PPAR{gamma} is not required for the inhibitory actions of PGJ2 on cytokine signaling in pancreatic {beta}-cells

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

Submitted 29 August 2003 ; accepted in final form 24 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peroxisome proliferator-activated receptor (PPAR){gamma} agonists, such as 15-deoxy-{Delta}12,14-prostaglandin J2 (PGJ2) and troglitazone, have been shown to elicit anti-inflammatory effects in pancreatic {beta}-cells that include inhibition of cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expression and production of nitric oxide. In addition, these ligands impair IL-1-induced NF-{kappa}B and MAPK as well as IFN-{gamma}-stimulated signal transducer and activator of transcription (STAT)1 activation in {beta}-cells. The purpose of this study was to determine if PPAR{gamma} activation participates in the anti-inflammatory actions of PGJ2 in {beta}-cells. Pretreatment of RINm5F cells for 6 h with PGJ2 results in inhibition of IL-1-stimulated I{kappa}B degradation and IFN-{gamma}-stimulated STAT1 phosphorylation. Overexpression of a dominant-negative (dn) PPAR{gamma} mutant or treatment with the PPAR{gamma} antagonist GW-9662 does not modulate the inhibitory actions of PGJ2 on cytokine signaling in RINm5F cells. Although these agents fail to attenuate the inhibitory actions of PGJ2 on cytokine signaling, they do inhibit PGJ2-stimulated PPAR{gamma} response element reporter activity. Consistent with the inability to attenuate the inhibitory actions of PGJ2 on cytokine signaling, neither dnPPAR{gamma} nor GW-9662 prevents the inhibitory actions of PGJ2 on IL-1-stimulated iNOS gene expression or nitric oxide production by RINm5F cells. These findings support a PPAR{gamma}-independent mechanism by which PPAR{gamma} ligands impair cytokine signaling and iNOS expression by islets.

peroxisome proliferator-activated receptor-{gamma}; 15-deoxy-{Delta}12,14-prostaglandin J2; inducible nitric oxide synthase; islet; inflammation


INSULIN-DEPENDENT DIABETES MELLITUS is an autoimmune disease characterized by an inflammatory reaction in and around pancreatic islets of Langerhans that results in the selective destruction of insulin-secreting {beta}-cells (13). Cytokines produced during this inflammatory reaction, specifically IL-1, produced by macrophages, and IFN-{gamma}, produced by infiltrating T cells, are believed to participate in {beta}-cell dysfunction and islet destruction (11, 15, 19, 26). In vitro studies have shown that IL-1, alone or in combination with IFN-{gamma}, impairs {beta}-cell function by inducing inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by {beta}-cells (8, 11, 16, 33). The signaling mechanism by which IL-1 stimulates iNOS expression in {beta}-cells includes the degradation of inhibitory protein {kappa}B (I{kappa}B), an event that allows nuclear factor (NF)-{kappa}B to translocate to the nucleus to activate gene expression (11, 23). IFN-{gamma} augments the stimulatory actions of IL-1 on iNOS expression by increasing the sensitivity of {beta}-cells to IL-1 >10-fold (16, 18). The inhibitory effects of IL-1 on glucose-stimulated insulin secretion and islet viability are mediated, in part, by the ability of NO to disrupt iron- and sulfur-containing enzymes within the mitochondria, such as aconitase and the electron transport chain complexes I and II (5, 6, 38). As a result, treatment of islets with IL-1{beta} results in the inhibition of glucose oxidation to CO2- and a fourfold reduction in the cellular levels of ATP, effects prevented by inhibitors of NO synthase (7).

In an effort to identify mechanisms to prevent cytokine-mediated {beta}-cell damage, we have shown that IL-1 fails to activate iNOS gene expression and NO production by RINm5F cells and rat islets treated simultaneously with the PPAR{gamma} agonist 15-deoxy-{Delta}12,14-prostaglandin J2 (PGJ2) (25, 37). We have also shown that PGJ2 inhibits signaling cascades initiated by the proinflammatory cytokines IL-1{beta} and IFN-{gamma} by a second mechanism that is associated with activation of a stress response. In a time-dependent manner, PGJ2 stimulates the expression of heat shock protein (hsp) 70 that is first detectable after a 3- to 6-h incubation (37). Importantly, when islets express hsp 70 either in response to PGJ2 treatment or after heat shock, IL-1-induced NF-{kappa}B and IFN-{gamma}-stimulated signal transducer and activator of transcription (STAT)1 activation are attenuated (25, 32, 37). Although the inhibitory actions of PGJ2 on cytokine signaling correlate with elevated levels of hsp 70, antisense depletion of hsp 70 does not affect the inhibitory actions of PGJ2 in {beta}-cells (37).

The peroxisome proliferator-activated receptors (PPARs) represent a family of nuclear receptor ligand-activated transcription factors that have been implicated in maintenance of lipid and glucose homeostasis, control of cell growth and differentiation, and regulation of inflammation (10, 28, 30). Agonists of PPAR{gamma}, such as PGJ2 and synthetic thiazolidinediones (troglitazone as an example), have been shown to attenuate proinflammatory cytokine, iNOS, and inducible cyclooxygenase (COX-2) expression by LPS-treated macrophages (3, 4, 20, 35); however, the specific role of PPAR{gamma} in this inhibition is less clear. PGJ2 has been shown to inhibit LPS-induced NF-{kappa}B activation in RAW 264.7 cells deficient in PPAR{gamma}, and transfection of PPAR{gamma} into these cells increases their sensitivity to the inhibitory actions of PGJ2 on iNOS reporter gene activation (35). PPAR{gamma} agonists also prevent IFN-{gamma}-induced iNOS and COX-2 expression in PPAR{gamma}-/- macrophages generated from blastocysts isolated from PPAR{gamma} heterozygous matings (3). These findings suggest that PGJ2 inhibits inflammatory gene activation by PPAR{gamma}-dependent and -independent mechanisms. PPAR{gamma}-independent inhibition of inflammatory gene activation has been associated with the direct inhibition of I{kappa}B kinase (IKK) and the inhibition of RelA DNA binding activity (29, 34). Also, the ability of PPAR{gamma} agonists to inhibit inflammatory gene expression appears to be cell type dependent, as macrophages are more sensitive to PGJ2 than HeLa cells or insulinoma RINm5F cells (25, 35).

The focus of the current study was to determine whether PPAR{gamma} activation is required for PGJ2 to inhibit cytokine signaling in pancreatic {beta}-cells. To address this question, a dominant-negative (dn) PPAR{gamma} mutant containing a single amino acid substitution in the ligand-binding domain of the PPAR{gamma} receptor was generated. Although this mutation is sufficient to inhibit PGJ2-stimulated PPAR{gamma} transcriptional activation from a PPAR{gamma} response element (PPRE) reporter, it does not modulate the inhibitory actions of PGJ2 on IL-1-stimulated iNOS expression, NO production, NF-{kappa}B activation, or IFN-{gamma}-stimulated STAT1 activation. These findings support a PPAR{gamma}-independent mechanism by which PGJ2 inhibits cytokine signaling and cytokine-stimulated inflammatory gene expression in islets.


    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) and CV-1 (monkey kidney) cells were obtained from the Washington University Tissue Culture Support Center (St. Louis, MO). RPMI 1640 containing L-glutamine, CMRL-1066, and DMEM tissue culture media, L-glutamine, penicillin, streptomycin, and rat recombinant IFN-{gamma} were from GIBCO-BRL (Grand Island, NY). FCS was obtained from Hyclone Laboratories (Logan, UT). Superfect reagent was obtained from Qiagen (Valencia, CA). Human recombinant IL-1{beta} was obtained from Cistron Biotechnology (Pine Brook, NJ). PGJ2 and GW-9662 were from Cayman Chemical (Ann Arbor, MI). The mouse PPAR{gamma} cDNA subcloned into the pcDNA3.1 mammalian expression plasmid and the PPRE (3xFATP)-luciferase reporter plasmid were generous gifts of Heidi Camp (Pfizer, Ann Arbor, MI). Horseradish peroxidase-conjugated donkey anti-rabbit and donkey anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit anti-phospho-STAT1 was from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-STAT1 and rabbit anti-I{kappa}B were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-iNOS antiserum was a gift from Dr. Pam Manning (Pharmacia, St. Louis, MO). Mouse anti-hsp 70 was from StressGen (Victoria, British Columbia, Canada). Rabbit anti-PPAR{gamma} antiserum was a gift from Todd Leff (Wayne State University, Detroit MI). 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 (27). Islets were cultured overnight in complete CMRL-1066 (containing 2 mM L-glutamine, 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin) at 37°C under an atmosphere of 95% air-5% CO2 before experimentation.

Luciferase and {beta}-galactosidase reporter assays. RINm5F or CV-1 cells were transiently transfected with 1 µg of PPRE-luciferase and 0.5 µg of the pCMV-SPORT-{beta}-galactosidase control plasmid using the Qiagen Superfect reagent according to the manufacturer's specifications and as described previously (1, 37). After a 48-h transfection, experiments were initiated as indicated in the legends for Figs. 1, 2, 3, 4, 5, 6; the cells were then harvested and lysed in Reporter Lysis Buffer (Promega, Madison, WI). {beta}-Galactosidase activity was determined as previously described (1, 24), and luciferase activity was determined using the Luciferase Assay Substrate according to manufacturer's instructions (Promega). Luciferase activity is reported as relative light units and is normalized to {beta}-galactosidase activity for each condition. Transfection efficiencies of 70-80%, as determined by cotransfection of {beta}-galactosidase, were routinely obtained using this protocol.



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Fig. 1. Q284P peroxisome proliferator-activated receptor (PPAR){gamma} mutant inhibits PGJ2-stimulated PPAR{gamma} reporter activity. dn, Dominant negative. CV-1 cells (7.5 x 105/2 ml DMEM) were transfected with 1 µg PPAR{gamma} response element (PPRE)-luciferase reporter plasmid DNA and 0.5 µg {beta}-galactosidase ({beta}-gal) in the presence or absence of 2 µg Q284P PPAR{gamma} for 48 h or pretreated for 30 min with PPAR{gamma} antagonist GW-9662 (1 µM). Control cells were transfected with the empty pcDNA3.1 vector DNA for 48 h. Cells were then treated with PGJ2 at the indicated concentrations for 24 h, at which time the cells were washed and luciferase and {beta}-galactosidase activity was determined. Luciferase activity (relative light units, RLU) was normalized to {beta}-galactosidase activity. The results are representative of 3 independent experiments.

 


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Fig. 2. PGJ2 inhibits IFN-{gamma} signaling in {beta}-cells expressing dn PPAR{gamma}. RINm5F cells (7.5 x 105/2 ml complete CMRL-1066; A-C) or rat islets (150:400 µl of complete CMRL-1066; D) transfected with 2 µg Q284P PPAR{gamma} for 48 h or pretreated with 1 µM GW-9662 for 30 min were treated with PGJ2 (30 µM) for 6 h. IFN-{gamma} (150 U/ml) was added, and the cells/islets were cultured for 30 additional min. A, B, and D: cells or islets were isolated, and the levels of phosphorylated (P-) STAT1 and total STAT1 (loading control) were determined by Western blot analysis. C: alternatively, RINm5F cells were collected, nuclear fractions were isolated, and gel shift analysis was used to examine the binding of STAT1 to its consensus DNA binding sequence. E: densitometry was performed on results shown in D, and percent inhibition of STAT1 phosphorylation was determined by comparing relative densities of P-STAT1 with total STAT1 for each condition. Results are representative of 3 independent experiments (A-D) or the average ± SE of 3 independent experiments (E). Ctrl, control; GW, GW-9662.

 


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Fig. 3. PGJ2 inhibits IL-1-stimulated I{kappa}B degradation in {beta}-cells expressing a dnPPAR{gamma}. RINm5F cells (7.5 x 105/2 ml complete CMRL-1066), transfected with 2 µg Q284P PPAR{gamma} DNA for 48 h or pretreated with 1 µM GW-9662 or DMSO vehicle control for 30 min, were treated with PGJ2 (15 µM) for 6 h. IL-1 (1 U/ml) was then added, and the cells were cultured for an additional 30 min. A and B: cells were collected, and levels of inhibitory protein {kappa}B (I{kappa}B), heat shock protein (hsp) 70, and signal transducer and activator of transcription (STAT)1 (loading control) were analyzed by Western blot analysis. C: alternatively, RINm5F cells were collected, nuclear fractions were isolated, and gel shift analysis was performed to detect binding of NF-{kappa}B to its consensus DNA binding sequence. Results are representative of 3 independent experiments.

 


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Fig. 4. PGJ2 inhibits IL-1-stimulated iNOS protein expression and nitrite accumulation in {beta}-cells expressing a dnPPAR{gamma}. RINm5F cells (7.5 x 105/2 ml complete CMRL-1066), transfected with 2 µg Q284P PPAR{gamma} for 48 h (A) or pretreated with GW-9662 (1 µM) for 30 min (B), were treated with PGJ2 (30 µM) + IL-1 (1 U/ml) for 24 h. Supernatants were collected, and nitrite production was determined using the Griess assay. Cells were isolated, and Western blot analysis was used to examine iNOS protein expression. Results are averages ± SE of 3 independent experiments for nitrite formation or representative of 3 independent experiments for protein expression.

 


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Fig. 5. PGJ2 inhibits IL-1-stimulated iNOS expression in RINm5F cells expressing dnPPAR{gamma}. RINm5F cells (7.5 x 105/2 ml complete CMRL-1066), transfected with 2 µg Q284P PPAR{gamma} for 48 h (A) or pretreated with GW-9662 (1 µM) for 30 min (B), were treated with PGJ2 (30 µM) + IL-1 (1 U/ml) for 4 h. Cells were then harvested, and total RNA was isolated and used to evaluate iNOS, hsp 70, and GAPDH (loading control) mRNA accumulation by RT-PCR. Results are representative of at least 3 independent experiments.

 


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Fig. 6. Expression and activity of PPAR{gamma} in RINm5F. A: RINm5F or CV-1 cells (7.5 x 105/2 ml complete CMRL-1066 or DMEM) were transfected with 1 µg PPRE-luciferase reporter plasmid DNA and 0.5 µg {beta}-galactosidase expression plasmid DNA for 48 h. Control cells were transfected with the empty pcDNA3.1 vector DNA for 48 h (data not shown). Cells were then treated with PGJ2 at the indicated concentrations for 24 h, at which time the cells were washed and luciferase and {beta}-galactosidase activity determined. Luciferase activity (RLU) was normalized to {beta}-galactosidase activity. B: RINm5F (750 x 105/400 µl complete CMRL-1066) were cultured in growth media or transfected with 2 µg Q284P PPAR{gamma} expression plasmid DNA for 48 h. Alternatively, rat islets (150:400 ml complete CMRL) were cultured overnight at 37°C. The cells or islets were isolated, and the levels of PPAR{gamma} and STAT1 (loading control) were determined by Western blot analysis. Purified PPAR{gamma} protein was included as a positive control for antibody specificity. Results are representative of at least 3 independent experiments.

 

Nitrite determination. Nitrite formation was measured by mixing 50 µl of culture medium with 50 µl of the Griess reagent, as previously described (14). Absorbance was measured at 540 nm, and nitrite concentrations were calculated from a sodium nitrite standard curve.

Electrophoresis and Western blot analysis. Cellular proteins were separated by SDS-PAGE and transferred to High Bond enhanced chemiluminescence (ECL) nitrocellulose membranes (Amersham), as previously described (18). Antibody dilutions were as follows: rabbit anti-PPAR{gamma}, 1:500; rabbit anti-phospho-STAT1, 1:800; rabbit anti-STAT1, 1:1,000; rabbit anti-I{kappa}B{alpha}, 1:1,000; rabbit anti-iNOS, 1:1,000; mouse anti-hsp 70, 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 extracts were isolated, and electrophoretic mobility shift analysis was performed as previously described (17) using a 32P end-labeled probe containing the consensus sequence for STAT1 binding (5'-CATGTTATGCATATTCCTGTAAGTG-3') or NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3'). Binding reactions were run on a 4% nondenaturing gel in a Trisglycine buffer system.

Expression plasmids. Mouse PPAR{gamma} cDNA in the pcDNA3.1 vector was used as the template to generate a dnPPAR{gamma} expression plasmid. The glutamine (Q) residue 284 was substituted with a proline (P) residue by mutating codon CAG(Q) to CCC(P) within the mouse PPAR{gamma} gene using the Stratagene QuikChange Site-Directed Mutagenesis kit (La Jolla, CA) per the manufacturer's protocol. The Q284P PPAR{gamma} was sequence verified at the Department of Biochemistry and Molecular Biology's Core Sequencing Center.

Densitometry. 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).


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of a dnPPAR{gamma} mutant. To examine the role of PPAR{gamma} in mediating the inhibitory actions of PGJ2 on cytokine signaling, a dn mutant was generated. Glutamine residue 284 within the ligand-binding domain of the mouse PPAR{gamma} cDNA was mutated to a proline (Q284P PPAR{gamma}). This mutation is homologous to a human somatic mutation shown to inhibit PPRE reporter activity in transfection assays (31). To confirm that this mutation inhibits PPAR{gamma} activity, the effects of overexpression of this mutant on PGJ2-stimulated PPRE reporter activity in CV-1 cells was examined. PPAR{gamma} activity has been extensively characterized in this cell line (12, 22). In these experiments, CV-1 cells were transfected with the Q284P PPAR{gamma} and the PPRE reporter construct for 48 h, and then the effects of increasing concentrations of PGJ2 on PPRE reporter activity were examined. As shown in Fig. 1, PGJ2 stimulates a concentration-dependent increase in PPRE reporter activity over a concentration range starting at 3 µM and is maximal at 10-15 µM. Importantly, coexpression of the Q284P PPAR{gamma} mutant attenuates PGJ2-stimulated reporter gene activity in CV-1 cells. Cotransfection of CV-1 kidney cells with the empty pcDNA3.1 vector does not affect the ability of PGJ2 to stimulate PPRE reporter activity in these cells. Similar to the Q284P PPAR{gamma} mutant, the PPAR{gamma} antagonist GW-9662 also attenuates PPRE reporter activity (Fig. 1). These findings confirm that the Q284P PPAR{gamma} mutant and the PPAR{gamma} antagonist GW-9662 are capable of inhibiting PPAR{gamma}-mediated transcriptional activation in response to ligand stimulation at ligand concentrations that inhibit cytokine signaling in {beta}-cells (25, 37).

PPAR{gamma} inhibition does not attenuate the inhibitory actions of PGJ2 on cytokine signaling. We have recently shown that PGJ2 inhibits IFN-{gamma}-stimulated STAT1 phosphorylation, nuclear localization, and DNA binding activity in RINm5F cells and rat islets (37). The inhibitory actions of PGJ2 on cytokine signaling are time dependent, with the initial inhibition observed after a 3-h pretreatment and maximal inhibition after a 6-h PGJ2 pretreatment before IFN-{gamma} stimulation (37). To determine if PPAR{gamma} participates in, or mediates, the inhibitory actions of PGJ2 on the activation of STAT1 by IFN-{gamma}, RINm5F cells were transfected with the dnPPAR{gamma} for 48 h, PGJ2 was added for 6 h, and the cells were then stimulated for 30 min with IFN-{gamma}. The cells were harvested, and IFN-{gamma}-induced STAT1 phosphorylation was examined by Western blot analysis using phosphospecific antisera. As shown in Fig. 2A, incubation of RINm5F cells for 30 min with IFN-{gamma} results in the phosphorylation of STAT1, and STAT1 phosphorylation is prevented if the cells are pretreated for 6 h with PGJ2 before IFN-{gamma} stimulation. Importantly, PGJ2 pretreatment inhibits IFN-{gamma}-stimulated STAT1 phosphorylation in cells transfected with the empty pcDNA3.1 vector (data not shown) or the Q284P PPAR{gamma} mutant (Fig. 2A). Consistent with a lack of an effect of the dnPPAR{gamma} mutant, the PPAR{gamma} antagonist GW-9662 does not modulate the inhibitory actions of PGJ2 on STAT1 phosphorylation in RINm5F cells and rat islets (Fig. 2, B and D). As analyzed by densitometry, PGJ2 inhibits IFN-{gamma}-stimulated STAT1 phosphorylation by >90% in rat islets in the presence or absence of GW-9662 (Fig. 2E). In addition, gel shift analysis was used to show that GW-9662 does not modulate the inhibitory actions of PGJ2 on IFN-{gamma}-stimulated STAT1 nuclear localization and DNA binding activity in RINm5F cells (Fig. 2C). 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 complex, and excess cold oligonucleotide probe inhibits STAT1-DNA complex formation (data not shown).

Treatment of pancreatic {beta}-cells with IL-1 results in the degradation of I{kappa}B, NF-{kappa}B nuclear translocation, and the expression of NF-{kappa}B-dependent inflammatory genes, such as iNOS and COX-2 (15). In previous studies, we have shown that PGJ2 inhibits each of these IL-1-stimulated events (25, 37). Similar to the IFN-{gamma}-stimulated signaling pathways, overexpression of the dnPPAR{gamma} Q284P mutant does not prevent the inhibitory actions of a 6-h pretreatment of RINm5F cells with PGJ2 on IL-1-induced I{kappa}B degradation (Fig. 3A). In a similar manner, the PPAR{gamma} antagonist GW-9662 does not modulate the ability of PGJ2 to inhibit IL-1-stimulated I{kappa}B degradation, as determined by Western blot analysis (Fig. 3B), or nuclear localization and DNA binding of NF-{kappa}B, as determined by gel shift analysis in RINm5F cells (Fig. 3C). Super-shift analysis and binding reactions performed in the presence of excess cold probe were used to confirm the presence of NF-{kappa}B in these DNA-protein complexes (data not shown). Taken together, these findings suggest that PPAR{gamma} is not required for the inhibitory actions of PGJ2 on IL-1- and IFN-{gamma}-stimulated signaling events.

We have previously shown that the inhibitory actions of PGJ2 on cytokine signaling correlate with increased expression of hsp 70 (37). To determine if PPAR{gamma} participates in the regulation of hsp 70 expression, the effects of GW-9662 on PGJ2-stimulated hsp 70 expression were examined by Western blot analysis. As shown in Fig. 3B, GW-9662 pretreatment does not attenuate the stimulatory actions of PGJ2 on hsp 70 expression by RINm5F cells. These findings suggest that the increased expression of this stress response protein by PGJ2 does not require PPAR{gamma} activation.

PPAR{gamma} inhibition does not attenuate the inhibitory actions of PGJ2 on cytokine-induced iNOS expression or NO production. Pancreatic {beta}-cells respond to the proinflammatory cytokine IL-1 by expressing iNOS and producing the free radical NO, and NO is known to mediate {beta}-cell damage stimulated by IL-1 (reviewed in Ref. 15). It has been previously reported that PGJ2 prevents the stimulatory actions of IL-1 on iNOS mRNA accumulation, protein expression, and NO production by rat islets and RINm5F cells (25, 37). Overexpression of dnPPAR{gamma} in RINm5F cells was used to determine whether PPAR{gamma} mediates the inhibitory actions of PGJ2 on IL-1-induced iNOS expression and NO production. In these experiments, RINm5F cells were transfected with the dnPPAR{gamma} expression plasmid for 48 h, the cells were then treated with PGJ2, and the effects of a 24-h incubation with IL-1 on iNOS expression and nitrite production were examined. As shown in Fig. 4A, PGJ2 inhibits to similar levels IL-1-stimulated iNOS expression (Western blot analysis, inset) and nitrite production by RINm5F cells expressing the vector control or the dnPPAR{gamma} mutant. In a similar manner, the PPAR{gamma} antagonist GW-9662 does not modulate the ability of PGJ2 to inhibit IL-1-induced iNOS expression or nitrite production by RINm5F cells (Fig. 4B). Consistent with the inhibition of iNOS protein expression, PGJ2 attenuates the stimulatory effects of a 4-h incubation with IL-1 on iNOS mRNA accumulation. Importantly, neither overexpression of dnPPAR{gamma} nor treatment with GW-9662 prevents the inhibitory actions of PGJ2 on iNOS mRNA accumulation by RINm5F cells (Fig. 5, A and B).

The inhibitory actions of PGJ2 on cytokine signaling and iNOS expression correlate with induction of a stress response, as evidenced by increased hsp 70 expression (25, 37). Recently, PGJ2-stimulated expression of hsp 70 has been shown to correlate with the inhibition of cytokine signaling; however, depletion of hsp 70 (by antisense) does not alter the inhibitory actions of PGJ2 on cytokine signaling, and hsp 70 overexpression does not inhibit cytokine signaling in RINm5F cells (37). To examine whether PPAR{gamma} is required for the induction of hsp 70 expression, the effects of GW-9622 on PGJ2-stimulated hsp 70 mRNA accumulation were examined by RT-PCR. As shown in Fig. 5B, GW-9662 treatment does not attenuate the stimulatory actions of PGJ2 on hsp 70 mRNA accumulation by RINm5F cells. This result is consistent with the lack of an inhibitory action of dnPPAR{gamma} overexpression on PGJ2-stimulated hsp 70 expression shown in Fig. 3B. These findings suggest that PPAR{gamma} activation is not required for induction of the stress response of hsp 70 expression by PGJ2 in RINm5F cells.

Endogenous PPAR{gamma} expression. Because dnPPAR{gamma} and the PPAR{gamma} antagonist GW-9662 do not modulate the inhibitory actions of PGJ2 on cytokine signaling in RINm5F cells and rat islets, we examined whether these cells express the PPAR{gamma} receptor by Western blot analysis and RT-PCR and compared expression levels with the levels of dnPPAR{gamma} expressed after transfection. As shown in Fig. 5A, the levels of PPAR{gamma} expressed in RINm5F cells are below the limits of detection by RT-PCR using primers that amplify mouse, human, and rat isoforms of PPAR{gamma}; however, the expression of the PPAR{gamma} is readily detectable in RINm5F cells transfected with the Q284P dnPPAR{gamma} mutant. Similar to the lack of PPAR{gamma} mRNA accumulation in RINm5F cells, PPAR{gamma} protein accumulation is only detectable in cells expressing the Q284P dnPPAR{gamma} mutant, as determined by Western blot analysis using antisera cross-reactive with human, mouse, and rat isoforms of PPAR{gamma} (Fig. 6B). With the same antibody, we are also unable to detect PPAR{gamma} expression in isolated rat islets (Fig. 6B).

Consistent with a lack of detectable PPAR{gamma} in RINm5F cells, treatment of these cells with increasing concentrations of PGJ2 was not sufficient to induce PPRE reporter gene activity. In these studies, RINm5F cells or CV-1 cells (as a positive control) were transfected with the PPRE-luciferase reporter plasmid and the {beta}-galactosidase control plasmid for 48 h, then cells were cultured with the indicated concentrations of PGJ2 for an additional 24 h. Cells were collected, and luciferase and {beta}-galactosidase activities were determined. Under these conditions, PGJ2, in a concentration-dependent fashion, stimulates PPRE reporter activity in CV-1 cells; however, PGJ2 fails to activate PPRE reporter activity in RINm5F cells (Fig. 6A). Although PGJ2 fails to stimulate PPRE activity in RINm5F cells, {beta}-galactosidase activity was readily detectable (data not shown). These studies indicate that RINm5F cells do not express detectable levels of PPAR{gamma} mRNA or protein, and that PGJ2, at concentrations that inhibit cytokine signaling, does not stimulate PPAR{gamma} transcriptional activation in these cells.


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PGJ2 and the synthetic thiazolidinediones (e.g., troglitazone and rosiglitazone), agonists of the PPAR{gamma} nuclear receptor, have been shown to possess anti-inflammatory activities that include attenuation of proinflammatory gene expression (9). The mechanisms by which PPAR{gamma} ligands inhibit inflammatory gene transcription have been associated with the ability of these cyclopentenone prostaglandins to inhibit the activation and DNA binding of the transcription factor NF-{kappa}B (35). Specifically, this class of prostanoids has been shown attenuate the activity of the regulatory enzyme IKK (29, 34). The PPAR{gamma} nuclear receptor does not appear to mediate the inhibitory actions of PGJ2 on NF-{kappa}B activation, since PGJ2 inhibits the activation of NF-{kappa}B in response to LPS in PPAR{gamma}-deficient macrophages (35). Conversely, overexpression of PPAR{gamma} in PPAR{gamma}-deficient macrophages increases the sensitivity of these cells to the inhibitory actions of PGJ2 on TNF-induced iNOS gene expression (35). Therefore, evidence supports a PPAR{gamma}-independent and PPAR{gamma}-dependent mechanism by which PPAR{gamma} ligands function as anti-inflammatory agents.

The aim of the current study was to evaluate whether PPAR{gamma} participates in the inhibitory actions of PGJ2 on cytokine signaling in RINm5F cells and rat islets. To address this aim, a dnPPAR{gamma} mutant containing a point mutation in the ligand-binding domain was generated. Expression of this mutant in CV-1 cells results in the inhibition of PGJ2-stimulated PPRE reporter gene activity. Although this mutant is an effective inhibitor of PPRE reporter gene activation, this mutant fails to prevent the inhibitory actions of PGJ2 on IFN-{gamma}-stimulated STAT1 phosphorylation and DNA binding and IL-1-stimulated NF-{kappa}B activation. In a similar fashion, the PPAR{gamma} antagonist GW-9662, which also inhibits PPRE reporter activity, does not attenuate the inhibitory actions of PGJ2 on IL-1 and IFN-{gamma} signaling in RINm5F cells or rat islets. Consistent with the inhibitory effects on cytokine signaling, PGJ2 also prevents IL-1-stimulated iNOS gene expression. This inhibitory action appears to be the result of PPAR{gamma}-independent mechanisms, as dnPPAR{gamma} Q284P mutant overexpression and PPAR{gamma} antagonism using GW-9662 fails to prevent these inhibitory effects of PGJ2 on IL-1-stimulated iNOS expression by {beta}-cells.

Recently, we have identified a third potential pathway by which PPAR{gamma} ligands inhibit inflammatory gene expression, and this pathway is associated with the activation of a stress response. PGJ2 and troglitazone stimulate the time-dependent expression of hsp 70, and, under conditions in which islets and RINm5F cells express hsp 70, IL-1 fails to activate NF-{kappa}B or JNK, and IFN-{gamma} fails to stimulate STAT1 phosphorylation and nuclear localization (25, 37). Although hsp 70 expression correlates with the inhibition of cytokine signaling, hsp 70 itself does not appear to mediate the anti-inflammatory actions of PGJ2. Antisense depletion of hsp 70 does not prevent the inhibitory actions of PGJ2 on IL-1-induced NF-{kappa}B activation or IFN-{gamma}-stimulated STAT1 activation. In addition, overexpression of hsp 70 in RINm5F cells does not inhibit cytokine signaling (37). Although hsp 70 expression correlates with the inhibitory actions of PGJ2 on cytokine signaling, the expression of hsp 70 in RINm5F cells also appears to occur by PPAR{gamma}-independent mechanisms. Neither overexpression of dnPPAR{gamma} nor treatment with the PPAR{gamma} antagonist GW-9662 modulates the ability of PGJ2 to stimulate hsp 70 expression by RINm5F cells.

In this study, we show that the inhibitory actions of PGJ2 on cytokine signaling do not require the activation of the PPAR{gamma} nuclear receptor. In previous studies, we have shown that the inhibitory actions of PPAR{gamma} ligands on cytokine signaling correlate with increased expression of hsp 70. Although hsp 70 does not mediate this cellular response (37), the ability of PPAR{gamma} ligands to stimulate hsp 70 may provide mechanistic insights as to how these ligands prevent cytokine signaling and inflammatory gene expression. One candidate cellular response is activation of endoplasmic reticulum (ER) stress, as studies have shown cyclopentenone prostaglandins colocalize with the ER (36), and the ER stress-induced pathways results in the increased expression of chaperones such as hsp 70 (21). Consistent with a role for ER stress are the recent findings that cyclopentenone prostaglandins attenuate protein synthesis (2). Importantly, one hallmark of ER stress activation is the phosphorylation of eukaryotic initiation factor (eIF)-2{alpha} and the inhibition of translation. Recently, we have shown that PGJ2,at concentrations that inhibit cytokine signaling, stimulates eIF-2{alpha} phosphorylation in RINm5F cells, and this phosphorylation event is associated with the inhibition of translation (S. M. Weber and J. A. Corbett, unpublished observations). These findings suggest that many of the anti-inflammatory actions of PPAR{gamma} ligands are mediated by PPAR{gamma}-independent mechanisms. Currently, we are examining the potential role of ER stress stimulated by these ligands, as one mechanism by these ligands attenuates cytokine signaling.


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


    ACKNOWLEDGMENTS
 
We thank Colleen Kelly Bratcher for expert technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Corbett, Dept. of Biochemistry and Molecular Biology, St. Louis Univ. School of Medicine, 1402 S. Grand Ave., 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.


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