2',3',4'-Trihydroxychalcone changes estrogen receptor α regulation of genes and breast cancer cell proliferation by a reprogramming mechanism.

BACKGROUND: Menopausal hormone therapy (MHT) is recommended for only five years to treat vasomotor symptoms and vulvovaginal atrophy because of safety concerns with long-term treatment. We investigated the ability of 2',3',4'-trihydroxychalcone (2',3',4'-THC) to modulate estrogen receptor (ER)-mediated responses in order to find drug candidates that could potentially prevent the adverse effects of long-term MHT treatment. METHODS: Transfection assays, real time-polymerase chain reaction, and microarrays were used to evaluate the effects of 2',3',4'-THC on gene regulation. Radioligand binding studies were used to determine if 2',3',4'-THC binds to ERα. Cell proliferation was examined in MCF-7 breast cancer cells by using growth curves and flow cytometry. Western blots were used to determine if 2',3',4'-THC alters the E2 activation of the MAPK pathway and degradation of ERα. Chromatin immunoprecipitation was used to measure ERα binding to genes. RESULTS: The 2',3',4'-THC/E2 combination produced a synergistic activation with ERα on reporter and endogenous genes in human U2OS osteosarcoma cells. Microarrays identified 824 genes that we termed reprogrammed genes because they were not regulated in U2OS-ERα cells unless they were treated with 2',3',4'-THC and E2 at the same time. 2',3',4'-THC blocked the proliferation of MCF-7 cells by preventing the E2-induced activation of MAPK and c-MYC transcription. The antiproliferative mechanism of 2',3',4'-THC differs from selective estrogen receptor modulators (SERMs) because 2',3',4'-THC did not bind to the E2 binding site in ERα like SERMs. CONCLUSION: Our study suggests that 2',3',4'-THC may represent a new class of ERα modulators that do not act as a direct agonists or antagonists. We consider 2',3',4'-THC to be a reprogramming compound, since it alters the activity of ERα on gene regulation and cell proliferation without competing with E2 for binding to ERα. The addition of a reprogramming drug to estrogens in MHT may offer a new strategy to overcome the adverse proliferative effects of estrogen in MHT by reprogramming ERα as opposed to an antagonist mechanism that involves blocking the binding of estrogen to ERα.

Cross talk between glucocorticoid and estrogen receptors occurs at a subset of proinflammatory genes.

Glucocorticoids exert potent anti-inflammatory effects by repressing proinflammatory genes. We previously demonstrated that estrogens repress numerous proinflammatory genes in U2OS cells. The objective of this study was to determine if cross talk occurs between the glucocorticoid receptor (GR) and estrogen receptor (ER)α. The effects of dexamethasone (Dex) and estradiol on 23 proinflammatory genes were examined in human U2OS cells stably transfected with ERα or GR. Three classes of genes were regulated by ERα and/or GR. Thirteen genes were repressed by both estradiol and Dex (ER/GR-repressed genes). Five genes were repressed by ER (ER-only repressed genes), and another five genes were repressed by GR (GR-only repressed genes). To examine if cross talk occurs between ER and GR at ER/GR-repressed genes, U2OS-GR cells were infected with an adenovirus that expresses ERα. The ER antagonist, ICI 182780 (ICI), blocked Dex repression of ER/GR-repressed genes. ICI did not have any effect on the GR-only repressed genes or genes activated by Dex. These results demonstrate that ICI acts on subset of proinflammatory genes in the presence of ERα but not on GR-activated genes. ICI recruited ERα to the IL-8 promoter but did not prevent Dex recruitment of GR. ICI antagonized Dex repression of the TNF response element by blocking the recruitment of nuclear coactivator 2. These findings indicate that the ICI-ERα complex blocks Dex-mediated repression by interfering with nuclear coactivator 2 recruitment to GR. Our results suggest that it might be possible to exploit ER and GR cross talk for glucocorticoid therapies using drugs that interact with ERs.

Estrogenic plant foods of red colobus monkeys and mountain gorillas in Uganda.

Phytoestrogens, or naturally occurring estrogen-mimicking compounds, are found in many human plant foods, such as soybeans (Glycine max) and other legumes. Because the consumption of phytoestrogens may result in both health benefits of protecting against estrogen-dependent cancers and reproductive costs of disrupting the developing endocrine system, considerable biomedical research has been focused on the physiological and behavioral effects of these compounds. Despite this interest, little is known about the occurrence of phytoestrogens in the diets of wild primates, nor their likely evolutionary importance. We investigated the prevalence of estrogenic plant foods in the diets of two folivorous primate species, the red colobus monkey (Procolobus rufomitratus) of Kibale National Park and mountain gorilla (Gorilla beringei) of Bwindi Impenetrable National Park, both in Uganda. To examine plant foods for estrogenic activity, we screened 44 plant items (species and part) comprising 78.4% of the diet of red colobus monkeys and 53 plant items comprising 85.2% of the diet of mountain gorillas using transient transfection assays. At least 10.6% of the red colobus diet and 8.8% of the gorilla diet had estrogenic activity. This was mainly the result of the red colobus eating three estrogenic staple foods and the gorillas eating one estrogenic staple food. All estrogenic plants exhibited estrogen receptor (ER) subtype selectivity, as their phytoestrogens activated ERß, but not ERα. These results demonstrate that estrogenic plant foods are routinely consumed by two folivorous primate species. Phytoestrogens in the wild plant foods of these two species and many other wild primates may have important implications for understanding primate reproductive ecology.

Estrogen receptor beta binds to and regulates three distinct classes of target genes.

Estrogen receptor beta (ERbeta) has potent antiproliferative and anti-inflammatory properties, suggesting that ERbeta-selective agonists might be a new class of therapeutic and chemopreventive agents. To understand how ERbeta regulates genes, we identified genes regulated by the unliganded and liganded forms of ERalpha and ERbeta in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ERalpha, whereas many genes were regulated by estradiol (E(2)). These results demonstrated that ERalpha requires a ligand to regulate a single class of genes. In contrast, ERbeta regulated three classes of genes. Class I genes were regulated primarily by unliganded ERbeta. Class II genes were regulated only with E(2), whereas class III genes were regulated by both unliganded ERbeta and E(2). There were 453 class I genes, 258 class II genes, and 83 class III genes. To explore the mechanism whereby ERbeta regulates different classes of genes, chromatin immunoprecipitation-sequencing was performed to identify ERbeta binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in class I genes, whereas ERE, NFkappaB1, and SP1 sites were more enriched in class II genes. ERbeta bound to all three classes of genes, demonstrating that ERbeta binding is not responsible for differential regulation of genes by unliganded and liganded ERbeta. The coactivator NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ERbeta regulate three classes of genes by interacting with different transcription factors and coactivators.

Estrogen receptor ß causes a G2 cell cycle arrest by inhibiting CDK1 activity through the regulation of cyclin B1, GADD45A, and BTG2.

The role of estrogen receptor beta (ERß) in breast cancer is unclear. ERß is considered to have a protective role in breast cancer development based on findings demonstrating that ERß expression inhibits ERα-mediated proliferation of breast cancer cells. We previously demonstrated that ERß causes a ligand independent G2 cell cycle arrest in MCF-7 cells. To study the mechanisms of the ERß-mediated G2 cell cycle arrest, we investigated its effects on the regulatory pathways responsible for the G2/M phase transition. We found that ERß inhibits CDK1 activity, which is the critical determinant of the G2/M progression. CDK1 activity is modulated by both stimulatory and inhibitory factors. Cyclin B1 is the major activator of CDK1. ERß inhibited the cell cycle-dependent stimulation of cyclin B1 mRNA and protein. GADD45A and BTG2 are two major inhibitors of CDK1, which have been implicated in breast tumor formation. Based on these findings, we explored if the expression pattern of GADD45A and BTG2 is affected by ERß. We found that ERß stimulates GADD45A and BTG2 mRNA levels. The induction of these two genes is caused by ERß binding directly to these genes and recruiting c-jun and NCOA2. Our findings demonstrated that unliganded ERß causes a G2 cell cycle arrest by inactivating CDK1 through the repression of cyclin B1 and stimulation of GADD45A and BTG2 expression. These results provide evidence that drugs that stimulate the production of unliganded ERß may be effective new therapies to prevent breast cancer.

Selective concomitant inhibition of mTORC1 and mTORC2 activity in estrogen receptor negative breast cancer cells by BN107 and oleanolic acid.

Hormonal, targeted and chemotherapeutic strategies largely depend on the expression of their cognate receptors and are often accompanied by intolerable toxicities. Effective and less toxic therapies for estrogen receptor negative (ER-) breast cancers are urgently needed. Here, we present the potential molecular mechanisms mediating the selective pro-apoptotic effect induced by BN107 and its principle terpene, oleanolic acid (OA), on ER- breast cancer cells. A panel of breast cancer cell lines was examined and the most significant cytotoxic effect was observed in ER- breast lines. Apoptosis was the major cellular pathway mediating the cytotoxicity of BN107. We demonstrated that sensitivity to BN107 was correlated to the status of ERalpha. Specifically, the presence of functional ERalpha protected cells from BN107-induced apoptosis and absence of ERalpha increased the sensitivity. BN107, an extract rich in OA derivatives, caused rapid alterations in cholesterol homeostasis, presumably by depleting cholesterol in lipid rafts (LRs), which subsequently interfered with signaling mediated by LRs. We showed that BN107 or OA treatment in ER- breast cancer cells resulted in rapid and specific inhibition of LR-mediated survival signaling, namely mTORC1 and mTORC2 activities, by decreasing the levels of the mTOR/FRAP1, RAPTOR and RICTOR. Cotreatment with cholesterol abolished the proapoptotic effect and restored the disrupted mTOR activities. This is the first report demonstrating possible concomitant inhibition of both mTORC1 and mTORC2 activities by modulating the levels of protein constituents present in these signaling complexes, and thus provides a basis for future development of OA-based mTOR inhibitors.

Cell type- and estrogen receptor-subtype specific regulation of selective estrogen receptor modulator regulatory elements.

Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene can act as estrogen receptor (ER) antagonists or agonists depending on the cell type. The antagonistic action of tamoxifen has been invaluable for treating breast cancer, whereas the agonist activity of SERMs also has important clinical applications as demonstrated by the use of raloxifene for osteoporosis. Whereas the mechanism whereby SERMs function as antagonists has been studied extensively very little is known about how SERMs produce agonist effects in different tissues with the two ER types; ERalpha and ERbeta. We examined the regulation of 32 SERM-responsive regions with ERalpha and ERbeta in transiently transfected MCF-7 breast, Ishikawa endometrial, HeLa cervical and WAR-5 prostate cancer cells. The regions were regulated by tamoxifen and raloxifene in some cell types, but not in all cell lines. Tamoxifen activated similar number of regions with ERalpha and ERbeta in the cell lines, whereas raloxifene activated over twice as many regions with ERbeta compared to ERalpha. In Ishikawa endometrial cancer cells, tamoxifen activated 17 regions with ERalpha, whereas raloxifene activated only 2 regions, which might explain their different effects on the endometrium. Microarray studies also found that raloxifene regulated fewer genes than tamoxifen in U2OS bone cancer cells expressing ERalpha, whereas tamoxifen was equally effective at regulating genes with ERalpha and ERbeta. Our studies indicate that tamoxifen is a non-selective agonist, whereas raloxifene is a relative ERbeta-selective agonist, and suggest that ERbeta-selective SERMs might be safer for treating clinical conditions that are dependent on the agonist property of SERMs.

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.

Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist.

After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

Multiple transcription factor elements collaborate with estrogen receptor alpha to activate an inducible estrogen response element in the NKG2E gene.

Estrogen receptors (ERs) regulate transcription by interacting with regulatory elements in target genes. However, known ER regulatory elements cannot explain the expression profiles of genes activated by estradiol (E2) and selective estrogen receptor modulators (SERMs). We previously showed that the killer cell lectin-like receptor (NKG2E) gene is regulated by E2, tamoxifen, and raloxifene. Here we used the NKG2E gene as a model to investigate the mechanism whereby target genes are regulated by E2 and SERMs with ERalpha. The ER regulatory element in the NKG2E promoter was mapped to the -1825 and -1686 region. Full activation of the NKG2E promoter required the collaboration between a transcription factor cluster containing c-jun, heat-shock factor 2, and CCAAT/enhancer-binding protein beta and a unique variant estrogen response element (ERE) that has only a two nucleotide spacer between half sites. The cluster elements and the variant ERE were inactive on their own, but the regulation by E2 and SERMs was restored when the c-jun, heat-shock factor-2, and CCAAT/enhancer-binding protein beta cluster was placed upstream of the variant ERE. The activation of the NKG2E gene by E2 and selective ER modulators was associated with the recruitment of the p160 coactivators glucocorticoid receptor-interacting protein 1 and amplified in breast cancer 1 but not steroid receptor coactivator 1. These studies identified one of the most complex ER regulatory units thus far reported and demonstrate that a cluster of flanking transcription factors collaborate with ER to induce a functional ERE in the NKG2E promoter.

Selective activation of estrogen receptor-beta transcriptional pathways by an herbal extract.

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes.

Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta.

Estrogens and selective estrogen receptor modulators (SERMs) interact with estrogen receptor (ER) alpha and beta to activate or repress gene transcription. To understand how estrogens and SERMs exert tissue-specific effects, we performed microarray analysis to determine whether ERalpha or ERbeta regulate different target genes in response to estrogens and SERMs. We prepared human U2OS osteosarcoma cells that are stably transfected with a tetracycline-inducible vector to express ERalpha or ERbeta. Western blotting, immunohistochemistry, and immunoprecipitation studies confirmed that U2OS-ERalpha cells synthesized only ERalpha and that U2OS-ERbeta cells expressed exclusively ERbeta. U2OS-ERalpha and U2OS-ERbeta cells were treated either with 17beta-estradiol (E2), raloxifene, and tamoxifen for 18 h. Labeled cRNAs were hybridized with U95Av2 GeneChips (Affymetrix). A total of 228, 190, and 236 genes were significantly activated or repressed at least 1.74-fold in U2OS-ERalpha and U2OS-ERbeta cells by E2, raloxifene, and tamoxifen, respectively. Most genes regulated in ERalpha cells in response to E2, raloxifene, and tamoxifen were distinct from those regulated in ERbeta cells. Only 38 of the 228 (17%) genes were regulated by E2 in both U2OS-ERalpha and U2OS-ERbeta cells. Raloxifene and tamoxifen regulated only 27% of the same genes in both the ERalpha and ERbeta cells. A subset of genes involved in bone-related activities regulated by E2, raloxifene, and tamoxifen were also distinct. Our results demonstrate that most genes regulated by ERalpha are distinct from those regulated by ERbeta in response to E2 and SERMs. These results indicate that estrogens and SERMs exert tissue-specific effects by regulating unique sets of targets genes through ERalpha and ERbeta

Tumor necrosis factor-alpha down-regulates human Cu/Zn superoxide dismutase 1 promoter via JNK/AP-1 signaling pathway.

Overexpression of Cu/Zn superoxide dismutase 1 (SOD1) in monocytes blocks reactive oxygen species-induced inhibition of cell growth and apoptosis and renders cells resistant to the toxic effect of tumor necrosis factor (TNF)-alpha, suggesting that TNF-alpha represses the SOD1 gene in these cells. We herein show that TNF-alpha decreases SOD1 mRNA, protein, and promoter activity in U937 cells. Electrophoretic mobility-shift assays (EMSA) show that TNF-alpha decreased binding of three different complexes. Ectopic Sp1 overexpression markedly increased SOD1-basal promoter activity and partially antagonized the TNF-alpha inhibitory effect. In contrast, ectopic c-Jun overexpression mimics TNF-alpha inhibitory effects and antagonizes Sp1 stimulatory effects. In agreement with these findings, EMSA shows a TNF-alpha-induced increase in AP-1 and a decrease in Sp1 DNA binding. Disruption of the C/EBP site decreases, whereas mutation in the Sp1/Egr-1 site completely abolishes DNA-binding and promoter activity. A JNK inhibitor antagonized the negative effects of TNF-alpha on SOD1 promoter activity, suggesting that JNK signaling through c-Jun protein activation is critical for the TNF-alpha-dependent SOD1 repression. A greater understanding of the mechanisms of TNF-alpha-induced SOD1 repression could facilitate the design and development of novel therapeutic drugs for inflammatory conditions.

Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest.

Studies indicate that estrogen receptor (ER) alpha mediates breast cancer-promoting effects of estrogens. The role of ERbeta in breast cancer is unknown. Elucidating the role of ERbeta in the pathogenesis of breast cancer is important because many human breast tumors express both ERalpha and ERbeta. We show that adenovirus-mediated expression of ERbeta changes the phenotype of ERalpha-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERalpha. In contrast, introducing ERbeta into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERbeta inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21(Cip1) and p27(Kip1), which leads to a G(2) cell cycle arrest. These results demonstrate that ERalpha and ERbeta produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ERbeta-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells.

BACKGROUND: Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. OBJECTIVES: We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). METHODS: The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. RESULTS: Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. CONCLUSION: Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. CITATION: Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM, Yaswen P, Vulpe CD, Leitman DC. 2016. Parabens and human epidermal growth factor receptor ligand cross-talk in breast cancer cells. Environ Health Perspect 124:563-569; http://dx.doi.org/10.1289/ehp.1409200.

Differential response of estrogen receptor subtypes to 1,3-diarylindene and 2,3-diarylindene ligands.

Estrogen receptors (ERs) control transcription of genes important for normal human development and reproduction. The signaling networks are complex, and there is a need for a molecular level understanding of the roles of receptor subtypes ERalpha and ERbeta in normal physiology and as therapeutic targets. We synthesized two series of ER ligands, based on a common indene scaffold, in an attempt to develop compounds that can selectively modulate ER-mediated transcription. The 3-ethyl-1,2-diarylindenes, utilizing an amide linker for the 1-aryl extension, bind weakly to the ERs. The 2,3-diarylindenes bind with high affinity to the ER subtypes and demonstrate a range of different biological activities, both in transcriptional reporter gene assays and inhibition of estradiol-stimulated proliferation of MCF-7 cells. Several ligands differentiate between ERalpha and ERbeta subtypes at an estrogen response element (ERE), displaying various levels of partial to full agonist activity at ERalpha, while antagonizing estradiol action at ERbeta.

Estrogens activate bone morphogenetic protein-2 gene transcription in mouse mesenchymal stem cells.

Estrogens exert their physiological effects on target tissues by interacting with the estrogen receptors, ERalpha and ERbeta. Estrogen replacement is one the most common and effective strategies used to prevent osteoporosis in postmenopausal women. Whereas it was thought that estrogens work exclusively by inhibiting bone resorption, our previous results show that 17beta-estradiol (E2) increases mouse bone morphogenetic protein (BMP)-2 mRNA, suggesting that estrogens may also enhance bone formation. In this study, we used quantitative real-time RT-PCR analysis to demonstrate that estrogens increase BMP-2 mRNA in mouse mesenchymal stem cells. The selective ER modulators, tamoxifen, raloxifene, and ICI-182,780 (ICI), failed to enhance BMP-2 mRNA, whereas ICI inhibited E2 stimulation of expression. To investigate if estrogens increase BMP-2 expression by transcriptional mechanisms and if the response is mediated by ERalpha and/or ERbeta, we studied the effects of estrogens on BMP-2 promoter activity in transient transfected C3H10T1/2 cells. E2 produced a dose-dependent induction of the mouse -2712 BMP-2 promoter activity in cells cotransfected with ERalpha and ERbeta. At a dose of 10 nM E2, ERalpha induced mouse BMP-2 promoter activity 9-fold, whereas a 3-fold increase was observed in cells cotransfected with ERbeta. Tamoxifen and raloxifene were weak activators of the mouse BMP-2 promoter via ERalpha, but not via ERbeta. ICI blocked the activation of BMP-2 promoter activity by E2 acting via both ERalpha and ERbeta, indicating that mouse BMP-2 promoter activation is ER dependent. In contrast to E2 and selective ER modulators, the phytoestrogen, genistein was more effective at activating the mouse BMP-2 promoter with ERbeta, compared with ERalpha. Using a deletion series of the BMP-2 promoter, we determined that AP-1 or Sp1 sites are not required for E2 activation. A mutation in a sequence at -415 to -402 (5'-GGGCCActcTGACCC-3') that resembles the classical estrogen-responsive element abolished the activation of the BMP-2 promoter in response to E2. Our studies demonstrate that E2 activation of mouse BMP-2 gene transcription requires ERalpha or ERbeta acting via a variant estrogen-responsive element binding site in the promoter, with ERalpha being the more efficacious regulator. Estrogenic compounds may enhance bone formation by increasing the transcription of the BMP-2 gene.

MF101: a multi-component botanical selective estrogen receptor beta modulator for the treatment of menopausal vasomotor symptoms.

INTRODUCTION: The Women's Health Initiative Estrogen Plus Progestin clinical trial demonstrated the risks exceeded the benefits which have led to a decline in menopausal hormone therapy (MHT) by greater than 50%. MHT use was initiated long before there was a significant understanding of the molecular mechanisms of estrogens. It has become clear that the problem with the current estrogens in MHT is they act non-selectively as an agonist in all tissues that contain estrogen receptors. MF101 is an oral, botanically derived extract that was designed to selectively regulate estrogen receptor beta (ERß) because the increased risk of breast and endometrial cancer is due to the activation of estrogen receptor alpha (ERα) by estrogens. Preclinical and clinical data support a role for selective ERß agonists, such as MF101, for vasomotor symptoms without increasing cancer risks. AREAS COVERED: The review covers the biological, pharmacological and clinical advantages of MF101, and the unique ability of MF101 to selectively target the ERß pathway for the treatment of hot flashes (HF). EXPERT OPINION: Preclinical and clinical studies indicate that MF101, a selective estrogen receptor beta agonist, represents a new class of drugs that is safe and effective for treating HF and nighttime awakenings.

Tissue-specific regulation of genes by estrogen receptors.

Estrogens are frequently used in reproductive medicine. The Women's Health Initiative trial found that the risks of menopausal hormone therapy (MHT) exceed the benefits. The estrogens in MHT, however, were introduced prior to our understanding of the mechanism of action of estrogens. Estrogen signaling is highly complex, involving various DNA regulatory elements to which estrogen receptors bind. Numerous transcription factors and co-regulatory proteins modify chromatin structure to further regulate gene transcription. With a greater understanding of estrogen action, the major problem with the current estrogens in MHT appears to be that they are nonselective. This produces beneficial effects in bone, brain, and adipose tissue but increases the risk of breast and endometrial cancer and thromboembolism. Resurrecting MHT for long-term therapy will require the development of more selective estrogens, such as estrogen receptor (ER)ß-selective estrogens and tissue-selective ERα agonists. These compounds will offer the best prospects to expand the indications of MHT and thus prevent the chronic conditions associated with menopause.

Estrogenic plant extracts reverse weight gain and fat accumulation without causing mammary gland or uterine proliferation.

Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERα). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women.