A PPAR-independent pathway to PUFA-induced COX-2 expression

doi:10.1016/j.mce.2008.02.015

Molecular and Cellular Endocrinology

Volume 287, Issues 1–2, 11 June 2008, Pages 65-71

https://doi.org/10.1016/j.mce.2008.02.015 Get rights and content

Abstract

Polyunsaturated fatty acids (PUFAs) induce COX-2 in bovine endometrial stromal cells through activation of peroxisome-proliferator-activated receptor alpha (PPARα). We have investigated alternative (PPAR-independent) pathways to COX-2 induction using a reporter construct driven by a COX-2 gene promoter sequence lacking a PPAR response element. This construct was induced by PUFAs, but not by PPAR agonists. PPAR-independent reporter gene expression occurred 6 h after PPAR-dependent induction of the endogenous COX-2 gene. In contrast to PPAR-dependent COX-2 induction, which is not affected by NF-κB inhibitors, the PPAR-independent pathway was blocked by the NF-κB inhibitor MG132 or following deletion of NF-κB sites in the COX-2 promoter. The PPAR-independent effect of PUFA was mimicked by the PKC activators 4β-PMA and prostaglandin F_2α, but was not blocked by the PKC inhibitor RO318425. The results demonstrate a pathway to the induction of COX-2 by PUFAs requiring NF-κB but not PPAR or PKC.

Introduction

Various hormones interact with G-protein-linked cell surface receptors to activate phospholipases and raise intracellular levels of polyunsaturated fatty acids (PUFAs). A good example is the effect of oxytocin exerted via oxytocin receptors on the epithelium of the uterine endometrium, which leads to activation of phospholipases C and A, releasing arachidonic acid and other PUFAs esterified in phospholipids (Flint et al., 1986). PUFAs produced in this way may then act as second messengers, both through activation of protein kinases and via other metabolic products (Khan et al., 1995, Maloberti et al., 2005).

PUFAs may also be secreted by the cells in which they are generated, to act on neighbouring cells as paracrine messengers (Cooke et al., 1991, Ronco et al., 2002). This has been suggested to occur in the endometrium in relation to both activation of cyclic AMP phosphodiesterase (Cheng et al., 2007) and induction of cyclooxygenase 2 (COX-2; also known as prostaglandin endoperoxide synthase 2; Sheldrick et al., 2007). Evidence for an effect on COX-2 includes the observations that PUFAs raise COX-2 levels in rat uterine stromal cells (Prigent-Tessier et al., 1996) and in bovine endometrial epithelial cells (Parent et al., 2003) through increased COX-2 gene expression and increased levels of COX-2 transcripts (Asselin et al., 1997, Meade et al., 1999). Furthermore, the oxytocin receptor is expressed in the bovine endometrial epithelium at a time when it is not expressed in the stroma (Robinson et al., 1999). Arachidonic acid has therefore been proposed to act as a paracrine signal released during the oestrous cycle by epithelial cells to induce COX-2 in the stroma (Sheldrick et al., 2007). The experiments described here were carried out to investigate the pathways by which PUFAs induce COX-2 gene expression in endometrial stromal cells.

Previous experiments on the mechanism by which arachidonic acid raises COX-2 levels suggest that peroxisome-proliferator-activated receptors (PPARs) mediate PUFA-induced COX-2 expression in the bovine endometrium (MacLaren et al., 2006, Sheldrick et al., 2007). The isoform PPARα appears to be involved in bovine endometrial stromal cells. PPARα is activated directly by a range of PUFAs, including arachidonic acid. However, PPARα function is dependent on phosphorylation by protein kinase C (PKC; Blanquart et al., 2004), which is also activated by PUFAs (Shinomura et al., 1991, Khan et al., 1995). Furthermore, in some cells PKC induces COX-2 through a second pathway, involving NF-κB (Paik et al., 2000, Wu and Wiltbank, 2001, Yan et al., 2002). As a result arachidonic acid may potentially induce COX-2 directly via PPARα and indirectly through PKC/NF-κB.

To distinguish between PPAR-dependent and -independent pathways we have used a reporter construct under the control of a naturally occurring sequence derived from the bovine COX-2 promoter. This construct, which includes most of the important transcription regulators present in the COX-2 promoter but lacks an identifiable peroxisome-proliferator response element (PPRE), allows the study of pathways to COX-2 induction in which PPARs are not involved. Furthermore we have investigated the role of NF-κB in the function of the promoter by sequential removal of the NF-κB sites from this construct.

Section snippets

Materials

All PUFAs; synthetic PPAR agonists; the NSAIDs, indomethacin and NS398; prostaglandin A₁ (PGA₁); the phorbol ester 4β-PMA and its inactive analogue 4α-phorbol 12,13-didecanoate (4α-PDD); the protein kinase C inhibitor, RO 318425, and the NF-κB inhibitor, MG132 were obtained from either Sigma (Poole, UK) or Calbiochem (Nottingham, UK). Conjugated linoleic acid (Sigma catalogue number O 5507) was a mixture of cis- and trans-9,11- and -10,12-octadecadienoic acids. The PUFAs, indomethacin and

Structure of the COX-2 promoter

The nucleotide sequence of the bovine COX-2 gene promoter is shown in Fig. 1. PPARs form heterodimers with the 9-cis retinoic acid receptor (RXR) to activate target genes by binding to specific response elements (PPREs). PPREs contain two direct repeats of PuGGTCA half-sites separated by one base (usually A) with an additional 4 bases (usually AACT) comprising the 5′ part of binding site, giving the consensus sequence AACTAGGNCAAAGGTCA (the hexameric half-sites being italicised; Ijpenberg et

Discussion

Previous experiments showed that one pathway to induction of COX-2 in uterine stromal cells treated with arachidonic acid involves PPARα (Sheldrick et al., 2007). However, in previous experiments it was not possible to distinguish between an effect of arachidonic acid as a PPAR agonist and that involving activation of PKC and NF-κB. Inhibitors were used to block either PKC or NF-κB, but blocking PKC led to inhibition of the PPAR pathway (as expected based on the requirement for PPARs to be

Acknowledgements

We thank Pat Fisher for isolating the endometrial stromal cells. This work was funded by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.

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A PPAR-independent pathway to PUFA-induced COX-2 expression

Abstract

Introduction

Section snippets

Materials

Structure of the COX-2 promoter

Discussion

Acknowledgements

Mol. Cell. Endocrinol.

Biochim. Biophys. Acta

Free Rad. Biol. Med.

FEBS Lett.

Anal. Biochem.

J. Steroid Biochem. Mol. Biol.

Dom. Anim. Endocrinol.

J. Biol. Chem.

Cell. Signal.

J. Biol. Chem.

J. Biol. Chem.

Dom. Anim. Endocrinol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines

Biochem. J.

Cellular mechanisms involved during oxytocin-induced prostaglandin F2α production in endometrial epithelial cells in vitro: role of cyclooxygenase-2

Endocrinology

Cellular mechanisms involved during oxytocin-induced prostaglandin F_2α production in endometrial epithelial cells in vitro: role of cyclooxygenase-2