Mediator Of Estrogen Action Research Articles

GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway

The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

Molecular Effects of Topical Estrogen on Vaginal Granulation Tissue in Postpartum Women.

The aims of this study were to evaluate the biomolecular properties of vaginal and perineal granulation tissue in postpartum women and assess the potential impact of vaginal estrogen application. We prospectively identified women referred to a subspecialty peripartum clinic between September 2016 and April 2018 who developed symptomatic perineal or vaginal granulation tissue. As part of routine clinical care, granulation tissue was excised from each participant by a urogynecologist and subjected to RNA extraction, real-time quantitative polymerase chain reaction, histologic evaluation, and immunohistochemistry. Serum steroid hormone levels were measured. Comparisons were made between participants who used topical vaginal estradiol (E2) and those who did not (non-E2 controls). Sixteen postpartum women were recruited for this pilot study. More than 30% of patients (n = 5, 31%) had used topical vaginal estradiol (E2) during their postpartum recovery. Histological appearance of granulation tissue evaluated by hematoxylin and eosin staining was similar in women treated with vaginal E2 and non-E2 controls. Both estrogen receptor α (ERα) and ERβ mRNA and ERα protein were readily detectable in the granulation tissue of E2-treated women. Although not statistically significant, participants who used topical E2 developed granulation tissue that exhibited local estrogen-responsive gene upregulation. Serum levels of estrone, E2, dehydroepiandrosterone, progesterone, and testosterone did not differ between vaginal E2-treated patients and controls. Estrogen receptor α seems to be the predominant receptor mediating estrogen action in postpartum perineal and vaginal granulation tissue. Vaginal E2 use does not seem to affect serum levels of estrone, E2, dehydroepiandrosterone, progesterone, and testosterone in postpartum women.

Role of GPER-Mediated Signaling in Testicular Functions and Tumorigenesis.

Estrogen signaling plays important roles in testicular functions and tumorigenesis. Fifteen years ago, it was discovered that a member of the G protein-coupled receptor family, GPR30, which binds also with high affinity to estradiol and is responsible, in part, for the rapid non-genomic actions of estrogens. GPR30, renamed as GPER, was detected in several tissues including germ cells (spermatogonia, spermatocytes, spermatids) and somatic cells (Sertoli and Leydig cells). In our previous review published in 2014, we summarized studies that evidenced a role of GPER signaling in mediating estrogen action during spermatogenesis and testis development. In addition, we evidenced that GPER seems to be involved in modulating estrogen-dependent testicular cancer cell growth; however, the effects on cell survival and proliferation depend on specific cell type. In this review, we update the knowledge obtained in the last years on GPER roles in regulating physiological functions of testicular cells and its involvement in neoplastic transformation of both germ and somatic cells. In particular, we will focus our attention on crosstalk among GPER signaling, classical estrogen receptors and other nuclear receptors involved in testis physiology regulation.

Estrogens in Human Male Gonadotropin Secretion and Testicular Physiology From Infancy to Late Puberty.

Several reports in humans as well as transgenic mouse models have shown that estrogens play an important role in male reproduction and fertility. Estrogen receptor alpha (ERα) and beta (ERβ) are expressed in different male tissues including the brain. The estradiol-binding protein GPER1 also mediates estrogen action in target tissues. In human testes a minimal ERα expression during prepuberty along with a marked pubertal up-regulation in germ cells has been reported. ERβ expression was detected mostly in spermatogonia, primary spermatocytes, and immature spermatids. In Sertoli cells ERβ expression increases with age. The aromatase enzyme (cP450arom), which converts androgens to estrogens, is widely expressed in human tissues (including gonads and hypothalamus), even during fetal life, suggesting that estrogens are also involved in human fetal physiology. Moreover, cP450arom is expressed in the early postnatal testicular Leydig cells and spermatogonia. Even though the aromatase complex is required for estrogen synthesis, its biological relevance is also related to the regulation of the balance between androgens and estrogens in different tissues. Knockout mouse models of aromatase (ArKO) and estrogen receptors (ERKOα, ERKOβ, and ERKOαβ) provide an important tool to study the effects of estrogens on the male reproductive physiology including the gonadal axis. High basal serum FSH levels were reported in adult aromatase-deficient men, suggesting that estrogens are involved in the negative regulatory gonadotropin feedback. However, normal serum gonadotropin levels were observed in an aromatase-deficient boy, suggesting a maturational pattern role of estrogen in the regulation of gonadotropin secretion. Nevertheless, the role of estrogens in primate testis development and function is controversial and poorly understood. This review addresses the role of estrogens in gonadotropin secretion and testicular physiology in male humans especially during childhood and puberty.

A loss of estrogen signaling in the aromatase deficient mouse penis results in mild hypospadias

Hypospadias is the abnormal opening of the urethra on the underside of the penis and occurs in approximately 1/125 live male births worldwide. The incidence rate of hypospadias has dramatically increased over the past few decades. This is now attributed, at least in part, to our exposure to endocrine-disrupting chemicals (EDCs) which alter the hormonal signals required for development of the penis. In humans androgens are the main drivers of fusion of the urethral folds to form the urethra within the shaft of the penis, a process required for termination of the urethra in its normal location at the tip of the penis. However, recent research has suggested that estrogen also plays a role in this process. To better understand how EDCs impact urethral development it is essential that we understand the normal function of hormones during development of the penis. To define the role of estrogen in urethral development we examined development of the penis in the aromatase (Cyp19a1) Knockout (ArKO) mouse strain in which endogenous estrogen production is completely ablated. We found that the ArKO penis had a mild hypospadias phenotype. The developing ArKO postnatal penis displayed an early disruption in preputial development, which likely causes the mild hypospadias observed in adults. Using qPCR, we found altered expression of keratin genes and key urethral patterning genes in response to the disrupted estrogen signaling. The hypospadias phenotype was almost identical to that reported for the estrogen receptor α (ERα) knockout confirming that ERα is the predominant receptor for mediating estrogen action during development of the mouse penis. Our results show that estrogen is required for normal prepucial development and placement of the mature urethral opening at the distal aspect of the penis. We also identified several genes which are potential downstream targets of estrogen during normal urethral closure. With this knowledge, we can now better understand how anti-estrogenic as well as estrogenic EDCs disrupt urethral closure to cause mild hypospadias in both mice and humans.

Association of ESR1 polymorphisms (rs3020314 and rs1514348) with breast cancer in Sudanese women. A pilot study

ESR1 encodes estrogen receptor alpha, which mediates estrogen action. Estrogen exposure is a central risk for breast cancer (BC); therefore, ESR1 variants are likely to affect BC susceptibility. We evaluated the association of ESR1 rs3020314 and rs1514348 with BC among Sudanese women, and correlated genotype with BC risk factors and clinicopathologic features. A total of 144 women were recruited in this case-control study (71 women with BC, and 73 healthy controls). ESR1 rs3020314 and rs1643821 –a proxy SNP for rs1514348–were genotyped using PCR-RFLP.Genotype frequencies differed significantly between cases and controls. For rs3020314, women carrying the heterozygous genotype CT had increased BC risk (OR: 2.67; 95%CI: 1.19–6.01; p = 0.02). Conversely, for rs1643821 women with the heterozygous genotype CT showed a decreased BC risk (OR: 0.41, 95%CI: 0.19–0.89; p = 0.02). Allele frequencies were not different between the two groups. In the subgroups of BMI ≥ 25 kg/m2 likewise postmenopausal women, patients carrying the rs3020314 genotype CT had 7-times and 6-times BC risk than controls, respectively. There was no association between genotype and clinicopathologic features.Our data suggest that ESR1 rs3020314 and rs1514348 might modify BC risk in Sudanese women. However, these findings need further testing in a larger number of patients.

In silico Molecular Modelling of Selected Natural Ligands and their Binding Features with Estrogen Receptor Alpha.

Breast cancer is one of the most common cancers diagnosed among women. It is now recognized that two receptors mediate estrogen action and the presence of estrogen receptor alpha (ERα) correlates with better prognosis and the likelihood of response to hormonal therapy. ERα is an attractive target for the treatment of breast cancer. Most of the drugs currently used for the breast cancer treatment have numerous side effects and they are often unsuccessful in removing the tumour completely. Hence, we focused on natural compounds like flavonoids, polyphenols, etc. which do not exhibit any high toxic effects against normal cells. To identify the potential natural inhibitors for BCa through an optimised in silico approach. Structural modification and molecular docking-based screening approaches were imposed to identify the novel natural compounds by using Schrödinger (Maestro 9.5). The Qikprop v3.5 was used for the evaluation of important ADME parameters and its permissible ranges. Cytotoxicity of the compounds was evaluated by MTT assay against MCF-7 Cell lines. From the docking studies, we found that the compounds, Myricetin, Quercetin, Apigenin, Luteolin and Baicalein showed the highest Glide Scores -10.78, -9.48, -8.92, -8.87 and -8.82 kcal mol-1 respectively. Of these, Luteolin and Baicalein showed the significant IC50 values (25 ± 4.0 and 58.3 ± 4.4 µM, respectively) against MCF-7 cell line. The ADME profiling of the test compounds was evaluated to find the drug-likeness and pharmacokinetic parameters. We mainly focused on in silico study to dock the compounds into the human estrogen receptor ligand binding domain (hERLBD) and compare their predicted binding affinity with known antiestrogens. Myricetin, Quercetin, Apigenin, Luteolin and Baicalein were identified as the most promising among all. Of these, Luteolin and Baicalein showed significant anticancer activities against MCF-7 cell line. These findings may provide basic information for the development of anti-breast cancer agents.

GREB1 is an estrogen receptor-regulated tumour promoter that is frequently expressed in ovarian cancer

Estrogenic hormone replacement therapy increases the risk of developing ovarian cancer, and estrogen promotes tumour initiation and growth in mouse models of this disease. GREB1 (Growth regulation by estrogen in breast cancer 1) is an ESR1 (estrogen receptor 1)-upregulated protein which may mediate estrogen action. GREB1 knockdown prevents hormone-driven proliferation of several breast and prostate cancer cell lines and prolongs survival of mice engrafted with ovarian cancer cells, but its mechanism of action remains unclear. In this study, we explored GREB1 function in ovarian cancer. GREB1 overexpression in ovarian cancer cell lines increased cell proliferation and migration and promoted a mesenchymal morphology associated with increased Col1a2, which encodes a collagen I subunit. GREB1 knockdown inhibited proliferation and promoted an epithelial morphology associated with decreased Col1a2. In human tissues, GREB1 was expressed in all ESR1-expressing tissues throughout the normal female reproductive tract, in addition to several tissues that did not show ESR1 expression. In a TMA of ovarian cancer cases, GREB1 was expressed in 75–85% of serous, endometrioid, mucinous, and clear cell carcinomas. Serous, endometrioid, and mucinous ovarian cancers were almost always positive for either ESR1 or GREB1, suggesting a possible reliance on signalling through ESR1 and/or GREB1. Targeting GREB1 may inhibit tumour-promoting pathways both downstream and independent of ESR1 and is therefore a possible treatment strategy worthy of further investigation.

Hepatocyte estrogen receptor alpha mediates estrogen action to promote reverse cholesterol transport during Western-type diet feeding

ObjectiveHepatocyte deletion of estrogen receptor alpha (LKO-ERα) worsens fatty liver, dyslipidemia, and insulin resistance in high-fat diet fed female mice. However, whether or not hepatocyte ERα regulates reverse cholesterol transport (RCT) in mice has not yet been reported. Methods and resultsUsing LKO-ERα mice and wild-type (WT) littermates fed a Western-type diet, we found that deletion of hepatocyte ERα impaired in vivo RCT measured by the removal of 3H-cholesterol from macrophages to the liver, and subsequently to feces, in female mice but not in male mice. Deletion of hepatocyte ERα decreased the capacity of isolated HDL to efflux cholesterol from macrophages and reduced the ability of isolated hepatocytes to accept cholesterol from HDL ex vivo in both sexes. However, only in female mice, LKO-ERα increased serum cholesterol levels and increased HDL particle sizes. Deletion of hepatocyte ERα increased adiposity and worsened insulin resistance to a greater degree in female than male mice. All of the changes lead to a 5.6-fold increase in the size of early atherosclerotic lesions in female LKO-ERα mice compared to WT controls. ConclusionsEstrogen signaling through hepatocyte ERα plays an important role in RCT and is protective against lipid retention in the artery wall during early stages of atherosclerosis in female mice fed a Western-type diet.

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1\u03b1/VEGF pathway in the breast tumor microenvironment

BackgroundThe G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells.MethodsWe analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1.ResultsWe first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation.ConclusionsThese findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies.

Protective Role of GPER Agonist G‐1 on Cardiotoxicity Induced by Doxorubicin

The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose-limiting cardiotoxicity in approximately 20% of patients. The G-protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G-1 reduced myocardial inflammation, improved immunosuppression, triggered pro-survival signaling cascades, improved myocardial mechanical performance, and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand-activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G-1 (50 μg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF-α, IL-1β, LDH, and ROS levels. Western blotting analysis of cardiac homogenates indicated that G-1 prevents the increase in p-c-jun, BAX, CTGF, iNOS, and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK, and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G-1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G-1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox-induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. J. Cell. Physiol. 232: 1640-1649, 2017. © 2016 Wiley Periodicals, Inc.

Estrogen Receptor β as a Therapeutic Target in Breast Cancer Stem Cells.

Background: Breast cancer cells with tumor-initiating capabilities (BSCs) are considered to maintain tumor growth and govern metastasis. Hence, targeting BSCs will be crucial to achieve successful treatment of breast cancer. Methods: We characterized mammospheres derived from more than 40 cancer patients and two breast cancer cell lines for the expression of estrogen receptors (ERs) and stem cell markers. Mammosphere formation and proliferation assays were performed on cells from 19 cancer patients and five healthy individuals after incubation with ER-subtype selective ligands. Transcriptional analysis was performed to identify pathways activated in ERβ-stimulated mammospheres and verified using in vitro experiments. Xenograft models (n = 4 or 5 per group) were used to study the role of ERs during tumorigenesis. Results: We identified an absence of ERα but upregulation of ERβ in BSCs associated with phenotypic stem cell markers and responsible for the proliferative role of estrogens. Knockdown of ERβ caused a reduction of mammosphere formation in cell lines and in patient-derived cancer cells (40.7%, 26.8%, and 39.1%, respectively). Gene set enrichment analysis identified glycolysis-related pathways (false discovery rate < 0.001) upregulated in ERβ-activated mammospheres. We observed that tamoxifen or fulvestrant alone was insufficient to block proliferation of patient-derived BSCs while this could be accomplished by a selective inhibitor of ERβ (PHTPP; 53.7% in luminal and 45.5% in triple-negative breast cancers). Furthermore, PHTPP reduced tumor initiation in two patient-derived xenografts (75.9% and 59.1% reduction in tumor volume, respectively) and potentiated tamoxifen-mediated inhibition of tumor growth in MCF7 xenografts. Conclusion: We identify ERβ as a mediator of estrogen action in BSCs and a novel target for endocrine therapy.

Effect of estradiol benzoate injection to male rabbits on glucose, total protein, albumin, calcium concentrations and prostate tissue

Objective: This study was carried out to investigate the effects of estradiol benzoate (EB) injection to male rabbits on glucose, total-protein, albumin, calcium concentrations and prostate tissue, and the role of prolactin as an important mediator of estrogen action in prostate. Materials and methods: Fifty four adult male rabbits were used in this study. The rabbits were randomly divided into two groups. Group A contained 36 male rabbits, which were further randomly divided into four sub-groups, three of them contained 10 rabbits and one sub-group contained 6 rabbits as control. Group B contained 18 male rabbits, which were divided randomly into three equal sub-groups. Three sub-groups of Groups A and B were treated once each on alternative day with the intramuscular injections of EB dosed at 40, 80 and 120 µg/rabbit, respectively for 20 days, whereas the fourth sub-group of Group A received no estradiol, and Group B received 1 mg Bromocriptine Mesilate in addition to EB through oral route on each alternative day. Blood samples were collected for measuring glucose, t-protein, albumin and calcium levels. Prostate tissue samples were collected from all the rabbits for histological studies. Results: Glucose was significantly ( P ?0.05) increased as a result of 80 µg EB injection, while significantly ( P ?0.05) decreased due to 40 and 120 µg EB injection. Total protein significantly ( P ?0.05) increased due to injection of 40 µg EB, however t-protein was not changed due to 40 and 120 µg injection. On the other hand, the results of albumin and calcium were not affected ( P ?0.05) by EB. In prostate tissues, EB induced hyperplasia with dysplasia or dysplasia only, but this effect was mild due to inhibition of prolactin. Conclusion: The injection of EB to male rabbits increased or decreased glucose level, increased t-protein level mildly or not changed, while albumin and calcium levels were not affected. EB induced hyperplasia on prostate tissue, and this effect was reduced by prolactin inhibition indicating that prolactin might have a role on the action of estrogen. http://doi.org/10.5455/javar.2017.d184

Molecular characterization and expression profile of the estrogen receptor α gene during different reproductive phases in Monopterus albus.

To understand the molecular mechanism of estrogen and to evaluate the role of the estrogen receptor in mediating estrogen action, the full-length cDNA of estrogen receptor α (ERα) was cloned from Monopterus albus, and its expression pattern and distribution were investigated. The ERα cDNA of M. albus includes an open reading frame of 1863 bp, a 140-bp 5’-untranslated region and a 797-bp 3’-untranslated region. Amino acid sequence homology analysis showed that the Monopterus albus ERα has a moderate degree of similarity with Sebastes schlegelii, Zoarces viviparus and Haplochromis burtoni (81.1%, 80.7% and 80.4%, respectively). Quantitative PCR results showed that the highest level of ERα expression was in the liver; the next highest level of expression was observed in the gonads, where it was expressed at high levels particularly in the ovary in developmental stages IV and V and in the testis in developmental stage II/III. Immunohistochemistry analysis showed that ERα was present as slender particles distributed mainly in the membranes of spermatocytes and oocytes in the testis and ovary, whereas no positive signal was observed in the cytoplasm of sperm cells. This report describes the first molecular characterization of full-length ERα and its tissue-specific distribution in M. albus.

Activated Platelets Induce Estrogen Receptor β Expression in Endometriotic Stromal Cells

Background/Aims: Endometriosis is viewed first and foremost as an estrogen-dependent disease, featuring not only excessive estrogen production but also aberrant expression of estrogen receptors (ERs), particularly ERβ, that mediate the estrogen action. ERβ is the predominant ER in mediating estrogen action in endometriosis, and estrogen plays a vital role in the development of endometriosis; thus, ERβ is viewed as a strong candidate for therapeutic targeting. Given our recent finding that platelets aggregate in endometriotic lesions, we sought to investigate whether activated platelets can upregulate ERβ. Methods: Using primary endometriotic stromal cells derived from patients with ovarian endometriomas and platelets harvested from healthy donors, we performed real-time RT-PCR analysis of mRNA abundance (n = 8) and Western blot analysis of protein expression (n = 8) of ERα and ERβ when co-cultured with phosphate-buffered saline, platelets, thrombin alone, and platelets plus thrombin for 48 h. Results: Treatment of endometriotic stromal cells with activated platelets resulted in the upregulation of ERβ gene and protein expression. Conclusion: In the presence of aggregated and thus activated platelets in endometriotic lesions, ERβ, but not ERα, is upregulated in endometriotic stromal cells. Our result suggests that the use of antiplatelet therapy may have potential in the treatment of endometriosis.

Gene-environment Interaction Between Estrogen Receptor (ER) SS, ApoE and Air Pollution On Cognitive Performance in an Elderly, Female Cohort.

Introduction In the brain, estrogen plays an important role in neural plasticity and its decline during menopause might result in cognitive impairment and predispose to the development of dementia. Two estrogen receptors – α and s – mediate estrogen actions either as ligand-activated dimeric transcription factors or as membrane-embedded ER monomers. Objective To analyze ERs-variants and ApoE-genotype in the population-based SALIA cohort of 834 non-demented, elderly women. Aims We aimed to explore, whether ERs variants may impact on cognitive performance in interaction with air pollution and APOE. Methods DNA was isolated from lymphocytes. ApoE-epsilon-variants and ERs SNPs rs944045, rs1256062, rs10144225 and rs2274705 were determined by LCG /KBioscience (Hoddesdon, UK). All participants were assessed for cognitive impairment by the CERAD-Plus test battery. Subtests were merged to a total z-score, that corrected for gender, age and time of education. Long-term exposure to air pollution was backextrapolated from data collected by monitoring stations using a geographic information system. Results SNP analysis revealed that 3 ERs variants (rs1256062, rs10144225 and rs2274705) were significantly correlated with lower CERAD z total scores and especially with decline in episodic memory. These effects were strengthened in APOE-epsilon-E4 carriers. Air pollution (NO2, PM2,5) did not worsen the CERAD z total score of the women, but interacting with the ERs variants PM2,5 affected their visuoconstructive practice and NO2 their semantic memory. Conclusion In this population-based, prospective study a gene-gene interaction effect of ApoE and ERs on cognitive performance appeared. Moreover, significant gene-environment interactions on cognition were found for air pollution.

Polymorphisms in estrogen receptors predict the risk of male infertility: a meta-analysis.

BackgroundEstrogen receptors play an important role in mediating estrogen action on target tissues, and the estrogen is relevant to male infertility. Single nucleotide polymorphisms (SNPs) in estrogen receptors may be associated with the risk of male infertility. A variety of case control studies have been published evaluating this association. However, the accumulated studies have shown inconsistent conclusions.MethodsTo further determine the potential association between the four common SNPs (rs2234693, rs9340799, rs1256049 and rs4986938) in estrogen receptors gene and male infertility, this meta-analysis was performed according to the 10 published case control studies. The odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the strength of the associations.ResultsIt was revealed that the sub-group analysis by the ethnicity, for the rs2234693, a significant association in the comparison of CC vs. TT (OR = 0.61, 95% CI: 0.40-0.93), CT vs. TT (OR = 0.67, 95% CI: 0.49-0.93) and CC + CT vs. TT (OR = 0.66, 95% CI: 0.49-0.89) in the Asian population with male infertility. For rs9340799 polymorphism, increased risks were observed for the comparison of AA vs. GG (OR = 1.75, 95% CI: 1.15-2.68) and AA vs. GA + GG (OR = 1.38, 95% CI: 1.02-1.88). For rs1256049 polymorphism, the comparison of the GA vs. GG (OR = 1.52, 95% CI: 1.00-2.31) and AA + GA vs. GG (OR = 1.74, 95% CI: 1.03-2.94), also increased risks present in Asian and Caucasian population, respectively.ConclusionsThe rs2234693C allele was associated with the decreased risk for male infertility; however, the rs9340799AA genotype and the rs1256049GA genotype were associated with an increased risk for male infertility.Electronic supplementary materialThe online version of this article (doi:10.1186/1477-7827-12-79) contains supplementary material, which is available to authorized users.

Significance of microRNA targeted estrogen receptor in male fertility.

Estrogen receptor-alpha (ERα) mediates estrogen action in regulation of different levels of the hypothalamic-pituitary-testis axis. It has a key role in spermatogenesis. Estrogen receptor alpha knock-out (ER koα) male mice were infertile and severe impairment in spermatogenesis and seminiferous tubules was observed. Recently, it has been reported that microRNA (miRNA) mir-100 and let-7b were predicted to target ERα gene. MiRNA are small, endogenous, single stranded RNA molecules that regulate gene expression and have been implicated in various disease states. It has been proved that some miRNAs expression is tissue- and disease-specific, giving potential for identifying miRNAs as a diagnostic tool. In this study, the change in the expression levels of mir-100, let-7b and ERα expression levels were evaluated in oligospermic infertile patients (n=43) compared to control fertile subjects (n=43). After washing and separating sperms, total RNA was isolated and then cDNA was synthesized. The expression levels of mir-100 and let-7b and ERα were evaluated by real time PCR. Mir-100, let-7b levels were significantly higher than those in control group (P=0.008 and P=0.009, respectively). We have found that, ERα level was significantly decreased in comparison with normal group (P< 0.0001). Changes in mir-100, let-7b and ERα expression levels in oligospermic patients may be associated with the susceptibility and progression of infertility. The results of this study indicate that miRNA can have a key role in spermatogenesis and might have a diagnostic and prognostic value in men infertility.

GPER signaling in spermatogenesis and testicular tumors

Estrogens play important roles in the regulation of testis development and spermatogenesis. Moreover, several evidences suggest that estrogen signaling can be involved in testicular tumorigenesis. The physiological effects of estrogen are mediated by the classical nuclear estrogen receptors ESR1 and 2, which regulate both genomic and rapid signaling events. In the recent years, a member of the seven-transmembrane G protein-coupled receptor family, GPR30 (GPER), has been identified to promote estrogen action in target cells including testicular cells. Ours and other studies reported that GPER is expressed in normal germ cells (spermatogonia, spermatocytes, spermatids), somatic cells (Sertoli and Leydig cells), and it is also involved in mediating estrogen action during spermatogenesis and testis development. In addition, GPER seems to be involved in modulating estrogen-dependent testicular cancer cell growth. However, in this context, the effects of GPER stimulation on cell survival and proliferation appear to be cell type specific. This review summarizes the current knowledge on the functions regulated by estrogens and mediated by GPER in normal and tumor testicular cells.

Abstract LB-152: Local estrogen formation and signaling through amphiregulin/EGFR may be implicated in human hepatocellular carcinoma: a unifying hypothesis.

Abstract Although there is evidence that hints at a potential role of estrogen in hepatocellular carcinoma (HCC), the use of antiestrogens has failed to improve survival of patients. We have investigated aromatase-driven estrogen formation in nontumoral and malignant human liver tissues and cells, also in relation to the expression of estrogen receptor (ER) α, ERβ and their splicing variants and of amphiregulin (AREG), an EGFR ligand, aiming to get insights in the potential role of estrogen and the underlying mechanism(s) in HCC. Chromatographic and exon-specific RT-PCR analyses were used to assess activity and expression of aromatase and the expression of wild-type (hERα66, hERβ1) and variant (hERα46, hERα36; hERβ2/Cx, hERβ5) ER, both in vivo and in vitro. Following incubation of tissue minces or cells with either testosterone or androstenedione used as precursor, human HCC tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates of 20% at 24h and &amp;gt;95% at 72h. By contrast, no aromatase activity could be detected in nontumoral hepatic tissues and HA22T liver cancer. Cirrhotic samples exhibited variable enzyme activity, with average estrogen formation rates of 4-8%, while Huh7 HCC cells gave rise to 34% estrogen formation. Markedly lower aromatase mRNA levels were observed in HA22T cells and nontumoral liver tissues, as compared to HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels. Interestingly, no or low expression of wild type ERα and ERβ could be detected in liver cancer cells and malignant tissues. Conversely, equivalent amounts of the hERα46 variant were observed in both cells and tissues, while hERα36 expression was highest in HepG2 cells and HCC samples, intermediate in Huh7 cells and cirrhotic tissues, and very low in HA22T cells and nontumoral liver. Only occasional expression of the two ERβ variants was observed in liver cancer cells and tissues. It is noteworthy that the pattern of hERα36 expression was strictly related to that of aromatase, suggesting that this ERα variant may be primarily implicated as a mediator of estrogen action in the malignant liver. AREG expression was consistently associated with that of aromatase, with nearly 3-fold and 8-fold higher levels being seen in HepG2 cells than in Huh7 cells (P=0,002) and HA22T cells (P=0,0014), respectively. This evidence suggests that AREG expression may be upregulated by estrogens in human HCC by binding to the membrane-associated ERα36 variant. Based on present and previous experimental evidence, we propose here a unifying hypothetical model whereby locally elevated, aromatase-driven, estrogen formation, combined with a shift in ER wild type/variant expression and the activation of rapid AREG signaling, may be primarily implicated in development and/or progression of human HCC and other neoplastic or chronic human diseases. Citation Format: Giuseppe Carruba, Vitale Miceli, Letizia Cocciadiferro, Lucia M. Polito, Biagio Agostara, Orazia M. Granata. Local estrogen formation and signaling through amphiregulin/EGFR may be implicated in human hepatocellular carcinoma: a unifying hypothesis. [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 LB-152. doi:10.1158/1538-7445.AM2013-LB-152