G Protein-Coupled Estrogen Receptor Mediates Cell Proliferation through the cAMP/PKA/CREB Pathway in Murine Bone Marrow Mesenchymal Stem Cells.
ABSTRACT: Estrogen is an important hormone to regulate skeletal physiology via estrogen receptors. The traditional estrogen receptors are ascribed to two nuclear estrogen receptors (ERs), ER? and ER?. Moreover, G protein-coupled estrogen receptor-1 (GPER-1) was reported as a membrane receptor for estrogen in recent years. However, whether GPER-1 regulated osteogenic cell biology on skeletal system is still unclear. GPER-1 is expressed in growth plate abundantly before puberty but decreased abruptly since the very late stage of puberty in humans. It indicates GPER-1 might play an important role in skeletal growth regulation. GPER-1 expression has been confirmed in osteoblasts, osteocytes and chondrocytes, but its expression in mesenchymal stem cells (MSCs) has not been confirmed. In this study, we hypothesized that GPER-1 is expressed in bone MSCs (BMSC) and enhances BMSC proliferation. The cultured tibiae of neonatal rat and murine BMSCs were tested in our study. GPER-1-specific agonist (G-1) and antagonist (G-15), and GPER-1 siRNA (siGPER-1) were used to evaluate the downstream signaling pathway and cell proliferation. Our results revealed BrdU-positive cell counts were higher in cultured tibiae in the G-1 group. The G-1 also enhanced the cell viability and proliferation, whereas G-15 and siGPER-1 reduced these activities. The cAMP and phosphorylation of CREB were enhanced by G-1 but inhibited by G-15. We further demonstrated that GPER-1 mediates BMSC proliferation via the cAMP/PKA/p-CREB pathway and subsequently upregulates cell cycle regulators, cyclin D1/cyclin-dependent kinase (CDK) 6 and cyclin E1/CDK2 complex. The present study is the first to report that GPER-1 mediates BMSC proliferation. This finding indicates that GPER-1 mediated signaling positively regulates BMSC proliferation and may provide novel insights into addressing estrogen-mediated bone development.
Project description:BACKGROUND:Breast cancer is the most frequently diagnosed malignancy among women and the second leading cause of cancer death worldwide. Among which nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. Recently, membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor has been documented to take essential part in the development and treatment of breast cancer. The present study was designed to investigate the anti nER negative breast cancer effect of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen and its possible molecular pathway. METHODS:The following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay respectively. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT. RESULTS:The results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT could also exert antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G-1 and attenuated by G-15. Results of western blot and immunofluorescence showed that expression of PI3K and p-AKT could be downregulated by CPT and such effects were mediated by GPER which were further demonstrated by gene silence test. CONCLUSION:The current study showed that the antiproliferative action of CPT on SKBR-3 cells was realized by inhibition of GPER mediated PI3K/AKT pathway. These findings provide further validation of GPER serving as useful therapeutic target.
Project description:17?-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor ?. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology.
Project description:BACKGROUND:The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS:Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS:We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS:In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.
Project description:The G protein-coupled estrogen receptor (GPER, GPR30) represents a promising target for the treatment of estrogen receptor ? and ? negative breast cancers. Previously reported agonists of GPER have shown that activation of GPER inhibits breast cancer cell proliferation. We report herein a new GPER agonist scaffold based upon in silico pharmacophore screening. Three of these compounds were found to increase cAMP at similar levels as the known GPER-selective agonist G-1. Compound 5 was found to be selective for GPER (over estrogen receptor ? and ?) and inhibit breast cancer cell proliferation at levels consistent with G-1. Docking studies go on to suggest that both 5 and G-1 bind within the same binding pocket in GPER and point to possible key residues that are important in GPER activation.
Project description:Background: Insulin resistance (IR) has been well studied in the initiation and development of endometrial endometrioid carcinoma (EEC). As yet, it has been largely neglected for estrogen sensitivity in local endometrium in hyperinsulinemia-induced systemic microenvironment. The aim of this study was to investigate the role of insulin in regulating estrogen sensitivity and explore the potential mechanisms in insulin-driven inflammatory microenvironment. Methods: We first investigated the effect of insulin on estradiol-driven endometrial cancer cells proliferation in vitro to address the roles of insulin in modulating estrogen sensitivity. Then GPER, ER? and TET1 in EEC samples with or without insulin resistance were screened by immunohistochemistry to confirm whether insulin resistance regulates estrogen receptors. Further mechanism analysis was carried out to address whether TET1 was mediated epigenetic modulation of GPER in insulin-induced microenvironment. Results: Insulin enhanced estradiol-driven endometrial cancer cells proliferation by up-regulating G-protein-coupled estrogen receptor (GPER) expression, but not ER? or ER?. Immunohistochemistry of EEC tissues showed that GPER expression was greatly increased in endometrial tissues from EEC subjects with insulin resistance and was positively correlated with Ten-eleven-translocation 1 (TET1) expression. Mechanistically, insulin up-regulates TET1 expression, and the latter, an important DNA hydroxymethylase, could up-regulate GPER expression through epigenetic modulation. Conclusion: This study identified TET1 as the upstream regulator of GPER expression and provides a possible mechanism that insulin-induced positive regulation of estrogen sensitivity in endometrial cancer cells. Increasing expression of GPER through TET1-mediated epigenetic modulation may emerge as the main regulator to enhance the response of endometrial cancer to estrogen in insulin-driven inflammatory microenvironment.
Project description:The estrogen receptor ER? is the predominant ER subtype expressed in normal well-differentiated colonic epithelium. However, ER? expression is lost under the hypoxic microenvironment as colorectal cancer (CRC) malignancy progresses. This raises questions about the role of signalling through other estrogen receptors such as ER? or G-protein coupled estrogen receptor (GPER, GPR30) by the estrogen 17?-estradiol (E2) under hypoxic conditions after ER? is lost in CRC progression. We tested the hypothesis that E2 or hypoxia can act via GPER to contribute to the altered phenotype of CRC cells. GPER expression was found to be up-regulated by hypoxia and E2 in a panel of CRC cell lines. The E2-modulated gene, Ataxia telangiectasia mutated (ATM), was repressed in hypoxia via GPER signalling. E2 treatment enhanced hypoxia-induced expression of HIF1-? and VEGFA, but repressed HIF1-? and VEGFA expression under normoxic conditions. The expression and repression of VEGFA by E2 were mediated by a GPER-dependent mechanism. E2 treatment potentiated hypoxia-induced CRC cell migration and proliferation, whereas in normoxia, cell migration and proliferation were suppressed by E2 treatment. The effects of E2 on these cellular responses in normoxia and hypoxia were mediated by GPER. In a cohort of 566 CRC patient tumor samples, GPER expression significantly associated with poor survival in CRC Stages 3-4 females but not in the stage-matched male population. Our findings support a potentially pro-tumorigenic role for E2 in ER?-negative CRC under hypoxic conditions transduced via GPER and suggest a novel route of therapeutic intervention through GPER antagonism.
Project description:Estrogens play important roles in the development and progression of multiple tumor types. Accumulating evidence points to the significance of estrogen action not only in tumors of hormonally regulated tissues such as the breast, endometrium and ovary, but also in the development of colorectal cancer (CRC). The effects of estrogens in physiological and pathophysiological conditions are mediated by the nuclear estrogen receptors ? and ?, as well as the membrane-bound G protein-coupled estrogen receptor (GPER). The roles of GPER in CRC development and progression, however, remain poorly understood. Studies on the functions of GPER in the colon have shown that this estrogen receptor regulates colonic motility as well as immune responses in CRC-associated diseases, such as Crohn's disease and ulcerative colitis. GPER is also involved in cell cycle regulation, endoplasmic reticulum stress, proliferation, apoptosis, vascularization, cell migration, and the regulation of fatty acid and estrogen metabolism in CRC cells. Thus, multiple lines of evidence suggest that GPER may play an important role in colorectal carcinogenesis. In this review, we present the current state of knowledge regarding the contribution of GPER to colon function and CRC.
Project description:Cadmium (Cd) is a ubiquitous environmental metal that is reported to be a "metalloestrogen." Uterine leiomyomas (fibroids) are estrogen-responsive gynecologic neoplasms that can be the target of xenoestrogens. Previous epidemiology studies have suggested Cd may be associated with fibroids. We have shown that Cd can stimulate proliferation of human uterine leiomyoma (ht-UtLM) cells, but not through classical estrogen receptor (ER) binding. Whether nongenomic ER pathways are involved in Cd-induced proliferation is unknown. In the present study, by evaluating G protein-coupled estrogen receptor (GPER), ER?36, and phospho-epidermal growth factor receptor (EGFR) expression in human tissues, we found that GPER, ER?36 and phospho-EGFR were all highly expressed in fibroids compared to patient-matched myometrial tissues. In ht-UtLM cells, cell proliferation was increased by low doses of Cd (0.1 µM and 10 µM), and this effect could be inhibited by GPER-specific antagonist (G15) pretreatment, or silencing (si) GPER, but not by siER?36. Cd-activated MAPK was dependent on GPER/EGFR transactivation, through significantly increased phospho-Src, matrix metalloproteinase-2 (MMP2) and MMP9, and heparin-binding EGF-like growth factor (HB-EGF) expression/activation. Also, phospho-Src could interact directly to phosphorylate EGFR. Overall, Cd-induced proliferation of human fibroid cells was through a nongenomic GPER/p-src/EGFR/MAPK signaling pathway that did not directly involve ER?36. This suggests that Cd may be a risk factor for uterine fibroids through cross talk between hormone and growth factor receptor pathways.
Project description:GPER is a membrane-associated estrogen receptor of the family of G-protein coupled receptors. For breast cancer, the contribution of GPER to promoting the proliferation and migration of both carcinoma cells and cancer-associated fibroblasts (CAFs) in response to estrogen and other agonists has extensively been investigated. Intriguingly, GPER was previously found to be localized to the nucleus in one isolate of breast CAFs. Moreover, this nuclear GPER was shown to bind regulatory sequences of cancer-relevant target genes and to induce their expression. We decided to find out what induces the nuclear localization of GPER, how general this phenomenon is, and what its functional significance is. We discovered that interfering with N-linked glycosylation of GPER, either by mutation of the predicted glycosylation sites or pharmacologically with tunicamycin, drives GPER into the nucleus. Surveying a small set of CAFs from breast cancer biopsies, we found that a relatively common single nucleotide polymorphism, which results in the expression of a GPER variant with the amino acid substitution P16L, is associated with the nuclear localization of GPER. GPER with P16L fails to be glycosylated, presumably because of a conformational effect on the nearby glycosylation sites. GPER P16L is defective for membrane-associated signaling, but instead acts like an estrogen-stimulated transcription factor. In CAFs, it induces the secretion of paracrine factors that promote the migration of carcinoma cells. This raises the possibility that the GPER P16L polymorphism could be a risk factor for breast cancer.
Project description:The pesticide atrazine does not bind to or activate the classical estrogen receptor (ER), but it up-regulates the aromatase activity in estrogen-sensitive tumor cells. The G protein estrogen receptor (GPR30/GPER) has been reported to be involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity.We aimed to evaluate the potential of atrazine to trigger GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs).Using gene reporter assays in diverse types of cancer cells, we found that atrazine did not transactivate endogenous ER? or chimeric proteins that encode the ER? and ER? hormone binding domains. Conversely, atrazine was able to bind to GPER to induce ERK activation and the expression of estrogen target genes, which, interestingly, appeared to rely on both GPER and ER? expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells that depend on GPER and ER?, as evidenced by gene silencing experiments and the use of specific signaling inhibitors. Of note, through GPER, atrazine elicited ERK phosphorylation, gene expression, and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment.Our results suggest a novel mechanism through which atrazine may exert relevant biological effects in cancer cells and CAFs. On the basis of our data, atrazine should be included among the environmental contaminants that may elicit estrogenic activity through GPER-mediated signaling.