G Protein-coupled Estrogen Receptor mRNA Research Articles

Sex-specific epigenetics drive low GPER expression in gastrointestinal smooth muscles in type 2 diabetic mice

Type 2 diabetes mellitus (T2D) causes gastroparesis, delayed intestinal transit, and constipation, for unknown reasons. Complications are predominant in women than men (particularly pregnant and postmenopausal women), suggesting a female hormone-mediated mechanism. Low G-protein coupled estrogen receptor (GPER) expression from epigenetic modifications may explain it. We explored sexually differentiated GPER expression and gastrointestinal symptoms related to GPER alterations in wild-type (WT) and T2D mice (db/db). We also created smooth muscle-specific GPER knockout (GPER KO) mice to phenotypically explore the effect of GPER deficiency on gastrointestinal motility. GPER mRNA and protein expression, DNA methylation and histone modifications were measured from stomach and colon samples of db/db and WT mice. Changes in gut motility were also evaluated as daily fecal pellet production patterns. We found that WT female tissues have the highest GPER mRNA and protein expressions. The expression is lowest in all db/db. GPER downregulation is associated with promoter hypermethylation and reduced enrichment of H3K4me3 and H3K27ac marks around the GPER promoter. We also observed sex-specific disparities in fecal pellet production patterns of the GPER KO mice compared to WT. We thus, conclude that T2D impairs gut GPER expression, and epigenetic sex-specific mechanisms matter in the downregulation.

Current progress and prospects for G protein-coupled estrogen receptor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a biologically and clinically heterogeneous disease. The G protein-coupled estrogen receptor (GPER) plays a crucial role in mediating the effect of estrogen and estrogen-like compounds in TNBC cells. Compared with other subtypes, GPER has a higher expression in TNBC. The GPER mechanisms have been thoroughly characterized and analyzed in estrogen receptor α (ERα) positive breast cancer, but not in TNBC. Our previous work revealed that a higher expression of GPER mRNA indicates a better prognosis for ERα-positive breast cancer; however, its effects in TNBC differ. Whether GPER could serve as a predictive prognostic marker or therapeutic target for TNBC remains unclear. In this review, we provide a detailed introduction to the subcellular localization of GPER, the different effects of various ligands, and the interactions between GPER and closely associated factors in TNBC. We focused on the internal molecular mechanisms specific to TNBC and thoroughly explored the role of GPER in promoting tumor development. We also discussed the interaction of GPER with specific cytokines and chemokines, and the relationship between GPER and immune evasion. Additionally, we discussed the feasibility of using GPER as a therapeutic target in the context of existing studies. This comprehensive review highlights the effects of GPER on TNBC, providing a framework and directions for future research.

Dihydrotestosterone induces arterial stiffening in female mice

BackgroundAndrogens are important sex hormones in both men and women and are supplemented when endogenous levels are low, for gender transitioning, or to increase libido. Androgens also circulate at higher levels in women with polycystic ovarian syndrome, a condition that increases the risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via downregulation of GPER.MethodsThe impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice at 15–16 weeks of age were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n = 8–9/group).ResultsIn cultured vascular smooth muscle cells, GPER mRNA was decreased by DHT (P = 0.001) but was not impacted by dexamethasone or medroxyprogesterone. In contrast, ERα expression in cultured cells was significantly suppressed by all three hormones (P < 0.0001). In control mice or mice treated with a single or double dose of DHT, a dose-dependent increase in body weight was observed (control 22 ± 2 g, single dose 24 ± 2 g, double dose 26 ± 2 g; P = 0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9 ± 0.3 m/s, single dose 4.3 ± 0.8 m/s, double dose 4.8 ± 1.0 m/s). This increase in arterial stiffness occurred independent of changes in blood pressure (P = 0.59). Histological analysis of aortic sections using Masson’s trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P = 0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20 ± 5, single dose 10.5 ± 5.6, double dose 10 ± 4 copies/ng; P = 0.001) and ERα (control 54 ± 2, single dose 24 ± 13, and double dose 23 ± 12 copies/ng; P = 0.003).ConclusionsThese findings indicate that androgen promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

G-Protein-Coupled Estrogen Receptor Expression in Rat Uterine Artery Is Increased by Pregnancy and Induces Dilation in a Ca2+ and ERK1/2 Dependent Manner.

Increasing levels of estrogens across gestation are partly responsible for the physiological adaptations of the maternal vasculature to pregnancy. The G protein-coupled estrogen receptor (GPER) mediates acute vasorelaxing effects in the uterine vasculature, which may contribute to the regulation of uteroplacental blood flow. The aim of this study was to investigate whether GPER expression and vasorelaxation may occur following pregnancy. Elucidation of the functional signalling involved was also investigated. Radial uterine and third-order mesenteric arteries were isolated from non-pregnant (NP) and pregnant rats (P). GPER mRNA levels were determined and—concentration–response curve to the GPER-specific agonist, G1 (10−10–10−6 M), was assessed in arteries pre-constricted with phenylephrine. In uterine arteries, GPER mRNA expression was significantly increased and vasorelaxation to G1 was significantly enhanced in P compared with NP rats. Meanwhile, in mesenteric arteries, there was a similar order of magnitude in NP and P rats. Inhibition of L-type calcium channels and extracellular signal-regulated kinases 1/2 significantly reduced vasorelaxation triggered by G1 in uterine arteries. Increased GPER expression and GPER-mediated vasorelaxation are associated with the advancement of gestation in uterine arteries. The modulation of GPER is exclusive to uterine arteries, thus suggesting a physiological contribution of GPER toward the regulation of uteroplacental blood flow during pregnancy.

Proliferation and apoptosis regulation by G protein-coupled estrogen receptor in glioblastoma C6 cells.

Glioblastoma is the most frequent primary tumor in the human brain. Glioblastoma cells express aromatase and the classic estrogen receptors ERα and ERβ and can produce estrogens that promote tumor growth. The membrane G protein-coupled estrogen receptor (GPER) also plays a significant role in numerous types of cancer; its participation in glioblastoma tumor development is not entirely known. The present study investigated the effect of the agonists [17β-estradiol (E2) and G1] and antagonist (G15) of GPER on proliferation and apoptosis of C6 glioblastoma cells. GPER expression was evaluated by immunofluorescence, western blotting and reverse transcription-quantitative PCR. Cell proliferation was determined using Ki67 immunopositivity. Cell viability was examined using the MTT assay and apoptosis using caspase-3 immunostaining and ELISA. C6 cells express GPER, and the immunopositivity increased after exposure to E2, G1, or their combination. GPER protein expression increased after treatment with E2 combined with G1. However, GPER mRNA expression decreased in treated cells compared with control. The percentage of Ki67 immunopositive C6 cells increased under the effect of E2 in combination with G1 or G1 alone. G15 significantly reduced Ki67 immunopositivity. Pearson's correlation analysis revealed a positive relationship between GPER and Ki67 immunopositivity across the study conditions. Additionally, the MTT assay showed a significant reduction in C6 cell viability after G15 treatment, alone or in combination with G1. The exposure to G15 increased the percentage of caspase-3 immunopositivity cells and caspase-3 levels. Pearson's correlation analysis demonstrated a negative correlation between GPER and caspase-3 immunopositivity across the study conditions. Glioblastoma C6 cells express GPER, and this receptor modulates cell proliferation and apoptosis. The GPER agonists E2 and G1 favored cell proliferation; meanwhile, the antagonist G15 reduced cell proliferation, viability and favored apoptosis. Therefore, GPER may be used as a biomarker of glioblastoma and as a target to develop new therapeutic strategies for glioblastoma treatment.

Phytoestrogen-like effect of Siwu Decoction and molecular mechanism: based on network pharmacology and in vivo experiment

This study explored the phytoestrogen-like effect of Siwu Decoction(SWD) and the estrogen receptor(ER)-mediated molecular mechanism based on network pharmacology and in vivo experiment. The active components and targets of SWD were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and related targets of &quot;estrogen&quot; from GeneCards and Online Mendelian Inheritance in Man(OMIM). Cytoscape and STRING were employed to construct the protein-protein interaction(PPI) network and &quot;chemical component-target-disease&quot; network and core targets were identified, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the core targets by R software. For the in vivo experiment, the 22-day-old SD female rats were treated(ig) with SWD for 4 days. Via hematoxylin-eosin(HE) staining, the morphological changes of rat uterus were observed. Reverse transcriptase-polymerase chain reaction(RT-PCR) was performed to detect mRNA expression of ER subtypes, estrogen-related targets, and the main regulatory factors in the estrogen signaling pathway. The results indicated 74 targets of SWD exerted phytoestrogen-like effect. KEGG pathway enrichment result suggested that estrogen signaling pathway was closely related to the phytoestrogen-like effect of SWD. Rats in SWD group demonstrated significantly thickened endometrium and significantly decreased expression of ERα, ERβ, and G protein-coupled estrogen receptor(GPER) mRNA in ovarian tissue. In addition, significant lowering of ERα and ERβ mRNA expression and significant rise of GPER mRNA expression in uterine tissue were observed in the SWD group. The expression of mitogen-activated protein kinase(MAPK) p38, MEK1/2 and extracellular signal-regulated kinase(ERK)1/2 mRNA was significantly low while that of epidermal growth factor receptor(EGFR) mRNA was significantly high in both ovarian and uterine tissues of SWD group compared with those in the control group. In conclusion, the phytoestrogen-like effect of SWD is closely related to the estrogen signaling pathway. The result lays a basis for revealing molecular mechanism of SWD in the treatment of gynecological diseases.

Expression of G-Protein-Coupled Estrogen Receptor (GPER) in Whole Testicular Tissue and Laser-Capture Microdissected Testicular Compartments of Men with Normal and Aberrant Spermatogenesis.

Simple SummaryNowadays, there is no doubt that estrogens play an important role in male reproduction, affecting testicular cell differentiation, proliferation, apoptosis and metabolism. It is also widely believed that intratesticular balance of androgens and estrogens is crucial for the testicular development and function and that the increased testicular estrogen production may be associated with spermatogenic failure. There is also growing epidemiological evidence that the exposure of men to endocrine disruptors demonstrating estrogenic activity (xenoestrogens) may lead to impairment of male fertility via interference with estrogen signaling pathways. Besides the two classical nuclear estrogen receptors, the membrane-bound G protein-coupled estrogen receptor (GPER) was described in human testicular tissue. However, there are little data on its expression in testes with disturbed spermatogenesis. In this study, we investigated the GPER expression pattern in biopsies of azoospermic men with complete and aberrant spermatogenesis. Our results showed an increased expression of the GPER in testes with impaired spermatogenesis. Moreover, they indicate a possible involvement of estrogen signaling through GPER in disturbed function of Sertoli cells—the cells that support spermatogenic process.In this study, we retrospectively investigated GPER expression in biopsies of azoospermic men with complete (obstructive azoospermia—OA) and aberrant spermatogenesis (nonobstructive azoospermia—NOA). Each biopsy was histologically evaluated with morphometry. The testicular GPER expression was analyzed by the immunohistochemistry and RT-PCR technique in the whole testicular tissue and in seminiferous tubules and Leydig cells after laser-capture microdissection. In laser-microdissected compartments, we also analyzed transcriptional expression of selected Leydig (CYP17A1, HSD17B3, StAR) and Sertoli cell (AMH, SCF, BMP4) function markers. Immunohistochemical staining revealed expression of GPER in the cytoplasm of Leydig and Sertoli cells. Its stronger intensity was observed in Sertoli cells of NOA biopsies. The RT-PCR analysis of the GPER mRNA level unequivocally showed its increased expression in seminiferous tubules (i.e., Sertoli cells), not Leydig cells in NOA biopsies. This increased expression correlated positively with the transcriptional level of AMH—a marker of Sertoli cell immaturity, as well as FSH serum level in NOA but not in the OA group. Our results clearly demonstrate altered GPER expression in testes with primary spermatogenic impairment that might be related to Sertoli cell maturity/function.

A novel transcript variant of human G-protein coupled estrogen receptor.

The G-protein coupled estrogen receptor (GPER) mediates short-term non-genomic effects of estrogen in diverse cell types and tissues. According to the NCBI nucleotide database, three variants of GPER are known. They are NM_001505.2 (GPER-v2), NM_001039966.1 (GPER-v3), and NM_001098201.1 (GPER-v4). Investigations on GPER expression are key to understand its physiological and pathological roles. However, most studies on GPER mRNA expression have considered total GPER mRNA expression regardless of the individual variants. The present study is motivated by a novel transcript observed in the UCSC Genome Browser (uc010ksd.1), which is annotated as GPER. The novel variant is similar to the known transcriptvariants of GPER in terms of the protein-coding sequence and the 3'UTR. However, it has a unique 5'UTR, which distinguishes it from other GPER variants. Using primers specific for uc010ksd.1, we have performed RT-PCR to show that the novel GPER transcript (hereafter referred to as GPER-v5) is expressed in human cancer cell lines, such as MCF-7, SW-620, COLO-205, and HT-29. Preliminary evidences indicate that GPER-v5 is a novel GPER mRNA variant. The expression of GPER-v5 in primary cells and tissues should be investigated before probing into its role and relevance in physiological and pathological conditions.

OR24-04 Ovarian Follicle Survival Is Determined by Follicle-Stimulating Hormone Receptor (FSHR) and Estrogen Receptor (GPER) Heteromers

Mechanisms regulating the selection of antral ovarian follicles are poorly understood and supposed to rely on low estrogen levels, decline of follicle-stimulating hormone (FSH) levels and receptor (FSHR) expression. These concepts are challenged in vitro, where apoptosis of human granulosa cells (hGLC) and transfected cell lines is induced by high doses of FSH or FSHR overexpression, while estrogens induce anti-apoptotic signals via nuclears and a G protein-coupled estrogen receptor (GPER). Therefore, in vitro data suggest that antral follicle selection may be driven by underestimated, FSH/FSHR-dependent apoptotic signals due to transiently maximized FSHR expression and overload of cAMP signalling, prevailing on estrogen-dependent signals. Here we demonstrate how FSHR/GPER physical interaction rescue ovarian follicles from FSH-mediated death. 10 nM FSH induces high intracellular levels of cAMP, measured by bioluminescence resonance energy transfer (BRET), and apoptosis in cultured hGLC under conditions where GPER levels are depleted by siRNA. This result was confirmed in transfected HEK293 cells overexpressing FSHR. Using BRET, photo-activated localization microscopy (PALM) and bioinformatics prodiction, we also demonstrate FSHR/GPER heteromers at the cell surface. The role of FSHR/GPER heteromers may be relevant to inhibit FSH-induced death signals, since increasing GPER expression levels in HEK293 cells co-expressing FSHR results in displacement of the Gαs-protein to FSHR, blockade of FSH-induced cAMP production and inhibition of apoptosis. However, in HEK293 cells coexpressing GPER/FSHR, FSH-induced activation of the anti-apoptotic AKT-pathway via a Gβγ-dependent mechanism, as demonstrated by Western blotting in cells treated using the inhibitor gallein. Inhibition of both FSH-induced cAMP production and apoptosis was lost when FSHR is coexpressed together with a mutant GPER, unable to heteromerize with FSHR, as well as in KO HEK293 cells unable to produce a molecular complex associated with GPER inhibiting cAMP. GPER/FSHR coexpression is confirmed in secondary follicles from paraffin-embedded tissues of human ovary by immunohistochemistry, suggesting that FSHR-GPER heterodimers could be physiologically relevant in vivo for inhibiting cAMP-linked apoptosis. Most importantly, FSHR and GPER co-expression correlates in hGLC from FSH-normo-responder women undergoing assisted reproduction, while it is not in hGLC from FSH-poor-responders, where increasing FSHR mRNA levels do not correspond to increasing GPER mRNA levels. We demonstrate that death signals in atretic follicles are delivered through overexpressed FSHR and inhibited by FSHR/GPER heteromerization, activating anti-apoptotic pathways. This finding unveils a novel working model of the physiology of dominant follicle selection and the relationship between FSH and estrogens.

G protein-coupled estrogen receptor-1 agonist induces chemotherapeutic effect via ER stress signaling in gastric cancer

G protein-coupled estrogen receptor (GPER) is known to play an important role in hormone-associated cancers. G-1, a novel synthetic GPER agonist, has been reported to exhibit anti-carcinogenic properties. However, the chemotherapeutic mechanism of GPER is yet unclear. Here, we evaluated GPER expression in human gastric cancer tissues and cells. We found that G-1 treatment attenuates GPER expression in gastric cancer. GPER expression increased G-1-induced antitumor effects in mouse xenograft model. We analyzed the effects of knockdown/overexpression of GPER on G-1-induced cell death in cancer cells. Increased GPER expression in human gastric cancer cells increased G-1-induced cell death via increased levels of cleaved caspase-3, -9, and cleaved poly ADP-ribose polymerase. Interestingly, during G-1-induced cell death, GPER mRNA and protein expression was attenuated and associated with ER stress-induced expression of PERK, ATF-4, GRP-78, and CHOP. Furthermore, PERK-dependent induction of ER stress activation increased G-1-induced cell death, whereas PERK silencing decreased cell death and increased drug sensitivity. Taken together, the data suggest that the induction of ER stress via GPER expression may increase G-1-induced cell death in gastric cancer cells. These results may contribute to a new paradigm shift in gastric cancer therapy.

Downregulation of G Protein-Coupled Estrogen Receptor (GPER) is Associated with Reduced Prognosis in Patients with Gastric Cancer.

BackgroundThis study is aimed to investigate the prognostic significance of the expression of G protein-coupled estrogen receptor (GPER) in gastric cancer tissue using bioinformatics data and immunohistochemistry.Material/MethodsExpression of GPER mRNA in gastric cancer tissues and normal adjacent tissues was investigated using data from The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and Oncomine database. Kaplan-Meier Plotter identified the association between GPER mRNA and prognosis. Correlation between GPER mRNA and DNA methylation used the cBioPortal for Cancer Genomics and the MethHC website. Genes co-expressed with GPER were identified from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) underwent FunRich analysis. Immunohistochemistry and Western blot evaluated GPER protein expression in tissue microarrays (TMAs) and gastric cancer cell lines.ResultsGPER mRNA and protein levels were significantly lower in gastric cancer tissue and cells lined when compared with normal tissues and cells. The results from GSE15459 showed that patients with low levels of GPER mRNA had a reduced overall survival (OS) (P=0.013) and disease-free survival (DFS) (P=0.019). A negative correlation (r=−0.611) between GPER mRNA and DNA methylation was found using the cBioPortal and MethHC. Co-expressed epithelial-mesenchymal transformation (EMT) genes were enriched with GPER (P<0.0001). Cox regression analysis showed that GPER protein expression was an independent prognostic factor (P=0.035)ConclusionsDownregulation of GPER predicts poor prognosis in gastric cancer. GPER may act as a tumor suppressor through the regulation of EMT in gastric cancer.

Effects of hypoxia exposure on apoptosis and expression of membrane steroid receptors, ZIP9, mPRα, and GPER in Atlantic croaker ovaries

Hypoxia exposure causes endocrine disruption in croaker resulting in impairment of ovarian growth and oocyte development, but the effects of hypoxia on non-classical steroid actions in fish ovaries in vivo remain largely unknown. Membrane androgen receptor (ZIP9) enhances apoptosis of cultured Atlantic croaker ovarian follicle cells via upregulation of p53 and Bax, and increased caspase 3 activity through non-classical steroid signaling. Conversely, apoptosis is inhibited in these cells by non-classical steroid mechanisms through membrane progestin receptor alpha (mPRα) and G protein-coupled estrogen receptor (GPER). Apoptosis and the expression of ZIP9, mPRα, and GPER were investigated in ovaries of croakers that had been exposed to normoxia (7.0 mg DO/L) or hypoxia (2.0 mg DO/L) for 6 weeks during their period of gonadal recrudescence. The proportion of tertiary yolk stage oocytes was decreased, and the proportions of atretic and apoptotic ovarian follicles were increased in ovaries from hypoxia-exposed fish compared to controls. Ovarian expression of all three receptors was altered after hypoxia exposure. Expression of mPRα and GPER mRNA and protein levels were decreased after hypoxia exposure, consistent with a decline in anti-apoptosis. In contrast, ZIP9 mRNA and protein levels were upregulated 4-fold in hypoxia-exposed fish compared to normoxic controls. The enhanced apoptosis in ovaries of hypoxia-exposed fish was associated with increased caspase-3/7 activity and elevated expression of the pro-apoptotic genes, Bax and p53. The finding that ZIP9 expression was increased in ovaries of croaker exposed to hypoxia provides in vivo evidence supporting ZIP9's proposed function in mediating ovarian follicle cell apoptosis.

G protein-coupled estrogen receptor (GPER) in adult boar testes, epididymis and spermatozoa during epididymal maturation

The G protein-coupled estrogen receptor (GPER) is a transmembrane receptor considered as a mediator of rapid non-genomic responses. GPER has been found in the male reproductive tract of many mammalian species. However, in adult boars, GPER has been reported only in ejaculated spermatozoa. Therefore, we focused on GPER detection in testicular and epididymal tissues and sperm cells in adult boars. We found GPER in Leydig cells and seminiferous tubules of boar testes and in the secretory epithelium of epididymis. A weaker signal was visible in smooth muscle cells and spermatozoa in the epididymal tubule. In spermatozoa isolated from epididymal parts, GPER was found to localize mainly in the sperm acrosome and flagellum. We immunodetected several protein bands in the extracts of the tissues and epididymal spermatozoa. A significantly higher amount of GPER mRNA was detected in the spermatozoa from caput epididymis, whereas the mRNA expression was lower in tissues of testes and caput epididymal. Our results showed the first evidence of GPER in boar epididymal spermatozoa. Moreover, the GPER localization in adult boar testes, epididymis, and mature spermatozoa suggests the involvement of estrogens via transmembrane receptor and rapid non-genomic signaling in both the sperm development and post-testicular maturation.

Sex Differences in the Central and Peripheral Manifestations of Heart Failure in Rats

Heart failure (HF) is a leading cause of morbidity and mortality for both men and women. Increased sympathetic nerve activity (SNA) is a hallmark of HF and correlates with poor prognosis. We and others have demonstrated that neuroinflammation and renin‐angiotensin system activity are elevated in the cardiovascular‐related regions of the brain in HF, contributing to SNA and the progression of HF. However, most studies that have examined these sympathoexcitatory mediators in the brain in HF have been performed in male animals. The goal of this study was to determine whether sex differences exist in neuroinflammation and renin‐angiotensin system activity in the hypothalamic paraventricular nucleus (PVN), a key cardiovascular‐related center of the brain that regulates SNA and extracellular fluid volume, in rats with HF. Male and female SD rats at ~13 weeks of age underwent coronary artery ligation (CL) to induce HF and were euthanized for anatomical and molecular studies 4 weeks later. Age‐matched male and female rats served as controls. There were no differences between male and female rats in echocardiography‐defined ischemic zone or left ventricular ejection fraction at 24 hours and at 4 weeks after CL. Female rats had lower body weight than male rats, but there was no difference in body weight between HF and control rats in either sex. At 4 weeks, lung weight was similar but right ventricular (RV) and left ventricular (LV) weights were smaller in female control than male control rats. Compared with their respective controls, male HF but not female HF rats had increases in lung weight/tibial length and RV weight/tibial length. There was no difference in LV dP/dtmax and LV end‐diastolic pressure between male and female controls. LV dP/dtmax was decreased in both male HF rats and female HF rats, but male HF rats had a lower dP/dtmax. Compared with their respective control rats, male but not female HF rats had increased LV end‐diastolic pressure. In the PVN, mRNA for tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), angiotensin receptor subtype 1a receptor (AT1aR) and estrogen receptors α and β (ER‐α, ER‐β) was similar in male and female controls. mRNA for G protein‐coupled estrogen receptor (GPER) was higher and mRNA for arginine vasopressin (AVP) was lower in female control than male control rats. Both male HF and female HF rats had increases in TNF‐α, IL‐1β, AT1aR and AVP mRNA in the PVN, compared with their respective controls, but the levels were higher in the male HF rats. ER‐α and ER‐β mRNA was decreased and GPER mRNA was unchanged in male HF and female HF rats, compared with respective controls. There was no difference in ERK1/2 activity in the PVN between male and female control rats, and ERK1/2 activity in the PVN was equally increased in both male and female HF rats. Plasma angiotensin II, AVP and norepinephrine levels were similar in male and female control rats. The plasma levels of these humoral factors were higher in HF rats in either sex, but the AVP and norepinephrine levels were greater in male HF rats. These preliminary results suggest that female rats with HF following CL have less central neural inflammation and renin‐angiotensin system activity, less peripheral sympathetic and vasopressinergic activity and less hemodynamic compromise than male HF rats with the same degree of initial ischemic cardiac injury. The mechanisms responsible for these sex differences remain to be determined.Support or Funding InformationSupported by NIH HLR01HL136149 and R01HL073986.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of G Protein‐coupled Estrogen Receptor Promotes Endothelin‐dependent Natriuresis in Female Rats

The novel estrogen receptor, G protein‐coupled estrogen receptor (GPER), is a membrane‐bound receptor linked to acute signaling pathways. GPER activation elicits cardiovascular and nephroprotective effects against salt‐induced complications. However, its specific role in renal sodium handling is not yet defined. We hypothesized that activation of GPER in the renal medulla facilitates sodium excretion. Isosmotic saline was infused into the renal medullary interstitium (500 μl/h) of anesthetized male and female Sprague‐Dawley rats for an 80 min equilibration period and 20 min baseline urine collection period. Then, G1 (GPER agonist, 50 pmol/kg/min) or vehicle was infused into the renal medulla for an additional 20 min period. Compared with vehicle, G1 significantly increased urinary sodium excretion in females (from 0.6 ± 0.1 to 1.0 ± 0.1 μmol/min n=9, p&lt;0.05). G1 did not change blood pressure or urinary potassium excretion in comparison with vehicle control (106 ± 3 vs. 105 ± 4 mmHg, 0.5 ± 0.1 vs. 0.4 ± 0.1 μmol/min, n=9). Medullary blood flow remained unchanged during these experiments. In males, G1 did not change urinary sodium excretion (from 0.6 ± 0.1 to 0.7 ± 0.1 μmol/min, n=9). No sex‐differences in medullary GPER mRNA expression was observed, suggesting that sex‐differences in G1 effects on sodium excretion could be related to GPER downstream signaling cascade. Given that endothelin‐1 (ET‐1) is a well‐established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an ET‐1‐dependent pathway. To test this idea, we studied the effect of medullary infusion of G1 after dual blockade of ETA and ETB receptors achieved by i.v. bolus injection of ABT‐627 (5 mg/kg) and A‐192621 (10 mg/kg), respectively. We found that the natriuretic response to medullary GPER activation in females was inhibited by dual blockade of ET‐1 receptors (from 0.5 ± 0.2 to 0.5 ± 0.2 μmol/min n=5). These data uncover a novel role for renal medullary GPER in promoting sodium excretion via an ET‐1‐dependent pathway in females, but not in males.Support or Funding InformationFunded by AHA 15POST25090329 to EYG and P01 HL136267 to DMP and JSPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression Pattern of G-Protein-Coupled Estrogen Receptor in Myometrium of Uteri with and without Adenomyosis.

Objective To compare the expression of G-protein-coupled estrogen receptor (GPER) in the junctional zone and outer myometrium of the proliferative and secretory phases of women with and without adenomyosis. Methods A total of 76 women were included in this study, 42 with adenomyosis (proliferative phase, n = 23; secretory phases, n = 19) and 34 controls (proliferative phase, n = 16; secretory phases, n = 18). Protein and total RNA were extracted from the junctional zone (JZ) and outer myometrium (OM). GPER protein and mRNA expression levels were evaluated by the use of western blotting and real-time quantitative polymerase chain reaction (RT-qPCR). Results The expression of GPER protein and mRNA in women with adenomyosis was significantly higher than that of control subjects, both in the junctional zone and in the outer myometrium and both in the proliferative and in the secretory phases. Conclusion The significant and consistent increase in GPER expression in adenomyosis compared with control subjects, regardless of whether it was in the proliferative or secretory phases and regardless of whether it was in the JZ or OM, suggests that GPER plays an important role in the pathogenesis of the adenomyosis.

Evaluation of Steroid Receptors mRNA Fingerprints in Two Groups of Normozoospermic Patients: Men from Unexplained Infertility Couples vs. Men from Couples with Tubal Factor Infertility

The study of sperm cellular components at molecular level is crucial for the diagnosis of male unexplained infertility. The aim of the study was to compare the molecular profile of steroid receptors and aromatase in spermatozoa obtained from two normozoospermic groups of patients issued from couples treated for infertility. We investigated 46 male patients from unexplained infertility couples and from men, 38 where female partners presented with tubal infertility. Sperm ERs (estrogen receptors: alpha and beta), GPER (G protein-coupled estrogen receptor), AR (androgen receptor) and aromatase mRNA expression levels by TaqMan qPCR were analyzed. AR transcript level was significantly lower in sperm of men from unexplained infertility couples vs. men from couples with tubal factor infertility (P = 0.04). Although the AR mRNA expression level did not had any effect on embryo development and its implantation, a significant correlation between AR mRNA levels and clinical pregnancy in unexplained infertility patients was observed. Taken together, AR transcript presence in ejaculated spermatozoa could be a potential marker for unexplained infertility.

G protein-coupled estrogen receptor deficiency accelerates liver tumorigenesis by enhancing inflammation and fibrosis

G protein-coupled estrogen receptor (GPER) is a novel estrogen-binding receptor involved in many pathological conditions, including cancer. In this study, we investigated the effect of GPER on hepatocellular carcinoma (HCC). Our data show GPER knockout in a diethylnitrosamine (DEN)-induced mouse tumor model significantly accelerated liver tumorigenesis, accompanied by enhanced immune cell infiltration, fibrosis, and the production of inflammatory factors, such as interleukin-6 (IL-6). We further delineated the function of GPER in macrophages and hepatic stellate cells (HSCs). Treatment with the selective GPER agonist, G-1, decreased the expression of IL-6 in bone marrow-derived macrophages, which was abrogated upon deficiency of GPER. In a HSC line (LX2), G-1 treatment downregulated the expression of α-smooth muscle actin. In addition, both GPER mRNA and protein levels were significantly lower in HCC compared with matched non-tumor tissues. However, modulating GPER expression did not affect the viability and proliferation of hepatoma cells in vitro. Together our results indicate that GPER protects against HCC tumorigenesis through regulating inflammatory responses rather than directly acting on tumor cells. Therefore, GPER activation may be a potential strategy for prevention and treatment of HCC.

NHERF1, a novel GPER associated protein, increases stability and activation of GPER in ER-positive breast cancer.

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC.

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes.