Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells.

BACKGROUND: Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERß, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. METHODS: The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. RESULTS: After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. CONCLUSIONS: Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

Activation of G Protein-Coupled Estrogen Receptor (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway.

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-ß-estradiol (E2, 10 nM) or G-1 (1 µM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 µM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

Dental pulp stem cell-derived exosomes revitalize salivary gland epithelial cell function in NOD mice via the GPER-mediated cAMP/PKA/CREB signaling pathway.

BACKGROUND: Restoration of salivary gland function in Sjogren's syndrome (SS) is still a challenge. Dental pulp stem cells (DPSCs) derived exosomes had shown anti-inflammatory, anti-oxidative, immunomodulatory, and tissue function restorative abilities. However, the salivary gland function restoration potential of DPSCs-derived exosomes (DPSC-Exos) during SS has not been investigated yet. METHODS: DPSC-Exos was isolated by ultracentrifugation methods and characterized. Salivary gland epithelial cells (SGEC) were treated with interferon-gamma (IFN-γ) to mimic SS in vitro and cultured with or without DPSC-Exos. SGEC survival and aquaporin 5 (AQP5) expression were analyzed. mRNA sequencing and bioinformatics analysis were performed in IFN-γ vs. DPSC-Exos+ IFN-γ treated SGEC. Non-obese diabetic (NOD)/ltj female mice (SS model), were intravenously administered with DPSC-Exos, and salivary gland functions and SS pathogenicity were analyzed. Furthermore, the mRNA sequencing and bioinformatics predicted mechanism of the therapeutic effect of DPSC-Exos was further investigated both in vitro and in vivo using RT-qPCR, Western blot, immunohistochemistry, immunofluorescence, flowcytometry analysis. RESULTS: DPSC-Exos partially rescued IFN-γ triggered SGEC death. IFN-γ inhibited AQP5 expression in SGEC and DPSC-Exos reversed this effect. Transcriptome analysis showed GPER was the upregulated DEG in DPSC-Exos-treated SGEC with a positive correlation with salivary secretion-related DEGs. Pathway enrichment analysis revealed that DEGs were mainly attributed to estrogen 16 alpha-hydroxylase activity, extracellular exosome function, cAMP signaling, salivary secretion, and estrogen signaling. Intravenous injection of DPSC-Exos in NOD/ltj mice alleviated the SS syndrome as indicated by the increased salivary flow rate, attenuated glandular inflammation, and increased AQP5 expression. GPER was also upregulated in the salivary gland of DPSC-Exos-treated NOD/ltj mice compared with the PBS-treated NOD/ltj mice. IFN-γ+DPSC-Exos-treated SGEC showed higher expression of AQP5, p-PKA, cAMP, and intracellular Ca2+ levels compared with IFN-γ-treated SGEC. These effects were reversed by the inhibition of GPER. CONCLUSIONS: Our results showed that DPSC-Exos revitalize salivary gland epithelial cell function during SS via the GPER-mediated cAMP/PKA/CREB pathway suggesting the possible therapeutic potential of DPSC-Exos in SS-treatment.

Comparison of the mechanisms of estrogen disrupting effects between triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP).

As the typical aryl-organophosphate flame retardants (OPFRs), triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to be estrogen disruptors. However, estrogen receptor α (ERα) binding experiments could not explain their biological effects. In this study, their action on ERα, G protein-coupled estrogen receptor (GPER) and the synthesis of 17ß-estradiol (E2) were investigated using in vitro assays and molecular docking. The results showed that TPhP acted as an ERα agonist and recruited steroid receptor co-activator 1 (SRC1) and 3 (SRC3), which was found for the first time. Unlike TPhP, TDCIPP acted as an ERα antagonist. However, both TPhP and TDCIPP activated the estrogen pathway by GPER in SKBR3 cells which were lack of ERα. Although molecular docking results revealed that both TPhP and TDCIPP could dock into ERα and GPER, their substituent groups and combination mode might affect the receptor activation. In addition, by using estrogen biosynthesis assay in H295R cells, both of TPhP and TDCIPP were found to promote E2 synthesis and E2/T ratio involving their different alteration on levels of progesterone, testosterone and estrone, and expression of various key genes. Our data proposed estrogen-disrupting mechanism frameworks of TPhP and TDCIPP. Moreover, our results will contribute to future construction of adverse outcome pathway (AOP) framework of endocrine disruptors.

A mechanistic evaluation of the potential for octamethylcyclotetrasiloxane to produce effects via endocrine modes of action.

This review is a hypothesis driven, mechanistic evaluation of the potential for octamethylcyclotetrasiloxane (D4) to produce any effects via endocrine modes of action. D4 is a volatile, lipophilic liquid used in the production of high molecular weight dimethylsiloxane polymers. These are used in a variety of industrial, medical, cleaning, and personal care products, and they may contain low levels of residual D4. Low concentrations of D4 are found in the environment and there is potential for low level human exposure. All of the measured environmental and workplace levels of D4 fall below no observed effect levels (NOEL). Most of the effects of high dose D4 involve the female reproductive system. In the mature intact female rat following chronic high dose exposure, D4 may cause inhibition of mating and ovulation, decreased live litter sizes, small increases in the estrogen to progesterone ratio primarily through decreases in progesterone, and increases in uterine hyperplasia. When endogenous estrogens are very low, high dose D4 causes increases in some uterine parameters. To assess whether these high dose effects can be attributed to an endocrine mode of action, endpoints are ranked for relevance and strength, consistent with published concepts. When sufficient information is available the level of activity of D4 for producing the observed effect is compared with that of potent endocrines. The conclusions reached are that all of the effects of D4 fall well short of any established criteria for D4 to be capable of producing any adverse effect via an endocrine mode of action.

HPTE-Induced Embryonic Thymocyte Death and Alteration of Differentiation Is Not Rescued by ERα or GPER Inhibition but Is Exacerbated by Concurrent TCR Signaling.

Organochlorine pesticides, such as DDT, methoxychlor, and their metabolites, have been characterized as endocrine disrupting chemicals (EDCs); suggesting that their modes of action involve interaction with or abrogation of endogenous endocrine function. This study examined whether embryonic thymocyte death and alteration of differentiation induced by the primary metabolite of methoxychlor, HPTE, rely upon estrogen receptor binding and concurrent T cell receptor signaling. Estrogen receptor inhibition of ERα or GPER did not rescue embryonic thymocyte death induced by HPTE or the model estrogen diethylstilbestrol (DES). Moreover, adverse effects induced by HPTE or DES were worsened by concurrent TCR and CD2 differentiation signaling, compared with EDC exposure post-signaling. Together, these data suggest that HPTE- and DES-induced adverse effects on embryonic thymocytes do not rely solely on ER alpha or GPER but may require both. These results also provide evidence of a potential collaborative signaling mechanism between TCR and estrogen receptors to mediate adverse effects on embryonic thymocytes, as well as highlight a window of sensitivity that modulates EDC exposure severity.

The estrogen receptor coactivator AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative invasive lobular carcinoma of the breast.

PURPOSE: According to the 2017 St Gallen surrogate definitions of the intrinsic subtypes, Ki67, progesterone receptor (PR) and Nottingham histological grade (NHG) are used for prognostic classification of estrogen receptor (ER) positive/HER2-negative breast cancer into luminal A- or luminal B-like. The aim of the present study was to investigate if additional biomarkers, related to endocrine signaling pathways, e.g., amplified in breast cancer 1 (AIB1), androgen receptor (AR), and G protein-coupled estrogen receptor (GPER), can provide complementary prognostic information in a subset of ER-positive/HER-negative invasive lobular carcinoma (ILC). METHODS: Biomarkers from 224 patients were analyzed immunohistochemically on tissue microarray. The primary endpoint was breast cancer mortality (BCM), analyzed with 10- and 25-year follow-up (FU). In addition, the prognostic value of gene expression data for these biomarkers was analyzed in three publicly available ILC datasets. RESULTS: AIB1 (high vs. low) was associated to BCM in multivariable analysis (adjusted for age, tumor size, nodal status, NHG, Ki67, luminal-like classification, and adjuvant systemic therapy) with 10-year FU (HR 6.8, 95% CI 2.3-20, P = 0.001) and 25-year FU (HR 3.0, 95% CI 1.1-7.8, P = 0.03). The evidence of a prognostic effect of AIB1 could be confirmed by linking gene expression data to outcome in independent publicly available ILC datasets. AR and GPER were neither associated to BCM with 10-year nor with 25-year FU (P > 0.33). Furthermore, Ki67 and NHG were prognostic for BCM at both 10-year and 25-year FU, whereas PR was not. CONCLUSIONS: AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative ILC.

Less Exploited GPCRs in Precision Medicine: Targets for Molecular Imaging and Theranostics.

Precision medicine relies on individually tailored therapeutic intervention taking into account individual variability. It is strongly dependent on the availability of target-specific drugs and/or imaging agents that recognize molecular targets and patient-specific disease mechanisms. The most sensitive molecular imaging modalities, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), rely on the interaction between an imaging radioprobe and a target. Moreover, the use of target-specific molecular tools for both diagnostics and therapy, theranostic agents, represent an established methodology in nuclear medicine that is assuming an increasingly important role in precision medicine. The design of innovative imaging and/or theranostic agents is key for further accomplishments in the field. G-protein-coupled receptors (GPCRs), apart from being highly relevant drug targets, have also been largely exploited as molecular targets for non-invasive imaging and/or systemic radiotherapy of various diseases. Herein, we will discuss recent efforts towards the development of innovative imaging and/or theranostic agents targeting selected emergent GPCRs, namely the Frizzled receptor (FZD), Ghrelin receptor (GHSR-1a), G protein-coupled estrogen receptor (GPER), and Sphingosine-1-phosphate receptor (S1PR). The pharmacological and clinical relevance will be highlighted, giving particular attention to the studies on the synthesis and characterization of targeted molecular imaging agents, biological evaluation, and potential clinical applications in oncology and non-oncology diseases. Whenever relevant, supporting computational studies will be also discussed.

Heart Disease in Women: Unappreciated Challenges, GPER as a New Target.

Heart disease in women remains underappreciated, underdiagnosed and undertreated. Further, although we are starting to understand some of the social and behavioral determinants for this, the biological basis for the increased rate of rise in atherosclerosis risk in women after menopause remains very poorly understand. In this review we will outline the scope of the clinical issues related to heart disease in women, the emerging findings regarding the biological basis underlying the increased prevalence of atherosclerotic risk factors in postmenopausal women (vs. men) and the role of the G protein-coupled estrogen receptor (GPER) and its genetic regulation as a determinant of these sex-specific risks. GPER is a recently appreciated GPCR that mediates the rapid effects of estrogen and aldosterone. Recent studies have identified that GPER activation regulates both blood pressure. We have shown that regulation of GPER function via expression of a hypofunctional GPER genetic variant is an important determinant of blood pressure and risk of hypertension in women. Further, our most recent studies have identified that GPER activation is an important regulator of low density lipoprotein (LDL) receptor metabolism and that expression of the hypofunctional GPER genetic variant is an important contributor to the development of hypercholesterolemia in women. GPER appears to be an important determinant of the two major risk factors for coronary artery disease-blood pressure and LDL cholesterol. Further, the importance of this mechanism appears to be greater in women. Thus, the appreciation of the role of GPER function as a determinant of the progression of atherosclerotic disease may be important both in our understanding of cardiometabolic function but also in opening the way to greater appreciation of the sex-specific regulation of atherosclerotic risk factors.

Tamoxifen Resistance: Emerging Molecular Targets.

17ß-Estradiol (E2) plays a pivotal role in the development and progression of breast cancer. As a result, blockade of the E2 signal through either tamoxifen (TAM) or aromatase inhibitors is an important therapeutic strategy to treat or prevent estrogen receptor (ER) positive breast cancer. However, resistance to TAM is the major obstacle in endocrine therapy. This resistance occurs either de novo or is acquired after an initial beneficial response. The underlying mechanisms for TAM resistance are probably multifactorial and remain largely unknown. Considering that breast cancer is a very heterogeneous disease and patients respond differently to treatment, the molecular analysis of TAM's biological activity could provide the necessary framework to understand the complex effects of this drug in target cells. Moreover, this could explain, at least in part, the development of resistance and indicate an optimal therapeutic option. This review highlights the implications of TAM in breast cancer as well as the role of receptors/signal pathways recently suggested to be involved in the development of TAM resistance. G protein-coupled estrogen receptor, Androgen Receptor and Hedgehog signaling pathways are emerging as novel therapeutic targets and prognostic indicators for breast cancer, based on their ability to mediate estrogenic signaling in ERα-positive or -negative breast cancer.

The role of hypothalamic estrogen receptors in metabolic regulation.

Estrogens regulate key features of metabolism, including food intake, body weight, energy expenditure, insulin sensitivity, leptin sensitivity, and body fat distribution. There are two 'classical' estrogen receptors (ERs): estrogen receptor alpha (ERS1) and estrogen receptor beta (ERS2). Human and murine data indicate ERS1 contributes to metabolic regulation more so than ESR2. For example, there are human inactivating mutations of ERS1 which recapitulate aspects of the metabolic syndrome in both men and women. Much of our understanding of the metabolic roles of ERS1 was initially uncovered in estrogen receptor α-null mice (ERS1(-/-)); these mice display aspects of the metabolic syndrome, including increased body weight, increased visceral fat deposition and dysregulated glucose intolerance. Recent data further implicate ERS1 in specific tissues and neuronal populations as being critical for regulating food intake, energy expenditure, body fat distribution and adipose tissue function. This review will focus predominantly on the role of hypothalamic ERs and their critical role in regulating all aspects of energy homeostasis and metabolism.

The G Protein Estrogen Receptor (GPER) is involved in the resistance to the CDK4/6 inhibitor palbociclib in breast cancer.

BACKGROUND: The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients. METHODS: BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored. RESULTS: Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs. CONCLUSIONS: Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.

The Role of G Protein-Coupled Estrogen Receptor (GPER) in Vascular Pathology and Physiology.

OBJECTIVE: Estrogen is indispensable in health and disease and mainly functions through its receptors. The protection of the cardiovascular system by estrogen and its receptors has been recognized for decades. Numerous studies with a focus on estrogen and its receptor system have been conducted to elucidate the underlying mechanism. Although nuclear estrogen receptors, including estrogen receptor-α and estrogen receptor-ß, have been shown to be classical receptors that mediate genomic effects, studies now show that GPER mainly mediates rapid signaling events as well as transcriptional regulation via binding to estrogen as a membrane receptor. With the discovery of selective synthetic ligands for GPER and the utilization of GPER knockout mice, significant progress has been made in understanding the function of GPER. In this review, the tissue and cellular localizations, endogenous and exogenous ligands, and signaling pathways of GPER are systematically summarized in diverse physiological and diseased conditions. This article further emphasizes the role of GPER in vascular pathology and physiology, focusing on the latest research progress and evidence of GPER as a promising therapeutic target in hypertension, pulmonary hypertension, and atherosclerosis. Thus, selective regulation of GPER by its agonists and antagonists have the potential to be used in clinical practice for treating such diseases.

GPER/PKA-Dependent Enhancement of Hormone-Sensitive Lipase Phosphorylation in 3T3-L1 Adipocytes by Piceatannol.

We previously reported that piceatannol (PIC) had an anti-obesity effect only in ovariectomized (OVX) postmenopausal obesity mice. PIC was found to induce the phosphorylation of hormone-sensitive lipase (pHSL) in OVX mice. To elucidate the mechanism by which PIC activates HSL, we investigated the effect of PIC using 3T3-L1 adipocytes. PIC induced HSL phosphorylation at Ser563 in 3T3-L1 cells, as in vivo experiments showed. pHSL (Ser563) is believed to be activated through the ß-adrenergic receptor (ß-AR) and protein kinase A (PKA) pathways; however, the addition of a selective inhibitor of ß-AR did not inhibit the effect of PIC. The addition of a PKA inhibitor with PIC blocked pHSL (Ser563), suggesting that the effects are mediated by PKA in a different pathway than ß-AR. The addition of G15, a selective inhibitor of the G protein-coupled estrogen receptor (GPER), reduced the activation of HSL by PIC. Furthermore, PIC inhibited insulin signaling and did not induce pHSL (Ser565), which represents its inactive form. These results suggest that PIC acts as a phytoestrogen and phosphorylates HSL through a novel pathway that activates GPER and its downstream PKA, which may be one of the inhibitory actions of PIC on fat accumulation in estrogen deficiency.

Improvement of Cardiovascular Function in Aging Females by the Prolonged Activation of G Protein-Coupled Estrogen Receptor.

Ample evidence suggests that estrogen replacement therapy is associated with beneficial effects with regard to cardiovascular diseases when the therapy is initiated temporally close to menopause but not when it is initiated later. Little is known about the complex interactions between hormone receptors after menopause. Coronary artery function and cardiac function were measured in rats that had either received no treatment or had been pretreated with an androgen receptor (AR) antagonist and/or a GPER agonist G-1. ICI 182,780 was used to block the classical estrogen receptors (ERs) to investigate their complex interactions with GPER. The beneficial effects of GPER were only observed by blocking ARs and classical ERs in aged female rats. The results demonstrate that GPER activation is a potential therapeutic target for the inhibition of age-dependent coronary artery dysfunction and cardiac dysfunction under the condition of blocking ARs and classical ERs after menopause. CLINICAL RELEVANCE: The risk of cardiovascular disease in postmenopausal women significantly increased. The role of sex hormones and their receptors during this process is still complicated. Our present study demonstrated that the imbalance of androgen and estrogen may contribute to the impairment of vascular reactivity and subsequent cardiac function. Treatment with GPER agonist G1 combined with the inhibition of ERα and ERß could improve vascular function and reduce the myocardial ischemia reperfusion injury. These findings may provide the novel and effective strategy for the treatment of cardiovascular diseases in postmenopausal women.

Sodium butyrate treatment and fecal microbiota transplantation provide relief from ulcerative colitis-induced prostate enlargement.

The ability to regulate the gut environment has resulted in remarkable great breakthroughs in the treatment of several diseases. Several studies have found that the regulation of the gut environment might provide relief from the symptoms of benign prostatic hyperplasia. However, the correlation between the gut microenvironment and the colon and prostate glands is still unknown. We found that ulcerative colitis (UC) induced an increase in prostate volumes that could be reversed by sodium butyrate (NaB) and fecal microbiota transplantation (FMT). The mechanism by which UC induced changes in the prostate gland was examined via RNA-Seq. The results show that the expression level of GPER was significantly lower in the prostate gland of UC mices than in normal mices. The expression of GPER could be increased via treatment with NaB or FMT. We found that prostate tissues exhibited higher butryic acid levels after they were treated with NaB or FMT. In experiments conducted in vitro, NaB or the fecal filtrate (FF) from healthy mice up-regulated of the expression of GPER, inhibited cell growth, and induced apoptosis in BPH-1 cells. These changes could be alleviated by treatment with the G15 or in GPER-silenced cells.

Differential Effects of the G-Protein-Coupled Estrogen Receptor (GPER) on Rat Embryonic (E18) Hippocampal and Cortical Neurons.

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERß. Studies related to the extranuclear, membrane-bound G-protein-coupled ER (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic day 18 (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and nonselective agonist, 17ß-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca2+ rise in hippocampal neurons involves internal Ca2+ store release via activation of phospholipase C (PLC) and extracellular entry via Ca2+ channels. Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet real-time qPCR (RT-qPCR) and Western blotting showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen's action in early neuronal development.

The Interplay of GPER1 with 17ß-Aminoestrogens in the Regulation of the Proliferation of Cervical and Breast Cancer Cells: A Pharmacological Approach.

The G-protein-coupled receptor for estrogen (GPER1) is a transmembrane receptor involved in the progression and development of various neoplasms whose ligand is estradiol (E2). 17ß-aminoestrogens (17ß-AEs) compounds, analogs to E2, are possible candidates for use in hormone replacement therapy (HRT), but our knowledge of their pharmacological profile is limited. Thus, we explored the molecular recognition of GPER1 with different synthetic 17ß-AEs: prolame, butolame, and pentolame. We compared the structure and ligand recognition sites previously reported for a specific agonist (G1), antagonists (G15 and G36), and the natural ligand (E2). Then, the biological effects of 17ß-AEs were analyzed through cell viability and cell-cycle assays in two types of female cancer. In addition, the effect of 17ß-AEs on the phosphorylation of the oncoprotein c-fos was evaluated, because this molecule is modulated by GPER1. Molecular docking analysis showed that 17ß-AEs interacted with GPER1, suggesting that prolame joins GPER1 in a hydrophobic cavity, similarly to G1, G15, and E2. Prolame induced cell proliferation in breast (MCF-7) and cervical cancer (SIHA) cells; meanwhile, butolame and pentolame did not affect cell proliferation. Neither 17ß-AEs nor E2 changed the activation of c-fos in MCF-7 cells. Meanwhile, in SIHA cells, E2 and 17ß-AEs reduced c-fos phosphorylation. Thus, our data suggest that butolame and pentolame, but not prolame, could be used for HRT without presenting a potential risk of inducing breast- or cervical-cancer-cell proliferation. The novelty of this work lies in its study of compound analogs to E2 that may represent important therapeutic strategies for women in menopause, with non-significant effects on the cell viability of cancer cells. The research focused on the interactions of GPER1, a molecule recently associated with promoting and maintaining various neoplasms.

Novel GPER Agonist, CITFA, Increases Neurite Growth in Rat Embryonic (E18) Hippocampal Neurons.

Numerous studies have reported neuroprotective and procognitive effects of estrogens. The estrogen 17ß-estradiol (E2) activates both the classical nuclear estrogen receptors ERα and ERß as well as the G protein-coupled estrogen receptor (GPER). The differential effects of targeting the classical estrogen receptors over GPER are not well-understood. A limited number of selective GPER compounds have been described. In this study, 10 novel compounds were synthesized and exhibited half-maximal effective concentration values greater than the known GPER agonist G-1 in calcium mobilization assays performed in nonadherent HL-60 cells. Of these compounds, 2-cyclohexyl-4-isopropyl-N-((5-(tetrahydro-2H-pyran-2-yl)furan-2-yl)methyl)aniline, referred to as CITFA, significantly increased axonal and dendritic growth in neurons extracted from embryonic day 18 (E18) fetal rat hippocampal neurons. Confirmation of the results was performed by treating E18 hippocampal neurons with known GPER-selective antagonist G-36 and challenging with either E2, G-1, or CITFA. Results from these studies revealed an indistinguishable difference in neurite outgrowth between the treatment and control groups, exhibiting that neurite outgrowth in response to G-1 and CITFA originates from GPER activation and can be abolished with pretreatment of an antagonist. Subsequent docking studies using a homology model of GPER showed unique docking poses between G-1 and CIFTA. While docking poses differed between the ligands, CIFTA exhibited more favorable distance, bond angle, and strain for hydrogen-bonding and hydrophobic interactions.

Modulatory Effects of Estradiol and Its Mixtures with Ligands of GPER and PPAR on MAPK and PI3K/Akt Signaling Pathways and Tumorigenic Factors in Mouse Testis Explants and Mouse Tumor Leydig Cells.

The present study was designed to evaluate how estradiol alone or in combination with G protein-coupled estrogen receptor (GPER) agonists and GPER and peroxisome proliferator-activated receptor (PPAR) antagonists alter the expression of tumor growth factor ß (TGF-ß), cyclooxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) in mouse testis explants and MA-10 mouse tumor Leydig cells. In order to define the hormone-associated signaling pathway, the expression of MAPK and PI3K/Akt was also examined. Tissue explants and cells were treated with estradiol as well as GPER agonist (ICI 182,780), GPER antagonist (G-15), PPARα antagonist (GW6471), and PPARγ antagonist (T00709072) in various combinations. First, we showed that in testis explants GPER and PPARα expressions were activated by the GPER agonist and estradiol (either alone or in mixtures), whereas PPARγ expression was activated only by GPER agonist. Second, increased TGF-ß expression and decreased COX-2 expression were found in all experimental groups of testicular explants and MA-10 cells, except for up-regulated COX-2 expression in estradiol-treated cells, compared to respective controls. Third, estradiol treatment led to elevated expression of HIF-1α and VEGF, while their lower levels versus control were noted in the remaining groups of explants. Finally, we demonstrated the up-regulation of MAPK and PI3Kp85/Akt expressions in estradiol-treated groups of both ex vivo and in vitro models, whereas estradiol in mixtures with compounds of agonistic or antagonistic properties either up-regulated or down-regulated signaling kinase expression levels. Our results suggest that a balanced estrogen level and its action together with proper GPER and PPAR signaling play a key role in the maintenance of testis homeostasis. Moreover, changes in TGF-ß and COX-2 expressions (that disrupted estrogen pathway) as well as disturbed GPER-PPAR signaling observed after estradiol treatment may be involved in testicular tumorigenesis.