GPER1 Expression Research Articles

Local sex steroid hormone milieu in the bovine oviduct ipsilateral and contralateral to preovulatory follicle or corpus luteum during the periovulatory phase

Estradiol-17β (E2) and progesterone (P4) regulate oviductal functions, providing a suitable environment for the transport and maturation of gametes, fertilization, and embryonic development. In addition to the E2 and P4 nuclear receptors, estrogen receptor (ESR) α and β, nuclear progesterone receptor (PGR), nongenomic mechanisms through G protein-coupled estrogen receptor (GPER1), and progesterone receptor membrane component (PGRMC) 1 and 2 mediate E2 and P4 actions. This study aimed to characterize the local endocrine environment of the oviduct by examining the oviductal E2 and P4 concentrations and their receptors' mRNA expression during the periovulatory phase. The bovine oviducts were collected in a slaughterhouse and the days postovulation were estimated according to state of the ovaries and the uterus. Samples of the ampulla and isthmus ipsilateral and contralateral to the preovulatory follicle or corpus luteum were collected on Days 19 to 21, Days 0 to 1, Days 2 to 4, and Days 5 to 7 of the estrous cycle. The effects of the estrous cycle phase and oviductal region (ampulla and isthmus) and side (ipsilateral and contralateral) were analyzed by 3-way ANOVA. Moreover, to clarify the regulatory mechanisms of the mRNA expression of hormone receptors, the effects of E2 and P4 on mRNA expression in the oviduct were examined by multiple linear regression. The oviductal endocrine milieu on Days 19 to 21 was characterized by an E2-dominant environment with high E2 and low P4, high ESR1 and PGR mRNA expression, and low ESR2, GPER1, and PGRMC2 mRNA expression, whereas the corresponding on Days 0 to 1 was characterized by the endocrine milieu without hormone dominance. The environment on Days 2 to 4 and Day 5 to 7 was characterized by opposite tendency of oviductal hormone concentrations and their receptors' mRNA expression to Days 19 to 21. Additionally, the ipsilateral oviduct had the more P4-dominant endocrine milieu, with lower E2 and higher P4 concentrations, and different expression of ESR1/2, GPER1, PGR, and PGRMC2 mRNA when compared with the contralateral oviduct on Days 2 to 4 and Days 5 to 7, except for PGRMC1. Although oviductal E2 and P4 influenced the mRNA expression of ESR1/2, GPER1, PGR, and PGRMC1/2, their effects were different between regions and sides. In summary, the oviductal endocrine milieu varies according to the estrous cycle phase and the oviductal region and side, which may be involved in the estrous cycle phase-specific and oviductal region-specific and side-specific functions.

GPER1 Signaling Initiates Migration of Female V-SVZ-Derived Cells.

SummaryIn the rodent ventricular-subventricular zone (V-SVZ) neurons are generated throughout life. They migrate along the rostral migratory stream (RMS) into the olfactory bulb before their final differentiation into interneurons and integration into local circuits. Estrogen receptors (ERs) are steroid hormone receptors with important functions in neurogenesis and synaptic plasticity. In this study, we show that the ER GPER1 is expressed in subsets of cells within the V-SVZ of female animals and provide evidence for a potential local estrogen source from aromatase-positive astrocytes surrounding the RMS. Blocking of GPER1 in Matrigel cultures of female animals significantly impairs migration of V-SVZ-derived cells. This outgrowth is accompanied by regulation of phosphorylation of the actin-binding protein cofilin by GPER1 signaling including an involvement of the p21-Ras pathway. Our results point to a prominent role of GPER1 in the initiation of neuronal migration from the V-SVZ to the olfactory bulb.

GPER-1 expression is associated with a decreased response rate to primary tamoxifen therapy of breast cancer patients.

Endocrine therapies using tamoxifen and/or aromatase inhibitors are important therapeutic options for the targeted treatment of hormone-responsive breast cancer. In addition to nuclear estrogen receptors ERα and β, G-protein-coupled estrogen receptor GPER-1 is a third receptor-mediating estrogen effects in breast cancer cells. The aim of this study was to examine to what extent GPER-1 expression might affect the efficacy of primary endocrine treatment of breast cancer. GPER-1 expression was determined in tissue samples from patients with early breast cancer by means of immunohistochemistry and a GPER-1 score of ≥ 3 was considered to be positive. In a total of 165 patients, the response to a primary therapy with tamoxifen (TAM) or aromatase inhibitors (AI) was assessed by ultrasound imaging for up to 6months. The primary endpoint of this study was the response to treatment evaluated by RECIST 1.1 criteria. GPER-1 expression was observed in 127 (77.0%) out of 165 cases. Based on GPER-1 expression and the type of endocrine treatment, the patients were divided into 4 groups: GPER-1 negative/TAM (12.1%), GPER-1 negative/AI (10.9%), GPER-1 positive/TAM (44.8%), and GPER-1 positive/AI (32.1%). The groups were well balanced regarding different clinical and pathological factors. After 4 and 6months of treatment, a high level of stable disease or progressive disease was observed in the GPER-1 positive/TAM group only (p < 0.0001), whereas in the other three groups of patients, the most common objective response was classified as partial response. We observed a continuous reduction of mean tumor size in patients treated with aromatase inhibitors irrespective of the GPER-1 status and in GPER-1 negative patients treated with TAM. In contrast, in GPER-1 positive patients treated with TAM, a reduction of mean tumor size was observed only in the first 2months after beginning of treatment. Four and six months after start of treatment, no reduction, but even a slight increase of tumor size was observed in this patients group. GPER-1 expression is significantly associated with a reduced effect of primary treatment with tamoxifen in breast cancer patients.

Clinical candidate and genistein analogue AXP107-11 has chemoenhancing functions in pancreatic adenocarcinoma through G protein-coupled estrogen receptor signaling.

Despite advances in cancer therapeutics, pancreatic cancer remains difficult to treat and often develops resistance to chemotherapies. We have evaluated a bioavailable genistein analogue, AXP107‐11 which has completed phase Ib clinical trial, as an approach to sensitize tumor cells to chemotherapy. Using organotypic cultures of 14 patient‐derived xenografts (PDX) of pancreatic ductal adenocarcinoma, we found that addition of AXP107‐11 indeed sensitized 57% of cases to gemcitabine treatment. Results were validated using PDX models in vivo. Further, RNA‐Seq from responsive and unresponsive tumors proposed a 41‐gene treatment‐predictive signature. Functional and molecular assays were performed in cell lines and demonstrated that the effect was synergistic. Transcriptome analysis indicated activation of G‐protein‐coupled estrogen receptor (GPER1) as the main underlying mechanism of action, which was corroborated using GPER1‐selective agonists and antagonists. GPER1 expression in pancreatic tumors was indicative of survival, and our study proposes that activation of GPER1 may constitute a new avenue for pancreatic cancer therapeutics.

Autocrine motility factor promotes endometrial cancer progression by targeting GPER-1

BackgroundAutocrine motility factor (AMF) is a critical factor regulating aggressiveness of endometrial cancer (EC). Multiple pieces of evidence indicate that it is through G protein coupled estrogen receptor (GPER) signaling pathway that some growth factors promoted the migration and proliferation of tumor cells. The aim of this study is to explore the role of GPER-1 in AMF mediated regulatory mechanisms of EC recurrence and progression.MethodsReal-Time Cell Analysis (RTCA) assays were performed to assess whether AMF depends on Autocrine motility factor recepter (AMFR) signaling in EC cells. A genome-wide expression microarray and Yeast Two-Hybrid assay were used to detect AMF and GPER-1 interaction in the context of AMFR depletion, and co-immunoprecipitation and immunofluorescence experiments were performed to confirm the physical interaction. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) analysis was used for the identification of the target pathway activated by AMF-GPER-1 interaction. Cohorts of mice harboring xenografts derived from modified SPEC2 cell lines were treated with or without exogenous AMF to validate the results of previous experiments. Immunohistochemistry was performed to assess AMF and GPER-1 expression in endometrial cancer specimens and normal endometrium.ResultsOur data showed that GPER-1 binds to AMF and the formed complex translocates from the plasma membrane to the cytoplasm. Mechanistic investigations demonstrated that interaction between AMF and GPER-1 triggers phosphoinositide-3-kinase signaling and promotes EC cell growth. More importantly, through animal experiments and human tissue experiments, we found that AMF contributes to GPER-1-mediated EC progression, which is consistent with the above observations.ConclusionsOur work not only delineated the regulatory mechanisms of endometrial cancer progression by AMF-GPER-1-AKT signaling cascade but also laid the foundation of targeting this pathway for treating endometrial cancer.

ALDOSTERONE INDUCES CYP11B2 GENE EXPRESSION VIA GPER-1 ACTIVATION IN HUMAN ADRENOCORTICAL CELL (HAC15) AND STRIPS FROM ALDOSTERONE PRODUCING ADENOMA

Objective: We previously reported that GPER-1 is mostly expressed in the zona glomerulosa of normal human adrenal gland and in aldosterone-producing adenoma (APA). In the adrenocortical carcinoma cell line HAC15 17b-estradiol induces aldosterone synthesis via GPER-1 activation. Since GPER-1 was found to bind not only 17b-estradiol, but also aldosterone in endothelial and vascular smooth muscle cells, we wondered if aldosterone binds GPER-1 receptor also in the adrenocortical cells, thereby modulating its own synthesis. Design and method: APA strips and adrenocortical carcinoma cell line HAC15 were exposed to [100 nM] aldosterone for 12 hours in the presence or absence of the selective mineralocorticoid receptor (MR) antagonist canrenone and/or of the selective GPER-1 antagonist G36. HAC15 cells were silenced for GPER-1 with specific small interfering RNA (siRNA) and then exposed to aldosterone. The changes of aldosterone synthase (CYP11B2) mRNA and protein levels were measured by real time qRT-PCR and immunoblotting. Results: Aldosterone increased CYP11B2 gene and protein expression (+200% and +130%, respectively) in HAC15 cells (p < 0.001 vs untreated). Pretreatment with canrenone did not prevent such increase, while G36 abolished aldosterone-induced CYP11B2 activation (p < 0.01). In APA strips aldosterone increased CYP11B2 gene expression (+330%, p < 0.01 vs untreated); pre-treatment with G36 blunted this effect of aldosterone. Silencing of GPER-1 with siRNA not only reduced GPER-1 expression (p < 0.01), but also abolished the CYP11B2 increase in response to aldosterone. Conclusions: Aldosterone enhances the expression of CYP11B2 acting via GPER-1, but not MR. Binding of aldosterone to GPER-1 could contribute to perpetuating the autonomous aldosterone excess, which is a hallmark of primary aldosteronism, via an autocrine-paracrine positive feed-back loop.

Immunolocalization of G Protein-Coupled Estrogen Receptor in the Pig Epididymis.

The presence of estrogen in the genital ducts of different mammalian species has been extensively studied and the estrogen influence on the functional activity of the male genital tract has been hypothesized. Conversely, very few data have been reported on pig excurrent ducts: the localization of classical estrogen receptors (ERα and ERβ) is scarcely known, while the expression of the G protein-coupled receptor (GPER1), a membrane estrogen receptor, is still unknown in pig. The aim of the present study was to evaluate GPER1 expression in the different regions of the mature pig epididymis, using immunohistochemistry, western blot and RT-PCR analyses. The results showed that GPER1 is mainly expressed in the epithelial cells of the corpus epididymis compared to the caput and the cauda, while muscle cells are moderately immunostained and stromal cells are unstained. The presence of GPER1 was confirmed by Western blot and RT-PCR analyses. In our study, we have demonstrated for the first time the GPER1 expression in male porcine epididymis, revealing a new mediator of estrogen signaling at this site. In conclusion, these new data suggest that estrogen action via GPER1 may contribute to sperm maturation in the corpus and sperm protection/storage in the cauda. Interestingly, the presence of GPER1 in the muscle layer may be indicative of a possible GPER1 involvement in the estrogen regulation of duct contractility. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Prenatal exposure to benzophenone-3 (BP-3) induces apoptosis, disrupts estrogen receptor expression and alters the epigenetic status of mouse neurons

Current evidence indicates that benzophenone-3 (BP-3) can pass through the placental and blood–brain barriers and thus can likely affect infant neurodevelopment. Despite widespread exposure, data showing the effects of BP-3 on the developing nervous system are scarce. This study revealed for the first time that prenatal exposure to BP-3 led to apoptosis and neurotoxicity, altered the levels of estrogen receptors (ERs) and changed the epigenetic status of mouse neurons. In the present study, subcutaneous injections of pregnant mice with BP-3 at 50 mg/kg, which is an environmentally relevant dose, evoked activation of caspase-3 and lactate dehydrogenase (LDH) release as well as substantial loss of mitochondrial membrane potential in neocortical cells of their embryonic offspring. Apoptosis-focused microarray analysis of neocortical cells revealed up-regulation of 22 genes involved in apoptotic cell death. This effect was supported by increased BAX and CASP3 mRNA and protein levels, as evidenced by qPCR, ELISAs and western blots. BP-3-induced apoptosis and neurotoxicity were accompanied by decreases in the mRNA and protein expression levels of ESR1 and ESR2 (also known as ERα and ERβ), with a simultaneous increase in GPER1 (also known as GPR30) expression. In addition to the demonstration that treatment of pregnant mice with BP-3 induced apoptosis, caused neurotoxicity and altered ERs expression levels in neocortical cells of their embryonic offspring, we showed that prenatal administration of BP-3 inhibited global DNA methylation as well as reduced DNMTs activity. BP-3 also caused specific hypomethylation of the genes Gper1 and Bax, an effect that was accompanied by increased mRNA and protein expression levels. In addition, BP-3 caused hypermethylation of the genes Esr1, Esr2 and Bcl2, which could explain the reduced mRNA and protein levels of the estrogen receptors. This study demonstrated for the first time that prenatal exposure to BP-3 caused severe neuronal apoptosis that was accompanied by impaired ESR1/ESR2 expression, enhanced GPER1 expression, global DNA hypomethylation and altered methylation statuses of apoptosis-related and ERs genes. We suggest that the effects of BP-3 in embryonic neurons may be the fetal basis of the adult onset of nervous system disease.

Abstract 2432: GPER1 expression is modulation by D-glucose concentration in estrogen-responsive cancer and tumor cells

Abstract G protein-coupled estrogen receptor 1 (GPER1, aka GPR30), is a 7-transmembrane receptor that mediates rapid cell signaling events stimulated by 17β-estradiol (E2) in cancer and tumor cells. In breast cancer cells, GPER1 mediates E2-induced CTGF-dependent cell migration and cyclin E expression resulting in cell cycle progression. GPER1 has also been shown to mediate antiproliferative cell signaling such as p53-dependent inhibition of cell cycle progression in breast cancer cells treated with the GPER1-specific agonist G1 and the inhibition of IGF-1R signaling in tamoxifen-treated breast cancer cells. In addition to E2, both natural and synthetic compounds have been shown to activate GPER1 in breast cancer cells. GPER1 has also been shown to regulate cell signaling in cells that lack ERα. For example, GPER1 is antiproliferative in triple-negative breast cancer cells. While the importance of GPER1 in the modulation of E2-responsive tumor and cancer cells as been demonstrated, there is very little information regarding the regulation of GPER1 expression in these cell types. The work presented here is aimed at determining the molecular mechanisms that regulate GPER1 expression. To determine if GPER1 expression is sensitive to D-glucose concentration in breast cancer cells, MCF-7 and T47D human breast cancer cells were cultured in media containing increasing concentrations of D-glucose and GPER1 expression was measured using real-time PCR and immunoblot. Data from these experiments showed that GPER1 expression was significantly increased in breast cancer cells cultured in media containing low D-glucose concentrations (0 and 2.5mM) and significantly reduced in media containing high D-glucose (25 mM). Since low D-glucose concentration is known to activate the energy-sensing AMP kinase (AMPK), the observed GPER1 induction in low D-glucose conditions was determined after pretreatment with the AMPK inhibitor compound C. GPER1 expression was inhibited in both MCF-7 and T47D breast cancer cells cultured in low D-glucose media when pretreated with compound C. Additionally, the AMPK activator metformin induced GPER1 expression in MCF-7 and T47D breast cancer cells cultured in high D-glucose (25mM) conditions. These data suggest that AMPK mediates GPER1 expression in cells cultured in low D-glucose. The D-glucose sensitivity of GPER1 expression was also determined in the Eker rat-derived uterine leiomyoma cells (ELT-3 and ELT-6) which are characterized by hyperactive AMPK. In both ELT-3 and ELT-6 cells, GPER1 expression was not sensitive to D-glucose concentration nor was the activation state of AMPK sensitive to D-glucose concentrations. However, the inhibition of AMPK by compound C in both ELT-3 and ELT-6 cells resulted in decreased GPER1 expression. These findings reveal a previously unknown mechanism that regulates GPER1 expression in E2-responsive tumor and cancer cells. Citation Format: Yan Zheng, Kevin D. Houston. GPER1 expression is modulation by D-glucose concentration in estrogen-responsive cancer and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2017-2432

GPER1 in sand rat epididymis: Effects of seasonal variations, castration and efferent ducts ligation

Estrogen plays a crucial role in regulating epididymal function and development. Estrogen signaling is mediated via two main receptors essentially involved in the genomic regulating pathway: ERα and ERβ. Recent studies revealed the contribution of a novel estrogen receptor involved in the non-genomic pathway: GPER1. This receptor belongs to the family of seven-transmembrane G-protein-coupled receptors and it triggers rapid cellular responses. Immuno-histochemical studies and Western Blot analyses were performed to investigate the GPER1 expression in the caput and cauda epididymis of free-ranging fat sand rats (Psammomys obesus) captured during the breeding and resting seasons. We also investigated the effect of castration (C), castration followed by testosterone treatment (C+T), and ligation of the efferent ducts (L). During the breeding season, a marked positive GPER1 immunoreactivity was detected in the cytoplasm of principal cells and basal cells; this signal persisted during the resting season, attenuated however, meanwhile the clear cells were not immuno-reactive. In C animals, the immuno-histochemical staining underwent nuclear translocation. In C+T animals, this response became nuclear and cytoplasmic. In the L group, the expression of the GPER1 was mainly located in the cytoplasm of principal cells and in the nuclei of basal cells; the sperm was also immune-positive in the cauda epididymis. Western blot analysis showed that GPER1 has a molecular weight of 55kDa in the caput and cauda epididymis during the breeding season, and it persisted during the resting season in the caput epididymis with a decrease in the cauda epididymis. These results suggest that GPER1 mediate a specific cellular estrogen signaling with marked differences between the breeding and resting seasons. Experimental groups suggest that testosterone is involved in the regulation of the expression of GPER1, in addition to other estrogen signalization pathways.

Analysis of GPER1, CXCR1 and HER-2 Immuno Expression in Follicular Thyroid Lesions and their Possible Role as Diagnostic Markers

Analysis of GPER1, CXCR1 and HER-2 Immuno Expression in Follicular Thyroid Lesions and their Possible Role as Diagnostic Markers

Deficiency of β-arrestin 2 in mice-induced bone remodeling was associated with down-regulation of GPER1 expression

Objective To determine the role of β-arrestin 2 on bone mineral density and microarchitecture, and its underlying mechanisms. Methods Ex vivo dual-energy-X-ray-absorptiometry(DXA) was used to measure the total body bone mineral density (BMD) and micro-CT scan was performed on mouse femora from female mice of wild type(WT) and β-arrestin 2 knockout(KO) at 16, 26, 30 weeks of age. RT-PCR was applied for the mRNA expression. Results Started from 16 weeks of age, total body BMD and the bone volume/total volume(BV/TV), trabecular number(Tb.N) in the femoral metaphysis of β-arrestin 2 KO female mice decreased dramatically. BMD decreased by 8.10%, 7.53%, 8.56%, BV/TV by 53.3%, 49.4%, 55.7% and Tb.N by 53.2%, 52.1%, 49.3% at 16, 26, 30 weeks of age, respectively, compared with those in WT mice(all P<0.05). In contrast, the trabecular pattern factor(Tb.Pf), trabecular separation(Tb.Sp), and structure model index(SMI) of the mice increased gradually with age, and reached statistically significant at age of 30 weeks(increased by 32.0%, 54.3%, and 23.7%, respectively, all P<0.05). RT-PCR showed that mRNA expression levels of osteoprotegrin, RANKL, and G protein-coupled estrogen receptor 1 in tibia from β-arrestin 2 KO mice were significantly lower than those in WT mice. Conclusion The effects of β-arrestin 2 on bone remodeling might be associated with G protein-coupled estrogen receptor1 signaling pathway. (Chin J Endocrinol Metab, 2015, 31: 897-901) Key words: β-Arrestin 2; Bone mineral density; Microarchitecture; Osteoprotegrin; G protein-coupled estrogen receptor 1

G protein-coupled estrogen receptor 1 (GPER 1) mediates estrogen-induced, proliferation of leiomyoma cells

G protein-coupled estrogen receptor 1 (GPER-1, formerly known as GPR30) has been proposed as the receptor for estrogen-induced, growth of leiomyomas though its precise mechanisms of action are not clear. We obtained leiomyoma cells (LC) and normal smooth muscle cells from 28 women (n = 28, median age 38 years, median parity 1.0). We incubated them with 17-β estradiol (E2), after blocking, or upregulating, expression of GPER-1 with ICI182,780 (a GPER-1 agonist) and siGPR30, respectively. We evaluated the role of GPER-1 in the mitogen-activated protein kinase (MAPK) signaling pathway using Western blot analysis. We studied cell proliferation with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, and, mitotic activity with phosphohistone H3 (PPH3) expression in leiomyoma, and, matched, normal, smooth muscle tissues using standard immunohistochemistry. Downregulation of GPER-1 expression with siGPR30 partially attenuated the E2-activated MAPK signaling pathway (p < 0.01). Upregulation of GPER-1 with ICI182,780 enhanced the E2-activated MAPK signaling pathway (p < 0.01). ICI182,780 enhanced E2-induced proliferation of LC (p < 0.01), while knock down of the GPER-1 gene with GPER-1 small interfering RNA partially inhibited E2-induced cell proliferation (p < 0.01). There were no significant differences in PPH3 expression between LCs and normal smooth muscle tissues (p > 0.05). Neither ICI182,780 nor siGPR30 increased mitosis in LCs (p > 0.05). Our results indicate that GPER-1 mediates proliferation of estrogen-induced, LC by activating the MAPK pathway, and, not by promoting mitosis.

An acute exposure to glyphosate-based herbicide alters aromatase levels in testis and sperm nuclear quality

Roundup is the major pesticide used in agriculture worldwide; it is a glyphosate-based herbicide. Its molecular effects are studied following an acute exposure (0.5%) of fifteen 60-day-old male rats during an 8-day period. Endocrine (aromatase, estrogen and androgen receptors, Gper1 in testicular and sperm mRNAs) and testicular functions (organ weights, sperm parameters and expression of the blood–testis barrier markers) were monitored at days 68, 87, and 122 after treatment, spermiogenesis and spermatogenesis. The major disruption is an increase of aromatase mRNA levels at least by 50% in treated rats at all times, as well as the aromatase protein. We have also shown a similar increase of Gper1 expression at day 122 and a light modification of BTB markers. A rise of abnormal sperm morphology and a decrease of the expression of protamine 1 and histone 1 testicular in epididymal sperm are observed despite a normal sperm concentration and motility.

Activation of GPER-1 Estradiol Receptor Downregulates Production of Testosterone in Isolated Rat Leydig Cells and Adult Human Testis

PurposeEstradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis.Materials and MethodsIsolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay.ResultsGPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20–30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs.ConclusionsOur results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

GPER-1 Expression Decreases During Breast Cancer Tumorigenesis

GPER-1 protein expression was immunohistochemically examined in 164 primary breast cancer specimens and their matched normal breast epithelium. GPER-1 down-regulation correlated significantly with increased histological grading (p = .015), lymph node metastases (p = .032), and negative estrogen receptor status (p = .018). The decrease of GPER-1 expression in breast cancer tissue, relative to normal tissue, was associated with poor overall survival (p = .043) and disease-free survival (p = .037) and remained a significant unfavorable factor in multivariate analysis for DFS (HR = 1.569; 95% CI, 1.024–2.797; p = .041) and OS (HR = 2.082; 95% CI, 1.248–4.773; p = .039). Thus aberrant GPER-1 expression seems to be an important factor in breast cancer progression.

GPER-1 acts as a tumor suppressor in ovarian cancer

BackgroundIt is known that the new membrane-bound estrogen receptor GPER-1 acts suppressive in breast cancer cells and its expression decreases during disease progression. This study was conducted to evaluate the GPER-1 expression in ovarian cancer and its correlation with progression. Its function was tested in vitro in ovarian cancer cells.Patients and methodsGPER-1 expression was analyzed by immunohistochemistry in 35 benign ovarian tumors, 35 tumors of low-malignant potential and in 124 ovarian cancers. GPER-1 expression was correlated to the prospectively evaluated disease-free survival of ovarian cancer patients. We also tested GPER-1 expression in ovarian cancer cells and the effect of GPER-1 stimulation on cell growth.ResultsGPER-1 expression was significantly lower in ovarian cancer tissue than in benign and low-malignant ovarian tumors. GPER-1 expression was observed in 83.1% of malignant tumors and was higher in early stage cancers and tumors with high histological differentiation. GPER-1 expression was associated with favourable clinical outcome. The difference in 2-year disease-free survival by GPER-1 expression was significant, 28.6% for GPER-1 negative and 59.2% for GPER-1 positive cases (p = 0.002). GPER-1 expression was observed in SKOV-3 and OVCAR-3 ovarian cancer cell lines. G-1, a selective GPER-1 agonist, suppressed proliferation of the two cell types via inhibition of cell cycle progression in G2/M phase and stimulation of caspase-dependent apoptosis. The blockade in G2/M phase was associated with increased expression of cyclin B1 and Cdc2 and phosphorylation of histone 3.ConclusionGPER-1 emerges as a new tumor suppressor with unsuspected therapeutic potential for ovarian cancer.

Minireview: G Protein-Coupled Estrogen Receptor-1, GPER-1: Its Mechanism of Action and Role in Female Reproductive Cancer, Renal and Vascular Physiology

Using cDNA cloning strategies commonly employed for G protein-coupled receptors (GPCR), GPCR-30 (GPR30), was isolated from mammalian cells before knowledge of its cognate ligand. GPR30 is evolutionarily conserved throughout the vertebrates. A broad literature suggests that GPR30 is a Gs-coupled heptahelical transmembrane receptor that promotes specific binding of naturally occurring and man-made estrogens but not cortisol, progesterone, or testosterone. Its "pregenomic" signaling actions are manifested by plasma membrane-associated actions familiar to GPCR, namely, stimulation of adenylyl cyclase and Gβγ-subunit protein-dependent release of membrane-tethered heparan bound epidermal growth factor. These facts regarding its mechanism of action have led to the formal renaming of this receptor to its current functional designate, G protein-coupled estrogen receptor (ER) (GPER)-1. Further insight regarding its biochemical action and physiological functions in vertebrates is derived from receptor knockdown studies and the use of selective agonists/antagonists that discriminate GPER-1 from the nuclear steroid hormone receptors, ERα and ERβ. GPER-1-selective agents have linked GPER-1 to physiological and pathological events regulated by estrogen action, including, but not limited to, the central nervous, immune, renal, reproductive, and cardiovascular systems. Moreover, immunohistochemical studies have shown a positive association between GPER-1 expression and progression of female reproductive cancer, a relationship that is diametrically opposed from ER. Unlike ER knockout mice, GPER-1 knockout mice are fertile and show no overt reproductive anomalies. However, they do exhibit thymic atrophy, impaired glucose tolerance, and altered bone growth. Here, we discuss the role of GPER-1 in female reproductive cancers as well as renal and vascular physiology.