Nanomolar and Micromolar Effects of 15-Deoxy- - prostaglandin J2 on Amnion-Derived WISH Epithelial Cells: Differential Roles of Peroxisome Proliferator-Activated Receptors and and Nuclear Factor B

15-Deoxy -prostaglandin J2 (15d-PGJ2), an activator of peroxisome proliferator-activated receptor (PPAR)and , is a prostanoid metabolite with anti-inflammatory actions. In intrauterine tissues, proinflammatory cytokines and prostaglandins have been identified as playing key roles in the maintenance of pregnancy and the onset of labor. We investigated and compared the early ( 3 h) effects of 15d-PGJ2 with rosiglitazone (PPARligand) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5yl)-methylsulfanyl)phenoxy-acetic acid (GW501516) (PPARligand) on interleukin (IL)-1 –induced prostaglandin and cytokine production by amnion-derived WISH cells. We show that 15dPGJ2 exerts differential effects depending on concentration. At low concentrations ( 0.1 M), 15d-PGJ2 inhibited IL-1 – stimulated prostaglandin E2 (PGE2) but not cytokine (IL-6/IL-8) production or cyclooxygenase-2 (COX-2) expression. This effect was attenuated by a PPARinhibitor [2-chloro-5-nitro-N-phenylbenzamide (GW9662)], by transfection with a dominant-negative PPAR construct, and was reproduced by the PPARligand rosiglitazone. At higher concentrations (1–10 M), 15d-PGJ2 inhibited IL-1 –stimulated PGE2 and cytokine production and COX-2 expression, and this effect was not blocked by GW9662. Rosiglitazone at high concentrations (1–10 M) stimulated PGE2 production in the absence or presence of the dominant-negative PPAR. The PPARligand GW501516 also inhibited IL-1 –stimulated PGE2 production but only at high concentrations (1 M). IL-1 – induced nuclear factorB (NFB) DNA binding activity was significantly inhibited by 15d-PGJ2 (10 M) and GW501516 (1 M) but increased with 10 M rosiglitazone. We conclude that 1) at low concentrations, 15d-PGJ2 acts through a PPARsignaling pathway; b) at higher concentrations, its actions are mediated most likely through other pathways such as activation of PPARand/or inhibition of NFB; and 3) rosiglitazone exerts PPAR-independent effects at high concentrations ( 1 M). Proinflammatory cytokines have been shown to play crucial roles in the maintenance of human pregnancy and the initiation of parturition (Romero et al., 1993; Mitchell et al., 1995). The presence of intrauterine infection has been shown to result in the local expression and secretion of proinflammatory cytokines such as interleukin (IL)-1 , tumor necrosis factor, IL-6, and IL-8 (Romero et al., 1993; Dudley et al., 1996; Keelan et al., 1999a), which act locally on intrauterine cells to induce the release of inflammatory mediators, extracellular matrix-remodeling enzymes (So et al., 1992; Draper et al., 1995), and prostaglandins (PGs) through altered expression of prostanoid biosynthetic enzymes including fatty acid cyclooxygenase-2 (COX-2) (Trautman et al., 1996; Hansen et al., 1999; Kniss, 1999; Rauk and Chiao, 2000). Although most studies to date have focused on the producThis study was funded by grants from the Health Research Council of New Zealand, Royal Society of New Zealand Marsden Fund, New Zealand Lottery Health Grants Board, University of Auckland Research Committee, National Research Centre for Growth and Development, and Auckland Medical Research Foundation. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.009449. ABBREVIATIONS: IL, interleukin; 15d-PGJ2, 15-deoxy -prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; rosiglitazone, 5-(4-[2-(N-methyl-N-(2-piridyl)amino)ethoxy]benzyl thiazolidine-2,4-dione maleic acid salt; COX-2, cyclooxygenase-2; NFB, nuclear factor B; GW9662, 2-chloro-5-nitro-N-phenyl-benzamide; GW501516, 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid; Bay 11–7085, (E)3-[(4-t-butylphenyl)sulfonyl]-2-propenenitrile; PG, prostaglandin; iNOS, inducible nitric-oxide synthase; ERK, extracellular signal-related kinase; HRPO, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyl transferase; DTT, dithiothreitol; ANOVA, analysis of variance; PPRE, peroxisome proliferator-activated receptor response element; PPAR D/N, peroxisome proliferator-activated receptor dominant-negative construct. 0026-895X/05/6801-169–178$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 1 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 9449/3038262 Mol Pharmacol 68:169–178, 2005 Printed in U.S.A. 169 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from tion of uterotonic PGs such as PGE2 and PGF2 , there is evidence of an abundance of PGD2 in the intrauterine environment during labor (Mitchell et al., 1982; Berryman et al., 1987). PGD2, synthesized from PGH2 via the action of PGD synthases (Helliwell et al., 2004a), is readily converted nonenzymatically into PGJ2 and its metabolites 9-deoxy, 13,14-dihydroprostaglandin D2 and 15-deoxy-prostaglandin J2 (15d-PGJ2) (Fitzpatrick and Wynalda, 1983; Kikawa et al., 1984; Shibata et al., 2002). These metabolites have been postulated to regulate a number of different cellular processes, including cell proliferation (Chinery et al., 1999), differentiation (Forman et al., 1995; Kliewer et al., 1995), apoptosis (Kim et al., 1993; Hashimoto et al., 2002), and inflammation (Harris et al., 2002). 15d-PGJ2 can induce apoptosis in several cell types (Bishop-Bailey and Hla, 1999; Rohn et al., 2001; Chen et al., 2002; Hashimoto et al., 2002; Rovin et al., 2002), including trophoblast (Schaiff et al., 2000), amnion-derived WISH cells (Keelan et al., 2001), and JEG3 choriocarcinoma cells (Keelan et al., 1999b). It is also reported to inhibit the expression of proinflammatory cytokines (Jiang et al., 1998; Ricote et al., 1998; Asada et al., 2004), inducible nitric-oxide synthase (iNOS) (Colville-Nash et al., 1998; Petrova et al., 1999), and COX-2 expression (Boyault et al., 2001; Tsubouchi et al., 2001; Mendez and LaPointe, 2003). However, its mechanism of action is controversial. Some have reported that 15d-PGJ2 acts as an endogenous ligand for the peroxisome proliferator activated receptor (PPAR)(Jiang et al., 1998; Ricote et al., 1998; Tsubouchi et al., 2001), whereas others have argued that its main effects are mediated through the inhibition of the transcription factor NFB (Rossi et al., 2000; Straus et al., 2000; Cernuda-Morollon et al., 2001) and modulation of the mitogen-activated protein kinase pathway (Hortelano et al., 2000; Rossi et al., 2000), such as inhibition of ERK phosphorylation (Relic et al., 2004). In gestational tissues, PPARhas been localized to the amnion, choriodecidual, and placental membranes (Marvin et al., 2000; Waite et al., 2000; Dunn-Albanese et al., 2004) and plays an important role in trophoblast differentiation and placental vascularization (Barak et al., 1999). NFB is a crucial transactivator of multiple proinflammatory and antiapoptotic genes (Lawrence et al., 2002). Recent studies have demonstrated that 15d-PGJ2 inhibits the signaling steps leading to NFB activation by sequestering coactivators needed for transcription (Li et al., 2000), by inhibition of I Bkinase activity (Mercurio and Manning, 1999; Rossi et al., 2000), and through the formation of covalent bonds with cysteine residues of the DNA binding domain of NFB subunits (Rossi et al., 2000; Straus et al., 2000; CernudaMorollon et al., 2001). Most of the studies to date have investigated the effect of 15d-PGJ2 at micromolar concentrations. It is interesting that Emi et al. (2004) reported recently that 15d-PGJ2 exhibits biphasic effects that are concentration-dependent. At 3 M, it was shown to induce cell proliferation, but at 10 M, it was an inducer of apoptosis. In gestational tissues, 15d-PGJ2 ( 10 M) has been shown to inhibit extravillous cytotrophoblast invasion and differentiation (Schaiff et al., 2000; Tarrade et al., 2001; Pavan et al., 2003b), leading to trophoblast apoptosis (Schaiff et al., 2000). A recent study also showed that at a high concentration ( 10 M), 15d-PGJ2 exhibited anti-inflammatory properties by reducing lipopolysaccharide-stimulated IL-6, IL-8, and tumor necrosis factorproduction by amnion, choriodecidual, and placental cells in vitro, possibly through the inhibition of NFB activity (Lappas et al., 2002). The present study was conducted as part of an evaluation of the role of 15d-PGJ2 in gestational tissues. We investigated the early effects ( 3 h) of 15d-PGJ2, rosiglitazone (a more potent and specific pharmacological PPARagonist), and GW501516 (a PPARagonist) on basal and IL-1 –induced PG and cytokine production in the WISH cell line (which has been used extensively in the past as an amnion epithelial cell model) (Pavan et al., 2003a) to clarify the effects and the mechanism(s) of action of 15d-PGJ2 at low (0.001–0.1 M) and high (0.1–10 M) concentrations. Specific inhibitors were used to clarify the respective roles of PPARs and NFB as targets for 15d-PGJ2–induced effects. Materials and Methods Reagents. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Ham’s F-12/Dulbecco’s modified Eagle’s media were obtained from Irvine Scientific (Santa Ana, CA), and penicillin/streptomycin/glutamine, fetal calf serum, normal horse serum, trypsin-EDTA, and 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide were purchased from Invitrogen NZ Limited (Auckland, New Zealand). Hybond-P nitrocellulose membranes were purchased from Amersham Biosciences Inc. (Auckland, New Zealand). Roche complete protease inhibitor tablets and human recombinant IL-1 were purchased from Roche Diagnostics (Auckland, New Zealand) and Immunex (Seattle, WA), respectively. The PPAR dominant-negative construct (pSG5hPPAR 500) was a gift from Dr. Joel Berger (Department of Molecular Endocrinology, Merck Research Laboratories, Rahway, NJ). Anti-actin, COX-2, and NFB p50 and p65 antibodies were purchased from Abcam Limited (Cambridge, UK), BD Biosciences (San Jose, CA), and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Goat anti-rabbit IgG-horseradish peroxidase (HRPO) antibody was purchased from Sigma-Aldrich. 15d-PGJ2, rosiglitazone, and GW501516 were purchased from Cayman Chemical (Ann Arbor, MI). GW9662 and valinomycin were generous gifts from Dr. Tim Willson (GlaxoSmithKline, Uxbridge, Middlesex, UK) and Dr Mark McKeage (Department of Pharmacology, University of Auckland, Auckland, New Zealand), respectively. Cell Culture. WISH cells (American Type Culture Collection, Manassas, VA) were maintained in Ham’s F-12/Dulbecco’s modified Eagle’s culture media supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin/glutamine at 37°C in 95% air/5% CO2. Cells were plated in 24-well plates and treated with various test agents in triplicate. A 3-h time point was chosen to pre-empt the apoptotic changes observed in morphology of WISH cells after treatment with 15d-PGJ2 (10 M) for 8 h (Keelan et al., 2001). At the end of each treatment, media were collected for PGE2 and cytokine measurements, and cells were lysed with lysis buffer (2% SDS, 8% glycerol, and 62.5 mM Tris, pH 6.8, protease inhibitor solution) for Western blotting. Cellular protein concentrations were measured using Bio-Rad DC protein assay (Bio-Rad Laboratories Pty Ltd, Auckland, New Zealand) according to the manufacturer’s in-