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 To cite this version: 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] © 2007 The Authors Journal compilation © 2007 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 May 2007 as manuscript BJ20070089 2 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 © 2007 The Authors Journal compilation © 2007 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 May 2007 as manuscript BJ20070089 3 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. © 2007 The Authors Journal compilation © 2007 Biochemical Society Biochemical Journal Immediate Publication. Published on 22 May 2007 as manuscript BJ20070089 4 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 © 2007 The Authors Journal compilation © 2007 Biochemical Society Biochemical Journal Immediate Publication.