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Activation of Peroxisome Proliferator-activated Receptor γ Inhibits Interleukin-1β-induced Membrane-associated Prostaglandin E2 Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1

Activation of Peroxisome Proliferator-activated Receptor γ Inhibits Interleukin-1β-induced... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 21, Issue of May 21, pp. 22057–22065, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Activation of Peroxisome Proliferator-activated Receptor  Inhibits Interleukin-1-induced Membrane-associated Prostaglandin E Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1* Received for publication, March 12, 2004 Published, JBC Papers in Press, March 15, 2004, DOI 10.1074/jbc.M402828200 Saranette Cheng‡§, Hassan Afif‡, Johanne Martel-Pelletier‡, Jean-Pierre Pelletier‡, Xinfang Li‡§, Katherine Farrajota‡, Martin Lavigne , and Hassan Fahmi‡§ From the ‡Osteoarthritis Research Unit, Centre Hospitalier de l’Universite ´ de Montre ´al, Ho ˆ pital Notre-Dame, the §Department of Medecine, Universite ´ de Montre ´al, and the Ho ˆ pital Maisonneuve-Rosemont, Montre ´al, Que ´bec H2L 4M1, Canada Prostaglandin (PG) E is an important modulator of many Membrane-associated prostaglandin (PG) E syn- thase-1 (mPGES-1) catalyzes the conversion of PGH to physiological and pathophysiological conditions, including cell PGE , which contributes to many biological processes. growth, vascular homeostasis, inflammation, immune regula- Peroxisome proliferator-activated receptor  (PPAR) tion, cancer, and arthritis (1, 2). The biosynthesis of PGE is a ligand-activated transcription factor and plays an requires two enzymes. Cyclooxygenase (COX; also termed PGH important role in growth, differentiation, and inflamma- synthase) converts arachidonic acid into PGH . Subsequently, tion in different tissues. Here, we examined the effect of PGE synthase (PGES) converts COX-2-derived PGH to PGE . 2 2 PPAR ligands on interleukin-1 (IL-1)-induced mPGES-1 Two isoforms of COX exist, COX-1 and COX-2, with similar expression in human synovial fibroblasts. PPAR ligands enzymatic properties but distinctly different biological func- 12,14 15-deoxy- prostaglandin J (15d-PGJ ) and the thiazo- 2 2 tions. COX-1 is expressed in most tissues and is responsible for lidinedione troglitazone (TRO), but not PPAR ligand physiological production of PGs. COX-2, in contrast, is almost Wy14643, dose-dependently suppressed IL-1-induced undetectable under physiological conditions but is strongly in- PGE production, as well as mPGES-1 protein and mRNA 2 duced in response to pro-inflammatory stimuli, growth factors, expression. 15d-PGJ and TRO suppressed IL-1-induced and mitogens (1–3). activation of the mPGES-1 promoter. Overexpression of At least three distinct PGES isoforms have been identified, wild-type PPAR further enhanced, whereas overexpres- including microsomal PGES-1 (mPGES-1), which was origi- sion of a dominant negative PPAR alleviated, the suppres- nally designated MGST1-L-1 (for membrane-bound GST1- sive effect of both PPAR ligands. Furthermore, pretreat- like-1) (4, 5), mPGES-2 (6), and cytosolic PGES (cPGES, or the ment with an antagonist of PPAR, GW9662, relieves the heat shock protein-associated protein p23) (7). cPGES is con- suppressive effect of PPAR ligands on mPGES-1 protein stitutively and ubiquitously expressed and is preferentially expression, suggesting that the inhibition of mPGES-1 ex- coupled with COX-1, promoting immediate production of PGE pression is mediated by PPAR. We demonstrated that (7, 8). By contrast, mPGES-1 is markedly up-regulated by PPAR ligands suppressed Egr-1-mediated induction of the pro-inflammatory stimuli and is functionally coupled with activities of the mPGES-1 promoter and of a synthetic re- COX-2, promoting delayed PGE synthesis (4, 5, 9). mPGES-2 porter construct containing three tandem repeats of an is ubiquitously expressed in diverse tissues (6); however, its Egr-1 binding site. The suppressive effect of PPAR ligands role remains elusive. Studies with deletion of mPGES-1 dem- was enhanced in the presence of a PPAR expression plas- onstrate that this isoform is largely responsible for the produc- mid. Electrophoretic mobility shift and supershift assays tion of PGE both in vitro and in vivo (10, 11). for Egr-1 binding sites in the mPGES-1 promoter showed mPGES-1 protein expression is induced in vitro in several that both 15d-PGJ and TRO suppressed IL-1-induced cell types after treatment with the pro-inflammatory cytokines, DNA-binding activity of Egr-1. These data define mPGES-1 interleukin (IL)-1, and tumor necrosis factor (TNF)- and is and Egr-1 as novel targets of PPAR and suggest that inhi- down-regulated by anti-inflammatory glucocorticoids (5, 12, bition of mPGES-1 gene transcription may be one of the 13). Moreover, mPGES-1 was shown to be up-regulated in vivo mechanisms by which PPAR regulates inflammatory in animal models of rheumatoid arthritis (9) and lipopolysac- responses. charide-induced pyresis (5). Increased levels of mPGES-1 mRNA and protein were also detected in symptomatic athero- sclerotic plaques, as well as various cancer cell lines and car- * This work was supported in part by the Canadian Institutes of Health Research Grant IMH-63168, the Fonds de Recherche en Sante ´ du Que ´ bec (FRSQ) Subvention d’Etablissement de Jeune Chercheur The abbreviations used are: PG, prostaglandin; 15d-PGJ , 15-deoxy- 12,14 # JC2836, and the Fonds de la Recherche du Centre de Recherche du  -prostaglandin J ; COX, cyclooxygenase; cPGES, cytosolic prosta- Centre Hospitalier de l’Universite ´ de Montre ´ al. The costs of publication glandin E synthase; EMSA, electrophoretic mobility shift assay; HSF, of this article were defrayed in part by the payment of page charges. human synovial fibroblast; IL, interleukin; iNOS, inducible nitric-oxide This article must therefore be hereby marked “advertisement”inac- synthase; MMP, metalloproteinase; mPGES, membrane-associated cordance with 18 U.S.C. Section 1734 solely to indicate this fact. prostaglandin E synthase; PPAR, peroxisome proliferator-activated re- A Research Scholar of FRSQ. To whom correspondence should be ceptor; TRO, troglitazone; TNF, tumor necrosis factor; OA, osteoar- addressed: Osteoarthritis Research Unit, Centre Hospitalier de thritic; NF-B, nuclear factor-B; PMSF, phenylmethylsulfonyl fluo- l’Universite ´ de Montre ´ al, Ho ˆ pital Notre-Dame, 1560 Sherbrooke St. ride; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf East, Montre ´ al, Que ´ bec H2L 4M1, Canada. Tel.: 514-890-8000 (ext. serum; DTT, dithiothreitol; DN, dominant negative; PPRE, PPAR-re- 28910); Fax: 514-412-7583; E-mail: [email protected]. sponsive element. This is an Open Access article under the CC BY license. This paper is available on line at http://www.jbc.org 22057 22058 Inhibition of mPGES-1 Gene Expression by PPAR Ligands ent in the cell preparation. In addition, flow cytometric analysis (Epic cinoma (14 –18), suggesting that aberrant expression of this II, Coulter, Miami, FL) using the anti-CD14 (fluorescein isothiocya- enzyme could contribute to the pathogenesis of these disorders. nate) antibody confirmed that no monocyte/macrophages were present Therefore, mPGES-1 may constitute a potential target for ther- in the synovial fibroblast preparation. The cells were seeded in tissue apeutic intervention. culture flasks and cultured until confluence in DMEM supplemented Peroxisome proliferator-activated receptors (PPARs) are a with 10% FCS and antibiotics at 37 °C in a humidified atmosphere of family of ligand-activated transcription factors belonging to the 5% CO /95% air. Only cells between passages 3 and 7 were used. PGE Assays—At the end of the incubation period, the culture me- nuclear receptor superfamily (19). To date, three PPAR sub- dium was collected and stored at 80 °C. Levels of PGE were deter- types have been identified: PPAR, PPAR/, and PPAR. mined using a PGE enzyme immunoassay kit from Cayman Chemical. PPAR is highly expressed in the liver, heart, kidney, and The detection limit and sensitivity was 9 pg/ml. All assays were per- intestinal mucosa, where its regulates lipid metabolism. formed in duplicate. PPAR is predominantly expressed in adipose tissue and reg- Western Blot Analysis—Cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 g/ml ulates adipocyte differentiation. PPAR is activated by eico- each of aprotinin, leupeptin, and pepstatin, 1% Nonidet P-40, 1 mM sanoids, fatty acids, and the hypolipidemic drug Wy14643 (se- sodium orthovanadate (Na VO ), and 1 mM NaF). Lysates were soni- 3 4 lective for PPAR). PPAR is activated by the prostaglandin cated on ice and centrifuged at 12,000 rpm for 15 min. The protein 12,14 D metabolite 15-deoxy- -PGJ (15d-PGJ ) and synthetic 2 2 2 concentration of the supernatant was determined using the bicincho- anti-diabetic thiazolidinedione drugs (e.g. troglitazone) (19). ninic acid method (Pierce). 20 g of total cell lysate or nuclear extracts There is accumulating evidence that PPAR and PPAR are was subjected to SDS-polyacrylamide gel electrophoresis and electro- transferred to a nitrocellulose membrane (Bio-Rad). After blocking in implicated as important regulators of immune and inflamma- 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.1% Tween 20, and tory responses. PPAR activation inhibits inflammatory medi- 5% (w/v) nonfat dry milk, blots were incubated overnight at 4 °C with ators release from several cell types (20 –22). In addition, primary antibodies and washed with wash buffer (Tris-buffered saline, PPAR-deficient mice exhibit exacerbated inflammatory re- pH 7.5, with 0.1% Tween 20). The blots were then incubated with sponses (23). PPAR activation results in the inhibition of horseradish peroxidase-conjugated secondary antibody (Pierce), washed again, incubated with SuperSignal Ultra Chemiluminescent various inflammatory events, such as the production of IL-1, reagent (Pierce), and finally exposed to X-Omat film (Eastman Kodak TNF-, and IL-6 in monocytes/macrophages as well as the Ltd., Rochester, NY). proliferation and the production of IL-2 by T lymphocytes (24 – RNA Extraction and cDNA Synthesis—Total RNA was isolated 26). Moreover, we have observed that PPAR ligands can sup- from HSFs using the TRIzol reagent (Invitrogen) and dissolved in 20 press the expression of the inducible nitric-oxide synthase l of diethylpyrocarbonate-treated H O. 1 g of total RNA was treated with RNase-free DNase and reverse-transcribed using Molo- (iNOS), MMP-13, and COX-2 in human chondrocytes and the ney Murine Leukemia Virus reverse transcriptase (Fermentas, Bur- expression of MMP-1 in human synovial fibroblasts (19, 27, 28). lington, Ontario, Canada) as detailed in the manufacturer’s guide- These actions of PPAR ligands were proven through repres- lines. One-fiftieth of the reverse transcriptase reaction was analyzed sion of activities of many transcription factors, including nu- by real-time PCR as described below. The following primers were clear factor-B (NF-B), activator protein 1, signal transducers used: mPGES-1, sense 5-GAAGAAGGCCTTTGCCAAC-3 and anti- and activators of transcription, and nuclear factor of activated sense 5-GGAAGACCAGGAAGTGCATC-3; cPGES, sense 5-GCAA- AGTGGTACGATCGAAGG-3 and antisense 5-TGTCCGTTCTTTTA- T cells (19, 24 –28). TGCTTGG-3; and glyceraldehyde-3-phosphate dehydrogenase, sense To date, limited information is available on the effect of PPAR 5-CAGAACATCATCCCTGCCTCT-3 and antisense 5-GCTTGACA- ligands on the induction of mPGES-1. Here, we analyze the effect AAGTGGTCGTTGAG-3. of two PPAR ligands, 15d-PGJ and troglitazone, on IL-1- Real-time Quantitative PCR—Quantitative PCR analysis was per- induced-mPGES-1 expression in human synovial fibroblasts and formed in a total volume of 50 l containing cDNA template, 200 nM of sense and antisense primers, and 25 l of SYBR® Green master mix investigate the mechanisms underlying this regulation. (Qiagen). Incorporation of SYBR® Green dye into PCR products was monitored in real time using a Gene Amp 5700 sequence detector EXPERIMENTAL PROCEDURES (Applied Biosystems) allowing determination of the threshold cycle (C ) Materials—Human recombinant IL-1 was obtained from R&D Sys- at which exponential amplification of PCR products begins. After incu- tems Inc. 15d-PGJ , troglitazone (TRO), Wy14643, GW9226, and en- bation at 95 °C for 10 min to activate the AmpliTaq Gold enzyme, the zyme immunoassay reagents for PGE assays were purchased from mixtures were subjected to 40 amplification cycles (15 s at 95 °C for Cayman Chemical. Aprotinin, leupeptin, pepstatin, and phenylmethyl- denaturation and 1 min for annealing and extension at 60 °C). After sulfonyl fluoride (PMSF) were from Sigma-Aldrich. Dulbecco’s modified PCR, dissociation curves were generated with one peak, indicating the Eagle’s medium (DMEM), penicillin and streptomycin, fetal calf serum specificity of the amplification. A threshold cycle (C value) was ob- (FCS), and TRIzol reagent were supplied by Invitrogen. [ P]ATP was tained from each amplification curve using the software provided by the from Amersham Biosciences. Plasmid DNA was prepared using a kit manufacturer (Applied Biosystems). Data were expressed as -fold from Qiagen. FuGENE 6 transfection reagent was from Roche Applied changes relative to control conditions (unstimulated cells) using the Science. The luciferase reporter assay system was from Promega. All C method as detailed in the manufacturer’s guidelines (Applied other chemicals were purchased from either Fisher Scientific or Bio- Biosystems). A C value was first calculated by subtracting the C T T Rad. Anti-mPGES-1 antibody was from Cayman Chemical, whereas value for the housekeeping gene glyceraldehyde-3-phosphate dehydro- anti-cPGES antibodies were from Cayman Chemical or Affinity BioRe- genase from the C value for each sample. A C value was then T T agents. Antibodies against Egr-1, PPAR, and -actin were purchased calculated by subtracting the C value of the control from the C T T from Santa Cruz Biotechnology Inc. Polyclonal rabbit anti-mouse IgG value of each treatment. The -fold changes compared with the control coupled with horseradish peroxidase and polyclonal goat anti-rabbit (unstimulated cells) were then determined by raising 2 to the C IgG with horseradish peroxidase were from Pierce. power. Each PCR reaction generated only the expected specific ampli- Specimen Selection and Cell Culture—HSFs were isolated from sy- con as shown by the melting-temperature profiles of the final product novial membranes obtained from osteoarthritic (OA) patients undergo- and by gel electrophoresis of test PCR reactions. Each PCR was per- ing total knee joint replacement. All OA patients were evaluated by a formed in triplicate on two separate occasions from at least three certified rheumatologist and diagnosed on criteria developed by the independent experiments. American College of Rheumatology Diagnostic Subcommittee for OA Plasmids and Transient Transfection—The human mPGES-1 pro- (29). Briefly, synovial fibroblasts were released by sequential enzymatic moter construct (538/28) was kindly provided by Dr. Terry J. Smith digestion with 1 mg/ml Pronase (Roche Applied Science) for 1 h, fol- (University of California, Los Angeles) (13). The human expression lowed by a 6-h incubation with 2 mg/ml collagenase (Type IA, Sigma) at vectors for wild type and dominant negative PPAR were a kind gift 37 °C in DMEM supplemented with 10% heat-inactivated FCS, 100 from Dr. Krishna K. Chatterjee (University of Cambridge, Cambridge, units/ml penicillin, and 100 g/ml streptomycin. Cells were incubated UK) (30). The human Egr-1 expression vector and the pEgr-1Mutx3- for1hat37 °C in tissue culture flasks (Primaria 3824, Falcon, Lincoln TK-Luc reporter construct were generously provided by Dr. Yuqing E. Park, NJ) allowing the adherence of non-fibroblastic cells possibly pres- Chen (Morehouse School of Medicine, Atlanta, GA) (31). A -galacto- Inhibition of mPGES-1 Gene Expression by PPAR Ligands 22059 sidase reporter vector under the control of SV40 promoter (pSV40-- galactosidase) was from Promega. Transient transfection experiments were performed using FuGENE 6(1 g of DNA:4 l of FuGENE 6) (Roche Applied Science) according to the manufacturer’s recommended protocol. Briefly, HSFs were seeded and grown to 50 – 60% confluence. The cells were transfected with 1 g of the reporter construct and 0.5 g of the internal control pSV40-- galactosidase. In cotransfection experiments the amount of transfected DNA was kept constant by using a corresponding empty vector. Six hours later, the medium was replaced with DMEM containing 1% FCS. The next day, the cells were treated for another 14 h with or without IL-1 in the absence or presence of 15d-PGJ or TRO. After harvesting, luciferase activity was determined and normalized to -galactosidase activity (27). Nuclear Extract Preparation and Electrophoretic Mobility Shift As- say—Nuclear extracts were prepared as previously described (28). Briefly, HSFs were washed in ice-cold phosphate-buffered saline and gently scrapped in ice-cold hypotonic buffer containing 10 mM HEPES- KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl , 0.5 mM DTT, 1 mM PMSF, 1 2 FIG.1. Effect of PPAR agonists on IL-1-induced PGE produc- mM Na VO , and 10 g/ml each of aprotinin, leupeptin, and pepstatin. tion in HSF. Confluent HSF were treated with increasing concentra- 3 4 The cells were allowed to swell on ice, and the nuclei were recovered by tions of 15d-PGJ , TRO, or Wy14643 for 30 min before incubation in the absence or the presence of 100 pg/ml IL-1 for 18 h. The culture media brief centrifugation. The pellets were resuspended in high salt buffer were collected and PGE production was determined. Data are ex- containing 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.2 mM MgCl , 0.5 mM 2 2 pressed as mean  S.E. from four independent experiments. *, p  0.05 DTT, 0.2 mM EDTA, 25% glycerol, 0.5 mM PMSF, 1 mM Na VO , and 10 3 4 compared with cells treated with IL-1 alone (control). g/ml each of aprotinin, leupeptin, and pepstatin, followed by incuba- tion on ice for 20 min. The nuclear extracts were recovered by centrif- ugation, and protein concentration was determined by the method of 2, A and B). By contrast, the PPAR-specific activator, Bradford (Bio-Rad). A synthetic double-stranded oligonucleotide, corresponding to the Wy14643, had no effect on IL-1-induced mPGES-1 expression Egr-1 motifs in the human mPGES-1 promoter (5-GTGGGG- (Fig. 2C). As shown in Fig. 2 (lower panels), cPGES protein was CGGGGCGTGGGCGGTGCT-3), was end-labeled by T4 polynucle- constitutively expressed in HSFs, and its expression was not otide kinase in the presence of [- P]ATP. The mutant competitor significantly altered by these treatments. These results indi- oligonucleotide had the following sequence with a 4-bp substitution cate that PPAR ligands can inhibit IL-1-induced mPGES-1 (underlined): 5-GTGGTTCGGGGCGTGTTCGGTGCT-3. The bind- expression. The concentrations of 15d-PGJ and TRO that in- ing buffer consisted of 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl , 4% glycerol, and 2.5 gofpoly- hibited IL-1-induced mPGES-1 expression and PGE produc- (dI-dC). Binding reactions were conducted with 5 g of nuclear ex- tion had no effect on cell viability as determined by Trypan blue tract and 100,000 cpm P-labeled oligonucleotide probe at 22 °Cfor exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- 20 min in a final volume of 10 l. In supershift assays, the antibody lium bromide assays (data not shown). to Egr-1 (1 g/reaction) was incubated with the reaction mixture for 32 Activation of PPAR Inhibits IL-1-induced mPGES-1 Ex- 1hat4 °C before the addition of P-labeled oligonucleotide. In cold pression at the Transcriptional Level—To further elucidate the competition assays, 100-fold molar excess of cold wild-type or mutant oligonucleotide was used. Binding complexes were resolved on non- mechanism responsible for the changes in amounts of denaturating 6% polyacrylamide gel electrophoresis in a Tris borate mPGES-1 protein, we measured the steady-state level of buffer system, after which the gels were fixed, dried, and subjected to mPGES-1 mRNA by quantitative reverse transcription-PCR. autoradiography. Treatment with IL-1 (100 pg/ml) enhanced the expression of Statistical Analysis—All results were calculated as the mean  S.E. mPGES-1 mRNA (Fig. 3A). Both 15d-PGJ and TRO dose-de- of independent experiments. Statistics were analyzed using Student’s pendently suppressed IL-1-induced mPGES-1 mRNA expres- 2-tailed t test. p values of 0.05 were considered significant. sion (Fig. 3A). In contrast, and in agreement with the data in RESULTS Fig. 2C, the PPAR-specific ligand Wy14643 had no significant Effect of PPAR Ligands on IL-1-induced PGE Production effect on IL-1-induced mPGES-1 mRNA expression (Fig. 3A). and mPGES-1 Protein Expression in HSF—We initially exam- As expected, the level of cPGES mRNA was not altered as a ined the effect of three distinct classes of PPAR ligands on consequence of treatment with IL-1 alone or in combination IL-1-induced PGE production in HSF: 15d-PGJ and TRO, with either PPAR ligands (Fig. 3B). Thus, the level of 2 2 natural and synthetic PPAR activators, respectively, and mPGES-1 and cPGES mRNA expression mirrors the pattern of Wy14643, a selective PPAR activator. Quiescent HSF were their respective protein expression. stimulated with IL-1 (100 pg/ml) in the absence or presence of To determine whether the regulation of IL-1-induced increasing concentrations of 15d-PGJ (5, 10, and 20 M), TRO, mPGES-1 mRNA by 15d-PGJ and TRO occurred at the level of 2 2 or Wy14643 (10, 25, and 50 M), and PGE production was transcription, we carried out transient transfection studies. determined. Under control cell culture conditions, HSFs re- Synovial fibroblasts were transfected with a human mPGES-1 leased low levels of PGE , and stimulation with IL-1 led to promoter (538 to 28) region/luciferase reporter gene con- severalfold increase in PGE production (Fig. 1). Pretreatment struct and stimulated with IL-1 in the absence or presence of with increasing concentrations of the PPAR ligands 15d-PGJ PPAR ligands. As shown in Fig. 4, IL-1 increased the lucif- or TRO suppressed IL-1-induced PGE production in a dose- erase activity of the mPGES-1 promoter, and this activation dependent manner. Conversely, the selective PPAR activator was dose-dependently reduced by 15d-PGJ (Fig. 4A). Simi- had no effect on IL-1-induced PGE production (Fig. 1). larly, treatment with TRO prevented IL-1-mediated activa- To determine whether these changes in PGE release were tion of the mPGES-1 promoter (Fig. 4B). Thus, PPAR ligands related to differences in amounts of mPGES-1, Western blot- suppress IL-1-induced mPGES-1 promoter activity, suggest- ting of cell lysate protein was carried out. As expected, treat- ing that PPAR ligands exert their inhibitory effects on ment with IL-1 resulted in a strong induction of mPGES-1 mPGES-1 expression through a transcriptional mechanism. protein expression (16 kDa) (Fig. 2, A–C). Interestingly, both Suppression of mPGES-1 Expression by 15d-PGJ and PPAR ligands, 15d-PGJ and TRO, suppressed IL-1-induced TRO Is Mediated by PPAR—PPAR ligands were reported mPGES-1 protein expression in a dose-dependent manner (Fig. to exert their transcriptional effects through PPAR-depend- 22060 Inhibition of mPGES-1 Gene Expression by PPAR Ligands FIG.3. Effect of PPAR agonists on IL-1-induced mPGES-1 mRNA expression in HSFs. Confluent HSFs were treated with in- creasing concentrations of 15d-PGJ , TRO, or Wy14643 for 30 min before incubation in the absence or the presence of 100 pg/ml IL-1 for 12 h. Total RNA was isolated; cDNA was synthesized; and mPGES-1, cPGES, and glyceraldehyde-3-phosphate dehydrogenase mRNAs were quantified using real-time quantitative PCR. The results are expressed FIG.2. Effect of PPAR agonists on IL-1-induced mPGES-1 as -fold changes, considering 1 as the value of untreated cells. All protein expression in HSFs. Confluent HSFs were treated with experiments were performed in triplicate, and negative controls with- increasing concentrations of 15d-PGJ (A), TRO (B), or Wy14643 (C) for out template RNA were included in each experiment. The results are 30 min before incubation in the absence or the presence of 100 pg/ml the mean  S.E. of three independent experiments. *, p  0.05; com- IL-1 for 18 h. Cell lysates were prepared and analyzed for mPGES-1 pared with cells treated with IL-1 alone (control). protein by Western blotting. In the middle panel, the blots were stripped and reprobed with a specific anti--actin antibody. The expres- sion of cPGES was also analyzed (lower panel). These blots are repre- PPAR-DN on its own had no significant effect on IL-1- sentative of similar results obtained from four independent induced mPGES-1 promoter activation (bar 2 versus bar 3), it experiments. relieved the suppressive effects of both 15d-PGJ (20 M)(bar 4 versus bar 6) and TRO (50 M)(bar 5 versus bar 7) (Fig. 5B), ent and -independent mechanisms (19). To evaluate the role suggesting that the inhibition of IL-1-induced mPGES-1 of PPAR in the suppressive effect of 15d-PGJ and TRO on expression by 15d-PGJ and TRO is mediated by PPAR.To 2 2 IL-1-induced mPGES-1 expression, we performed addi- confirm the involvement of PPAR in the suppressive effects tional transient transfection experiments using wild-type or of 15d-PGJ and TRO on IL-1-induced mPGES-1 expression, dominant negative (DN) PPAR expression plasmids. As we examined the action of GW9662, a selective and irrevers- shown in Fig. 5A, IL-1-induced activation of the mPGES-1 ible PPAR antagonist. HSFs were preincubated with in- promoter was reduced by 15d-PGJ (5 M)(bar 2 versus bar 4) creasing concentrations of GW9662 (1, 5, and 10 M) for 30 or TRO (10 M)(bar 2 versus bar 5). Cotransfection with an min prior to the addition of 15d-PGJ (20 M) or TRO (50 M) expression plasmid encoding PPAR also reduced IL-1-in- and were subsequently stimulated with IL-1 (100 pg/ml) for duced activation of the mPGES-1 promoter (bar 2 versus bar 18 h. Western blot analysis revealed that GW9662 dose-de- 3). Moreover, this effect was further enhanced with the ad- pendently relieved the suppressive effect of 15d-PGJ (Fig. dition of either 15d-PGJ (bar 3 versus bar 6)orTRO(bar 3 6A) and TRO (Fig. 6B) on IL-1-induced mPGES-1 protein versus bar 7). The possibility that PPAR is involved in the expression. GW9662 on its own had no significant effect on repression of mPGES-1 was further tested using the PPAR mPGES-1 expression (Fig. 6, last three lanes). As expected, double mutant (L468A/E471A), which was reported to exert the level of cPGES protein expression was not altered by powerful inhibitory action on endogenous PPAR. It contains these treatments (Fig. 6, lower panels). Taken together, these mutations in the AF-2 ligand-dependent domain, resulting in results suggest that 15d-PGJ and TRO inhibit IL-1-in- a marked impairment of coactivator recruitment and tran- duced mPGES-1 expression at the transcriptional level in a scriptional activation (30). The dominant negative activity of PPAR-dependent mechanism. this construct was confirmed in transient transfection exper- PPAR Activation Inhibits Transcriptional Activation by iments using wild-type PPAR and a luciferase reporter plas- Egr-1—The transcription factor Egr-1 had been shown to play mid consisting of three copies of the PPAR-responsive ele- a crucial role in the transcription of mPGES-1 (32, 33). There- ment (PPRE) (data not shown). Although overexpression of fore, we investigated the effect of 15d-PGJ and TRO on Egr- 2 Inhibition of mPGES-1 Gene Expression by PPAR Ligands 22061 FIG.4. 15d-PGJ and TRO inhibit the transcriptional activity of mPGES-1 promoter. HSFs were cotransfected with 1 g/well of the human mPGES-1 promoter (538/28) construct ligated to luciferase and 0.5 g of the internal control pSV40--galactosidase, using Fu- GENE 6 transfection reagent. The next day, transfected cells were incubated with increasing concentrations of 15d-PGJ (A) or TRO (B)in the absence or presence of IL-1 (100 pg/ml) for 14 h. Luciferase activity values were determined on cell extracts and normalized to -galacto- sidase activity. Results are expressed as -fold induction, considering 1 as the value of unstimulated cells, and represent the mean  S.E. of four independent experiments. *, p  0.05; compared with cells treated FIG.5. Effect of PPAR and dominant negative PPAR on with IL-1 alone (control). 15d-PGJ - and TRO-mediated suppression of mPGES-1 pro- moter activity. HSFs were cotransfected with the mPGES-1 promoter (1 g/well), the internal control pSV40--galactosidase (0.5 g/well), and 0.5 g of vectors expressing PPAR (A) or DN-PPAR (B). The total 1-mediated activation of mPGES-1 promoter. As shown in Fig. amount of transfected DNA was kept constant by addition of empty 7, the activity of the mPGES-1 promoter was enhanced by vector. The next day, cells were treated with the indicated concentra- cotransfection with a human Egr-1 expression plasmid (bar 1 tion of 15d-PGJ or TRO in the absence or presence of 100 pg/ml IL-1 for 14 h. Luciferase activity values were determined on cell extracts and versus bar 2). However, the activation of the mPGES-1 pro- normalized to -galactosidase activity. Results are expressed as -fold moter by Egr-1 was significantly attenuated by cotransfection induction, considering 1 as the value of unstimulated cells and repre- with the human PPAR expression plasmid (bar 2 versus bar sent the mean  S.E. of four independent experiments. *, p  0.05; 3). The activity of mPGES-1 promoter was also reduced by compared with cells treated with IL-1 alone (control). either 15d-PGJ (5 M)(bar 2 versus bar 4) or TRO (10 M)(bar 2 versus bar 5). Moreover, the suppressive effect of PPAR was sus bar 3). 15d-PGJ (bar 2 versus bar 4) and TRO (bar 2 versus further enhanced in the presence of 15d-PGJ (bar 3 versus bar 2 bar 5) also reduced the transcriptional activity induced by 6) or TRO (bar 3 versus bar 7). Egr-1. Again, the suppressive effect of PPAR was further Next, we sought to confirm that the inhibition of Egr-1 tran- enhanced in the presence of 15d-PGJ (bar 3 versus bar 6)or scriptional activity is essential in the suppression of mPGES-1 TRO (bar 3 versus bar 7). Taken together, these data suggest by PPAR. To this end, we analyzed the effect of PPAR on the that PPAR activation inhibits mPGES-1 promoter activation transcriptional activation of a synthetic luciferase reporter con- by interfering with the Egr-1 transcriptional activity. struct containing three tandem repeats of the putative Egr-1 PPAR Ligands Inhibit Egr-1 Binding Activity—Egr-1 has binding sequence, pEgr-1  3-TK-Luc (31). As shown in Fig. been shown to bind to the GC box of the mPGES-1 promoter 7B, overexpression of the human Egr-1 cDNA induced a robust (32, 33). To establish whether IL-1 could induce binding of increase in the transcriptional activity of the above construct Egr-1 to the mPGES-1 promoter and whether this was altered (bar 1 versus bar 2). This activation was attenuated by cotrans- by PPAR ligands, we investigated the effect of 15d-PGJ and fection with the human PPAR expression plasmid (bar 2 ver- TRO on the binding activity of Egr-1 using nuclear extracts 22062 Inhibition of mPGES-1 Gene Expression by PPAR Ligands FIG.6. PPAR antagonist (GW9662) alleviates the suppressive effect of 15d-PGJ and TRO on IL-1-induced mPGES-1 expres- sion. Confluent HSFs were pretreated with increasing concentrations of GW9662 for 30 min. Then, the cells were treated with or without IL-1 (100 pg/ml) for 18 h in the absence or the presence of 20 M 15d-PGJ (A)or50 M TRO (B). Cell lysates were prepared and ana- lyzed for mPGES-1 protein by Western blotting as described under “Experimental Procedures.” In the lower panel, the blots were stripped and reprobed with a specific anti--actin antibody. These blots are representative of similar results obtained from four independent experiments. from HSFs and a radiolabeled oligonucleotide corresponding to the Egr-1 binding sites in the mPGES-1 promoter. As shown in Fig. 8A, the binding of Egr-1 was strongly induced by IL-1 (lane 1 versus lane 2). When the cells were treated with 15d- FIG.7. PPAR ligands inhibit mPGES-1 gene transcription by interfering with promoter transactivation by Egr-1. HSFs were PGJ (lanes 3–5) or TRO (lanes 6 – 8) the formation of the cotransfected with mPGES-1-Luc (A) or p3xEgr-1-Luc (B) and an ex- Egr-1-DNA complex decreased in a dose-dependent manner. pression plasmid for Egr-1 with or without PPAR. The total amount of This binding was specific, because it could be completely abol- transfected DNA was kept constant by addition of empty vector. The ished by coincubation with a 100-fold molar excess of unlabeled next day, the cells were incubated with the indicated concentration of probe (lane 9). Coincubation, with a 100-fold molar excess of 15d-PGJ or TRO for 14 h. Luciferase activity values were determined on cell extracts and normalized to -galactosidase activity. Results are the mutant probe, did not affect Egr-1 DNA-binding activity expressed as -fold induction, considering 1 as the value of cells trans- (lane 10). The specificity of this interaction was further ob- fected with the reporter construct alone, and represent the mean  S.E. served by the supershift assays, showing a further retardation of four independent experiments. *, p  0.05; compared with cells in the electrophoretic mobility of the Egr-1-DNA complex in the transfected with Egr-1 alone (control). presence of a specific anti-Egr-1 antibody (lane 11). These re- sults suggest that PPAR ligands inhibit IL-1-induced of PPAR ligands on mPGES-1 expression does not involve mPGES-1 expression by reducing Egr-1 DNA-binding activity inhibition of Egr-1 protein expression. to the promoter sequence. DISCUSSION To determine whether the reduction of Egr-1 DNA-binding activity by PPAR ligands in HSFs was due to inhibition of An expanding body of evidence indicates that PPAR and its Egr-1 expression, we examined the effects of 15d-PGJ and ligands play an important role in the regulation of multiple TRO on IL-1-induced Egr-1 protein expression. The cells were inflammatory processes (19, 24 –28). In the present study, we pretreated with increasing concentrations of 15d-PGJ or TRO have extended these observations by showing that both natural prior to stimulation with IL-1. In quiescent HSFs, protein and synthetic PPAR ligands inhibit IL-1-induced mPGES-1 levels of Egr-1 were very low. Treatment with IL-1 (100 pg/ml) expression in HSFs. Furthermore, we elucidate the molecular caused a robust induction of Egr-1. Interestingly, neither 15d- mechanism underlying this effect. We demonstrate that this PGJ nor TRO altered IL-1-induced Egr-1 (Fig. 8B). These suppressive effect is transcriptional and PPAR-dependent. data suggest that 15d-PGJ and TRO are not general inhibitors Moreover, PPAR activation inhibited the transcriptional and of IL-1-induced gene expression and that the inhibitory effect DNA binding activities of Egr-1. Taken together, our results Inhibition of mPGES-1 Gene Expression by PPAR Ligands 22063 mPGES-1 promoter activation. Finally, pretreatment with an irreversible pharmacological PPAR antagonist, GW9662, overcame the inhibitory effect of PPAR ligands on mPGES-1 protein expression. However, inhibition of PPAR (via GW9662 or a DN), almost completely restored the suppressive effect of TRO, whereas the suppressive effect of 15d-PGJ was only partially restored, suggesting that 15d-PGJ can activate other PPAR-independent signaling pathways to inhibit mPGES-1 expression. In this context, several studies reported that 15d- PGJ inhibits many inflammatory responses by mechanisms that are independent of PPAR, such as the expression of iNOS in microglial cells and astrocytes (34), the  -integrin-depend- ent oxidative burst in human neutrophils (35) and the expres- sion of CD95 ligand in T lymphocytes (36). In addition, it was demonstrated that 15d-PGJ inhibits NF-B signaling at dif- ferent levels, including modification of I-B kinase activity, which reduces the NF-Bp65 nuclear translocation, and by direct modification of the DNA binding domain of NF-Bp50 (37, 38). Finally, Chawla et al. (39) examined inflammatory responses in macrophages derived from PPAR embryonic stem cells and reported that PPAR ligands still repress LPS- induced iNOS and COX-2 expression. Elucidation of PPAR- independent mechanisms of PPAR ligands needs further study. The transcriptional induction of mPGES-1 is controlled pri- marily by Egr-1 through two Egr-1 binding motifs identified in the proximal promoter region of the mPGES-1 region (32, 33). We hypothesized that inhibition of Egr-1 activity by PPAR could be the mechanism by which PPAR exerts its repressive effect on mPGES-1 transcription. By using reporter gene as- says, we found that Egr-1 indeed activated the mPGES-1 pro- moter, and this activation was reduced by cotransfection with an expression vector for PPAR. Moreover, 15d-PGJ and TRO inhibited Egr-1-mediated mPGES-1 promoter activation and this inhibition was further enhanced in the presence of a PPAR expression plasmid. PPAR activation also inhibited Egr-1-induced activation of a synthetic luciferase reporter con- struct containing three tandem repeats of Egr-1 motif, suggest- ing that PPAR inhibits Egr-1 transcriptional activity in a promoter-independent manner. This is the first evidence that PPAR activation inhibits Egr-1 transcriptional activity in HSFs. In EMSA and supershift assays, we observed that PPAR ligands reduced DNA binding of Egr-1 to a radiolabeled FIG.8. PPAR ligands inhibit DNA-binding activity of Egr-1. A, confluent HSFs were pretreated with increasing concentrations of 15d- oligonucleotide corresponding to the Egr-1 binding sites in the PGJ or TRO for 4 h, followed by the addition of IL-1 (100 pg/ml) for mPGES-1 promoter. Altogether, these results strongly suggest 1 h. Nuclear extracts (5 g) were incubated with a P-labeled oligonu- that PPAR-mediated repression of mPGES-1 results from de- cleotide containing the two Egr-1 binding sites of the mPGES-1 pro- creased Egr-1 binding activity. moter. Specificity of binding was confirmed using 100-fold molar excess of unlabeled oligonucleotides containing wild type (wt) or mutated (mt) Several mechanisms can explain the repression of Egr-1 Egr-1 binding sites. Positions of Egr-1-DNA complex (Egr-1), nonspe- activities by PPAR. One possibility is that PPAR activation cific binding (NS), and supershifted band (SS) are indicated. A repre- suppresses Egr-1 expression. Indeed, PPAR ligands were re- sentative result of four independent experiments is shown. B, confluent ported to inhibit hypoxia-induced Egr-1 expression in mononu- HSF were treated with increasing concentrations of 15d-PGJ or TRO or for 4 h, followed by the addition of IL-1 (100 pg/ml) for 1 h. Nuclear clear phagocytes (40). However, in our study, 15d-PGJ and extracts were prepared and analyzed for Egr-1 protein by Western TRO had no effect on IL-1-induced Egr-1 expression in HSFs, blotting. This blot is representative of similar results obtained from suggesting that PPAR ligands inhibit Egr-1 transcriptional three independent experiments. and DNA binding activities in HSFs by distinct mechanisms. A second mechanism could be competition between PPAR and reveal a novel function of PPAR, further supporting its role in Egr-1 for binding to response elements. This possibility is prob- the control of inflammatory responses. ably unlikely, because: (i) EMSA analysis showed no binding of Several lines of evidence indicate that the inhibitory effects PPAR to an oligonucleotide corresponding to the Egr-1 bind- of 15d-PGJ and TRO on IL-1-induced mPGES-1 expression ing sites in the mPGES-1 promoter (data not shown); (ii) the are likely to act through PPAR activation. First, treatment human mPGES-1 promoter construct used in this study con- with the specific PPAR activator, Wy14653, had no effect on IL-1-induced mPGES-1 expression. Second, overexpression of tains no consensus PPRE sequence; and (iii) PPAR activation inhibited Egr-1 transcriptional activity in a promoter-indepen- PPAR suppressed transcriptional activation of the mPGES-1 promoter, which was enhanced by the addition of PPAR li- dent manner. Alternatively, PPAR may inhibit Egr-1 activity by directly binding to Egr-1 and inhibiting its DNA binding gands. 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Immunol. 168, 2508 –2515 (2003) Arthritis Rheum. 48, 3544 –3556 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Activation of Peroxisome Proliferator-activated Receptor γ Inhibits Interleukin-1β-induced Membrane-associated Prostaglandin E2 Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 21, Issue of May 21, pp. 22057–22065, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Activation of Peroxisome Proliferator-activated Receptor  Inhibits Interleukin-1-induced Membrane-associated Prostaglandin E Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1* Received for publication, March 12, 2004 Published, JBC Papers in Press, March 15, 2004, DOI 10.1074/jbc.M402828200 Saranette Cheng‡§, Hassan Afif‡, Johanne Martel-Pelletier‡, Jean-Pierre Pelletier‡, Xinfang Li‡§, Katherine Farrajota‡, Martin Lavigne , and Hassan Fahmi‡§ From the ‡Osteoarthritis Research Unit, Centre Hospitalier de l’Universite ´ de Montre ´al, Ho ˆ pital Notre-Dame, the §Department of Medecine, Universite ´ de Montre ´al, and the Ho ˆ pital Maisonneuve-Rosemont, Montre ´al, Que ´bec H2L 4M1, Canada Prostaglandin (PG) E is an important modulator of many Membrane-associated prostaglandin (PG) E syn- thase-1 (mPGES-1) catalyzes the conversion of PGH to physiological and pathophysiological conditions, including cell PGE , which contributes to many biological processes. growth, vascular homeostasis, inflammation, immune regula- Peroxisome proliferator-activated receptor  (PPAR) tion, cancer, and arthritis (1, 2). The biosynthesis of PGE is a ligand-activated transcription factor and plays an requires two enzymes. Cyclooxygenase (COX; also termed PGH important role in growth, differentiation, and inflamma- synthase) converts arachidonic acid into PGH . Subsequently, tion in different tissues. Here, we examined the effect of PGE synthase (PGES) converts COX-2-derived PGH to PGE . 2 2 PPAR ligands on interleukin-1 (IL-1)-induced mPGES-1 Two isoforms of COX exist, COX-1 and COX-2, with similar expression in human synovial fibroblasts. PPAR ligands enzymatic properties but distinctly different biological func- 12,14 15-deoxy- prostaglandin J (15d-PGJ ) and the thiazo- 2 2 tions. COX-1 is expressed in most tissues and is responsible for lidinedione troglitazone (TRO), but not PPAR ligand physiological production of PGs. COX-2, in contrast, is almost Wy14643, dose-dependently suppressed IL-1-induced undetectable under physiological conditions but is strongly in- PGE production, as well as mPGES-1 protein and mRNA 2 duced in response to pro-inflammatory stimuli, growth factors, expression. 15d-PGJ and TRO suppressed IL-1-induced and mitogens (1–3). activation of the mPGES-1 promoter. Overexpression of At least three distinct PGES isoforms have been identified, wild-type PPAR further enhanced, whereas overexpres- including microsomal PGES-1 (mPGES-1), which was origi- sion of a dominant negative PPAR alleviated, the suppres- nally designated MGST1-L-1 (for membrane-bound GST1- sive effect of both PPAR ligands. Furthermore, pretreat- like-1) (4, 5), mPGES-2 (6), and cytosolic PGES (cPGES, or the ment with an antagonist of PPAR, GW9662, relieves the heat shock protein-associated protein p23) (7). cPGES is con- suppressive effect of PPAR ligands on mPGES-1 protein stitutively and ubiquitously expressed and is preferentially expression, suggesting that the inhibition of mPGES-1 ex- coupled with COX-1, promoting immediate production of PGE pression is mediated by PPAR. We demonstrated that (7, 8). By contrast, mPGES-1 is markedly up-regulated by PPAR ligands suppressed Egr-1-mediated induction of the pro-inflammatory stimuli and is functionally coupled with activities of the mPGES-1 promoter and of a synthetic re- COX-2, promoting delayed PGE synthesis (4, 5, 9). mPGES-2 porter construct containing three tandem repeats of an is ubiquitously expressed in diverse tissues (6); however, its Egr-1 binding site. The suppressive effect of PPAR ligands role remains elusive. Studies with deletion of mPGES-1 dem- was enhanced in the presence of a PPAR expression plas- onstrate that this isoform is largely responsible for the produc- mid. Electrophoretic mobility shift and supershift assays tion of PGE both in vitro and in vivo (10, 11). for Egr-1 binding sites in the mPGES-1 promoter showed mPGES-1 protein expression is induced in vitro in several that both 15d-PGJ and TRO suppressed IL-1-induced cell types after treatment with the pro-inflammatory cytokines, DNA-binding activity of Egr-1. These data define mPGES-1 interleukin (IL)-1, and tumor necrosis factor (TNF)- and is and Egr-1 as novel targets of PPAR and suggest that inhi- down-regulated by anti-inflammatory glucocorticoids (5, 12, bition of mPGES-1 gene transcription may be one of the 13). Moreover, mPGES-1 was shown to be up-regulated in vivo mechanisms by which PPAR regulates inflammatory in animal models of rheumatoid arthritis (9) and lipopolysac- responses. charide-induced pyresis (5). Increased levels of mPGES-1 mRNA and protein were also detected in symptomatic athero- sclerotic plaques, as well as various cancer cell lines and car- * This work was supported in part by the Canadian Institutes of Health Research Grant IMH-63168, the Fonds de Recherche en Sante ´ du Que ´ bec (FRSQ) Subvention d’Etablissement de Jeune Chercheur The abbreviations used are: PG, prostaglandin; 15d-PGJ , 15-deoxy- 12,14 # JC2836, and the Fonds de la Recherche du Centre de Recherche du  -prostaglandin J ; COX, cyclooxygenase; cPGES, cytosolic prosta- Centre Hospitalier de l’Universite ´ de Montre ´ al. The costs of publication glandin E synthase; EMSA, electrophoretic mobility shift assay; HSF, of this article were defrayed in part by the payment of page charges. human synovial fibroblast; IL, interleukin; iNOS, inducible nitric-oxide This article must therefore be hereby marked “advertisement”inac- synthase; MMP, metalloproteinase; mPGES, membrane-associated cordance with 18 U.S.C. Section 1734 solely to indicate this fact. prostaglandin E synthase; PPAR, peroxisome proliferator-activated re- A Research Scholar of FRSQ. To whom correspondence should be ceptor; TRO, troglitazone; TNF, tumor necrosis factor; OA, osteoar- addressed: Osteoarthritis Research Unit, Centre Hospitalier de thritic; NF-B, nuclear factor-B; PMSF, phenylmethylsulfonyl fluo- l’Universite ´ de Montre ´ al, Ho ˆ pital Notre-Dame, 1560 Sherbrooke St. ride; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf East, Montre ´ al, Que ´ bec H2L 4M1, Canada. Tel.: 514-890-8000 (ext. serum; DTT, dithiothreitol; DN, dominant negative; PPRE, PPAR-re- 28910); Fax: 514-412-7583; E-mail: [email protected]. sponsive element. This is an Open Access article under the CC BY license. This paper is available on line at http://www.jbc.org 22057 22058 Inhibition of mPGES-1 Gene Expression by PPAR Ligands ent in the cell preparation. In addition, flow cytometric analysis (Epic cinoma (14 –18), suggesting that aberrant expression of this II, Coulter, Miami, FL) using the anti-CD14 (fluorescein isothiocya- enzyme could contribute to the pathogenesis of these disorders. nate) antibody confirmed that no monocyte/macrophages were present Therefore, mPGES-1 may constitute a potential target for ther- in the synovial fibroblast preparation. The cells were seeded in tissue apeutic intervention. culture flasks and cultured until confluence in DMEM supplemented Peroxisome proliferator-activated receptors (PPARs) are a with 10% FCS and antibiotics at 37 °C in a humidified atmosphere of family of ligand-activated transcription factors belonging to the 5% CO /95% air. Only cells between passages 3 and 7 were used. PGE Assays—At the end of the incubation period, the culture me- nuclear receptor superfamily (19). To date, three PPAR sub- dium was collected and stored at 80 °C. Levels of PGE were deter- types have been identified: PPAR, PPAR/, and PPAR. mined using a PGE enzyme immunoassay kit from Cayman Chemical. PPAR is highly expressed in the liver, heart, kidney, and The detection limit and sensitivity was 9 pg/ml. All assays were per- intestinal mucosa, where its regulates lipid metabolism. formed in duplicate. PPAR is predominantly expressed in adipose tissue and reg- Western Blot Analysis—Cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 g/ml ulates adipocyte differentiation. PPAR is activated by eico- each of aprotinin, leupeptin, and pepstatin, 1% Nonidet P-40, 1 mM sanoids, fatty acids, and the hypolipidemic drug Wy14643 (se- sodium orthovanadate (Na VO ), and 1 mM NaF). Lysates were soni- 3 4 lective for PPAR). PPAR is activated by the prostaglandin cated on ice and centrifuged at 12,000 rpm for 15 min. The protein 12,14 D metabolite 15-deoxy- -PGJ (15d-PGJ ) and synthetic 2 2 2 concentration of the supernatant was determined using the bicincho- anti-diabetic thiazolidinedione drugs (e.g. troglitazone) (19). ninic acid method (Pierce). 20 g of total cell lysate or nuclear extracts There is accumulating evidence that PPAR and PPAR are was subjected to SDS-polyacrylamide gel electrophoresis and electro- transferred to a nitrocellulose membrane (Bio-Rad). After blocking in implicated as important regulators of immune and inflamma- 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.1% Tween 20, and tory responses. PPAR activation inhibits inflammatory medi- 5% (w/v) nonfat dry milk, blots were incubated overnight at 4 °C with ators release from several cell types (20 –22). In addition, primary antibodies and washed with wash buffer (Tris-buffered saline, PPAR-deficient mice exhibit exacerbated inflammatory re- pH 7.5, with 0.1% Tween 20). The blots were then incubated with sponses (23). PPAR activation results in the inhibition of horseradish peroxidase-conjugated secondary antibody (Pierce), washed again, incubated with SuperSignal Ultra Chemiluminescent various inflammatory events, such as the production of IL-1, reagent (Pierce), and finally exposed to X-Omat film (Eastman Kodak TNF-, and IL-6 in monocytes/macrophages as well as the Ltd., Rochester, NY). proliferation and the production of IL-2 by T lymphocytes (24 – RNA Extraction and cDNA Synthesis—Total RNA was isolated 26). Moreover, we have observed that PPAR ligands can sup- from HSFs using the TRIzol reagent (Invitrogen) and dissolved in 20 press the expression of the inducible nitric-oxide synthase l of diethylpyrocarbonate-treated H O. 1 g of total RNA was treated with RNase-free DNase and reverse-transcribed using Molo- (iNOS), MMP-13, and COX-2 in human chondrocytes and the ney Murine Leukemia Virus reverse transcriptase (Fermentas, Bur- expression of MMP-1 in human synovial fibroblasts (19, 27, 28). lington, Ontario, Canada) as detailed in the manufacturer’s guide- These actions of PPAR ligands were proven through repres- lines. One-fiftieth of the reverse transcriptase reaction was analyzed sion of activities of many transcription factors, including nu- by real-time PCR as described below. The following primers were clear factor-B (NF-B), activator protein 1, signal transducers used: mPGES-1, sense 5-GAAGAAGGCCTTTGCCAAC-3 and anti- and activators of transcription, and nuclear factor of activated sense 5-GGAAGACCAGGAAGTGCATC-3; cPGES, sense 5-GCAA- AGTGGTACGATCGAAGG-3 and antisense 5-TGTCCGTTCTTTTA- T cells (19, 24 –28). TGCTTGG-3; and glyceraldehyde-3-phosphate dehydrogenase, sense To date, limited information is available on the effect of PPAR 5-CAGAACATCATCCCTGCCTCT-3 and antisense 5-GCTTGACA- ligands on the induction of mPGES-1. Here, we analyze the effect AAGTGGTCGTTGAG-3. of two PPAR ligands, 15d-PGJ and troglitazone, on IL-1- Real-time Quantitative PCR—Quantitative PCR analysis was per- induced-mPGES-1 expression in human synovial fibroblasts and formed in a total volume of 50 l containing cDNA template, 200 nM of sense and antisense primers, and 25 l of SYBR® Green master mix investigate the mechanisms underlying this regulation. (Qiagen). Incorporation of SYBR® Green dye into PCR products was monitored in real time using a Gene Amp 5700 sequence detector EXPERIMENTAL PROCEDURES (Applied Biosystems) allowing determination of the threshold cycle (C ) Materials—Human recombinant IL-1 was obtained from R&D Sys- at which exponential amplification of PCR products begins. After incu- tems Inc. 15d-PGJ , troglitazone (TRO), Wy14643, GW9226, and en- bation at 95 °C for 10 min to activate the AmpliTaq Gold enzyme, the zyme immunoassay reagents for PGE assays were purchased from mixtures were subjected to 40 amplification cycles (15 s at 95 °C for Cayman Chemical. Aprotinin, leupeptin, pepstatin, and phenylmethyl- denaturation and 1 min for annealing and extension at 60 °C). After sulfonyl fluoride (PMSF) were from Sigma-Aldrich. Dulbecco’s modified PCR, dissociation curves were generated with one peak, indicating the Eagle’s medium (DMEM), penicillin and streptomycin, fetal calf serum specificity of the amplification. A threshold cycle (C value) was ob- (FCS), and TRIzol reagent were supplied by Invitrogen. [ P]ATP was tained from each amplification curve using the software provided by the from Amersham Biosciences. Plasmid DNA was prepared using a kit manufacturer (Applied Biosystems). Data were expressed as -fold from Qiagen. FuGENE 6 transfection reagent was from Roche Applied changes relative to control conditions (unstimulated cells) using the Science. The luciferase reporter assay system was from Promega. All C method as detailed in the manufacturer’s guidelines (Applied other chemicals were purchased from either Fisher Scientific or Bio- Biosystems). A C value was first calculated by subtracting the C T T Rad. Anti-mPGES-1 antibody was from Cayman Chemical, whereas value for the housekeeping gene glyceraldehyde-3-phosphate dehydro- anti-cPGES antibodies were from Cayman Chemical or Affinity BioRe- genase from the C value for each sample. A C value was then T T agents. Antibodies against Egr-1, PPAR, and -actin were purchased calculated by subtracting the C value of the control from the C T T from Santa Cruz Biotechnology Inc. Polyclonal rabbit anti-mouse IgG value of each treatment. The -fold changes compared with the control coupled with horseradish peroxidase and polyclonal goat anti-rabbit (unstimulated cells) were then determined by raising 2 to the C IgG with horseradish peroxidase were from Pierce. power. Each PCR reaction generated only the expected specific ampli- Specimen Selection and Cell Culture—HSFs were isolated from sy- con as shown by the melting-temperature profiles of the final product novial membranes obtained from osteoarthritic (OA) patients undergo- and by gel electrophoresis of test PCR reactions. Each PCR was per- ing total knee joint replacement. All OA patients were evaluated by a formed in triplicate on two separate occasions from at least three certified rheumatologist and diagnosed on criteria developed by the independent experiments. American College of Rheumatology Diagnostic Subcommittee for OA Plasmids and Transient Transfection—The human mPGES-1 pro- (29). Briefly, synovial fibroblasts were released by sequential enzymatic moter construct (538/28) was kindly provided by Dr. Terry J. Smith digestion with 1 mg/ml Pronase (Roche Applied Science) for 1 h, fol- (University of California, Los Angeles) (13). The human expression lowed by a 6-h incubation with 2 mg/ml collagenase (Type IA, Sigma) at vectors for wild type and dominant negative PPAR were a kind gift 37 °C in DMEM supplemented with 10% heat-inactivated FCS, 100 from Dr. Krishna K. Chatterjee (University of Cambridge, Cambridge, units/ml penicillin, and 100 g/ml streptomycin. Cells were incubated UK) (30). The human Egr-1 expression vector and the pEgr-1Mutx3- for1hat37 °C in tissue culture flasks (Primaria 3824, Falcon, Lincoln TK-Luc reporter construct were generously provided by Dr. Yuqing E. Park, NJ) allowing the adherence of non-fibroblastic cells possibly pres- Chen (Morehouse School of Medicine, Atlanta, GA) (31). A -galacto- Inhibition of mPGES-1 Gene Expression by PPAR Ligands 22059 sidase reporter vector under the control of SV40 promoter (pSV40-- galactosidase) was from Promega. Transient transfection experiments were performed using FuGENE 6(1 g of DNA:4 l of FuGENE 6) (Roche Applied Science) according to the manufacturer’s recommended protocol. Briefly, HSFs were seeded and grown to 50 – 60% confluence. The cells were transfected with 1 g of the reporter construct and 0.5 g of the internal control pSV40-- galactosidase. In cotransfection experiments the amount of transfected DNA was kept constant by using a corresponding empty vector. Six hours later, the medium was replaced with DMEM containing 1% FCS. The next day, the cells were treated for another 14 h with or without IL-1 in the absence or presence of 15d-PGJ or TRO. After harvesting, luciferase activity was determined and normalized to -galactosidase activity (27). Nuclear Extract Preparation and Electrophoretic Mobility Shift As- say—Nuclear extracts were prepared as previously described (28). Briefly, HSFs were washed in ice-cold phosphate-buffered saline and gently scrapped in ice-cold hypotonic buffer containing 10 mM HEPES- KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl , 0.5 mM DTT, 1 mM PMSF, 1 2 FIG.1. Effect of PPAR agonists on IL-1-induced PGE produc- mM Na VO , and 10 g/ml each of aprotinin, leupeptin, and pepstatin. tion in HSF. Confluent HSF were treated with increasing concentra- 3 4 The cells were allowed to swell on ice, and the nuclei were recovered by tions of 15d-PGJ , TRO, or Wy14643 for 30 min before incubation in the absence or the presence of 100 pg/ml IL-1 for 18 h. The culture media brief centrifugation. The pellets were resuspended in high salt buffer were collected and PGE production was determined. Data are ex- containing 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.2 mM MgCl , 0.5 mM 2 2 pressed as mean  S.E. from four independent experiments. *, p  0.05 DTT, 0.2 mM EDTA, 25% glycerol, 0.5 mM PMSF, 1 mM Na VO , and 10 3 4 compared with cells treated with IL-1 alone (control). g/ml each of aprotinin, leupeptin, and pepstatin, followed by incuba- tion on ice for 20 min. The nuclear extracts were recovered by centrif- ugation, and protein concentration was determined by the method of 2, A and B). By contrast, the PPAR-specific activator, Bradford (Bio-Rad). A synthetic double-stranded oligonucleotide, corresponding to the Wy14643, had no effect on IL-1-induced mPGES-1 expression Egr-1 motifs in the human mPGES-1 promoter (5-GTGGGG- (Fig. 2C). As shown in Fig. 2 (lower panels), cPGES protein was CGGGGCGTGGGCGGTGCT-3), was end-labeled by T4 polynucle- constitutively expressed in HSFs, and its expression was not otide kinase in the presence of [- P]ATP. The mutant competitor significantly altered by these treatments. These results indi- oligonucleotide had the following sequence with a 4-bp substitution cate that PPAR ligands can inhibit IL-1-induced mPGES-1 (underlined): 5-GTGGTTCGGGGCGTGTTCGGTGCT-3. The bind- expression. The concentrations of 15d-PGJ and TRO that in- ing buffer consisted of 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl , 4% glycerol, and 2.5 gofpoly- hibited IL-1-induced mPGES-1 expression and PGE produc- (dI-dC). Binding reactions were conducted with 5 g of nuclear ex- tion had no effect on cell viability as determined by Trypan blue tract and 100,000 cpm P-labeled oligonucleotide probe at 22 °Cfor exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- 20 min in a final volume of 10 l. In supershift assays, the antibody lium bromide assays (data not shown). to Egr-1 (1 g/reaction) was incubated with the reaction mixture for 32 Activation of PPAR Inhibits IL-1-induced mPGES-1 Ex- 1hat4 °C before the addition of P-labeled oligonucleotide. In cold pression at the Transcriptional Level—To further elucidate the competition assays, 100-fold molar excess of cold wild-type or mutant oligonucleotide was used. Binding complexes were resolved on non- mechanism responsible for the changes in amounts of denaturating 6% polyacrylamide gel electrophoresis in a Tris borate mPGES-1 protein, we measured the steady-state level of buffer system, after which the gels were fixed, dried, and subjected to mPGES-1 mRNA by quantitative reverse transcription-PCR. autoradiography. Treatment with IL-1 (100 pg/ml) enhanced the expression of Statistical Analysis—All results were calculated as the mean  S.E. mPGES-1 mRNA (Fig. 3A). Both 15d-PGJ and TRO dose-de- of independent experiments. Statistics were analyzed using Student’s pendently suppressed IL-1-induced mPGES-1 mRNA expres- 2-tailed t test. p values of 0.05 were considered significant. sion (Fig. 3A). In contrast, and in agreement with the data in RESULTS Fig. 2C, the PPAR-specific ligand Wy14643 had no significant Effect of PPAR Ligands on IL-1-induced PGE Production effect on IL-1-induced mPGES-1 mRNA expression (Fig. 3A). and mPGES-1 Protein Expression in HSF—We initially exam- As expected, the level of cPGES mRNA was not altered as a ined the effect of three distinct classes of PPAR ligands on consequence of treatment with IL-1 alone or in combination IL-1-induced PGE production in HSF: 15d-PGJ and TRO, with either PPAR ligands (Fig. 3B). Thus, the level of 2 2 natural and synthetic PPAR activators, respectively, and mPGES-1 and cPGES mRNA expression mirrors the pattern of Wy14643, a selective PPAR activator. Quiescent HSF were their respective protein expression. stimulated with IL-1 (100 pg/ml) in the absence or presence of To determine whether the regulation of IL-1-induced increasing concentrations of 15d-PGJ (5, 10, and 20 M), TRO, mPGES-1 mRNA by 15d-PGJ and TRO occurred at the level of 2 2 or Wy14643 (10, 25, and 50 M), and PGE production was transcription, we carried out transient transfection studies. determined. Under control cell culture conditions, HSFs re- Synovial fibroblasts were transfected with a human mPGES-1 leased low levels of PGE , and stimulation with IL-1 led to promoter (538 to 28) region/luciferase reporter gene con- severalfold increase in PGE production (Fig. 1). Pretreatment struct and stimulated with IL-1 in the absence or presence of with increasing concentrations of the PPAR ligands 15d-PGJ PPAR ligands. As shown in Fig. 4, IL-1 increased the lucif- or TRO suppressed IL-1-induced PGE production in a dose- erase activity of the mPGES-1 promoter, and this activation dependent manner. Conversely, the selective PPAR activator was dose-dependently reduced by 15d-PGJ (Fig. 4A). Simi- had no effect on IL-1-induced PGE production (Fig. 1). larly, treatment with TRO prevented IL-1-mediated activa- To determine whether these changes in PGE release were tion of the mPGES-1 promoter (Fig. 4B). Thus, PPAR ligands related to differences in amounts of mPGES-1, Western blot- suppress IL-1-induced mPGES-1 promoter activity, suggest- ting of cell lysate protein was carried out. As expected, treat- ing that PPAR ligands exert their inhibitory effects on ment with IL-1 resulted in a strong induction of mPGES-1 mPGES-1 expression through a transcriptional mechanism. protein expression (16 kDa) (Fig. 2, A–C). Interestingly, both Suppression of mPGES-1 Expression by 15d-PGJ and PPAR ligands, 15d-PGJ and TRO, suppressed IL-1-induced TRO Is Mediated by PPAR—PPAR ligands were reported mPGES-1 protein expression in a dose-dependent manner (Fig. to exert their transcriptional effects through PPAR-depend- 22060 Inhibition of mPGES-1 Gene Expression by PPAR Ligands FIG.3. Effect of PPAR agonists on IL-1-induced mPGES-1 mRNA expression in HSFs. Confluent HSFs were treated with in- creasing concentrations of 15d-PGJ , TRO, or Wy14643 for 30 min before incubation in the absence or the presence of 100 pg/ml IL-1 for 12 h. Total RNA was isolated; cDNA was synthesized; and mPGES-1, cPGES, and glyceraldehyde-3-phosphate dehydrogenase mRNAs were quantified using real-time quantitative PCR. The results are expressed FIG.2. Effect of PPAR agonists on IL-1-induced mPGES-1 as -fold changes, considering 1 as the value of untreated cells. All protein expression in HSFs. Confluent HSFs were treated with experiments were performed in triplicate, and negative controls with- increasing concentrations of 15d-PGJ (A), TRO (B), or Wy14643 (C) for out template RNA were included in each experiment. The results are 30 min before incubation in the absence or the presence of 100 pg/ml the mean  S.E. of three independent experiments. *, p  0.05; com- IL-1 for 18 h. Cell lysates were prepared and analyzed for mPGES-1 pared with cells treated with IL-1 alone (control). protein by Western blotting. In the middle panel, the blots were stripped and reprobed with a specific anti--actin antibody. The expres- sion of cPGES was also analyzed (lower panel). These blots are repre- PPAR-DN on its own had no significant effect on IL-1- sentative of similar results obtained from four independent induced mPGES-1 promoter activation (bar 2 versus bar 3), it experiments. relieved the suppressive effects of both 15d-PGJ (20 M)(bar 4 versus bar 6) and TRO (50 M)(bar 5 versus bar 7) (Fig. 5B), ent and -independent mechanisms (19). To evaluate the role suggesting that the inhibition of IL-1-induced mPGES-1 of PPAR in the suppressive effect of 15d-PGJ and TRO on expression by 15d-PGJ and TRO is mediated by PPAR.To 2 2 IL-1-induced mPGES-1 expression, we performed addi- confirm the involvement of PPAR in the suppressive effects tional transient transfection experiments using wild-type or of 15d-PGJ and TRO on IL-1-induced mPGES-1 expression, dominant negative (DN) PPAR expression plasmids. As we examined the action of GW9662, a selective and irrevers- shown in Fig. 5A, IL-1-induced activation of the mPGES-1 ible PPAR antagonist. HSFs were preincubated with in- promoter was reduced by 15d-PGJ (5 M)(bar 2 versus bar 4) creasing concentrations of GW9662 (1, 5, and 10 M) for 30 or TRO (10 M)(bar 2 versus bar 5). Cotransfection with an min prior to the addition of 15d-PGJ (20 M) or TRO (50 M) expression plasmid encoding PPAR also reduced IL-1-in- and were subsequently stimulated with IL-1 (100 pg/ml) for duced activation of the mPGES-1 promoter (bar 2 versus bar 18 h. Western blot analysis revealed that GW9662 dose-de- 3). Moreover, this effect was further enhanced with the ad- pendently relieved the suppressive effect of 15d-PGJ (Fig. dition of either 15d-PGJ (bar 3 versus bar 6)orTRO(bar 3 6A) and TRO (Fig. 6B) on IL-1-induced mPGES-1 protein versus bar 7). The possibility that PPAR is involved in the expression. GW9662 on its own had no significant effect on repression of mPGES-1 was further tested using the PPAR mPGES-1 expression (Fig. 6, last three lanes). As expected, double mutant (L468A/E471A), which was reported to exert the level of cPGES protein expression was not altered by powerful inhibitory action on endogenous PPAR. It contains these treatments (Fig. 6, lower panels). Taken together, these mutations in the AF-2 ligand-dependent domain, resulting in results suggest that 15d-PGJ and TRO inhibit IL-1-in- a marked impairment of coactivator recruitment and tran- duced mPGES-1 expression at the transcriptional level in a scriptional activation (30). The dominant negative activity of PPAR-dependent mechanism. this construct was confirmed in transient transfection exper- PPAR Activation Inhibits Transcriptional Activation by iments using wild-type PPAR and a luciferase reporter plas- Egr-1—The transcription factor Egr-1 had been shown to play mid consisting of three copies of the PPAR-responsive ele- a crucial role in the transcription of mPGES-1 (32, 33). There- ment (PPRE) (data not shown). Although overexpression of fore, we investigated the effect of 15d-PGJ and TRO on Egr- 2 Inhibition of mPGES-1 Gene Expression by PPAR Ligands 22061 FIG.4. 15d-PGJ and TRO inhibit the transcriptional activity of mPGES-1 promoter. HSFs were cotransfected with 1 g/well of the human mPGES-1 promoter (538/28) construct ligated to luciferase and 0.5 g of the internal control pSV40--galactosidase, using Fu- GENE 6 transfection reagent. The next day, transfected cells were incubated with increasing concentrations of 15d-PGJ (A) or TRO (B)in the absence or presence of IL-1 (100 pg/ml) for 14 h. Luciferase activity values were determined on cell extracts and normalized to -galacto- sidase activity. Results are expressed as -fold induction, considering 1 as the value of unstimulated cells, and represent the mean  S.E. of four independent experiments. *, p  0.05; compared with cells treated FIG.5. Effect of PPAR and dominant negative PPAR on with IL-1 alone (control). 15d-PGJ - and TRO-mediated suppression of mPGES-1 pro- moter activity. HSFs were cotransfected with the mPGES-1 promoter (1 g/well), the internal control pSV40--galactosidase (0.5 g/well), and 0.5 g of vectors expressing PPAR (A) or DN-PPAR (B). The total 1-mediated activation of mPGES-1 promoter. As shown in Fig. amount of transfected DNA was kept constant by addition of empty 7, the activity of the mPGES-1 promoter was enhanced by vector. The next day, cells were treated with the indicated concentra- cotransfection with a human Egr-1 expression plasmid (bar 1 tion of 15d-PGJ or TRO in the absence or presence of 100 pg/ml IL-1 for 14 h. Luciferase activity values were determined on cell extracts and versus bar 2). However, the activation of the mPGES-1 pro- normalized to -galactosidase activity. Results are expressed as -fold moter by Egr-1 was significantly attenuated by cotransfection induction, considering 1 as the value of unstimulated cells and repre- with the human PPAR expression plasmid (bar 2 versus bar sent the mean  S.E. of four independent experiments. *, p  0.05; 3). The activity of mPGES-1 promoter was also reduced by compared with cells treated with IL-1 alone (control). either 15d-PGJ (5 M)(bar 2 versus bar 4) or TRO (10 M)(bar 2 versus bar 5). Moreover, the suppressive effect of PPAR was sus bar 3). 15d-PGJ (bar 2 versus bar 4) and TRO (bar 2 versus further enhanced in the presence of 15d-PGJ (bar 3 versus bar 2 bar 5) also reduced the transcriptional activity induced by 6) or TRO (bar 3 versus bar 7). Egr-1. Again, the suppressive effect of PPAR was further Next, we sought to confirm that the inhibition of Egr-1 tran- enhanced in the presence of 15d-PGJ (bar 3 versus bar 6)or scriptional activity is essential in the suppression of mPGES-1 TRO (bar 3 versus bar 7). Taken together, these data suggest by PPAR. To this end, we analyzed the effect of PPAR on the that PPAR activation inhibits mPGES-1 promoter activation transcriptional activation of a synthetic luciferase reporter con- by interfering with the Egr-1 transcriptional activity. struct containing three tandem repeats of the putative Egr-1 PPAR Ligands Inhibit Egr-1 Binding Activity—Egr-1 has binding sequence, pEgr-1  3-TK-Luc (31). As shown in Fig. been shown to bind to the GC box of the mPGES-1 promoter 7B, overexpression of the human Egr-1 cDNA induced a robust (32, 33). To establish whether IL-1 could induce binding of increase in the transcriptional activity of the above construct Egr-1 to the mPGES-1 promoter and whether this was altered (bar 1 versus bar 2). This activation was attenuated by cotrans- by PPAR ligands, we investigated the effect of 15d-PGJ and fection with the human PPAR expression plasmid (bar 2 ver- TRO on the binding activity of Egr-1 using nuclear extracts 22062 Inhibition of mPGES-1 Gene Expression by PPAR Ligands FIG.6. PPAR antagonist (GW9662) alleviates the suppressive effect of 15d-PGJ and TRO on IL-1-induced mPGES-1 expres- sion. Confluent HSFs were pretreated with increasing concentrations of GW9662 for 30 min. Then, the cells were treated with or without IL-1 (100 pg/ml) for 18 h in the absence or the presence of 20 M 15d-PGJ (A)or50 M TRO (B). Cell lysates were prepared and ana- lyzed for mPGES-1 protein by Western blotting as described under “Experimental Procedures.” In the lower panel, the blots were stripped and reprobed with a specific anti--actin antibody. These blots are representative of similar results obtained from four independent experiments. from HSFs and a radiolabeled oligonucleotide corresponding to the Egr-1 binding sites in the mPGES-1 promoter. As shown in Fig. 8A, the binding of Egr-1 was strongly induced by IL-1 (lane 1 versus lane 2). When the cells were treated with 15d- FIG.7. PPAR ligands inhibit mPGES-1 gene transcription by interfering with promoter transactivation by Egr-1. HSFs were PGJ (lanes 3–5) or TRO (lanes 6 – 8) the formation of the cotransfected with mPGES-1-Luc (A) or p3xEgr-1-Luc (B) and an ex- Egr-1-DNA complex decreased in a dose-dependent manner. pression plasmid for Egr-1 with or without PPAR. The total amount of This binding was specific, because it could be completely abol- transfected DNA was kept constant by addition of empty vector. The ished by coincubation with a 100-fold molar excess of unlabeled next day, the cells were incubated with the indicated concentration of probe (lane 9). Coincubation, with a 100-fold molar excess of 15d-PGJ or TRO for 14 h. Luciferase activity values were determined on cell extracts and normalized to -galactosidase activity. Results are the mutant probe, did not affect Egr-1 DNA-binding activity expressed as -fold induction, considering 1 as the value of cells trans- (lane 10). The specificity of this interaction was further ob- fected with the reporter construct alone, and represent the mean  S.E. served by the supershift assays, showing a further retardation of four independent experiments. *, p  0.05; compared with cells in the electrophoretic mobility of the Egr-1-DNA complex in the transfected with Egr-1 alone (control). presence of a specific anti-Egr-1 antibody (lane 11). These re- sults suggest that PPAR ligands inhibit IL-1-induced of PPAR ligands on mPGES-1 expression does not involve mPGES-1 expression by reducing Egr-1 DNA-binding activity inhibition of Egr-1 protein expression. to the promoter sequence. DISCUSSION To determine whether the reduction of Egr-1 DNA-binding activity by PPAR ligands in HSFs was due to inhibition of An expanding body of evidence indicates that PPAR and its Egr-1 expression, we examined the effects of 15d-PGJ and ligands play an important role in the regulation of multiple TRO on IL-1-induced Egr-1 protein expression. The cells were inflammatory processes (19, 24 –28). In the present study, we pretreated with increasing concentrations of 15d-PGJ or TRO have extended these observations by showing that both natural prior to stimulation with IL-1. In quiescent HSFs, protein and synthetic PPAR ligands inhibit IL-1-induced mPGES-1 levels of Egr-1 were very low. Treatment with IL-1 (100 pg/ml) expression in HSFs. Furthermore, we elucidate the molecular caused a robust induction of Egr-1. Interestingly, neither 15d- mechanism underlying this effect. We demonstrate that this PGJ nor TRO altered IL-1-induced Egr-1 (Fig. 8B). These suppressive effect is transcriptional and PPAR-dependent. data suggest that 15d-PGJ and TRO are not general inhibitors Moreover, PPAR activation inhibited the transcriptional and of IL-1-induced gene expression and that the inhibitory effect DNA binding activities of Egr-1. Taken together, our results Inhibition of mPGES-1 Gene Expression by PPAR Ligands 22063 mPGES-1 promoter activation. Finally, pretreatment with an irreversible pharmacological PPAR antagonist, GW9662, overcame the inhibitory effect of PPAR ligands on mPGES-1 protein expression. However, inhibition of PPAR (via GW9662 or a DN), almost completely restored the suppressive effect of TRO, whereas the suppressive effect of 15d-PGJ was only partially restored, suggesting that 15d-PGJ can activate other PPAR-independent signaling pathways to inhibit mPGES-1 expression. In this context, several studies reported that 15d- PGJ inhibits many inflammatory responses by mechanisms that are independent of PPAR, such as the expression of iNOS in microglial cells and astrocytes (34), the  -integrin-depend- ent oxidative burst in human neutrophils (35) and the expres- sion of CD95 ligand in T lymphocytes (36). In addition, it was demonstrated that 15d-PGJ inhibits NF-B signaling at dif- ferent levels, including modification of I-B kinase activity, which reduces the NF-Bp65 nuclear translocation, and by direct modification of the DNA binding domain of NF-Bp50 (37, 38). Finally, Chawla et al. (39) examined inflammatory responses in macrophages derived from PPAR embryonic stem cells and reported that PPAR ligands still repress LPS- induced iNOS and COX-2 expression. Elucidation of PPAR- independent mechanisms of PPAR ligands needs further study. The transcriptional induction of mPGES-1 is controlled pri- marily by Egr-1 through two Egr-1 binding motifs identified in the proximal promoter region of the mPGES-1 region (32, 33). We hypothesized that inhibition of Egr-1 activity by PPAR could be the mechanism by which PPAR exerts its repressive effect on mPGES-1 transcription. By using reporter gene as- says, we found that Egr-1 indeed activated the mPGES-1 pro- moter, and this activation was reduced by cotransfection with an expression vector for PPAR. Moreover, 15d-PGJ and TRO inhibited Egr-1-mediated mPGES-1 promoter activation and this inhibition was further enhanced in the presence of a PPAR expression plasmid. PPAR activation also inhibited Egr-1-induced activation of a synthetic luciferase reporter con- struct containing three tandem repeats of Egr-1 motif, suggest- ing that PPAR inhibits Egr-1 transcriptional activity in a promoter-independent manner. This is the first evidence that PPAR activation inhibits Egr-1 transcriptional activity in HSFs. In EMSA and supershift assays, we observed that PPAR ligands reduced DNA binding of Egr-1 to a radiolabeled FIG.8. PPAR ligands inhibit DNA-binding activity of Egr-1. A, confluent HSFs were pretreated with increasing concentrations of 15d- oligonucleotide corresponding to the Egr-1 binding sites in the PGJ or TRO for 4 h, followed by the addition of IL-1 (100 pg/ml) for mPGES-1 promoter. Altogether, these results strongly suggest 1 h. Nuclear extracts (5 g) were incubated with a P-labeled oligonu- that PPAR-mediated repression of mPGES-1 results from de- cleotide containing the two Egr-1 binding sites of the mPGES-1 pro- creased Egr-1 binding activity. moter. Specificity of binding was confirmed using 100-fold molar excess of unlabeled oligonucleotides containing wild type (wt) or mutated (mt) Several mechanisms can explain the repression of Egr-1 Egr-1 binding sites. Positions of Egr-1-DNA complex (Egr-1), nonspe- activities by PPAR. One possibility is that PPAR activation cific binding (NS), and supershifted band (SS) are indicated. A repre- suppresses Egr-1 expression. Indeed, PPAR ligands were re- sentative result of four independent experiments is shown. B, confluent ported to inhibit hypoxia-induced Egr-1 expression in mononu- HSF were treated with increasing concentrations of 15d-PGJ or TRO or for 4 h, followed by the addition of IL-1 (100 pg/ml) for 1 h. Nuclear clear phagocytes (40). However, in our study, 15d-PGJ and extracts were prepared and analyzed for Egr-1 protein by Western TRO had no effect on IL-1-induced Egr-1 expression in HSFs, blotting. This blot is representative of similar results obtained from suggesting that PPAR ligands inhibit Egr-1 transcriptional three independent experiments. and DNA binding activities in HSFs by distinct mechanisms. A second mechanism could be competition between PPAR and reveal a novel function of PPAR, further supporting its role in Egr-1 for binding to response elements. This possibility is prob- the control of inflammatory responses. ably unlikely, because: (i) EMSA analysis showed no binding of Several lines of evidence indicate that the inhibitory effects PPAR to an oligonucleotide corresponding to the Egr-1 bind- of 15d-PGJ and TRO on IL-1-induced mPGES-1 expression ing sites in the mPGES-1 promoter (data not shown); (ii) the are likely to act through PPAR activation. First, treatment human mPGES-1 promoter construct used in this study con- with the specific PPAR activator, Wy14653, had no effect on IL-1-induced mPGES-1 expression. Second, overexpression of tains no consensus PPRE sequence; and (iii) PPAR activation inhibited Egr-1 transcriptional activity in a promoter-indepen- PPAR suppressed transcriptional activation of the mPGES-1 promoter, which was enhanced by the addition of PPAR li- dent manner. Alternatively, PPAR may inhibit Egr-1 activity by directly binding to Egr-1 and inhibiting its DNA binding gands. 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