Novel role of estrogen receptor-α on regulating chondrocyte phenotype and response to mechanical loading.

OBJECTIVE: In knee cartilage from patients with osteoarthritis (OA), both preserved cartilage and damaged cartilage are observed. In this study, we aim to compare preserved with damaged cartilage to identify the molecule(s) that may be responsible for the mechanical loading-induced differences within cartilage degradation. METHODS: Preserved and damaged cartilage were harvested from the same OA knee joint. RNA Sequencing was performed to examine the transcriptomic differences between preserved and damaged cartilage cells. Estrogen receptor-α (ERα) was identified, and its function of was tested through gene knockin and knockout. The role of ERα in mediating chondrocyte response to mechanical loading was examined via compression of chondrocyte-laded hydrogel in a strain-controlled manner. Findings from the studies on human samples were verified in animal models. RESULTS: Level of estrogen receptor α (ERα) was significantly reduced in damaged cartilage compared to preserved cartilage, which were observed in both human and mice samples. Knockdown of ESR1, the gene encoding ERα, resulted in an upregulation of senescence- and OA-relevant markers in chondrocytes. Conversely, knockin of ESR1 partially reversed the osteoarthritic and senescent phenotype of OA chondrocytes. Using a three-dimensional (3D) culture model, we demonstrated that mechanical overload significantly suppressed ERα level in chondrocytes with concomitant upregulation of osteoarthritic phenotype. When ESR1 expression was suppressed, mechanical loading enhanced hypertrophic and osteogenic transition. CONCLUSION: Our study demonstrates a new estrogen-independent role of ERα in mediating chondrocyte phenotype and its response to mechanical loading, and suggests that enhancing ERα level may represent a new method to treat osteoarthritis.

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Oestrogen receptor α in T cells controls the T cell immune profile and glucose metabolism in mouse models of gestational diabetes mellitus.

AIMS/HYPOTHESIS: The imbalance between maternal insulin resistance and a relative lack of insulin secretion underlies the pathogenesis of gestational diabetes mellitus (GDM). Alterations in T cell subtypes and increased levels of circulating proinflammatory cytokines have been proposed as potential mechanisms underlying the pathophysiology of insulin resistance in GDM. Since oestrogen modulates T cell immunity, we hypothesised that oestrogen plays a homeostatic role in visceral adipose tissue by coordinating T cell immunity through oestrogen receptor α (ERα) in T cells to prevent GDM. METHODS: Female CD4-cre ERαfl/fl (KO) mice on a C57BL/6 background with ERα ablation specifically in T cells, and ERαfl/fl (ERα-floxed [FL]) mice were fed 60 kJ% high-fat diet (HFD) for 4 weeks. Female mice mated with male BALB/c mice to achieve allogenic pregnancy and were maintained on an HFD to generate the GDM model. Mice were divided into four experimental groups: non-pregnant FL, non-pregnant KO, pregnant FL (FL-GDM) and pregnant KO (KO-GDM). GTTs and ITTs were performed on day 12.5 or 13.5 and 16.5 after breeding, respectively. On day 18.5 after breeding, mice were killed and T cell subsets in the gonadal white adipose tissue (gWAT) and spleen were analysed using flow cytometry. Histological examination was also conducted and proinflammatory gene expression in gWAT and the liver was evaluated. RESULTS: KO mice that mated with BALB/c mice showed normal fertility rates and fetal weights as compared with FL mice. Body and tissue weights were similar between FL and KO mice. When compared with FL-GDM mice, KO-GDM mice showed decreased insulin secretion (serum insulin concentration 15 min after glucose loading: 137.3 ± 18.3 pmol/l and 40.1 ± 36.5 pmol/l, respectively; p < 0.05), impaired glucose tolerance (glucose AUC in GTT: 2308.3 ± 54.0 mmol/l × min and 2620.9 ± 122.1 mmol/l × min, respectively; p < 0.05) and increased numbers of T helper (Th)17 cells in gWAT (0.4 ± 0.0% vs 0.8 ± 0.1%; p < 0.05). However, the contents of Th1 and regulatory T cells (Tregs) in gWAT remained similar between FL-GDM and KO-GDM. Glucose-stimulated insulin secretion was similar between isolated islets derived from FL and KO mice, but was reduced by IL-17A treatment. Moreover, the levels of proinflammatory gene expression, including expression of Emr1 and Tnfa in gWAT, were significantly higher in KO-GDM mice than in FL-GDM mice (5.1-fold and 2.7-fold, respectively; p < 0.01 for both). Furthermore, KO-GDM mice showed increased expression of genes encoding hepatokines, Ahsg and Fgf21 (both were 2.4-fold higher vs FL-GDM mice; p < 0.05 and p = 0.09, respectively), with no changes in inflammatory gene expression (e.g., Tnfa and Ifng) in the liver compared with FL-GDM mice. CONCLUSIONS/INTERPRETATION: Deletion of ERα in T cells caused impaired maternal adaptation of insulin secretion, changes in hepatokine profiles, and enhanced chronic inflammation in gWAT alongside an abnormal increase in Th17 cells. These results suggest that the ERα-mediated oestrogen signalling effects in T cells regulate T cell immunity and contribute to glucose homeostasis in pregnancy.

The Role of 17ß-Estrogen in Escherichia coli Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation.

Estrogen, the predominant sex hormone, has been found to be related to the occurrence of vaginal infectious diseases. However, its role in the occurrence and development of bacterial vaginitis caused by Escherichia coli is still unclear. The objective of this study was to investigate the role of 17ß-estrogen in E. coli adhesion on human vaginal epithelial cells. The vaginal epithelial cell line VK2/E6E7 was used to study the molecular events induced by estrogen between E. coli and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation with cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by Western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα-FAK signaling cascade. The results showed that, following stimulation with 1,000 nM estrogen for 48 h, transient activation of ERα and FAK was observed, as was an increased average number of E. coli cells adhering to vaginal epithelial cells. In addition, estrogen-induced activation of ERα and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 µM concentration and acted for 1 h, and a decrease in the number of adherent E. coli cells was observed simultaneously. However, this inhibitory effect diminished as the concentration of estrogen increased. In conclusion, FAK and ERα signaling cascades were associated with the increasing E. coli adherence to vaginal epithelial cells, which was promoted by a certain concentration of estrogen.

ERα36-GPER1 Collaboration Inhibits TLR4/NFκB-Induced Pro-Inflammatory Activity in Breast Cancer Cells.

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.

Estrogen Receptors Alpha and Beta Mediate Synaptic Transmission in the PFC and Hippocampus of Mice.

Distinct from ovarian estradiol, the steroid hormone 17ß-estradiol (E2) is produced in the brain and is involved in numerous functions, particularly acting as a neurosteroid. However, the physiological role of E2 and the mechanism of its effects are not well known. In hippocampal slices, 17ß-estradiol has been found to cause a modest increase in fast glutamatergic transmission; because some of these effects are rapid and acute, they might be mediated by membrane-associated receptors via nongenomic action. Moreover, activation of membrane estrogen receptors can rapidly modulate neuron function in a sex-specific manner. To further investigate the neurological role of E2, we examined the effect of E2, as an estrogen receptor (ER) agonist, on synaptic transmission in slices of the prefrontal cortex (PFC) and hippocampus in both male and female mice. Whole-cell recordings of spontaneous excitatory postsynaptic currents (sEPSC) in the PFC showed that E2 acts as a neuromodulator in glutamatergic transmission in the PFC in both sexes, but often in a cell-specific manner. The sEPSC amplitude and/or frequency responded to E2 in three ways, namely by significantly increasing, decreasing or having no response. Additional experiments using an agonist selective for ERß, diarylpropionitrile (DPN) showed that in males the sEPSC and spontaneous inhibitory postsynaptic currents sIPSC responses were similar to their E2 responses, but in females the estrogen receptor ß (ERß) agonist DPN did not influence excitatory transmission in the PFC. In contrast, in the hippocampus of both sexes E2 potentiated the gluatmatergic synaptic transmission in a subset of hippocampal cells. These data indicate that activation of E2 targeting probably a estrogen subtypes or different downstream signaling affect synaptic transmission in the brain PFC and hippocampus between males versus females mice.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

The transcriptional co-activator NCOA6 promotes estrogen-induced GREB1 transcription by recruiting ERα and enhancing enhancer-promoter interactions.

Estrogen and its cognate receptor, ERα, regulate cell proliferation, differentiation, and carcinogenesis in the endometrium by controlling gene transcription. ERα requires co-activators to mediate transcription via mechanisms that are largely uncharacterized. Herein, using growth-regulating estrogen receptor binding 1 (GREB1) as an ERα target gene in Ishikawa cells, we demonstrate that nuclear receptor co-activator 6 (NCOA6) is essential for estradiol (E2)/ERα-activated GREB1 transcription. We found that NCOA6 associates with the GREB1 promoter and enhancer in an E2-independent manner and that NCOA6 knockout reduces chromatin looping, enhancer-promoter interactions, and basal GREB1 expression in the absence of E2. In the presence of E2, ERα bound the GREB1 enhancer and also associated with its promoter, and p300, myeloid/lymphoid or mixed-lineage leukemia protein 4 (MLL4), and RNA polymerase II were recruited to the GREB1 enhancer and promoter. Consequently, the levels of the histone modifications H3K4me1/3, H3K9ac, and H3K27ac were significantly increased; enhancer and promoter regions were transcribed; and GREB1 mRNA was robustly transcribed. NCOA6 knockout reduced ERα recruitment and abolished all of the aforementioned E2-induced events, making GREB1 completely insensitive to E2 induction. We also found that GREB1-deficient Ishikawa cells are much more resistant to chemotherapy and that human endometrial cancers with low GREB1 expression predict poor overall survival. These results indicate that NCOA6 has an essential role in ERα-mediated transcription by increasing enhancer-promoter interactions through chromatin looping and by recruiting RNA polymerase II and the histone-code modifiers p300 and MLL4. Moreover, GREB1 loss may predict chemoresistance of endometrial cancer.

A critical role for estrogen signaling in penis development.

Hypospadias, a developmental defect of the penis, is one of the most common congenital malformations in humans. Its incidence has rapidly increased over recent decades, and this has been largely attributed to our increased exposure to endocrine-disrupting chemicals. Penis development is primarily an androgen-driven process; however, estrogen and xenoestrogens are known to affect penis development in both humans and mice. Here, we investigated the role of estrogen in the developing penis. Using a novel penis culture system, we showed that exogenous estrogen directly targets the developing penis in utero to cause hypospadias. In addition, we also uncovered an unexpected endogenous role for estrogen in normal postnatal penis development and showed that a loss of estrogen signaling results in a mild hypospadias phenotype, the most common manifestation of this disease in humans. Our findings demonstrated that both androgen and estrogen signaling are intrinsically required for normal urethral closure. These findings confirmed that penis development is not an entirely androgen-driven process but one in which endogenous estrogen signaling also plays a critical role.-Govers, L. C., Phillips, T. R., Mattiske, D. M., Rashoo, N., Black, J. R., Sinclair, A., Baskin, L. S., Risbridger, G. P., Pask, A. J. A critical role for estrogen signaling in penis development.

Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Polycystic ovary syndrome (PCOS) is a hypothalamic-pituitary-gonadal (HPG) axis disorder. PCOS symptoms most likely result from a disturbance in the complex feedback regulation system of the HPG axis, which involves gonadotrophic hormones and ovarian steroid hormones. However, the nature of this complex and interconnecting feedback regulation makes it difficult to dissect the molecular mechanisms responsible for PCOS phenotypes. Global estrogen receptor α (ERα) knockout (KO) mice exhibit a disruption of the HPG axis, resulting in hormonal dysregulation in which female ERα KO mice have elevated levels of serum estradiol (E2), testosterone, and LH. The ERα KO females are anovulatory and develop cystic hemorrhagic ovaries that are thought to be due to persistently high circulating levels of LH from the pituitary. However, the role of ERα in the pituitary is still controversial because of the varied phenotypes reported in pituitary-specific ERα KO mouse models. Therefore, we developed a mouse model where ERα is reintroduced to be exclusively expressed in the pituitary on the background of a global ERα-null (PitERtgKO) mouse. Serum E2 and LH levels were normalized in PitERtgKO females and were comparable to wild-type serum levels. However, the ovaries of PitERtgKO adult mice displayed a more overt cystic and hemorrhagic phenotype when compared with ERα KO littermates. We determined that anomalous sporadic LH secretion caused the severe ovarian phenotype of PitERtgKO females. Our observations suggest that pituitary ERα is involved in the estrogen negative feedback regulation, whereas hypothalamic ERα is necessary for the precise control of LH secretion. Uncontrolled, irregular LH secretion may be the root cause of the cystic ovarian phenotype with similarities to PCOS.-Arao, Y., Hamilton, K. J., Wu, S.-P., Tsai, M.-J., DeMayo, F. J., Korach, K. S. Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

The Role of ERα36 in Development and Tumor Malignancy.

Estrogen nuclear receptors, represented by the canonical forms ERα66 and ERß1, are the main mediators of the estrogen-dependent pathophysiology in mammals. However, numerous isoforms have been identified, stimulating unconventional estrogen response pathways leading to complex cellular and tissue responses. The estrogen receptor variant, ERα36, was cloned in 2005 and is mainly described in the literature to be involved in the progression of mammary tumors and in the acquired resistance to anti-estrogen drugs, such as tamoxifen. In this review, we will first specify the place that ERα36 currently occupies within the diversity of nuclear and membrane estrogen receptors. We will then report recent data on the impact of ERα36 expression and/or activity in normal breast and testicular cells, but also in different types of tumors including mammary tumors, highlighting why ERα36 can now be considered as a marker of malignancy. Finally, we will explain how studying the regulation of ERα36 expression could provide new clues to counteract resistance to cancer treatments in hormone-sensitive tumors.

Role of Differential Estrogen Receptor Activation in Airway Hyperreactivity and Remodeling in a Murine Model of Asthma.

Evidence suggests that airway hyperresponsiveness (AHR) is a characteristic feature of asthma. Epidemiological studies have confirmed that the severity of asthma is greater in women, suggesting a critical role of female sex steroid hormones (especially estrogen). Very few in vivo studies have examined the role of sex steroid hormones in asthma, and the sequence of events that occur through differential activation of estrogen receptors (ERs) remains to be determined in asthmatic airways. Our recent in vitro findings indicated that ERß had increased expression in asthmatic airway smooth muscle (ASM), and that its activation by an ERß-specific agonist downregulated airway remodeling. In this study, we translated the in vitro findings to a murine asthma model and examined the differential role of ER activation in modulating lung mechanics. C57BL/6J male, female, and ovariectomized mice were exposed to mixed allergen (MA) and subcutaneously implanted with sustained-release pellets of placebo, an ERα agonist (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [PPT]), and/or an ERß agonist (WAY-200070). We then evaluated the effects of these treatments on airway mechanics, biochemical, molecular, and histological parameters. Mice exposed to MA showed a significant increase in airway resistance, elastance, and tissue damping, and a decrease in compliance; pronounced effects were observed in females. Compared with PPT, WAY treatment significantly reversed the MA-induced changes. The increased mRNA/protein expression of ERα, ERß, and remodeling genes observed in MA-treated mice was significantly reversed in WAY-treated mice. This novel study indicates that activation of ERß signaling downregulates AHR and airway remodeling, and is a promising target in the development of treatments for asthma.

Single-cell profiling reveals that eRNA accumulation at enhancer-promoter loops is not required to sustain transcription.

Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer-promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ERα, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer-promoter loops is not required to sustain target gene transcription.

Lupeol, the major compound of the dichloromethane extract of Millettia macrophylla Benth (Fabaceae), displays estrogenic effects in ovariectomized rats.

The present work deals with the assessment of the in vitro and in vivo estrogenic effects of the triterpenoids (lupenone, lupeol, and stigmastenone) isolated from Millettia macrophylla extract. The in vitro estrogenicity was performed by a reporter gene assay and estrogen receptor-α (ERα) target gene expression, whereas the in vivo estrogenicity was evaluated by a 3-day uterotrophic assay in ovariectomized rats. As results, lupenone and lupeol did not transactivate ERα as well as ERß of human embryonic kidney 293T (HEK293T) cells. However, lupeol seems to be antagonistic to estrogen (E2) only in HEK293T-ERα (10-9 and 10-8  µM). Furthermore, lupeol slightly upregulated GREB1 gene expression at the concentration of 1 µM, suggesting a weak activation of endogenous ERα. In vivo, only lupeol at a dose of 1 mg/kg significantly increased the uterine wet weight (p < 0.05), uterine (p < 0.05), and vaginal (p < 0.01) epithelial heights. The concomitant administration of lupeol (1 mg/kg) with a pure antiestrogen fulvestrant abrogated its effects only in the vagina, whereas in combination with E2, lupeol exhibited a significant antiestrogen-like effect in uterine wet weight and synergistic effects on endometrium. Lupeol has estrogenic effects that is partly through ERs transcriptional activity and does involve alternative mechanisms that are still to be uncovered.

Placental supernatants' enhancement of the metastatic potential of breast cancer cells: is estrogen receptor (ERα) essential for this phenomenon?

PURPOSE: Pregnancy-associated breast cancer (PABC) is usually diagnosed at an advanced stage in comparison to non-pregnant women. The placenta secretes hormones and cytokines, which affect breast cancer progression. Previously, we demonstrated that human placental secretome facilitates the survival and migration of ERα+ breast cancer cells (BCCL), but pregnant women have a relatively high frequency of ERα-negative tumors. In the current study, we analyzed the effect of placental secretome on ERα-negative BCCL. METHODS: BCCL [MCF-7(estrogen/progesterone receptor positive (ERα+/PR+), ERα reduced MCF-7 (siRNA, MCF-7 ERα-), HS-578 and BT-549 cells (both ER-/PR-)] were exposed to supernatants (collected from first trimester human placental explants and from control BCCL) or to E2 + P4 (estrogen + progesterone) in placental supernatant concentrations and then tested for cell proliferation (number, cell cycle, PCNA), cell-death, cell migration, STAT3 pathway activation and functionality. RESULTS: Silencing ERα in the MCF-7 cells negated the placental supernatant and E2 + P4 enhancement of cell migration (> 130%, p < 0.05), number (> 120%) and survival (~ 130%). However, it had no such effect on MCF-7-ER- migration, which was still elevated in the presence of placental secretome. ER-/PR- BCCL were unaffected by the hormones, but placental secretome significantly elevated their migration (115%), number (140-170%), STAT3 phosphorylation (~ 180%) and BT-549 STAT3 level. These effects were negated by the STAT3 inhibitor. CONCLUSIONS: Placental supernatant facilitates BCCL malignant characteristics by activating ERα in estrogen responsive cells and STAT3 in ERα- BCCL. This indicates a possible mechanism that may underlie PABC's advanced state and suggests STAT3 pathway as a therapeutic target for PABC.

Impact of estrogens and estrogen receptor-α in brain lipid metabolism.

Estrogens and their receptors play key roles in regulating body weight, energy expenditure, and metabolic homeostasis. It is known that lack of estrogens promotes increased food intake and induces the expansion of adipose tissues, for which much is known. An area of estrogenic research that has received less attention is the role of estrogens and their receptors in influencing intermediary lipid metabolism in organs such as the brain. In this review, we highlight the actions of estrogens and their receptors in regulating their impact on modulating fatty acid content, utilization, and oxidation through their direct impact on intracellular signaling cascades within the central nervous system.

Interaction of WBP2 with ERα increases doxorubicin resistance of breast cancer cells by modulating MDR1 transcription.

BACKGROUND: Surgery combined with new adjuvant chemotherapy is the primary treatment for early stage invasive and advanced stage breast cancer. Growing evidence indicates that patients with ERα-positive breast cancer show poor response to chemotherapeutics. However, ERα-mediated drug-resistant mechanisms remain unclear. METHODS: Levels of WW domain-binding protein 2 (WBP2) and drug-resistant gene were determined by western blotting and RT-PCR, respectively. Cell viability was measured by preforming MTT assay. CD243 expression and apoptosis rate were evaluated by flow cytometry. Interactions of WBP2/ERα and ERα/MDR1 were detected by co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assay, respectively. RESULTS: There was an intrinsic link between WBP2 and ERα in drug-resistant cancer cells. Upregulation of WBP2 in MCF7 cells increased the chemoresistance to doxorubicin, while RNAi-mediated knockdown of WBP2 in MCF7/ADR cells sensitised the cancer cells to doxorubicin. Further investigation in in vitro and in vivo models demonstrated that WBP2 expression was directly correlated with MDR1, and WBP2 could directly modulate MDR1 transcription through binding to ERα, resulting in increased chemotherapy drug resistance. CONCLUSIONS: Our finding provides a new mechanism for the chemotherapy response of ERα-positive breast tumours, and WBP2 might be a key molecule for developing new therapeutic strategies to treat chemoresistance in breast cancer patients.

Timing during translation matters: synonymous mutations in human pathologies influence protein folding and function.

Ribosomes translate mRNAs with non-uniform speed. Translation velocity patterns are a conserved feature of mRNA and have evolved to fine-tune protein folding, expression and function. Synonymous single-nucleotide polymorphisms (sSNPs) that alter programmed translational speed affect expression and function of the encoded protein. Synergistic advances in next-generation sequencing have led to the identification of sSNPs associated with disease penetrance. Here, we draw on studies with disease-related proteins to enhance our understanding of mechanistic contributions of sSNPs to functional alterations of the encoded protein. We emphasize the importance of identification of sSNPs along with disease-causing mutations to understand genotype-phenotype relationships.

Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse.

Estrogen receptor α (ERα) regulates gene transcription through two activation functions (ERα-AF1 and ERα-AF2). We recently found that the protection conferred by 17ß-estradiol against obesity and insulin resistance requires ERα-AF2 but not ERα-AF1. However, the interplay between the two ERα-AFs is poorly understood in vivo and the metabolic influence of a specific ERα-AF1 action remains to be explored. To this end, wild-type, ERα-deficient, or ERα-AF1-deficient ovariectomized female mice were fed a high-fat diet and concomitantly administered with vehicle or tamoxifen, a selective ER modulator that acts as a ERα-AF1 agonist/ERα-AF2 antagonist. In ovariectomized wild-type mice, tamoxifen significantly reduced food intake and totally prevented adiposity, insulin resistance, and steatosis. These effects were abolished in ERα-deficient and ERα-AF1-deficient mice, revealing the specific role of ERα-AF1 activation. Finally, hepatic gene expression changes elicited by tamoxifen in wild-type mice were abrogated in ERα-AF1-deficient mice. The combination of pharmacologic and transgenic approaches thus indicates that selective ERα-AF1 activation by tamoxifen is sufficient to elicit metabolic protection, contrasting with the specific requirement of ERα-AF2 in the metabolic actions of 17ß-estradiol. This redundancy in the ability of the two ERα-AFs to separately mediate metabolic prevention strikingly contrasts with the contribution of both ERα-AFs in breast cancer proliferation, shedding new light on the therapeutic potential of selective ER modulation.

Is the membrane estrogen receptor, GPER1, a promiscuous receptor that modulates nuclear estrogen receptor-mediated functions in the brain?

Contribution to Special Issue on Fast effects of steroids. Estrogen signals both slowly to regulate transcription and rapidly to activate kinases and regulate calcium levels. Both rapid, non-genomic signaling as well as genomic transcriptional signaling via intracellular estrogen receptors (ER)s can change behavior. Rapid non-genomic signaling is initiated from the plasma membrane by a G-protein coupled receptor called GPER1 that binds 17ß-estradiol. GPER1 or GPR30 is one of the candidates for a membrane ER (mER) that is not only highly expressed in pathology i.e. cancers but also in several behaviorally-relevant brain regions. In the brain, GPER1 signaling, in response to estrogen, facilitates neuroprotection, social behaviors and cognition. In this review, we describe several notable characteristics of GPER1 such as the ability of several endogenous steroids as well as artificially synthesized molecules to bind the GPER1. In addition, GPER1 is localized to the plasma membrane in breast cancer cell lines but may be present in the endoplasmic reticulum or the Golgi apparatus in the hippocampus. Unusually, GPER1 can also translocate to the perinuclear space from the plasma membrane. We explore the idea that subcellular localization and ligand promiscuity may determine the varied downstream signaling cascades of the activated GPER1. Lastly, we suggest that GPER1 can act as a modulator of ERα-mediated action on a convergent target, spinogenesis, in neurons that in turn drives female social behaviors such as lordosis and social learning.