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Endocrinology, doi:10.1210/en.2007-0438
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Endocrinology Vol. 149, No. 6 3046-3053
Copyright © 2008 by The Endocrine Society

The MAPK Kinase Kinase-1 Is Essential for Stress-Induced Pancreatic Islet Cell Death

Dariush Mokhtari, Jason W. Myers and Nils Welsh

Department of Medical Cell Biology (D.M., N.W.), Uppsala University, S-751 23 Uppsala, Sweden; and Department of Biochemistry (J.W.M.), Stanford University School of Medicine, Stanford California 94305-5307

Address all correspondence and requests for reprints to: Nils Welsh, Department of Medical Cell Biology, Uppsala University, Biomedicum, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: nils.welsh{at}mcb.uu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of the present investigation was to characterize the role of the MAPK kinase kinase-1 (MEKK-1) in stress-induced cell death of insulin producing cells. We observed that transient overexpression of the wild type MEKK-1 protein in the insulin-producing cell lines RIN-5AH and βTC-6 increased c-Jun N-terminal kinase (JNK) phosphorylation and augmented cell death induced by diethylenetriamine/nitroso-1-propylhydrazino)-1-propanamine (DETA/NO), streptozotocin (STZ), and hydrogen peroxide (H2O2). Furthermore, DETA/NO or STZ induced a rapid threonine phosphorylation of MEKK-1. Silencing of MEKK-1 gene expression in βTC-6 and human dispersed islet cells, using in vitro-generated diced small interfering RNA, resulted in protection from DETA/NO, STZ, H2O2, and tunicamycin induced cell death. Moreover, in DETA/NO-treated cells diced small interfering RNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK but not p38 and ERK. Inhibition of JNK by treatment with SP600125 partially protected against DETA/NO- or STZ-induced cell death. In summary, our results support an essential role for MEKK-1 in JNK activation and stress-induced β-cell death. Increased understanding of the signaling pathways that augment or diminish β-cell MEKK-1 activity may aid in the generation of novel therapeutic strategies in the treatment of type 1 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE-1 (INSULIN DEPENDENT) diabetes is an autoimmune disease characterized by the selective destruction of the insulin producing β-cells located in the islets of Langerhans (1). Proinflammatory cytokines such as IL-1β, TNF-{alpha}, and interferon-{gamma} and the free radical nitric oxide (NO) have been proposed as possible mediators of pancreatic β-cell destruction (2, 3). Cytokines induce β-cell apoptosis and necrosis in vitro (2), and in rodents this effect is caused by increased NO production mediated by inducible nitric oxide synthase. At low concentrations NO has been shown to have protective effects against proapoptotic stimuli. However, when produced in higher concentrations, NO induces apoptosis (4). Multiple signaling pathways have been proposed to mediate NO-induced cell death. It has been shown that NO activates both p53 and caspases and down-regulates the antiapoptotic Bcl-2 protein (5, 6). In addition, inducible NO synthase induction leads to inhibition of aconitase, a decrease in mitochondrial membrane potential, inhibition of mitochondrial ATP production and endoplasmic reticulum (ER) stress (7, 8, 9, 10).

The MAPKs ERK, c-Jun N-terminal kinase (JNK), and p38 control various cellular processes such as differentiation, proliferation, and apoptosis (11). MAPKs are activated by dual phosphorylation of conserved threonine and tyrosine residues and are organized in signaling cascades in which a MAPK kinase kinase [MAP3K, MKKK, or mitogen-activated protein/ERK kinase (MEKK)] phosphorylates and activates a MAPK kinase [MAPK kinase (MKK) or MAPK kinase)], which then activates a MAPK (12). In β-cells, MAPKs are rapidly activated in response to IL-1β, TNF-{alpha}, and the NO donor diethylenetriamine/nitroso-1-propylhydrazino)-1-propanamine (DETA/NO) (8, 13, 14, 15), and inhibition of JNK or p38 has been reported to protect against cytokine-induced β-cell death (14, 16, 17, 18, 19).

MEKK-1 is a 196-kDa serine/threonine protein kinase that has been shown to play an important role in both apoptosis and cell survival. In various cell types, it has been reported that MEKK-1 promotes apoptosis in response to genotoxic stimuli, such as cisplatin, UV irradiation, and etoposide (20) and nongenotoxic stimuli, including anoikis and Fas stimulation (21, 22). When challenged by a proapoptotic stimulus, the full-length MEKK-1 protein is cleaved by Asp-Glu-Val-Asp caspases into an 80- to 95-kDa proapoptotic fragment (20, 21, 22). On the other hand, the full-length MEKK-1 has been shown to be antiapoptotic in cardiomyocytes (23, 24).

MEKK-1 was initially discovered as an activator of the ERK pathway (25). However, subsequent studies demonstrated that MEKK-1 preferentially activates the JNK pathway through phosphorylation of MKK4 (26, 27, 28). Furthermore, MEKK-1 has also been implicated in p38 signaling (29, 30) and the regulation of nuclear factor-{kappa}B and p53 (31, 32, 33).

To our knowledge the effect of MEKK-1 signaling in β-cells has not yet been established. Therefore, the aim of the present study was to investigate the putative role of MEKK-1 in pancreatic islet cells. We report here that treatment with the β-cell-specific toxin streptozotocin (STZ) (34) or the NO donor DETA/NO increased MEKK-1 phosphorylation in insulin-producing cells. Whereas overexpression of MEKK-1 sensitized insulin-producing cells to treatments with DETA/NO, STZ, and H2O2, knockdown of MEKK-1 mRNA in primary islet cells resulted in protection against these agents. In addition, the MEKK-1 down-regulation mediated protection against DETA/NO-induced cell death was paralleled by a lowered JNK activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat insulinoma cells (RIN-5AH) at passage 20–30 were maintained in RPMI 1640 medium (Sigma Chemicals, St. Louis, MO) supplemented with 10% fetal calf serum (Sigma), 2 mM L-glutamine, streptomycin (0.1 mg/ml), and benzylpenicillin (100 U/ml) (WS). Murine βTC-6 cells (American Type Culture Collection, Manassas, VA) at passage numbers 20–30 were maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented as above. Islets from NMRI mice (Naval Medical Research Institute-established, Mölle and Bomholt gård, Denmark) were isolated by collagenase digestion as described previously (35) and incubated free floating in WS. Isolated human pancreatic islets were kindly provided by Dr. Olle Korsgren (Department of Radiology, Oncology, and Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden). For human islet culture, the glucose concentration of the WS was lowered to 5.6 mM. All cells were grown at 37 C in a humidified air incubator with 5% CO2.

Plasmids
PcDNA3 (Invitrogen, Carlsbad, CA) plasmids containing the full-length mouse MEKK-1 cDNA was kindly provided by Pär Gerwins (Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden). The MEKK-1 plasmid was sequenced at insertion sites and the mutation site to verify the integrity of construct before experimentation. For green fluorescent protein (GFP) expression, the pd2EGFP-N1 (CLONTECH Laboratories Inc., Mountain View, CA), vector was used.

Overexpression of MEKK-1 in RIN-5AH and βTC-6 cells
During transfection, cells were maintained in serum-free RPMI 1640. The cells were transfected by adding a transfection mixture of 10 µl Lipofectamine 2000 (Invitrogen, San Diego, CA) reagent (2 mg/ml) to 1 µg DNA according to the instructions of the supplier. The DNA was pd2EGFP-N1 or a combination of pdEGFP-N1 and wild-type (wt) MEKK-1 [1:4, (wt/wt)]. Cells were centrifuged for 10 min at 550 x g and incubated for 1.5 h at 37 C. After the incubation, the serum-free RPMI 1640 + transfection mix was replaced with WS. The next day (12–15 h after transfection), GFP-positive cells were sorted using a FACSCalibur flow cytometer (Becton Dickinson, Oxford, UK). After sorting, the cells were pelleted by centrifugation for 2 min at 600 x g. The cells were then washed in PBS and plated onto culture dishes as described above.

Diced-small interfering-RNA (d-siRNA)-mediated down regulation of MEKK-1 in human islet cells
Human islets, in groups of 100, were trypsinized (0.5%) for 5 min at 37 C and then treated with DNase I (Amersham Life Science, Piscataway, NJ) (30 U/ml) for 2 min. The resulting free islet cells were placed in nonattachment plates and transfected with 100 ng of d-siRNA directed against mouse MEKK-1 or GL3 (a firefly luciferase siRNA plasmid). d-siRNA directed against the 3'-end of coding region of the mouse MEKK-1 gene and 5'-end of Photinus Pyralis GL3 luciferase gene was synthesized as described previously (36). The in vitro transcription templates were amplified from cloned cDNAs using PCR and the following primers: MEKK-1 forward, 5'-GCGTAATACGACTCACTATAGGGCTG AAGTTCTAAGCAGCGCACG-3'; MEKK-1 reverse, 5'-GCGTAATACGACTCA CTATAGGGAGACAGGATATGCAACCGGGAG-3'; GL3-forward, 5'-GCGTAATACG ACTCACTATAGGAACAATTGCTTTTACAGATGC-3'; GL3-reverse, 5'-GCGTAAT ACGACTCACTATAGGAGGCAGACCAGTAGATCC-3'. The T7 polymerase promoter sequence is shown in bold. d-siRNA was introduced into islet cells during a 2-h incubation using Lipofectamine 2000 in 200 µl of serum-free culture medium. The transfection medium was then replaced by full culture medium and the cells were cultured for 24–48 h.

RNA isolation and cDNA synthesis
Total RNA was isolated from mouse and human pancreatic islet cells using the Ultraspec RNA isolation system reagent (Biotecx Laboratories, Houston, TX). Two micrograms of RNA was used for cDNA synthesis. cDNA was synthesized using the Muloney murine leukemia virus reverse transcriptase (Finnzymes, Espoo, Finland) and oligo-dT primers according to the manufacturer’s instructions.

Real-time PCR
PCR amplification was performed using the Lightcycler instrument (Roche Diagnostics, Lewes, UK) and the Lightcycler FastStart DNA master SYBR Green I kit (Roche Diagnostics). The following primers were used: glyceraldehyde-3-phosphate dehydrogenase (human) (GAPDH), 5'-ACCACAGTCCATGCCATCAC (forward), 5'-TCCACCACCCTGTTGCTGTA (reverse), β-actin (mouse), 5'-GCTCTGGCTCCTAGGACC, (forward), 5'-CCACCGATCCACACAGAGTACTTG (reverse), MEKK-1 (mouse and human), 5'-GGTTGGTGGTGTCGATTACG (forward), 5'-GGCTCAGCTTGTGAGACATC (reverse). Semiquantitative data for MEKK-1 expression were calculated using the formula: 2crossing point GAPDH or β-actin-crossing point MEKK-1. The different groups were expressed as percent of GL3 d-siRNA transfected cells. PCR products were analyzed by agarose gel electrophoresis and SYBR green staining to verify that the correct PCR products were obtained.

In vitro treatment of cells and evaluation of cell viability
Transfected cells were either left untreated or treated with DETA/NO, [2,2'-(hydroxynitrosohydrazono) bis-ethanamine] (Cayman Chemical, Ann Arbor, MI) (2.5 mM, 12–15 h), STZ (Sigma) (15 mM, 12–15 h), H2O2 (125 µM, 12–15 h), tunicamycin (Sigma) (5 µg/ml, 12–15 h), or SP600125 (Tocris, Bristol, UK) (1 µM, added 30 min before other additions). Cell viability for RIN-5AH and βTC-6 cells was determined by staining the cells with propidium iodide (Sigma) (20 µg/ml) for 10 min at 37 C. After careful washing, cells were trypsinized and analyzed for red fluorescence (FL-3) using flow cytometry (37). Human islet cells were stained with propidium iodide (20 µg/ml) and bisbenzimide (Sigma) (5 µg/ml) for 10 min at 37 C and analyzed by fluorescence microscopy using Openlab 3.0.4 software (Improvision, Waltham, MA). Total number of cells, as well as propidium iodide-positive cells, were counted using the NIH Image 1.63 software (National Institute of Health, Bethesda, MD) by investigators not aware of sample identity.

Immunoprecipitation of MEKK-1
Cells (108) were washed twice in ice-cold PBS and resuspended in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2 mM EDTA, 20 mM β-glycerophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM sodium diphosphate decahydrate, and 0.2% protease inhibitor cocktail (Sigma)] on ice for 20 min. The lysed cells were cleared by centrifugation, and remaining extracts were incubated with 5 µg MEKK-1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or 5 µg of purified rabbit IgG (control) for 2.5 h on ice. Immune complexes were purified by binding to 50 µl protein A Sepharose (Amersham Biosciences, Buckinghamshire, UK) for 1 h at 4 C and thereafter washed three times with lysis buffer and once with H2O. The Sepharose beads were resuspended in SDS sample buffer [2% SDS, 0.15 M Tris (pH 8.8), 10% glycerol, 5% β-mercaptoethanol, bromophenol blue, and 2 mM phenylmethylsulfonylflouride] and immunoprecipitates were resolved by electrophoresis.

Immunoblotting
Cells (106) were washed with ice-cold PBS and directly lysed in SDS sample buffer, boiled for 5 min, and separated on SDS-PAGE. Proteins were electrophoretically transferred to Immobilon filters (Amersham Biosciences). Filters were blocked in 5% BSA for 1 h, after which they were probed with anti-MEKK-1 (c22) threonine phosphorylation (P-Thr) (Santa Cruz Biotechnology), phosphorylated (P)-JNK, P-ERK, P-p38, total JNK, and total ERK (Cell Signaling, Beverly, MA) antibodies. Bound antibodies were removed from Immobilon filters (Amersham Biosciences) by incubating for 40 min at 55 C in 2% (wt/vol) SDS and 0.1 mM β-mercaptoethanol. Horseradish peroxidase-linked goat antirabbit was used as secondary antibody. The immunodetection was performed as described for the ECL immunoblotting detection system (Amersham Biosciences) and using the Imagestation 4000MM (Eastman Kodak, Rochester, NY). The intensities of the bands were quantified by densitometric scanning using Kodak Digital Science ID software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lipofection and fluorescence-activated cell sorting of RIN-5AH and βTC-6 cells
To study the effect of MEKK-1 genetic gain of function, two insulin-producing cell lines were transiently transfected with an enhanced GFP (EGFP) expression vector alone or EGFP together with wt MEKK-1. Transfection of the rat cell line RIN-5AH and the mouse cell line βTC-6 using Lipofectamine 2000 (Invitrogen) usually results in a transfection efficiency of only 5–15% (results not shown). However, it is possible to enrich the transfected cells by fluorescence-activated cell sorting (38). Using this approach the percentage of GFP-positive cells was increased to 60–80% (results not shown). Indeed, in the GFP-enriched cell population, we observed strong MEKK-1 immunoreactivity in cells transfected with wt MEKK-1 expression vector 24 and 48 h after the transfection procedure when comparing with cells transfected with GFP alone (Fig. 1AGo).


Figure 1
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  		FIG. 1. Transient MEKK-1 overexpression in RIN-5AH and βTC-6 cells increases stress-induced cell death. A, βTC-6 cells and RIN-5AH cells were transiently transfected with EGFP alone or EGFP together with wt MEKK-1. Forty-eight hours after transfection, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with MEKK-1 and ERK antibodies. A representative blot from one of three experiments is shown, and the position of the 250-kDa molecular mass marker is given to the left. RIN-5AH (B) and βTC-6 cells (C) were transfected with EGFP either alone or together with wt MEKK-1. Forty-eight hours after transfection, the cells were either left untreated or treated with DETA/NO (2.5 mM, 15 h), STZ (15 mM, 15 h), or H2O2 (125 µM, 15 h) and assayed for propidium iodide-positive nuclei by flow cytometry. Data are presented as means ± SEM for four to six individual experiments. *, P < 0.05 (two-way ANOVA followed by Tukey’s post-ANOVA test). D, βTC-6 cells were transfected and treated with DETA/NO and STZ as given above. Cells were then lysed and used for immunoblotting with MEKK-1, phospho-JNK, and total JNK antibodies. The figure is representative of two independent experiments.

 
MEKK-1 overexpression increases cell death and JNK phosphorylation in response to DETA/NO, STZ, and H2O2
Having established that this approach generates a strong increase in MEKK-1 immunoreactivity, we wanted to investigate whether overexpression of wt MEKK-1 affected cell viability in RIN-5AH and βTC-6 cells when subjected to different stressful stimuli. Therefore, RIN-5AH and βTC-6 cells transfected with GFP alone or together with wt MEKK-1 were either left untreated (control) or treated with DETA/NO (2.5 mM, 12–15 h), STZ (15 mM, 12–15 h), or H2O2 (125 µM, 12–15 h) and assayed for propidium iodide-positive nuclei using flow cytometry. In both RIN-5AH (Fig. 1BGo) and βTC-6 (Fig. 1CGo), all treatments significantly increased cell death when compared with no treatment. Cell death in response to all treatments was further increased in cells overexpressing wt MEKK-1 when compared with GFP-expressing cells (Fig. 1Go, B and C). MEKK-1 overexpression did not affect cell death rates in untreated cells. We also analyzed JNK phosphorylation in βTC-6 cells overexpressing MEKK-1. It was observed that JNK phosphorylation, induced by treatment with DETA/NO or STZ, was augmented in MEKK-1 overexpressing cells, compared with non-MEKK-1-overexpressing cells (Fig. 1DGo). The results suggest that MEKK-1 has a death-promoting role in insulin-producing cells and that this effect may be mediated by JNK phosphorylation.

MEKK-1 is phosphorylated in response to STZ and DETA/NO
Because overexpression of wt MEKK-1 augmented DETA/NO-, STZ-, and H2O2-induced cell death, we next studied whether MEKK-1 is activated by these treatments. One way to determine MEKK-1 activation is by measuring MEKK-1 P-Thr using the immunoblotting technique (39). βTC-6 cells were therefore left untreated or treated with DETA/NO (2.5 mM, 1 h) or STZ (15 mM, 1 h) followed by immunoprecipitation of the MEKK-1 protein. Both STZ and DETA/NO promoted increased P-Thr of MEKK-1 when compared with untreated cells (Fig. 2AGo). We could not observe any increase in 80- to 95-kDa MEKK-1 fragments at 3, 6, or 12 h after STZ or DETA/NO exposure (Fig. 2BGo), indicating that DETA/NO and STZ did not promote caspase-mediated cleavage of MEKK-1.


Figure 2
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  		FIG. 2. STZ and DETA/NO treatment induces MEKK-1 phosphorylation in βTC-6 cells without signs of MEKK-1 cleavage. A, βTC-6 cells were either left untreated or treated with STZ (15 mM) or DETA/NO (2.5 mM) for 1 h. After treatment, the cells were lysed and immunoprecipitated using MEKK-1 antibody or rabbit IgG as control. The immunoprecipitates were separated by SDS gel electrophoresis and analyzed by immunoblotting using P-Thr and MEKK-1 antibodies (upper panel). Results from immunoblots as the one shown in upper panel were quantified by densitometry (lower panel). MEKK-1 phosphorylation was determined by relating P-Thr bands to those of MEKK-1. Data are presented as means ± SEM for five to six individual experiments. *, P < 0.05 vs. control using one-way ANOVA and Tukey’s test. B, βTC-6 cells were either left untreated or treated with STZ (15 mM) or DETA/NO (2.5 mM) for 1 h. The cells were then harvested at the indicated times, and MEKK-1 and ERK immunoblot analysis was performed as described above. The figure is representative of three separate experiments.

 
MEKK-1 knockdown protects βTC-6 cells and human islets
Next we investigated the effects of genetic MEKK-1 loss of function in βTC-6 cells and primary human islet cells. We have previously shown that successful knockdown in dispersed primary islet cells can be achieved by using d-siRNA (40). Instead of using predesigned 21- to 23-nucleotide-long synthetic siRNA molecules, the d-siRNA technique uses the in vitro activity of recombinant dicer to yield a pool of dicer-generated siRNA sequences (d-siRNA) (36). βTC-6 cells and dispersed human islet cells were transfected with either GL3 Luciferase (GL3 d-siRNA) or MEKK-1 d-siRNA. Forty-eight hours after d-siRNA transfection, we observed a knockdown of MEKK-1 mRNA in human islet cells treated with GL3 d-siRNA (Fig. 3AGo). To verify knockdown of MEKK-1 on the protein level, GL3 or MEKK-1 d-siRNA transfected human islet cells were lysed and analyzed for MEKK-1 immunoreactivity 48 h after transfection. In human islet cells treated with MEKK-1 d-siRNA, a knockdown of the MEKK-1 protein was observed when compared with cells transfected with GL-3 d-siRNA (Fig. 3BGo).


Figure 3
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  		FIG. 3. MEKK-1 mRNA knockdown protects βTC-6 and dispersed human islet cells from STZ, DETA/NO, H2O2, and tunicamycin-induced cell death. A, Semiquantitative analysis of MEKK-1 mRNA. Human islet cells were transfected for 3 h with either GL3 d-siRNA (100 nM) or MEKK-1 d-siRNA (100 ng). RNA isolation and cDNA synthesis were performed 48 h after transfection. Upper panel, The 170-bp MEKK-1 real-time PCR product. Middle panel: The GAPDH real time PCR product. Lower panel, Data are presented as means ± SEM for four individual experiments. **, P < 0.01 (Student’s t test). B, Western blot showing MEKK-1 immunoreactivity in dispersed human islet cell extracts treated with either GL-3 or MEKK-1 d-siRNA. A representative blot from one of two experiments is shown. C, βTC-6 cells were transfected with either GL3 or MEKK-1 d-siRNA. Forty-eight hours after d-siRNA treatment, the islet cells were either left untreated or subjected to STZ (15 mM, 15–20 h), DETA-NO (2 mM, 15–20 h) H2O2 (125 µM, 12–15 h), or tunicamycin (5 µg/ml, 12 h) and analyzed for propidium iodide-positive nuclei by fluorescence microscopy. Results are means ± SEM for three separate observations. +, P < 0.05 vs. the GL3 d-siRNA control not exposed to any treatment using two-way ANOVA and Dunnet’s test; *, P < 0.05 using two-way ANOVA followed by Dunnet’s post-ANOVA test. D, Human islets were dispersed and transfected with either GL3 or MEKK-1 d-siRNA. Forty-eight hours after d-siRNA treatment, the islet cells were either left untreated or subjected to STZ (15 mM, 15–20 h), DETA-NO (2 mM, 15–20 h), or tunicamycin (5 µg/ml, 12 h) and analyzed for propidium iodide-positive nuclei by fluorescence microscopy (C). Results are means ± SEM for five separate observations. +, P < 0.05 vs. the GL3 d-siRNA control not exposed to any treatment using two-way ANOVA and Dunnet’s test; *, P < 0.05 using two-way ANOVA followed by Dunnet’s post-ANOVA test.

 
To determine whether knockdown of MEKK-1 mRNA affects βTC-6 and human islet cell viability, GL3 or MEKK-1 d-siRNA transfected βTC-6 and human islet cells were either left untreated (control) or exposed to STZ (15 mM, 12–15 h), DETA/NO (2 mM, 12–15 h), H2O2 (125 µM, 12–15 h), or tunicamycin (5 µg/ml, 12–15 h) and analyzed for propidium iodide-positive nuclei by fluorescence microscopy. Tunicamycin inhibits N-linked glycosylation and is used as an inducer of endoplasmic reticulum stress (41). All the compounds promoted significant cell death in βTC-6 and human islets cells (Fig. 3Go, C and D). MEKK-1 knockdown protected βTC-6 completely or partially from STZ, DETA/NO, H2O2, and tunicamycin (Fig. 3CGo) and human islet cells from DETA/NO, STZ, and tunicamycin-induced cell death (Fig. 3DGo). These data support a necessary role of MEKK-1 in the death of primary human islet cells.

MEKK-1 is required for DETA/NO-induced JNK phosphorylation in human islet cells
MEKK-1 has in different cell types previously been shown to activate all three major MAPK pathways, i.e. the JNK, ERK, and p38 pathways (25). Having observed that MEKK-1 is activated in response to DETA/NO and is a necessary death-promoting event in primary human islet cells, we wanted to study the effect of MEKK-1 on JNK, ERK, and p38 activation. Therefore, human islet cells transfected with GL3 or MEKK-1 d-siRNA were either left untreated or treated with DETA/NO. We found that 30 min of DETA/NO (2.5 mM) treatment induced phosphorylation of all three MAPKs (Fig. 4AGo) and that DETA/NO-induced JNK phosphorylation was abolished in human islet cells treated with MEKK-1 d-siRNA (Fig. 4AGo). No effect of MEKK-1 knockdown could be observed on p38 and ERK activation (Fig. 4AGo). To verify that the MEKK-1/JNK pathway actually promotes β-cell death in response to DETA/NO or STZ, we treated βTC-6 cells with the JNK-inhibitor SP600125 before (30 min) and during exposure to DETA/NO or STZ. We observed a significantly lower cell death rate in SP600125-treated cells when challenged with DETA/NO or STZ (Fig. 4BGo). In summary, our findings suggest that DETA/NO-induced MEKK-1 signaling involves activation of JNK but not p38 and ERK, an event that leads to increased cell death.


Figure 4
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  		FIG. 4. DETA/NO-induced JNK phosphorylation requires MEKK-1 in primary human islet cells. A, Dispersed human islet cells were transfected with GL3 d-siRNA or MEKK-1 d-siRNA and either left untreated or treated DETA/NO (2.5 mM). After 30 min of DETA/NO exposure, the cells were lysed and separated by SDS gel electrophoresis and analyzed by immunoblotting with phospho-specific and total JNK, ERK, and p38 antibodies. The results were quantified by densitometric scanning of three immunoblots and data are presented as means ± SEM for three individual experiments. *, P < 0.05 vs. using two-way ANOVA followed by Dunnet’s post-ANOVA test. B, βTC-6 cells were supplemented with vehicle only [dimethylsulfoxide (DMSO)] or 1 µM SP600125 30 min before addition of DETA/NO (1.5 mM) or STZ (15 mM). The culture was continued overnight, and the SP600125 was present in the culture media throughout this period. The cells were then stained with propidium iodide and analyzed for cell death using fluorescence microscopy as given above. *, P < 0.05 vs. corresponding control using two-way ANOVA and Dunnet’s post hoc test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that a prolonged and pronounced activation of JNK, in response to, for example, islet isolation, amyloid formation, or proinflammatory cytokines, leads to β-cell apoptosis and/or necrosis (42, 43, 44). Therefore, it is highly motivated to obtain a better understanding of the events leading to JNK activation in pancreatic β-cells. In cell types other than β-cells, it has been observed that different MAP3Ks, such as apoptosis signal-regulating kinase 1, TGFβ-activated kinase 1, mixed-lineage protein kinase 3, and MEKK-1, promote JNK activation in a cell type- and stimulus-specific manner (27, 45, 46, 47). In β-cells, however, the events preceding JNK activation are much less characterized. It has been reported that expression of the kinase domain of MEKK-1 promotes JNK activation (44) and that JNK-activation is negatively modulated by the scaffold protein islet-brain1/ JNK-interacting protein-1 (48), but the main JNK-activating MAP3K has not yet been identified. In the present paper, we propose an essential role for MEKK-1 in islet cell apoptosis as a JNK-activating MAP3K.

First, we demonstrate that overexpression of the wt MEKK-1 protein augments DETA/NO-, STZ-, and H2O2-induced cell death in rat RIN-5AH and mouse βTC-6 cells. MEKK-1 overexpression resulted also in increased JNK phosphorylation in βTC-6. Interestingly, untreated cells overexpressing wt MEKK-1 did not show increased cell death, which indicates that elevated MEKK-1 levels per se is not a sufficient event to promote β-cell death. Second, we observed that down-regulation of the MEKK-1 gene in βTC-6 cells and human islet cells mediated protection against DETA/NO, STZ, and tunicamycin. These findings strongly support a necessary role for MEKK-1 in islet cell death. Third, d-siRNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK, but not p38 and ERK, in DETA/NO-treated cells, which indicates that MEKK-1 may be the main JNK-activating MAP3K in islet cells. This finding is also in line with studies performed on non-β-cells showing that increased MEKK-1 activity leads to the preferential activation of JNK over p38 and ERK (49). In this context, it should also be noted that β-cells seem to use a TGFβ-activated protein kinase 1-binding protein-dependent, MKK3/6-independent p38 autophosphorylation mechanism to activate p38 in response to NO (19, 50). Interestingly, inhibition of either JNK or p38 often leads to improved β-cell survival (14, 16, 17, 18, 19). This indicates that a pronounced activation of one of the two MAPKs is required for certain forms of β-cell death.

Several studies have reported caspase-mediated cleavage of the MEKK-1 protein into an 80- to 95-kDa proapoptotic fragment and that this leads to subcellular relocalization of the fragment from membrane bound to the cytosolic compartment (21, 22, 23). However, we were not able to detect any early cleavage of MEKK-1 in βTC-6 cells in response to the different treatments. This strengthens the previously proposed possibility that MEKK-1 cleavage is not a prerequisite for its ability to activate downstream MAPKs and participate in proapoptotic signaling (51).

MEKK-1 was phosphorylated on one or several threonine residues in response to DETA/NO or STZ. Although we could not determine which threonine residues that were phosphorylated due to lack of commercially available phospho-specific antibodies, it is likely to be at the two described T1383 or T1395 sites, which are both considered as activation sites (52, 53). The mechanisms by which MEKK-1 is phosphorylated and subsequently activated are not well understood, but it has been suggested that ras-related C3 botulinum toxin substrate (Rac1) (54), receptor-interacting protein (55), TNF receptor-associated factor (56), and RhoA (57) by a direct interaction promote MEKK-1 activation.

The precise downstream events to MEKK-1/JNK activation are unclear but may involve phosphorylation of transcription factors c-Jun, Elk, and activating transcription factor (44); induction of p53 and reactive oxygen species (58); release of second mitochondria-derived activator of caspase direct IAP binding protein with low pI (Smac/Diablo) from mitochondria (59); release of the Bcl-2 inhibitor nur77 from nuclei (60); or ubiquitination and degradation of c-Jun (61). It is also possible that MEKK-1 interacts with the ER-stress pathway. Indeed, down-regulation of MEKK-1 presently counteracted islet cell death evoked by the ER-stress inducer tunicamycin. In line with this notion, it has been observed that apoptosis signal-regulating kinase 1, which is also a known activator of JNK, is involved in ER-stress signaling (62). Finally, it has been observed that the β-cell antiapoptotic protein kinase Akt (63) antagonizes the proapoptotic action of MEKK-1 (60), which might indicate that the Akt and MEKK1 pathways are inversely linked to each other.

In summary, our results support an essential role for MEKK-1 in JNK activation and stress-induced β-cell death. Increased understanding of the signaling pathways upstream and downstream MEKK-1 will hopefully improve our understanding of the pathogenesis of type 1 diabetes.


   Footnotes
 
This work was supported in part by the Swedish Research Council (2006-11590), the Swedish Diabetes Association, the family Ernfors Fund, and the Novo-Nordisk Fund.

Disclosure Statement: All of the authors have nothing to disclose.

First Published Online February 28, 2008

Abbreviations: DETA/NO, Diethylenetriamine/nitroso-1-propylhydrazino)-1-propanamine; d-siRNA, diced-small interfering-RNA; EGFP, enhanced GFP; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; MEKK, mitogen-activated protein/ERK kinase; MKK, MAPK kinase; NO, nitric oxide; P, phosphorylated; P-Thr, threonine phosphorylation; SDS, sodium dodecyl sulfate; STZ, streptozotocin; WS, working solution; wt, wild type.

Received April 4, 2007.

Accepted for publication February 15, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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