₂DCFDA, by fluorescence microscopy (Fig. 1A). Each of the three culture conditions evoked DCF fluorescence, and the fluorescence intensity at LG was comparable to that evoked by the other stressors (Fig. 1, A and B, and supplemental Fig. 2). Under the LG culture, intracellular ROS began to accumulate at 8 h and became prominent at 24 h (Fig. 1B). A quantitative H₂O₂ assay further revealed that the LG culture generated a 1.5-fold increase of H₂O₂ accumulation for 12 h, and a 3.9-fold increase for 24 h in the MIN6 cell lysates (Fig. 1C). These results were consistent with the previous observation that LG exposure caused redox imbalance, then induced superoxide formation and mitochondrial dysfunction in β-cells (8).

View larger version (13K): [in this window] [in a new window]   FIG. 2. Effect of long-term LG culture on the viability of cultured β-cells. A, The viability of MIN6 cells was measured in the LG or HG culture for up to 48 h, using Trypan blue dye-exclusion assay. Data are representative of four independent experiments. B, The viability of INS-1 cells was measured under a variety of glucose culture concentrations for 24 h. Data are representative of three independent experiments.

We then examined whether the increased ROS production was implicated in the cell death. MIN6 cells were exposed to the LG culture in the absence or presence of ROS scavengers, and the cell death rate was then determined. Both the SOD mimetic MnTBAP (10 µM) and the compound NAC (2 mM) scavenged ROS accumulation (Fig. 3A), and significantly blocked the cell death (Fig. 3B). This suggests that ROS accumulation contributes to the LG-induced MIN6 cell death.

View larger version (22K): [in this window] [in a new window]   FIG. 3. ROS scavengers prevent ROS generation and LG-induced cell death. A, MIN6 cells were cultured in LG and HG for 24 h in the presence or absence of 10 µM MnTBAP or 2 mM NAC. Intracellular ROS generation was observed with a ROS detection probe, CM-H2DCFDA. Experiments were repeated three times with reproducible results. B, The MIN6 cell death rate in LG for 36 h was determined by Trypan blue dye-exclusion assay under the same conditions as in A. Data are representative of three independent experiments. DMSO, Dimethylsulfoxide.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Effect of long-term LG culture on the phosphorylation of p38 and JNK. A, MIN6 cells were cultured in LG for up to 8 h. Cell lysates were prepared and subjected to immunoblot analysis for detecting the total protein and the phosphorylated form of p38 or JNK. B, MIN6 cells were cultured in LG for up to 36 h. The total protein and the phosphorylated form of p38 or JNK, and -tubulin were detected as in A. C and D, Intensity of each band was quantified with the densitometric imager, and the results from three independent experiments are presented as a fold increase ± SEM compared with time zero (*, P < 0.05; **, P < 0.01). C, Total and the phosphorylated form of p38. D, Total and the phosphorylated form of JNK.

View larger version (13K): [in this window] [in a new window]   FIG. 5. Overexpression of p38 reduces the LG-induced cell death. MIN6 cells were infected with adenoviruses integrating LacZ, wild-type p38, or nonphosphorylatable p38-AGF for 12 h. The cells were then cultured in LG for 36 h. A, The cell death rate was determined for MIN6 cells overexpressed with LacZ, p38, and p38-AGF, by Trypan blue dye-exclusion assay. Data are representative of three independent experiments. B, Cell lysates at time points zero and 24 h were collected and subjected to immunoblot analysis with antibodies to the total protein and the phosphorylated form of p38. Experiments were repeated three times with reproducible results.

View larger version (17K): [in this window] [in a new window]   FIG. 7. Effects of inhibitors of p38 or JNK on the LG-induced cell death. The LG-induced cell death of MIN6 cells was evaluated with the intervention of p38 inhibitors. Inhibitors were applied for 24 or 36 h, and the cell death rate was measured by Trypan blue dye-exclusion assay. A, The p38 inhibitors were SB203580 (20 µM), SB202190 (10 µM), or PD169316 (10 µM). Dimethylsulfoxide (DMSO) was used as a control. B, The JNK inhibitors were JNKI-1 (1.0 µM, dissolved in d-H2O) and SP600125 (5.0 µM). JNKI-1 was added to the culture again at the 24-h point in the 36-h culture to supplement any degraded JNKI-1 peptides. Dimethylsulfoxide- or d-H2O-supplemented culture was used as a control. Values represent the means of four independent experiments.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Overexpression of JNK increases the LG-induced cell death. MIN6 cells were infected with adenoviruses integrating LacZ, wild-type JNK, or nonphosphorylatable JNK-APF for 12 h. After infection the cells were cultured in LG for 36 h. A, The cell death rate was determined for MIN6 cells overexpressed with LacZ, JNK, and JNK-APF, by Trypan blue dye-exclusion assay. Data are representative of three independent experiments. B, Cell lysates at 24 h were collected and subjected to immunoblot analysis with antibodies to the total protein and the phosphorylated form of JNK. Experiments were repeated three times with reproducible results.

Next, we used immunoblotting to examine a specific phosphorylation state of proteins constituting the JNK signaling cascade. Similar to JNK phosphorylation, the number of its downstream molecules, ATF-2 and c-Jun, in their phosphorylated form gradually increased at about the 12-h point. In contrast, no correlative activation with JNK was observed for the upstream kinase MKK4 (Fig. 8). Therefore, we next examined the involvement of JNK phosphatases, which derive from the MKP-family phosphatases.

View larger version (17K): [in this window] [in a new window]   FIG. 8. Phosphorylation states of upstream and downstream JNK pathway molecules during the LG culture. A, MIN6 cell lysates from the LG culture up to 36 h were prepared and subjected to immunoblot analysis as in Fig. 3B. The phosphorylation states were analyzed with antibodies to phospho-JNK, phospho-MKK4, phospho-ATF-2, and phospho-c-Jun. -Tubulin is an internal control. Experiments were repeated three times with reproducible results. B, Intensity of each band was quantified with the densitometric imager, and the results from three independent experiments are presented as a fold increase ± SEM compared with time zero.

MKPs share the protein tyrosine phosphatase (PTP) signature motif at their catalytic pocket. A key feature of this motif is the catalytic cysteine, which is highly sensitive to oxidation due to its low pKa (28). Thus, we examined the oxidation of MKP-1 in the LG-exposed MIN6 cells using an EMSA. As a control, MIN6 cells were treated with 10 µM H₂O₂ for 10 min, and cell lysates were prepared for separation by SDS-PAGE in the absence of β-ME. Immunoblotting revealed a marked EMS of MKP-1 in H₂O₂-treated cells to high molecular weight (HMW) species; however, incubation of the cell lysates with β-ME abolished the EMS (Fig. 9A). A previous study showed that oxidized MKP-1 forms a complex as HMW proteins by interdisulfide bridge formation (20). Thus, the HMW complex reflects an oxidation state of MKP-1 protein.

View larger version (22K): [in this window] [in a new window]   FIG. 9. MKP-1 is oxidized in LG-cultured MIN6 cells. A, We examined the oxidative product of MKP-1 (Oxf) after treating the cells with or without 10 µM H2O2 in the presence of MG132 (50 µM), a proteasome inhibitor. MKP-1 oxidization was monitored by EMSA. β-ME was used to reduce an oxidized form of MKP-1 (Red). B, MIN6 cells were cultured in LG for an indicated time up to 36 h. Cells were lysed in a lysis buffer containing 10 mM N-ethylmaleimide to prevent oxidization of cysteine during a sample preparation. MKP-1 oxidization was monitored by EMSA as in A. The blot was separated to two parts: the left panel is 0–24 h, and the right panel is 24 and 36 h, after the LG culture. Experiments were repeated three times with reproducible results. C, Intensity of each aggregated MKP-1 band was quantified with the densitometric imager, and the results from three independent experiments are presented as a fold increase ± SEM compared with time zero (*, P < 0.05). C, Control; ns, nonspecific band.

View larger version (22K): [in this window] [in a new window]   FIG. 10. ROS scavengers prevent MKP-1 inactivation and JNK activation. A, MIN6 cells were cultured in LG for 24 h in the presence or absence of 10 µM MnTBAP or 2 mM NAC. LG-cultured MIN6 cell lysates were prepared, and the oxidation product of MKP-1 was detected by EMSA, as in Fig. 9. Right panel, Intensity of each aggregated MKP-1 band was quantified with the densitometric imager, and the results from three independent experiments are presented as a fold increase ± SEM compared with H2O. B, The cell lysates prepared in A were subjected to immunoblot analysis with anti-phospho-JNK and anti-JNK antibodies. Right panel, Intensity of each phosphorylated JNK band was quantified with the densitometric imager, and the results from three independent experiments are presented as a fold increase ± SEM compared with H2O. DMSO, Dimethylsulfoxide.

View larger version (17K): [in this window] [in a new window]   FIG. 11. Effect of overexpressed MKPs on the LG-induced cell death. MIN6 cells were infected with adenoviruses integrating MKP-1, MKP-7, Wip1, or MKP3 for 12 h. A, The exogenous expression of each protein was detected by immunoblotting with anti-FLAG antibody. The asterisk indicates a nonspecific band. B, Adenovirus-infected cells were cultured in LG for 36 h. The death rate was determined by Trypan blue-exclusion assay. Data are representative of four independent experiments. C, Cell lysates at the 8- and 36-h points were immunoblotted with antibodies to the total protein, and the phosphorylated form of p38 (upper panel) and JNK (lower panel). Experiments were repeated three times with reproducible results.

View larger version (17K): [in this window] [in a new window]   FIG. 12. Effect of JNK and MKP-1 on the LG-induced primary β-cell apoptosis. Dispersed mouse islet cells were incubated under conditions of RPMI 1640 with 0.5% BSA containing 11.1 mM glucose or LG (2.0 mM) separately for 2–4 d. A, The apoptosis index (mean ± SEM) of primary β-cell was measured by nuclear characteristics as described in Materials and Methods. The representative photos are in B, denoting apoptotic β-cells with condensed or fragmented nuclei in insulin-positive cells cultured in LG. Bar, 10 µm. C, Dispersed mouse islet cells were infected separately with adenoviruses integrating LacZ, JNK, JNK-APF, and MKP-1 at MOI of five plaque-forming units per cell. After 12-h infection, they were cultured in LG for 3 d. The apoptosis was determined as in A, and the results are presented as a fold increase ± SEM compared with uninfected cells (mock). All data are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Oxidative stress induces damage to β-cells by activating stress-sensing pathways exemplified by the SAPKs, p38 and JNK (2, 13). Although both p38 and JNK are categorized as SAPKs, their action modes appear to be distinct (15). p38 is activated immediately after the β-cells are placed under the LG culture, whereas that of JNK takes many hours, based on observation of the phosphorylation forms (Fig. 4). Furthermore, phosphorylated p38 has a suppressive effect on β-cell death under the LG culture for 36 h, whereas phosphorylated JNK enhances it (Figs. 5–7). These differences may be due to their phosphorylation substrates. Downstream of p38, there are reportedly nuclear factor B, CCAAT/enhancer binding protein homologous protein (CHOP), and MAPK-activated protein kinase-2, and further downstream are heat shock protein 25 and cAMP response element-binding protein (CREB), whereas downstream of JNK, c-Jun is specific (13). Thus, the distinct action of JNK may occur from c-Jun phosphorylation (Fig. 8). In addition, the activity of p38 appears to be cell-type specific. For example, in human islets, cytokines such as IL-1β, TNF-, and IL-6 are highly produced in resident macrophages; cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are expressed in resident endothelial cells, and SB203580, a p38 inhibitor, suppresses cytokine production and cyclooxygenase-2 and inducible nitric oxide synthase expression (45). It may be that SB203580 improves islet graft function by suppressing p38 in resident macrophages and in endothelial cells.

Previously, we reported that MKPs are important for modulating MAPK functions in β-cells, and that adenoviral expression of MKP-1 in MIN6 cells increases insulin gene expression by decreasing JNK phosphorylation and the resultant c-Jun phosphorylation (29). We identified five MKPs in the previous report, and here, we have added two more: a dual-specific phosphatase MKP-7 and a serine/threonine-specific phosphatase Wip-1, to β-cells. The adenoviral expression of MKP-1, MKP-7, and Wip-1 decreased β-cell death significantly (Fig. 11). The dual-specific phosphatases, MKP-1 and MKP-7, efficiently decreased phosphorylated p38 at 8 h after the start of LG culture and phosphorylated JNK at 36 h after the start. However, only p38 was significantly phosphorylated at the 8-h point during the LG culture (Fig. 4). Because the phosphorylation state is a balance between upstream kinase action to MAPKs and dephosphorylation action by MKPs, the dephosphorylation of p38 by MKPs may be inefficient at the 8-h point. At the 36-h point, JNK and c-Jun were phosphorylated at higher rates (Figs. 4 and 8), and conversely, the immunoblot intensity of MKP-1 was attenuated, perhaps due to the rapid increase in dead cells at the 36-h point (supplemental Fig. 4B).

Recently, Kamata et al. (20) demonstrated that intracellular H₂O₂ accumulation inactivates MKPs by oxidation of their catalytic cysteine, which leads to sustained activation of JNK. We confirmed the presence of this oxidized MKP-1 in MIN6 cells under the LG culture, and the oxidized form disappeared after treatment with two antioxidants, MnTBAP and NAC (Figs. 9 and 10). Although we could not confirm the oxidized form of MKP-7 by the lack of the appropriate antibody, JNK-specific MKPs seem to act as ROS sensors for regulating MAPK activity. Thioredoxin has been suggested to act as a ROS sensor for ASK1, which activates JNK and p38 (18). Because MKK4 is a middle component of the ASK1 to JNK pathway (17), we examined MKK4 phosphorylation under the LG culture but found no correlation with the appearance of phosphorylated JNK for up to 36 h in the LG culture (Fig. 8). Our findings suggest that thioredoxin/ASK1 does not function as a ROS sensor under a LG culture.

Our data indicate that ROS accumulation induced by LG culture leads β-cells to death through MKP-1 inactivation and JNK activation. However, ROS scavengers were not sufficient to completely block the LG-induced β-cell death (Fig. 3B). Treatment of MnTBAP or NAC could not prevent the LG-induced ROS accumulation completely (Fig. 3A), and other reagents (diphenyleneiodonium or edaravone) displayed a limited effect (data not shown). Application with a higher dose or combination of different scavengers also did not achieve a complete inhibition of ROS accumulation (data not shown). Because ROS consists of several oxygen molecules and their generation is mutually interacted, active oxygen molecules other than hydrogen peroxide and superoxide may be involved in the LG-induced β-cell death.

ROS-induced β-cell death is controlled by the interaction between a number of phosphorylation and dephosphorylation cascades. Among these, SAPKs, JNK, and p38, along with their corresponding MKPs, are representative regulators of cell death. In our system, only 25% of cell death reduction was achieved by the treatment of JNK inhibitors (Fig. 7). We have verified that JNK inhibitors were effective and completely prevented phosphorylation of downstream substrates induced by the LG culture (data not shown). This suggests that JNK activity contributes to the LG-induced β-cell death, but other molecules and signaling pathways are also involved in the cell death regulation. Although transcription factor c-myc and mitochondrial membrane protein hyperglycemia/hypoxia-inducible mitochondrial protein have recently been involved in the LG-induced β-cell death (5, 46), molecular interaction of those proteins with JNK/MKP is unclear at present. Most recently, Cai et al. (47) showed that caspase activation and Bcl-2 cleavage were detected in LG-cultured β-cells, after ROS formation and JNK phosphorylation. This suggests that LG-induced β-cell death consists of apoptosis. Interestingly, we noted that LG-induced β-cell death was reduced with a caspase inhibitor zVAD and an autophagy inhibitor 3-methyladenine by 42 and 40%, respectively (data not shown). Inhibition of apoptosis by caspase inhibitors often causes ROS production and other types of cell death such as autophagic death and necrosis (48), and several cell death pathways are suggested to be mutually associated (48, 49). Thus, treatment of JNK inhibitors may result in activation of another cell death pathway independent of JNK activity. The LG-induced cell death pathway appears to be complicated because both surviving signals (p38 contributed) and death signals (JNK contributed) were implicated simultaneously.

Together with the essential role of JNK by MKPs in the expression of the insulin gene (29), we suggest that JNK-dephosphorylatable MKPs, such as MKP-1 and MKP-7, protect β-cells from apoptotic cell death by interrupting JNK/c-Jun-mediated pathways. Although the regulatory mechanisms of MKP-1 have been well investigated, including its cAMP-mediated expression (29) and its oxidative inactivation (20), those of other MKPs remain to be investigated. Because other MKPs correspond to specific MAPKs, MKP type-specific modification should be essential for regulating full-functioning survival of pancreatic β-cells.

	Acknowledgments

 
We thank Ms. M. Kosaki and Ms. M. Hosoi for their technical support.

	Footnotes

 
This work was supported by the Global Center of Excellence Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 10, 2008

Abbreviations: ASK1, Apoptosis signal-regulating kinase 1; CM-H₂DCFDA, 5-(and-6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; DAPI, 4',6-diamidino-2-phenylindole; DCF, dichlorodihydrofluorescein; FBS, fetal bovine serum; HG, high glucose; HMW, high molecular weight; JNK, c-jun N-terminal kinase; LacZ, β-galactosidase; LG, low glucose; β-ME, β-mercaptoethanol; MKP, MAPK phosphatase; MnTBAP, manganese(III)tetrakis(4-benzoic acid) porphyrin; MOI, multiplicity of infection; NAC, N-acetylcysteine; ROS, reactive oxygen species; SAPK, stress-activated protein kinase; SOD, superoxide dismutase; VHR, vaccinia H1-related phosphatase.

Received July 18, 2007.

Accepted for publication December 28, 2007.

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