Inhibition of IFN-gamma -induced STAT1 activation by 15- deoxy-Delta 12,14-prostaglandin J2

Sarah M. Weber, Anna L. Scarim, and John A. Corbett

Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The inhibitory actions of 15-deoxy-Delta 12,14-prostaglandin J2 (PGJ2) on inflammatory gene expression have been attributed to the ability of this prostaglandin to inhibit the activation of NF-kappa B. In this study, we have identified an additional signaling pathway sensitive to inhibition by PGJ2. We show that PGJ2 inhibits interferon (IFN)-gamma -stimulated phosphorylation and DNA-binding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1- to 2-h incubation and are maximal after a 6-h incubation with PGJ2, and they correlate with the expression of heat shock protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ2 on inducible nitric oxide synthase (iNOS) expression and NF-kappa B activation in response to IL-1 with the increased expression of HSP70. Using overexpression and antisense depletion, we provide evidence that HSP70 does not mediate the inhibitory actions of PGJ2 on IL-1-induced NF-kappa B or IFN-gamma -induced STAT1 activation or cytokine-stimulated iNOS expression by beta -cells. Last, we show that the inhibitory actions of a short 6-h pulse with PGJ2 on IL-1 plus IFN-gamma -stimulated iNOS expression and NO production by beta -cells are persistent for extended periods (<= 48 h). These findings suggest that PGJ2 inhibits multiple cytokine-signaling pathways (IL-1 and IFN-gamma ), that the inhibitory actions are persistent for extended periods, and that increased HSP70 expression correlates with, but does not appear to mediate, the inhibitory actions of PGJ2 on IL-1 and IFN-gamma signaling in beta -cells.

interferon-gamma ; signal transducer and activator of transcription-1; peroxisome proliferator-activated receptor-gamma


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

AUTOIMMUNE DIABETES IS CHARACTERIZED by islet inflammation followed by the selective destruction of insulin-secreting beta -cells found in pancreatic islets of Langerhans (14). Cytokines released from inflammatory macrophages and T cells are believed to participate in beta -cell damage during diabetes development (3, 35). Treatment of rat islets with IL-1 results in a time- and concentration-dependent inhibition of glucose-stimulated insulin secretion that is followed by islet degeneration (8, 22, 29). The inhibitory and destructive actions of IL-1 on beta -cell function correlate with the time-dependent expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide and can be prevented by inhibitors of iNOS (10, 12, 36). Alone, interferon (IFN)-gamma does not appear to modulate beta -cell function; however, IFN-gamma has been shown to both prime and potentiate the stimulatory actions of IL-1 on iNOS expression by rat islets (19, 21). In addition, a combination of IL-1 and IFN-gamma is required to stimulate iNOS expression by mouse and human beta -cells (6, 41). Nitric oxide, produced following iNOS expression, impairs beta -cell function by inhibiting mitochondrial oxidative metabolism, and this inhibition in mitochondrial function, coupled with the ability of nitric oxide to induce DNA damage, appears to mediate islet cell death (12, 18, 24).

In an effort to identify mechanisms to protect beta -cells from cytokine-mediated damage, we have shown that IL-1 fails to inhibit glucose-stimulated insulin secretion by rat islets heat shocked before cytokine stimulation (33). The protective actions of heat shock on beta -cell function are associated with the accumulation of heat shock protein (HSP)70, the inhibition of IL-1-induced NF-kappa B activation, and the inhibition of IL-1-induced iNOS expression (33). Consistent with the protective actions of heat shock, liposomal delivery of HSP70 has been shown to prevent the inhibitory actions of IL-1 on glucose-stimulated insulin secretion (27), and the overexpression of HSP70 in insulinoma cell lines prevents the damaging actions of nitric oxide, delivered exogenously, on cell viability (1). These findings suggest that HSP70 may protect beta -cells from the damaging actions of cytokines by inhibiting signaling pathways activated by cytokine treatment or by attenuating the damaging actions of free radicals on beta -cell function.

Peroxisome proliferator-activated receptor-gamma (PPARgamma ) agonists are known insulin sensitizers that have been shown to stimulate the expression of HSP70 in a number of cell types, including RINm5F cells and islets (5, 11, 26). We have shown that a 6-h pretreatment of RINm5F cells or rat islets with the naturally occurring PPARgamma agonist 15-deoxy-Delta 12,14-prostaglandin J2 (PGJ2) and the synthetic agonist troglitazone stimulates HSP70 expression and that HSP70 expression correlates with an impairment of IL-1-induced NF-kappa B activation and JNK phosphorylation (26). In contrast, a 30-min preincubation of islets or RINm5F cells with either agonist does not stimulate HSP70 expression and also does not inhibit the ability of IL-1 to stimulate JNK phosphorylation or NF-kappa B activation (26). Importantly, NF-kappa B activation is required for beta -cell expression of iNOS in response to IL-1 treatment (16, 17, 25). Although previous studies have shown that PGJ2 attenuates NF-kappa B activation by directly inhibiting Ikappa B kinase (IKK) as well as inhibiting NF-kappa B DNA binding activity (30, 31, 38), this direct inhibition of IKK or NF-kappa B DNA binding activity does not appear to account for the inhibitory actions of PGJ2 on NF-kappa B activation in RINm5F cells or rat islets, as a 30-min pretreatment with PGJ2 does not affect the ability of IL-1 to stimulate the activation of NF-kappa B (26).

In this study, the potential role of PGJ2 as an inhibitor of IFN-gamma -stimulated signaling pathways and whether the inhibitory actions of PGJ2 are mediated by the increased expression of HSP70 in isolated rat islets as well as insulinoma RINm5F cells have been examined. Evidence is presented indicating that pretreatment of RINm5F cells and rat islets for 6 h with PGJ2 results in the attenuation of IFN-gamma -induced STAT1 phosphorylation, nuclear localization, and DNA binding. Although the inhibitory actions of PGJ2 on IFN-gamma -stimulated STAT1 activation correlate with the time-dependent expression of HSP70, antisense depletion of HSP70 does not influence the ability of PGJ2 to inhibit IFN-gamma -stimulated STAT1 activation. In addition, overexpression of HSP70 in RINm5F cells does not modulate the stimulatory actions of IL-1 on NF-kappa B or IFN-gamma on STAT1 activation. These findings suggest that the inhibitory actions of PGJ2 on proinflammatory cytokine signaling are not limited to the inhibition of NF-kappa B activation in response to IL-1, as PGJ2 appears to be a potent inhibitor of IFN-gamma -induced STAT1 activation in islets and RINm5F cells. In addition, these studies indicate that, although HSP70 expression correlates with an inhibition of cytokine signaling, HSP70 does not appear to mediate the inhibitory actions of PGJ2 on cytokine signaling in RINm5F cells.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials and animals. Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). RPMI 1640 containing 1× L-glutamine, CMRL-1066 tissue culture medium, L-glutamine, penicillin, streptomycin, and rat recombinant IFN-gamma were from GIBCO-BRL (Grand Island, NY). Fetal calf serum was obtained from Hyclone Laboratories (Logan, UT). Superfect reagent was obtained from Qiagen (Valencia, CA). Human recombinant IL-1beta was obtained from Cistron Biotechnology (Pine Brook, NJ). PGJ2 was from Cayman Chemicals (Ann Arbor, MI). [gamma -32P]dATP and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The HSP70 gene, cloned into the pBluescript plasmid, was a generous gift from Dr. Ken Polonsky (Washington University School of Medicine, St. Louis, MO). Horseradish peroxidase-conjugated donkey anti-rabbit and donkey anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse anti-rabbit HSP70 was from StressGen (Victoria, BC, Canada). Rabbit anti-phospho-STAT1 was from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-STAT1 and rabbit anti-Ikappa B were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-iNOS antiserum was a gift from Dr. Pam Manning (Parmacia, St. Louis, MO). All other reagents were from commercially available sources.

Islet isolation. Islets were isolated from 250- to 300-g male Sprague-Dawley rats by collagenase digestion as previously described (28). 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 mg/ml streptomycin) at 37°C under an atmosphere of 95% air-5% CO2 before experimentation.

Nitrite determination. Nitrite formation was measured by mixing 50 µl of culture media with 50 µl of the Griess reagent (15) as previously described. Absorbance was measured at 540 nm, and nitrite concentrations were calculated from a sodium nitrite standard curve.

Electrophoresis and Western blotting. Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to Highbond ECL nitrocellulose membranes (Amersham) as previously described (33). Antibody dilutions were as follows: rabbit anti-phospho-STAT1, 1:800; rabbit anti-STAT1, 1:1,000; mouse anti-HSP70, 1:1,000; rabbit anti-Ikappa Balpha , 1:1,000; rabbit anti-iNOS, 1:1,000; horseradish peroxidase-conjugated donkey anti-mouse, 1:5,000; and horseradish peroxidase-conjugated donkey anti-rabbit, 1:7,000. Antigens were detected by ECL (Amersham Pharmacia Biotech).

Gel shift analysis. Nuclear proteins were isolated, and gel shift analysis was performed as previously described (20) using a 32P end-labeled probe containing the consensus sequence for STAT1 binding (5'-CATGTTATGCATATTCCTGTAAGTG-3') or NF-kappa B binding (5'-AGTTGAGGGGACTTTCCCAGGC-3'). Binding reactions were run on a 4% nondenaturing gel in a Tris-glycine buffer system.

Transfections. RINm5F cells were transiently transfected using Superfect reagent according to the manufacturer's specifications. Briefly, RINm5F cells were plated at 4 × 105 cells/2 ml complete CMRL-1066 and grown to ~50% confluence (~24 h at 37°C under 95% air-5% CO2). Plasmid DNA (2 µg) was incubated in 100 µl of CMRL-1066 (containing 2 mM L-glutamine) for 5 min at room temperature, 6 µl of Superfect was added, and the mixture was incubated for an additional 15 min at room temperature followed by the addition of 600 µl of complete CMRL-1066. The cells were washed once with sterile PBS, and then the DNA-Superfect mixture was added dropwise onto cells. After a 4- to 6-h incubation at 37°C, 1.3 ml of complete CMRL-1066 was added to the cells followed by an overnight incubation at 37°C. Transfection efficiencies of 70-80%, as determined by cotransfection of beta -galactosidase, were routinely obtained using this protocol.

Expression plasmids. Antisense or sense HSP70 cDNA was subcloned into the pDEST12.2 expression plasmid under control of the CMV promoter using the Gateway Expression recombination system according to the manufacturer's guidelines (Life Technologies, Rockville, MD).

Densitometry and statistical analysis. Autoradiograms were scanned using a COHU high-performance charge-coupled device camera, and densities were determined using NIH Image version 1.59 software (Brookefield, WI). Statistical comparisons were made between groups using a one-way analysis of variance (ANOVA). Significant differences between groups (P < 0.05) were determined by Bonferroni post hoc analysis.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PGJ2 inhibits IFN-gamma -induced STAT1 activation in RINm5F cells and rat islets. Previous studies have shown that pretreatment of rat islets and RINm5F cells with PGJ2 and troglitazone for 6 h results in an inhibition of IL-1-induced NF-kappa B activation and JNK phosphorylation, and these inhibitory actions correlate with the expression of HSP70 (26). To determine whether signaling pathways in addition to IL-1 are sensitive to inhibition by PGJ2, the effects of this PPARgamma agonist on IFN-gamma -induced STAT1 phosphorylation and nuclear localization were examined in rat islets and RINm5F cells. Treatment of RINm5F cells (Fig. 1A) or isolated rat islets (Fig. 1B) with IFN-gamma for 30 min results in STAT1 phosphorylation. Alone, a 6-h pretreatment with PGJ2 does not stimulate STAT1 phosphorylation; however, this PGJ2 pretreatment attenuates the stimulatory actions of IFN-gamma on STAT1 phosphorylation in both RINm5F cells and rat islets. Similar to the attenuation of IL-1 signaling (26), the inhibitory actions of a 6-h pretreatment with PGJ2 on IFN-gamma -induced STAT1 phosphorylation correlate with the increased expression of HSP70 by rat islets and RINm5F cells (Fig, 1, A and B). In addition, a 6-h preincubation of RINm5F cells with PGJ2 results in an inhibition of IFN-gamma -induced STAT1 nuclear localization and DNA binding to gamma -activated sequences as determined by gel shift analysis (Fig. 1C). To confirm the presence of STAT1 in these DNA-protein complexes, inclusion of STAT1 antiserum in the DNA binding reaction results in a reduction in the mobility of the STAT1-DNA protein complex, and excess cold oligonucleotide probe inhibits the formation of the STAT1-DNA probe complex (data not shown). These findings indicate that PGJ2 inhibits IFN-gamma -induced STAT1 phosphorylation, nuclear localization, and DNA binding and that these inhibitory actions of PGJ2 correlate with an increase in HSP70 expression.


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Fig. 1.   15-Deoxy-Delta 12,14-prostaglandin J2 (PGJ2) inhibits interferon (IFN)-gamma -induced STAT1 activation in RINm5F cells and rat islets. A: RINm5F cells (4 × 105/400 µl of complete CMRL-1066 tissue culture medium) were pretreated with 10 µg/ml of PGJ2 for 6 h, IFN-gamma (150 U/ml) was added, and the cells were cultured for an additional 30 min at 37°C. B: rat islets (150/400 µl of complete CMRL-1066) were treated with 10 µg/ml of PGJ2 for 6 h, IFN-gamma (150 U/ml) was added, and the islets were cultured for an additional 30 min. After treatment, heat shock protein (HSP)70, phosphorylated STAT1 (P-STAT1), and total STAT1 levels were determined by Western blot analysis. C: RINm5F cells (5 × 106/400 µl of complete CMRL-1066) were treated with PGJ2 and IFN-gamma as described in A. Cells were isolated, nuclei were extracted, and STAT1 nuclear translocation was determined by gel shift analysis. D: RINm5F cells (4 × 105/400 µl of complete CMRL-1066) were pretreated with 10 µg/ml PGJ2 for increasing time intervals from 1 to 6 h as indicated, IFN-gamma (150 U/ml) was added, and the cells were cultured for an additional 30 min at 37°C. Cells were isolated, and HSP70, P-STAT1, and total STAT1 levels were determined by Western blot analysis. Results for A-D are representative of >= 3 independent experiments.

To determine whether the time-dependent inhibitory actions of PGJ2 on IFN-gamma -induced STAT1 phosphorylation correlate with HSP70 expression, RINm5F cells were pretreated for 0-6 h with PGJ2, IFN-gamma was added, and cells were cultured for an additional 30 min. The cells were then isolated, and HSP70 and STAT1 expression and STAT1 phosphorylation were examined by Western blot analysis. As shown in Fig. 1D, the stimulatory actions of PGJ2 on HSP70 expression correlate with the inhibitory actions on IFN-gamma -induced STAT1 phosphorylation. After 1-2 h of incubation, PGJ2 stimulates the initial increase in HSP70 expression, and under these conditions the initial inhibitory actions of PGJ2 on IFN-gamma -stimulated STAT1 phosphorylation are first observed. After a 6-h incubation, PGJ2 stimulates high levels of HSP70 expression and a nearly complete inhibition of IFN-gamma -stimulated STAT1 phosphorylation. These findings indicate that the inhibitory actions of PGJ2 on cytokine signaling correlate with the increased expression of HSP70 by beta -cells and that cytokine signaling pathways in addition to IL-1 are also inhibited by PGJ2.

Role of HSP70 in mediating the inhibitory actions of PPARgamma agonists on IFN-gamma signaling. Because the time-dependent inhibition of IFN-gamma signaling in PGJ2-treated RINm5F cells parallels the induction of HSP70, and HSP70 overexpression has been shown to protect islets from cytokine-induced damage (27, 33), we examined whether HSP70 depletion would modulate the inhibitory actions of PGJ2 on IFN-gamma signaling in RINm5F cells. For these experiments, RINm5F cells were transiently transfected with antisense HSP70 or the pDEST 12.2 empty vector for 48 h at 37°C. The cells were then incubated with PGJ2 for 6 h followed by a 30-min stimulation with IFN-gamma . The cells were isolated, and HSP70 and STAT1 expression and STAT1 phosphorylation were evaluated by Western blot analysis. As shown in Fig. 2A, expression of antisense HSP70 reduces PGJ2-stimulated HSP70 expression by >90% at all time points examined (1-6 h). Importantly, the attenuation of HSP70 accumulation does not modulate the ability of PGJ2 to inhibit STAT1 phosphorylation. The initial inhibitory actions of PGJ2 on IFN-gamma -stimulated STAT1 phosphorylation are first observed after a 2-h pretreatment and are maximal after a 5- to 6-h pretreatment with this PPARgamma agonist (Fig. 2A). Figure 2B shows that, to control for nonspecific actions of the expression vector, transfection with the pDEST 12.2 empty vector does not modulate 1) the ability of IFN-gamma to stimulate STAT1 phosphorylation following a 30-min incubation, 2) the inhibitory actions of a 6-h PGJ2 pretreatment on IFN-gamma -induced STAT1 phosphorylation, or 3) the stimulatory actions of a 6-h PGJ2 pretreatment on HSP70 expression.


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Fig. 2.   Depletion of HSP70 does not prevent the inhibitory actions of PGJ2 on IFN-gamma -induced STAT1 phosphorylation. A: RINm5F cells (4 × 105/2 ml of complete CMRL-1066) were transfected with 2 µg of antisense HSP70 expression plasmid DNA for 48 h at 37°C. Cells were then treated with 10 µg/ml of PGJ2 for increasing time intervals from 1 to 6 h followed by a 30-min incubation with IFN-gamma (150 U/ml). Cells were harvested and HSP70, P-STAT1, and total STAT1 levels were determined by Western blot analysis. B: to confirm that the DNA expression vector does not affect IFN-gamma signaling, effects of a 6-h pretreatment of RINm5F cells (4 × 105/2 ml of complete CMRL-1066) transfected with 2 µg of the empty-expression plasmid pDEST 12.2 and 1 µg of beta -galactosidase, and STAT1 phosphorylation was examined. C: RINm5F cells (4 × 105/2 ml of complete CMRL-1066) were transfected with 2 µg of pDEST-HSP70 for 48 h at 37°C, IFN-gamma (150 U/ml) was added, and the cells were cultured for an additional 30 min at 37°C. Cells were harvested, and P-STAT1, STAT1, and HSP70 levels were determined by Western blot analysis. Results for A-C are representative of 3 independent experiments.

Overexpression of HSP70 in RINm5F cells was used to confirm that increased expression of this stress protein does not inhibit IFN-gamma -induced STAT1 phosphorylation. In these experiments, RINm5F cells transfected with wild-type HSP70 (pDEST HSP70) or the pDEST 12.2 empty vector were treated for 30 min with IFN-gamma , and STAT1 phosphorylation was then examined by Western blot analysis. As shown in Fig. 2C, IFN-gamma stimulates STAT1 phosphorylation to similar levels in RINm5F cells or RINm5F cells overexpressing HSP70. The levels of HSP70 expressed following transient transfection were similar in magnitude to the levels induced after a 6-h treatment with PGJ2 in nontransfected cells (data not shown). These findings suggest that HSP70 does not mediate the inhibitory actions of PGJ2 on IFN-gamma -stimulated STAT1 phosphorylation and that HSP70 overexpression, in the absence of PGJ2, is not capable of inhibiting IFN-gamma signaling in RINm5F cells.

Effects of PGJ2 and HSP70 overexpression on IL-1 signaling in RINm5F cells. Because the inhibitory actions of PGJ2 on IL-1 signaling correlate with HSP70 expression (26), and induction of HSP70 by heat stress results in impairment in IL-1 signaling (33), the effects of HSP70 overexpression on IL-1-induced Ikappa B degradation and NF-kappa B nuclear localization were examined. Treatment of RINm5F cells for 30 min with IL-1 results in Ikappa B degradation and NF-kappa B nuclear localization as determined by Western blot and gel shift analysis, respectively (Fig. 3). Consistent with our previous findings, a 6-h pretreatment with PGJ2 results in the inhibition of IL-1-induced Ikappa B degradation, and these inhibitory actions correlate with the increased expression of HSP70 (Fig. 3A). To determine whether HSP70 alone is sufficient to attenuate IL-1-stimulated Ikappa B degradation and NF-kappa B nuclear localization, RINm5F cells were transiently transfected with HSP70 and then stimulated with IL-1 for 30 min. As shown in Fig. 3, HSP70 overexpression does not attenuate IL-1-induced Ikappa B degradation or NF-kappa B nuclear localization and DNA binding activity. The levels of HSP70 expressed by transient transfection are similar in magnitude to the levels induced by a 6-h treatment with PGJ2, indicating that the lack of an inhibitory effect of HSP70 overexpression on cytokine signaling is not due to differences in expression of this stress response protein. Also, transfection of RINm5F cells with the pDEST 12.2 empty vector does not attenuate IL-1-induced Ikappa B degradation or NF-kappa B nuclear localization in RINm5F cells. Taken together, these findings suggest that the increased expression of HSP70 does not inhibit cytokine signaling and that HSP70 does not appear to mediate the inhibitory actions of PGJ2 on cytokine signaling in beta -cells.


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Fig. 3.   Effects of HSP70 overexpression on IL-1-induced Ikappa B degradation and NF-kappa B nuclear localization. A: RINm5F cells (4 × 105/400 µl of complete CMRL-1066) were preincubated with 10 µg/ml PGJ2 for 6 h at 37°C, IL-1 (1 U/ml) was added, and the cells were cultured for an additional 30 min at 37°C. B: RINm5F cells (4 × 105/2 ml of complete CMRL-1066) were transfected with 2 µg of pDEST-HSP70 plasmid DNA for 48 h at 37°C, IL-1 (1 U/ml) was added, and the cells were cultured for an additional 30 min at 37°C. Cells (for A and B) were harvested, and HSP70, STAT1, and Ikappa B levels were determined by Western blot analysis. STAT1 levels are shown as a loading control. C: RINm5F cells (5 × 106/2 ml complete CMRL-1066) were transfected with pDEST-HSP70 plasmid DNA as described in B, IL-1 (1 U/ml) was added, and the cells were cultured for an additional 30 min at 37°C. Cells were then isolated, nuclear fractions were extracted, and gel shift analysis was performed to detect binding of NF-kappa B to its consensus DNA-binding element. Results for A-C are representative of 3 independent experiments.

Effects of HSP70 overexpression on IL-1-induced iNOS expression. Although HSP70 overexpression does not attenuate cytokine signaling, increased HSP70 expression has been associated with the inhibition of iNOS expression by islets and RINm5F cells (33). Therefore, the effects of HSP70 overexpression on IL-1-induced iNOS expression and nitrite production by RINm5F cells were examined. As shown in Fig. 4, IL-1 stimulates the expression of iNOS and the production of nitrite to similar levels in RINm5F cells and RINm5F cells overexpressing HSP70. In addition, HSP70 overexpression does not modulate the stimulatory actions of IL-1 plus IFN-gamma on iNOS expression and nitric oxide production by RINm5F cells (data not shown). These findings are consistent with the lack of an inhibitory action of HSP70 overexpression on the intracellular signal transduction pathways activated by these cytokines and suggest that HSP70 does not mediate the inhibitory actions of heat shock on cytokine-induced iNOS expression by islets.


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Fig. 4.   Effects of HSP70 overexpression on IL-1-induced inducible nitric oxide synthase (iNOS) expression and nitrite production by RINm5F cells. RINm5F cells (4 × 105/2 ml complete CMRL-1066) were transfected with 2 µg of pDEST-HSP70 plasmid DNA for 48 h, IL-1 (1 U/ml) was added, and the cells were cultured for an additional 18 h. Nitrite production was determined on the culture supernatants (A), and HSP70 and iNOS expression was determined by Western blot on the cell lysates (B). Results for nitrite production are the average ± SE of 3 independent experiments, and HSP70 and iNOS expression is representative of 3 independent experiments.

PPARgamma agonists inhibit IL-1 plus IFN-gamma -stimulated iNOS expression and nitric oxide accumulation. Although HSP70 does not appear to mediate the inhibitory actions of PGJ2 on cytokine signaling and cytokine-stimulated iNOS expression, we present evidence in Fig. 5 to confirm that, under conditions in which cytokine signaling is impaired, cytokine-induced iNOS expression and nitric oxide production by rat islets and RINm5F cells are also inhibited. As shown in Fig. 5, the stimulatory actions of IL-1 plus IFN-gamma on iNOS expression (inset) and nitrite production by RINm5F cells (Fig. 5A) and isolated rat islets (Fig. 5B) are prevented by a 6-h pretreatment with PGJ2 before cytokine stimulation. These findings show that, under conditions in which PGJ2 inhibits cytokine signaling, cytokine-induced iNOS expression is also impaired.


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Fig. 5.   Effects of PGJ2 on IL-1- plus IFN-gamma -induced iNOS expression and nitrite production by RINm5F cells and rat islets. RINm5F cells (4 × 105/400 µl of complete CMRL-1066; A) or rat islets (150/400 µl of complete CMRL-1066; B) were pretreated with 10 µg/ml PGJ2 for 6 h at 37°C, IL-1 (1 U/ml) and IFN-gamma (150 U/ml) were added, and the cells and/or islets were cultured for an additional 24 h at 37°C. Nitrite production was determined on the culture, and iNOS expression was determined by Western blot of the cell lysates. Results for nitrite are the average ± SE of 3 independent experiments, and iNOS protein expression is representative of 3 independent experiments.

Persistence of the inhibitory actions of PGJ2 on cytokine-induced iNOS expression by islets. We next evaluated the duration of the protective response induced by PGJ2 that renders islets inert to cytokine signaling. In this experiment, rat islets were pulsed with 10 µg/ml PGJ2 for 6 h, washed, and then incubated in fresh media at 37°C for increasing time intervals before a 24-h incubation with IL-1. As shown in Fig. 6, IL-1 fails to stimulate iNOS expression or nitrite production by rat islets pretreated for 6 h with PGJ2 before cytokine stimulation. In a similar fashion, a 6-h pulse with PGJ2 prevents the subsequent stimulation of iNOS expression in response to IL-1 plus IFN-gamma for periods as long as 48 h (data not shown). These findings suggest that the inhibitory actions of a short 6-h pulse with PGJ2 on IL-1 and IFN-gamma -induced iNOS expression are persistent for extended periods of time up to 48 h.


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Fig. 6.   Persistence of the inhibitory actions of PGJ2 on IL-1-induced iNOS expression and nitrite production by rat islets. Rat islets (150/400 µl of complete CMRL-1066) were pulsed for 6 h with 10 µg/ml PGJ2. The islets were washed 3 times to remove the PGJ2 and cultured for 0, 24, or 48 h in complete CMRL-1066 tissue culture media. IL-1 was then added, and the islets were cultured for an additional 24 h. Production of nitrite was determined on culture supernatants, and iNOS expression was determined by Western blot analysis on the islet lysates. Results for nitrite production are the average ± SE of 3 independent experiments, and iNOS protein expression is representative of 3 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Agonists of PPARgamma such as PGJ2 and synthetic thiazolidinediones (TZD; e.g, troglitazone) have been shown to attenuate proinflammatory cytokine, iNOS, and inducible cyclooxygenase 2 expression by LPS-treated macrophages (4, 5, 23, 38), and we have shown that these agonists attenuate IL-1-induced iNOS expression by RINm5F cells and rat islets (26). The specific mechanisms by which these agonists inhibit inflammatory gene expression are associated with an inhibition of transcription factor activation, as these cyclopentonone prostaglandins and TZD drugs have been shown to directly inhibit IKK activation, NF-kappa B-DNA-binding activity (31, 37), and the activation of AP-1 (39). Activation of PPARgamma does not appear to be required for the inhibitory actions of PGJ2 on NF-kappa B activation, as PGJ2 has been shown to inhibit LPS-induced NF-kappa B activation in RAW 264.7 cells deficient in PPARgamma . However, PPARgamma may participate directly in the inhibitory actions of PGJ2 on inflammatory gene expression, as transfection of PPARgamma increases the sensitivity of RAW 264.7 cells to the inhibitory actions of PGJ2 on TNF-induced iNOS reporter gene activation (38). In the present study, we have identified the IFN-gamma -signaling pathway as an additional signaling pathway that is inhibited by PPARgamma agonists. Treatment of isolated rat islets or RINm5F cells with IFN-gamma for 30 min results in STAT1 phosphorylation and nuclear localization. In a time-dependent manner, PGJ2 prevents IFN-gamma -induced STAT1 phosphorylation and nuclear localization with the initial inhibitory actions observed following a 1- to 2-h pretreatment and maximal actions after a 6-h pretreatment before cytokine stimulation. The inhibitory actions of PGJ2 on IFN-gamma -induced STAT1 activation also correlate with induction of HSP70 expression by rat islets and RINm5F cells.

Previous studies have shown that, similar to the inhibition of IFN-gamma signaling, PGJ2 and troglitazone attenuate IL-1-induced Ikappa B degradation and JNK phosphorylation in isolated rat islets and RINm5F cells (26). In these studies, we showed that a 30-min pretreatment with PGJ2 or troglitazone does not modulate the ability of IL-1 to stimulate NF-kappa B activation or JNK phosphorylation; however, after a 6-h incubation, IL-1-induced Ikappa B degradation and JNK phosphorylation are significantly attenuated (26). The inhibitory actions of these agonists on NF-kappa B and JNK activation are associated with the induction of HSP70 expression or induction of a stress response. These findings suggest that PGJ2 and troglitazone may attenuate cytokine-mediated cell signaling and cytokine-induced inflammatory gene expression by an additional mechanism that is associated with the induction of HSP70 expression.

Overexpression of HSP70 has been shown to protect islets or insulinoma cells against cytokine-mediated damage at two levels: 1) the inhibition of IL-1-induced NF-kappa B activation and the subsequent inhibition of iNOS expression (33), and 2) the inhibition of nitric oxide donor compound-mediated damage (1, 2). Because PPARgamma agonists are known to stimulate HSP70 expression (5, 26) and under conditions in which beta -cells express HSP70 these cells are protected from cytokine-mediated damage (33), we examined the role of HSP70 in mediating the protective actions of PPARgamma agonists on cytokine signaling and cytokine-induced iNOS expression by rat islets and RINm5F cells. Using an antisense approach, we show that, in the absence of HSP70 accumulation, PGJ2 is still capable of impairing IFN-gamma -induced STAT1 activation. In addition, antisense depletion of HSP70 does not affect the ability of PGJ2 to attenuate IL-1-induced Ikappa B degradation in RINm5F cells (data not shown). Conversely, overexpression of HSP70 in RINm5F cells to levels similar in magnitude to those induced by PGJ2 does not result in an impairment of IL-1-induced NF-kappa B activation or IFN-gamma -induced STAT1 activation. In addition, IL-1 and IL-1 plus IFN-gamma stimulate iNOS expression and nitric oxide production to similar levels in RINm5F cells and RINm5F cells overexpressing HSP70. These findings suggest that the inhibitory effects of PGJ2 on cytokine signaling in rat islets and RINm5F cells are not mediated by HSP70 expression, although HSP70 expression does correlate with the impairment in cytokine signaling.

Cytokines such as IL-1 and IFN-gamma have been shown to impair beta -cell function by stimulating iNOS expression and nitric oxide production by beta -cells (18). Evidence to support a role for iNOS and nitric oxide in cytokine-mediated beta -cell damage includes the following: 1) the ability of iNOS inhibitors to prevent the inhibitory actions of these cytokines on insulin secretion, islet oxidative metabolism, and islet degeneration (7, 9, 36); 2) the inability of cytokines to impair glucose-induced insulin secretion by islets isolated from iNOS-deficient mice (13); and 3) the induction of diabetes in transgenic mice expressing iNOS under control of the rat insulin promoter (40). In the current study, we show that PGJ2 is a potent inhibitor of IL-1- and IFN-gamma -stimulated signaling pathways as well as the downstream activation of iNOS expression in islets and RINm5F cells. Importantly, the inhibitory actions of PGJ2 on cytokine-stimulated iNOS expression are persistent for extended periods. A short 6-h pretreatment of islets with PGJ2 renders these islets nonresponsive to IL-1 or IL-1 plus IFN-gamma for periods as long as 48 h. These new findings may have important implications for the attenuation of islet graft rejection in the transplantation setting. The recent success by the Edmonton group (32, 34) has provided new enthusiasm for the transplantation of islets, isolated from cadaver donors, as a method to treat brittle insulin-dependent diabetic patients. Notwithstanding this recent success, the lack of sufficient supply of islets and the need to transplant islets isolated from two donor organs are issues that will need to be addressed if islet transplantation is to be routinely used for the treatment of insulin-dependent diabetics. One potential method to reduce the islet mass required for a successful transplant may be to promote survival or protect islets from immune-mediated injury. Our findings that PGJ2 pretreatment provides an extended protection of islets from cytokine-stimulated iNOS expression suggest that the use of PGJ2 (or other PPARgamma agonists) to attenuate proinflammatory cytokine signaling and induction of inflammatory gene expression may provide a new strategy to promote islet graft survival and thereby reduce the islet mass required for a successful transplantation.


    ACKNOWLEDGEMENTS

We thank Colleen Kelly Bratcher for expert technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-52194 and AI-44458.

Address for reprint requests and other correspondence: J. A. Corbett, Edward A. Doisy Dept. of Biochemistry and Molecular Biology, Saint Louis Univ. School of Medicine, 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.

First published January 7, 2003;10.1152/ajpendo.00515.2002

Received 22 November 2002; accepted in final form 14 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Bellmann, K, Jaattela M, Wissing D, Burkart V, and Kolb H. Heat shock protein hsp70 overexpression confers resistance against nitric oxide. FEBS Lett 391: 185-188, 1996[ISI][Medline].

2.   Bellmann, K, Wenz A, Radons J, Burkart V, Kleemann R, and Kolb H. Heat shock induces resistance in rat pancreatic islet cells against nitric oxide, oxygen radicals and streptozotocin toxicity in vitro. J Clin Invest 95: 2840-2845, 1995[ISI][Medline].

3.   Bottazzo, GF, Dean BM, McNally JM, MacKay EH, Swift PG, and 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].

4.   Chawla, A, Barak Y, Nagy L, Liao D, Tontonoz P, and Evans RM. PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med 7: 48-52, 2001[ISI][Medline].

5.   Colville-Nash, PR, Qureshi SS, Willis D, and 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].

6.   Corbett, JA, Kwon G, Marino MH, Rodi CP, Sullivan PM, Turk J, and 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].

7.   Corbett, JA, Lancaster JR, Jr, Sweetland MA, and McDaniel ML. Interleukin-1 beta-induced formation of EPR-detectable iron-nitrosyl complexes in islets of Langerhans. Role of nitric oxide in interleukin-1 beta-induced inhibition of insulin secretion. J Biol Chem 266: 21351-21354, 1991[Abstract/Free Full Text].

8.   Corbett, JA, and McDaniel ML. Reversibility of interleukin-1 beta-induced islet destruction and dysfunction by the inhibition of nitric oxide synthase. Biochem J 299: 719-724, 1994[ISI][Medline].

9.   Corbett, JA, and McDaniel ML. Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Methods Enzymol 268: 398-408, 1996[ISI][Medline].

10.   Corbett, JA, Wang JL, Sweetland MA, Lancaster JR, Jr, and 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 Invest 90: 2384-2391, 1992[ISI][Medline].

11.   Debril, MB, Renaud JP, Fajas L, and Auwerx J. The pleiotropic functions of peroxisome proliferator-activated receptor gamma. J Mol Med 79: 30-47, 2001[ISI][Medline].

12.   Eizirik, DL, Flodstrom M, Karlsen AE, and 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].

13.   Flodstrom, M, Tyrberg B, Eizirik DL, and Sandler S. Reduced sensitivity of inducible nitric oxide synthase-deficient mice to multiple low-dose streptozotocin-induced diabetes. Diabetes 48: 706-713, 1999[Abstract].

14.   Gepts, W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes 14: 619-633, 1965[ISI][Medline].

15.   Green, LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, and Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 131-138, 1982[ISI][Medline].

16.   Grey, ST, Arvelo MB, Hasenkamp W, Bach FH, and Ferran C. A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets. J Exp Med 190: 1135-1146, 1999[Abstract/Free Full Text].

17.   Heimberg, H, Heremans Y, Jobin C, Leemans R, Cardozo AK, Darville M, and Eizirik DL. Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. Diabetes 50: 2219-2224, 2001[Abstract/Free Full Text].

18.   Heitmeier, MR, and Corbett JA. Cytotoxic role of nitric oxide in diabetes. In: Nitric Oxide Biology and Pathobiology. San Diego, CA: Academic, 2000, p. 785-810.

19.   Heitmeier, MR, Scarim AL, and 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].

20.   Heitmeier, MR, Scarim AL, and 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].

21.   Heitmeier, MR, Scarim AL, and 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].

22.   Hughes, JH, Colca JR, Easom RA, Turk J, and McDaniel ML. Interleukin 1 inhibits insulin secretion from isolated rat pancreatic islets by a process that requires gene transcription and mRNA translation. J Clin Invest 86: 856-863, 1990[ISI][Medline].

23.   Jiang, C, Ting AT, and Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82-86, 1998[ISI][Medline].

24.   Kay, TWH, Thomas HE, Harrison LC, and Allison J. The beta cell in autoimmune diabetes: many mechanisms and pathways of loss. Trends Endocrinol Metab 11: 11-15, 2000[ISI][Medline].

25.   Kwon, G, Corbett JA, Rodi CP, Sullivan P, and McDaniel ML. Interleukin-1 beta-induced nitric oxide synthase expression by rat pancreatic beta-cells: evidence for the involvement of nuclear factor kappa B in the signaling mechanism. Endocrinology 136: 4790-4795, 1995[Abstract].

26.   Maggi, LB, Jr, Sadeghi H, Weigand C, Scarim AL, Heitmeier MR, and 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].

27.   Margulis, BA, Sandler S, Eizirik DL, Welsh N, and 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].

28.   McDaniel, ML, Colca JR, Kotagal N, and Lacy PE. A subcellular fractionation approach for studying insulin release mechanisms and calcium metabolism in islets of Langerhans. Methods Enzymol 98: 182-200, 1983[ISI][Medline].

29.   Palmer, JP, Helqvist S, Spinas GA, Molvig J, Mandrup-Poulsen T, Andersen HU, and Nerup J. Interaction of beta-cell activity and IL-1 concentration and exposure time in isolated rat islets of Langerhans. Diabetes 38: 1211-1216, 1989[Abstract].

30.   Petrova, TV, Akama KT, and Van Eldik LJ. Cyclopentenone prostaglandins suppress activation of microglia: down-regulation of inducible nitric-oxide synthase by 15-deoxy-delta12,14-prostaglandin J2. Proc Natl Acad Sci USA 96: 4668-4673, 1999[Abstract/Free Full Text].

31.   Ricote, M, Li AC, Willson TM, Kelly CJ, and Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 391: 79-82, 1998[ISI][Medline].

32.   Ryan, EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, Rabinovitch A, Elliott JF, Bigam D, Kneteman NM, Warnock GL, Larsen I, and Shapiro AM. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50: 710-719, 2001[Abstract/Free Full Text].

33.   Scarim, AL, Heitmeier MR, and Corbett JA. Heat shock inhibits cytokine-induced nitric oxide synthase expression by rat and human islets. Endocrinology 139: 5050-5057, 1998[Abstract/Free Full Text].

34.   Shapiro, AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, and Rajotte RV. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343: 230-238, 2000[Abstract/Free Full Text].

35.   Sibley, RK, Sutherland DE, Goetz F, and 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].

36.   Southern, C, Schulster D, and Green IC. Inhibition of insulin secretion by interleukin-1 beta and tumour necrosis factor-alpha via an L-arginine-dependent nitric oxide generating mechanism. FEBS Lett 276: 42-44, 1990[ISI][Medline].

37.   Straus, DS, and Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev 21: 185-210, 2001[ISI][Medline].

38.   Straus, DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Ghosh G, and 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].

39.   Subbaramaiah, K, Lin DT, Hart JC, and 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].

40.   Takamura, T, Kato I, Kimura N, Nakazawa T, Yonekura H, Takasawa S, and Okamoto H. Transgenic mice overexpressing type 2 nitric-oxide synthase in pancreatic beta cells develop insulin-dependent diabetes without insulitis. J Biol Chem 273: 2493-2496, 1998[Abstract/Free Full Text].

41.   Thomas, HE, Darwiche R, Corbett JA, and 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].


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