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.
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
Expression of oestrogen receptor alpha and beta mRNA in corpus luteum of human subjects.
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
Expression of estrogen receptor beta1, beta2, and beta5 messenger RNAs in human breast tissue.
A triple-primer PCR assay was developed, based on the coamplification of estrogen receptor (ER)-beta1, -beta2, and -beta5 cDNAs, to investigate the relative expressions of the corresponding mRNAs in breast cancer lines and in 53 independent breast tumors. The expression of ER-beta2 and ER-beta5 mRNAs was higher than that of ER-beta1 mRNA in both cancer cell lines and breast tumors. In breast tumors, increases in the ER-beta2:ER-beta1 and ER-beta5:ER-beta1 mRNA expression ratios were observed, which positively correlated with the level of tumor inflammation and tumor grade, respectively. A trend toward an increase of these ratios was also found in tumors, as compared to the normal adjacent breast tissue available for 13 cases. Our data suggest that changes in the relative expression of ER-beta1, -beta2, and -beta5 mRNAs occur during breast tumorigenesis and tumor progression. (+info
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. (+info
Ligand-independent recruitment of SRC-1 to estrogen receptor beta through phosphorylation of activation function AF-1.
The estrogen receptors (ERs) alpha and beta possess a constitutive N-terminal activation function (AF-1) whose activity can be modulated by kinase signalling pathways. We demonstrate here that phosphorylation of AF-1 by MAP kinase (MAPK) leads to the recruitment of steroid receptor coactivator-1 (SRC-1) by ER beta in vitro. Enhancement of the interaction between SRC-1 and ER beta AF-1 is also observed in vivo in cells either treated with EGF or expressing activated Ras. Two serine residues in ER beta AF-1, of which one is contained within a motif present in other steroid receptors, are critical for physical interaction with SRC-1 and transcriptional activation. Our results establish a role for nuclear receptor phosphorylation in the recruitment of SRC-1 and provide a molecular basis for ligand-independent activation by ER beta via the MAPK pathway. (+info
Expression of human estrogen receptor-alpha and -beta, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells.
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
Variant estrogen and progesterone receptor messages in human vascular smooth muscle.
BACKGROUND: Estrogens stimulate growth of breast or uterine cells but have the opposite effect on vascular smooth muscle cells, in which they protect against coronary artery disease with or without concomitant administration of progesterone. A possible cause of differences in hormone action is variable tissue-specific expression of hormone receptor. Therefore, we analyzed the structure of estrogen receptors (ERs) and progesterone receptors (PRs) in human vascular smooth muscle. METHODS AND RESULTS: RNA was isolated from human vascular smooth muscle, and the functional domains of ER-alpha and PR were characterized by reverse transcriptase and polymerase chain reaction. Interestingly, in addition to wild-type ER-alpha and PR, 5 variant ER-alpha and 2 variant PR transcripts were found. These variants contained precise deletions of exons encoding regions of the hormone-binding domain. The PR transcripts lacked exon 4 (PRDelta4) and exon 6 (PRDelta6). The ER-alpha transcripts were missing exon 4 (ERDelta4), exon 5 (ERDelta5), exon 6 (ERDelta6), exon 7 (ERDelta7), and exons 6 and 7, (ERDelta6,7). ER-beta variants were also detected. The PR variants were functionally characterized, and PRDelta6 was found to be a dominant-negative transcription inhibitor of wild-type receptors. Variant PR was present in premenopausal women but absent in postmenopausal women. CONCLUSIONS: Variant PR and ER transcripts are extensively expressed in human vascular smooth muscle. The complex tissue-specific effects of sex hormones may be mediated by the expression of heterogeneous forms of their cognate receptors. The presence of variant ERs and PRs may be of importance in altering the physiological effects of estrogens or progestins in vascular smooth muscle. (+info
Oestrogen receptor alpha and beta mRNA expression in human endometrium throughout the menstrual cycle.
We examined the localization of oestrogen receptor (ER) beta mRNA in the human endometrium throughout the menstrual cycle using non-radioactive in-situ hybridization with Brigati-tailed oligonucleotides. The findings were compared with those of ERalpha in order to examine the possible biological significance of ERbeta in the human endometrium. Both ERalpha and ERbeta mRNA expression were detected in all major human uterine cell types, including glandular epithelial cells, stromal cells and smooth muscle cells of the uterine wall, at every menstrual cycle stage. However, ERalpha mRNA expression was more prominent than that of ERbeta in all cell types throughout the menstrual cycle. In proliferative phase endometrium, ERalpha mRNA was expressed in both glandular epithelial and stromal cells, while ERbeta mRNA was expressed predominantly in glandular epithelial cells. Although the same pattern was observed in the secretory phase, both the ERalpha and ERbeta mRNA expression was relatively weaker. These results suggest that oestrogenic effects occur predominantly through ERalpha, but that ERbeta may also play a role in the modulation of oestrogenic action, especially on glandular epithelial cells in the human endometrium throughout the menstrual cycle. (+info