GPER Agonist Research Articles

Abstract 1318: 17β-estradiol inhibits metastatic granulosa cell migration by repressing ERK1/2 pathway via an extra-nuclear mechanism of action.

Abstract Granulosa cell tumor (GCT) is a rare but severe form of ovarian tumor that is characterized by its tendency to recur years after surgical ablation. Because there is no efficient curative treatment beyond surgery, ∼20% of adult patients die of the consequences of their tumor. However, very little is known of the molecular etiology of this pathology. Although GCTs express the estrogen receptors (ER) ERα and ERβ, the effect of 17β-estradiol (E2) in disease progression remains elusive. The purpose of our study was to evaluate the potential role of E2 in GCT pathogenesis and to determine its underlying mechanisms of actions. To address this issue, molecular studies were performed using two human ER-positive GCT-derived cell lines, i.e., COV434 and KGN cells established from a primary tumor and from a metastatic recurrence, respectively. In both cell lines, E2 treatment (1 to 100 nM) up to 72 hours did not affect cell growth, as assessed by MTT assay and automatic cell counting. Importantly, horizontal migration assay showed that a 24-hour E2 treatment (10 nM) significantly inhibited KGN cell migration by ∼25%, in terms of migration distance and migrating cells. In contrast, no effect was observed in the primary tumor-derived COV434 cell line. Western blot studies revealed that in KGN, but not in COV434 cells, E2 treatment (10 nM) led to a rapid (as early as 5 minutes) and durable (up to 24 hours) decrease by ∼30% in the phosphorylation levels of ERK1/2, while it had no effect on ERK1/2 abundance. This was accompanied by the down-regulation of the expression of myosin light chain-2 (MLC-2), which is involved in stress fiber contraction during cell migration. ERK1/2 inhibition by the MEK1/2 inhibitor U0126 mimicked E2 actions, suggesting that the effect of E2 in KGN cells mostly results from the alteration of ERK1/2 pathway. To identify the ER involved in E2 action in KGN cells, a pharmacological approach with ER agonists was used. As none of the ER agonists could mimic E2 action, we investigated the possibility that the G protein coupled estrogen receptor (GPER or GPR30), which belongs to the G protein coupled receptor family, was involved. GPER expression was confirmed by RT-PCR and Western blot assays. Treatment of KGN cells with the GPER agonist G-1 (50 nM) rapidly and durably inhibited ERK1/2 pathway and cell migration. Pre-treatment with the GPER antagonist G15 (200 nM) completely prevented the E2-induced repression of ERK1/2 activity and inhibition of cell migration. Altogether, our study reveals that E2 would prevent metastasis spreading by inhibiting ERK1/2 activity to down-regulate MLC2 expression through a GPER-dependent mechanism. It, thus, brings evidence that extra-nuclear actions of E2 can be inhibitory to cancer pathways. Further, our observations may have translational implications, as they suggest the potential benefit of selective GPER agonists for patients with recurrent GCT. Citation Format: Charlotte M. François, Richard Wargnier, Joëlle Cohen-Tannoudji, Celine J. Guigon. 17β-estradiol inhibits metastatic granulosa cell migration by repressing ERK1/2 pathway via an extra-nuclear mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2013-1318

Activation of G protein‐coupled estrogen receptor 1 (GPER) induced coronary vasodilation by activation of MLCP via cAMP/PKA pathway

Activation of GPER exerts a protective effect in hypertension and ischemia‐reperfusion models, and relaxes arteries in vitro. However, our understanding of the mechanisms of GPER‐mediated vascular regulation is far from completed. We have investigated whether GPER‐mediated relaxation of porcine coronary arteries involves cAMP/PKA signaling. In these vessels, the selective GPER agonist, G‐1, increased cAMP production in a concentration‐dependent manner. Myograph studies demonstrated that inhibiting the activity of adenylyl cyclase (AC) with100 μM SQ‐22538 inhibited 0.3–3 μM G‐1‐induced relaxation of coronary arteries preconstricted with 1 μM PGF2α. Activating AC with forskolin also induced a concentration‐dependent relaxation. Furthermore, G‐1‐induced coronary vasodilation was attenuated by inhibiting PKA with either 5μM Rp‐8‐CPT‐cAMPS or 5μM PKI. To determine downstream signals of the cAMP/PKA cascade, we measured myosin light chain phosphatase (MLCP) activity in these artery rings and cultured smooth muscle cells with Western blot by detecting phosphorylation of myosin‐targeting subunit protein‐1 (pMYPT‐1), the MLCP regulatory subunit. Treatment with G‐1 decreased pMYPT‐1induced by PGF2α. Both Rp‐8‐CPT‐cAMPS and PKI attenuated the decrease of pMYPT‐1 by G‐1. These findings demonstrate that cAMP/PKA signaling is involved in GPER‐mediated coronary vasodilation by activating MLCP.

GPER1 agonist improves cerebral microvascular function after hypoxia/reoxygenation injury in male and female rats

Estrogen is a known neuro‐ and vaso‐protective agent in experimental cerebral ischemia. Recently, a novel G protein‐coupled estrogen receptor 1 (GPER1) has been identified. We investigated the mechanism of GPER1 mediated vasoreactivity and also its vasoprotective effect after hypoxia/reoxygenation (H/RO) injury. Rat cerebral penetrating arterioles from both genders were isolated, cannulated and pressurized. Vessel diameters were measured. To investigate vasomotor mechanism of the GPER1 agonist (G‐1), several inhibitors were tested. Ischemia/reperfusion (I/R) injury was simulated using H/RO. Vasomotor responses to adenosine tri‐phophate (ATP) after H/RO were measured with or without G‐1. G‐1 produced a vasodilatory response, which was partially dependent on endothelium‐derived nitric oxide (NO) but not arachidonic acid cascades and endothelial hyperpolarization factor. G‐1 treatment after H/RO injury fully restored arteriolar dilation to ATP compared to controls. In conclusion, GPER1 agonist elicited dilation, which partially caused by endothelial NO pathway and induced by direct relaxation of smooth muscle cells. Further, GPER1 agonist restored vessel function of arterioles after H/RO injury and may play an important role in estrogen fs ability to protect the cerebrovasculature against I/R injury. Support: NIH P01 NS32636, NIH R01 HL041250, NIH R01 NS30555, and KAKENHI 24890079.

G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons

Estradiol promotes neuritogenesis in developing hippocampal neurons by a mechanism involving the upregulation of neurogenin 3, a Notch-regulated transcription factor. In this study we have explored whether G-protein coupled estrogen receptor 1 (GPER) participates in this hormonal action. GPER agonists (17β-estradiol, G1, ICI 182,780) increased neurogenin 3 expression and neuritogenesis in mouse primary hippocampal neurons and this effect was blocked by the GPER antagonist G15 and by a siRNA for GPER. In addition, GPER agonists increased Akt phosphorylation in ser473, which is indicative of the activation of phosphoinositide-3-kinase (PI3K). G15 or GPER silencing prevented the estrogenic induction of Akt phosphorylation. Furthermore, the PI3K inhibitor wortmannin prevented the effect of G1 and estradiol on neurogenin 3 expression and the effect of estradiol on neuritogenesis. These findings suggest that GPER participates in the control of hippocampal neuritogenesis by a mechanism involving the activation of PI3K signaling.

G Protein–Coupled Estrogen Receptor Agonist Improves Cerebral Microvascular Function After Hypoxia/Reoxygenation Injury in Male and Female Rats

Reduced risk and severity of stroke in adult females are thought to depend on normal levels of endogenous estrogen, which is a known neuro- and vasoprotective agent in experimental cerebral ischemia. Recently, a novel G protein-coupled estrogen receptor (GPER, formerly GPR30) has been identified and may mediate the vasomotor and -protective effects of estrogen. However, the signaling mechanisms associated with GPER in the cerebral microcirculation remain unclear. We investigated the mechanism of GPER-mediated vasoreactivity and also its vasoprotective effect after hypoxia/reoxygenation (H/RO) injury. Rat cerebral penetrating arterioles from both sexes were isolated, cannulated, and pressurized. Vessel diameters were recorded by computer-aided videomicroscopy. To investigate vasomotor mechanism of the GPER agonist (G-1), several inhibitors with or without endothelial impairment were tested. Ischemia/reperfusion injury was simulated using H/RO. Vasomotor responses to adenosine triphophate after H/RO were measured with or without G-1 and compared with controls. G-1 produced a vasodilatory response, which was partially dependent on endothelium-derived nitric oxide (NO) but not arachidonic acid cascades and endothelial hyperpolarization factor. Attenuation of G-1-vasodilation by the NO synthase inhibitor and endothelium-impairment were greater in vessels from female than male animals. G-1 treatment after H/RO injury fully restored arteriolar dilation to adenosine triphophate compared with controls. GPER agonist elicited dilation, which was partially caused by endothelial NO pathway and induced by direct relaxation of smooth muscle cells. Further, GPER agonist restored vessel function of arterioles after H/RO injury and may play an important role in the ability of estrogen to protect the cerebrovasculature against ischemia/reperfusion injury.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10−7−5×10−6M), the main oestrogenic hormone, 17β-estradiol (10−9−10−4M), the NR3A1 agonist, PPT (10−8−10−5M), the NR3A2 agonist DPN (10−8−10−5M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10−8−3×10−6M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca2+ channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17β-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney.

G Protein-Coupled Estrogen Receptor-Selective Ligands Modulate Endometrial Tumor Growth

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the “ERα-selective” agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of “ER-targeted” therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth.

GPER-1 acts as a tumor suppressor in ovarian cancer

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

Estradiol, acting through estrogen receptor alpha, restores dimethylarginine dimethylaminohydrolase activity and nitric oxide production in oxLDL-treated human arterial endothelial cells

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. ADMA accumulation, mainly due to a decreased dimethylarginine dimethylaminohydrolase (DDAH) activity, has been related to the development of cardiovascular diseases. We investigate whether estradiol prevents the changes induced by oxidized low density lipoprotein (oxLDL) on the DDAH/ADMA/NO pathway in human umbilical artery endothelial cells (HUAEC). HUAEC were exposed to estradiol, native LDL (nLDL), oxLDL and their combinations for 24h. In some experiments, cells were also exposed to the unspecific estrogen receptor (ER) antagonist ICI 182780, the specific ERα antagonist MPP or specific agonists for ERα, ERβ and GPER. ADMA concentration was measured by HPLC and concentration of NO by amperometry. Protein expression and DDAH activity were measured by immunoblotting and an enzymatic method, respectively. oxLDL, but not nLDL, increased ADMA concentration with a concomitant decrease on DDAH activity. oxLDL reduced eNOS protein and NO production. Estradiol alone had no effects on DDAH/ADMA/NO pathway, but increased the attenuated endothelial NO production induced by oxLDL by reduction in ADMA and preventing loss of eNOS protein levels. ICI 182780 and MPP completely abolished these effects of estradiol on oxLDL-exposed cells. ERα agonist, but not ERβ and GPER agonists, mirrored estradiol effects on NO production. In conclusion, estradiol restores (1) DDAH activity, and therefore ADMA levels, and (2) NO production impaired by oxLDL in HUAEC acting through ERα.

Two Novel GPER Agonists Induce Gene Expression Changes and Growth Effects in Cancer Cells

Although the action of estrogens has been traditionally explained by the binding to and transactivation of the nuclear estrogen receptor (ER)α and ERβ, recently the G protein-coupled receptor GPR30/GPER has been involved in the rapid estrogen signaling. We investigated the ability of two original molecules, which were named GPER-L1 and GPERL2, to bind to and activate the GPER transduction pathway in cancer cells. Competition assays, docking simulations, transfection experiments, real-time PCR, immunoblotting, gene silencing technology and growth assays were performed to ascertain the selective action of GPER-L1 and GPER-L2 in activating the GPER-mediated signaling. Both compounds, which did not show any ability to bind to and activate the classical ERs, were able to bind to GPER and to trigger the rapid activation of the GPER/EGFR/ERK transduction pathway which led to the up-regulation of GPER-target genes. Notably, GPER-L1 and GPER-L2 induced the proliferation of SkBr3 breast and Ishikawa endometrial cancer cells at nM concentrations through GPER, hence providing further evidence on their capability to elicit relevant biological responses mediated by GPER. The identification and characterization of these novel compounds as selective GPER agonists represent a valuable tool to further dissect the pharmacology of this novel estrogen receptor and to better differentiate the specific functions elicited by each estrogen receptor subtype in cancer cells.

Abstract 3920: Off-target effects of the putative G-protein coupled estrogen receptor 1 (GPER) agonist G1 in ovarian and breast cancer cells

Abstract G-protein coupled estrogen receptor 1 (GPER or GPR30) plays important roles in mediating estrogen action in many different tissues and organs under both physiological and pathological conditions. G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone), the putative selective GPER agonist, has been reported to activate GPER, trigger several signaling pathways, and stimulate cell proliferation in ER-negative breast cancer, endometrial and ovarian cancer. More recently, reports showed that activation of GPER by estrogen or G1 can suppress certain cell proliferation. This study was initiated to clarify the function of GPER and its agonist on the granulosa cell tumor (GCT) cell proliferation using KGN cell as a cellular model. Well characterized epithelial ovarian cancer and breast cancer cell lines were used for comparison. GPER siRNA was used to knock down GPER. Western blot and immunohistochemistry were used to detect protein expression. We found that knockdown of GPER significantly (P < 0.05) suppressed KGN cell proliferation, suggesting that GPER may play positive roles on KGN cell proliferation. Low concentrations of G1 (<500nM) had no effect on KGN cell proliferation, whereas higher concentrations (>1μM) of G1 significantly suppressed KGN cell proliferation and arrested KGN cells in G2/M phase. These results indicated that G1 may suppress KGN cell proliferation in a GPER-independent manner. Supporting our hypothesis, knockdown of GPER did not block the inhibitory effect of G1on KGN cell proliferation. Moreover, G15, a potent GPER antagonist, failed to block G1-induced suppression of cell proliferation and G1-induced cell cycle arrest in KGN cells. In IGROV-1 epithelial ovarian cancer cells, G1 suppressed cell proliferation, a response which was not blocked by G15, even at concentrations 8-fold higher than that of G1. G1 treatment also suppressed the proliferation of MDA-MB-231 breast cancer cells, a cell line which reportedly expresses very low amounts of GPER. In HEK-293 cells, which are reported to have no expression of GPER, G1 also significantly suppressed the cell proliferation and induced cell cycle arrest. In conclusion, our results demonstrate that G1 is able to suppress ovarian and breast cancer cell proliferation in a GPER-independent manner. G1 may be a candidate for the development of drugs against ovarian and breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3920. doi:1538-7445.AM2012-3920

Activation of G protein‐coupled estrogen receptor 1 (GPER) induces coronary artery relaxation via cAMP/PKA pathway

Activation of the membrane estrogen receptor, GPER, may lower high blood pressure and preserve cardiac function after reperfusion. We reported that GPER activation induces a rapid relaxation of porcine coronary arteries by opening of BKCa channels. However, downstream signaling events in this pathway are poorly understood. The objective of our study was to investigate the mechanism of GPER‐mediated coronary artery relaxation. We found that the adenylyl cyclase (AC) inhibitor, SQ‐22538 (100 μM), attenuated G‐1 (GPER agonist)‐induced relaxation of pre‐constricted porcine coronary arteries, and shifted the G‐1 concentration‐relaxation curve to the left. These findings suggest that AC plays functional role in GPER action. Furthermore, G‐1 (1μM) stimulated cAMP production, and this stimulation was blocked by 5 μM G15 (GPER selective antagonist). Treating arteries with 5μM Rp‐8CPT‐cAMP (PKA selective inhibitor) significantly inhibited the G‐1 relaxation effect, and shifted the concentration‐response curve to the left. Lastly, 100 μM FMP‐API‐1 (an inhibitor of the interaction between PKA and the A kinase anchoring protein, AKAP) attenuated the relaxation effect of G‐1. In conclusion, G‐1 induces porcine coronary artery relaxation via cAMP/PKA signaling, which involves subcellular localization of PKA via interaction with AKAP.

Effects of GPER activation on intracellular Ca2+ stores in the vascular endothelium

The novel G protein‐coupled estrogen receptor 1 (GPER) has been demonstrated to mediate numerous beneficial cardiovascular effects. We have begun to investigate the effects of GPER activation on intracellular Ca2+ stores in the vascular endothelium. We observed that GPER is present in primary endothelial cells in a glycosylated form. Immunostaining locates GPER at both the plasma membrane and in the perinuclear region. In nominally Ca2+‐free condition, treatment with thapsigargin triggers the release of the ER's Ca2+ content. Subsequent addition of the GPER agonist G1 in Ca2+‐free condition surprisingly triggers a slow and sustained Ca2+ rise up to ~ 400 nM, a value comparable to agonist‐induced Ca2+ entry signals. Inhibition of PI3 kinase with wortmannin decreases G1‐triggered Ca2+ signal in Ca2+ free condition, but does not abolish it, suggesting that GPER activation in endothelial cells triggers the release of both IP3‐dependent and IP3‐independent Ca2+ stores. Pretreatment of endothelial cells with brefeldin A, a disruptor of the trans‐Golgi network, abolishes the slow and prolonged phase of the Ca2+ response to G1 treatment. Our data suggest that activation of GPER in primary vascular endothelial cells triggers the release of two different types of intracellular Ca2+ stores, namely the endoplasmic reticulum and the Golgi. The data also suggest that the Golgi represents a substantial intracellular Ca2+ store in the vascular endothelium.

The G protein-coupled oestrogen receptor 1 agonist G-1 disrupts endothelial cell microtubule structure in a receptor-independent manner

The G protein-coupled oestrogen receptor GPER1, also known as GPR30, has been implicated in oestrogen signalling, but the physiological importance of GPER1 is not fully understood. The GPER1 agonist G-1 has become an important tool to assess GPER1-mediated cellular effects. Here, we report that this substance, besides acting via GPER1, affects the microtubule network in endothelial cells. Treatment with G-1 (3 μM) for 24 h reduced DNA synthesis by about 60 % in mouse microvascular endothelial bEnd.3 cells. Treatment with 3 μM G-1 prevented outgrowth of primary endothelial cells from mouse aortic explants embedded in Matrigel. Treatment with G-1 (0.3-3 μM) for 24 h disrupted bEnd.3 cell and HUVEC microtubule structure in a concentration-dependent manner as assessed by laser-scanning confocal immunofluorescence microscopy. G-1-induced (3 μM) disruption of microtubule was observed also after acute (3 and 6 h) treatment and in the presence of the protein synthesis inhibitor cycloheximide. Disruption of microtubules by 3 μM G-1 was observed in aortic smooth muscle cells obtained from both GPER1 knockout and wild-type mice, suggesting that G-1 influences microtubules through a mechanism independent of GPER1. G-1 dose dependently (10-50 μM) stimulated microtubule assembly in vitro. On the other hand, microtubules appeared normal in the presence of 10-50 μM G-1 as determined by electron microscopy. We suggest that G-1-promoted endothelial cell anti-proliferation is due in part to alteration of microtubule organization through a mechanism independent of GPER1. This G-1-promoted mechanism may be used to block unwanted endothelial cell proliferation and angiogenesis such as that observed in, e.g. cancer.

17β-Estradiol induces nongenomic effects in renal intercalated cells through G protein-coupled estrogen receptor 1

Steroid hormones such as 17β-estradiol (E2) are known to modulate ion transporter expression in the kidney through classic intracellular receptors. Steroid hormones are also known to cause rapid nongenomic responses in a variety of nonrenal tissues. However, little is known about renal short-term effects of steroid hormones. Here, we studied the acute actions of E2 on intracellular Ca(2+) signaling in isolated distal convoluted tubules (DCT2), connecting tubules (CNT), and initial cortical collecting ducts (iCCD) by fluo 4 fluorometry. Physiological concentrations of E2 induced transient increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subpopulation of cells. The [Ca(2+)](i) increases required extracellular Ca(2+) and were inhibited by Gd(3+). Strikingly, the classic E2 receptor antagonist ICI 182,780 also increased [Ca(2+)](i), which is inconsistent with the activation of classic E2 receptors. G protein-coupled estrogen receptor 1 (GPER1 or GPR30) was detected in microdissected DCT2/CNT/iCCD by RT-PCR. Stimulation with the specific GPER1 agonist G-1 induced similar [Ca(2+)](i) increases as E2, and in tubules from GPER1 knockout mice, E2, G-1, and ICI 182,780 failed to induce [Ca(2+)](i) elevations. The intercalated cells showed both E2-induced concanamycin-sensitive H(+)-ATPase activity by BCECF fluorometry and the E2-mediated [Ca(2+)](i) increment. We propose that E2 via GPER1 evokes [Ca(2+)](i) transients and increases H(+)-ATPase activity in intercalated cells in mouse DCT2/CNT/iCCD.

Aberrant Ligand-Induced Activation of G Protein–Coupled Estrogen Receptor 1 (GPER) Results in Developmental Malformations During Vertebrate Embryogenesis

G protein-coupled estrogen receptor 1 (GPER) is a G protein-coupled receptor (GPCR) unrelated to nuclear estrogen receptors but strongly activated by 17β-estradiol in both mammals and fish. To date, the distribution and functional characterization of GPER within reproductive and nonreproductive vertebrate organs have been restricted to juvenile and adult animals. In contrast, virtually nothing is known about the spatiotemporal distribution and function of GPER during vertebrate embryogenesis. Using zebrafish as an animal model, we investigated the potential functional role and expression of GPER during embryogenesis. Based on real-time PCR and whole-mount in situ hybridization, gper was expressed as early as 1 h postfertilization (hpf) and exhibited strong stage-dependent expression patterns during embryogenesis. At 26 and 38 hpf, gper mRNA was broadly distributed throughout the body, whereas from 50 to 98 hpf, gper expression was increasingly localized to the heart, brain, neuromasts, craniofacial region, and somite boundaries of developing zebrafish. Continuous exposure to a selective GPER agonist (G-1)-but not continuous exposure to a selective GPER antagonist (G-15)-from 5 to 96 hpf, or within three developmental windows ranging from 10 to 72 hpf, resulted in adverse concentration-dependent effects on survival, gross morphology, and somite formation within the trunk of developing zebrafish embryos. Importantly, based on co-exposure studies, G-15 blocked severe G-1-induced developmental toxicity, suggesting that G-1 toxicity is mediated via aberrant activation of GPER. Overall, our findings suggest that xenobiotic-induced GPER activation represents a potentially novel and understudied mechanism of toxicity for environmentally relevant chemicals that affect vertebrate embryogenesis.

G protein-coupled estrogen receptor 1-mediated effects in the rat myometrium

G protein-coupled estrogen receptor 1 (GPER), also named GPR30, has been previously identified in the female reproductive system. In this study, GPER expression was found in the female rat myometrium by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Using GPER-selective ligands, we assessed the effects of the GPER activation on resting membrane potential and cytosolic Ca(2+) concentration ([Ca(2+)](i)) in rat myometrial cells, as well as on contractility of rat uterine strips. G-1, a specific GPER agonist, induced a concentration-dependent depolarization and increase in [Ca(2+)](i) in myometrial cells. The depolarization was abolished in Na(+)-free saline. G-1-induced [Ca(2+)](i) increase was markedly decreased by nifedipine, a L-type Ca(2+) channel blocker, by Ca(2+)-free or Na(+)-free saline. Intracellular administration of G-1 produced a faster and transitory increase in [Ca(2+)](i), with a higher amplitude than that induced by extracellular application, supporting an intracellular localization of the functional GPER in myometrial cells. Depletion of internal Ca(2+) stores with thapsigargin produced a robust store-activated Ca(2+) entry; the Ca(2+) response to G-1 was similar to the constitutive Ca(2+) entry and did not seem to involve store-operated Ca(2+) entry. In rat uterine strips, administration of G-1 increased the frequency and amplitude of contractions and the area under the contractility curve. The effects of G-1 on membrane potential, [Ca(2+)](i), and uterine contractility were prevented by pretreatment with G-15, a GPER antagonist, further supporting the involvement of GPER in these responses. Taken together, our results indicate that GPER is expressed and functional in rat myometrium. GPER activation produces depolarization, elevates [Ca(2+)](i) and increases contractility in myometrial cells.

Activation of G protein-coupled estrogen receptor induces endothelium-independent relaxation of coronary artery smooth muscle

Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERβ, estrogen may also stimulate G protein-coupled estrogen receptor 1 (GPER) in nonvascular tissue; however, a potential role for GPER in coronary arteries is unclear. The purpose of this study was to determine how GPER activity influenced coronary artery reactivity. In vitro isometric force recordings were performed on endothelium-denuded porcine arteries. These studies were augmented by RT-PCR and single-cell patch-clamp experiments. RT-PCR and immunoblot studies confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G-1, a selective GPER agonist, produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro. This response was attenuated by G15, a GPER-selective antagonist, or by inhibiting large-conductance calcium-activated potassium (BK(Ca)) channels with iberiotoxin, but not by inhibiting NO signaling. Last, single-channel patch-clamp studies demonstrated that G-1 stimulates BK(Ca) channel activity in intact smooth muscle cells from either porcine or human coronary arteries but had no effect on channels isolated in excised membrane patches. In summary, GPER activation relaxes coronary artery smooth muscle by increasing potassium efflux via BK(Ca) channels and requires an intact cellular signaling mechanism. This novel action of estrogen-like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.

Involvement of Epidermal Growth Factor Receptor Signaling in Estrogen Inhibition of Oocyte Maturation Mediated Through the G Protein-Coupled Estrogen Receptor (Gper) in Zebrafish (Danio rerio)1

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression.

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen‐dependent rapid pathways modulating expression of cyclin B1 and Bax

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERβ agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.