Dominant activity of activation function 1 (AF-1) and differential stoichiometric requirements for AF-1 and -2 in the estrogen receptor alpha-beta heterodimeric complex.
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Estrogenic responses are now known to be mediated by two forms of estrogen receptors (ER), ERalpha and ERbeta, that can function as homodimers or heterodimers. As homodimers the two have been recently shown to exhibit distinct transcriptional responses to estradiol (E2), antiestrogens, and coactivators, suggesting that the ER complexes are not functionally equivalent. However, because the three possible configurations of ER complexes all recognize the same estrogen response element, it has not been possible to evaluate the transcriptional properties of the ER heterodimer complex by transfection assays. Using ER subunits with modified DNA recognition specificity, we were able to measure the transcriptional properties of ERalpha-ERbeta heterodimers in transfected cells without interference from the two ER homodimer complexes. We first demonstrated that the individual activation function 1 (AF-1) domains act in a dominant manner within the ERalpha-ERbeta heterodimer: the mixed agonist-antagonist 4-hydroxytamoxifen acts as an agonist in a promoter- and cell context-dependent manner via the ERalpha AF-1, while activation of the complex by the mitogen-activated protein kinase (MAPK) pathway requires only the ERalpha- or ERbeta-responsive MAPK site. Using ligand-binding and AF-2-defective mutants, we further demonstrated that while the ERalpha-ERbeta heterodimer can be activated when only one E2-binding competent partner is present per dimer, two functional AF-2 domains are required for transcriptional activity. Taken together, the results of this study of a retinoid X receptor-independent heterodimer complex, the first such study, provide evidence of different stoichiometric requirements for AF-1 and -2 activity and demonstrate that AF-1 receptor-specific properties are maintained within the ERalpha-ERbeta heterodimer. (+info)
Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene.
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We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes. (+info)
Expression of oestrogen receptor alpha and beta mRNA in corpus luteum of human subjects.
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To investigate the role of oestrogen receptor beta (ERbeta) in the function of human ovarian corpus luteum, the levels of luteal ERalpha and ERbeta mRNA were determined using competitive reverse transcription-polymerase chain reaction-Southern blot analysis. The expression of ERalpha and ERbeta mRNA was detected in all luteal samples analysed. Luteal ERalpha and ERbeta mRNA levels were significantly lower (P<0.01 and P<0.05 respectively) at the late secretory phase than those at the early and mid-secretory phases of the endometrium. The ratio of ERalpha to ERbeta mRNA levels showed no change during the secretory phase of the endometrium. This study demonstrates that ERbeta is co-expressed with ERalpha in human corpus luteum and is likely to play a biological role in the regulation of steroidal action of the corpus luteum with ERalpha. (+info)
Oestrogen receptor-alpha variant mRNA expression in primary human breast tumours and matched lymph node metastases.
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We have shown previously that the relative expression of a truncated oestrogen receptor-alpha variant mRNA (ER clone 4) is significantly increased in axillary node-positive primary breast tumours compared with node-negative tumours. In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-alpha variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-alpha variant mRNAs appeared conserved between any primary and its matched secondary tumour. (+info)
Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor alpha knockout mouse uterus.
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Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor alpha knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor alpha modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus. (+info)
Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta.
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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. (+info)
A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland hyperplasia and tumorigenesis in mice lacking estrogen receptor-alpha.
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Estrogens have important functions in mammary gland development and carcinogenesis. To better define these roles, we have used two previously characterized lines of genetically altered mice: estrogen receptor-alpha (ER alpha) knockout (ERKO) mice, which lack the gene encoding ER alpha, and mouse mammary virus tumor (MMTV)-Wnt-1 transgenic mice (Wnt-1 TG), which develop mammary hyperplasia and neoplasia due to ectopic production of the Wnt-1 secretory glycoprotein. We have crossed these lines to ascertain the effects of ER alpha deficiency on mammary gland development and carcinogenesis in mice expressing the Wnt-1 transgene. Introduction of the Wnt-1 transgene into the ERKO background stimulates proliferation of alveolar-like epithelium, indicating that Wnt-1 protein can promote mitogenesis in the absence of an ER alpha-mediated response. The hyperplastic glandular tissue remains confined to the nipple region, implying that the requirement for ER alpha in ductal expansion is not overcome by ectopic Wnt-1. Tumors were detected in virgin ERKO females expressing the Wnt-1 transgene at an average age (48 weeks) that is twice that seen in virgin Wnt-1 TG mice (24 weeks) competent to produce ER alpha. Prepubertal ovariectomy of Wnt-1 TG mice also extended tumor latency to 42 weeks. However, pregnancy did not appear to accelerate the appearance of tumors in Wnt-1 TG mice, and tumor growth rates were not measurably affected by late ovariectomy. Small hyperplastic mammary glands were observed in Wnt-1 TG males, regardless of ER alpha gene status; the glands were similar in appearance to those found in ERKO/Wnt-1 TG females. Mammary tumors also occurred in Wnt-1 TG males; latency tended to be longer in the heterozygous ER alpha and ERKO males (86 to 100 weeks) than in wild-type ER alpha mice (ca. 75 weeks). We conclude that ectopic expression of the Wnt-1 proto-oncogene can induce mammary hyperplasia and tumorigenesis in the absence of ER alpha in female and male mice. The delayed time of tumor appearance may depend on the number of cells at risk of secondary events in the hyperplastic glands, on the carcinogenesis-promoting effects of ER alpha signaling, or on both. (+info)
Expression of human estrogen receptor-alpha and -beta, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells.
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Our understanding of the roles played by sex hormones in ovarian carcinogenesis has been limited by a lack of data concerning the mode of sex hormone action in human ovarian surface epithelial (HOSE) cells, the tissue of origin of >90% of ovarian cancers. We have compared the relative abundance of estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), and androgen receptor (AR) mRNA in four primary cultures of HOSE cells obtained from postmenopausal women to those found in late serous adenocarcinoma primary cell cultures and established ovarian cancer cell lines. We observed coexpression of ERalpha and ERbeta mRNA along with AR and PR transcripts in normal HOSE cells and disruption of ERalpha mRNA expression as well as dramatic down-regulation of PR and AR transcript expression in most ovarian cancer cells. In contrast, levels of ERbeta mRNA were unaffected by the malignant state. Additionally, a novel mutation involving a 32-bp deletion in exon 1 of ERalpha transcripts was detected in the SKOV3 cell line. This mutation would explain why SKOV3 was reported to be ER-positive but estrogen-insensitive. Taken together, these findings suggest that estrogens, signaling via either or both ER subtypes, may play an indispensable role in regulating normal HOSE cell functions. Therefore, loss of ERalpha, PR, and AR mRNA expression in HOSE cells may be responsible for neoplastic transformation in this cell type. In contrast, the roles played by ERbeta in normal and malignant HOSE cells remain elusive. Finally, the coexistence of mutated ERalpha mRNA and normal ERbeta transcripts in SKOV3 argues in favor of a dependency of ERbeta action on functional ERalphas. (+info)