Synthetic Estrogen 17a-Ethinyl Estradiol Induces Pattern of Uterine Gene Expression Similar to Endogenous Estrogen 17b-Estradiol

17a-Ethinyl estradiol is one of most widely prescribed estrogens. We compared the effects of this synthetic estrogen to those of the endogenous ovarian hormone 17b-estradiol on the expression of four estrogen-inducible genes in the rat uterus. The genes examined include c-fos, c-jun, vascular endothelial growth factor, and creatine kinase B, which are all known to be primary responses to estrogen administration. Both estrogens induced the four target genes with similar time courses and produced the same pattern of cell-specific expression of c-fos and vascular endothelial growth factor in the uterine epithelium and stroma, respectively. Dose-response studies established that the potency and efficacy of both estrogens in the uterus were the same for all four hormone-regulated genes. These studies suggest that 17a-ethinyl and 17b-estradiol produce similar if not identical patterns of gene expression in the uterus. Estrogens are some of the most commonly prescribed pharmacological agents, being used most frequently for hormone replacement therapy of postmenopausal women and as components of combination oral contraceptives, although they are also used for a variety of other purposes (Williams and Stancel, 1996). Most of the pharmacological actions of estrogens are thought to result from interactions with the classic nuclear estrogen receptor (ER), which is a ligand-activated transcription factor. The ER-ligand complex interacts with DNA binding sites, termed estrogen response elements (EREs), in target genes, and this complex recruits coactivators (or corepressors) and other regulatory proteins that form the active transcription complex (Shibata et al., 1997; White and Parker, 1998). A large number of steroidal and nonsteroidal compounds bind to this receptor and display hormonelike activity in humans and a number of experimental test systems (Anstead et al., 1997). There are substantial differences in the potencies of different estrogens that result primarily from differences in their receptor-binding affinities and their pharmacokinetic properties, including absorption, first-pass metabolism, plasma protein binding, and elimination (Williams and Stancel, 1996). However, it was thought until recently that the response to all estrogens was basically the same if these differences in receptor affinities and pharmacokinetics were accounted for. All estrogens were thought to produce their responses by a similar activation of the ER, which led to the same basic pattern of gene expression in target tissues. Conversely, competitive hormone antagonists were thought to produce their effects by blocking hormone binding to the receptor and/or by favoring the interaction of the ER-ligand complex with factors that repress rather than activate transcription. In most early models of estrogen action, the receptor was thus viewed as an on/off switch that was activated by agonist binding, with any pharmacological differences between various estrogens due primarily to differences in potencies (Stancel et al., 1995; Katzenellenbogen et al., 1996; McDonnell and Norris, 1997). Recently, results from a number of studies have called this unitary view of estrogen action into question (Katzenellenbogen et al., 1996; McDonnell and Norris; 1997; Stancel et al., 1995). At the biochemical level, protease digestion (McDonnell et al., 1995) and crystallographic studies (Brzozowski et al., 1997) have clearly established that the ER assumes very different conformations when occupied by different ligands, and functional studies have revealed that the pattern of gene expression produced by estrogens is both gene and context specific. For example, different ligands can produce distinct patterns of gene expression in cultured cells or in target tissues in vivo. The emerging paradigm of estrogen action is that different ligands cause the ER to assume different conReceived for publication January 12, 1999. 1 This study was supported by National Institutes of Health Grant HD08615. ABBREVIATIONS: ER, estrogen receptor; VEGF, vascular endothelial growth factor; CKB, creatine kinase B; ERE, estrogen response element; 17b-E2, 17b-estradiol; 17a-EE, 17a-ethinyl estradiol. 0022-3565/99/2902-0740$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290:740–747, 1999 740 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from formations that may selectively interact with different coactivators/corepressors, EREs, or other regulatory factors (Stancel et al., 1995; Katzenellenbogen et al., 1996; McDonnell and Norris, 1997). Thus, it can no longer be assumed that different estrogens produce identical responses in target tissues, especially considering that overall tissue responses to these hormones are likely to involve a primary activation of multiple genes. These considerations prompted us to consider whether commonly used estrogenic drugs produce the same patterns of gene expression in target tissues as endogenous ovarian hormones. On searching the literature, we did not uncover any reports that directly addressed this question, which prompted us to compare the in vivo responses of the uterus to 17a-ethinyl estradiol (17a-EE) and 17b-estradiol (17b-E2). We selected 17a-EE for this study because it is the most frequently used synthetic estrogen (Williams and Stancel, 1996). The genes we selected to compare the response of the two estrogenic compounds are c-fos (Loose-Mitchell et al., 1988), c-jun (Chiappetta et al., 1992; Nephew et al., 1994), vascular endothelial growth factor (VEGF) (Hyder et al., 1996), and creatine kinase B (CKB) (Pentecost et al., 1990). These genes were chosen because they are well characterized primary responses of the normal uterus to estrogenic stimulation. This allowed us to compare the dose-response patterns of endogenous genes produced by the synthetic and naturally occurring estrogens in a physiologically relevant test system. Experimental Procedures Animals. Immature (21 days old, 40–45 g) female Sprague-Dawley rats (Sasco, Omaha, NE) were ovariectomized under Chloropent anesthesia (Sigma, St. Louis, MO) 3 to 7 days before use. 17b-E2 and 17a-EE (dissolved in 0.5 ml 5% ethanol/95% saline) were administered via s.c. injection at a dose of 40 mg/kg (except for the doseresponse studies shown in Figs. 5 and 6). At the times indicated for individual experiments, uteri were removed under Chloropent anesthesia, and the animals were then sacrificed by decapitation while still anesthetized. Protocols for the care and use of these animals were approved by the University of Texas Animal Care and Use Committee, in accordance with the Guiding Principles for the Care and Use of Research Animals. Materials. Estradiol was obtained from Steraloids (Wilton, NH) and 17a-EE was purchased from Sigma. [P]UTP (800 Ci/mmol) was obtained from Amersham Radiochemicals (Arlington Heights, IL) and diluted to 400 Ci/mmol with radioinert UTP (Boehringer Mannheim, Indianapolis, IN) for synthesis of riboprobes. Guanidine isothiocyanate, CsCl, and formamide were obtained from International Biotechnologies (New Haven, CT). All other chemicals were obtained from Sigma and were the highest grade commercially available. RNA Preparation and Analysis. RNA was prepared as described previously (Loose-Mitchell et al., 1988; Chiappetta et al., 1992). Briefly, uteri were removed from anesthetized animals and immediately homogenized in 5 M guanidinium isothiocyanate with a Polytron homogenizer set at half-maximal power for 60 s. Uteri from two or three animals were pooled for the preparation of a single RNA sample. RNA was pelleted through 5.7 M CsCl, extracted twice with phenol/chloroform (1:1), once with chloroform, and precipitated with