Peroxisome Proliferator-Activated Receptors and the Control of Levels of Prostaglandin-Endoperoxide Synthase 2 by Arachidonic Ac

Peroxisome proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus E Linda R Sheldrick, Kamila Derecka, Elaine Marshall, Evonne C Chin, Louise Hodges, D Claire Wathes, D Robert E Abayasekara, Anthony Pf Flint

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E Linda R Sheldrick, Kamila Derecka, Elaine Marshall, Evonne C Chin, Louise Hodges, et al.. Peroxi- some proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus. Biochemical Journal, Portland Press, 2007, 406 (1), pp.175-183. 10.1042/BJ20070089. hal-00478738

HAL Id: hal-00478738 https://hal.archives-ouvertes.fr/hal-00478738 Submitted on 30 Apr 2010

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 22 May 2007 as manuscript BJ20070089

1 2 Peroxisome proliferator-activated receptors and the control of levels of 3 prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine 4 uterus 5 6 7 E Linda R Sheldrick, Kamila Derecka, Elaine Marshall, Evonne C Chin *, Louise Hodges *, 8 D Claire Wathes *, D Robert E Abayasekara *, and Anthony PF Flint 9 10 11 12 Division of Animal Physiology, School of Biosciences, University of Nottingham, Sutton 13 Bonington Campus, Loughborough, Leics LE12 5RD, UK and *Reproduction and Development 14 Group, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, 15 UK 16 17 18 19 20 21 Short title: Polyunsaturated fatty acids and PTGS2 22 23 Address correspondence including reprint requests to: Prof Tony Flint, Division of Animal 24 Physiology, School of Biosciences, University of Nottingham, Sutton Bonington Campus, 25 Loughborough, Leicestershire LE12 5RD, UK 26 27 Tel: (44)-115-9516300 Fax: (44)-115-9516302 Email: [email protected]

28 29 ABSTRACT 30 Arachidonic acid is a potential paracrine agent released by the uterine endometrial 31 epithelium to induce prostaglandin-endoperoxide synthase 2 (PTGS2) in the stroma. Bovine 32 endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid 33 in stromal cells, and to investigate the potential role of peroxisome proliferator-activated 34 receptors (PPARs) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 35 6 h. The cells expressed PPARA and PPARD (but not PPARG). PTGS2 protein level was raised 36 by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, prostaglandin

37 (PG) A 1 and non-steroidal anti-inflammatory drugs (NSAIDs) with a time course resembling 38 that of arachidonic acid. Use of agonists and antagonists indicated PPARA (but not PPARD or 39 PPARG) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not 40 require prostaglandin synthesis. PTGS2 levels were increased by the protein kinase C (PKC)

41 activators 4?-PMA and PGF2@, and the effects of arachidonic acid, NSAIDs, synthetic PPAR 42 ligands and 4?-PMA were blocked by PKC inhibitors. This is consistent with PPAR 43 phosphorylation by PKC. Induction of PTGS2 protein by 4?-PMA in the absence of a PPAR 44 ligand was reduced by the NF-AB inhibitors MG132 and parthenolide, suggesting that PKC 45 acted through NF-AB in addition to PPAR phosphorylation. Use of NF-AB inhibitors allowed the 46 action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The 47 data are consistent with the proposal that arachidonic acid acts via PPARA to increase PTGS2 48 levels in bovine endometrial stromal cells. 49 50 51 Key words: prostaglandin-endoperoxidase synthase 2, arachidonic acid, peroxisome proliferator- 52 activated receptors, endometrium, cow, paracrine. 53

54 INTRODUCTION 55 Prostaglandin-endoperoxidase synthases (PTGS) 1 and 2 (previously known as 56 cyclooxygenases or prostaglandin H synthases-1 and -2) are heme enzymes that catalyse the first 57 2 steps in the synthesis of prostanoids [1, 2]. Their principal substrates are 58 dihomogammalinolenic acid (DGLA; 20:3 n-6), arachidonic acid (20:4 n-6) and 59 eicosapentaenoic acid (20:5 n-3), essential fatty acids mostly esterified in membrane 60 phospholipids. Both enzymes have cyclooxygenase and peroxidase activities, sequentially 61 catalysing the cyclooxygenation of fatty acid to form prostaglandin (PG) G isomers, and the 62 reduction of PGG to PGH. PTGS1 is expressed constitutively in the gastrointestinal tract, 63 platelets, renal collecting tubules and seminal vesicles. PTGS2 is expressed constitutively in 64 brain, testis, tracheal epithelium and kidney, but is inducible in the uterus, amnion, ovary, kidney 65 and the central nervous system [1]. The two PTGS isoforms are the products of related genes; 66 mature processed human PTGS1 consists of 576 amino acids and PTGS2, 587 amino acids, and 67 they share 60 œ 65% amino acid sequence identity. Both enzymes are glycosylated to varying 68 degrees. 69 PTGS1 maintains normal physiological functions through production of prostanoids and 70 thromboxanes. PTGS2, being inducible in inflammation, fever and pain, produces the 71 prostanoids responsible for these processes. Both these enzymes have attracted attention as the 72 sites of action of non-steroidal anti-inflammatory drugs (NSAIDs) [1]. PTGS2 is also implicated 73 in the aetiology of cancers, notably of the colon and reproductive tract, and prostanoids play 74 important roles in all aspects of uterine function in pregnant and non-pregnant mammals, 75 including luteolysis, menstruation, implantation, the maternal recognition of pregnancy and 76 decidualisation, as well as in labour, parturition and post-partum uterine involution [3]. NSAIDs 77 have been used to treat a range of disorders of these processes in women, including 78 dysmenorrhoea, menorrhagia and preterm labour. 79 The role of PGs in luteolysis is well-established in ruminants. The uterine secretion of

80 luteolytic episodes of PGF2@ in response to activation of the oxytocin receptor is accompanied 81 by a rise in PTGS2 concentration in the endometrium [4,5]. This increase is most marked in the 82 stroma [4]. At the time of luteolysis endometrial oxytocin receptor expression is limited to the 83 epithelium [6], and the question arises as to how up-regulation of PTGS2 occurs in the stroma.

84 The experiments described here were designed to investigate the control of PTGS2 expression in 85 bovine endometrial stromal cells by a potential paracrine agent, arachidonic acid. 86 Paracrine relationships between the epithelium and the stroma have been suggested to 87 control various uterine functions [4,7,8,9], including expression of PTGS2. For instance in the 88 rat, interleukin-1@ acts in a paracrine manner to induce PTGS2 in stromal cells [9]. A second 89 potential agent for this role is arachidonic acid. Arachidonic acid is a good candidate for a 90 paracrine inducer of PTGS2 expression in the stroma at luteolysis, as it is produced in response 91 to phospholipase C activation by oxytocin [10] and induces PTGS2 in other tissues, including 92 mammary epithelial cells [11], bovine endometrial epithelial cells [12] and rat uterine stromal 93 cells [13]. Arachidonic acid also plays paracrine and autocrine roles elsewhere in the 94 reproductive system [14,15]. 95 The PTGS2 gene upstream region contains numerous sequences controlling gene 96 expression. Among these are sites activated by peroxisome proliferator-activated receptors (the 97 peroxisome proliferator response element, PPRE) and nuclear factor-AB (NF-AB), as well as 98 C/EBP, AP-2, CRE and E-box sequences [11,16]. NF-AB sites are responsible for induction of 99 PTGS2 expression by lipopolysaccharide and proinflammatory cytokines [17]. PTGS2 is also 100 induced following activation of protein kinase C (PKC) through NF-AB, C/EBP, CRE and E-box 101 sites [18]. These enhancers are not all active in all tissues, and in some cases their functions 102 differ between cell types. 103 The control of PTGS2 expression by PPARs has been studied in detail. PPREs mediate 104 increases in PTGS2 expression in a variety of cell lines [11,17,19]. PPARs are activated by their 105 ligands, among which are arachidonic acid and other polyunsaturated fatty acids (PUFAs) [20- 106 22], non-steroidal anti-inflammatory drugs (NSAIDs) [23] and cyclopentenone prostaglandins

107 (such as PGA 1 and PGJ 2) [17]. There are at least 3 PPARs, PPARA, PPARD and PPARG of 108 which the PPARA and PPARD isoforms are expressed in the bovine endometrium, although 109 whether they are expressed in the stroma is not known [24]. Therefore activation of a PPAR is 110 one mechanism by which arachidonic acid may induce PTGS2. 111 The transactivation function of PPARA is affected by phosphorylation [25,26]. PPARA is 112 activated through phosphorylation by PKA at serine residues principally in the DNA-binding 113 domain [27] and by PKC at threonine and serine residues between the DNA-binding and ligand- 114 binding domains [28]. Use of inhibitors and non-phosphorylatable mutant PPARs shows that

115 phosphorylation at these sites is a prerequisite for PPAR transactivation function, and that if 116 phosphorylation by PKC is blocked then PPAR ligands do not induce target gene transcription. 117 PKC is activated by arachidonic acid and other PUFAs [29,30], and these compounds may 118 therefore induce PTGS2 through increased PPAR phosphorylation in addition to their action as 119 PPAR ligands. 120 We show here that arachidonic acid induces PTGS2 in endometrial stromal cells, and we 121 further test the hypothesis that PPARs are responsible for PTGS2 induction by arachidonic acid, 122 determine which PPAR isoforms may be involved and investigate whether the effect of 123 arachidonic acid as a PPAR ligand can be differentiated from its actions as an activator of PKC. 124 Endometrial stromal cells of bovine origin have been used because of the role of oxytocin in 125 luteolysis in this species [6], and since oxytocin receptor occupancy generates arachidonic acid 126 [10]. The effects of the agents used were determined by measurement of protein levels, and no 127 attempt was made to differentiate between effects on PTGS2 gene expression and PTGS2 128 transcript or protein turnover. 129 130 MATERIALS AND METHODS 131 Cell culture 132 Bovine endometrial stromal cells were isolated from a day 16 cyclic Holstein-Friesian cow using 133 pancreatin and dispase in calcium- and magnesium-free medium [31], and were maintained in 134 Dulbecco Modified Eagles Medium (DMEM; Sigma, Poole, UK) containing 10% fetal bovine 135 serum and 1% antibiotic-antimycotic (ABAM). These cells, which were phenotypically stable, 136 were purified and maintained free of epithelial cell contamination by differential trypsinization, 137 as confirmed by cytokeratin immunocytochemistry. They were grown in flasks to 60 œ 80% 138 confluence and passaged at intervals of 3-4 days. For testing effects of PUFAs and other agents, 139 the cells were transferred to 24 well plates and medium was changed to DMEM containing 10% 140 dextran-coated charcoal-stripped fetal bovine serum and 1% ABAM. In contrast to bovine

141 endometrial epithelial cells, which produce PGF 2@ more rapidly than PGE 2 in culture, stromal

142 cells produce more PGE 2 than PGF 2@ [32]. The ratio of prostaglandin E 2 : F 2@ produced (Fig. 1a) 143 therefore confirmed the stromal origin of the cells. 144 The following reagents (from Sigma or Calbiochem, Nottingham, UK) were added to 145 culture media to activate or inhibit PTGS2 expression, at the concentrations given in the figure

146 legends: the PUFAs arachidonic acid, DGLA, linoleic acid and conjugated linoleic acid; the 147 synthetic PPAR agonists WY14643, ciprofibrate, methylclofenapate, bezafibrate, SB400455, 148 ciglitazone and pioglitazone [33]; the PPARG antagonists bisphenol A diglycidyl ether 149 (BADGE) [34] and GW9662 [35]; the NSAIDs indomethacin and NS398; the prostaglandins

150 PGF 2@ and PGA 1; the phorbol ester 4?-PMA and its inactive analogue 4@-phorbol-12,13- 151 didecanoate; the protein kinase C inhibitors RO 318425 and calphostin C, and the NF-AB 152 inhibitors MG 132 [36], parthenolide [37] and sulfasalazine [38]. Conjugated linoleic acid 153 (Sigma catalog number O 5507) was a mixture of cis- and trans-9,11- and -10,12- 154 octadecadienoic acids. The PUFAs, indomethacin and prostaglandins were dissolved in ethanol; 155 all other compounds were added to culture medium in DMSO. Vehicle controls were used as

156 appropriate. The PUFAs were stored in ethanol under N 2 at -20 èC in darkness. 157 In preparation for immunoblotting, the medium was removed from cells at the end of the 158 incubation period, centrifuged for 10 min at 13,000 x g and the supernatants stored at -20 èC for 159 prostaglandin assay. Cells were washed once in 1 ml ice-cold phosphate-buffered saline 160 containing 0.2 mM sodium orthovanadate and lysed for 20 min on ice in 0.1 ml lysis buffer 161 containing 10% (w/v) glycerol, 2% (w/v) sodium dodecyl sulphate, 60 mg/ml leupeptin, 1 mM 162 4-(2-aminoethyl)-benzenesulphonyl fluoride and 2 mM sodium orthovanadate in 63.5 mM Tris- 163 HCl, pH 6.8. A sample (60 ml) of lysed cell extract was diluted with 4 ml mercaptoethanol and 2 164 ml 2% (w/v) bromophenol blue, heated in a boiling water bath for 5 min and stored at -20 èC. 165 The remaining lysate was used for protein assay. 166 167 Polymerase chain reaction 168 Total RNA extracted from stromal cells [39] was used in random hexamer- or specific primer- 169 initiated reverse transcription (BIOLINE) and subsequently used for PCR. Pairs of PCR primers 170 spanning different exons were designed to distinguish between the PPAR isoforms. For PPARA 171 (accession no. BT020756), a fragment of 105 bp was amplified using primers PPARA forward 172 (F; position in the sequence: base pairs 682-701) and PPARA reverse (R; 786-765), annealing 173 temperature 51 èC; PPARD (accession no. AF 229357) gave a sequence of 159 bp using primers 174 PPARD (F, 351-371) and PPARD (R, 509-490) at an annealing temperature of 55 èC. 175 For PPARG 2 (accession no. Y12420) three sets of primers were used: PPARG-5 (F, 972- 176 992) with PPARG-2 (R, 1117-1136) or PPARG-6 (R, 1178-1199) and, located further 5‘,

177 PPARG-7 (F, 383-393) and PPARG-8 (R, 606-626). Annealing temperature was 54 èC in all 178 cases. All 3 primer sets were used directly in PCR from RNA reverse transcribed using random 179 hexamers or the specific reverse primers. A nested PCR approach was also used with cDNA 180 generated using random hexamers or PPARG-6, followed by PCR with a combination of 181 primers PPARG-7 and -6. This pair of primers generated a product of 840 bp, which was 182 subsequently used as a template in nested PCR with primers PPARG-7 or -8 and -5 or -6. 183 All PCR reactions were run for 30 cycles. PPARA and PPARD PCR products obtained 184 from stromal cell RNA, and PPARG products obtained from spleen (the positive control tissue) 185 were confirmed by sequencing. 186 187 Immunoblotting 188 Cell lysates (10 mg protein) were subjected to electrophoresis on 10% acrylamide gels (5% 189 stacking gels) before electroblotting onto Optitran BA-S 83 membrane (Schleicher and Schuell, 190 Anderman and Company, Kingston-upon-Thames, UK) in 25 mM Tris pH 8.3 containing 148 191 mM glycine and 20% (v/v) methanol. For detection of PTGS2 bands, membranes were probed 192 with goat polyclonal PTGS2 antibody IgG (C-20; SC 1745, Santa Cruz, obtained through 193 Autogen Bioclear, Calne, UK; 1:250 dilution in a solution containing 1% (w/v) Marvel milk 194 powder and 0.5% (w/v) Tween 20 in phosphate buffered saline). PTGS1 antibody IgG was C-20 195 (SC 1752, Santa Cruz). Second antibody in both cases was donkey anti-goat IgG-horseradish 196 peroxidase (SC 2020; Santa Cruz; 1:11,000 dilution in 3% (w/v) Marvel, 0.5% (w/v) Tween 20 197 in phosphate buffered saline), and visualization was by enhanced chemiluminescence 198 (Amersham, Little Chalfont, UK) using Kodak BioMax Light film. Colour markers (molecular 199 masses 29 œ 205 kDa; Sigma) were used to identify molecular weights of proteins. Expected 200 molecular masses of bovine PTGS1 and -2 were 70 and 72 kDa respectively [5]. PTGS2 201 blocking peptide (SC 1745P, Santa Cruz) was incubated with the first antibody at room 202 temperature overnight. Band intensities were quantified using Kodak 1D digital image analysis 203 software.

204 PPARG antibody was PPARG2 G-18 (SC22020, Santa Cruz), a goat polyclonal antibody

205 raised against a peptide from the N-terminal end of the mouse PPARG2. A sheep kidney extract 206 was used as positive control for PPARG immunoblotting. Pre-stained SeeBlue plus 2 markers 207 (Invitrogen, Paisley, UK; molecular masses 50, 64, 98 and 148 kDa) were used to identify

208 molecular weights. PPARG blocking peptide (SC 22020P, Santa Cruz) was incubated with the 209 first antibody at room temperature overnight. 210 Prostaglandin assays

211 Prostaglandin E 2 and F 2@ were assayed in spent culture media by radioimmunoassay [40]. Intra-

212 assay coefficients of variation were 3.5 and 4.5% for PGE 2 and PGF 2@ assays respectively, and 213 all samples were measured in a single assay to eliminate inter-assay variation. 214 215 Protein assay 216 Protein concentrations in lysates prepared for electrophoresis were measured by the BCA 217 method (Perbio, Cramlingham, UK). 218 219 Experimental design and analysis 220 Experiments were performed in 24 well plates, with replicate treatments of between 3 and 8 221 wells. In time course experiments compounds were added at varying times before cell lysis, in 222 order that all cells should be cultured for the same length of time after plating. To account for 223 differences in band intensity between immunoblots, all blots included at least 2 control samples, 224 and experimental treatments were related to the control bands on each gel. All immunoblot 225 bands were included for analysis. All experiments were performed at least three times. Statistical 226 analysis of treatment effects was by analysis of variance using Genstat version 8 (Lawes 227 Agricultural Trust; obtained through VSN International Ltd, Hemel Hempstead, UK). Where 228 data from more than one experiment have been pooled, this is indicated in the figure legends. 229 Where significant effects were detected (P<0.05) individual differences were tested using 230 Bonferroni‘s multiple comparison post-hoc test. Values are given as means ± s.e.m. In the 231 figures significant treatment effects (from controls or zero-time treatments) are indicated by 232 different letters above bars. 233 234 RESULTS 235 Prostanoid production, PTGS isoforms and effects of arachidonic acid 236 Endometrial stromal cells were tested for production of PGs and responses to PUFAs.

237 Stromal cells released PGE 2 and PGF 2@ into the culture medium, quantities of PGE 2 exceeding

238 those of PGF 2@ by up to 100-fold (Fig. 1a), and PTGS2 was detected at molecular mass 72 kDa

239 by immunoblotting (Fig. 1b). The antiserum was specific for PTGS2 as shown using blocking 240 peptide (Fig. 1c) and as reported before in bovine endometrium [5], PTGS1 was undetectable. 241 Arachidonic acid raised PTGS2 levels in stromal cells (Fig. 1d), the response to arachidonic acid 242 peaking 6 h after addition to the medium and then declining, so that by 48 h the level of PTGS2 243 was close to that before treatment. The response to arachidonic acid was consistent but variable; 244 in the 7 experiments in which it was tested (Figs. 1d, 1e, 2a-d, 4b and 5b), the response ranged 245 from 1.3- to 7.5-fold. This variation did not reflect passage number. In experiments to 246 investigate whether PUFAs other than arachidonic acid increased PTGS2 levels, the effect was 247 also observed with linoleic and conjugated linoleic acids [11,20-22,41], which induced PTGS2 248 within 6 h (Figs. 1e) and there was a tendency for DGLA to have a similar effect. In agreement 249 with previous observations [11,12,20], PUFAs were effective at QM concentrations; dose 250 response experiments in the range 1 œ 100 QM showed the effects of arachidonic acid, DGLA 251 and conjugated linoleic acid were maximal at 50 QM, whereas that of linoleic acid peaked at 2 252 QM (data not shown). 253 254 Effects of PPAR agonists and antagonists 255 To test the hypothesis that PPARs are responsible for the induction of PTGS2 by PUFAs, a 256 variety of established PPAR agonists [11,17,20,33] were used to determine whether they 257 mimicked the effects of PUFAs (Fig. 2). The synthetic PPARA and PPARG agonists WY14643, 258 ciprofibrate and methylclofenapate increased PTGS2 levels up to 2.2-fold at 6 h (Fig. 2a). The 259 effect of methylclofenapate was not significant at 6 h, but reached significance (P<0.02) 260 between 6 and 24 h (data not shown). The PPARD agonist bezafibrate (which is also a poor 261 PPARA agonist) [33] and the selective PPARD agonist SB400455 were ineffective, as were the 262 PPARG agonists ciglitazone (50 QM) and pioglitazone (1 QM). The PPARG antagonists 263 BADGE and GW9662 [34,35] did not block the effect of arachidonic acid (data not shown). 264 Therefore it appeared that PPARA was responsible for induction of PTGS2. Consistent with 265 this, the NSAIDs indomethacin and NS398, which are mixed PPARA and PPARG agonists [23, 266 33], increased PTGS2 expression approximately 3-fold within 6 h (Fig. 2b). The effects of 267 arachidonic acid and the NSAIDs were additive rather than synergistic (i.e. there were no 268 significant interactions, P > 0.05; Fig. 2b); at the concentrations tested those of NS398 and 269 indomethacin were additive (data not shown). The time courses of the effects of arachidonic acid

270 and indomethacin were identical (Fig. 2c), and with the exception of methylclofenapate the 271 effects of the synthetic ligands used in Fig. 2a were also higher at 6 than at 24 h (data not

272 shown). The cyclopentanone prostanoid PGA 1 [17] increased PTGS2 by 2.2- and 2.9-fold at 0.3 273 and 3 mM respectively at 6 h (Fig. 2d). As in rat endometrial stromal cells [13], the increase in 274 PTGS2 in response to arachidonic acid was not due to increased prostanoid synthesis, as it

275 occurred in the presence of NSAIDs (Fig. 2b) when PGE 2 synthesis was low (PGE 2 production, 276 pmol/mg protein/h: controls, 465; with arachidonic acid, 4791; with arachidonic acid and 277 indomethacin, 162). 278 279 PPAR expression 280 Polymerase chain reaction showed that PPARA and PPARD transcripts were expressed in 281 bovine endometrial stromal cells (Fig. 3a). There was no evidence for expression of PPARG. 282 None of the pairs of PPARG primers (PPARG5/6, -5/2 and -7/8) generated products in direct 283 PCR from RNA of stromal cells, in contrast to a positive control tissue (bovine spleen; Fig. 3b). 284 Primers PPARG7/6 generated a longer PCR product of 840 bp if used with spleen cDNA, which 285 was confirmed to be derived from PPARG by sequencing. In contrast there was no product with 286 stromal cell cDNA regardless of whether specific primers or random hexamers were used as 287 primers to generate cDNA. In nested PCR, where a larger product of 840 bp was used to 288 generate a smaller internal product of 222 to 260 bp, again no amplification of PPARG was seen 289 in stromal cell samples. Absence of PPARG in stromal cells was confirmed by immunoblotting 290 (Fig. 3c). Two bands were present in the positive control lane, which were consistent with 291 PPARG (molecular mass 52 kDa) and a dimer (with molecular mass 92 kDa). Both bands 292 disappeared on adding PPARG blocking peptide (data not shown). 293 294 Role of protein kinases in induction of PTGS2 295 The additive effects of arachidonic acid and NSAIDs (Fig. 2b) suggested that they may act 296 via different mechanisms. This is consistent with the fact that PPAR activation requires 297 phosphorylation [25-28], as arachidonic acid is an activator of PKC [29,30], one of the protein 298 kinases reported to phosphorylate PPARA [28]. An effect of arachidonic acid via PKC was 299 supported by the results of treating cells with compounds which activate or inhibit PKC (Fig. 4). 300 The phorbol ester 4?-PMA increased levels of PTGS2 protein by up to 7.6-fold (mean of 4

301 experiments, 3.4-fold). The inactive isomer 4@-phorbol-12,13-didecanoate (2 QM) was

302 ineffective (data not shown). Prostaglandin F 2@ (which activates PKC via the FP receptor) [42] 303 increased PTGS2 protein by 2.2-fold. The effect of 4?-PMA was blocked by RO318425 (Fig. 304 4a). The effect of arachidonic acid was blocked by RO318425 and calphostin C (500 nM; Fig. 305 4b). RO318425 also prevented induction of PTGS2 by indomethacin or NS398 (Fig. 4c), and by 306 WY14643 or ciprofibrate (Fig. 4d). 307 308 PKC induces PTGS2 independently of PPARs 309 On the basis of the experiments described above it was not possible to dissociate a potential 310 effect of arachidonic acid as a PPAR ligand from an effect on PPAR phosphorylation via basal 311 or activated PKC. However in addition to phosphorylating PPARs, in the absence of an 312 exogenous PPAR ligand PKC appeared to act through a second pathway to induce PTGS2. This 313 was revealed by blocking the PTGS2 induction pathway downstream of PKC. When cells were 314 treated with inhibitors of the NF-AB pathway [36-38], the effect of 4?-PMA was reduced by 315 approximately 70 % by both MG132 and parthenolide (Fig. 5a). In contrast, none of the NF-AB 316 inhibitors used (MG132, parthenolide and sulfasalazine) affected PTGS2 induction by 317 arachidonic acid (Fig. 5b). In these experiments none of the NF-AB inhibitors reduced the basal 318 level of PTGS2 when added alone (data not shown). 319 320 DISCUSSION 321 Arachidonic acid induced PTGS2 in endometrial stromal cells, as in epithelial cells [12]. 322 This effect provides an explanation for the rise in PTGS2 in endometrial stroma at luteolysis [4], 323 and is consistent with the proposed paracrine action of arachidonic acid within the endometrium. 324 Five separate pieces of evidence support the hypothesis that PPARs are involved in the 325 effect of arachidonic acid on PTGS2 levels: (a) a wide variety of PPAR agonists mimicked the

326 action of arachidonic acid on PTGS2, including other PUFAs, PGA 1, NSAIDs and synthetic 327 pharmaceutical agents; (b) these compounds acted with time courses similar to that of 328 arachidonic acid, leading to raised PTGS2 levels 6 h after treatment; (c) agents expected to act

329 via PPAR phosphorylation (4?-PMA and PGF 2@) also raised PTGS2; (d) consistent with PPAR 330 function requiring phosphorylation, effects of arachidonic acid and NSAIDs were both inhibited 331 by RO318425. Finally (e) the effect of arachidonic acid was not blocked by NF-AB inhibitors,

332 whereas in the absence of a PPAR ligand, that of 4?-PMA was prevented, suggesting that 333 arachidonic acid acted independently of PKC as well as via PKC. Linoleic acid raised PTGS2 334 levels at a lower concentration than other PUFAs, possibly reflecting its greater efficacy in 335 causing PPARA heterodimerisation [20]. Although a PPAR response element has not yet been 336 identified upstream of the coding sequence in the bovine PTGS2 gene, these findings are 337 consistent with the hypothesis that PUFAs induce PTGS2 expression in stromal cells via PPARs, 338 as in other cell lines. The interactions postulated to occur in the experiments described here are 339 summarized in Fig. 6. 340 PPARA and PPARD were expressed in stromal cells, but there was no evidence for 341 expression of PPARG either from polymerase chain reaction or immunoblotting. This is 342 consistent with the previous demonstration of PPARA and PPARD, and the absence of PPARG, 343 in whole bovine endometrium and in an endometrial epithelial cell line (BEND cells) by 344 northern blotting [24]. The PPARG probe used by MacLaren et al. [24] was derived by PCR 345 from adipose tissue cDNA using primers corresponding closely to the PPARG-5/PPARG-2 or 346 PPARG-5/PPARG-6 primer pairs used here. The overlap between the 2 products was 347 approximately 150 out of 240 bp. The absence of PPARG in endometrium is consistent with this 348 isoform being principally found in adipose tissue [33]. 349 Synthetic PPARD agonists were ineffective in raising PTGS2 and therefore, since PPARG 350 was absent, PPARA was most likely responsible for the effect on PTGS2 levels. This was 351 confirmed by showing that PTGS2 levels increased in response to the PPARA agonists 352 WY14643, methylclofenapate and ciprofibrate, but not in response to appropriate concentrations 353 of the PPARG agonists, ciglitazone and pioglitazone. The lack of involvement of PPARD was 354 compatible with the effect of indomethacin, which activates PPARA and PPARG, but not 355 PPARD [33]. The conclusion that PPARA modulates PTGS2 levels in stromal cells is consistent 356 with the observation that WY14643 induces PTGS2 in a bovine endometrial epithelial cell line 357 [24]. 358 The effects of the agonists and antagonists used were generally (but not always) consistent 359 with their reported specificities. For instance the PPARG agonist pioglitazone, which is also a 360 weak PPARA agonist [43], raised PTGS2 at 50 QM but not at 1 QM. The PPARG antagonists 361 BADGE and GW9662 also acted as partial PPARA agonists [34,44,45], but did not block the 362 stimulatory effect of arachidonic acid. 3-[1-(4-Chlorobenzyl)-5-(isopropyl)-3-tert -

363 butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK886), which is a PPARA antagonist in 364 some systems [46], augmented rather than prevented the effects of arachidonic acid and 365 indomethacin, and stimulated PTGS2 levels when added alone at 1 - 50 QM (data not shown). 366 Indomethacin and MK886 are both substituted chlorophenyl indole derivatives, and it is 367 therefore not surprising that they have similar effects in some cells. Furthermore MK886 is a 368 potent lipoxygenase inhibitor, and may have mimicked other lipoxygenase inhibitors such as 369 nordihydroguaiaretic acid, which also raised PTGS2 in stromal cells (EL Sheldrick, unpublished 370 observations), as in rat fibroblasts and murine keratinocytes [47,48]. Clearly, as noted previously 371 [49], lipoxygenase inhibitors have cell- and tissue-specific effects on PPARs. Unfortunately, 372 more specific PPARA antagonists which could be used to test the effect of inhibiting PPARA 373 are not currently available. 374 PPAR transactivation function depends upon phosphorylation. The principal protein kinases 375 responsible are mitogen activated protein kinase, PKA and PKC [25-28], and PUFAs are 376 activators of PKC [29,30]. The consensus amino acid sequences subject to phosphorylation in 377 PPARs have been studied mostly in the human and mouse, but potential phosphorylation sites 378 for each of these serine/threonine kinases are conserved in the corresponding bovine proteins. 379 There are 2 PKC consensus sites in bovine PPARA (accession no. BT020756) at Thr 131 380 (FFRR TIRLK), and Ser 348 (EFLK SLRKP) which are identical in the human and bovine 381 proteins. Therefore bovine PPARA would be expected to undergo phosphorylation by PKC in 382 the same way as the human protein [25,28]. Consistent with this, the effects of arachidonic acid, 383 NSAIDs and PPAR activators were blocked by the PKC inhibitors RO318425 and (in the case 384 of arachidonic acid) calphostin. This is consistent with the requirement for a basal level of 385 phosphorylation for a PPAR response to ligand occupancy. We did not attempt to determine

386 which of the many isoforms of PKC mediated the effects of 4?-PMA and PGF 2@. 387 The data described above were insufficient to differentiate between an action of arachidonic 388 acid as a PPAR ligand [20,21] and an effect through activation of PKC [29,30], and arachidonic 389 acid and other PUFAs may have acted via both mechanisms. Actions of PUFAs exerted through 390 2 mechanisms (i.e. as PPAR ligands and through activation of PKC) might be expected to cause 391 additive responses, and these were observed when indomethacin or NS398 were added with 392 arachidonic acid (Fig. 2b). In certain cell types 4?-PMA-activated PKC induces PTGS2 393 expression via NF-AB [17], in addition to any effects resulting from phosphorylation of PPARs.

394 We therefore tested whether inhibitors of NF-AB (MG132 and parthenolide) prevented the effect 395 of 4?-PMA on PTGS2 levels in stromal cells. The results showed that both inhibitors reduced 396 the response to 4?-PMA (Fig. 5). In contrast the effect of arachidonic acid was unaffected by the 397 NF-AB blockers MG132, parthenolide or sulfasalazine (which was not tested with 4?-PMA). 398 Inhibitors of NF-AB could therefore be used to dissociate the action of arachidonic acid from 399 that of 4?-PMA. Since 4?-PMA is thought to be specific in activating PKC, this shows that 400 arachidonic acid increases PTGS2 levels by more than one mechanism. Although NSAIDs of the 401 fenamate group (but not indomethacin) block PTGS2 expression induced by tumor necrosis 402 factor-@ or lipopolysaccharide in HT-29 human colon adenocarcinoma cells through inhibition 403 of NF-AB [17], it is unlikely that the NSAIDs used here (indomethacin and NS398) affected NF- 404 AB. 405 The concentrations at which prostanoids were added to culture medium (0.3 - 3 mM) were 406 comparable to levels achieved through endogenous prostanoid production by the cells 407 themselves (up to 0.3 QM; Fig. 1). As a result it is possible that, if they acted via cell-surface 408 receptors, endogenous prostaglandins may have affected PTGS2 levels in these experiments.

409 They are unlikely to have acted as PPAR ligands, as prostaglandins F 2@ and E 2 are poor in this 410 respect [11]. 411 The increase in PTGS2 level in the mammary gland in response to a high dietary n-6 PUFA 412 intake [50] raises the possibility that the effect of arachidonic acid on PTGS2 expression in 413 uterine cells may occur in vivo . If, as proposed here, uterine PTGS2 levels are controlled by 414 PPARs, then NSAIDs administered to treat reproductive disorders may result in detrimental 415 rises in PTGS2 in the tissues in which they are intended to reduce prostanoid synthesis. 416 417 418 419 Acknowledgments 420 421 We thank Dr David Bell, University of Nottingham, for kindly providing certain PPAR agonists. 422 We thank Martin Bishton, Matthew Elmes, Mandeep Kandola and Yasmin Mussaddeq for 423 assistance in the laboratory, and Pat Fisher for deriving the cells. This work was funded by the 424 Biotechnology and Biological Sciences Research Council and the Wellcome Trust.

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586 Figure legends 587 588 Figure 1 Prostaglandin production, PTGS levels and effects of PUFAs

589 (a) Prostaglandin E 2 and F 2@ production by bovine endometrial stromal cells. Prostaglandin 590 concentrations were measured in culture medium after 48 h. (b) Immunoblotting demonstrated 591 the presence of the molecular mass 72 kDa PTGS2. PTGS1 was not detectable. (c) 592 Dihomogammalinolenic acid (DGLA; 50 QM) increased PTGS2 levels and the PTGS2 blocking 593 peptide confirmed the identity of the immunoblotting signal. (d) Arachidonic acid (AA; 50 QM) 594 increased PTGS2 levels, the effect peaking at 6 h. (e) In a separate experiment PTGS2 levels 595 were raised at 6 h in cells treated with AA (50 QM), DGLA (50 QM), linoleic acid (LA; 2 QM) 596 and conjugated linoleic acid (CLA; 50 QM). Representative immunoblots shown in b, c and d as 597 examples of data obtained at least in triplicate. 598 599 Figure 2 Effects of PPAR agonists and antagonists on PTGS2 levels 600 (a) Bovine endometrial stromal cells were cultured with or without arachidonic acid (50QM) and 601 the synthetic PPARA agonists WY14643 (50 QM), ciprofibrate (50 QM) and methylclofenapate 602 (50 QM), the PPARD agonists bezafibrate (50 QM) and SB400455 (50 nM), and the PPARG 603 agonists ciglitazone (50 QM) and pioglitazone (1 QM). These concentrations have been shown to 604 be effective in other cell types. PTGS2 was measured by immunoblotting after 6 h. The effects 605 of WY14643 and ciprofibrate were statistically significant (P<0.05); the effect of 606 methylclofenapate was not significant at 6 h, but reached significance (P<0.02) between 6 and 607 24 h (data not shown). Data of 3 experiments. (b) Effects of PPARA/PPARG agonist NSAIDs

608 on PTGS2 level. Closed bars, PTGS2 levels; open bars, PGE 2 production. Stromal cells were 609 cultured for 6 h with or without the following additions: arachidonic acid (AA; 50 QM), NS398 610 (50 QM) or indomethacin (10 QM). PTGS2 level was increased by arachidonic acid (P<0.001),

611 NS398 (P<0.001) and indomethacin (P<0.01). The increase in PGE 2 production in response to 612 arachidonic acid was blocked by NSAIDs. Data of 4 experiments. (c) The time courses of the 613 effects of arachidonic acid (50 QM; open bars) and indomethacin (10 QM; closed bars) were

614 identical. (d) Effects of the PPARA/PPARD/PPARG agonist prostaglandin A 1 on PTGS2 levels. 615 Bovine endometrial stromal cells were cultured for 6 h with or without arachidonic acid (50

616 QM), PGA 1 (0.3 QM) or PGA 1 (3 QM). PTGS2 levels were increased in the presence of PGA 1 617 (P<0.001). 618 Figure 3 Identification of PPARs 619 Total RNA extracted from stromal cells or bovine spleen (positive control) was analysed by 620 PCR. (a) Initial analysis for PPAR isoforms in stromal cells. PPARA and PPARD were 621 expressed but not PPARG. The expected products of 105 bp and 159 bp for PPARA and 622 PPARD, obtained using the PPARA and PPARD primers described under Materials and 623 Methods, were verified by sequencing. No product was obtained for PPARG using primers 624 PPARG-5 and -2. Right panel represents control reactions without reverse transcriptase. (b) 625 Further analysis for PPARG. Total RNA from stromal cells (BST) and spleen was reverse 626 transcribed using the specific primer PPARG-6 (Specific) or random hexamers (Random), and 627 subsequently used in direct amplification (upper panel), or in nested PCR (lower panel) with 628 primers PPARG-5/6 or -7/8. In direct amplification spleen gave products of the expected sizes, 629 which were verified by sequencing, but no product was obtained with stromal cells. To confirm 630 lack of amplification in stromal cells, a nested approach was subsequently used. The first round 631 of nested PCR (lower panel) using primers PPARG-7/6 generated a product of 840 bp, which 632 was then used as a template with primers PPARG-5/6 or PPARG-7/8. RNA from spleen 633 generated a positive reaction but stromal cells (BST) failed to generate a product of the 634 appropriate size. MWM, molecular weight markers; -RT, controls without reverse transcriptase; 635 -DNA, controls without cDNA with primer pairs 5/6 and 7/8. (c) Immunoblotting for PPARG. 636 Lane 1, molecular mass markers (kDa); lane 2, control extract of ovine kidney tissue; lane 3, 637 untreated stromal cells. 638

639 Figure 4 Effects of arachidonic acid, 4;-PMA, prostaglandin F 2< , NSAIDs and synthetic 640 PPARA agonists with and without protein kinase C inhibitors 641 Bovine endometrial stromal cells were cultured for 6 h with or without additions and PTGS2 642 was measured by immunoblotting. a) Effects of 4?-phorbol myristate acetate (PMA; 2QM),

643 prostaglandin F 2@ (PGF 2@ ; 3 QM) and RO318425 (RO; 500 nM). In the absence of PPAR ligand

644 PTGS2 level was increased by 4?-PMA (P<0.001) and PGF 2@ (P<0.05), and the effect of 4?- 645 PMA was blocked by RO318425 (P<0.005). Data of 4 experiments. (b) PTGS2 level was 646 increased by arachidonic acid (P<0.001), and the effect of arachidonic acid was blocked by

647 RO318425 (P<0.001) and calphostin C (Cal; 500 nM). Data of 6 experiments. Neither 648 RO318425 (Fig. 4a) nor calphostin C (Fig.4b) affected PTGS2 when added alone. (c) 649 Indomethacin and NS398 increased PTGS2 levels (P<0.001 in both cases). The effects of 650 NSAIDs were blocked by RO318425. Data of 5 experiments. (d) The effects of the synthetic 651 PPARA agonists WY14643 (200 QM) and ciprofibrate (200 QM) were blocked by RO318425 652 (500 nM). Data of 3 experiments. 653 654 Figure 5 The effect of 4;-PMA on PTGS2 level, but not that of arachidonic acid, was 655 reduced by NF-=B inhibitors 656 (a) Bovine endometrial stromal cells were cultured in the absence of exogenous PPAR ligand for 657 6 h with 4?-PMA (2 QM) with or without the NF-AB inhibitors MG132 (MG; 30 QM) and 658 parthenolide (P; 4 QM). Data of 3 experiments. (b) The effect of arachidonic acid on PTGS2 659 level (P<0.01) was not blocked by NF-AB inhibitors. Cells were cultured for 6 h with or without 660 arachidonic acid (50 QM) and MG (30 QM), P (4 QM) or sulfasalazine (S; 1 QM). Data of 6 661 experiments. 662 663 Scheme 1 Diagram illustrating proposed mechanisms controlling PTGS2 levels in bovine 664 endometrial stromal cells

665 a) PUFAs and NSAIDs are PPARA ligands. b) PUFAs, PGF 2@ and 4?-PMA activate PKC. c)

666 PUFAs are converted into prostanoids including PGF 2@. d) PKC phosphorylates PPARA, a step 667 required for subsequent activation of PPARA by ligand occupancy; if PKC is inhibited PPAR 668 ligands do not induce PTGS2. e) PKC also activates NF-AB, and if NF-AB is inhibited in the 669 absence of PPAR ligand, activation of PKC does not induce PTGS2. f) PPARA and NF-AB 670 increase PTGS2 levels.

Figure 1 24

(a) PTGS2 (b) PTGS1 50

40 72kDa 70kDa 30

Prostaglandin secretion (nmol/mg secretion Prostaglandin protein) PGF 2aaa PGE 2

(c)

72kDa

Control DGLA Control DGLA + PTGS2 blocking peptide

(d)

(e) 72kDa 600 b b 1000 b 500 b a 750 400 a,b b 300 500

200 a a 250 b a a 100

0 PTGS2 band intensity (% intensity control) band PTGS2 0 0 3 6 12 24 36 48 PTGS2 band intensity (percent control) (percent intensity band PTGS2 AA LA Time after addition of arachidonic acid (h) CLA DGLA Control 671 672 673 674

675 676 Figure 2 677 678

(a)

(b) 275 b a

c PGE 250 1400 c 6000

225 b 2 secreted protein/h) (pmol/mg 1200 5000 200 b a 175 1000 4000 150 a 800 a 125 a a 3000 100 600 b 2000 75 400 b b 50 200 1000 25 a PTGS2 band intensity band (%PTGS2 control)

0 (%intensity control) band PTGS2 0 0 l d 5 8 n A ro t 643 ate rate 45 cid 39 ci A n 4 b a a o c aci 1 ap fi th C i Y n 400 itazone ic NS 98+ n fe za B l Control n 3 o W S g o id Be Ci d NS h Ciprofibrate Pioglitazone dome In rac A Arachi Methylclo Indomethacin+AA

250 b 150 b 200

100 150 a 100 50 50 PTGS2 band intensity (% band control) PTGS2

PTGS2 band intensity (%intensity control) band PTGS2 0 0 0 3 6 12 24 36 48 d rol M) M) mmm mmm .3 Time (h) Cont 0 (3 ( 1 idonic aci 1 h PGA PGA Arac 679 680 681 682 683 684

26 Figure 3a

PPARA PPARD PPARG PPARA PPARD PPARG MWM -RT reactions MWM

300

200

100

Figure 3b 27

Direct amplification, cDNA used directly in PCR with: primers 5/6 or primers 7/8

cDNA obtained with primer: MWM Specific Random -RT MWM Specific Random -RT

200 bp

BST spleen BST spleen

Nested PCR, primary PCR product (obtained with primers 7/6) of 840 bp subjected to nested reaction with: primers 5/6 or primers 7/8

cDNA obtained with primer: MWM Specific Random MWM Specific Random MWM -DNA controls

200 bp

BST spleen BST spleen

28 Mol. Mass Kidney Stromal Figure 3 c Markers cells

Mol. Mass es :

98 kDa

64 kDa

50 kDa

Figure 4 29

(b)

(a) 180 b 160 400 b 140

120 300 a a a b 100 a 200 80 a a 60 a 100 40 20 PTGS2 band intensity (%intensity band control) PTGS2

PTGS2 band intensity (% band control) PTGS2 0 0 l ol A aaa O O A a O M 2 R A C ntr -P o bbb PGF A+R A+R C 4 M Control A AA+Cal -P bbb 4b (d) (c) 180 b 800 b 160 b 700 140

600 120 a b a a 500 100 a 400 80

300 60 200 a 40 100 a a a 20

0 0 PTGS2 band intensity (% band PTGS2 control) PTGS2 band intensity (percent control) (percent intensity band PTGS2 l l O 3 O in 4 RO tro R c RO RO 6 +R n + S398 + e tha in 8 fibrate Co e c N 9 Contro a WY14 S3 ipro th N e C rofibrat Indom m WY14643+ROp o Ci Ind

(a)

400 b

300

a,b a 200

a 100

PTGS2 band intensity (% band control) PTGS2 0 l o A th M MG -P + bbb Contr 4 -PMA bbb -PMA+Par 4 bbb 4b

(b)

200 a,b

b b 150 b

a 100

PTGS2 band intensity (%band PTGS2 control) 0

h S AA rt + trol a A P on + A C AA+MG A A

Scheme 1

PUFAs NSAIDs a c a b PGF 2: PPARA b f d PKC PTGS2 e f b 4<-PMA NF-=B