AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E1052-E1061, 2007. First published December 5, 2006; doi:10.1152/ajpendo.00274.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/E1052    most recent
00274.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chambers, K. T.
Right arrow Articles by Corbett, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chambers, K. T.
Right arrow Articles by Corbett, J. A.

PGJ2-stimulated beta-cell apoptosis is associated with prolonged UPR activation

Kari T. Chambers, Sarah M. Weber, and John A. Corbett

Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri

Submitted 7 June 2006 ; accepted in final form 16 November 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) ligands have been shown to possess anti-inflammatory properties that include the inhibition of transcription factor activation and the expression of inflammatory genes. Using pancreatic beta-cells, we have shown that PPAR{gamma} ligands such as 15-deoxy-{Delta}12,14-prostaglandin J2 (PGJ2) attenuate interferon-{gamma}-induced signal transducer and activator of transcription 1 activation and interleukin (IL)-1beta-induced nuclear factor-{kappa}B activation by a pathway that correlates with endoplasmic reticulum stress and the induction of the unfolded protein response (UPR). The UPR is a conserved cellular response activated by a number of cell stressors and is believed to alleviate the stress and promote cell survival. However, prolonged activation of the UPR results in cellular death by apoptosis. In this report, we have examined the effects of PGJ2 on UPR activation and the consequences of this activation on cell survival. Consistent with induction of a cell death pathway, treatment of rat islets and RINm5F cells for 24 h with PGJ2 results in caspase-3 activation and caspase-dependent beta-cell death. The actions of these ligands do not appear to be selective for beta-cells, because PGJ2 stimulates macrophage apoptosis in a similar fashion. Associated with cell death is the enhanced phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), and in cells expressing a mutant of eIF2{alpha} that cannot be phosphorylated, the stimulatory actions of PGJ2 on caspase-3 activation are augmented. These findings suggest that, whereas PGJ2-induced UPR activation is associated with an inhibition of cytokine signaling, prolonged UPR activation results in cell death, and that eIF2{alpha} phosphorylation may function in a protective manner to attenuate cell death.

15-deoxy-{Delta}12,14-prostaglandin J2; unfolded protein response; signaling

INSULIN-DEPENDENT DIABETES mellitus is characterized by islet inflammation followed by the selective destruction of insulin-secreting pancreatic beta-cells found within the islets of Langerhans (20). Cytokines produced during islet inflammation, such as macrophage-derived interleukin-1 (IL-1) and T-cell-derived interferon-{gamma} (IFN-{gamma}), have been implicated in beta-cell dysfunction and destruction during the development of autoimmune diabetes (7, 17, 37, 49). Nitric oxide is a primary mediator of the inhibitory and destructive actions of cytokines on beta-cells. Alone, IL-1 is sufficient to stimulate inducible nitric oxide synthase (iNOS) expression by rat beta-cells, whereas a combination of IL-1 and IFN-{gamma} is required to stimulate iNOS expression and nitric oxide production by mouse and human beta-cells (13, 26, 27, 54). Nitric oxide impairs glucose-stimulated insulin secretion by the targeted disruption of iron- and sulfur-containing enzymes, such as those in electron transport chain complexes I and II and the tricarboxylic acid cycle enzyme aconitase (14, 58), resulting in reduced cellular levels of ATP. While the role of nitric oxide as a mediator of cytokine-induced beta-cell death has been somewhat controversial (16, 18, 28, 33, 36), two recent studies provide compelling molecular and biochemical evidence that cytokines induce beta-cell death by nitric oxide-dependent necrosis (11, 50).

Because cytokines are believed to participate in beta-cell damage during the development of autoimmune diabetes and in the rejection of transplanted islets (24, 42), recent studies have focused on methods to attenuate cytokine-mediated damage (2, 35, 39, 55, 56). Significant attention has been focused on the anti-inflammatory actions of ligands of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). In vitro, these ligands have been shown to attenuate inflammatory gene expression (10, 12, 15, 29, 52) and inhibit cytokine signaling (29, 35, 52, 56). In addition, these ligands inhibit the natural development of autoimmune diabetes in the nonobese diabetic (NOD) mouse (1) and attenuate neuronal damage in stroke models (4). There are at least two distinct mechanisms by which these ligands attenuate inflammatory gene expression. Initial reports showed that cyclopentenone PPAR{gamma} ligands such as 15-deoxy-{Delta}12,14-prostaglandin J2 (PGJ2) inhibit inflammatory gene expression by inhibiting I{kappa}B kinase (IKK)-dependent activation of nuclear factor-{kappa}B (NF-{kappa}B) (45, 46, 51). A second mechanism of action was required to explain the ability of PGJ2 to attenuate IFN-{gamma}-induced signal transducer and activator of transcription 1 (STAT1) activation (56) and IL-1-stimulated activator protein-1 (AP-1)-dependent gene activation (53). In a series of three reports, our laboratory provided evidence that the inhibitory actions of PGJ2 on cytokine signaling correlate with the activation of a stress response in pancreatic beta-cells as evidenced by the enhanced expression of the heat shock protein 70 (HSP70) (55–57). More recently, our laboratory and others have shown that these ligands are capable of activating the UPR (19, 55) and that, in cells in which the UPR is activated using classic inducers, IL-1 and IFN-{gamma} fail to signal to downstream targets (57).

The UPR is a conserved cellular response activated by endoplasmic reticulum (ER) stresses, such as nutrient deprivation, virus infection, the accumulation of unfolded or misfolded proteins, and reagents such as tunicamycin, thapsigargin, and dithiothreitol that disrupt protein maturation, disrupt ER homeostasis, or prevent normal protein folding, respectively (31). Activation of the UPR results in attenuation of protein synthesis due to double-stranded RNA-dependent kinase-like ER kinase (PERK)-mediated phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}). This event is believed to decrease the load of nascent proteins entering the ER. In addition to PERK, inositol-requiring enzyme-1{alpha} (IRE1{alpha}) and activating transcription factor 6 (ATF6) are also known transducers of the UPR that, along with PERK, participate in the increased transcription of UPR-associated genes, such as chaperones and the ER-associated degradation machinery. UPR activation first allows the cell the opportunity to recover from the stressful insult; however, if the insult is too overwhelming or the UPR is activated for extended periods of time, then apoptotic signaling pathways ensue (47). Although the pathways associated with ER stress-induced apoptosis are not fully characterized, caspase-3 is an executioner caspase that is activated by both intrinsic and extrinsic apoptotic signaling cascades that appears to participate in UPR-mediated cellular apoptosis (5, 59).

In the present study, we have evaluated the effects of PGJ2 and other PPAR{gamma} ligands on the viability of insulinoma RINm5F cells and isolated rat islets. We show that PGJ2 stimulates beta-cell death in a concentration- and time-dependent fashion that correlates with the activation of the UPR and caspase-3. Pharmacological inhibition of caspase-3 activity results in an attenuation of RINm5F cell death in response to PGJ2 treatment. The proapototic effects of PGJ2 do not appear to be cell-type selective because this ligand stimulates the death of macrophages in a similar concentration-dependent manner. Furthermore, expression of a nonfunctional eIF2{alpha} S51A mutant in RINm5F cells results in an enhancement in PGJ2-stimulated caspase-3 activation. These findings suggest that PGJ2 activates the UPR in beta-cells and that prolonged activation results in beta-cell death by apoptosis. In addition, these findings provide evidence that the phosphorylation of eIF2{alpha} may serve to attenuate cell death under conditions associated with UPR activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and animals. Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). RINm5F (rat insulinoma) cells and RAW 264.7 cells (murine macrophage-derived cell line) were obtained from Washington University Tissue Culture Support Center (St. Louis, MO). RPMI 1640, DMEM, CMRL-1066 tissue culture medium, L-glutamine, penicillin, and streptomycin were from GIBCO-BRL (Grand Island, NY). Fetal calf serum was obtained from Hyclone Laboratories (Logan, UT). Human recombinant IL-1beta was obtained from PeproTech (Rocky Hill, NJ). PGJ2, troglitazone, and ciglitazone were from Cayman Chemicals (Ann Arbor, MI). Enhanced chemiluminescence (ECL) reagent was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red, camptothecin, tunicamycin, and staurosporine were obtained from Sigma (St. Louis, MO). In situ death detection kit was obtained from Roche (Indianapolis, IN). Caspase-3 Fluorometric Assay was from R&D Systems (Minneapolis, MN). Caspase-3 inhibitors IV and V were from Calbiochem (San Diego, CA). Rabbit anti-eIF2{alpha}, rabbit anti-phospho-eIF2{alpha}, and mouse anti-HSP70 were obtained from Stressgen (San Diego, CA). Rabbit anti-caspase-3 and rabbit anti-active caspase-3 were obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-STAT1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit and donkey anti-mouse were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). The plasmid pETFVA–-eIF2{alpha} (S51A) was a generous gift from Dr. Randal Kaufman (University of Michigan). All other reagents were obtained from commercially available sources.

Islet isolation and cell culture. Islets were isolated from 250- to 300-g male Sprague-Dawley rats by collagenase digestion as previously described (32). Islets were cultured overnight in complete CMRL-1066 (containing 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin) at 37°C under an atmosphere of 95% air and 5% CO2 before experimentation. RINm5F cells and RAW 264.7 cells were removed from growth flasks by treatment with 0.05% trypsin and 0.02% EDTA for 5 min at 37°C; washed two times with RPMI 1640 or DMEM, respectively; and plated at the indicated density.

Cell viability. Two methods were used to assess cell viability. The MTT assay is based on the ability of viable cells to reduce MTT to insoluble purple formazan crystals (40). For this assay, RINm5F cells (2.0 x 105 cells/400 µl RPMI), RAW 264.7 cells (2.0 x 105 cells/400 µl DMEM), or rat islets (150 islets/400 µl complete CMRL-1066) were washed with culture medium following a 24-h treatment with PGJ2, tunicamycin, or camptothecin, the medium was replaced, and MTT was added at a final concentration of 0.5 mg/ml. Following a 1-h incubation at 37°C, the culture medium was removed and the cells were lysed with 500 µl isopropanol for 1 h. The optical density of the formazan product was determined at a wavelength of 562 nm. Cell viability is presented as changes in optical density (OD).

The accumulation of neutral red dye in viable cells was also used to assess cell viability (6). For this assay, RINm5F cells (1.0 x 105 cells/200 µl RPMI) or RAW 264.7 cells (1.0 x 105 cells/200 µl DMEM) were cultured for 24 h with the indicated concentration of PGJ2, camptothecin, or tunicamycin. The culture supernatants were removed and discarded, and the cells were incubated in fresh medium containing neutral red (50 µg/ml) at 37°C for an additional 2 h. The neutral red solution was removed, cells were washed and fixed with 1% formaldehyde-1% CaCl2 solution, and the intracellular accumulation of neutral red dye was extracted in 100 µl of a 50% ethanol-1% acetic acid lysing solution. The accumulation of neutral red dye in the lysing solution was measured at a wavelength of 540 nm. The percentage of viable cells was determined by comparing neutral red uptake of treated samples to uptake in untreated controls and is presented as percent viable or changes in OD.

TUNEL staining. RINm5F cells and RAW 264.7 cells (2.0 x 105 cells/400 µl culture media) were treated as indicated in the figure legends. The cells were removed from tissue culture plates and centrifuged onto glass slides. DNA damage was determined by transferase-mediated dUTP nick-end labeling (TUNEL) staining according to the manufacturer's instructions using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IN).

Caspase-3 activity. RINm5F cells or RAW 264.7 cells (2.0 x 105 cells/400 µl culture medium) were treated with PGJ2, tunicamycin, and camptothecin for 24 h. Cells were isolated, lysed in 55 µl of supplied lysis buffer, and caspase-3 activity in 50 µl of the cell lysate was determined according to the manufacturer's instructions (Caspase-3 Fluorometric Assay Kit; R&D Systems, Minneapolis, MN). The relative fluorescence units were normalized to total protein content of each sample as determined by the BCA assay (Pierce, Rockford, IL). Samples were then compared with controls, and data are presented as fold increase over control.

Western blot analysis. Protein lysates prepared from RINm5F cells, RAW 264.7 cells, or rat islets were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) under semidry transfer conditions as previously described (26). Antibody dilutions of 1:1,000 were used for the following antibodies: rabbit anti-phospho-eIF2{alpha}, rabbit anti-eIF2{alpha}, mouse anti-HSP70, rabbit anti-caspase-3, rabbit anti-active caspase-3, and rabbit anti-STAT1. HRP-conjugated donkey anti-rabbit and donkey anti-mouse secondary antibodies were used at 1:7,000 and 1:5,000, respectively. Antigens were detected by ECL according to the manufacturer's specifications (Amersham Pharmacia Biotech).

RT-PCR and real-time PCR. Total RNA was isolated from cells using the RNeasy kit (Qiagen, Valencia, CA). First-strand cDNA synthesis was performed using oligo(dT) and reverse transcriptase Superscript Preamplification System (Invitrogen) according to the manufacturer's instructions. Standard PCR was performed as previously described (56). Real-time PCR was performed by inclusion of SYBR Green in standard 25-µl PCR reaction using a Research DNA Engine Opticon II thermocycler with continuous fluorescence detection (MJ Research) per manufacturer's protocol. The fold change in UPR-associated gene expression was determined by comparing unstimulated cells to those treated with 30 µM PGJ2 or 2 µg/ml tunicamycin and normalized to the housekeeping gene GAPDH. Primer sequences for GAPDH and CCAAT/enhancer-binding protein homologous protein (CHOP) have been previously described (25, 55).

Transfection. RINm5F cells were transiently transfected using the Amaxa Nucleofector electroporator (Amaxa Biosystems, Gaithersburg, MD). Briefly, RINm5F cells were removed from growth flasks by treatment with 0.05% trypsin and 0.02% EDTA for 5 min at 37°C, washed two times with RPMI 1640, and incubated for 1 h at 37°C in a 50-ml conical tube. The cells (2.0 x 106) were harvested by centrifugation and resuspended in 100 µl of Amaxa buffer V. Two micrograms each of enhanced green fluorescent protein (pEGFP) and pETFVA–-eIF2{alpha} (S51A) or 2 µg of pEGFP and pETFVA– were added, and electroporation was performed using program G-16. Immediately following electroporation, the cells were transferred to six-well tissue culture plate containing 1 ml of RPMI and cultured overnight at 37°C. The cells were washed three times with 1x PBS and then cultured for an additional 24 h in 2 ml of fresh RPMI before initiation of experiments. Using this method, we routinely obtain a transfection efficiency of >50% as determined by EGFP expression.

Statistics. Statistical comparisons were made between groups using one-way ANOVA. Significant differences between groups, treated compared with unstimulated controls (P < 0.05), were determined by Newman-Keuls post hoc analysis.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PGJ2 treatment induces beta-cell death. Our laboratory has previously reported that PPAR{gamma} ligands stimulate UPR activation in pancreatic beta-cells, and this activation is associated with the inhibition of cytokine signaling (55). Because prolonged activation of the UPR can result in cell death, the effects of PGJ2 on RINm5F cell viability were examined. Treatment of RINm5F cells with PGJ2 for 24 h results in morphological changes that include cell shrinkage, rounding, and detachment from the tissue culture plates. These morphological changes are consistent with the effects of 24-h incubation with the topoisomerase inhibitor and apoptosis inducer camptothecin, suggesting that PGJ2 stimulates RINm5F cell death by apoptosis. Consistent with the effects of PGJ2 and camptothecin, the NH2-linked glycoslyation inhibitor and UPR activator tunicamycin also induces morphological changes (Fig. 1A).


Figure 1
View larger version (59K):
[in this window]
[in a new window]

  		Fig. 1. 15-Deoxy-{Delta}12,14-prostaglandin J2 (PGJ2) stimulates DNA damage and morphological changes in RINm5F consistent with apoptotic cell death. RINm5F cells (2 x 105/400 µl RPMI) were treated with tunicamycin (2 µg/ml), camptothecin (25 µM), or PGJ2 (30 µM) for 24 h and then examined by phase microscopy for morphological alterations (A). Apoptotic cells are indicated by arrows. Under these same conditions, RINm5F cell DNA damage was examined by transferase-mediated dUTP nick-end labeling (TUNEL) staining (B). Results are representative of 3 independent experiments.

 
To further explore the mechanisms of death, the effects of PGJ2, camptothecin, and tunicamycin on the integrity of RINm5F cell DNA were examined by TUNEL staining. As shown in Fig. 1B, PGJ2 stimulates high levels of DNA damage in RINm5F cells as evidenced by the increased level of TUNEL-positive staining. In a similar fashion, camptothecin and tunicamycin also stimulate DNA damage in RINm5F cells. These findings suggest that PGJ2 stimulates RINm5F cell death, and the morphological changes are consistent with the changes stimulated by known inducers of apoptosis and activators of the UPR.

The effects of PGJ2 on beta-cell viability were quantified using the MTT and neutral red assays of cell viability. Consistent with the morphological changes shown in Fig. 1A, there was an ~67% decrease in the ability of RINm5F cells to reduce MTT to formazan crystals when treated for 24 h with PGJ2 (Fig. 2A). The destructive actions of PGJ2 are concentration dependent, as ~91% of RINm5F cells maintain the ability to reduce MTT in response to 3 µM, and this decreases to ~22% in response to 30 µM PGJ2 (Fig. 2B). Similar levels of RINm5F cell death were observed in response to the UPR activator tunicamycin (~55%), whereas the apoptosis inducer camptothecin attenuated MTT reduction by ~76%. To confirm that the decrease in MTT activity in response to PGJ2 is due to cell death and not reduced cell proliferation, a trypan blue exclusion assay was performed. Treatment of RINm5F cells with PGJ2 for 24 h resulted in a 40% loss of cell viability, while tunicamycin and camptothecin reduced cell viability by 30 and 43%, respectively (data not shown). These findings indicate that the reductions in MTT activity in response to PGJ2 treatment are associated with the loss of RINm5F cells viability.


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 2. Biochemical analysis of beta-cell viability following PGJ2 treatment. RINm5F cells (2 x 105/400 µl RPMI; A–C) or isolated rat islets (150 islets/400 µl cCMRL-1066; D) were treated with PGJ2 for 24 h at 37°C. As positive controls, RINm5F cells were also treated with 2 µg/ml tunicamycin (Tuni), a known endoplasmic reticulum stress inducer, and 25 µM camptothecin (Campto), an inducer of apoptosis, for 24 h at 37°C. Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (A, B, and D) and the neutral red uptake assay (C). Results are averages ± SE of 3 independent experiments. Stauro, staurosporine. *P < 0.05. OD, optical density.

 
An additional biochemical assay of cell viability, based on the ability of live cells to accumulate neutral red dye due to an intact plasma membrane, whereas nonviable cells fail to retain neutral red (3), was used to confirm the results obtained using the MTT assay. Somewhat surprisingly, PGJ2 decreases RINm5F cell viability by 91% following a 24-h incubation, a loss of cell viability that is significantly larger than the levels observed in response to tunicamycin (~75%) and camptothecin (~38%) (Fig. 2C).

To confirm that primary beta-cells respond in a manner similar to RINm5F cells, the effects of PGJ2 on islet cell viability were examined using the MTT assay. Similar to RINm5F cells, PGJ2 induces an ~29% decrease in the ability of islet cells to reduce MTT to formazan crystals following a 24-h incubation (Fig. 2D). UPR activation following treatment with tunicamycin also results in an ~33% reduction in the ability of islet cells to reduce MTT, whereas the apoptosis inducer staurosporine stimulates an ~48% decrease in MTT activity of islet cells following a 24-h incubation (Fig. 2D). Staurosporine, a classic inducer of apoptosis (3), was used in place of camptothecin as previous reports have shown that camptothecin is not an effective inducer of islet cell death most likely due to the quiescent nature of primary islets (50). Overall, these findings demonstrate that prolonged incubation of rat islets or RINm5F cells with anti-inflammatory concentrations of PGJ2 results in beta-cell death as evidenced by morphological changes, DNA damage, and two biochemical assays of cell viability.

PGJ2 activates the UPR in beta-cells. Recently, our laboratory provided evidence that UPR activation correlates with an inhibition of cytokine signaling in beta-cells, providing a novel mechanism by which PPAR{gamma} ligands inhibit inflammatory gene expression (55). To further explore whether UPR activation correlates with cell death in response to prolonged incubation with PGJ2, we examined the ability of this ligand to activate PERK, a UPR transducer that has been proposed to participate in ER stress-mediated cell death (23). The expression of CHOP under conditions of ER stress is regulated in part by the activation of PERK and subsequent phosphorylation of eIF2{alpha} (21, 22). Treatment of RINm5F cells with PGJ2 results in both a time- and concentration-dependent increase in the accumulation of CHOP mRNA, which reaches a maximal level of accumulation in response to 30 µM PGJ2 following a 6-h incubation (Fig. 3, A and B). Lower concentrations of PGJ2 (3 µM) also stimulate CHOP mRNA accumulation (2.5-fold increase following a 3-h incubation) to levels comparable to the levels induced by 3-h incubation with tunicamycin (Fig. 3B). Similar changes in the expression of a second PERK-regulated gene, ATF4, were also observed (data not shown). Consistent with the accumulation of CHOP and ATF4 mRNA, PGJ2 stimulates the phosphorylation of eIF2{alpha} (1.9-fold increase over control cells when normalized to STAT1 expression levels) to levels similar to the levels induced by the ER stress activator tunicamycin (2.0-fold increase; Fig. 3C). In addition, a second PPAR{gamma} ligand, troglitazone, also stimulates eIF2{alpha} phosphorylation (2.4-fold increase over control cells), a finding consistent with our laboratory's previous report showing that this ligand also stimulates CHOP expression in RINm5F cells and human islets (55). The stimulatory actions of PGJ2 on eIF2{alpha} phosphorylation are time dependent, first occurring following a 3-h (11-fold increase over control) incubation and persisting for up to 9 h (40-fold increase over control cells; Fig. 3D). In addition, eIF2{alpha} phosphorylation correlates with the activation of a stress response as evidenced by enhanced expression of HSP70 (Fig. 3D). In contrast to the actions of PGJ2, tunicamycin fails to stimulate HSP70 expression (data not shown), whereas it is an effective activator of eIF2{alpha} phosphorylation (Fig. 3C). These findings correlate activation of UPR transducer PERK (or an eIF2{alpha} kinase) with PGJ2-stimulated death of RINm5F cells and rat islets. However, the stress pathways activated by PGJ2 appear to be more complicated than strictly the PERK transducer, because tunicamycin fails to stimulate hsp 70 expression, whereas PGJ2 is effective at stimulating the expression of this heat shock protein.


Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 3. PGJ2 activates the unfolded protein response in RINm5F cells and isolated rat islets. RINm5F cells (A and B) (2 x 105/400 µl RPMI) were treated with the indicated concentrations of PGJ2 and tunicamycin for 3 or 6 h at 37°C. Total RNA was isolated, and CCAAT/enhancer-binding protein homologous protein (CHOP) mRNA accumulation was determined by RT-PCR or real-time PCR (A and B). mRNA accumulation of target genes was normalized to GAPDH mRNA, which is used as a control. RINm5F cells (2 x 105/400 µl RPMI) were treated with PGJ2 (30 µM), troglitazone (Trog; 50 µM), or tunicamycin (2 µg/ml) for 6 h, and eukaryotic initiation factor 2{alpha} (eIF2{alpha}) phosphorylation was determined by Western blot analysis (C). RINm5F cells (2 x 105/400 µl RPMI) were treated with PGJ2 (30 µM) for 3–12 h. The cells were isolated, and heat shock protein 70 (HSP70), phosphorylated eIF2{alpha} (P-eIF2{alpha}), and total eIF2{alpha} (eIF2{alpha}) levels were determined by Western blot analysis (D). Results are representative of 3 independent experiments. STAT1, signal transducer and activator of transcription 1.

 
PGJ2 stimulates caspase-3-dependent beta-cell death. Because PGJ2 induces UPR activation and beta-cell death, and it has been reported that prolonged UPR activation results in cell death by an apoptotic mechanism, the effects of PGJ2 on the processing and enzymatic activity of caspase-3 were examined. Caspase-3 was chosen as a target because this effector caspase participates in both the intrinsic and extrinsic signaling pathways of apoptosis and it has been shown to participate in ER stress-mediated cell death (5, 47, 59). In response to PGJ2, caspase-3 is processed from the inactive pro form (35 kDa) to the mature and active (17 kDa) protease, as determined by Western blot analysis using antibodies specific for the cleaved fragment (17 kDa) and pro-caspase-3 (37 kDa; Fig. 4A). The cleavage of caspase-3 in response to PGJ2 is concentration-dependent and correlates with the levels of cell death induced by this PPAR{gamma} ligand (see Fig. 1D). The maximal cleavage of caspase-3 was observed in response to a 24 h incubation with 30 µM PGJ2, and the levels of cleavage are similar in magnitude to the levels stimulated by the apoptosis inducer camptothecin (Fig. 4A). The UPR activator tunicamycin also stimulates caspase-3 cleavage to levels comparable to the levels induced by 15 µM PGJ2. As an additional control, we show that IL-1 fails to stimulate caspase-3 cleavage in RINm5F cells following a 24-h incubation. The latter finding is consistent with recent evidence suggesting that beta-cell death in response to IL-1 occurs by caspase-3-independent necrosis (11, 50).


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 4. PGJ2 stimulates the activation of caspase-3. RINm5F cells (2 x 105/400 µl RPMI) were treated with indicated concentrations of PGJ2, tunicamycin, camptothecin, and interleukin-1 (IL-1) for 24 h at 37°C. The cells were isolated, and active caspase-3 (17-kDa, cleavage product) and pro-caspase-3 (35 kDa) levels were determined by Western blot analysis (A). Caspase-3 enzymatic activity was determined using the fluorometric caspase-3 activity kit (R&D Systems) in RINm5F cells treated for 24 h with PGJ2, tunicamycin, or camptothecin in the presence or absence of the caspase-3 inhibitor IV (Casp-3 Inh IV; B). RINm5F cells (2 x 105/400 µl RPMI) were pretreated with 50 µM caspase-3 inhibitor V for 30 min, PGJ2 was added, and cell viability was determined by MTT assay following a 24-h incubation (C). Results are the representative (A) or the average ± SE of 2 (C) or 3 independent experiments (B).

 
The effects of PGJ2 on caspase-3 activity were determined to confirm that caspase-3 cleavage correlates with activation of this protease. In a concentration-dependent manner, PGJ2 activates caspase-3 with a maximal fivefold increase in activity in response to 30 µM (Fig. 4B). Caspase-3 activity following treatment with PGJ2 is sensitive to caspase-3 inhibitor IV (Ac-DQMD-CHO). Similar to the effects of PGJ2 on caspase-3 activity, both the UPR inducer tunicamycin and the apoptosis inducer camptothecin stimulate caspase 3-activity, and this activity is also sensitive to this caspase-3 inhibitor (Fig. 4B).

Because PGJ2-mediated beta-cell death (Fig. 2) correlates with the cleavage and activation of caspase-3 (Fig. 4, A and B), the effects of pharmacological inhibition of caspase-3 on viability of beta-cells following PGJ2 treatment were examined using the MTT assay. Treatment of RINm5F cells for 24 h with PGJ2 (15 µM) results in the death of ~55% of beta-cells. When the cells are pretreated for 30 min with 50 µM caspase-3 inhibitor V [Z-D(OMe)-QMD(Ome)-CH2F], beta-cell death in response to PGJ2 is reduced by >50%. At a concentration of 50 µM, the caspase inhibitor alone induces the death of ~15% of beta-cells, suggesting that the protective actions against PGJ2-mediated beta-cell death may be underrepresented in this experiment (Fig. 4C). In response to higher concentrations of PGJ2 (30 µM), this caspase inhibitor fails to prevent PGJ2-mediated beta-cell death (data not shown). Based on the observations that >70% of beta-cells are killed in response to 30 µM PGJ2 (Fig. 2) and that the caspase inhibitor prevents caspase-3 activation under these conditions, it is likely that the robust nature of the PGJ2-stimulated apoptotic signals at this concentration of PGJ2 results in the activation of multiple caspases that function to mediate beta-cell apoptosis.

Ligand- and cell-type specificity of cell death. To determine whether the damaging actions of PGJ2 are selective for this PPAR{gamma} ligand, the effects of additional thiazolidinediones (TZDs) on beta-cell viability were examined. Our laboratory has previously reported that troglitazone and ciglitazone at concentrations of 50 µM stimulate UPR activation as evidenced by an increase in UPR gene expression and the phosphorylation of eIF2{alpha} (55). We now show that, following a 24-h incubation, each of these ligands induce the death of ~25–35% of RINm5F cells (Fig. 5). While the levels of cell death in response to troglitazone and ciglitazone are lower than the levels induced by PGJ2, these findings provide evidence that multiple members of this ligand class, including TZDs, known to activate the UPR (19, 55) also induce beta-cell death.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 5. Effects of additional peroxisome proliferator-activated receptor-{gamma} ligands on beta-cell viability. RINm5F cells (2 x 105/400 µl RPMI) were treated with the indicated concentrations of PGJ2, troglitazone, ciglitazone, or tunicamycin for 24 h at 37°C, and cell viability was determined by the MTT assay. Results are averages ± SE of 4 independent experiments. *P < 0.05.

 
To examine cell-type specificity, the effects of PGJ2 on macrophage viability and UPR activation were examined. In a concentration-dependent manner, PGJ2 induces RAW 264.7 cell death that is first apparent at 3 µM (~15%) and maximal in response to 30 µM PGJ2 (~75%) following a 24-h incubation (Fig. 6A). The levels of cell death induced by PGJ2 are comparable to the levels induced by tunicamycin and camptothecin (Fig. 6A). Similar results were obtained using neutral red uptake as a second assay of cell death (Fig. 6B). Consistent with the effects of PGJ2 on RINm5F cell death, treatment of RAW 264.7 cells with PGJ2 for 24 h results in an approximately fivefold increase in caspase-3 activity, and the levels of caspase-3 activity induced by PGJ2 are approximately twofold higher than the levels induced by tunicamycin and twofold lower than the level induced by camptothecin (Fig. 6C). These findings indicate that PGJ2 stimulates macrophage cell death in a concentration-dependent manner that is associated with enhanced caspase-3 activation.


Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 6. PGJ2 stimulates caspase-3 activation and induces RAW 264.7 cell death. RAW 264.7 cells (2 x 105/400 µl DMEM) were treated with the indicated concentrations of PGJ2, tunicamycin, and camptothecin for 24 h at 37°C, and cell viability was then evaluated by the MTT assay (A) and the neutral red uptake assay (B). The effects of PGJ2, tunicamycin, and camptothecin on the activation of caspase-3 in RAW 264.7 cells were determined using the fluorometric substrate kit (R&D Systems; C). Results are averages ± SE of 3 independent experiments. *P < 0.05.

 
Role of eIF2{alpha} phosphorylation in PGJ2-induced beta-cell death. Because the attenuation of translation has been associated with apoptotic cell death (23), we hypothesized that eIF2{alpha} phosphorylation and the subsequent attenuation of translation may contribute to PGJ2-mediated beta-cell death. Additionally, eIF2{alpha} phosphorylation has previously been shown to have a protective role in cells (8, 30, 34). To examine this possibility, RINm5F cells were transiently transfected with a nonphosphorylatable eIF2{alpha} mutant (S51A), PGJ2 was added, and caspase-3 activation was examined following a 24-h incubation. In this experiment, 15 µM PGJ2 fails to induce caspase-3 activity in RINm5F cells transfected with the vector control. However, in RINm5F cells expressing the S51A mutant, PGJ2 stimulates an approximately twofold increase in caspase-3 activity (Fig. 7). In a similar fashion, there is an approximately threefold increase in caspase-3 activation by 30 µM PGJ2 in RINm5F cells expressing the S51A eIF2{alpha} mutant compared with vector control. Furthermore, tunicamycin-stimulated caspase-3 activity is also enhanced in RINm5F cells expressing this nonphosphorylatable eIF2{alpha} mutant compared with vector controls. In contrast to our original hypothesis that the inhibition of translation by eIF2{alpha} phosphorylation may attenuate PGJ2-stimulated cell death, translation inhibition may provide a protective mechanism against ER stress-mediated apoptotic cell death. In support of this view, PGJ2-induced caspase-3 activation is enhanced in cells expressing the S51A eIF2{alpha} mutant.


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 7. Expression of an S51A eIF2{alpha} mutant enhances PGJ2-stimulated caspase-3 activity in RINm5F cells. RINm5F cell were transfected with the plasmid (p) ETFVA vector containing the S51A eIF2{alpha} mutant and enhanced green fluorescent protein (pEGFP) or pETFVA empty vector and pEGFP. Cells were then treated with the indicated concentrations of PGJ2 and tunicamycin for 24 h, and caspase-3 activity was determined. Results are averages ± SE of 3 independent experiments. Statistically significant differences between S51A mutant compared with empty vector-expressing cells are as indicated (*P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Agonists of PPAR{gamma}, such as PGJ2, have been shown to attenuate the proinflammatory effects that are associated with lipopolysaccharide treatment of macrophages, including the inhibition of inflammatory gene expression and the activation of NF-{kappa}B (10, 12, 15, 29, 52). The ability of PGJ2 to inhibit NF-{kappa}B activation is associated with attenuation in the activation of IKK as well as repression of proinflammatory genes (9, 52). Additional mechanisms of action must exist because PGJ2 attenuates IL-1-induced mitogen-activating protein kinase activation (35, 55), IL-1-induced AP-1 reporter activity (53), and IFN-{gamma}-induced STAT1 phosphorylation (55–57). One such mechanism appears to be the induction of cellular stress. PGJ2 has been shown to stimulate HSP70 expression in a number of cell types, and our laboratory has correlated the inhibitory actions of PGJ2 on cytokine signaling with the enhanced expression of HSP70 (55–57). Importantly, HSP70 does not mediate the inhibitory actions of PGJ2 on cytokine signaling, because antisense depletion of HSP70 fails to attenuate the inhibitory actions of this ligand on IL-1 or IFN-{gamma} signaling in RINm5F cells (56). In addition, the inhibitory actions of PGJ2 on cytokine signaling occur by PPAR{gamma}-independent mechanisms, as expression of dominant negative PPAR{gamma} mutants that attenuate PPAR{gamma} reporter activity fail to attenuate the inhibitory actions of PGJ2 on cytokine signaling (57).

In an effort to identify the mechanisms by which PGJ2 attenuates cytokine signaling, we observed that this ligand attenuates protein synthesis in RINm5F cells and isolated rat islets (55). The attenuation of protein synthesis is one hallmark of UPR activation, as ER stress stimulates eIF2{alpha} phosphorylation, resulting in a translational blockade (21–23). It has now been reported in a number of cell types that PGJ2 is capable of stimulating the expression of ER stress-responsive genes such as CHOP and glucose-regulated protein 78 (BiP), the phosphorylation of eIF2{alpha}, and the attenuation of translation (19, 38, 48, 55).

Because prolonged UPR activation results in cell death and PGJ2 stimulates the activation of the UPR, the effects of PGJ2 on beta-cell viability were examined. Using multiple biochemical (MTT and neutral red assays) and morphological (TUNEL and phase-contrast microscopy) assays, we show that PGJ2 stimulates beta-cell death in a fashion that mimics death in response to classic activators of the UPR (tunicamycin) and apoptosis (camptothecin). beta-Cell death in response to PGJ2 is associated with UPR activation as evidenced by enhanced CHOP expression and eIF2{alpha} phosphorylation. PGJ2 appears to stimulate beta-cell death by a caspase-3-dependent apoptotic process, as evidenced by the processing of pro-caspase-3 and a fivefold increase in caspase-3 activity in RINm5F cells treated with PGJ2. Importantly, PGJ2-mediated beta-cell death is attenuated by the pharmacological inhibition of caspase-3. The deleterious actions of PGJ2 on cell viability do not appear to be selective for beta-cells because PGJ2 also induces death of RAW 264.7 cell, a murine macrophage-derived cell line.

The identity of the UPR transducer responsible for triggering caspase-3-dependent beta-cell death in response to PGJ2 is currently unknown. We have examined the potential role of eIF2{alpha} kinase-mediated phosphorylation of eIF2{alpha} using a well characterized Ser-Ala mutant that is incapable of being phosphorylated (41). In this experiment, overexpression of this mutant enhanced PGJ2-stimulated caspase-3 activity as well as tunicamycin-induced caspase-3 activity. These results suggest that eIF2{alpha} phosphorylation and attenuation of translation do not function as a trigger for caspase-dependent cell death. In contrast, we interpret these result to suggest that eIF2{alpha} phosphorylation may provide protection from ER stress-mediated apoptosis and that additional arms of the UPR are responsible for the induction of apoptosis. We are currently examining the potential roles of ATF6 and IRE1{alpha} as mediators of PGJ2 stimulated beta-cell death.

The mechanisms by which PGJ2 stimulates UPR activation are also currently unknown. Previous studies have shown that PGJ2 is capable of stimulating UPR activation in RINm5F cells expressing a dominant negative PPAR{gamma} mutant or in cells treated with the PPAR{gamma} antagonist GW-9622 (57). These findings support a PPAR{gamma}-independent mechanism of UPR activation. PGJ2 has been shown to localize to the ER, and this localization is associated with enhanced expression of GRP78 (BiP) and protein disulfide isomerase (43, 44), suggesting that the actions of PGJ2 are ER directed. Our findings suggest that PGJ2 may exert effects directly or indirectly at the plasma membrane as the levels of cell death induced by PGJ2 significantly differ depending on the biochemical assay being performed. In response to a 24-h incubation with PGJ2 or tunicamycin, ~60 or ~55%, respectively, of RINm5F cells are killed as determined by the MTT assay. Using the neutral red assay <25% of the RINm5F cells survive a 24-h treatment of PGJ2, yet 75% of the cells survive in response to tunicamycin (Fig. 2). Because the neutral red assay is a measure of plasma membrane integrity, these findings suggest that the effects of PGJ2 on UPR activation and cell death may be a consequence of changes that occur at the plasma membrane. One potential target enzyme that may mediate this response is tissue transglutaminase, because the activation of this enzyme would result in the cross-linking of plasma membrane proteins to levels that could be sufficient to activate the UPR. Consistent with this potential mechanism of action, in preliminary studies we have observed a greater than twofold increase in tissue transglutaminase activity in RINm5F cells treated for 6 h with PGJ2 (unpublished observation, S. M. Weber and J. A. Corbett). We are currently exploring the role of tissue transglutaminase as a potential mediator of UPR activation in RINm5F cells in response to PGJ2 treatment.

It is clear that the stress response activated by PGJ2 differs from the response stimulated by the classic UPR activator tunicamycin. As described in the preceding paragraph, the levels of cell death induced by PGJ2 and tunicamycin differ dramatically when assessed by neutral red uptake, but the levels are very similar when evaluated by MTT. In addition, PGJ2 stimulates, whereas tunicamycin fails to stimulate, HSP70 expression in beta-cells. Whereas the mechanisms of UPR activation and the type of response stimulated by PGJ2 may differ from that of the classic UPR inducer tunicamycin, the net outcome of UPR activation by each stimulus is very similar. Our laboratory has previously shown that induction of ER stress and UPR activation by both tunicamycin and PGJ2 results in attenuation in the ability of cytokines to signal to downstream targets (55). In the current study, we show that both PGJ2 and tunicamycin stimulate beta-cell apoptosis due to prolonged activation of the UPR, and that attenuation of PERK/eIF2{alpha} activation augments cell death in response to both stimuli.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-52194, DK-068839, and AI-44458 (to J. A. Corbett). K. T. Chambers was supported by an American Heart Association predoctoral fellowship award.


   ACKNOWLEDGMENTS
 
We thank Colleen Kelly Bratcher for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Corbett, Saint Louis University School of Medicine, Dept. of Biochemistry and Molecular Biology, 1402 South Grand Blvd., St. Louis, MO 63104 (e-mail: corbettj{at}slu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Beales PE, Liddi R, Giorgini AE, Signore A, Procaccini E, Batchelor K, Pozzilli P. Troglitazone prevents insulin dependent diabetes in the non-obese diabetic mouse. Eur J Pharmacol 357: 221–225, 1998.[CrossRef][ISI][Medline]
  2. Bellmann K, Jaattela M, Wissing D, Burkart V, Kolb H. Heat shock protein hsp70 overexpression confers resistance against nitric oxide. FEBS Lett 391: 185–188, 1996.[CrossRef][ISI][Medline]
  3. Bertrand R, Solary E, O'Connor P, Kohn KW, Pommier Y. Induction of a common pathway of apoptosis by staurosporine. Exp Cell Res 211: 314–321, 1994.[CrossRef][ISI][Medline]
  4. Blanco M, Moro MA, Davalos A, Leira R, Castellanos M, Serena J, Vivancos J, Rodriguez-Yanez M, Lizasoain I, Castillo J. Increased plasma levels of 15-deoxyDelta prostaglandin J2 are associated with good outcome in acute atherothrombotic ischemic stroke. Stroke 36: 1189–1194, 2005.[Abstract/Free Full Text]
  5. Boatright KM, Salvesen GS. Mechanisms of caspase activation. Curr Opinion Cell Biol 15: 725–731, 2003.[CrossRef][ISI][Medline]
  6. Borenfreund E, Shopsis C. Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica 15: 705–711, 1985.[ISI][Medline]
  7. Bottazzo GF, Dean BM, McNally JM, MacKay EH, Swift PG, Gamble DR. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N Engl J Med 313: 353–360, 1985.[Abstract]
  8. Boyce M, Bryant KF, Jousse C, Long K, Harding HP, Scheuner D, Kaufman RJ, Ma D, Coen DM, Ron D, Yuan J. A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307: 935–939, 2005.[Abstract/Free Full Text]
  9. Bradshaw G, Gutierrez A, Miyake JH, Davis KR, Li AC, Glass CK, Curtiss LK, Davis RA. Facilitated replacement of Kupffer cells expressing a paraoxonase-1 transgene is essential for ameliorating atherosclerosis in mice. Proc Natl Acad Sci USA 102: 11029–11034, 2005.[Abstract/Free Full Text]
  10. Chawla A, Barak Y, Nagy L, Liao D, Tontonoz P, Evans RM. PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation [see comment]. Nat Med 7: 48–52, 2001.[CrossRef][ISI][Medline]
  11. Collier JJ, Fueger PT, Hohmeier HE, Newgard CB. Pro- and antiapoptotic proteins regulate apoptosis but do not protect against cytokine-mediated cytotoxicity in rat islets and beta-cell lines. Diabetes 55: 1398–1406, 2006.[Abstract/Free Full Text]
  12. Colville-Nash PR, Qureshi SS, Willis D, Willoughby DA. Inhibition of inducible nitric oxide synthase by peroxisome proliferator-activated receptor agonists: correlation with induction of heme oxygenase 1. J Immunol 161: 978–984, 1998.[Abstract/Free Full Text]
  13. Corbett JA, Kwon G, Marino MH, Rodi CP, Sullivan PM, Turk J, McDaniel ML. Tyrosine kinase inhibitors prevent cytokine-induced expression of iNOS and COX-2 by human islets. Am J Physiol Cell Physiol 270: C1581–C1587, 1996.[Abstract/Free Full Text]
  14. Corbett JA, Wang JL, Sweetland MA, Lancaster JR Jr, McDaniel ML. Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide. J Clin Investig 90: 2384–2391, 1992.[ISI][Medline]
  15. Delerive P, Fruchart JC, Staels B. Peroxisome proliferator-activated receptors in inflammation control. J Endocrinol 169: 453–459, 2001.[Abstract]
  16. Eizirik DL, Darville MI. Beta-cell apoptosis and defense mechanisms: lessons from type 1 diabetes. Diabetes 50, Suppl 1: S64–S69, 2001.[ISI][Medline]
  17. Eizirik DL, Flodstrom M, Karlsen AE, Welsh N. The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic beta cells. Diabetologia 39: 875–890, 1996.[ISI][Medline]
  18. Eizirik DL, Mandrup-Poulsen T. A choice of death—the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia 44: 2115–2133, 2001. [Corrigenda. Diabetologia 45: June 2002, p. 936.][CrossRef][ISI][Medline]
  19. Gardner OS, Shiau CW, Chen CS, Graves LM. Peroxisome proliferator-activated receptor gamma-independent activation of p38 MAPK by thiazolidinediones involves calcium/calmodulin-dependent protein kinase II and protein kinase R: correlation with endoplasmic reticulum stress. J Biol Chem 280: 10109–10118, 2005.[Abstract/Free Full Text]
  20. Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes 14: 619–633, 1965.[ISI][Medline]
  21. Harding HP, Calfon M, Urano F, Novoa I, Ron D. Transcriptional and translational control in the mammalian unfolded protein response. Annu Rev Cell Dev Biol 18: 575–599, 2002.[CrossRef][ISI][Medline]
  22. Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6: 1099–1108, 2000.[CrossRef][ISI][Medline]
  23. Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 5: 897–904, 2000.[CrossRef][ISI][Medline]
  24. Heitmeier MR, Corbett JA. Cytotoxic role of nitric oxide in diabetes. In: Nitric Oxide Biology and Pathobiology. San Diego, CA: Academic, 2000, p. 785–810.
  25. Heitmeier MR, Scarim AL, Corbett JA. Double-stranded RNA-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine macrophages requires NF-kappaB activation. J Biol Chem 273: 15301–15307, 1998.[Abstract/Free Full Text]
  26. Heitmeier MR, Scarim AL, Corbett JA. Interferon-gamma increases the sensitivity of islets of Langerhans for inducible nitric-oxide synthase expression induced by interleukin 1. J Biol Chem 272: 13697–13704, 1997.[Abstract/Free Full Text]
  27. Heitmeier MR, Scarim AL, Corbett JA. Prolonged STAT1 activation is associated with interferon-gamma priming for interleukin-1-induced inducible nitric-oxide synthase expression by islets of Langerhans. J Biol Chem 274: 29266–29273, 1999.[Abstract/Free Full Text]
  28. Heller B, Wang ZQ, Wagner EF, Radons J, Burkle A, Fehsel K, Burkart V, Kolb H. Inactivation of the poly(ADP-ribose) polymerase gene affects oxygen radical and nitric oxide toxicity in islet cells. J Biol Chem 270: 11176–11180, 1995.[Abstract/Free Full Text]
  29. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82–86, 1998.[CrossRef][Medline]
  30. Jiang HY, Wek RC. GCN2 phosphorylation of eIF2alpha activates NF-kappaB in response to UV irradiation. Biochem J 385: 371–380, 2005.[CrossRef][ISI][Medline]
  31. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest 110: 1389–1398, 2002.[CrossRef][ISI][Medline]
  32. Kelly CB, Blair LA, Corbett JA, Scarim AL. Isolation of islets of Langerhans from rodent pancreas. Methods Mol Med 83: 3–14, 2003.[Medline]
  33. Liu D, Pavlovic D, Chen MC, Flodstrom M, Sandler S, Eizirik DL. Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS–/–). Diabetes 49: 1116–1122, 2000.[Abstract]
  34. Lu PD, Jousse C, Marciniak SJ, Zhang Y, Novoa I, Scheuner D, Kaufman RJ, Ron D, Harding HP. Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2. EMBO J 23: 169–179, 2004.[CrossRef][ISI][Medline]
  35. Maggi LB Jr, Sadeghi H, Weigand C, Scarim AL, Heitmeier MR, Corbett JA. Anti-inflammatory actions of 15-deoxy-delta 12,14-prostaglandin J2 and troglitazone: evidence for heat shock-dependent and -independent inhibition of cytokine-induced inducible nitric oxide synthase expression. Diabetes 49: 346–355, 2000.[Abstract]
  36. Mandrup-Poulsen T. Beta-cell apoptosis: stimuli and signaling. Diabetes 50, Suppl 1: S58–S63, 2001.[ISI][Medline]
  37. Mandrup-Poulsen T. The role of interleukin-1 in the pathogenesis of IDDM. Diabetologia 39: 1005–1029, 1996.[Medline]
  38. Maniratanachote R, Minami K, Katoh M, Nakajima M, Yokoi T. Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines. Toxicol Sci 83: 293–302, 2005.[Abstract/Free Full Text]
  39. Margulis BA, Sandler S, Eizirik DL, Welsh N, Welsh M. Liposomal delivery of purified heat shock protein hsp70 into rat pancreatic islets as protection against interleukin 1 beta-induced impaired beta-cell function. Diabetes 40: 1418–1422, 1991.[Abstract]
  40. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63, 1983.[CrossRef][ISI][Medline]
  41. Murtha-Riel P, Davies MV, Scherer BJ, Choi SY, Hershey JW, Kaufman RJ. Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis. J Biol Chem 268: 12946–12951, 1993.[Abstract/Free Full Text]
  42. Oberholzer J, Shapiro AM, Lakey JR, Ryan EA, Rajotte RV, Korbutt GS, Morel P, Kneteman NM. Current status of islet cell transplantation. Adv Surg 37: 253–282, 2003.[Medline]
  43. Odani N, Negishi M, Takahashi S, Ichikawa A. Induction of protein disulfide isomerase mRNA by delta 12-prostaglandin J2. Biochem Biophys Res Commun 220: 264–268, 1996.[CrossRef][ISI][Medline]
  44. Odani N, Negishi M, Takahashi S, Kitano Y, Kozutsumi Y, Ichikawa A. Regulation of BiP gene expression by cyclopentenone prostaglandins through unfolded protein response element. J Biol Chem 271: 16609–16613, 1996.[Abstract/Free Full Text]
  45. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391: 79–82, 1998.[CrossRef][Medline]
  46. Rossi A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, Santoro MG. Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase. Nature 403: 103–108, 2000.[CrossRef][Medline]
  47. Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress. Trends Cell Biol 14: 20–28, 2004.[CrossRef][ISI][Medline]
  48. Satoh T, Toyoda M, Hoshino H, Monden T, Yamada M, Shimizu H, Miyamoto K, Mori M. Activation of peroxisome proliferator-activated receptor-gamma stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells. Oncogene 21: 2171–2180, 2002. [Corrigendum. Oncogene 21: November 2002, p. 8220.][CrossRef][ISI][Medline]
  49. Sibley RK, Sutherland DE, Goetz F, Michael AF. Recurrent diabetes mellitus in the pancreas iso- and allograft. A light and electron microscopic and immunohistochemical analysis of four cases. Lab Invest 53: 132–144, 1985.[ISI][Medline]
  50. Steer SA, Scarim AL, Chambers KT, Corbett JA. Interleukin-1 stimulates beta-cell necrosis and release of the immunological adjuvant HMGB1. PLoS Med 3: e17, 2006.[CrossRef][Medline]
  51. Straus DS, Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Medicinal Res Rev 21: 185–210, 2001.[CrossRef][ISI][Medline]
  52. Straus DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Ghosh G, Glass CK. 15-Deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway. Proc Natl Acad Sci USA 97: 4844–4849, 2000.[Abstract/Free Full Text]
  53. Subbaramaiah K, Lin DT, Hart JC, Dannenberg AJ. Peroxisome proliferator-activated receptor gamma ligands suppress the transcriptional activation of cyclooxygenase-2. Evidence for involvement of activator protein-1 and CREB-binding protein/p300. J Biol Chem 276: 12440–12448, 2001.[Abstract/Free Full Text]
  54. Thomas HE, Darwiche R, Corbett JA, Kay TW. Interleukin-1 plus gamma-interferon-induced pancreatic beta-cell dysfunction is mediated by beta-cell nitric oxide production. Diabetes 51: 311–316, 2002.[Abstract/Free Full Text]
  55. Weber SM, Chambers KT, Bensch KG, Scarim AL, Corbett JA. PPAR{gamma} ligands induce ER stress in pancreatic beta-cells: ER stress activation results in attenuation of cytokine signaling. Am J Physiol Endocrinol Metab 287: E1171–E1177, 2004.[Abstract/Free Full Text]
  56. Weber SM, Scarim AL, Corbett JA. Inhibition of IFN-{gamma}-induced STAT1 activation by 15-deoxy-{Delta}12,14-prostaglandin J2. Am J Physiol Endocrinol Metab 284: E883–E891, 2003.[Abstract/Free Full Text]
  57. Weber SM, Scarim AL, Corbett JA. PPAR{gamma} is not required for the inhibitory actions of PGJ2 on cytokine signaling in pancreatic beta-cells. Am J Physiol Endocrinol Metab 286: E329–E336, 2004.[Abstract/Free Full Text]
  58. Welsh N, Eizirik DL, Bendtzen K, Sandler S. Interleukin-1 beta-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase. Endocrinology 129: 3167–3173, 1991.[Abstract]
  59. Wilson MR. Apoptotic signal transduction: emerging pathways. Biochem Cell Biol 76: 573–582, 1998.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/E1052    most recent
00274.2006v1