Molecular Mechanisms Of Estrogen Action Research Articles

Molecular mechanisms of estrogen action in female genital tract development

The development of the female reproductive tract can be divided into three parts consisting of Müllerian duct organogenesis, pre-sexual maturation organ development, and post-sexual maturation hormonal regulation. In primates, Müllerian duct organogenesis proceeds in an estrogen independent fashion based on transcriptional pathways that are suppressed in males by the presence of AMH and SRY. However, clinical experience indicates that exposure to xenoestrogens such as diethylstilbestrol (DES) during critical periods including late organogenesis and pre-sexual maturational development can have substantial effects on uterine morphology, and confer increased risk of disease states later in life. Recent evidence has demonstrated that these effects are in part due to epigenetic regulation of gene expression, both in the form of aberrant CpG methylation, and accompanying histone modifications. While xenoestrogens and selective estrogen receptor modulators (SERMS) both can induce non-canonical binding confirmations in estrogen receptors, the primate specific fetal estrogens Estriol and Estetrol may act in a similar fashion to alter gene expression through tissue specific epigenetic modulation.

Exogenous Hormonal Regulation in Breast Cancer Cells by Phytoestrogens and Endocrine Disruptors

Observations on the role of ovarian hormones in breast cancer growth, as well as interest in contraception, stimulated research into the biology of estrogens. The identification of the classical receptors ERα and ERβ and the transmembrane receptor GPER and the resolution of the structure of the ligand bound to its receptor established the principal molecular mechanisms of estrogen action. The presence of estrogen-like compounds in many plants used in traditional medicine or ingested as food ingredients, phytoestrogens, as well as the estrogenic activities of many industrial pollutants and pesticides, xenoestrogens, have prompted investigations into their role in human health. Phyto- and xenoestrogens bind to the estrogen receptors with a lower affinity than the endogenous estrogens and can compete or substitute the hormone. Xenoestrogens, which accumulate in the body throughout life, are believed to increase breast cancer risk, especially in cases of prenatal and prepuberal exposure whereas the role of phytoestrogens is still a matter of debate. At present, the application of phytoestrogens appears to be limited to the treatment of post-menopausal symptoms in women where the production of endogenous estrogens has ceased. In this review we discuss chemistry, structure and classification, estrogen signaling and the consequences of the interactions of estrogens, phytoestrogens and xenoestrogens with their receptors, the complex interactions of endogenous and exogenous ligands, the evaluation of the health risks related to xenoestrogens, and the perspectives toward the synthesis of potent third generation selective estrogen receptor modulators (SERMs).

Molecular cloning, expression, and regulation of estrogen receptors in pigeon oviduct epithelial cells

Estrogen regulates reproductive behavior and drives the proliferation and differentiation of several cell types. These physiological functions of estrogen are mediated by estrogen receptors (ERs), and each ER isoform plays a distinct role. To clarify the molecular mechanism of estrogen action and to evaluate the effect of ERs on the secretion of ovalbumin (OVA) in pigeon oviduct epithelial cells (POECs), we determined the complete coding sequences encoding ER alpha (ERα) and ER beta (ERβ) in pigeons. The abundance of pigeon ERα and ERβ mRNA was detected using quantitative polymerase chain reaction. These results revealed that pigeon ERα is highly expressed in the oviduct, while pigeon ERb is highly expressed in the ovary and kidney. We hypothesize that ERα mRNA predominates over that of ERβ in the oviduct. The expression of ERα can be down-regulated by 17β-estradiol, and the knockdown of ERα promoted OVA mRNA expression in cultured POECs, indicating that ERα may play an important role in OVA secretion.

ERβ-dependent effects on uterine endothelial cells are cell specific and mediated via Sp1

STUDY QUESTIONWhat are the in vitro effects of estrogen receptor β (ERβ) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)?SUMMARY ANSWERStudies conducted in vitro demonstrate that the ERβ agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ERβ-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus.WHAT IS KNOWN ALREADYEstrogens, acting via ERs (ERα and ERβ), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ERβ.STUDY DESIGN, SIZE, DURATIONCells were treated with a vehicle control or the ERβ agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 5).PARTICIPANT/MATERIALS, SETTING, METHODSAnalysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERβ-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERβ signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes.MAIN RESULTS AND THE ROLE OF CHANCEHEECs and UtMVECs were ERβ+/ERα−. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ≤ 0.001) but an increase in UtMVECs (P ≤ 0.05). Genomic analysis identified opposite changes in ERβ target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ERβ target genes via association with the transcription factor (‘tethered’ mechanism).LIMITATIONS, REASONS FOR CAUTIONThe study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out.WIDER IMPLICATIONS OF THE FINDINGSThese results have implications for our understanding of the mechanisms responsible for ERβ-dependent changes in EC gene expression in hormone-dependent disorders.STUDY FUNDING/COMPETEING INTEREST(S)The study was funded by a Medical Research Council Programme Grant. E.G. is the recipient of an MRC Career Development Fellowship. The authors have nothing to disclose.

Cloning, expression and functional characterization of carp,Cyprinus carpio, estrogen receptors and their differential activations by estrogens

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα, ERβ1 and ERβ2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERα, ERβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs β1), 49% (α vs β2) and 53% (β1 vs β2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution.

Estrogen receptors in medaka ( Oryzias latipes) and estrogenic environmental contaminants: An in vitro– in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka ( Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E 2) and op’-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E 2 treatment where we observed a trigger in ERβ2 expression upon E 2 exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin ( VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor–ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.

Cloning and functional characterization of Chondrichthyes, cloudy catshark, Scyliorhinus torazame and whale shark, Rhincodon typus estrogen receptors

Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor–ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark ( Scyliorhinus torazame) and whale shark ( Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17β-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor–ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERα and ERβ) from the Japanese firebelly newt ( Cynops pyrrhogaster), Tokyo salamander ( Hynobius tokyoensis), axolotl ( Ambystoma mexicanum), and Raucous toad ( Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5′ and 3′ rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

C-fos gene and protein expression in pelvic endometriosis: a local marker of estrogen action

Endometriosis is an estrogen-dependent disease, causing pelvic pain and infertility. c-fos is an early transcription factor that has been reported to be related to estradiol-dependent cell proliferation. The aim of the present study was to assess the c-fos gene and protein expression in pelvic endometriotic implants in comparison to normal endometrium from infertile women. An open, prospective and controlled study included 15 infertile women with endometriosis and 19 control infertile women. Endometrial and endometriotic biopsies were performed at the follicular phase and the samples were processed for RT-PCR and immunohistochemistry. ERalpha mRNA levels were similar in the endometriotic implants/eutopic endometrium from women with endometriosis and in normal tissue (P = 0.649). The aromatase gene, however, was not expressed in the eutopic endometrium from either control or endometriosis groups, and was only expressed in 50% of endometriotic implants (P = 0.044). c-fos gene expression was higher in endometriotic implants (1.32 +/- 0.13; P = 0.011) than in eutopic endometrium from patients with endometriosis (0.97 +/- 0.11) or from the control group (0.91 +/- 0.05). In addition, immunohistochemistry showed a more abundant distribution of c-Fos in the stroma of endometriotic tissue compared to eutopic endometrium. These data suggest that c-fos may play a role in the molecular mechanisms of estrogen action on the induction, promotion or progression of endometriosis.

Estradiol represses Insulin-like 3 expression and promoter activity in MA-10 Leydig cells

There are increasing evidence in the literature reporting the detrimental effects of endocrine disruptors on the development and function of the male reproductive system. One example is cryptorchidism, or undescended testis, caused by exposure to excessive estrogens. Estrogens, acting through the estrogen receptor α (ERα), have been shown to repress expression of the gene encoding insulin-like 3 (INSL3), a small peptide produced by testicular Leydig cells that is essential for normal testis descent. The molecular mechanism of estrogen/ER action on Insl3 expression, however, remains poorly understood. Here we report estradiol (E 2) represses Insl3 mRNA levels in MA-10 cells, a Leydig cell line model. We also found that E 2 represses the activity of the human and mouse Insl3 promoter in these cells. The E 2-responsive region of the human INSL3 promoter was located to the proximal INSL3 promoter. This region does not contain a consensus estrogen response element indicating an indirect mechanism of action. In agreement with this, we found that E 2-responsiveness was lost when two previously characterized binding sites for the nuclear receptors NUR77 and SF1 were mutated. Finally we show that the E 2 repressive effect could be overcome by cotreatment with testosterone, a positive regulator of Insl3 transcription. Collectively our data provide important new insights into the molecular mechanism of estrogen action in Insl3 transcription in Leydig cells.

ER-α36, a variant of ER-α, is the estrogen receptor that mediates mitogenic estrogen signaling in breast cancer cells.

Abstract Abstract #18 The role of endogenous estrogens in breast cancer etiology has been well established. Dysregulated estrogen signaling increases the rate of cell proliferation and thus the risk of breast cancer. However, despite widespread agreement that estrogens are involved in breast cancer etiology, the molecular mechanisms of estrogen action, especially those by which estrogen signaling stimulates mammary tumorigenesis and breast cancer progression have not been well established.
 Recently, we have identified and cloned a 36-kDa novel isoform of estrogen receptor α (ER-α36) that is generated from a promoter located in the first intron of the original 66-kDa ER-α (ER-α66) gene. The ER-α36 differs from the ER-α66 by lacking both transcriptional activation domains (AF-1 and AF-2) but retains the DNA-binding, dimerization and most of the ligand-binding domains. ER-α36 is mainly expressed on the plasma membrane and mediates membrane-initiated estrogen signaling that activates the MAPK/ERK pathway and stimulates cell proliferation. ER-α36 inhibits the genomic estrogen signaling mediated by the AF1 and AF2 domains of ER-α66 and ligand-dependent and -independent transactivation activities of ER-β. We designed and constructed shRNA expression vectors to express shRNAs specific for ER-α36 or ER-α66. We found that ER-positive breast cancer cells MCF7 and T47D expressing ER-α36 shRNA that successfully knockdown about 90% ER-α36 expression failed to respond to estrogen stimulation. However, estrogen strongly stimulated cell growth in MCF7 and T47D cells with ER-α66 knockdown. We found that some estrogen responsive genes such as c-Myc, CyclinD1 and progesterone receptor (PR) were induced through the extra-nuclear estrogen signaling pathway while pS2 was induced by the nuclear pathway. Our results for the first time demonstrated that ER-α36, not the original ER-α36 is the estrogen receptor that actually mediates estrogen-stimulated cell proliferation in breast cancer cells.
 Among 800 cases of human breast cancer specimens examined, ER-α36 is expressed in most of the breast cancer specimens, and even in 40% of ER-negative breast cancer cases that lack expression of ER-α66. In addition, ER-α36 expressing patients responded poorly to anti-estrogen Tamoxifen treatment. In vitro study revealed that breast cancer cells that highly express ER-α36 exhibited estrogen hypersensitivity and Tamoxifen resistance, consistent with the clinical evidence that breast cancers expressing ER-α36 exhibit higher degree of malignancy and poorer survival rate.
 Taken together, our results demonstrated that ER-α36 is a novel player in mitogenic estrogen signaling that plays an important role in estrogen stimulated mammary carcinogenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 18.

Stimulatory effect of estrogen on the growth of endometrial cancer cells is regulated by cell-cycle regulators

Estrogen is known as a major risk factor in tumorigenesis of the endometrium. The aim of this study is to establish stable estrogen-responsive endometrial cancer cell lines and to investigate the mechanism of estrogen action, focusing on cell-cycle regulation. Human wild-type estrogen receptor cDNA was transfected into endometrial cancer cells (Ishikawa) and estrogen-responsive cell lines were cloned. Their estrogen responsiveness was evaluated by the effect of estrogen on cellular growth and progesterone receptor expression. It was quantitatively estimated by immunocytochemistry or immunoblotting how the expression of cell-cycle regulators such as cyclin D1, cyclin E, Cyclin A, p53, p21 and p27 was regulated by estrogen. A cell line stably responsive to estrogen was established, and cells proliferated and the glandular structure was formed by estrogen stimulation. Cyclin D1 expression increased at 6–24 h and cyclin A gradually increased until 48 h of estrogen treatment compared with untreated cells. On the other hand, p53 and p21 expressions decreased at 6–24 h, and p27 gradually decreased until 24 h by estrogen. Our results show that the stimulatory effect of estrogen on cell proliferation may be regulated by the up-regulation of cyclin D1 and cyclin A, and down-regulation of p53, p21 and p27. This cell line is useful to clarify the molecular mechanism of estrogen action on endometrial cancer.

Molecular cloning of estrogen receptor alpha (ERα; ESR1) of the Japanese giant salamander, Andrias japonicus

Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander ( Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5′ and 3′ rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERα (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERα mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

Molecular cloning of estrogen receptor α of the Nile crocodile

Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile ( Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor α (ERα) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ERα cDNA was obtained using 5′ and 3′ rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ERα showed high identity to the American alligator ERα (98%), caiman ER (98%), lizard ER (82%) and chicken ERα (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ERα was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ERα in crocodiles and expands our knowledge of estrogen receptor evolution.

Gene expression signature of estrogen receptor α status in breast cancer

BackgroundEstrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor α (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts.ResultsWe identified 520 transcripts differentially expressed between ERα-positive (+) and ERα-negative (-) primary breast tumors (Fold change ≥ 2; p < 0.05). Furthermore, we identified 220 high-affinity Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERα (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERα status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERα associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERα (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR.ConclusionThe integration of the breast cancer comparative transcriptome analysis based on ERα status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy.

Insights into the molecular biology of the estrogen receptor define novel therapeutic targets for breast cancer.

Evidence for a role of ovarian factors in the growth of metastatic breast cancer was first recognized over 100 years ago. Today, anti-estrogens are central to the treatment of breast cancer of all stages. We now understand that the action of estrogen is mediated by the estrogen receptors (ER) which are members of the nuclear receptor family of ligand-regulated transcription factors. In this article we review the molecular mechanisms through which ER activates transcription of target genes and through which available anti-estrogens mediate their therapeutic effects. We discuss possible mechanisms of failure of treatment with current anti-estrogens and how newer anti-estrogens under development attempt to address these problems. In addition an expanded view of the molecular mechanisms of estrogen action is leading to the development of novel selective ER modulators or SERMs.

Molecular cloning of the estrogen and progesterone receptors of the American alligator

Steroid hormones perform many essential roles in vertebrates during embryonic development, reproduction, growth, water balance, and responses to stress. The estrogens are essential for normal reproductive activity in female and male vertebrates and appear to have direct actions during sex determination in some vertebrates. To begin to understand the molecular mechanisms of estrogen action in alligators, we have isolated cDNAs encoding the estrogen receptors (ER) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify alligator ovary RNA. Two different DNA fragments (ERα and ERβ) were obtained and the full-length alligator ERα cDNA was obtained using 5 ′ and 3 ′ RACE. The inferred amino acid sequence of alligator ERα (aERα) was very similar to the chicken ERα (91% identity), although phylogenetic analyses suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. We also isolated partial DNA fragments encoding ERβ and the progesterone receptor (PR) of the alligator, both of which show strong sequence similarities to avian ERβ and PR. We examined the expression levels of these three steroid receptors (ERα, ERβ, and PR) in the ovary of juvenile alligators and observed detectable levels of all three receptors. Quantitative RT-PCR showed that gonadal ERα transcript levels in juvenile alligators decreased after E 2 treatment whereas ERβ and PR transcripts were not changed. These results provide tools that will allow future studies examining the regulation and ontogenic expression of steroid receptors in alligators and expand our knowledge of vertebrate steroid receptor evolution.

Molecular mechanism of estrogen action and its role in bone metabolism and brain functions.

Molecular mechanism of estrogen action and its role in bone metabolism and brain functions.

Identification of Estrogen-Responsive Genes by Complementary Deoxyribonucleic Acid Microarray and Characterization of a Novel Early Estrogen-Induced Gene:EEIG1

Estrogen receptors (ERs) are nuclear transcription factors that regulate gene expression in response to estrogen and estrogen-like compounds. Identification of estrogen-regulated genes in target cells is an essential step toward understanding the molecular mechanisms of estrogen action. Using cDNA microarray examinations, 19 genes were identified as induced by 17 beta-estradiol in MCF-7 cells, 10 of which have been reported previously to be estrogen responsive or to be linked with ER status. Five known estrogen-regulated genes, E2IG4, IGFBP4, SLC2A1, XBP1 and B4GALT1, and AFG3L1, responded quickly to estrogen treatment. A novel estrogen-responsive gene was identified and named EEIG1for early estrogen-induced gene 1. EEIG1 was clearly induced by 17 beta-estradiol within 2 h of treatment, and was widely responsive to a group of estrogenic compounds including natural and synthetic estrogens and estrogenic environmental compounds. EEIG1 was expressed in ER-positive but not in ER-negative breast cancer cell lines. EEIG1 expression was repressed by antiestrogens 4-OH-tamoxifen and ICI 182,780 but not by protein synthesis inhibitors cycloheximide and puromycin. These results provide evidence that some estrogenic compounds differentially enhance the transcription of estrogen-regulated genes and suggest a role for EEIG1 in estrogen action.

Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta.

Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERbeta in vivo. In further support of ERbeta-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERbeta, but not by ERalpha. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.