Treatment of Rats with 17β-Estradiol or Relaxin Rapidly Inhibits Uterine Estrogen Receptor β1 and β2 Messenger Ribonucleic Acid Levels

BIOLOGY OF REPRODUCTION 67, 1919–1926 (2002) Published online before print 04 October 2002. DOI 10.1095/biolreprod.102.003392 Treatment of Rats with 17b-Estradiol or Relaxin Rapidly Inhibits Uterine Estrogen Receptor b1 and b2 Messenger Ribonucleic Acid Levels1 Suresh B. Pillai,3 Jenny M. Jones,3 and Robert D. Koos2 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 ABSTRACT estradiol, estradiol receptor, gene regulation, relaxin, uterus INTRODUCTION The diverse effects of estrogen on a wide range of target tissues are mediated by estrogen receptors (ERs), nuclear transcription factors that are activated either upon binding of estrogen and estrogen-like ligands or via cross-talk, most likely through phosphorylation of the unliganded receptors, with various other signal transduction pathways [1–4]. Recently, a second form of ER, designated ERb to differenThis research was supported by NCI/NIH grant CA45055 and by NICHD/ NIH cooperative agreement U54 HD36207 as part of the Specialized Cooperative Centers Program in Reproduction Research. J.M.J. was supported by NICHD/NIH T32 HD07170. A preliminary report of this work was presented at The Endocrine Society’s 81st Annual Meeting, June 12– 15, 1999, San Diego, CA. 2 Correspondence: Robert D. Koos, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201-1559. FAX: 410 706 8341; e-mail: 3 S.B.P. and J.M.J. contributed equally and should be considered co-first authors. 1 Received: 15 January 2002. First decision: 4 February 2002. Accepted: 1 July 2002. Q 2002 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 1919 Estrogen regulates the growth and differentiation of the uterus via binding to estrogen receptors (ERs), members of the nuclear receptor family of transcription factors. Two forms of ER exist: ERa and ERb. The former is a well-characterized mediator of estrogen-induced transcription, but the function of the latter is unclear. Recent in vitro studies suggest that both splicing forms of ERb expressed in rat tissues, b1 and b2, may function as inhibitors of ERa transcriptional activity. To gain insight into the role of ERb in estrogen action, we examined the effects of estrogen and relaxin, a ligand-independent activator of ERs, on the expression of ERb1 and ERb2 mRNA in the uterus in vivo. Eighteen-day-old female rats were ovariectomized and, after recovery, treated with 17b-estradiol, relaxin, or vehicle. Quantitative reverse transcription-polymerase chain reaction analyses of uterine RNA from estrogen-treated animals revealed marked decreases in the steady-state levels of the mRNAs for both ERb1 and ERb2 at 3, 6, and 24 h after treatment. Relaxin induced a similar effect. Neither hormone had any significant effect on ERa mRNA levels. To determine if endogenous estrogen exerts this effect, we examined the expression of ERbs in the uterus during the estrous cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). These data suggest that down-regulation of ERb expression may be required for estrogen to exert its full trophic effects on the uterus. tiate it from the original ER (consequently called ERa), was discovered [5–7]. A further layer of complexity was added when a second common splicing variant of ERb was identified in tissues of both the rat and mouse [8–12]. Designated ERb2 (with the original form becoming ERb1), it contains a 54-base pair (bp)/18-aa insert within the ligandbinding domain. The physiological function of ERb is presently unclear, but its discovery has necessitated a complete reevaluation of estrogen signaling pathways in target cells. The cyclic growth and differentiation of the uterine endometrium and other tissues of the reproductive tract are controlled in large part by estrogen [2, 4, 13]. While the predominant ER in the uterus is ERa, numerous investigators have reported that ERb is also expressed there, albeit at a markedly lower level [14–20]. ERb2, the longer splice variant present in rodents, has been reported to coexist with ERa and ERb1 in all tissues examined, including the uterus, prostate, and ovary [10], and the level of its expression is similar to, in some cases greater than, that of ERb1 [8, 9]. ERb protein has also been detected in the rodent uterus by immunohistochemistry [19, 20]. Thus, a substantial body of evidence indicates that both ERb mRNA and protein are expressed in the uterus. With regard to function, it has been repeatedly demonstrated in transfection studies that ERb1 is capable of mediating estrogen-induced reporter gene transcription in vitro, but it is significantly less active than ERa [7, 21]. Furthermore, it remains to be definitively shown that endogenous ERb1 acts as a transcriptional activator of endogenous genes at physiological estrogen concentrations in vivo. ERb2 binds estrogen with still lower affinity than ERb1 and is a much weaker inducer of transcriptional activity [10–12]. On the other hand, there is mounting evidence, also from in vitro transfection studies, that both isoforms of ERb could actually serve to inhibit ERa signaling in target tissues [10, 12, 21]. ERb2 was first shown to inhibit estrogen-dependent, ERa-mediated expression of estrogen response element (ERE)-containing reporter genes in COS1 cells [10]. Subsequently, Hall and McDonnell [21] demonstrated that human ERb (homologous to rat/mouse ERb1) also repressed ERa activity in HepG2 cells in response to subsaturating concentrations of 17b-estradiol, which is physiologically relevant because estrogen’s uterotrophic effects require only 5–20% receptor occupancy [13]. The inhibitory effect of ERbs could involve formation of heterodimers with ERa [11, 12, 21–23], an interaction that is enhanced by estrogen [11, 21]. In addition, ERb1, unlike ERa, appears to interact with the ERE in the absence of ligand [21], suggesting that it may also competitively inhibit ERa binding to DNA. Such interactions would, of course, require that ERa and ERb be coexpressed in cells. Several studies indicate that both are expressed in uterine epithelial and stromal cells [16, 17, 19, 20, 24, 25]. That 1920 PILLAI ET AL. MATERIALS AND METHODS Animals and Experimental Design Animal studies were conducted in accordance with mandated standards of humane care as described in the Guide for the Care and Use of Laboratory Animals (National Research Council) and approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. To examine the effect of estrogen on ERb1, ERb2, ERa, and VEGF (vascular endothelial growth factor) expression in the uterus, female Sprague-Dawley rats (Crl:CD[SD] BR strain; Charles River, Wilmington, MA) were ova (...truncated)