Dual activation of PPARα and PPARγ by mono-(2-ethylhexyl) phthalate in rat ovarian granulosa cells

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Abstract

Peroxisome proliferator-activated receptors (PPARs) are key regulators of lipid metabolism and cell differentiation. The plasticizer di-(2-ethylhexyl) phthalate is a peroxisome proliferator, and its active metabolite mono-(2-ethylhexyl) phthalate (MEHP) activates PPARα and PPARγ in cell transactivation assays. MEHP is a female reproductive toxicant and decreases activity, mRNA, and protein levels of aromatase, the rate-limiting enzyme that converts testosterone to estradiol in ovarian granulosa cells. To test the hypothesis that MEHP suppresses aromatase through PPAR pathways, granulosa cells were cultured with MEHP (50 μM) or selective activators of PPARγ or PPARα for 48 h and gene expression was analyzed by real time RT-PCR. Both PPARα and PPARγ activators significantly decreased aromatase mRNA and estradiol production like MEHP. The PPARγ-selective antagonist GR 259662 partially blocked the suppression of aromatase by MEHP, suggesting that MEHP acts through PPARγ, but not exclusively. MEHP and the PPARα-selective agonist GW 327647 induced expression of 17β-hydroxysteroid dehydrogenase IV, a known PPARα-regulated gene, and induction was maintained with addition of the PPARγ-selective antagonist. PPARα-selective activation also induced expression of aryl hydrocarbon receptor (AhR), CYP1B1, and epoxide hydrolase in the granulosa cell. These data support a model in which MEHP activates both PPARα and PPARγ to suppress aromatase and alter other genes related to metabolism and differentiation in the granulosa cell.

Introduction

Cell differentiation is executed by complex networks of signaling pathways activated by chemical messengers including endogenous hormones, fatty acids, pharmaceuticals, and environmental compounds. Pharmacological agents are designed to target specific pathways, while environmental toxicants are identified based on their ability to alter cell function through unknown pathways. Understanding perturbations of specific and promiscuous chemical messengers can provide important insights into how signaling networks are wired. In turn, identifying the biochemical and molecular mechanisms of action of environmental toxicants is useful in assessing their potential harm to exposed humans. Phthalates are ubiquitous environmental toxicants to which many people are exposed. Over 18 billion pounds of phthalates are used each year in plastic products (Blount et al., 2000a) and phthalate metabolites are measured in human urine, serving as useful biomarkers of phthalate exposure (Blount et al., 2000a, Blount et al., 2000b). In our studies of how one phthalate plasticizer affected ovarian function, we discovered that it activated two signal transduction pathways, resulting in disruption of ovarian granulosa cell estradiol production.

The commonly used plasticizer di-(2-ethylhexyl) phthalate (DEHP) is a peroxisome proliferator, liver carcinogen, and ovarian toxicant in rodents (Davis et al., 1994a, Lake et al., 1987, Reddy and Lalwai, 1983). Its active metabolite mono-(2-ethylhexyl) phthalate (MEHP) alters gene expression by activating peroxisome proliferator-activated receptors (PPARs), key regulators of lipid metabolism and cell differentiation (Maloney and Waxman, 1999). The liver toxicity of DEHP is dependent on PPARα, as mice lacking this receptor do not develop liver tumors or induce fatty acid metabolizing enzymes in response to DEHP (Macdonald et al., 2001, Ward et al., 1998). MEHP activates PPARα in cell transactivation assays (Corton et al., 2000, Hasmall et al., 2000, Maloney and Waxman, 1999) and increases PPARα-mediated gene expression in the liver (Fan et al., 1998, Macdonald et al., 2001). However, MEHP can work through pathways other than PPARα in some tissues, because testicular toxicity is maintained in PPARα null mice treated with DEHP (Ward et al., 1998). MEHP activation of PPARγ in transactivation assays (Maloney and Waxman, 1999) suggests that this isoform of PPAR may be responsible for DEHP-mediated toxicity in tissues other than the liver (Maloney and Waxman, 1999, Ward et al., 1998).

All known isoforms of PPAR are expressed in the rat ovary (Braissant et al., 1996), but their functions in this tissue remain unclear. While PPARγ is highly expressed in preovulatory granulosa cells, it is downregulated after ovulation, suggesting it may be involved in the differentiation of estrogen-producing granulosa cells to progesterone-producing luteal cells (Komar et al., 2001, Lohrke et al., 1998). Granulosa cell differentiation, like differentiation in other cells, is accompanied by cessation of proliferation, altered gene expression, and morphological changes (Nanbu-Wakao et al., 2000). A hallmark of terminal granulosa cell differentiation after ovulation is the rapid loss of cytochrome P450 aromatase (CYP 19), the rate-limiting enzyme that converts testosterone to estradiol (Fitzpatrick et al., 1997, Hickey et al., 1988). The mechanism triggering this shift in gene expression is unknown, but recent studies suggest a link between activation of PPARγ and suppression of aromatase. The PPARγ ligand troglitazone inhibits aromatase activity and mRNA levels in human ovarian granulosa cells (Mu et al., 2000). Combined treatment with both troglitazone and an RXR ligand causes a synergistic effect to decrease aromatase, suggesting that this effect is mediated by the PPARγ:RXR heterodimer (Mu et al., 2000).

DEHP causes anovulation by suppressing granulosa cell estradiol production in preovulatory follicles of adult female rats (Davis et al., 1994a). Suppression of estradiol is not due to chemically mediated apoptosis of granulosa cells as there is no morphological evidence of apoptosis and the numbers of follicular cells are comparable in DEHP-treated rats and vehicle-treated rats (Davis et al., 1994a). Moreover, DEHP-treated rats can ovulate in response to exogenous luteinizing hormone, suggesting that DEHP has specific effects on estradiol production (Davis et al., 1994a). In cultured granulosa cells, MEHP suppresses estradiol production by decreasing mRNA, protein, and activity of aromatase (Davis et al., 1994b, Lovekamp and Davis, 2001). The decrease in aromatase and estradiol is independent of cAMP stimulation (Davis et al., 1994b, Lovekamp and Davis, 2001). Additionally, the effect is relatively specific for aromatase because MEHP does not decrease mRNA levels of cholesterol side-chain cleavage cytochrome P450 (P450scc), another rate-limiting steroidogenic enzyme in the granulosa cell (Lovekamp and Davis, 2001).

We previously showed that the PPARα activator Wy-14643 decreases aromatase and estradiol similarly to MEHP in rat granulosa cells (Lovekamp and Davis, 2001). Here, we test the hypothesis that the suppression of aromatase mRNA by MEHP is mediated by both PPARα and PPARγ in the rat granulosa cell. We also investigate PPAR subtype specific effects on gene expression in the granulosa cell.

Section snippets

Materials

MEHP (CAS Number 4376-20-9) was obtained from TCI America (Portland, OR). Troglitazone was kindly provided by Dr Chris Nicol (National Cancer Institute, Bethesda, MD). The PPARα (GW 327647) and PPARγ (GW 347845) selective agonists, as well as the PPARγ antagonist (GR 259662) were provided by Dr T. Willson (GlaxoSmithKline, RTP, NC). The RXR-selective agonist (SR 11246) was provided by Dr M. Dawson (The Burnham Institute, La Jolla, CA). 9-cis retinoic acid was purchased from Sigma-Aldrich (St.

PPARα and PPARγ ligands and MEHP decrease aromatase and estradiol

Given that MEHP activates both PPARα and PPARγ in a COS-1 cell transactivation assay (Maloney and Waxman, 1999), we tested whether PPAR activation by receptor-selective ligands caused effects similar to those of MEHP in the granulosa cell. As shown in Fig. 1, both PPARα and PPARγ-selective agonists significantly decreased aromatase mRNA compared to control. MEHP (50 μM) decreased aromatase mRNA by 45%, while the PPARγ ligands troglitazone (1 μM), 15dPGJ2 (1 μM), and GW 347845 (2 μM) and the

Discussion

Here, we provide evidence indicating that MEHP employs both PPARα and PPARγ pathways to regulate key genes in granulosa cell differentiation and metabolism, and that activation of PPAR pathways has both redundant and receptor-specific effects on gene expression in the granulosa cell. A model is proposed to explain these effects based on our observations of the dual action of MEHP in activating both pathways and our identification of sets of genes regulated by each pathway (Fig. 6). Activation

Acknowledgements

The authors would like to thank Ms Glenda Corniffe for technical assistance and Dr Joe Haseman for help with statistical analysis. Thanks to Drs Trevor Archer and Cary Weinberger for critical review of this manuscript. This work is in partial fulfillment of a graduate thesis.

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