Membrane-bound Estrogen Receptor Research Articles

Role of estrogen and RAS signaling in repeated implantation failure.

In humans, hormonal regulation is crucial for the preparation of uterine environment leading to either successful implantation or menstrual cycle. Estrogen is a pivotal female steroid hormone that regulates the uterine dynamics along with progesterone in the estrous and menstrual cycles in humans. Estrogen signals act via nuclear estrogen receptor or membrane-bound receptor. The membrane-bound estrogen receptor plays a crucial role in the rapid response of estrogen in the uterine epithelium. Recently, RASD1 has received attention as a novel signal transducer of estrogen in various systems including female reproductive organs. In this review, we discuss the regulation of estrogen and RASD1 signaling in the uterus and also provide insights into RAS as a novel signaling molecule in repeated implantation failure.

Genistein, a Phytoestrogen in Soybean, Induces the Expression of Acetylcholinesterase via G Protein-Coupled Receptor 30 in PC12 Cells.

Genistein, 4′,5,7-trihydroxyisoflavone, is a major isoflavone in soybean, which is known as phytestrogen having known benefit to brain functions. Being a common phytestrogen, the possible role of genistein in the brain protection needs to be further explored. In cultured PC12 cells, application of genistein significantly induced the expression of neurofilaments (NFs), markers for neuronal differentiation. In parallel, the expression of tetrameric form of proline-rich membrane anchor (PRiMA)-linked acetyl-cholinesterase (G4 AChE), a key enzyme to hydrolyze acetylcholine in cholinergic synapses, was induced in a dose-dependent manner: this induction included the associated protein PRiMA. The genistein-induced AChE expression was fully blocked by the pre-treatment of H89 (an inhibitor of protein kinase A, PKA) and G15 (a selective G protein-coupled receptor 30 (GPR30) antagonist), which suggested a direct involvement of a membrane-bound estrogen receptor (ER), named as GPR30 in the cultures. In parallel, the estrogen-induced activation of GPR30 induced AChE expression in a dose-dependent manner. The genistein/estrogen-induced AChE expression was triggered by a cyclic AMP responding element (CRE) located on the ACHE gene promoter. The binding of this CRE site by cAMP response element-binding protein (CREB) induced ACHE gene transcription. In parallel, increased expression levels of miR132 and miR212 were found when cultured PC12 cells were treated with genistein or G1. Thus, a balance between production and destruction of AChE by the activation of GPR30 was reported here. We have shown for the first time that the activation of GPR30 could be one way for estrogen or flavonoids, possessing estrogenic properties, to enhance cholinergic functions in the brain, which could be a good candidate for possible treatment of neurodegenerative diseases.

Membrane-initiated estrogen signaling via Gq-coupled GPCR in the central nervous system

The last few decades have revealed increasing complexity and depth to our knowledge of receptor-mediated estrogen signaling. Nuclear estrogen receptors (ERs) ERα and ERβ remain the fundamental dogma, but recent research targeting membrane-bound ERs urges for a more expanded view on ER signaling. ERα and ERβ are also involved in membrane-delineated signaling alongside membrane-specific G protein-coupled estrogen receptor 1 (GPER1), ER-X, and the Gq-coupled membrane ER (Gq-mER). Membrane ERs are responsible for eliciting rapid responses to estrogen signaling, and their importance has been increasingly indicated in central nervous system (CNS) regulation of such functions as reproduction, energy homeostasis, and stress. While the Gq-mER signaling pathway is well characterized, the receptor structure and gene remains uncharacterized, although it is not similar to the nuclear ERα/β. This review will describe the current knowledge of this putative membrane ER and its selective ligand, STX, from its initial characterization in hypothalamic melanocortin circuitry to recent research exploring its role in the CNS outside of the hypothalamus.

A Membrane G-Protein-Coupled Estrogen Receptor Is Necessary but Not Sufficient for Sex Differences in Zebra Finch Auditory Coding.

Estradiol acts as a neuromodulator in brain regions important for cognition and sensory processing. Estradiol also shapes brain sex differences but rarely have these concepts been considered simultaneously. In male and female songbirds, estradiol rapidly increases within the auditory forebrain during song exposure and enhances local auditory processing. We tested whether G-protein-coupled estrogen receptor 1 (GPER1), a membrane-bound estrogen receptor, is necessary and sufficient for neuroestrogen regulation of forebrain auditory processing in male and female zebra finches (Taeniopygia guttata). At baseline, we observed that females had elevated single-neuron responses to songs vs males. In males, narrow-spiking (NS) neurons were more responsive to conspecific songs than broad-spiking (BS) neurons, yet cell types were similarly auditory responsive in females. Following acute inactivation of GPER1, auditory responsiveness and coding were suppressed in male NS yet unchanged in female NS and in BS of both sexes. By contrast, GPER1 activation did not mimic previously established estradiol actions in either sex. Lastly, the expression of GPER1 and its coexpression with an inhibitory neuron marker were similarly abundant in males and females, confirming anatomical similarity in the auditory forebrain. In this study, we found: (1) a role for GPER1 in regulating sensory processing and (2) a sex difference in auditory processing of complex vocalizations in a cell type-specific manner. These results reveal sex specificity of a rapid estrogen signaling mechanism in which neuromodulation accounts and/or compensates for brain sex differences, dependent on cell type, in brain regions that are anatomically similar in both sexes.

Bisphenol A accelerates meiotic progression in embryonic chickens via the estrogen receptor β signaling pathway

Bisphenol A (BPA) as an endocrine-disrupting chemical with weak estrogenic activity affects formation of primordial follicles. This study aimed to identify the potential effects and molecular mechanisms of BPA on meiosis and primordial follicle formation in chickens. The results suggest that the cortical layer was thickened and the number of germ cells that entered into meiosis was increased in BPA-treated ovaries. The percentage of γH2AX-positive cells increased significantly. In addition, up-regulated mRNA expression of meiotic genes, including stimulated by retinoic acid gene 8 (Stra8), disrupted meiotic cDNA 1 homologue (Dmc1) and synaptonemal complex protein 3 (Scp3) were observed in BPA-treated ovaries. Therefore, progression to meiosis prophase I was accelerated by exposure to BPA. Furthermore, the results demonstrated that injection of BPA resulted in hypomethylation of Dazl (Deleted in A Zoospermia-Like gene) and Stra8 and up-regulation mRNA expression of Dazl and Stra8 during meiotic onset. Finally, the relationship between estrogen receptor (ER) expression and BPA-induced meiosis was revealed using an in vitro ovarian culture system. BPA enhanced ERβ expression at the levels of mRNA and protein, while BPA exerted no significant effect on ERα and membrane-bound estrogen receptor (GPR30) expression. The inducing effects of BPA on meiosis were blocked by ER inhibitor. Collectively, these results demonstrate the dynamic ovarian response to BPA exposure, which indicate that BPA affects the formation of primordial follicles by promoting meiotic progression of oocytes via hypomethylation of Dazl and Stra8 and ERβ signaling pathways.

Estrogen Modulates ubc9 Expression and Synaptic Redistribution in the Brain of APP/PS1 Mice and Cortical Neurons.

Estrogen exerts multiple actions in the brain and is an important neuroprotective factor in a number of neuronal disorders. However, the underlying mechanism remains unknown. Studies demonstrate that ubiquitin-conjugating enzyme 9 (ubc9) has an integral role in synaptic plasticity and may contribute to the pathology of neuronal disorders. We aimed to investigate the effects of estrogen on ubc9 and in the Alzheimer's disease brain. Ubc9 protein and mRNA were significantly increased in the cortex and hippocampus of APP/PS1 mice with enhanced SUMOylation. Systemic estrogen administration led to reduced ubc9 expression in ovariectomized APP/PS1 mice and reduced SUMOylation. The inhibition of ubc9 expression by estrogen was found to be dose-dependent in cultured neurons. However, estrogen receptor (ER) antagonist ICI182780 did not block the inhibition of ubc9 expression by estrogen. Furthermore, the reduced expression of ubc9 was not mediated by ERα or ERβ agonists alone or in combination, but by the membrane-impermeable ER agonist E2-bovine serum albumin. The activation of the G protein-coupled ER mediated the inhibition of ubc9 expression of estrogen. A phosphoinositide 3-kinase (PI3K) inhibitor, rather than an extracellular signal-regulated kinase inhibitor, blocked the inhibition of ubc9 by estrogen. Estrogen treatment significantly increased the phosphorylation of PI3K, which suggests that activation of the PI3K pathway by estrogen is required for ubc9 regulation. Further, ubc9 interacted with the synaptic proteins post-synaptic density protein 95 (PSD95) and synaptophysin. Estrogen decreased the interaction of ubc9 with post-synaptic PSD95, but increased the interaction of ubc9 with pre-synaptic synaptophysin. These results suggest that a membrane-bound ER might mediate the estrogen inhibition of ubc9 in cortical neurons, where PI3K plays an important role. We also show that ubc9 can interact with synaptic proteins, which are subject to estrogen regulation.

Membrane-Bound Estrogen Receptor Alpha Initiated Signaling is Dynamin Dependent in MCF-7 Cells

Although membrane-associated estrogen receptors (mERs) have been known to play important role in steroid induced signal transmission, we still know little about their function in the estrogen induced proliferation of breast cancer cells. In our current work we tried to separate membrane initiated estrogen receptor (ERα,) signaling from the overall estrogenic effect in MCF7 breast carcinoma cells. Re-analyzing expression data from multiple microarray experiments, we selected a set of key regulatory genes involved in proliferation regulation and estrogen signaling. Our quantitative real time-PCR results confirmed that the selective membrane receptor agonist, estrogen-BSA induces similarly pronounced expression changes regarding these genes as 17β-estradiol. Our light and electronmicroscopical studies revealed that the membrane-bound form of classical estrogen receptor alpha is internalized after ligand binding via dynamin dependent, caveola-mediated endocytosis. Inhibition of this internalization with dynamin inhibitor, dynasore practically abolished the regulatory effect of E2-BSA, suggesting that interaction and internalization with the scaffold protein is necessary for effective signaling. The physiological role of plasma membrane estrogen receptor alpha is intensively studied, yet there are still several aspects of it to be resolved. The dynamin-dependent, ligand mediated internalization of mERs seems to play an important role in estrogen signaling. Our results may serve as another example how membrane initiated and nuclear receptor signaling well-orchestrated is and form an integrated system.

17β-estradiol-induced growth of triple-negative breast cancer cells is prevented by the reduction of GPER expression after treatment with gefitinib.

Triple-negative breast cancers (TNBCs) are neither susceptible to endocrine therapy due to a lack of estrogen receptorα expression nor trastuzumab. TNBCs frequently overexpress epidermal growth factor receptor (EGFR) and membrane bound estrogen receptor, GPER. To a certain extent the growth of TNBCs is stimulated by 17β-estradiol via GPER. We analyzed whether inhibition of EGFR by gefitinib reduces the expression of GPER and subsequent signal transduction in TNBC cells. Dependence of proliferation on 17β-estradiol was determined using Alamar Blue assay. Expression of GPR30 and activation of c-src, EGFR and cAMP-responsive element binding (CREB) protein by 17β-estradiol was analyzed by western blotting. Expression of c-fos, cyclinD1 and aromatase was determined using RT-PCR. Gefitinib reduced GPER expression concentration‑and time‑dependently. In HCC70 cells, GPER expression was reduced to 15±11% (p<0.05) after treatment with 200nM gefitinib for four days, and in HCC1806 cells GPER expression was reduced to 39±5% (p<0.01) of the control. 17β-estradiol significantly increased the percentage of HCC1806 cells within 7days to 145±29% of the control (HCC70, 110±8%). This increase in cell growth was completely prevented in both TNBC cell lines after GPR30 expression was downregulated by treatment with 200nM gefitinib. In HCC1806 cells, activation of c-src was increased by 17β-estradiol to 350±50% (p<0.01), and gefitinib reduced src activation to 110%. Similar results were obtained in the HCC70 cells. Phosphorylation of EGFR increased to 240±40% (p<0.05) in the HCC1806 cells treated with 17β-estradiol (HCC70,147±25%). Gefitinib completely prevented this activation. Phosphorylation of CREB and induction of c-fos, cyclin D1 and aromatase expression by 17β-estradiol were all prevented by gefitinib. These experiments conclusively show that reduction of GPER expression is a promising therapeutic approach for TNBC.

Glycone-rich Soy Isoflavone Extracts Promote Estrogen Receptor Positive Breast Cancer Cell Growth

ABSTRACTDue to the association of hormone replacement therapy (HRT) with breast cancer risk, estrogenically active soy isoflavones are considered as an HRT alternative to alleviate menopausal symptoms. However, several recent reports challenged the health benefits of soy isoflavones and associated them with breast cancer promotion. While glyconic isoflavones are the major constituents of soybean seeds, due to their low cell permeability, they are considered to be biologically inactive. The glyconic isoflavones may exert their effects on membrane-bound estrogen receptors or could be converted to aglycones by extracellular β-glucosidases. Therefore, we hypothesized that despite their low cell permeability, soybean cultivars with high glyconic isoflavones may promote breast cancer cell growth. To test this, composition and estrogenic activity of isoflavones from 54 commercial soybean cultivars were determined. Soybean seeds produced in identical climate and growth conditions were used to minimize the effects of extraneous factors on isoflavone profile and concentrations. The glyconic daidzin concentration negatively correlated with genistin and with other aglycones. Relative to control, isoflavone extracts from 51 cultivars were estrogenic and promoted the growth of estrogen receptor positive (ER+) breast cancer cell line MCF-7 from 1.14 to 4.59 folds and other three cultivars slightly reduced the growth. Among these, extracts from three cultivars were highly estrogenic and promoted MCF-7 cell growth by 2.59–4.64 folds (P<0.005). Among six isoflavones, daidzin was positively associated with MCF-7 cell growth (P<0.005, r = 0.13966), whereas the negative correlation between genistin and MCF-7 cell growth was nearly significant (P≤0.0562, r = −0.026141). Furthermore, in drug interaction studies daidzin-rich isoflavone extracts antagonized tamoxifen, an ER inhibitor. Taken together, our results suggest that the glyconic daidzin-rich soy isoflavone extracts may exert estrogenic effects and promote ER+ breast cancer growth.

The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens.

Hippocampal cytosolic estrogen receptors regulate fear generalization in females

Generalization of fear responses is a symptom of many anxiety disorders and we have previously demonstrated that female rats generalize fear to a neutral context at a faster rate compared to males. This effect is due in part, to activation of ER and modulation of memory retrieval mechanisms resulting in fear generalization. Given that the effects of estradiol on fear generalization required approximately 24h, our data suggested possible genomic actions on fear generalization. To determine whether these actions were due to cytosolic versus membrane bound receptors, female rats were given infusions of ICI 182,780, a cytosolic estrogen receptor antagonist, into the lateral ventricle or dorsal hippocampus simultaneously with estradiol treatment or with an ER agonist (DPN). Infusions of ICI into the lateral ventricle or the dorsal hippocampus blocked fear generalization induced by peripheral or central treatment with estradiol or DPN, suggesting that estradiol acts through cytosolic ERβ receptors. In further support of these findings, intracerebroventricular or intra-hippocampal infusions of bovine serum conjugated estradiol (E2-BSA), activating membrane-bound estrogen receptors only, did not induce fear generalization. Moreover, rats receiving intra-hippocampal infusions of the ERK/MAPK inhibitor, U0126, continued to display estradiol-induced generalization, again suggesting that membrane-bound estrogen receptors do not contribute to fear generalization. Overall, these data suggest that estradiol-induced enhancements in fear generalization are mediated through activation of cytosolic/nuclear ER within the dorsal hippocampus. This region seems to be an important locus for the effects of estradiol on fear generalization although additional neuroanatomical regions have yet to be identified.

Estrogen Effects on Cognitive and Synaptic Health Over the Lifecourse.

Estrogen facilitates higher cognitive functions by exerting effects on brain regions such as the prefrontal cortex and hippocampus. Estrogen induces spinogenesis and synaptogenesis in these two brain regions and also initiates a complex set of signal transduction pathways via estrogen receptors (ERs). Along with the classical genomic effects mediated by activation of ER α and ER β, there are membrane-bound ER α, ER β, and G protein-coupled estrogen receptor 1 (GPER1) that can mediate rapid nongenomic effects. All key ERs present throughout the body are also present in synapses of the hippocampus and prefrontal cortex. This review summarizes estrogen actions in the brain from the standpoint of their effects on synapse structure and function, noting also the synergistic role of progesterone. We first begin with a review of ER subtypes in the brain and how their abundance and distributions are altered with aging and estrogen loss (e.g., ovariectomy or menopause) in the rodent, monkey, and human brain. As there is much evidence that estrogen loss induced by menopause can exacerbate the effects of aging on cognitive functions, we then review the clinical trials of hormone replacement therapies and their effectiveness on cognitive symptoms experienced by women. Finally, we summarize studies carried out in nonhuman primate models of age- and menopause-related cognitive decline that are highly relevant for developing effective interventions for menopausal women. Together, we highlight a new understanding of how estrogen affects higher cognitive functions and synaptic health that go well beyond its effects on reproduction.

PD47-03 EXPRESSION OF CYTOPLASMIC ERβ1 AND NUCLEAR ERβ2 IS ASSOCIATED WITH POOR OUTCOMES FOLLOWING RADICAL PROSTATECTOMY FOR LOCALIZED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Localized VII1 Apr 2015PD47-03 EXPRESSION OF CYTOPLASMIC ERβ1 AND NUCLEAR ERβ2 IS ASSOCIATED WITH POOR OUTCOMES FOLLOWING RADICAL PROSTATECTOMY FOR LOCALIZED PROSTATE CANCER George R. Schade, Sarah K. Holt, Xiaotun Zhang, Jonathan L. Wright, Shan Shan Zhao, Suzanne Kolb, Hung-Ming Lam, Linda Levin, Yuet-Kin Leung, Shuk-Mei Ho, and Janet Stanford George R. SchadeGeorge R. Schade More articles by this author , Sarah K. HoltSarah K. Holt More articles by this author , Xiaotun ZhangXiaotun Zhang More articles by this author , Jonathan L. WrightJonathan L. Wright More articles by this author , Shan Shan ZhaoShan Shan Zhao More articles by this author , Suzanne KolbSuzanne Kolb More articles by this author , Hung-Ming LamHung-Ming Lam More articles by this author , Linda LevinLinda Levin More articles by this author , Yuet-Kin LeungYuet-Kin Leung More articles by this author , Shuk-Mei HoShuk-Mei Ho More articles by this author , and Janet StanfordJanet Stanford More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2747AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Data regarding the expression of estrogen receptors (ER) and prostate cancer (PCa) outcomes are limited. We evaluated the relationship of expression profiles of ERβ; isoforms and the membrane bound estrogen receptor GPR30 with patient factors and outcomes following radical prostatectomy (RP). METHODS Tissue microarrays constructed from 566 men who underwent RP for localized PCa were analyzed with immunohistochemistry targeting ERβ1, ERβ2, ERβ5 and GPR30. An experienced, blinded pathologist scored receptor distributions and staining intensities. Demographic and clinical data were evaluated with descriptive statistics. Correlations between PCa ERβ; isoform and GPR30 expression and clinicopathologic data were analyzed with χ2 tests. Cox proportional hazards models were used to examine associations between each ER's staining pattern and time to recurrence and prostate cancer-specific mortality (PCSM). Receptor sub-type staining patterns found to have a significant association with recurrence or PCSM were further analyzed using the Kaplan-Meier method. Significance was set at α <0.05, two-tailed test. RESULTS PCa cells had unique staining patterns with ERβ1 demonstrating predominantly nuclear localization, while ERβ2, ERβ5 and GPR30 were predominantly cytoplasmic. Median follow-up among was 10.5 years. After controlling for patient factors, increasing cytoplasmic ERβ1 (cERβ1) (HR 1.53, 95% CI 1.01-2.32, p=0.047) and nuclear ERβ2 (nERβ2) (HR 1.55, 95% CI 1.01-2.38, p=0.043) staining were associated with significantly worse 5-year recurrence-free survival (RFS). Increasing cERβ1 (HR 4.67, 95% CI 1.80 – 12.11, p=0.002) and nERβ2 (HR 2.49, 95% CI 1.08 – 5.80, p=0.033) expression were also associated with significantly increased risk of PCSM. Patients with cERβ1 and nERβ2 co-staining had significantly worse 15-year PCSM vs. patients expressing only cERβ1, only nERβ2, or neither (16.4% vs. 4.3% vs. 0.0% vs 2.0 %, respectively p=0.001) (Figure). CONCLUSIONS Increased cERβ1 and nERβ2 expression are associated with worse PCa-specific outcomes following RP. These findings suggest that evaluating tumor ERβ1 and ERβ2 staining patterns following RP may provide prognostic information © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e962 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information George R. Schade More articles by this author Sarah K. Holt More articles by this author Xiaotun Zhang More articles by this author Jonathan L. Wright More articles by this author Shan Shan Zhao More articles by this author Suzanne Kolb More articles by this author Hung-Ming Lam More articles by this author Linda Levin More articles by this author Yuet-Kin Leung More articles by this author Shuk-Mei Ho More articles by this author Janet Stanford More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PD47-04 PATHOLOGICAL FEATURES OF THE INDEX AND NON-INDEX LESIONS IN RADICAL PROSTATECTOMY SPECIMENS: IMPLICATIONS FOR FOCAL THERAPY.

INTRODUCTION AND OBJECTIVES: Data regarding the expression of estrogen receptors (ER) and prostate cancer (PCa) outcomes are limited. We evaluated the relationship of expression profiles of ERb; isoforms and the membrane bound estrogen receptor GPR30 with patient factors and outcomes following radical prostatectomy (RP). METHODS: Tissue microarrays constructed from 566 men who underwent RP for localized PCa were analyzed with immunohistochemistry targeting ERb1, ERb2, ERb5 and GPR30. An experienced, blinded pathologist scored receptor distributions and staining intensities. Demographic and clinical data were evaluated with descriptive statistics. Correlations between PCa ERb; isoform and GPR30 expression and clinicopathologic data were analyzed with c2 tests. Cox proportional hazards models were used to examine associations between each ER’s staining pattern and time to recurrence and prostate cancer-specific mortality (PCSM). Receptor sub-type staining patterns found to have a significant association with recurrence or PCSM were further analyzed using the Kaplan-Meier method. Significance was set at a <0.05, two-tailed test. RESULTS: PCa cells had unique staining patterns with ERb1 demonstrating predominantly nuclear localization, while ERb2, ERb5 and GPR30 were predominantly cytoplasmic. Median follow-up among was 10.5 years. After controlling for patient factors, increasing cytoplasmic ERb1 (cERb1) (HR 1.53, 95% CI 1.01-2.32, p1⁄40.047) and nuclear ERb2 (nERb2) (HR 1.55, 95% CI 1.01-2.38, p1⁄40.043) staining were associated with significantly worse 5-year recurrence-free survival (RFS). Increasing cERb1 (HR 4.67, 95% CI 1.80 e 12.11, p1⁄40.002) and nERb2 (HR 2.49, 95% CI 1.08 e 5.80, p1⁄40.033) expression were also associated with significantly increased risk of PCSM. Patients with cERb1 and nERb2 co-staining had significantly worse 15-year PCSM vs. patients expressing only cERb1, only nERb2, or neither (16.4% vs. 4.3% vs. 0.0% vs 2.0 %, respectively p1⁄40.001) (Figure). CONCLUSIONS: Increased cERb1 and nERb2 expression are associated with worse PCa-specific outcomes following RP. These findings suggest that evaluating tumor ERb1 and ERb2 staining patterns following RP may provide prognostic information Source of Funding: R01-CA056678, R01-CA092579, R03CA137799, and P50-CA097186

Inhibition of GPR30 by estriol prevents growth stimulation of triple-negative breast cancer cells by 17β-estradiol.

BackgroundDue to the lack of ERα, triple negative breast cancers (TNBCs) are not susceptible to endocrine therapy using antiestrogens. However, the majority of TNBCs express the membrane bound estrogen receptor GPR30. We have recently shown that knock-down of GPR30 expression prevented growth stimulation of TNBC cell lines by 17β-estradiol. Now we analyzed whether specific inhibition of GPR30 represents a new option for therapy of TNBC.MethodsGrowth of TNBC cells was assessed using Alamar-blue colorimetric assay. Activation of c-Src and EGF-receptor was assessed using Western blots. Expression of c-fos, cyclin D1 and aromatase was quantified by RT-PCR. Gα-specific signaling of GPR30 was analyzed by electrophoretic mobility shift assay.ResultsHCC1806 cells showed the highest GPR30 expression, in HCC70 cells it was clearly lower, in MDA-MB-231 cells it was lowest. 10-8 M 17β-estradiol significantly increased proliferation of HCC1806 cells to 134 ± 12% of control (p < 0.01). Proliferation of HCC70 cells was slightly increased to 116 ± 8% of control. Estriol significantly reduced cell number of HCC1806 cells to 16 ± 12% (p < 0.01). Cell number of HCC70 cells and of MDA-MB-231 cells was reduced to 68 ± 25% and to 61 ± 10%, respectively.Activity of Src kinase increased to 150 ± 10% (p < 0.05) by 10-8 M 17β-estradiol treatment in HCC1806 and to 220 ± 20% in HCC70 cells (p < 0.01). Estriol treatment completely inhibited 17β-estradiol-induced p-src activation. Transactivation of EGF-receptor increased by estradiol treatment to 350% in HCC1806 and to 280% in HCC70 cells. Estriol completely suppressed EGF-receptor transactivation. c-fos expression increased to 260% and to 190%, respectively. Estriol reduced this induction to 160% (HCC1806) and below control in HCC70 cells. Cyclin D1 was induced to 290% (HCC1806) and 170% (HCC70) and completely inhibited by estriol. 17β-estradiol increased CREB-phosphorylation to 400%. Binding of phospho-CREB to a CRE of cyclin D1 was enhanced to 320%.ConclusionSpecific pharmacological inhibition of GPR30 might become a promising targeted therapy for TNBC in future.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-935) contains supplementary material, which is available to authorized users.

Estrogen and its role in thyroid cancer.

Proliferative thyroid diseases are more prevalent in females than in males. Upon the onset of puberty, the incidence of thyroid cancer increases in females only and declines again after menopause. Estrogen is a potent growth factor both for benign and malignant thyroid cells that may explain the sex difference in the prevalence of thyroid nodules and thyroid cancer. It exerts its growth-promoting effect through a classical genomic and a non-genomic pathway, mediated via a membrane-bound estrogen receptor. This receptor is linked to the tyrosine kinase signaling pathways MAPK and PI3K. In papillary thyroid carcinomas, these pathways may be activated either by a chromosomal rearrangement of the tyrosine receptor kinase TRKA, by RET/PTC genes, or by a BRAF mutation and, in addition, in females they may be stimulated by high levels of estrogen. Furthermore, estrogen is involved in the regulation of angiogenesis and metastasis that are critical for the outcome of thyroid cancer. In contrast to other carcinomas, however, detailed knowledge on this regulation is still missing for thyroid cancer.

Bisphenol A—Why an adverse outcome pathway framework needs to be applied

Bisphenol A (BPA) is one of the most widely used and extensively studied chemicals. Numerous studies have reported in vitro effects or animal adverse findings at BPA doses lower than the no observed adverse effect levels (NOAELs) established in regulatory toxicity studies and used for human health risk assessment. Intensive discussions on the adequacy and relevance of test systems have not satisfactorily resolved whether positive or negative animal and/or in vitro findings are more relevant for human health risk assessment purposes.BPA imperfectly mimics endogenous estrogens at membrane-bound estrogen receptors in the fM–nM concentration range, and may have downstream pleiotropic effects such as human seminoma proliferation and mammary gland hyperplasia after in utero exposure which are not detectable in regulatory toxicology studies. We argue that a structured approach like the OECD Adverse Outcome Pathway (AOP) framework is needed to help researchers in designing relevant studies, and risk assessors in evaluating them.The huge amount of experimental data generated for BPA has highlighted data gaps in basic biology and the shortcomings of current approaches to hazard characterization and risk assessment. Establishing AOPs for BPA, and other endocrine active chemicals, will require major scientific as well as training investments by all responsible stakeholders.

GPER functions as a tumor suppressor in triple-negative breast cancer cells

The orphan, membrane-bound estrogen receptor (GPER) is expressed at high levels in a large fraction of breast cancer patients and its expression is favorable for patients' survival. We investigated the role of GPER as a potential tumor suppressor in triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 using cell cycle analysis and apoptosis assay. The constitutive activity of GPER was investigated. GPER-specific activation with G-1 agonist inhibited breast cancer cell growth in concentration-dependent manner via induction of the cell cycle arrest in G2/M phase, enhanced phosphorylation of histone H3 and caspase-3-mediated apoptosis. Analysis of the methylation status of the GPER promoter in the triple-negative breast cancer cells and in tissues derived from breast cancer patients revealed that GPER amount is regulated by epigenetic mechanisms and GPER expression is inactivated by promoter methylation. Furthermore, GPER expression was induced by stress factors, such as radiation, and GPER amount inversely correlated with the p53 expression level. Overall, our results establish the protective role in breast cancer tumorigenesis, and the cell surface expression of GPER makes it an excellent potential therapeutic target for triple-negative breast cancer.

GPER functions as a tumor suppressor in MCF-7 and SK-BR-3 breast cancer cells

The orphan, membrane-bound estrogen receptor (GPER) is expressed at high levels in a large fraction of breast cancer patients, and its expression is favorable for patients' survival. We investigated the role of GPER as a potential tumor suppressor in MCF-7 and SK-BR-3 breast cancer cells. The effect of GPER agonist G-1 in cell culture was used to determine whether GPER inhibit cell growth. The methylation status of GPER promoter was investigated by methylation-specific PCR. GPER-specific agonist G-1 inhibited breast cancer cell proliferation in concentration-dependent manner via induction of the cell cycle arrest in M-phase, enhanced phosphorylation of histone 3 and cell apoptosis. Analysis of the methylation status of the GPER promoter in MCF-7 and SK-BR-3 cells revealed that GPER expression is regulated by epigenetic mechanisms and GPER expression is inactivated by promoter methylation. Overall, our results are consistent with our recent findings in triple-negative breast cancer cells, and the cell surface expression of GPER makes it an excellent potential therapeutic target for non-triple-negative breast cancer.

Vasodilation by GPER in mesenteric arteries involves both endothelial nitric oxide and smooth muscle cAMP signaling

Our previous work showed that chronic activation of the membrane-bound estrogen receptor GPR30/GPER significantly lowers blood pressure in ovariectomized hypertensive mRen2.Lewis female rats which may, in part, reflect direct vasodilatory actions. The current study assessed the hypothesis that cyclic adenosine monophosphate (cAMP) signaling contributes to GPER-mediated vasorelaxation. In mesenteric resistance arteries from intact Lewis females, relaxation to 17-β-estradiol (E2; 47±3% of phenylephrine contraction vs. vehicle 89±2%, P<0.001) or G-1 (44±8%, P<0.001) was blunted to a similar extent by denuding (P<0.001) or the nitric oxide synthase inhibitor l-NAME (P<0.001). In contrast, the cyclooxygenase inhibitor indomethacin did not alter vasodilation (P>0.05). The cAMP analog Rp-cAMPS partially attenuated vasodilation (65±7%, P<0.001), while the combination of l-NAME and Rp-cAMPS exhibited additive effects to effectively abolish vasorelaxation (P>0.05 vs. vehicle). Pretreatment of endothelium-intact vessels with the adenylyl cyclase inhibitor SQ (63±6%) or the guanylyl cyclase inhibitor ODQ (62±9%) both partially inhibited the response to G-1 (P<0.01), while pretreatment with the both inhibitors completely abolished vasorelaxation (P>0.05 vs. vehicle). In denuded vessels only SQ reduced the response (88±3%, P<0.001). Moreover, G-1 significantly increased intracellular cAMP levels in cultured mesenteric smooth muscle cells (P<0.05). We conclude that GPER-dependent vasorelaxation apparently involves both endothelial release of nitric oxide which activates guanylyl cyclase and smooth muscle cell activation of adenylyl cyclase. Downstream production of cyclic nucleotides and stimulation of protein kinases may phosphorylate proteins to promote vascular smooth muscle cell relaxation. The ability of GPER to initiate these signaling pathways may contribute to the beneficial vascular effects of estrogen.