Regulation Of Estrogen Receptor Research Articles

Estrogen Receptor Activation of Phosphoinositide-3 Kinase, Akt, and Nitric Oxide Signaling in Cerebral Blood Vessels: Rapid and Long-Term Effects

Estrogen receptor regulation of nitric oxide production by vascular endothelium may involve rapid, membrane-initiated signaling pathways in addition to classic genomic mechanisms. In this study, we demonstrate using intact cerebral blood vessels that 17beta-estradiol rapidly activates endothelial nitric-oxide synthase (eNOS) via a phosphoinositide-3 (PI-3) kinase-dependent pathway. The effect is mediated by estrogen receptors (ERs), consistent with colocalization of ERalpha and caveolin-1 immunoreactivity at the plasma membrane of endothelial cells lining cerebral arteries. Treatment with 10 nM 17beta-estradiol for 30 min increased NO production, as measured by total nitrite assay, in cerebral vessels isolated from ovariectomized rats. This effect was significantly decreased by membrane cholesterol depletion with beta-methyl-cyclodextrin, the ER antagonist ICI 182,780 [fulvestrant (Faslodex)], and two inhibitors of PI-3 kinase: wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride]. In parallel with NO production, 17beta-estradiol treatment rapidly increased phosphorylation of both eNOS (p-eNOS) and Akt (p-Akt). PI-3 kinase inhibitors also blocked the latter effects; together, these data are consistent with ER activation of the PI-3 kinase-p-Akt-p-eNOS pathway. ERalpha protein (66 and 50 kDa) coimmunoprecipitated with eNOS as well as with the p85alpha regulatory subunit of PI-3 kinase, further implicating ERalpha in kinase activation of eNOS. Little is known regarding the effects of estrogen on cellular kinase pathways in vivo; therefore, we compared cerebral blood vessels isolated from ovariectomized rats that were either untreated or given estrogen replacement for 4 weeks. Long-term estrogen exposure increased levels of cerebrovascular p-Akt and p-eNOS as well as basal NO production. Thus, in addition to the rapid activation of PI-3 kinase, p-Akt, and p-eNOS, estrogen signaling via nontranscriptional, kinase mechanisms has long-term consequences for vascular function.

Strain dependence of receptor regulation on chemical preconditioning in mice hippocampus

While one current focus for studying mechanisms of disease is investigation of transgenic mice confounding effects of the background strain often are neglected. We investigated mRNA expression of known markers of hypoxic tolerance by a semiquantitative RT-PCR (adenosine receptors (A1 and A3), nitric oxide synthases (eNOS and nNOS), APP production, progesterone receptor, and estrogen receptors α and β) in CD-1, C3H, and B6 mice. We found differences in the baseline mRNA expression of adenosine A3 receptors in C3H mice and neuronal NOS in B6 mice as well as a distinct regulation of adenosine A3 receptors and estrogen receptor β (no changes in C3H and B6 compared to upregulation in CD-1) on treatment of animals with a low dosage of 3-nitropropionate (20 mg/kg body weight, i.p.). We conclude that the choice of background strain may confound interpretation of the effects of specific transgens in the study of the mechanisms of primary and induced hypoxic tolerance.

Role of estrogen receptor in regulation of polycyclic aromatic hydrocarbon metabolic activation in lung

Epidemiological and biochemical studies have indicated that females may be at greater risk of smoking associated lung cancer compared with males. Among lung cancer patients, female smokers have been found to have higher levels of PAH-related DNA adducts and CYP1A1 gene expression in their normal lung tissue compared to male smokers. A possible role of steroid hormones in these sex differences via interactions between aryl hydrocarbon receptor and estrogen receptor mediated cellular effects has been suggested. In the present study the impact of the estrogen receptor (ERα) on CYP1A1 and CYP1B1 gene expression was studied in vitro in human bronchial epithelial cells. Transient transfection of the BEP2D cell line with ERα influenced neither constitutive expression of CYP1A1 or CYP1B1 nor induction of these genes by TCDD as measured by real-time RT-PCR. ERα had no effect on the constitutive or TCDD-induced enzymatic activity of CYP1A1 (EROD). We also studied the effect of steroid hormones on lung PAH metabolic activation in A/J mice. Intact and ovariectomized female mice were orally exposed to a single dose of benzo[ a]pyrene. Ovariectomy did not influence the levels of either benzo[ a]pyrene-derived protein or DNA adducts in the lung tissue measured by HPLC and 32P-postlabeling, respectively. In conclusion, the present data do not support the hypothesis of a role of estrogen or the ERα in regulating the metabolic activation of polycyclic aromatic hydrocarbons in lung.

Differential regulation of estrogen receptor subtypes alpha and beta in human aortic smooth muscle cells by oligonucleotides and estradiol.

We investigated the mechanisms regulating estrogen receptor (ER) expression in human aortic smooth muscle cells (HASMCs) and the mechanisms by which estradiol inhibits HASMC growth. The autologous down-regulation pathway involves binding of liganded ER to the ER gene, thus suppressing transcription. Blockade of this pathway with sense and AS-OLIGOs to ERs up-regulated the expression of ERalpha but not ERbeta. Activation of the autologous down-regulation pathway with ER agonists down-regulated the expression of ERalpha but not ERbeta. The proteasomal degradation pathway entails ubiquination of liganded ER, followed by proteasome-mediated degradation. Blockade of the proteasomal degradation pathway increased the expression of ERbeta. Up-regulation of ERalpha by AS-OLIGOs did not increase the antimitogenic effects of estradiol on HASMCs; the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent inhibitors of HASMC growth, compared with estradiol; and blockade of metabolism of estradiol to hydroxyestradiols and methoxyestradiols abrogated the inhibitory effects of estradiol on HASMC growth. We conclude that, in HASMCs: 1) the expression of ERalpha is regulated by the autologous downregulation pathway; 2) the expression of ERbeta is governed by the proteasomal degradation pathway; and 3) the antigrowth effects of estradiol are not mediated by ERalpha, but rather by metabolism of estradiol to methoxyestradiols.

Regulation of the Estrogen Receptor α Minimal Promoter by Sp1, USF-1 and ERα

The exact molecular mechanisms regulating estrogen receptor alpha (ERalpha) expression in breast tumors are unclear, but studies suggest that they are partly at the level of transcription. We have focused on the transcription factors that regulate the ERalpha minimal promoter, which we have previously shown to reside within the first 245 bp of the 5'-flanking region of the gene. Within this region are several elements essential for full ERalpha promoter transcriptional activity, including a GC box and an imperfect E box. In earlier studies we demonstrated an essential function for the Sp1 family of transcription factors in the regulation of ERalpha expression. We have now identified both USF-1 and ERalpha itself as components of a multi-protein complex of transcription factors that interacts at the ERalpha minimal promoter and is essential for its full transcriptional activity. Electrophoretic mobility shift assays demonstrated that Sp1 and USF-1, but not ERalpha, bind directly to the ERalpha minimal promoter. We showed by GST pull-down assays that ERalpha is able to interact in vitro with USF-1, suggesting, in addition to a possible interaction between ERalpha and Sp1, a mechanism whereby ERalpha is able to interact with the protein complex. Combined exogenous expression of the components of the complex in MCF-7 breast cancer cells resulted in a synergistic effect on transactivation of the ERalpha minimal promoter, suggesting that the importance of the protein complex is in the interactions among the components. Based upon these findings, we propose a possible model for transcription from the ERalpha minimal promoter.

Drug targeting of estrogen receptor signaling in the cardiovascular system: preclinical and clinical studies.

Atherosclerosis and associated coronary heart disease events have lower prevalence in women than in men, especially during young adult years. Although multiple lines of evidence suggest that estrogens contribute to this difference, the efficacy of hormone replacement therapy for the prevention of cardiovascular disease in postmenopausal women is controversial. The protective action of estrogen in the cardiovascular system appears to be mediated indirectly by an effect on serum lipoprotein and triglyceride profiles and on the expression of coagulant and fibrinolytic proteins, and by a direct effect on the vessel wall itself. Estrogen has both rapid effects involving alteration of membrane ionic permeability and activation of membrane-bound enzymes and increases in endothelial cell nitric oxide synthase activity, as well as longer-term effects on gene expression that are mediated, at least in part, by the ligand-activated transcription factors, estrogen receptor alpha and beta. Compounds with pure antiestrogenic activity and selective estrogen receptor modulators that regulate estrogen receptor function in a tissue-specific manner have been developed in an attempt to achieve the cardioprotective effects of estrogens while minimizing the undesirable risks associated with hormone replacement therapy (e.g., endometrial and breast cancer). In this review, we will discuss recent developments on the mechanisms of estrogen action in the cardiovascular system. The results of clinical trials testing the long-term efficacy of hormone replacement therapy for the treatment of cardiovascular disease will also be discussed.

Sexually dimorphic regulation of estrogen receptor α mRNA in the ventromedial hypothalamus of adult whiptail lizards is testosterone dependent

Female, but not male, whiptail lizards will respond to exogenous estrogen with an increase in estrogen receptor α (ERα) mRNA in the ventromedial hypothalamus (VMH). The current study tested whether differences in long-term testosterone exposure may be responsible for this sex difference. In the first experiment, female whiptails were gonadectomized and implanted for 6 weeks with either a Silastic capsule containing testosterone or an empty capsule after which the implant was removed. In a second experiment, male whiptail lizards were castrated for 1 week or for 6 weeks. In both experiments animals were then injected with either estradiol benzoate or steroid suspension vehicle and the brains collected 24 h later. Brain mRNA expression was assayed using in situ hybridization. Chronic testosterone treatment reduced estrogen-induced ERα mRNA levels (number of grains per cell) in the VMH in females. In contrast, there were no significant effects of time after castration or estrogen treatment on induction of ERα mRNA in the VMH in males. The abundance of estrogen-induced ERα mRNA in the VMH, therefore, appears to be responsive to testosterone environment in the female but not in the male, suggesting a difference in this species between the sexes in the mechanism of steroid receptor regulation.

Regulation of hepatic progesterone and estrogen receptors in the female turtle, Chrysemys picta: relationship to vitellogenesis

Previous studies using the fresh water turtle Chrysemys picta have demonstrated the differential expression of the two progesterone receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with ER mRNA levels and hepatic vitellogenesis. During the inter-vitellogenic periods the ratio of PRB:PRA favors PRA, suggesting that the PRB:PRA ratio may be important in the regulation of vitellogenesis. Based on these and other studies we hypothesize that progesterone may have differential effects on the estrogen (E)-induced vitellogenin response (inhibitory or stimulatory), depending on the PRB:PRA ratio. In this study, we determined the expression pattern and the hormonal regulation of progesterone receptor (PR) isoforms in the liver, during the estrogen-induced vitellogenin response in female C. picta. Northern blot analysis was used to evaluate the changes in vitellogenin mRNA, estrogen receptor (ER) mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vitellogenin. The expression of PR isoforms in the liver of female turtles at the mRNA and protein levels was differentially regulated by estradiol and progesterone. Estradiol treatment enhanced the transcription of PR mRNA isoforms and possibly translation and/or increased stability of PRB protein in the female turtle liver. In contrast, this hormone decreased PRA protein levels. Progesterone alone down-regulated progesterone receptor isoform A (PRA) and progesterone receptor isoform B (PRB) proteins equally, but did not affect PR mRNA levels. Estradiol markedly increased hepatic ER mRNA, vtg mRNA, and vtg in plasma, but this effect was not impacted significantly by progesterone. The results presented here indicate that in female turtles hepatic PRB:PRA ratios are markedly changed by estradiol treatment due primarily to a decrease in PRA. The change in the PRB:PRA ratio after hormonal treatment confirms that progesterone and estrogen exposure will be a determinant in the regulation of vitellogenesis, and, in turn, that the regulation of vitellogenesis will be determined by the ratio of PR isoforms and the physiological levels of steroid hormones.

Hormonal regulation and localization of estrogen, progestin and androgen receptors in the endometrium of nonhuman primates: effects of progesterone receptor antagonists

This article reviews the effects of estradiol (E(2)), progesterone (P) and P receptor antagonists (PA) on the rhesus macaque endometrium. Ovariectomized macaques can be treated with implants of estradiol (E(2)) and P to induce precisely controlled, artificial menstrual cycles. During these cycles, treatment with E(2) alone induces an artificial proliferative phase marked by extensive endometrial epithelial cell proliferation and increased expression of stromal and epithelial estrogen receptor (ER) and P receptor (PR). Androgen receptor (AR) is also upregulated by E(2) but is expressed only by the endometrial stroma. Progesterone acts on the E(2) primed endometrium to induce secretory differentiation and causes suppression of epithelial and stromal ER, epithelial PR, and stromal AR in the functionalis zone. However, epithelial ER and PR are retained in the basalis zone during the secretory phase. When potent P antagonists (PA) are administered acutely at the end of an E(2) + P induced cycle, menses typically ensues similar to P withdrawal at the end of the menstrual cycle. When PAs are administered chronically there is significant blockage of all P- dependent effects including upregulation of ER, PR and AR and suppression of glandular secretory function. However, chronic PA administration also inhibits estrogen-dependent endometrial cell proliferation and growth. This endometrial antiproliferative effect is the basis of the clinical use of PA to control various diseases such as endometriosis.

Malignant mammary cells acquire independence from extracellular context for regulation of estrogen receptor alpha.

Interactions between luminal epithelial cells and their surrounding microenvironment govern the normal development and function of the mammary gland. Alterations of these interactions can induce abnormal intracellular signaling pathways that affect the development and progression of breast tumors. One critical component of mammary gland development, as well as breast cancer progression, is the expression of estrogen receptors. In a previous study using cultured nonmalignant mammary epithelial cells, we found that the basement membrane molecules, laminin-1 and collagen-IV, were involved in maintenance of estrogen receptor (ER) alpha expression, and that this response could be interfered with by disrupting cell-extracellular matrix adhesion. Here we use phenotypically normal mammary epithelial SCp2 cells to dissect the promoter region of the ERalpha that is involved in the selective response to basement membrane. We also analyze the alteration of this response in SCg6 cells, a malignant cell line that shares a common lineage with the SCp2 cells, to provide insight into the relative overexpression of ERalpha and the unresponsiveness to basement membrane regulation found in those malignant cells. Evidence is presented to show the relevance of the cross-talk between different signaling pathways in the constitution of a functional tissue organization and how this integration may be disrupted in the malignant phenotype.

Effects of estradiol on liver estrogen receptor-alpha and its mRNA expression in hepatic fibrosis in rats.

Estradiol treatment regulates estrogen receptor (ER) level in normal rat liver. However, little information is available concerning the role of estrogen in regulating liver ER in hepatic fibrosis in rats. The present study was conducted to determine whether estradiol treatment in CCl4-induced liver fibrosis of female and ovariectomized rats altered liver ERalpha and its mRNA expression, and to investigate the possible mechanisms. Seventy female rats were divided into seven groups with ten rats in each. The ovariectomy groups were initiated with ovariectomies and the sham operation groups were initiated with just sham operations. The CCl4 toxic fibrosis groups received 400 mL/L CCl4 subcutaneously at a dose of 2 mL/kg twice weekly. Estrogen groups were treated subcutaneously with estradiol 1 mg/kg, the normal control group and an ovariectomy group received injection of peanut oil vehicle twice weekly. At the end of 8 weeks, all the rats were killed to detect their serum and hepatic indicators, their hepatic collagen content, and liver ER and ER mRNA expression. Estradiol treatment in both ovariectomy and sham ovariectomy groups reduced liver levels of ALT (from 658 +/- 220 nkat/L to 311 +/- 146 nkat/L and 540 +/- 252 nkat/L to 314 +/- 163 nkat/L, P<0.05) and AST (from 697 +/- 240 nkat/L to 321 +/- 121 nkat/L and 631 +/- 268 nkat/L to 302 +/- 153 nkat/L, P<0.05), increased serum nitric oxide (NO) level (from 53.7 +/- 17.1 micromol/L to 93.3 +/- 24.2 micromol/L and 55.3 +/- 23.1 micromol/L to 87.5 +/- 23.6 micromol/L, P<0.05) and hepatic nitric oxide synthase (NOS) activity (from 1.73 +/- 0.71 KU/g to 2.49 +/- 1.20 KU/g and 1.65 +/- 0.46 KU/g to 2.68 +/- 1.17 KU/g, P<0.05), diminished the accumulation of hepatic collagen, decreased centrolobular necrotic areas as well as the inflammatory reaction in rats subjected to CCl4. The positive signal of ER and ER mRNA distributed in parenchymal and non-parenchymal hepatic cells, especially near the hepatic centrolobular and periportal areas. Ovariectomy decreased ER level (from 10.2 +/- 3.2 to 4.3 +/- 1.3) and ER mRNA expression (from 12.8 +/- 2.1 to 10.9 +/- 1.3) significantly (P<0.05). Hepatic ER and ER mRNA concentrations were elevated after treatment with estradiol in both ovariectomy (15.8 +/- 2.4, 20.8 +/- 3.1) and sham ovariectomy (18.7 +/- 3.8, 23.1 +/- 3.7) fibrotic groups (P<0.05). The increase in hepatic ER and mRNA expression may be part of the molecular mechanisms underlying the suppressive effect of estradiol on liver fibrosis induced by CCl4 administration.

Regulation of estrogen receptor (ER) isoform messenger RNA expression by different ER ligands in female rat pituitary.

Net estrogen sensitivity in target tissues critically depends on the regulated expression of full-length and alternately processed estrogen receptor (ER) isoforms. However, the molecular mechanisms for the control of pituitary responsiveness to estrogen remain partially unknown. In the present communication, we report the ability of different ligands, with distinct agonistic or antagonistic properties at the ER, to modulate the expression of the transcripts encoding ERalpha and ERbeta isoforms, as well as those for the truncated ERalpha product (TERP), and the variant ERbeta2, in pituitaries from ovariectomized rats, i.e., a background devoid of endogenous estrogen. Compared with expression levels at the morning of proestrus, ovariectomy (OVX) resulted in increased pituitary expression of ERbeta and ERbeta2 mRNAs, whereas it decreased TERP-1 and -2 levels without affecting those of ERalpha. Administration of estradiol benzoate (as potent agonist for alpha and beta forms of ER) or the selective ERalpha agonist, propyl pyrazole triol, fully reversed the responses to OVX, while the ERbeta ligand, diarylpropionitrile, failed to induce any significant effect except for a partial stimulation of TERP-1 and -2 mRNA expression levels. To note, the ERbeta agonist was also ineffective in altering pituitary expression of progesterone receptor-B mRNA, i.e., a major estrogen-responsive target. In all parameters tested, tamoxifen, a selective ER modulator with mixed agonist/antagonist activity, behaved as ERalpha agonist, although the magnitude of tamoxifen effects was significantly lower than those of the ERalpha ligand, except for TERP induction. In contrast, the pure antiestrogen RU-58668 did not modify the expression of any of the targets under analysis. Overall, our results indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, by a mechanism that is mostly conducted through ERalpha. Differential regulation of ER isoforms may represent a relevant system for the self-tuning of estrogen responsiveness in female pituitary.

Differential colocalization of Islet-1 and estrogen receptor alpha in the murine preoptic area and hypothalamus during development.

Estrogen receptor (ER) expression and regulation is vital to the correct functioning of the neuroendocrine brain. Islet-1 (Isl-1) is a LIM homeodomain-containing transcription factor that has been implicated in neuronal differentiation, is located in the hypothalamus, and can alter ER function in vitro. We have determined that Isl-1 is localized in several regions of the hypothalamus, including the ER rich areas of the ventromedial nucleus (VMH), the preoptic area, and the anterior hypothalamus. Using double-label immunocytochemistry, we examined the overlap between immunoreactive ERalpha and Isl-1 in these different hypothalamic brain regions. In the developing brain, almost 100% of VMH cells that contain immunoreactive ERalpha also contain Isl-1. However, in older animals, the percentage of double-label cells decreased below 70%. This change is due to a decrease in the number of cells containing Isl-1, because there was no difference in the number of ERalpha-containing cells. By contrast, in more anterior regions of the hypothalamus, cells containing both Isl-1 and ERalpha were less common, with the two populations adjacent to each other, rather than overlapping. These data suggest that, although Isl-1 and ERalpha can interact, they are not always found in the same cells and that regulation of ERalpha function is not under the same control in the VMH, preoptic area, and the anterior hypothalamus.

Overexpression of histone deacetylase HDAC1 modulates breast cancer progression by negative regulation of estrogen receptor alpha.

The interaction between 17beta-estradiol and estrogen receptor alpha (ER-alpha) plays an important role in breast carcinogenesis and breast cancer treatment. ER-alpha is a critical growth regulatory gene in breast cancer and its expression level is tightly linked to the prognosis and treatment outcomes of breast cancer patients. Loss of ER-alpha expression in breast epithelial cells is critical for breast cancer progression. The underlying molecular mechanisms for this loss, however, are poorly defined. Histone deacetylases (HDACs) are implicated in the alteration of chromatin assembly and tumorigenesis. We show that histone deacetylase 1 (HDAC1) interacts with ER-alpha in vitro and in vivo and suppresses ER-alpha transcription activity. The interaction of HDAC1 with ER-alpha was mediated by the AF-2 and DBD domains of ER-alpha. We observed an endogenous interaction of HDAC1 with ER-alpha in breast cancer cells, which was decreased in the presence of estrogen. Interestingly, overexpression of HDAC1 in stable transfected MCF-7 clones induced loss of ER-alpha and significantly increased cell proliferation and colony formation, as compared to the control MCF-7 cells, whereas treatment of stable MCF-7 clones with the HDAC specific inhibitor trichostatin A (TSA) induced re-expression of ER-alpha mRNA and protein. Our findings strongly suggest that HDAC1 affects breast cancer progression by promoting cellular proliferation in association with a reduction in both ER-alpha protein expression and transcriptional activity. Thus, HDAC1 may be a potential target for therapeutic intervention in the treatment of a subset of ER-negative breast cancers.

Modulation of ovarian follicle maturation in Long–Evans rats exposed to polychlorinated biphenyls (PCBs) in-utero and lactationally

Polychlorinated biphenyls (PCBs) are ubiquitous man-made toxicants capable of endocrine disruption. Studies in several species have shown that exposure to PCBs and their hydroxylated metabolites reduces fecundity and decreases circulating concentrations of thyroid hormones, causing serious reproductive and developmental defects. Thyroid hormones modulate both follicular development and steroidogenesis, and affect estrogen metabolism and the regulation of estrogen receptor. This study was designed (1) to determine whether exposure to a commercially prepared PCB mixture (Aroclor 1016) exerts detrimental effects on follicle maturation in the Long–Evans hooded rat; and (2) to determine whether the modulatory effects of Aroclor can be attenuated by levo-thyroxine sodium (T 4) supplementation. Animals were treated on gestation days 7–13 with a single daily intraperitoneal injection (2.5 mg/kg per day) of Aroclor. Half of the Aroclor-treated dams were also given T 4 supplements (2.89 μg/kg per day) via drinking water. Female pups were sacrificed on postnatal days 24/25, and the ovaries were excised, fixed for histology and analyzed. The analysis included a count, measurement and classification of healthy and atretic preantral and antral follicles in the greatest cross-sectional area. The results indicated that treatment with Aroclor significantly reduced the number of preantral follicles <50,000 μm 2 and the total number of antral follicles in the 50–100,000 and >100,000 μm 2 size classes. T 4 circumvented the Aroclor effect on the number of preantral follicles <50,000 μm 2; however, a significant reduction in the antral follicle number persisted in the 50–100,000 and >100,000 μm 2 size classes. In addition, we observed a significant increase in atresia in the Aroclor-treated ovaries in the antral <50,000 μm 2 size class, which was not present in ovaries exposed to both Aroclor and T 4. These data support the hypothesis that Aroclor reduces the number of preantral and antral follicles of certain size classes in rats exposed during the critical period of development, and that supplementation with T 4 can attenuate the effects of Aroclor on small, but not medium or large antral follicles. Atresia of small, antral follicles may constitute one of the underlying mechanisms by which folliculogenesis is modulated by Aroclor 1016.

Transcription profiling of estrogen target genes in young and old mouse uterus

The goal of this study was to identify age-related changes in the expression of estrogen target genes in mouse uterus. We developed a novel ‘estrogen response element (ERE) Chip’ microarray bearing 297 genes including both known estrogen target genes and genes identified by searching the mouse genome database to have EREs, AP-1 sites, and Sp1 sites, all targets of estrogen receptor (ER) regulation. 400–500 bp PCR products of these 297 genes were printed onto nylon membranes creating the ‘ERE Chip’ microarray. This microarray is unique because it is the first estrogen-responsive gene-specific microarray to identify changes in uterine gene expression in young versus old mice. Using this ERE microarray we identified 10 uterine genes whose expression was up-regulated in old mice, e.g. β-actin, calcium binding protein 45a, Sp1, and COUP-TFII. In contrast, the expression of only 4 uterine genes, i.e., complement C3, lactoferrin, Muc-1, and 17-beta-hydroxysteroid dehydrogenase 8 (H2-Ke6) was down-regulated in old mice. These changes may reflect an increase in stromal and a decrease in glandular epithelial gene expression, and may be associated with age-related changes in these tissue compartments within the uterus, possibly leading to the decline in reproductive function in C57Bl/6 mice.

Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells.

The expression level and functional activity of estrogen receptor alpha is an important determinant of breast physiology and breast cancer treatment. However, it has been difficult to identify the signals that regulate estrogen receptor because cultured mammary epithelial cells generally do not respond to estrogenic signals. Here, we use a combination of two- and three-dimensional culture systems to dissect the extracellular signals that control endogenous estrogen receptor alpha. Its expression was greatly reduced when primary mammary epithelial cells were placed on tissue culture plastic; however, the presence of a reconstituted basement membrane in combination with lactogenic hormones partially prevented this decrease. Estrogen receptor alpha expression in primary mammary fibroblasts was not altered by these culture conditions, indicating that its regulation is cell type specific. Moreover, estrogen receptor-dependent reporter gene expression, as well as estrogen receptor alpha levels, were increased threefold in a functionally normal mammary epithelial cell line when reconstituted basement membrane was added to the medium. This regulatory effect of reconstituted basement membrane was reproduced by two of its components, collagen-IV and laminin-1, and it was blocked by antibodies against alpha2, alpha6 and beta1 integrin subunits. Our results indicate that integrin-mediated response to specific basement membrane components, rather than cell rounding or cell growth arrest induced by reconstituted basement membrane, is critical in the regulation of estrogen receptor alpha expression and function in mammary epithelial cells.

Effect of estrogen and antiestrogens on the estrogen receptor content in the cochlea of ovariectomized rats

Older women in the normal population tend to develop less severe hearing loss as compared to males in the same age. In Turner syndrome (45,X), estrogen deficiency is one of the predominant problems. Ear and hearing problems are common among these patients. Does estrogen have an impact on the hearing organ? Twenty-four rats were ovariectomized and treated with vehicle (controls), estradiol or selective estrogen receptor modulators such as tamoxifen and ICI182780, in order to study the effects on the estrogen receptor levels and distribution in the inner ear. The cochleas were stained immunohistochemically using antibodies against estrogen receptor α and β. No major difference in estrogen receptor content in the cochleas was observed among groups. There was however a potential down regulation of estrogen receptor α in the marginal cells of stria vascularis in the rats that were substituted with ICI182780 (pure antiestrogen) as compared to those given estradiol or tamoxifen. When investigating the tissues with light microscopy no change in inner ear anatomy could be observed.

Differential regulation of proteasome-dependent estrogen receptor alpha and beta turnover in cultured human uterine artery endothelial cells.

Estrogen-induced loss of estrogen receptor (ER) alpha expression limits estrogen responsiveness in many target cells. However, whether such a mechanism contributes to changes in vascular endothelial ER alpha and/or ER beta levels is unclear. Using RT-PCR assays, we did not find any regulation of ER alpha or ER beta mRNA expression in human uterine artery endothelial cell (HUAEC) nuclear extracts on stimulation with 17 beta-estradiol for 1 or 2 h. By contrast, Western analysis on HUAEC extracts revealed that 17 beta-estradiol was capable of down-regulating both ER alpha and ER beta protein starting 1 h after treatment, an effect that can be blocked by pretreatment with tamoxifen as well as with the proteasome inhibitor lactacystin. The proteolysis inhibitors insulin, cycloheximide, and puromycin impede ER alpha, but not ER beta, turnover. Ubiquitin, but not its competitive inhibitor methyl-ubiquitin, induces rapid turnover of both ERs in a cell-free system of MCF-7 and HUAEC extracts. We, thus, propose the existence of estrogen-induced ER degradation that serves to control physiological responses in an estrogen target tissue, i.e. human vascular endothelium, by down- regulating ER alpha as well as ER beta through different proteasomal uptake mechanisms.

Expression and localization of estrogen receptor α, estrogen receptor β and progesterone receptor in the bovine oviduct in vivo and in vitro

This study examined the regulation and localization of estrogen receptors α and β (ERα, ERβ) and progesterone receptor (PR) in the bovine oviduct. Oviduct epithelial cells from cycling cows (in vivo) were investigated. In addition, the reactivity of a cell suspension culture stimulated with physiological doses of estradiol-17β (E2) or progesterone (P4) was tested (in vitro). The specific steroid receptor expression of oviductal cells was quantified for mRNA using real-time RT-PCR. Furthermore, steroid receptor proteins were analyzed by Western blotting and localized by immunohistochemistry in situ. Obvious cyclic changes of receptor expression in vivo were observed and concurrent expression patterns were detected in vitro. PR and ERα mRNA transcripts were elevated in vivo during the follicular phase. The highest PR and ERα protein expression was detected subsequently during the early-luteal phase. In vitro, E2-supplementation resulted in an upregulation of PR and ERα. Both ERβ mRNA and protein expression were highest during the luteal phase in vivo and elevated ERβ expression levels were observed in vitro after P4 treatment. Evidence is provided for a varying expression of ERα, ERβ and PR in bovine oviducts at different cycle stages in vivo, respectively under steroid supplementation in vitro. The region specific and cycle dependent expression differences point towards a functional importance of the three steroid receptors in the bovine oviduct, the site of fertilization and early embryonic development.