G Protein-coupled Estrogen Receptor Activation Research Articles

17β-Estradiol reduces inhibitory synaptic currents in entorhinal cortex neurons through G protein-coupled estrogen receptor-1 activation of extracellular signal-regulated kinase.

Estrogens are believed to modulate cognitive functions in part through the modulation of synaptic transmission in the cortex and hippocampus. Administration of 17β-estradiol (E2) can rapidly enhance excitatory synaptic transmission in the hippocampus and facilitate excitatory synaptic transmission in rat lateral entorhinal cortex via activation of the G protein-coupled estrogen receptor-1 (GPER1). To assess the mechanisms through which GPER1 activation facilitates synaptic transmission, we assessed the effects of acute 10 nM E2 administration on pharmacologically isolated evoked excitatory and inhibitory synaptic currents in layer II/III entorhinal neurons. Female Long-Evans rats were ovariectomized between postnatal day (PD) 63 and 74 and implanted with a subdermal E2 capsule to maintain continuous low levels of E2. Electrophysiological recordings were obtained between 7 and 20 days after ovariectomy. Application of E2 for 20 min did not significantly affect AMPA or NMDA receptor-mediated excitatory synaptic currents. However, GABA receptor-mediated inhibitory synaptic currents (IPSCs) were markedly reduced by E2 and returned towards baseline levels during the 20-min washout period. The inhibition of GABA-mediated IPSCs was blocked in the presence of the GPER1 receptor antagonist G15. GPER1 can modulate protein kinase A (PKA), but blocking PKA with intracellular KT5720 did not prevent the E2-induced reduction in IPSCs. GPER1 can also stimulate extracellular signal-regulated kinase (ERK), a negative modulator of GABAA receptors, and blocking activation of ERK with PD90859 prevented the E2-induced reduction of IPSCs. E2 can therefore result in a rapid GPER1 and ERK signaling-mediated reduction in GABA-mediated IPSCs. This provides a novel mechanism through which E2 can rapidly modulate synaptic excitability in entorhinal layer II/III neurons and may also contribute to E2 and ERK-dependent alterations in synaptic transmission in other brain areas.

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-β-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.

Endothelin receptor B is Required for the Blood Pressure lowering Effect of G Protein-coupled Estrogen Receptor 1 in Ovariectomized Rats.

Activation of the G protein-coupled estrogen receptor 1 (GPER1) elicits antihypertensive actions in different animal models. The endothelin-1 signaling system plays a fundamental role in blood pressure regulation. Lack of functional endothelin receptor B receptors (ETB) evokes hypertension and salt sensitivity. GPER1 and ETB interact to promote urinary sodium excretion in female rats. We hypothesized that activation of GPER1 protects against hypertension and salt sensitivity induced by ETB antagonism in female rats. Female Sprague Dawley rats were implanted with radiotelemetry. Then, animals were subjected to ovariectomy and simultaneously implanted with minipumps to deliver either the GPER1 agonist, G1, or its corresponding vehicle (Veh). Two weeks post-surgery, we initiated treatment of rats with the ETB antagonist, A-192621. Animals were maintained on a normal salt (NS) diet then challenged with a high salt (HS) diet for an additional 5 days. Assessment of mean arterial blood pressure revealed an increase in Veh-treated, but not G1-treated, rats in response to ovariectomy. A-192621 increased blood pressure in NS-fed Veh and G1-treated rats. G1 improved the circadian blood pressure rhythms which were disrupted in A-192621-treated ovariectomized rats. Thus, although systemic GPER1 activation did not protect ovariectomized rats from hypertension and salt sensitivity induced by ETB antagonism, it maintained the circadian blood pressure rhythms. Functional ETB is required to elicit the antihypertensive actions of GPER1. Additional studies are needed to improve our understanding of the interaction between G protein-coupled receptors in regulating circadian blood pressure rhythm.

Activation of GPER1 by G1 prevents PTSD-like behaviors in mice: Illustrating the mechanisms from BDNF/TrkB to mitochondria and synaptic connection.

G1 is a specific agonist of G protein-coupled estrogen receptor 1 (GPER1), which binds and activates GPER1 to exert various neurological functions. However, the preventive effect of G1 on post-traumatic stress disorder (PTSD) and its mechanisms are unclear. To evaluate the protective effect of G1 against synaptic and mitochondrial impairments and to investigate the mechanism of G1 to improve PTSD from brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) signaling. This study initially detected GPER1 expression in the hippocampus of single prolonged stress (SPS) mice, utilizing both Western blot and immunofluorescence staining. Subsequently, the effects of G1 on PTSD-like behaviors, synaptic, and mitochondrial functions in SPS mice were investigated. Additionally, the involvement of BDNF/TrkB signaling involved in the protection was further confirmed using GPER1 antagonist and TrkB inhibitor, respectively. The expression of GPER1 was reduced in the hippocampus of SPS mice, and G1 treatment given for 14 consecutive days significantly improved PTSD-like behaviors in SPS mice compared with model group. Electrophysiological local field potential (LFP) results showed that G1 administration for 14 consecutive days could reverse the abnormal changes in the gamma oscillation in the CA1 region of SPS mice. Meanwhile, G1 administration for 14 consecutive days could significantly improve the abnormal expression of synaptic proteins, increase the expression of mitochondria-related proteins, increase the number of synapses in the hippocampus, and ameliorate the damage of hippocampal mitochondrial structure in SPS mice. In addition, G15 (GPER1 inhibitor) and ANA-12 (TrkB inhibitor) blocked the ameliorative effects of G1 on PTSD-like behaviors and aberrant expression of hippocampal synaptic and mitochondrial proteins in SPS mice and inhibited the reparative effects of G1 on structural damage to hippocampal mitochondria, respectively. G1 improved PTSD-like behaviors in SPS mice, possibly by increasing hippocampal GPER1 expression and promoting BDNF/TrkB signaling to repair synaptic and mitochondrial functional impairments. This study would provide critical mechanism for the prevention and treatment of PTSD.

Selective Activation of G Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces ER Stress and Pyroptosis via AMPK Signaling Pathway in Experimental Subarachnoid Hemorrhage.

Neuroinflammation is a critical pathogenic event following hemorrhagic stroke. Endoplasmic reticulum (ER) stress-induced apoptosis and nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3(NLRP3)-associated pyroptosis can contribute to the escalation of neuroinflammatory responses, leading to increased brain damage. G protein-coupled estrogen receptor 1(GPER1), as the most extensively characterized brain-derived estrogen, was reported to trigger neuroprotective effects. However, the anti-apoptotic and anti-pyroptotic effect of GPER1 activation and the underlying mechanism has not been fully elucidated. We established the experimental SAH model by intravascular perforation. The GPER1 selective agonist G1 was intravenously administered 1h following SAH. For mechanistic exploration, the selective inhibitor of adenosine monophosphate-activated protein kinase (AMPK), dorsomorphin, was administered via intracerebroventricular injection 30min prior to SAH induction. Post-SAH assessments included SAH grade, the short-term and long-term neurological outcomes, brain edema, cerebral blood flow, transmission electron microscopy (TEM), western blot (WB), ELISA, TUNEL staining, Fluoro-Jade C staining (FJC), and immunofluorescence staining. The expression of GPER1 was observed to elevate at 6h and peaked at 24h subsequent to SAH, predominantly co-localized with neurons. Post-treatment with G1 markedly ameliorated both the short-term and long-term neurological deficits of SAH mouse, as well as inhibiting the expression of neuronal ER stress-associated apoptotic proteins (i.e., CHOP, GRP78, Caspase-12, Cleaved Caspase-3, Bax, Bcl2) and pyroptosis-associated proteins (i.e., NLRP3, ASC, Cleaved Caspase-1). Additionally, our research revealed that inhibition of AMPK with dorsomorphin attenuated the neuroprotective effects of G1. This was accompanied by modifications in the molecular pathways associated with ER stress-induced apoptosis and pyroptosis. These data herein elucidated that GPER1 exerted neuroprotective effects by mitigating neuroinflammation in an AMPK-dependent manner, which modulates neuronal ER stress-associated apoptosis and pyroptosis. Boosting the anti-apoptotic and anti-pyroptotic effect by activating GPER1 may be an efficient treatment strategy for SAH patients.

Estradiol contributes to sex differences in resilience to sepsis-induced metabolic dysregulation and dysfunction in the heart via GPER-1-mediated PPARδ/NLRP3 signaling

Background and aimClinically, septic males tend to have higher mortality rates, but it is unclear if this is due to sex differences in cardiac dysfunction, possibly influenced by hormonal variations. Cardiac dysfunction significantly contributes to sepsis-related mortality, primarily influenced by metabolic imbalances. Peroxisome proliferator-activated receptor delta (PPARδ) is a key player in cardiac metabolism and its activation has been demonstrated to favor sepsis outcomes. While estradiol (E2) is abundant and beneficial in females, its impact on PPARδ-mediated metabolism in the heart with regards to sex during sepsis remains unknown. Methods and resultsHere, we unveil that while sepsis diminishes PPARδ nuclear translocation and induces metabolic dysregulation, oxidative stress, apoptosis and dysfunction in the heart thereby enhancing mortality, these effects are notably more pronounced in males than females. Mechanistic experiments employing ovariectomized(OVX) mice, E2 administration, and G protein-coupled estrogen receptor 1(GPER-1) knockout (KO) mice revealed that under lipopolysaccharide (LPS)-induced sepsis, E2 acting via GPER-1 enhances cardiac electrical activity and function, promotes PPARδ nuclear translocation, and subsequently ameliorates cardiac metabolism while mitigating oxidative stress and apoptosis in females. Furthermore, PPARδ specific activation using GW501516 in female GPER-1−/− mice reduced oxidative stress, ultimately decreasing NLRP3 expression in the heart. Remarkably, targeted GPER-1 activation using G1 in males mirrors these benefits, improving cardiac electrical activity and function, and ultimately enhancing survival rates during LPS challenge. By employing NLRP3 KO mice, we demonstrated that the targeted GPER-1 activation mitigated injury, enhanced metabolism, and reduced apoptosis in the heart of male mice via the downregulation of NLRP3. ConclusionOur findings collectively illuminate the sex-specific cardiac mechanisms influencing sepsis mortality, offering insights into physiological and pathological dimensions. From a pharmacological standpoint, this study introduces specific GPER-1 activation as a promising therapeutic intervention for males under septic conditions. These discoveries advance our understanding of the sex differences in sepsis-induced cardiac dysfunction and also present a novel avenue for targeted interventions with potential translational impact.

GPER1 Activation Upregulates Renal Purinergic P2Y2 Receptor Expression and Lowers Blood Pressure in Ovariectomized Rats Fed High Salt Diet

Renal purinergic P2Y2 receptor promotes urinary Na+ excretion via inhibiting epithelial Na+ channel (ENaC) activity. Recent evidence implicates that ovarian hormones regulate P2Y2/ENaC natriuretic pathway, however the underlying mechanism remains unclear. Estradiol activates the G protein-coupled estrogen receptor 1 (GPER1), which promotes natriuresis in females. Taken together, we hypothesized that GPER1 activation regulates renal P2Y2 receptor expression and maintains blood pressure in ovariectomized (OVX) rats. To test our hypothesis, we determined the effect of pharmacologic activation GPER1 on (i) blood pressure and (ii) mRNA expression of renal P2Y2 receptor. In particular, female (16–20 weeks of age) Sprague Dawley rats were implanted with telemeters. After recording baseline blood pressure, rats were ovariectomized and simultaneously implanted with an osmotic minipump to deliver either G1, selective GPER1 agonist (OVX+G1), or Vehicle (OVX+Veh) for 3 weeks. Rats were fed a normal salt (NS, 0.49% NaCl) throughout the study and challenged with a high salt (HS, 4% NaCl) during the last week. Finally, animals were euthanized, renal cortex and medulla were assessed for P2Y2 and αENaC mRNA expression. We found that OVX+Veh rats fed a HS diet had greater mean arterial pressure (MAP) when compared to their baseline values prior to ovariectomy (113 ± 1 vs. 105 ± 1 mmHg; p < 0.01; n = 6). Interestingly, OVX+G1 rats fed a HS diet did not elicit significant changes in MAP when compared to their baseline values (106 ± 3 vs. 104 ± 2 mmHg; p = 0.25; n = 6). Moreover, renal cortical P2Y2 mRNA expression in OVX+G1 rats was higher compared to corresponding vehicle-treated rats (1.95 ± 0.21 vs. 1.00 ± 0.17 fold change from OVX+Veh; p < 0.01; n = 6-7/group). Renal outer and inner medullary P2Y2 mRNA expression elicited similar trends; however, it did not reach statistical significance. No differences were evident in the renal αENaC mRNA expression in response to G1 treatment. We also extended the study in female mice to determine the effect of genetic deletion of GPER1 on renal P2Y2 receptor mRNA expression. We found that P2Y2 mRNA expression in kidneys obtained from GPER1 KO mice was markedly lower when compared to wildtype litter mates. However, no differences were detected in the mRNA expression of αENaC in response to GPER1 deletion. The current results indicate that GPER1 activity upregulates the renal P2Y2 expression which may contribute to preventing the increase in blood pressure in HS-fed OVX animals, possibly by promoting natriuresis. Additional studies are required to improve our understanding for GPER1/P2Y2/ENaC signaling pathway in the postmenopausal female population. NIH/NIDDK R00DK119413 to EYG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of astrocyte GPER on the optic nerve inflammatory response following optic nerve injury in mice

Activated astrocytes are a primary source of inflammatory factors following traumatic optic neuropathy (TON). Accumulation of inflammatory factors in this context leads to increased axonal damage and loss of retinal ganglion cells (RGCs). Therefore, in the present study, we explored the role of the astrocyte G protein-coupled estrogen receptor (GPER) in regulating inflammatory factors following optic nerve crush (ONC), and analyzed its potential regulatory mechanisms. Overall, our results showed that GPER was abundantly expressed in the optic nerve, and co-localized with glial fibrillary acidic proteins (GFAP). Exogenous administration of G-1 led to a significant reduction in astrocyte activation and expression of inflammation-related factors (including IL-1β, TNF-α, NFκB, and p-NFκB). Additionally, it dramatically increased the survival of RGCs. In contrast, astrocytes were activated to a greater extent by exogenous G15 administration; however, RGCs survival was significantly reduced. In vitro, GPER activation significantly reduced astrocyte activation and the release of inflammation-related factors. In conclusion, activation of astrocyte GPER significantly reduced ONC inflammation levels, and should be explored as a potential target pathway for protecting the optic nerve and RGCs after TON.

Modulation of GPER1 alleviates early brain injury via inhibition of A1 reactive astrocytes activation after intracerebral hemorrhage in mice

BackgroundEarly brain injury (EBI) caused by inflammatory responses in acute phase of Intracerebral hemorrhage (ICH) plays a vital role in the pathological progression of ICH. Increasing evidences demonstrate A1 reactive astrocytes are associated with the severity of EBI. G-protein coupled estrogen receptor 1 (GPER1) has been proved mediating the neuroprotective effects of estrogen in central nervous system (CNS) disease. However, whether GPER1 plays a protective effect on ICH and A1 reactive astrocytes activation is not well studied. MethodsICH model was established by infused the autologous whole blood into the right basal ganglia in wild type and GPER1 knockout mice. GPER1 specific agonist G1 and antagonist G15 were administered by intraperitoneal injection at 1 h or 0.5 h after ICH. Neurological function was detected on day 1 and day 3 by open field test and corner turn test following ICH. Besides, A1 reactive astrocytes were determined by immunofluorescence staining after ICH on day 3. To further identify the possible mechanism of GPER1 mediated neuroprotective effect, Western blot assays was performed after ICH on day 3. ResultsAfter ICH, G1 treatment alleviated mice neurobehavior deficits on day 1 and day 3. Meanwhile, G1 treatment also significantly reduced the GFAP positive astrocytes and the C3 positive cells after ICH. Interestingly, G15 reversed the protective effect of G1 on the neurobehavior of ICH mice. Meanwhile, the expression of GFAP+C3+ A1 reactive astrocytes were also reduced by activation of GPER1. Mechanistic studies indicated TLR4 and NF-κB mediated the neuroprotective effect of GPER1. ConclusionGenerally, activation of GPER1 alleviated the EBI through inhibiting A1 reactive astrocytes activation via TLR4/NF-κB pathway after ICH in mice. Additionally, GPER1may be a promising target for ICH treatment.

G protein–coupled estrogen receptor 1 ameliorates nonalcoholic steatohepatitis through targeting AMPK-dependent signaling

Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), has emerged as a prevalent cause of liver cirrhosis and hepatocellular carcinoma, posing severe public health challenges worldwide. The incidence of NASH is highly correlated with an increased prevalence of obesity, insulin resistance, diabetes, and other metabolic diseases. Currently, no approved drugs specifically targeted for the therapies of NASH partially due to the unclear pathophysiological mechanisms. G protein-coupled estrogen receptor 1 (GPER1) is a membrane estrogen receptor involved in the development of metabolic diseases such as obesity and diabetes. However, the function of GPER1 in NAFLD/NASH progression remains unknown. Here, we show that GPER1 exerts a beneficial role in insulin resistance, hepatic lipid accumulation, oxidative stress, or inflammation in vivo and in vitro. In particular, we observed that the lipid accumulation, inflammatory response, fibrosis, or insulin resistance in mouse NAFLD/NASH models were exacerbated by hepatocyte-specific GPER1 knockout but obviously mitigated by hepatic GPER1 activation in female and male mice. Mechanistically, hepatic GPER1 activates AMP-activated protein kinase signaling by inducing cyclic AMP release, thereby exerting its protective effect. These data suggest that GPER1 may be a promising therapeutic target for NASH.

GPER activation attenuates cardiac dysfunction by upregulating the SIRT1/3-AMPK-UCP2 pathway in postmenopausal diabetic rats.

Postmenopausal diabetic women are at higher risk to develop cardiovascular diseases (CVD) compared with nondiabetic women. Alterations in cardiac cellular metabolism caused by changes in sirtuins are one of the main causes of CVD in postmenopausal diabetic women. Several studies have demonstrated the beneficial actions of the G protein-coupled estrogen receptor (GPER) in postmenopausal diabetic CVD. However, the molecular mechanisms by which GPER has a cardioprotective effect are still not well understood. In this study, we used an ovariectomized (OVX) type-two diabetic (T2D) rat model induced by high-fat diet/streptozotocin to investigate the effect of G-1 (GPER-agonist) on sirtuins, and their downstream pathways involved in regulation of cardiac metabolism and function. Animals were divided into five groups: Sham-Control, T2D, OVX+T2D, OVX+T2D+Vehicle, and OVX+T2D+G-1. G-1 was administrated for six weeks. At the end, hemodynamic factors were measured, and protein levels of sirtuins, AMP-activated protein kinase (AMPK), and uncoupling protein 2 (UCP2) were determined by Western blot analysis. In addition, cardiac levels of oxidative stress biomarkers were measured. The findings showed that T2D led to left ventricular dysfunction and signs of oxidative stress in the myocardium, which were accompanied by decreased protein levels of Sirt1/2/3/6, p-AMPK, and UCP2 in the heart. Moreover, the induction of the menopausal state exacerbated these changes. In contrast, treatment with G-1 ameliorated the hemodynamic changes associated with ovariectomy by increasing Sirt1/3, p-AMPK, UCP2, and improving oxidative status. The results provide evidence of the cardioprotective effects of GPER operating through Sirt1/3, p-AMPK, and UCP2, thereby improving cardiac function. Our results suggest that increasing Sirt1/3 levels may offer new therapeutic approaches for postmenopausal diabetic CVD.

Activation of GPER1 in macrophages ameliorates UUO-induced renal fibrosis

Numerous studies have proven the critical role of macrophages in the renal fibrosis process. Notably, G Protein-coupled Estrogen Receptor 1 (GPER1), a novel estrogen receptor, has been shown to play a ubiquitous role in regulating macrophage activities and proinflammatory pathways. However, the precise role of GPER1 in macrophage-mediated renal fibrosis is unknown. In this study, we aimed to investigate the function of macrophage GPER1 in the UUO-induced renal fibrosis model. Compared to vehicle-treated ovariectomized (OVX) female and male unilateral ureteral obstruction (UUO) models, we observed that G-1 (GPER1 agonist)-treated OVX female and male UUO mice had fewer renal fibrotic lesions and less M1 and M2 macrophage infiltration in the kidney tissues. Conversely, Gper1 deletion in male UUO mice accelerated renal fibrosis and increased inflammation. In vitro studies also revealed that GPER1 activation reduced M0 macrophage polarization towards M1 or M2 phenotypes. The RNA-sequencing analysis and immunoblotting indicated that GPER1 activation was primarily involved in downregulating immune pathways activation and inactivating MAPK pathways. Tubular epithelial cells co-cultured with G-1-pretreated M1 macrophages exhibited fewer injuries and immune activation. In addition, fibroblasts co-cultured with G-1-pretreated M2 macrophages showed downregulated extracellular matrix expression. Overall, this is the first study to demonstrate the effect of GPER1 on macrophage-mediated renal fibrosis via inhibition of M1 and M2 macrophage activation. These findings indicate that GPER1 may be a promising therapeutic target for treating renal fibrosis.

GPER1 Activation Exerts Anti-Tumor Activity in Multiple Myeloma.

G protein-coupled estrogen receptor 1 (GPER1) activation is emerging as a promising therapeutic strategy against several cancer types. While GPER targeting has been widely studied in the context of solid tumors, its effect on hematological malignancies remains to be fully understood. Here, we show that GPER1 mRNA is down-regulated in plasma cells from overt multiple myeloma (MM) and plasma cell leukemia patients as compared to normal donors or pre-malignant conditions (monoclonal gammopathy of undetermined significance and smoldering MM); moreover, lower GPER1 expression associates with worse overall survival of MM patients. Using the clinically applicable GPER1-selective agonist G-1, we demonstrate that the pharmacological activation of GPER1 triggered in vitro anti-MM activity through apoptosis induction, also overcoming the protective effects exerted by bone marrow stromal cells. Noteworthy, G-1 treatment reduced in vivo MM growth in two distinct xenograft models, even bearing bortezomib-resistant MM cells. Mechanistically, G-1 upregulated the miR-29b oncosuppressive network, blunting an established miR-29b-Sp1 feedback loop operative in MM cells. Overall, this study highlights the druggability of GPER1 in MM, providing the first preclinical framework for further development of GPER1 agonists to treat this malignancy.

Activation of G-Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces Progression of Vulvar Carcinoma Cells.

Whether G protein-coupled estrogen receptor 1 (GPER1) is tumor-promoting or tumor-suppressive depends in part on tumor entity. Little is known about the function of GPER1 in vulvar carcinoma. In this work, we aim to clarify what role GPER1 plays in vulvar cancer, tumor-promoting or tumor-suppressive. Localization of GPER1 in A431 and CAL-39 vulvar carcinoma cells was examined by immunofluorescence. Using a tissue microarray of vulvar neoplasias, the correlation between GPER1 expression and grade of malignancy was investigated. A431 and CAL-39 cells were treated either with GPER1 agonist G1 or antagonist G36. Proliferation was quantified by BrdU assay and viability examined using Resazurin assay. Morphological changes were analyzed by microscopy and measured using ImageJ. Cell migration was analyzed by gap closure assay. Clonogenic potential was tested by colony and sphere formation. Expression of estrogen receptors was examined by Western blot. GPER1 was found consistently expressed in vulvar neoplasia tissues. The immune-reactive score was found to be significantly higher in tissue samples of lymph node metastases and neoplasias with grade 3. In A431 and CAL-39 vulvar carcinoma cells, GPER1 expression was mainly found in the cytoplasm and nuclei. Treatment of A431 and CAL-39 cells with GPER1 agonist G1 resulted in a decrease in proliferation and migration. In addition, colony formation and tumor sphere formation were reduced. Furthermore, morphological signs of necrosis and reduction in cell viability after G1 treatment were observed. The GPER1 antagonist G36 did not have significant effects on vulvar carcinoma cells. Neither agonist G1 nor antagonist G36 treatment resulted in altered expression of estrogen receptors. Activation of GPER1 with GPER1 agonist G1 reduces the tumorigenic potential of the vulvar carcinoma cells. It can be deduced from this that GPER1 appears to have a tumor-suppressive effect in vulvar carcinoma.

The G protein-coupled estrogen receptor of the trigeminal ganglion regulates acute and chronic itch in mice.

Itch is an unpleasant sensation that severely impacts the patient's quality of life. Recent studies revealed that the G protein-coupled estrogen receptor (GPER) may play a crucial role in the regulation of pain and itch perception. However, the contribution of the GPER in primary sensory neurons to the regulation of itch perception remains elusive. This study aimed to investigate whether and how the GPER participates in the regulation of itch perception in the trigeminal ganglion (TG). Immunofluorescence staining results showed that GPER-positive (GPER+ ) neurons of the TG were activated in both acute and chronic itch. Behavioral data indicated that the chemogenetic activation of GPER+ neurons of the TG of Gper-Cre mice abrogated scratching behaviors evoked by acute and chronic itch. Conversely, the chemogenetic inhibition of GPER+ neurons resulted in increased itch responses. Furthermore, the GPER expression and function were both upregulated in the TG of the dry skin-induced chronic itch mouse model. Pharmacological inhibition of GPER (or Gper deficiency) markedly increased acute and chronic itch-related scratching behaviors in mouse. Calcium imaging assays further revealed that Gper deficiency in TG neurons led to a marked increase in the calcium responses evoked by agonists of the transient receptor potential ankyrin A1 (TRPA1) and transient receptor potential vanilloid V1 (TRPV1). Our findings demonstrated that the GPER of TG neurons is involved in the regulation of acute and chronic itch perception, by modulating the function of TRPA1 and TRPV1. This study provides new insights into peripheral itch sensory signal processing mechanisms and offers new targets for future clinical antipruritic therapy.

G protein-coupled estrogen receptor (GPER) selective agonist G1 attenuates the neurobehavioral, molecular and biochemical alterations induced in a valproic acid rat model of autism

AimsAutism Spectrum Disorder (ASD) is a neurodevelopmental disorder with a rising prevalence in boys rather than girls. G protein-coupled estrogen receptor (GPER) activation by its agonist G1 showed a neuroprotective effect, similar to estradiol. The present study aimed to examine the potential of the selective GPER agonist G1 therapy on the behavioral, histopathological, biochemical, and molecular alterations induced in a valproic acid (VPA)-rat model of autism. Main methodsVPA (500 mg/kg) was intraperitoneally administered to female Wistar rats (on gestational day 12.5) to induce the VPA-rat model of autism. The male offspring were intraperitoneally administered with G1 (10 and 20 μg/kg) for 21 days. After the treatment process, rats performed behavioral assessments. Then, sera and hippocampi were collected for biochemical and histopathological examinations and gene expression analysis. Key findingsGPER agonist G1 attenuated behavioral deficits, including hyperactivity, declined spatial memory and social preferences, anxiety, and repetitive behavior in VPA rats. G1 improved neurotransmission and reduced oxidative stress and histological alteration in the hippocampus. G1 reduced serum free T levels and interleukin-1β and up-regulated GPER, RORα, and aromatase gene expression levels in the hippocampus. SignificanceThe present study suggests that activation of GPER by its selective agonist G1 altered the derangements induced in a VPA-rat model of autism. G1 normalized free T levels via up-regulation of hippocampal RORα and aromatase gene expression. G1 provoked estradiol neuroprotective functions via up-regulation of hippocampal GPER expression. The G1 treatment and GPER activation provide a promising therapeutic approach to counteract the autistic-like symptoms.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector β-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/β-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/β-catenin signaling in OVX mice.

Molecular identification of bulbospinal ON neurons by GPER, which drives pain and morphine tolerance.

The rostral ventromedial medulla (RVM) exerts bidirectional descending modulation of pain attributable to the activity of electrophysiologically identified pronociceptive ON and antinociceptive OFF neurons. Here, we report that GABAergic ON neurons specifically express G protein-coupled estrogen receptor (GPER). GPER+ neurons exhibited characteristic ON-like responses upon peripheral nociceptive stimulation. Optogenetic activation of GPER+ neurons facilitated, but their ablation abrogated, pain. Furthermore, activation of GPER caused depolarization of ON cells, potentiated pain, and ameliorated morphine analgesia through desensitizing μ-type opioid receptor-mediated (MOR-mediated) activation of potassium currents. In contrast, genetic ablation or pharmacological blockade of GPER attenuated pain, enhanced morphine analgesia, and delayed the development of morphine tolerance in diverse preclinical pain models. Our data strongly indicate that GPER is a marker for GABAergic ON cells and illuminate the mechanisms underlying hormonal regulation of pain and analgesia, thus highlighting GPER as a promising target for the treatment of pain and opioid tolerance.

In vitro and in silico assessment of GPER-dependent neurocytotoxicity of emerging bisphenols

To rapidly assess the toxicity of bisphenols (BPs) via the activation of G protein-coupled estrogen receptor (GPER), eight BPs action on GPER were evaluated by molecular docking and molecular dynamics (MD) simulation and then confirmed with IMR-32 cells. The target BPs significantly promoted the production of reactive oxygen species (ROS), reduced cell viability, activated the expression of apoptosis-related proteins and increased the apoptosis rate of IMR-32 cells. Intracellular Ca2+ level increased significantly after the treatments with bisphenol A (BPA), bisphenol E (BPE), bisphenol C (BPC) and bisphenol AP (BPAP), suggesting the activation of GPER. Moreover, the stable binding conformations between GPER and BPA, BPE, BPC and BPAP and their dynamic changes of GPER-BPs via MD simulation also suggest that these BPs may activate GPER. The interaction between bisphenol G/bisphenol P/bisphenol PH and GPER are weak, which is consistent with their low GPER activity in vitro. Notably, after the pretreatment of GPER antagonist, Ca2+ accumulation and ROS production induced by BPA, BPE, BPC and BPAP in IMR-32 cells were attenuated. Overall, MD simulation and in vitro results mutually verified the activation of GPER by BPs, and MD simulation can rapidly evaluate the neurocytotoxicity of BPs.

LBODP005 Gper Modulates Multiple Functions In Adipose Tissue To Promote An Anti-obese Phenotype

Abstract Obesity and metabolic syndrome are major health issues of modern society and their incidence is increasing at alarming rates worldwide. Obesity and associated chronic diseases exhibit sexual disparity with pre-menopausal females displaying better protection compared to age-matched males. These beneficial effects on metabolism in females are predominantly attributed to the female hormone estrogen, specifically 17β-estradiol (E2). E2 exerts its actions via multiple receptors, nuclear and extra-nuclear estrogen receptor (ER) α and ERβ, and the G protein-coupled estrogen receptor (GPER). GPER is present in diverse cells types and tissues and has been implicated in numerous physiological processes via multiple cellular signaling pathways. We have combined in vivo studies in an ovariectomized (OVX) mouse model along with in vitro assays in 3T3-L1 cells to investigate the role of GPER in modulating adipocyte function. Our studies reveal that loss of GPER increased adiposity in female mice on high fat diet and activation of GPER by its selective agonist G-1 attenuates adiposity in OVX mice. G-1 activated AMPK, increased cellular respiration and expression of mitochondrial and antioxidant genes and reduced lipid deposition in adipocytes. Furthermore, G-1 treatment promoted an anti-inflammatory phenotype in mice. Thus, our findings establish a role of GPER agonist G-1 in exerting anti-lipogenic and anti-inflammatory effects in adipocytes, which may have potential therapeutic implications in treatment of obesity. Presentation: No date and time listed