Classical Estrogen Response Element Research Articles

The transcriptional regulation of an antimicrobial peptide hepcidin1 in Oryzias melastigma upon EE2 exposure involved in a new pathway with a novel transcriptional regulatory element HepERE

17α-ethinylestradiol (EE2) exerts endocrine disrupting effect and immunotoxic effect on marine animals, including modulation of hepcidin expression. The antimicrobial peptide hepcidin displays a crucial role in innate immunity in fish against invading pathogens. It is known that the transcription of hepcidin in mammals is individually regulated by many stimuli, including inflammation, iron overload, anemia or hypoxia, through several distinct molecular pathways. The canonical mechanism for endocrine disrupting effects is mediated by an estrogen receptor (ER) and estrogen responsive element (ERE), whereas the underlying mechanism for immunotoxic effect is still unclear. In this study, a hepcidin from Oryzias melastigma (OM-hep1) was found to be down-regulated upon EE2 exposure and was associated with ERα. Unlike the revealed signaling pathways for hepcidin regulation in mammals, it was revealed by promoter activity analysis that the OM-hep1 transcription was not associated with canonical immune-associated and hormone-associated regulatory elements, known as the nuclear factor κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), ERE and estrogen-related receptor responsive element (ERRE). Further analysis through a series of base mutations revealed a short fragment from −315 to −289 bp on the OM-hep1 promoter with high activity. This fragment was composed of a putative ERE-like element (23 bases) plus an adjacent down-streamed four bases motif GTGT. Replacement of either of the core bases (GGTCA) of ERE-like or GTGT motif showed non-activity and non-response to EE2 exposure, thus a new hepcidin-associated element named as HepERE was revealed. Evidences from electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) assay demonstrated that the EE2-mediated down-regulation of OM-hep1 expression was associated with ERα binding to HepERE but not classical ERE. Taken together, a novel signaling pathway was revealed and the regulatory mechanism associated with the ERα and HepERE element on immunomodulation of OM-hep1 expression upon EE2 exposure was first reported here.

Daidzein increases OPG/RANKL ratio and suppresses IL-6 in MG-63 osteoblast cells

Daidzein is a major dietary source of isoflavones found in Leguminosae, and belongs to the family of diphenolic compounds. The estrogenic effects of daidzein to prompt bone formation and prevent bone resorption have been observed in animal models and cultured cells. In our study, we studied the effects of daidzein, raloxifene and E2 on expression of the osteoblast-produced bone regulatory factors OPG, RANKL and IL-6 in human osteoblastic MG-63 cells. Results suggest that treatment with daidzein, raloxifene and E2 increased the levels of OPG and decreased those of RANKL and IL-6. The effects of daidzein on OPG and RANKL expression are mediated by both ERα and ERβ but those on IL-6 production primarily by ERα. Moreover, daidzein may promote activation of the classic estrogen response element (ERE) pathway through increasing ERα, ERβ and steroid hormone receptor coactivator (SRC)-1 expression. E2 was also able to enhance transcription derived from the ERE, while raloxifene has no effect on it. Raloxifene increased ERα protein and gene expression levels but had no effect on ERβ protein and gene expression at 0.1μM. E2 was found significantly increased the protein and mRNA levels of SRC-1, while raloxifene has no effect on it compared with control. This ability of daidzein to affect osteoblastic cells makes it a good candidate for the treatment of bone loss in postmenopausal women.

Estrogen receptor β exon 3-deleted mouse: The importance of non-ERE pathways in ERβ signaling

In 1998, an estrogen receptor β (ERβ) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ERβ mutant mouse was generated by deleting ERβ exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes of the two KO strains, have led to questions about the physiological function of ERβ. In the present study, we created an ERβ exon 3-deleted mouse (ERβ-Δex3) and confirmed that the only observable defect was anovulation. Despite the two in-frame stop codons introduced by splicing between exons 2 and 4, an ERβ protein was expressed in nuclei of prostate epithelial cells. Using two different anti-ERβ antibodies, we showed that an in-frame ligand binding domain and C terminus were present in the ERβ-Δex3 protein. Moreover, with nuclear extracts from ERβ-Δex3 prostates, there was an ERβ-dependent retardation of migration of activator protein-1 response elements in EMSA. Unlike the original knockout mouse, expression of Ki67, androgen receptor, and Dachshund-1 in prostate epithelium was not altered in the ERβ-Δex3 mouse. We conclude that very little of ERβ transcriptional activity depends on binding to classical estrogen response elements (EREs).

Puerarin concurrently stimulates osteoprotegerin and inhibits receptor activator of NF-κB ligand (RANKL) and interleukin-6 production in human osteoblastic MG-63 cells

Puerarin, a daidzein-8-C-glucoside, is the major isoflavone glycoside found in the Chinese herb radix of Pueraria lobata (Willd.) Ohwi, and has received increasing attention because of its possible role in the prevention of osteoporosis. In our previous studies, puerarin reduced the bone resorption of osteoclasts and promoted long bone growth in fetal mouse in vitro. Further study confirmed that puerarin stimulated proliferation and differentiation of osteoblasts in rat. However, the mechanisms underlying its actions on human bone cells have remained largely unknown. Here we show that puerarin concurrently stimulates osteoprotegerin (OPG) and inhibits receptor activator of nuclear factor-κB ligand (RANKL) and Interleukin-6 (IL-6) production by human osteoblastic MG-63 cells containing two estrogen receptor (ER) isotypes. Treatment with the ER antagonist ICI 182,780 abrogates the above actions of puerarin on osteoblast-derived cells. Using small interfering double-stranded RNAs technology, we further demonstrate that the effects of puerarin on OPG and RANKL expression are mediated by both ERα and ERβ but those on IL-6 production primarily by ERα. Moreover, we demonstrate that puerarin may promote activation of the classic estrogen response element (ERE) pathway through increasing ERα, ERβ and steroid hormone receptor coactivator (SRC)-1 expression. Therefore, puerarin will be a promising agent that prevents or retards osteoporosis.

Abstract 830: Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells.

Abstract Recent clinical trials have demonstrated that estrogen (E2) alone reduces breast cancer incidence in postmenopausal women and has therapeutic effects on aromatase inhibitor resistant patients which both are related with the effect of E2-induced apoptosis. We have shown that E2 induces apoptosis in long-term E2 deprived breast cancer cells (MCF-7:5C) through stress responses, but the molecular mechanism underlying E2-induced stress remains to be elucidated. Here, we report that E2 activated the sensors of unfolded protein response (UPR) inositol-requiring protein 1 alpha (IRE1α) and PRK-like endoplasmic reticulum kinase (PERK) within 24 hours. Knockdown of PERK and IRE-1α through small interferon RNAs (siRNA) partially prevented E2-induced apoptosis, which suggested that endoplasmic reticulum stress was involved in the E2-induced apoptosis. Further examination showed E2 activated both classical estrogen responsive element (ERE) pathway and nonclassical activating protein-1 (AP-1) pathway in MCF-7:5C cells. Classical ERE regulated genes were not directly involved in E2-induced apoptosis. However, the transcription factor c-Fos acted as a critical trigger involved in stress responses induced by E2 in MCF-7:5C cells. E2 immediately elevated c-Fos expression in MCF-7:5C cells but not in wild-type breast cancer cells and 4-hydroxytamoxifen blocked this stimulation which suggested that E2 activated c-Fos through estrogen receptor (ER). E2 increased phosphorylation of c-Jun after 24 hours treatment but did not significantly enhance the abundance of c-Jun as c-Fos. c-Fos protein forms stable heterodimers with c-Jun which preferentially bind to phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE). Interestingly, low doses of TPA which activated TRE activity could induce apoptosis and activated apoptosis-related genes similar to E2 in MCF-7:5C cells. Knockdown of c-Fos and c-Jun with specific siRNAs blocked E2-induced apoptosis, which confirmed the activation of AP-1 played a critical role in the process of apoptosis induced by E2. Although c-Fos closely associates with c-Jun to form a stable heterodimer, they had differential functions in modulation of UPR and other nuclear transcription factors. Knockdown of c-Fos but not c-Jun could inhibit the activation of UPR by E2 in MCF-7:5C cells. Furthermore, downregulation of c-Fos abolished the expression of multiple nuclear transcription factors such as SRC-3, NF-κB, and p300 etc in MCF-7:5C cells. Overall, these data illustrate that c-Jun might provide a dimerizing site for c-Fos, whereas c-Fos was identified as a pivotal regulatory target of E2 in MCF-7:5C cells to trigger the apoptotic cascades. This study provides an important rationale for further exploration of E2-induced apoptosis in endocrine resistant breast cancer to improve clinical benefit. Citation Format: Ping Fan, Fadeke Agboke, Obi L. Griffith, Russell E. McDaniel, Xiaojun Zou, Karen Creswell, Joe W. Gray, Virgil Craig Jordan. Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 830. doi:10.1158/1538-7445.AM2013-830

P38γ Mitogen-activated Protein Kinase (MAPK) Confers Breast Cancer Hormone Sensitivity by Switching Estrogen Receptor (ER) Signaling from Classical to Nonclassical Pathway via Stimulating ER Phosphorylation and c-Jun Transcription

Estrogen receptor (ER) α promotes breast cancer growth by regulating gene expression through classical estrogen response element (ERE) binding and nonclassical (interaction with c-Jun at AP-1 sites) pathways. ER is the target for anti-estrogens such as tamoxifen (TAM). However, the potential for classical versus nonclassical ER signaling to influence hormone sensitivity is not known. Moreover, anti-estrogens frequently activate several signaling cascades besides the target ER, and the implications of these "off-target" signaling events have not been explored. Here, we report that p38γ MAPK is selectively activated by treatment with TAM. This results in both phosphorylation of ER at Ser-118 and stimulation of c-Jun transcription, thus switching ER signaling from the classical to the nonclassical pathway leading to increased hormone sensitivity. Unexpectedly, phosphorylation at Ser-118 is required for ER to bind both p38γ and c-Jun, thereby promoting ER relocation from ERE to AP-1 promoter sites. Thus, ER/Ser-118 phosphorylation serves as a central mechanism by which p38γ regulates signaling transduction of ER with its inhibitor TAM.

LC, a novel estrone–rhein hybrid compound, promotes proliferation and differentiation and protects against cell death in human osteoblastic MG-63 cells

Estrogen analogs are promising drugs for postmenopausal osteoporosis, but because of their possible side effects, estrogens which exert their estrogenic effects selectively on bone are desired. Based on our previous studies that rhein had high affinity for the bone mineral, we synthesized estrone–rhein hybrid compounds and confirmed that one of these hybrid compounds, LC, exhibited a selective profile in the bone and prevented bone loss but had no effect on endometrium growth in ovariectomized rats. However, the mechanisms underlying its actions on human bone cells have remained largely unknown. Here we show that LC increases proliferation and differentiation and opposes cisplatin-induced apoptosis in human osteoblastic MG-63 cells containing two estrogen receptor (ER) isoforms. LC promotes proliferation by altering cell cycle distribution whereas LC-mediated survival may be associated with up-regulation of X-linked inhibitor of apoptosis (XIAP) expression. Treatment with the ER antagonist ICI 182,780 abolishes the above actions of LC on osteoblast-derived cells. Using small interfering double-stranded RNAs technology, we further demonstrate that the effects of LC on proliferation and survival are mediated by both ERα and ERβ but those on differentiation primarily by ERα. Moreover, we demonstrate that LC may promote activation of the classic estrogen response element (ERE) pathway through increasing steroid receptor coactivator (SRC)-3 expression. Meanwhile, we find that regulation of osteoblastic proliferation and survival by LC involves Ras/MEK/ERK and PI3K/Akt signaling. Therefore, using rhein for conjugating compounds is a promising method of effectively targeting estrogens to the bone.

LC, a novel estrone–rhein hybrid compound, concurrently stimulates osteoprotegerin and inhibits receptor activator of NF-κB ligand (RANKL) and interleukin-6 production by human osteoblastic cells

Estrogen analogues are promising drugs for postmenopausal osteoporosis, but because of their possible side effects such as increased risk of cancer, estrogens which exert their estrogenic effects selectively on bone are desired. It has been shown that rhein inhibits osteoclast formation and bone resorption activity and has an antitumor role in several types of cancers. Having found that rhein had high affinity for the bone mineral, we synthesized estrone–rhein hybrid compounds and confirmed that one of these hybrid compounds, LC, exhibited a selective profile in the bone and prevented bone loss but had no effect on endometrium growth in ovariectomized rats. However, the mechanisms underlying its actions on human bone cells have not been well defined. Here we show that LC concurrently stimulates osteoprotegerin (OPG) and inhibits receptor activator of nuclear factor-κB ligand (RANKL) and Interleukin-6 (IL-6) production by human osteoblastic MG-63 cells containing two estrogen receptor (ER) isotypes. Treatment with the ER antagonist ICI 182,780 abrogates the above actions of LC on osteoblast-derived cells. Using small interfering double-stranded RNAs (siRNA) technology, we further demonstrate that the effects of LC on IL-6 production are mediated by both ERα and ERβ but those on OPG and RANKL expression primarily by ERα. Furthermore, we also demonstrate that LC functions at least partially through activation of the classic estrogen response element (ERE) pathway as well as Ras/MEK/ERK and PI3K/Akt signaling. The effect of LC on bone is due to not only its estrogenic activity but also action of its rhein moiety. Also, this compound shows much weaker effect on breast epithelial cell growth than that of estrone. Therefore, using rhein for conjugating compounds is a promising method of effectively targeting estrogens to the bone.

The hinge region of the human estrogen receptor determines functional synergy between AF-1 and AF-2 in the quantitative response to estradiol and tamoxifen

Human estrogen receptors alpha and beta (ERalpha and ERbeta) greatly differ in their target genes, transcriptional potency and cofactor-binding capacity, and are differentially expressed in various tissues. In classical estrogen response element (ERE)-mediated transactivation, ERbeta has a markedly reduced activation potential compared with ERalpha; the mechanism underlying this difference is unclear. Here, we report that the binding of steroid receptor coactivator-1 (SRC-1) to the AF-1 domain of ERalpha is essential but not sufficient to facilitate synergy between the AF-1 and AF-2 domains, which is required for a full agonistic response to estradiol (E2). Complete synergy is achieved through the distinct hinge domain of ERalpha, which enables combined action of the AF-1 and AF-2 domains. AF-1 of ERbeta lacks the capacity to interact with SRC-1, which prevents hinge-mediated synergy between AF-1 and AF-2, thereby explaining the reduced E2-mediated transactivation of ERbeta. Transactivation of ERbeta by E2 requires only the AF-2 domain. A weak agonistic response to tamoxifen occurs for ERalpha, but not for ERbeta, and depends on AF-1 and the hinge-region domain of ERalpha.

Epigenetic regulation of the ERβ gene on the estrogen signal transfection pathway in colon cancer cells

We studied the regulatory effects of the estragen receptorbeta (ERbeta) gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved. A human ERbeta gene recombinant expression plasmid, pEGFP-C1-ERbeta, was constructed and transfected into the Caco-2 colon cancer cell line, a line with low ERbeta gene expression. The expression of ERbeta mRNA and protein was detected 72 h after transfection. RT-PCR was used to examine the expression levels of the progesterone recepror (PR) gene containing the classic estrogen response element (ERE), the C-fos oncogene containing the AP-1 site (a non-classical ER binding site), the epigenetic modifying genes, such as Dnmt1, Dnmt3a, Dnmt3b, and histone methyltransferase (HMT), and the human mismatch repair gene hMLH1. Methylation-specific PCR was used to detect the changes in the methylated sites of the CpG islands in the promoters of the ERbeta, PR, and C-fos genes. The results indicated that the human ERbeta gene recombinant expression plasmid pEGFP-C1-ERbeta was successfully constructed and transfected into Caco-2 cells. As compared with the control group, the mRNA and protein expression of ERbeta gene was increased significantly 72 h after the transfection of pEGFP-C1-ERbeta into the Caco-2 cells. As compared with the control group, the mRNA expression of the PR, C-fos, Dnmt3a and Dnmt3b genes was increased significantly 72 h after the transfection of pEGFP-C1-ERbeta into the Caco-2 cells, but the mRNA expression of the Dnmt1, HMT, and hMLH1 genes decreased significantly (P<0.05). As compared with the control group, different degrees of demethylation occurred in the promoters of the ERbeta, progesterone receptor (PR), and C-fos oncogene 72 h after the transfection of pEGFP-C1-ERbeta into the Caco-2 cells. The methylation index of the estrogen signal transfection pathway in Caco-2 cells was decreased significantly following the expression restoration of ERbeta gene (P<0.05). It is concluded that the restoration or up-regulation of the ERbeta gene in Caco-2 cells may significantly activate the expression of the related target genes in the downstream estrogen signal transfection pathway and may result in the demethylation changes of the pathway. During the process, the expression level and activity of the epigenetic modifying genes and the human mismatch repair gene have changed simultaneously. The regulatory effect of the ERbeta gene on the estrogen signal transfection pathway to a certain extent partly involves demethylation.

Estrogenic in vitro assay on mouse embryonic Leydig cells

We and others have reported that mouse embryonic testes contain a subpopulation of somatic cells expressing estrogen receptor alpha (ERalpha). In order to provide evidence for a possible direct estrogen effect on mammalian testes from the early stage of their differentiation, here we devised a method for the in vitro culture of the ERalpha-expressing cells from 12.5 days post coitum mouse testes and their transfection with plasmids containing the classical estrogen responsive element (ERE) or the alternative estrogen AP-1 responsive element upstream of the luciferase reporter gene (ERE-Luc and AP-1-Luc). StAR immunopositivity of the most part of the ERalpha+ cells grown in culture and subjected to the estrogenic assay, allowed their identification as embryonic Leydig cells. Maximum induction of the ERE-Luc activity was achieved with 10 nM 17-beta estradiol (E2), from 1.7 to 3-fold in such cells and from 2.3 to 5.7-fold in MCF-7 cells used for comparison; the anti-estrogen ICI 182.780 abolished such effects. AP-1-Luc was less sensitive to E2 in both cell types (10 nM E2, 1.2 to 2.7-fold increase in embryonic Leydig cells; about 3-fold in MCF-7 cells) and the effect was not ICI-dependent. Eventually, we stimulated the transfected cells with various xenoestrogens such as lindane, bisphenol A or mono-(2-ethylhexyl) pthalate and with the phytoestrogen zeralenone obtaining evidence for ERE-Luc, but not AP-1-Luc stimulation in embryonic Leydig cells. These results represent evidence of functional ERalpha-dependent genomic pathways in embryonic Leydig cells and describe an in vitro assay suitable for evaluating the activity of putative estrogenic compounds on such cells.

Postmenopausal hormone replacement therapy and the vascular wall: mechanisms of 17 beta-estradiol's effects on vascular biology.

17 beta-estradiol (E2) protects against atherosclerosis independent of changes in plasma lipoproteins in a variety of animal models, which is explained by direct effects of E2 on the vascular wall. E2 improves vasomotion by modulation of vasoconstrictor and vasodilator systems through endothelium-dependent and endothelium-independent mechanisms. E2 affects the remodeling of the vascular wall by inhibiting smooth muscle cell proliferation and accelerating reendothelialization of injured blood vessels. E2 modulates the vascular inflammatory response by inhibiting cytokine production, cytokine-induced expression of cell adhesion molecules and platelet aggregation/adhesion. This review focuses on the cellular and molecular mechanisms underlying these vasculoprotective actions of E2. E2 can act through nongenomic stimulation of membrane/intracellular mediators and/or the classical genomic pathway of steroid actions, which is dependent on transcription and protein synthesis. The existence of at least two nuclear estrogen receptor (ER) subtypes alpha and beta and a putative membrane ER present the potential of tissue-specific as well as biologically different E2 actions. Nuclear ERs act as ligand-activated transcription factors and can affect gene regulation by interaction with the classical estrogen response element or other nonreceptor transcription factors. The molecular basis of genomic E2 actions by identifying transcription factors and regulatory elements involved in the induction and inhibition of E2 regulated gene expression is only at the beginning of being understood. The impact of E2-mediated increased NO availability on the hemodynamic and antiatherosclerotic actions of E2 is still a debate of controversy.

Estrogen Receptor (ER) β Regulates ERα Expression in Stromal Cells Derived from Ovarian Endometriosis

Estradiol and its nuclear receptors, estrogen receptor (ER) alpha and ERbeta, play critical roles in endometrium and endometriosis. Levels of ERbeta, due to pathological hypomethylation of its promoter, are significantly higher in endometriotic vs. endometrial tissue and stromal cells, whereas ERalpha levels are lower in endometriosis. Estradiol regulates ERalpha gene expression via its alternatively used promoters A, B, and C. The aim of the study was to determine whether high levels of ERbeta in endometriotic stromal cells from ovarian endometriomas regulate ERalpha gene expression. ERbeta knockdown significantly increased ERalpha mRNA and protein levels in endometriotic stromal cells. Conversely, ERbeta overexpression in endometrial stromal cells decreased ERalpha mRNA and protein levels. ERbeta knockdown significantly decreased proliferation of endometriotic stromal cells. Chromatin immunoprecipitation assays demonstrated that estradiol enhanced ERbeta binding to nonclassical activator protein 1 and specificity protein 1 motifs in the ERalpha gene promoters A and C and a classic estrogen response element in promoter B in endometriotic stromal cells. High levels of ERbeta suppress ERalpha expression and response to estradiol in endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERalpha promoters. ERbeta also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells. We speculate that a significantly increased ratio of ERbeta:ERalpha in endometriotic tissues may also suppress progesterone receptor expression and contribute to progesterone resistance. Thus, ERbeta may serve as a significant therapeutic target for endometriosis.

Estrogen Receptor α Signaling Pathways Differentially Regulate Gonadotropin Subunit Gene Expression and Serum Follicle-Stimulating Hormone in the Female Mouse

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

The genome landscape of ERα- and ERβ-binding DNA regions

In this article, we have applied the ChIP-on-chip approach to pursue a large scale identification of ERα- and ERβ-binding DNA regions in intact chromatin. We show that there is a high degree of overlap between the regions identified as bound by ERα and ERβ, respectively, but there are also regions that are bound by ERα only in the presence of ERβ, as well as regions that are selectively bound by either receptor. Analysis of bound regions shows that regions bound by ERα have distinct properties in terms of genome landscape, sequence features, and conservation compared with regions that are bound by ERβ. ERβ-bound regions are, as a group, located more closely to transcription start sites. ERα- and ERβ-bound regions differ in sequence properties, with ERα-bound regions having an overrepresentation of TA-rich motifs including forkhead binding sites and ERβ-bound regions having a predominance of classical estrogen response elements (EREs) and GC-rich motifs. Differences in the properties of ER bound regions might explain some of the differences in gene expression programs and physiological effects shown by the respective estrogen receptors.

Determination of the differential estrogenicity of isoflavonoids by E2–ER–ERE-dependent gene expression in recombinant yeast and MCF-7 human breast cancer cells

The use of phytoestrogens-containing natural sources as alternative hormone replacement therapy (HRT) has been a subject of debate for decades. Development of assays to characterize these phytoestrogens is required. In this study, differential estrogenicities of five isoflavonoids found in red clover and soy, including biochanin A, daidzein, formononetin, genistein and glycitein were examined in a yeast-based screen system with a classical palindromic estrogen response element (ERE)-ADE2 reporter and in a MCF-7 cell culture system with mRNA levels of ER-dependent genes compared. In a yeast-based assay, five isoflavonoids showed various extents of estrogenic potencies. A collection of primary estrogen receptor (ER)-regulated genes by estradiol (E2), including hTERT, c-MYC, BCL2 and Ha-ras (oncogenic) and quinone reductase (QR), human complement 3 (C3) and COX7RP (non-oncogenic) were selected as marker genes for a MCF-7 cell-based endogenous gene expression assay. The results indicated that the mRNA levels of these E2–ER–ERE-dependent marker genes were regulated differentially by five isoflavonoids, leading to distinct expression patterns, which are also significantly different from that of E2. Moreover, the anti-estrogenic effects of biochanin A and formononetin on E2-induced transcriptions of marker genes in MCF-7 cells were also displayed. Taken together, these results are significant for these naturally occurring isoflavonoids regarding the issues of safety and efficacy.

Differential Regulation of Native Estrogen Receptor-Regulatory Elements by Estradiol, Tamoxifen, and Raloxifene

Estrogen receptors (ERs) regulate gene transcription by interacting with regulatory elements. Most information regarding how ER activates genes has come from studies using a small set of target genes or simple consensus sequences such as estrogen response element, activator protein 1, and Sp1 elements. However, these elements cannot explain the differences in gene regulation patterns and clinical effects observed with estradiol (E(2)) and selective estrogen receptor modulators. To obtain a greater understanding of how E(2) and selective estrogen receptor modulators differentially regulate genes, it is necessary to investigate their action on a more comprehensive set of native regulatory elements derived from ER target genes. Here we used chromatin immunoprecipitation-cloning and sequencing to isolate 173 regulatory elements associated with ERalpha. Most elements were found in the introns (38%) and regions greater than 10 kb upstream of the transcription initiation site (38%); 24% of the elements were found in the proximal promoter region (<10 kb). Only 11% of the elements contained a classical estrogen response element; 23% of the elements did not have any known response elements, including one derived from the naked cuticle homolog gene, which was associated with the recruitment of p160 coactivators. Transfection studies found that 80% of the 173 elements were regulated by E(2), raloxifene, or tamoxifen with ERalpha or ERbeta. Tamoxifen was more effective than raloxifene at activating the elements with ERalpha, whereas raloxifene was superior with ERbeta. Our findings demonstrate that E(2), tamoxifen, and raloxifene differentially regulate native ER-regulatory elements isolated by chromatin immunoprecipitation with ERalpha and ERbeta.

Ligands Differentially Modify the Nuclear Mobility of Estrogen Receptors α and β

Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor alpha (ERalpha) and ERbeta mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERalpha, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERalpha. Interestingly, we found that for ERbeta such a classification was not possible because ERbeta ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERalpha, whereas ERbeta retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERbeta retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.

A novel molecular assay to discriminate transcriptional effects caused by xenoestrogens

A phenotypic definition of the term estrogen has become increasingly problematic due to the multiple modes of estrogen action which can now be defined by differing nuclear and membrane receptors for the classic ligand, 17β-estradiol, and by the multiple signalling pathways that are consequently addressed. This has led to the term xenoestrogen being largely determined by whatever assay system is used for its definition. Here we describe a novel and simple matrix for a transfection system using MBA-MD231 and MCF-7 breast cancer cells as hosts. This matrix is able to vary the type of nuclear estrogen receptor used, and by varying the promoter-reporter construct between one using a classic estrogen response element (ERE) enhancer, and one using an enhancer element derived from the bovine oxytocin gene promoter binding an orphan nuclear receptor, direct classical effects can be neatly discriminated from non-classical and non-genomic actions of test substances. This assay matrix has been used to examine a selection of phytoestrogens and xenobiotics, thereby providing new information on the mechanism of action of some of these substances in breast cancer cells.

Effects of Loss of Classical Estrogen Response Element Signaling on Bone in Male Mice

The role of estrogen signaling in the male skeleton via estrogen receptor (ER)-alpha is now well established. ERalpha can elicit responses through either classical estrogen response elements (ERE) pathways or nonclassical, non-ERE pathways. In the present study, we examined the effects of either the attenuation or loss of classical ERalpha signaling on the murine male skeleton. To accomplish this, we crossed male mice heterozygous for a knock-in mutation [nonclassical ERalpha knock-in (NERKI)], which abolishes the ERE-mediated pathway with female heterozygous ERalpha knockout mice (ERalpha+/-) and studied the F1 generation ERalpha+/+, ERalpha+/-, ERalpha+/NERKI, and ERalpha-/NERKI male progeny longitudinally using bone density and histomorphometry. The only ERalpha allele present in ERalpha-/NERKI mice is incapable of classical ERE-mediated signaling, whereas the heterozygous ERalpha+/NERKI mice have both one intact ERalpha and one NERKI allele. As compared with ERalpha+/+ littermates (n=10/genotype), male ERalpha+/NERKI and ERalpha-/NERKI mice displayed axial and appendicular skeletal osteopenia at 6, 12, 20, and 25 wk of age, as demonstrated by significant reductions in total bone mineral density (BMD) at representative sites (areal BMD by dual-energy x-ray absorptiometry at the lumbar vertebrae and femur and volumetric BMD by peripheral quantitative computed tomography at the tibia; P<0.05-0.001 vs. ERalpha+/+). The observed osteopenia in these mice was evident in both trabecular and cortical bone compartments. However, these decreases were more severe in mice lacking classical ERalpha signaling (ERalpha-/NERKI mice), compared with mice in which one wild-type ERalpha allele was present (ERalpha+/NERKI mice). Collectively, these data demonstrate that classical ERalpha signaling is crucial for the development of the murine male skeleton.