Transdominant suppression of estrogen receptor signaling by progesterone receptor ligands in uterine leiomyoma cells

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Abstract

Uterine leiomyomas develop in reproductive-age women with high frequency and are dependent on the production of ovarian hormones. While it is generally accepted that these tumors are estrogen (E2)-responsive, the role of progesterone (P4) in modulating tumor growth is less clear. In the present study, an in vivo/in vitro rat model was used to characterize progesterone receptor (PR) isoform expression in uterine leiomyoma and investigate PR signaling using progestins and antiprogestins in the leiomyoma-derived cell line ELT-3. PR-A was the predominant isoform expressed in normal myometrium, leiomyomas and ELT3 cells. In the normal myometrium, PR-A and PR-B levels varied during the estrous cycle with low ratios of PR-A relative to PR-B (PR-A/PR-B) coinciding with times of cell proliferation. Although PR ligands had no effect on basal levels of uterine leiomyoma cell proliferation in vitro, both progestins and antiprogestins inhibited E2-stimulated cell proliferation. In addition, E2-stimulated transactivation of an estrogen-response-element reporter gene as well as E2-induced upregulation of the PR were also inhibited by PR ligands. These data indicate that PR ligands can transdominantly suppress estrogen receptor signaling and stimulation of uterine leiomyoma cell growth.

Introduction

Although benign, uterine leiomyomas represent a major gynecological health problem due to their extraordinarily high incidence and current lack of satisfactory surgical or chemotherapeutic options. Uterine leiomyomas are the most common gynecological neoplasm and the most frequent indication for hysterectomy in the United States in premenopausal women (Cramer and Patel, 1990; Easterday et al., 1983). GnRH therapy, which blocks ovarian steroid production, effectively reduces tumor volume but is unsuitable for long-term use due to side effects induced by a hypoestrogenic state. In addition, much of the observed decrease in uterine size is due to involution of the normal myometrium and the tumors themselves show little decrease in cellularity (Andreyko et al., 1987; Cohen et al., 1994). Several limited studies have shown that the antiprogestin RU486 is also effective in reducing the volume of leiomyomas in humans (Kettel et al., 1994; Murphy et al., 1993), although the long-term effects and mechanism behind its efficacy remain to be determined.

Uterine leiomyomas appear during the reproductive years, often change dramatically in volume during pregnancy and regress after menopause (Rein and Nowak, 1992). While it is generally accepted that uterine leiomyomas are responsive to ovarian hormones, the exact roles of estrogen (E2) and progesterone (P4) in the etiology of this disease remain to be elucidated. In the case of E2, there is significant evidence to support an important role for this hormone in the pathogenesis of uterine leiomyoma. Increased expression of estrogen receptors (ER) and an elevated transcriptional response to E2 have been observed in uterine leiomyoma compared to the normal myometrium (Andersen et al., 1995; Brandon et al., 1995; Englund et al., 1998; Rein et al., 1990). The progesterone receptor (PR) is an E2-responsive gene whose mRNA and protein expression is elevated in leiomyoma (Brandon et al., 1993; Englund et al., 1998; Viville et al., 1997). Recently, it has become increasingly clear that E2 and P4 signaling work in concert to regulate the normal myometrium and disruption of the normal signaling patterns of these two receptors could affect the growth and development of leiomyoma. For example, Shimomuro et al. have shown that in leiomyoma cultures, P4 upregulates EGF while E2 upregulates its receptor (Shimomura et al., 1998). Expression of Bcl-2, an antiapoptotic protein, has recently been shown to be upregulated by P4 but downregulated by E2 in leiomyoma cells (Matsuo et al., 1997). These studies suggest that in the uterine myometrium there are complex biochemical interactions between the signaling pathways of these two sex steroids and their receptors.

The PR has two isoforms, A and B, which are expressed from a single gene in rodents and humans (Feil et al., 1988; Lessey et al., 1988; Schneider et al., 1991). The relative expression of PR-A to PR-B has been shown to vary in the human endometrium during the menstrual cycle (Mangal et al., 1997) and also seasonally in the chick oviduct (Boyd and Spelsberg, 1979). The A and B isoforms of the PR have very distinct biochemical profiles which depend on the ligand, promoter and cell context. PR-A has been shown to act as a transdominant inhibitor of PR-B mediated transcriptional activity in some cell types (McDonnell and Goldman, 1994; Tung et al., 1993; Vegeto et al., 1993). Furthermore, PR signaling is capable of transdominant suppression of ER transactivation of E2-responsive promoters in human breast cancer cells and in rat uterine cells (Chalbos and Galtier, 1994; Kraus et al., 1995; Wen et al., 1994). However, transdominant suppression by PR is cell type-specific, ligand-specific (agonist or antagonist) and isoform specific. Therefore, in addition to absolute levels of PR, the ratio of PR-A relative to PR-B may be an important factor in determining whether PR ligands are able to mediate an agonist or antagonist response in a given tissue and may modulate signaling via other nuclear hormone receptors such as the ER.

In the Eker rat model of uterine leiomyoma, tumors arise spontaneously from the uterine myometrium and, as in humans, occur with high frequency and are responsive to endogenous and exogenous hormones (Fuchs-Young et al., 1996; Hodges et al., 2000; Howe et al., 1995a; Howe et al., 1995b; Hunter et al., 1999). Previous work from our laboratory has characterized the proliferative and apoptotic program of the normal and neoplastic uterine myometrium (Burroughs et al., 2000; Burroughs et al., 1997). The myometrium of young, cycling rats exhibits maximum proliferation during proestrus when E2 and P4 levels are highest, while maximum apoptosis is observed during estrus when steroid hormone levels are relatively low (Burroughs et al., 2000; Freeman, 1994). Thus, proliferation and apoptosis in young, cycling animals is tightly regulated during the estrous cycle by E2 and P4. Leiomyomas on the other hand, exhibit a dysregulation of tissue homeostasis, characterized by a high proliferative index at relatively low serum E2 levels and an apoptotic index that is reduced relative to age-matched myometrium.

In the present study, normal myometrium and neoplastic uterine myometrial cells from the Eker rat were used to evaluate the effect of PR ligands, both progestins and antiprogestins, on cell proliferation and E2-induced signaling. We observed that in the uterine myometrium, PR-A/B ratios fluctuate during the estrous cycle, with the lowest PR-A/B ratio coinciding with times in which high levels of circulating E2 and maximal proliferation are observed. In vitro, several PR ligands, both progestins and antiprogestins, inhibited E2-induced cell proliferation and exhibited transdominant suppression of ER-mediated gene transcription.

Section snippets

Cells

The ELT-3 rat uterine leiomyoma cell line has been characterized previously and was maintained in DF8 medium supplemented with 10% fetal calf serum (Hyclone Laboratories Inc., Logan, UT) at 37 °C, 5% CO2, as described (Howe et al., 1995c). Test compounds were added to the cells growing in serum-free, phenol red-free DF8-basal medium, containing 1% BSA (Sigma Chemical Co, St. Louis, MO).

Ligands

PR ligands used were R5020 (promegestone) (NEN, Boston, MA), 6α-methyl-17α-hydroxy-progesterone acetate (MPA)

Proliferation in the normal myometrium coincides with low PR-A to PR-B ratios

PR protein levels in the normal myometrium of 2–4-month-old cycling Eker rats were determined by western analysis and the levels of the individual PR isoforms quantitated (Fig. 1A,B). PR-A was the predominant isoform in the normal rat uterine myometrium throughout the estrous cycle. The levels of both PR isoforms were highest in the myometrium during proestrus, when circulating E2 and P4 levels peak, consistent with the known E2-responsiveness of this gene in other tissues such as the breast

Discussion

In the present study, an in vivo/in vitro rat model was used to characterize PR isoform expression in the normal and neoplastic myometrium and investigate the impact of PR signaling in leiomyoma cells using progestins and antiprogestins. PR-A was the predominant PR isoform expressed in normal myometrium, leiomyoma and in the leiomyoma-derived cell line, ELT-3. Low ratios of PR-A relative to PR-B (PR-A/PR-B) appeared to be permissive for myometrial proliferation during the estrous cycle,

Acknowledgements

This work was supported by grant ES08263 (CLW) from the National Institute of Environmental Health Science, Research Triangle Park, NC. We would like to thank Dr. Nancy Weigel at Baylor College of Medicine for critical review of this manuscript.

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