Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor alpha and estrogen receptor beta

The molecular mechanism used by environmental chemicals to exert their hormone-like actions is still only partially resolved. Although it generally is accepted that xenoestrogens act at the genomic level by binding to intracellular estrogen receptors, we have shown here that they trigger nongenomic effects in pancreatic beta cells. Both xenoestrogens and the circulating hormone, 17beta-estradiol, bind with high affinity to a common membrane binding site unrelated to the intracellular estrogen receptors ERalpha and ERbeta. This binding site is shared by dopamine, epinephrine, and norepinephrine and has the pharmacological profile of the gammaadrenergic receptor. This study provides an outline of the membrane receptor involved in rapid xenoestrogen actions.     

Oestrogen-mediated cardioprotection following ischaemia and reperfusion is mimicked by an oestrogen receptor (ER)alpha agonist and unaffected by an ER beta antagonist

Oestrogen protects the heart from ischaemic injury. The current study aims to characterise two novel oestrogen receptor (ER) ligands, an ER alpha agonist ERA-45 and an ER beta antagonist ERB-88, and then use them to investigate the roles of ER alpha and ER beta in mediating the cardioprotection by E from ischaemia-reperfusion injury in the rat. The ER ligands were characterised by gene transactivation assay using ER-transfected Chinese hamster ovary (CHO) cells and in bioavailability studies in vivo. Female rats (n=48) were ovariectomised and implanted with 17beta-oestradiol (17 beta E(2)) releasing or placebo pellets. ERA-45, ERB-88 or vehicle was administered for 5 days prior to ischaemia-reperfusion studies. Necrosis, neutrophil infiltration (myeloperoxidase activity) and oxidant stress production (electron paramagnetic resonance) from the area-at-risk were measured to assess reperfusion injury. The ER alpha agonist ERA-45 showed more than 35-fold selectivity for ER alpha compared with ER beta gene transactivation. In vitro, the ER beta antagonist ERB-88 inhibited transactivation by 17 beta E(2) via ER beta with 46-fold selectivity relative to inhibition via ER alpha. In vivo, 17 beta E(2) significantly reduced neutrophil infiltration, oxidant stress and necrosis following ischaemia and reperfusion. Cardioprotection by 17 beta E(2) was not inhibited by ERB-88 but was completely reproduced by ERA-45. In conclusion, protection of the rat heart after ischaemia-reperfusion by 17 beta E(2) is achieved through the reduction of cardiomyocyte death, neutrophil infiltration and oxygen-free radical availability.The results of this study indicate that these effects are primarily mediated via activation of ER alpha.     

Estrogen receptor (ER) beta modulates ERalpha responses to estrogens in the developing rat ventromedial nucleus of the hypothalamus

The mechanisms by which estradiol exerts specific actions on neural function are unclear. In brain the actions of estrogen receptor (ER) alpha are well documented, whereas the functions of ERbeta are not yet fully elucidated. Here, we report that ERbeta inhibits the activity of ERalpha in an anatomically specific manner within the neonatal (postnatal d 7) brain. Using selective agonists we demonstrate that the selective activation of ERalpha in the relative absence of ERbeta activation induces progesterone receptor expression to a greater extent than estradiol alone in the ventromedial nucleus, but not the medial preoptic nucleus, despite high ERalpha expression. Selective activation of ERbeta attenuates the ERalpha-mediated increase in progesterone receptor expression in the ventromedial nucleus but has no effect in medial preoptic nucleus. These results suggest that ERalpha/ERbeta interactions may regulate the effects of estrogens on neural development and reveal the neonatal brain as a unique model in which to study the specificity of steroid-induced gene expression.     

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs alpha and beta and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER alpha and ER beta ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.     

Studies on estrogen receptor (ER) alpha and beta responses on gene regulation in peripheral blood leukocytes in vivo using selective ER agonists

Major reproductive events such as menstruation, ovulation, implantation, and cervical ripening are characterized by an increased number of invading leukocytes in the tissues. Sex steroid hormones, particularly estrogens, play an important role in these dynamic changes in the female reproductive tract. Estrogens have also been implicated in the pathogenesis of many common pathological conditions associated with leukocyte infiltration and immunological dysfunction, such as auto-immune diseases and atherosclerosis. Although the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, have been found in different leukocyte populations in tissues and in peripheral blood, there is still very little known about functional activity and importance of ERs in blood cells. To elucidate the different roles for ERalpha and ERbeta in peripheral blood leukocytes, we used microarray gene expression profiling of rat peripheral blood leukocytes subjected to in vivo treatment with estradiol (E2), the selective ERalpha agonist 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), and the selective ERbeta agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). We report the identification of genes that were commonly regulated by E2, PPT, and DPN, and genes that were regulated either by the ERalpha or ERbeta agonist. Further confirmatory analyses of the selected regulated genes 12-lipoxygenase, fibulin-1, furin, and calgranulin B are also presented. These results were then compared with those from the uterine tissue of the same animals. Our study demonstrates that peripheral blood leukocytes are responsive to estrogens. E2 and selective ERalpha and ERbeta agonists regulate a number of genes that may contribute to inflammation and remodeling of the extracellular matrix.     

Intimal estrogen receptor (ER)beta, but not ERalpha expression, is correlated with coronary calcification and atherosclerosis in pre- and postmenopausal women

BACKGROUND: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) alpha and beta are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERalpha or ERbeta in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERalpha and ERbeta expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status. METHODS: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERalpha and ERbeta, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERalpha and ERbeta with smooth muscle actin, a marker of VSMCs. RESULTS: ERbeta and ERalpha were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERbeta expression exceeded ERalpha expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERbeta expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERalpha expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERalpha but not ERbeta declined with age (P < 0.01) in HT women only. Age had no effect on ERalpha or ERbeta expression in non-HT or premenopausal women. CONCLUSIONS: ERbeta is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERbeta expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.     

Estrogen receptor (ER)-beta isoforms: a key to understanding ER-beta signaling

Estrogen receptor beta (ER-beta) regulates diverse physiological functions in the human body. Current studies are confined to ER-beta1, and the functional roles of isoforms 2, 4, and 5 remain unclear. Full-length ER-beta4 and -beta5 isoforms were obtained from a prostate cell line, and they exhibit differential expression in a wide variety of human tissues/cell lines. Through molecular modeling, we established that only ER-beta1 has a full-length helix 11 and a helix 12 that assumes an agonist-directed position. In ER-beta2, the shortened C terminus results in a disoriented helix 12 and marked shrinkage in the coactivator binding cleft. ER-beta4 and -beta5 completely lack helix 12. We further demonstrated that ER-beta1 is the only fully functional isoform, whereas ER-beta2, -beta4, and -beta5 do not form homodimers and have no innate activities of their own. However, the isoforms can heterodimerize with ER-beta1 and enhance its transactivation in a ligand-dependent manner. ER-beta1 tends to form heterodimers with other isoforms under the stimulation of estrogens but not phytoestrogens. Collectively, these data support the premise that (i) ER-beta1 is the obligatory partner of an ER-beta dimer, whereas the other isoforms function as variable dimer partners with enhancer activity, and (ii) a single functional helix 12 in a dimer is sufficient for gene transactivation. Thus, ER-beta behaves like a noncanonical type-I receptor, and its action may depend on differential amounts of ER-beta1 homo- and heterodimers formed upon stimulation by a specific ligand. Our findings have provided previously unrecognized directions for studying ER-beta signaling and design of ER-beta-based therapies.     

Developmental expression of estrogen receptor (ER) alpha and ERbeta in the hamster ovary: regulation by follicle-stimulating hormone

Perinatal expression of estrogen receptor (ER) protein and mRNA and the influence of FSH on this process were examined by immunofluorescence and RT-PCR using ovaries from fetal (d 13-15 of gestation) and postnatal [postnatal d 1-15 (P1-P15)] hamsters and from 8-d-old hamsters exposed in utero to an anti-FSH serum on d 12 of gestation and saline or equine chorionic gonadotropin (eCG) on P1. A few somatic cells expressing ERalpha immunoreactivity appeared first on d 14 of gestation and increased markedly by P8-P15 in the interstitial cells and granulosa cells of primordial follicles. In contrast, appreciable ERbeta immunoreactivity was localized on d 13 of gestation, and more cells expressed ERbeta immunoreactivity by P1-P8. By P7, ERbeta immunoreactivity was present in cells adjacent to the oocytes, and by P8, ERbeta was preferentially localized in the granulosa cells. Receptor immunoreactivities decreased markedly in P8 ovaries exposed in utero to the FSH antiserum but were reversed with postnatal eCG replacement. Oocytes and somatic cells expressed ERalpha and ERbeta mRNA, and levels of ER mRNA in the ovary increased by P7-P8, corresponding to the appearance of primordial follicles. Thereafter, only ERbeta mRNA levels increased progressively with postnatal ovary development. Similar to ER protein, mRNA levels decreased significantly in FSH antiserum-treated ovaries but were restored by eCG. These results indicate that both ER subtypes are expressed in undifferentiated somatic cells and the oocytes during perinatal ovary development in the hamster; however, ERbeta expression segregates with the differentiation of granulosa cells. Furthermore, ER expression and differentiation of somatic cells to granulosa cells depend on perinatal FSH action.     

Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus

Estrogen is of great importance in the regulation of uterine function. The aim of this study was to examine the individual physiological roles of each of the two receptors for estradiol, estrogen receptor (ER) alpha and ERbeta, and their potential comodulatory effects on gene expression and uterine growth using recently developed ER subtype-selective agonist ligands. The use of ER subtype-selective ligands provides an alternative, complementary approach to the use of receptor knockout mice. Administration of the ERalpha-selective ligand propyl pyrazole triol (PPT) to immature mice resulted in a significant increase in uterine weight, as well as bromodeoxyuridine incorporation and proliferating cell nuclear antigen expression in luminal epithelial cells. PPT also increased complement component 3, lactoferrin, and glucose-6-phosphate dehydrogenase (G6PDH), and decreased androgen receptor (AR) and progesterone receptor (PR) mRNA levels in uterine tissue, as did estradiol (E(2)). However, when compared with E(2), PPT was less effective in stimulating uterine weight, complement component 3, and G6PDH expression but was as effective as E(2) in regulating lactoferrin, AR, and PR expression. In contrast to the action of the ERalpha agonist PPT, the ERbeta agonist diarylpropionitrile (DPN) did not increase uterine weight or luminal epithelial cell proliferation at a dose that reduced G6PDH and elicited a decrease in PR and AR mRNA and protein expression. Interestingly, DPN reduced the uterine weight stimulation by PPT, and enhanced the effect of PPT in decreasing uterine PR and AR mRNA. These findings with ER subtype-selective ligands indicate that ERalpha is the major regulator of estrogen function in the uterus, but that ERbeta does exert effects on some uterine markers of estrogen action. In addition, ERbeta can modulate ERalpha activity in a response-specific and dose-dependent manner.     

Differential regulation of estrogen receptor (ER)alpha and ERbeta in primate mammary gland

Estrogen, mainly estradiol (E2), and progesterone (P) are essential for the growth and differentiation of the breast, but their roles in breast cancer are highly debated. To understand how E2 and P influence cell proliferation and differentiation, it is essential to know how their receptors are regulated. Because of limited tissue availability, little is known about regulation of the two estrogen receptors (ERalpha and ERbeta) and the two progesterone receptor isoforms (PR-A and PR-B) in the normal human breast. What we know comes from rodent studies, which are not always pertinent for the human breast. We report now on regulation of gonadal hormone receptors during the menstrual cycle, pregnancy, and lactation in rhesus monkey mammary gland and on the relationship of these receptors to proliferation. We found that ERalpha but not ERbeta is down-regulated when E2 levels increase and when cells enter the cell cycle. PR-B but not PR-A is expressed in proliferating cells. Thus under normal conditions, the ratio of ERalpha to ERbeta in the breast depends on plasma concentrations of E2. Elevated expression of ERalpha (as occurs in postmenopausal women) is a normal response to loss of E2 and indicates nonproliferating cells. As selective receptor ligands become available, they will be helpful in delineation of the functions of these receptors.     

Estrogen receptor (ER) beta, a modulator of ERalpha in the uterus

Many of the effects of estrogens on the uterus are mediated by ERalpha, the predominant ER in the mature organ. Because of the poor reproductive capacity of ERbeta knockout (BERKO) female mice (small litter size, multiple-resorbed fetuses), the role of uterine ERbeta was explored. In the immature uterus, ERalpha and ERbeta are expressed at comparable levels in the epithelium and stroma, and 17beta-estradiol (E(2)) treatment decreases ERbeta in the stroma. The immature uterus of untreated BERKO mice exhibits elevated levels of progesterone receptor (PR) and the proliferation-associated protein, Ki-67. It also exhibits exaggerated responsiveness to E(2), as indicated by enlargement of the lumen, increase in volume and protein content of uterine secretion, induction of the luminal epithelial secretory protein, complement C3, and its regulatory cytokine IL-1beta, and induction of vascular endothelial growth factor and insulin-like growth factor 1 but not its receptor. As expected, E(2) increased PR in the stroma and decreased it in the luminal epithelium of wild-type mice. In the BERKO uterus, E(2) induced PR in the stroma but did not down-regulate it in the epithelium. Increased cell proliferation and exaggerated response to E(2) in BERKO suggest that ERbeta plays a role in modulation of the effects of ERalpha and in addition (or as a consequence of this) has an antiproliferative function in the immature uterus.     

In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER){alpha} and ER{beta} null mice indicate a role for ER{beta} in follicular maturation

Both estrogen receptor (ER) alpha and beta are expressed within the ovary and lack of either of these receptors affects ovarian function. In this study, the role of ERalpha and ERbeta in folliculogenesis and ovulation was further analyzed. Evaluation of ovarian follicle populations in wild-type and ERbeta knockout (betaERKO) ovaries revealed reduced late antral growth and ovulatory capacity of betaERKO follicles, indicated by reduced numbers of large antral follicles and corpora lutea and increased atresia of large antral follicles. An in vitro culture system was used to study growth, rupture, and luteinization of wild-type, ERalpha knockout (alphaERKO) and betaERKO ovarian follicles. alphaERKO follicles exhibited wild-type-like growth and ovulation rates but an increased capacity to synthesize estradiol. In contrast, betaERKO follicles showed a significant lack of progression from early antral to large antral stage, decreased estradiol production, and reduced ovulation. Expression patterns of several genes involved in follicle maturation and ovulation were analyzed in follicles grown in vitro. Ar, Pgr, and Has2 mRNA expression levels were the same among the three genotypes. However, betaERKO follicles showed reduced expression of Cyp19 mRNA during follicle maturation and reduced Lhcgr and Ptgs2 mRNA expression after human chorionic gonadotropin stimulus. Luteinization occurs normally in alphaERKO and betaERKO follicles, shown by increased progesterone secretion and increased cdkn1b mRNA expression after human chorionic gonadotropin. Collectively, these data indicate that ERbeta, but not ERalpha, plays a direct role in folliculogenesis. ERbeta appears to facilitate follicle maturation from the early antral to the preovulatory stage.