G Protein-coupled Estrogen Receptor Activation Research Articles

Bisphenol A induces focal adhesions assembly and activation of FAK, Src and ERK2 via GPER in MDA-MB-231 breast cancer cells

Bisphenol A (BPA) is an industrial synthetic chemical used in the production of polycarbonate plastics and epoxy resins. Human exposition to BPA is primarily through eating food, and drinking liquids, because BPA can leach from polycarbonate plastic containers, beverage cans and epoxy resins. BPA induces proliferation and migration in human breast cancer cells. The G protein-coupled estrogen receptor (GPER) is a G protein-coupled receptor coupled with Gs proteins that is activated by estrogen and estrogenic compounds and it is the receptor for BPA. However, the signal transduction pathways that mediate migration via BPA/GPER in triple negative breast cancer (TNBC) cells has not been studied in detail. Here, we demonstrate that BPA induces an increase of GPER expression and activation of FAK, Src and ERK2, and an increase of focal adhesion assembly via GPER in TNBC MDA-MB-231 cells. Moreover, BPA induces FAK and ERK2 activation, focal adhesion assembly and migration via epidermal growth factor receptor (EGFR) transactivation. Collectively our data showed that BPA via GPER and/or EGFR transactivation induces activation of signal transduction pathways that mediate migration in TNBC MDA-MB-231 cells.

Sex-dependent effects of GPER activation on neuroinflammation in a rat model of traumatic brain injury

The G protein-coupled estrogen receptor (GPER) plays a role in estrogen-mediated neuroprotection and has been considered a potential therapeutic target for treating various neurological diseases. It is increasingly recognized that sex is a biological variable affecting treatment outcomes and efficacy, and that neuroinflammation is a key secondary injury mechanism following brain injury, though it is unknown whether the neuroprotective effects exerted by GPER involve modulation of inflammatory processes. The aim of this study was to investigate whether activation of GPER has a sex-dependent effect on neuroinflammation following traumatic brain injury (TBI), a sexually dimorphic disease. In male and ovariectomized (OVX) female rats, the GPER agonist, G1, inhibited the upregulated expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), increased the expression of the anti-inflammatory cytokine IL-4, and shifted microglia/macrophage polarization toward the M2 phenotype. In gonadally-intact females, G1 caused more pro-inflammatory (IL-6 and TNF-α) and less anti-inflammatory cytokine (IL-4) production, without altering microglia/macrophage polarization. Estradiol supplementation blocked the effects of G1 in OVX females. We also found that post-injury GPER expression was increased in males and OVX females but not in intact females. G1 administration increased Akt phosphorylation in males and OVX females, but had no significant effect in intact females, while Akt inhibition blocked the effects of G1 in males and OVX females. These results indicate that G1 exerts anti-inflammatory effects in males and OVX females but not in intact females; these sex-specific effects are dependent on circulating estrogen levels and are partially mediated through Akt signaling. Future studies are needed to elucidate the relevant molecular mechanisms, especially in females. A better understanding of the sex differences in treatment efficacy with GPER agonists may help improve personalized therapeutic strategies for males and pre- and postmenopausal females with TBI.

Effects of the G Protein‐coupled Estrogen Receptor on Cardiovascular Remodeling Induced by Chronic Sympathetic Activation

Estrogens modulate cardiovascular function via the classical nuclear hormone receptors ERα and ERβ, and the more recently discovered G protein‐coupled estrogen receptor (GPER). While the activation of GPER elicits many cardioprotective effects, the mechanisms behind these effects are incompletely understood. Sympathetic nervous activities are essential for normal cardiac functions; however, chronic sympathetic activation is an important contributor to maladaptive cardiovascular remodeling that leads to cardiovascular diseases. Our recent data demonstrate that GPER activity moderates the increases in myocardial contraction and calcium dynamics stimulated by acute activation of the cardiac beta1 adrenergic receptor. However, the effects of GPER activation on the cardiac manifestations in response to chronic sympathetic activation are unknown. We hypothesized that GPER activation would reduce cardiovascular remodeling induced by chronic norepinephrine (NE) administration. In adult female ovariectomized mice, administration of NE (10 mg/kg/day) via osmotic pumps for 8 weeks increased heart weight, kidney weight, cardiomyocyte size as assessed by wheat germ agglutinin staining, left ventricular wall thickness, and aorta wall thickness. These changes occurred without an apparent increase in blood pressure assessed by tail cuff measurements. These effects were reduced by the co‐administration of GPER agonist G‐1 (120 mg/kg/day), while administration of G‐1 alone at the same dose had no effect. These initial data indicate that the GPER agonist G‐1 can reduce cardiovascular remodeling caused by chronic sympathetic activation.Support or Funding InformationDMU IOER 05‐19‐06

G Protein‐coupled Estrogen Receptor Protects Against Aging‐Induced Vascular Dysfunction in Females but Not Males.

G protein‐coupled estrogen receptor (GPER) promotes the vasoprotective effects of estrogen, and we previously published that resistance vessels from middle‐aged female rodents are less responsive to selective GPER activation compared with adult vessels. Therefore, we hypothesized that aging or global GPER deletion would induce similar dysfunction in the vasculature. Mesenteric resistance arteries were isolated from adult (21–25 weeks old) and middle‐aged (52–57 weeks old) male and female wildtype (WT) and GPER knockout (KO) mice. Vessel reactivity was measured via wire myography in response to increasing doses of vasoconstrictors phenylephrine (PE) and prostaglandin F2α (PGF2α) and vasodilators acetylcholine (Ach) and sodium nitroprusside (SNP). PGF2α‐induced constriction was significantly decreased with aging in WT males (446 ± 15% vs. 378 ± 14%; P=0.02) but not in vessels from KO males (434 ± 24% vs. 416 ± 30%; ns). PE‐induced constriction was comparable between male middle‐aged and adult WT (401 ± 19% vs. 416 ± 22%; ns) and KO vessels (416 ± 22% vs. 398 ± 19%; ns). Relaxation to SNP in male animals was comparable between age groups and unaltered by GPER deletion (WT Middle‐Aged: 26 ± 6% vs. Adult: 15 ± 4%; ns; KO Middle‐Aged: 14 ± 3% vs. Adult: 11 ± 2%; ns). However, GPER deletion enhanced relaxation to Ach in middle‐aged males (WT: 42 ± 7% vs. KO: 18 ± 5%; P<0.01) but not adult males (WT: 37 ± 9% vs. KO: 28 ± 12%; ns). In females, PGF2α‐induced vasoconstriction was significantly greater in middle‐aged versus adult WT vessels (446 ± 18% vs. 388 ± 32%; ns) and abolished with GPER deletion (463 ± 12% vs. 413 ± 25%; ns). PE‐induced constriction was comparable between female middle‐aged and adult WT vessels (454 ± 17% vs. 387 ± 18%; ns) and unaltered by GPER deletion (474 ± 25% vs. 412 ± 26%; ns). In females, relaxation to SNP was not altered by aging or GPER deletion (WT Middle‐Aged: 8 ± 1% vs. Adult: 11 ± 3%; ns; KO Middle‐Aged: 13 ± 3% vs. Adult: 20 ± 6%; ns). However, relaxation to Ach was impaired by GPER deletion in adult female vessels (WT: 7 ± 1% vs. KO: 26 ± 10%; P=0.01) but not in middle‐aged vessels (WT: 6 ± 1% vs. KO: 20 ± 5%; ns). The current study demonstrates that the role of GPER in resistance artery function is dependent on sex and age. In males, GPER deletion enhanced vasoconstriction in adult animals but improved endothelial‐dependent vasodilation in middle‐age. In females, GPER deletion was detrimental for endothelial function in adulthood and enhanced vasoconstriction in middle‐age. Therefore, GPER function may contribute to vascular protection in aging women but not men.Support or Funding InformationThis study was funded by NIH grants HL133619 and U54 GM104940.

17β-Estradiol Inhibits PCSK9-Mediated LDLR Degradation Through GPER/PLC Activation in HepG2 Cells.

Plasma levels of PCSK9 are significantly higher in postmenopausal women. Pharmacologically increased estrogen levels have been shown to lower PCSK9 and LDL-C levels in animals and humans. The action of estrogen suggests that it has the ability to prevent PCSK9-mediated LDLR degradation in liver cells. However, little is known about how estrogen alters PCSK9-mediated LDLR degradation. Here, we report that 17β-estradiol (βE2) reduces PCSK9-mediated LDLR degradation by a mechanism that involves activation of the G protein-coupled estrogen receptor (GPER). In cultured HepG2 cells, βE2 prevented the internalization of PCSK9, which subsequently lead to PCSK9-mediated LDLR degradation. The altered LDLR levels also resulted in an increase in LDL uptake that was not observed in the absence of PCSK9. In addition, we showed that clathrin was rapidly increased in the presence of PCSK9, and this increase was blocked by βE2 incubation, suggesting rapid recruitment of clathrin in HepG2 cells. PLCγ activation and intracellular Ca2+ release were both increased due to the rapid effect of estrogen. By using a GPER antagonist G15, we demonstrated that the GPER mediates the action of estrogen. Together, the data from this in vitro study demonstrate that estrogen can regulate LDLR levels mainly through GPER activation, which prevents PCSK9-dependent LDLR degradation in HepG2 cells.

Pharmacologic Activation of the G Protein-Coupled Estrogen Receptor Inhibits Pancreatic Ductal Adenocarcinoma.

Background & AimsFemale sex is associated with lower incidence and improved clinical outcomes for most cancer types including pancreatic ductal adenocarcinoma (PDAC). The mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that PDAC lacks classic nuclear estrogen receptors.MethodsHere we used murine syngeneic tumor models and human xenografts to determine that signaling through the nonclassic estrogen receptor G protein-coupled estrogen receptor (GPER) on tumor cells inhibits PDAC.ResultsActivation of GPER with the specific, small molecule, synthetic agonist G-1 inhibited PDAC proliferation, depleted c-Myc and programmed death ligand 1 (PD-L1), and increased tumor cell immunogenicity. Systemically administered G-1 was well-tolerated in PDAC bearing mice, induced tumor regression, significantly prolonged survival, and markedly increased the efficacy of PD-1 targeted immune therapy. We detected GPER protein in a majority of spontaneous human PDAC tumors, independent of tumor stage.ConclusionsThese data, coupled with the wide tissue distribution of GPER and our previous work showing that G-1 inhibits melanoma, suggest that GPER agonists may be useful against a range of cancers that are not classically considered sex hormone responsive and that arise in tissues outside of the reproductive system.

The activation of the G protein-coupled estrogen receptor (GPER) prevents and regresses cardiac hypertrophy

Ventricular hypertrophy is a risk factors for arrhythmias, ischemia and sudden death. It involves cellular modifications leading to a pathological remodeling and is associated with heart failure. The activation of the G protein-coupled estrogen receptor (GPER) mediates beneficial actions in the cardiovascular system. Our goal was to prevent and regress the hypertrophy by the activation of GPER in neonatal cardiac myocytes (NRCM) and SHR male rats. Aldosterone increased the neonatal cardiomyocytes cell surface area after 48 h of incubation. The aldo-induced hypertrophy was blocked by the mineralocorticoid receptor (MR) inhibitor Eplererone or the reduction of MR expression by siRNA. The activation of GPER by the agonist G-1 totally prevented the increase surface area by Ald. The transfection of neonatal rat cardiac myocytes with a siRNA against GPER or the incubation with GPER blockers G-15 and G-36 inhibited the protection of G-1. The significant increase of cell surface area after 48 h of incubation with Ald was totally regressed in 24 h by the presence of G-1, indicating that the activation of GPER not only prevent the hypertrophy but also regress the hypertrophy when it is already established. In the in vivo model, G-1 or Vehicle was constantly infused via the minipump to SHR. The reduction of the hypertrophy by G-1 was evident by the cross-sectional area, BNP and ANP markers and by echocardiography. In this studied we demonstrated that the activation of GPER prevented and regressed the hypertrophy induced by Ald in NRCM and regressed hypertrophy in SHR rats.

Activation of the G Protein-Coupled Estrogen Receptor Elicits Store Calcium Release and Phosphorylation of the Mu-Opioid Receptors in the Human Neuroblastoma SH-SY5Y Cells.

Estrogens exert extensive influences on the nervous system besides their well-known roles in regulation of reproduction and metabolism. Estrogens act via the nuclear receptor ERα and ERβ to regulate gene transcription (classical genomic effects). In addition, estrogens are also known to cause rapid non-genomic effects on neuronal functions including inducing fast changes in cytosolic calcium level and rapidly desensitizing the μ type opioid receptor (MOR). The receptors responsible for the rapid actions of estrogens remain uncertain, but recent evidence points to the G protein-coupled estrogen receptor (GPER), which has been shown to be expressed widely in the nervous system. In the current study, we test the hypothesis that activation of GPER may mediate rapid calcium signaling, which may promote phosphorylation of MOR through the calcium-dependent protein kinases in neuronal cells. By qPCR and immunocytochemistry, we found that the human neuroblastoma SH-SY5Y cells endogenously express GPER and MOR. Activation of GPER by 17β-estradiol (E2) and G-1 (GPER selective agonist) evoked a rapid calcium rise in a concentration-dependent manner, which was due to store release rather than calcium entry. The GPER antagonist G15, the PLC inhibitor U73122 and the IP3 receptor inhibitor 2-APB each virtually abolished the calcium responses to E2 or G-1. Activation of GPER stimulated translocation of PKC isoforms (α and ε) to the plasma membrane, which led to MOR phosphorylation. Additionally, E2 and G-1 stimulated c-Fos expression in SH-SY5Y cells in a PLC/IP3-dependent manner. In conclusion, the present study has revealed a novel GPER-mediated estrogenic signaling in neuroblastoma cells in which activation of GPER is followed by rapid calcium mobilization, PKC activation and MOR phosphorylation. GPER-mediated rapid calcium signal may also be transmitted to the nucleus to impact on gene transcription. Such signaling cascade may play important roles in the regulation of opioid signaling in the brain.

Abstract B21: Pharmacologic activation of G protein-coupled estrogen receptor inhibits pancreatic ductal adenocarcinoma

Abstract LNS8801 is a novel, orally bioavailable, small-molecule agonist of the G Protein Coupled Estrogen Receptor (GPER) being developed for the treatment of pancreatic ductal adenocarcinoma (PDAC) and other advanced cancers. Women have a lower incidence and improved clinical outcomes for most cancer types, including PDAC. The mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that PDAC lacks classic nuclear estrogen receptors. Here we used murine syngeneic tumor models and human xenografts to determine that agonizing GPER on tumor cells inhibits PDAC. Systemically administered GPER agonists were well tolerated, induced tumor regression, significantly prolonged survival, and markedly increased the efficacy of PD-1 targeted immune therapy. Complete responses, observed with the GPER agonist alone or in combination with PD-1 targeted immune therapy, were associated with durable immune memory that protected against tumor rechallenge. Activation of GPER depleted c-Myc and programmed death ligand 1 (PD-L1) and increased tumor cell immunogenicity. These data, coupled with the wide tissue distribution of GPER and previous work showing that GPER activation inhibits other cancer types, suggest that GPER agonists may be useful against a range of cancers that are not classically considered sex hormone responsive, and that arise in tissues outside of the reproductive system. Citation Format: Christopher Natale, Jinyang Li, Tzvete Dentchev, Brian Capell, John Seykora, Ben Stanger, Todd Ridky. Pharmacologic activation of G protein-coupled estrogen receptor inhibits pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B21.

Activation of the G Protein-Coupled Estrogen Receptor (GPER) Increases Neurogenesis and Ameliorates Neuroinflammation in the Hippocampus of Male Spontaneously Hypertensive Rats.

It is known that spontaneously hypertensive rats (SHR) present a marked encephalopathy, targeting vulnerable regions such as the hippocampus. Abnormalities of the hippocampus of SHR include decreased neurogenesis in the dentate gyrus (DG), partial loss of neurons in the hilus of the DG, micro and astrogliosis and inflammation. It is also known that 17β-estradiol (E2) exert neuroprotective effects and prevent hippocampal abnormalities of SHR. The effects of E2 may involve a variety of mechanisms, including intracellular receptors of the ERα and ERβ subtypes or membrane-located receptors, such as the G protein-coupled estradiol receptor (GPER). We have now investigated the protective role of GPER in SHR employing its synthetic agonist G1. To accomplish this objective, 5month-old male SHR received 150μg/day of G1 during 2weeks. At the end of this period, we analyzed neuronal progenitors by staining for doublecortin (DCX), and counted the number of glial fibrillary acidic protein (GFAP)-labeled astrocytes and Iba1-stained microglial cells by computerized image analysis. We found that G1 activation of GPER increased DCX+ cells in the DG and reduced GFAP+ astrogliosis and Iba1+ microgliosis in the CA1 region of hippocampus. We also found that the high expression of proinflammatory makers IL1β and cyclooxygenase 2 (COX2) of SHR was decreased after G1 treatment, which correlated with a change of microglia phenotype from the activated to a resting morphology. Additionally, G1 treatment increased the anti-inflammatory factor TGFβ in SHR hippocampus. Altogether, our results suggest that activation of GPER plays a neuroprotective role on the encephalopathy of SHR, an outcome resembling E2 effects but avoiding secondary effects of the natural hormone.

Dorsal Hippocampal Actin Polymerization Is Necessary for Activation of G-Protein-Coupled Estrogen Receptor (GPER) to Increase CA1 Dendritic Spine Density and Enhance Memory Consolidation.

Activation of the membrane estrogen receptor G-protein-coupled estrogen receptor (GPER) in ovariectomized mice via the GPER agonist G-1 mimics the beneficial effects of 17β-estradiol (E2) on hippocampal CA1 spine density and memory consolidation, yet the cell-signaling mechanisms mediating these effects remain unclear. The present study examined the role of actin polymerization and c-Jun N-terminal kinase (JNK) phosphorylation in mediating effects of dorsal hippocampally infused G-1 on CA1 dendritic spine density and consolidation of object recognition and spatial memories in ovariectomized mice. We first showed that object learning increased apical CA1 spine density in the dorsal hippocampus (DH) within 40 min. We then found that DH infusion of G-1 increased both CA1 spine density and phosphorylation of the actin polymerization regulator cofilin, suggesting that activation of GPER may increase spine morphogenesis through actin polymerization. As with memory consolidation in our previous work (Kim et al., 2016), effects of G-1 on CA1 spine density and cofilin phosphorylation depended on JNK phosphorylation in the DH. Also consistent with our previous findings, E2-induced cofilin phosphorylation was not dependent on GPER activation. Finally, we found that infusion of the actin polymerization inhibitor, latrunculin A, into the DH prevented G-1 from increasing apical CA1 spine density and enhancing both object recognition and spatial memory consolidation. Collectively, these data demonstrate that GPER-mediated hippocampal spinogenesis and memory consolidation depend on JNK and cofilin signaling, supporting a critical role for actin polymerization in the GPER-induced regulation of hippocampal function in female mice.SIGNIFICANCE STATEMENT Emerging evidence suggests that G-protein-coupled estrogen receptor (GPER) activation mimics effects of 17β-estradiol on hippocampal memory consolidation. Unlike canonical estrogen receptors, GPER activation is associated with reduced cancer cell proliferation; thus, understanding the molecular mechanisms through which GPER regulates hippocampal function may provide new avenues for the development of drugs that provide the cognitive benefits of estrogens without harmful side effects. Here, we demonstrate that GPER increases CA1 dendritic spine density and hippocampal memory consolidation in a manner dependent on actin polymerization and c-Jun N-terminal kinase phosphorylation. These findings provide novel insights into the role of GPER in mediating hippocampal morphology and memory consolidation, and may suggest first steps toward new therapeutics that more safely and effectively reduce memory decline in menopausal women.

Role of estrogen receptors in modulating aldosterone biosynthesis and blood pressure

Blood pressure is lower in premenopausal women than in age-matched men; after menopause blood pressure values and the prevalence of hypertension show opposite trends indicating that estrogens contribute to maintaining normal blood pressure values in women. In experimental studies menopause increases aldosterone levels, an effect alleviated by estrogen treatment. We have recently discovered a role of estrogen receptors (ER) in controlling aldosterone biosynthesis in the human adrenocortical zona glomerulosa, which expresses both the classical ERα and β receptors and G protein-coupled estrogen receptor (GPER). We have also identified that GPER mediates an aldosterone-induced aldosterone response. We found that 17 β-estradiol exerts a dual effect: it blunts aldosterone production via ERβ, but displays a potent aldosterone secretagogue effect via GPER activation after ERβ blockade. Thus, in premenopausal women high estrogen levels might tonically blunt aldosterone synthesis via ERβ, thereby maintaining normal blood pressure; after menopause loss of this estrogen-mediated inhibition can contribute to increasing blood pressure via GPER-mediated aldosterone release. The additional findings that GPER mediates an aldosterone-induced stimulation of aldosterone biosynthesis and that GPER predominates in aldosterone-producing adenomas strongly involves this receptor in the pathophysiology of primary aldosteronism. Our purpose here was to provide an update on estrogen receptor function in the normal adrenal cortex and its relevance for the sex differences in blood pressure in light of the newly discovered role of GPER in regulating aldosterone synthesis. The implications of the novel knowledge for the treatment of estrogen-dependent malignancies with ER modulators are also discussed.

Estrogen receptors modulate ectonucleotidases activity in hippocampal synaptosomes of male rats

Extracellular adenine nucleotides and nucleosides, such as adenosine-5'-triphosphate (ATP) and adenosine, are among least investigated signaling factors that participate in 17β-estradiol (E2)-mediated synaptic rearrangements in rodent hippocampus. Their levels in the extrasynaptic space are tightly controlled by ecto-nucleoside triphosphate diphosphohydrolases1-3 (NTPDase1-3)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, the aim of the present study was to get closer insight in the E2-induced decrease in NTPDase and eN activity in the hippocampal synaptic compartment of male rats and to identify estradiol receptors (ERs i.e. ERα, ERβ or GPER1) responsible for the observed effects of E2. In this study we show indiscriminate participation of estradiol receptor α (ERα), -β (ERβ) and G- protein coupled estrogen receptor 1 (GPER1) in the mediation of E2 actions in hippocampal synaptosomes of male rats. Synaptic NTPDase1-3 activities are modulated only through activation of ERβ, while activation of ERα, -β and/or non-classical GPER1 decreases synaptic eN activity. Since both ATP and adenosine function as neuromodulators in the hippocampal networks, influencing its function, profound knowledge of mechanisms by which ectonucleotidases are regulated/modulated is of great importance.

GPER-induced signaling is essential for the survival of breast cancer stem cells.

G protein‐coupled estrogen receptor‐1 (GPER), a member of the G protein‐coupled receptor (GPCR) superfamily, mediates estrogen‐induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non‐BCSCs of three patient‐derived xenografts of ER−/PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER‐mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD‐Ser118 to sustain BCSC characteristics. Transfection with a dominant‐negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs.

Abstract 876: Chronic GPER1 Cardioprotection in H9c2 Myoblasts is Mediated by an MST1-YAP Pathway and Mitochondrial Function and Dynamics

Introduction: We recently showed that cardiac tissue treated acutely with estrogen (17β-estradiol; E2), pre- and post-ischemia, exhibited reduced myocardial infarct size and better functional recovery after ischemia /reperfusion via the G protein-coupled estrogen receptor 1 (GPER1). These E2-GPER1 cardioprotective effects have been found to involve the preservation of mitochondrial structure and function. Here, we investigate the impact and mechanisms underlying chronic GPER1 activation in H9c2 cardiac myoblasts treated with a cytotoxic agent, H 2 O 2 . Methods: H9c2 cells were treated with 200 μM H 2 O 2 , for 24 h and reoxygenated in no-serum conditions for 4 and 8 days with the addition of E2 (80nM) and GPER1 agonist (G1; 100nM) and GPER1 antagonist (G15; 1μM). We measured cell death and proliferation, as well as gene expression, mitochondrial morphology and function using flow cytometry, cell assays, qRT-PCR, along with fluorescence and electron microscopy. We also analyzed cell lysates by Western blot to assess the mechanisms involved. Results: We found that chronic treatment of cardiac myoblasts with E2 and G1 results in reduced cell death. The surviving cells showed improved mitochondrial function as evidenced by better cristae morphology, increased ATP production and increased resistance to opening of the mitochondrial permeability transition pore. Particularly chronic GPER1 activation led to reduced cell cycle activity along with changes in mitochondrial dynamics of fusion and fission. Additionally, we found that chronic GPER1 activation signaling involves decreased phosphorylation of mammalian sterile-20-like kinase (MST1) and increased translocation of the transcription coactivator, yes-associated protein (YAP), to the nucleus for upregulation of pro-survival genes. These effects were mainly prevented by the G15, suggesting a GPER1-dominant mechanism. Conclusion: Chronic GPER1 activation in H9c2 cardiac myoblasts treated with oxidative stress results in reduced cell death by preserving mitochondrial structural integrity and function by increasing mitochondrial dynamics. This chronic GPER1 cardioprotection involves activation of MST1/YAP signaling pathways as well as upregulation of pro-survival gene.

Aldosterone Stimulates Its Biosynthesis Via a Novel GPER-Mediated Mechanism.

The G protein-coupled estrogen receptor (GPER) mediates an aldosterone secretagogue effect of 17β-estradiol in human HAC15 adrenocortical cells after estrogen receptor β blockade. Because GPER mediates mineralocorticoid receptor-independent aldosterone effects in other cell types, we hypothesized that aldosterone could modulate its own synthesis via GPER activation. HAC15 cells were exposed to aldosterone in the presence or absence of canrenone, a mineralocorticoid receptor antagonist, and/or of the selective GPER antagonist G36. Aldosterone synthase (CYP11B2) mRNA and protein levels changes were the study end points. Similar experiments were repeated in strips obtained ex vivo from aldosterone-producing adenoma (APA) and in GPER-silenced HAC15 cells. Aldosterone markedly increased CYP11B2 mRNA and protein expression (vs untreated samples, P < 0.001) in both models by acting via GPER, because these effects were abolished by G36 (P < 0.01) and not by canrenone. GPER-silencing (P < 0.01) abolished the aldosterone-induced increase of CYP11B2, thus proving that aldosterone acts via GPER to augment the step-limiting mitochondrial enzyme (CYP11B2) of its synthesis. Angiotensin II potentiated the GPER-mediated effect of aldosterone on CYP11B2. Coimmunoprecipitation studies provided evidence for GPER-angiotensin type-1 receptor heterodimerization. We propose that this autocrine-paracrine mechanism could enhance aldosterone biosynthesis under conditions of immediate physiological need in which the renin-angiotensin-aldosterone system is stimulated as, for example, hypovolemia. Moreover, as APA overexpresses GPER this mechanism could contribute to the aldosterone excess that occurs in primary aldosteronism in a seemingly autonomous fashion from angiotensin II.

Dissociable involvement of estrogen receptors in perirhinal cortex-mediated object-place memory in male rats

Estrogens and the estrogen receptors (ER) - ERα, ERβ, and the G-protein coupled estrogen receptor (GPER) - are implicated in various forms of hippocampus (HPC)-dependent memory. However, the involvement of ER-related mechanisms in perirhinal cortex (PRh), which is necessary for object memory, remains much less clear. Moreover, there is a paucity of data assessing ER contributions to cognition in males,despite documented sex differences at the cellular level.We hypothesized that estrogens in PRh are important for object memory in males, assessingthe role of 17-βestradiol (E2), ERα, ERβ, GPER, and their downstream signaling pathways, in PRh-mediated object-in-place (OiP) memory in gonadally-intact male rats. Intra-PRh administration of E2 enhanced both long-term memory (LTM; 24 h) and short-term memory (STM; 20 min). Conversely, aromatase inhibition with letrozole impaired LTM and STM. The semi-selective ER inhibitor ICI 182780 impaired LTM, but not STM. This effect may be due to inhibition of ERβ, as the ERβagonist DPN, but not ERαagonist PPT, enhanced LTM. GPER was also found to be necessary in PRh, as the antagonist G15 impaired both LTM and STM. Western blot analyses demonstrated that phosphorylation levels of the extracellular signal-related kinase (ERK2 isoform), awell-establisheddownstream signaling pathway activated by estrogens through ERα/ERβ, was elevated in PRh 5 min following OiP learning.We also reportincreased levels of c-Jun N-terminal kinase (JNK; p46 and p54 isoforms) phosphorylation in PRh 5 min following learning,consistent with recent research linking GPER activation and JNK signaling in the HPC. This effect was abolished by intra-PRh administration of G15, but not letrozole, suggesting that JNK signaling is triggered via GPER activation during OiP learning, and is possibly E2-independent, similar to findings in the HPC. These results, therefore, reveal interesting dissociations between the roles of various ERs, possibly involving both estrogen-dependent and independent mechanisms, in PRh-mediated object-place learning in male rats.

NLRP3 inhibition improves heart function in GPER knockout mice

The molecular mechanisms of postmenopausal heart diseases in women may involve the loss of estrogen-deactivation of its membrane receptor, G-protein coupled estrogen receptor (GPER), and subsequent activation of the cardiac NLRP3 inflammasome, a component of the innate immune system. To study the potential effects of cardiac GPER on NLRP3-mediated inflammatory pathways, we characterized changes in innate immunity gene transcripts in hearts from 6-month-old cardiomyocyte-specific GPER knockout (KO) mice and their GPER-intact wild type (WT) littermates using RT2 Profiler™ real-time PCR array. GPER deletion in cardiomyocytes decreased %fractional shortening (%FS) and myocardial relaxation (e′), and increased the early mitral inflow filling velocity-to-early mitral annular descent velocity ratio (E/e′), determined by echocardiography, and increased the mRNA levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), determined by real-time PCR. Of the 84 inflammasome-related genes tested, 9 genes were upregulated, including NLRP3 and IL-18, while 1 gene, IL-12a, was downregulated in GPER KO when compared to WT. The importance of NLRP3 upregulation in GPER KO-induced heart failure was further confirmed by an in vivo study showing that, compared to vehicle-treated KO mice, 8 weeks of treatment with a NLRP3 inhibitor, MCC950 (10 mg/kg, i.p., 3 times per week), significantly limited hypertrophic remodeling, defined by reductions in heart weight/body weight, and improved systolic and diastolic functional indices, including increases in %FS and e′, and decreases E/e′ (P < 0.05). Both ANF and BNP mRNA levels were also significantly reduced by chronic MCC950 treatment. The findings from this study point toward a new understanding for the increased occurrence of heart diseases in women following loss or absence of estrogenic protection and GPER activation that involves cardiac NLRP3 inflammatory pathways.

G protein-coupled estrogen receptor mediates anti-inflammatory action in Crohn\u2019s disease

Estrogens exert immunomodulatory action in many autoimmune diseases. Accumulating evidence highlights the meaningful impact of estrogen receptors in physiology and pathophysiology of the colon. However, the significance of G protein-coupled estrogen receptor (GPER) on Crohn’s disease (CD), one of the inflammatory bowel disease (IBD) types, is still elusive. Our study revealed GPER overexpression at the mRNA and protein levels in patients with CD. To evaluate the effects of GPER activation/inhibition on colitis development, a murine 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced model of CD was used. We showed that activation of GPER reduces mortality, improves macroscopic and microscopic scores and lowers C-reactive protein (CRP) level. The impact of estrogen signaling on the suppression of the intestinal inflammation was proved by immunohistochemistry. It was demonstrated that GPER activation is accompanied by modulation of extracellular-signal regulated kinase (ERK) signaling pathway and expression level of genes involved in signal transmission and immune response as well as the expression of some microRNAs (miR-145, miR-148-5p and miR-592). Our study revealed that the membrane-bound estrogen receptor GPER mediates anti-inflammatory action and seems to be a potent therapeutic target in maintaining remission in CD.

SAT-LB035 The G Protein-Coupled Estrogen Receptor GPER Plays a Dominant Role in Human Islet Cell Proliferation upon High Glucose

Estrogen exerts its effects through genomic and rapid extra-nuclear signaling pathways. The classic estrogen receptors, ERα and ERβ have demonstrated favorable effects on beta-cell function and survival but their clinical utility has been questioned owing to oncogenic potential and concerns regarding feminizing effects in men. Recent work indicates, however, that the G-protein coupled estrogen receptor (GPER), expressed in pancreatic islet cells retains the estrogenic beta-cell protective effects, without the shortcomings of the classic ERs. GPER activation enhances glucose stimulated insulin secretion, reduces islet apoptosis upon insult and has been implicated in beta-cell mass expansion during pregnancy in rodent islets. Since GPER can thus comprise an attractive treatment target in hyperglycemic states we examined the effects of the three ERs on proliferation of human pancreatic islets in the presence of hyperglycemia. Islets from eight non diabetic human donors (6 males and 2 females) were grown at glucose concentrations of either 11mM or 25mM for 24 hours and examined for DNA synthesis after treatment with E2 (at increasing concentrations) and with specific agonists for ERα, ERβ and GPER (PPT 10nM, DPN 10nM and G1 100nM, respectively). At a glucose concentration of 11mM, E2 and all three ER agonists induced a significant ~2 folds increase in 3[H]-thymidine incorporation. However, under still higher glucose conditions (HG; 25mM) only the GPER agonist G1 increased proliferation equipotently (~2.5 folds; p<0.001), while stimulation of ERα and ERβ enhanced proliferation by ~1.4 folds only (p<0.05 for ERα). In search for an explanation for the differential effects, we examined expression of the three ERs at glucose concentrations of 11mM and 25mM, by qRT-PCR. High glucose reduced expression of ERα and ERβ (by 15%; p<0.05) but not the expression of GPER. Although beta-cells comprise the majority (~70%) of cells in human islets, expression of GPER has also been demonstrated in alpha and delta cells and thus the full effect of hyperglycemia on GPER expression in beta-cells may be masked. Hence, extending previous reports, our results suggest that GPER may comprise an attractive target in the therapy of human diabetes and point to the phenomenon of species specificity regarding effects of glucose on estrogen receptor subtype expression and activity. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.