Activation Of GPR30 Research Articles

G-protein coupled receptor 30 attenuates myocardial hypertrophy by reducing oxidative stress and apoptosis in Ang II-treated mice

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors that mediate the effects of cardiac diseases. GPR30, also named G-protein-coupled estrogen receptor, shows beneficial effect on female patients with heart failure. This research aimed to probe the role and mechanism of GPR30 in myocardial hypertrophy. The model of cardiac hypertrophy was induced by infusion of angiotensin (Ang) II in mice, and was induced by Ang II treatment in neonatal rat cardiomyocyte (NRCM). The mouse model of myocardial hypertrophy was induced by angiotensin (Ang) Ⅱ, and the neonatal rat cardiomyocyte (NRCM) was induced by Ang Ⅱ treatment. GPR30 agonist G1 reduced cardiac hypertrophy induced by Ang II in mice, and reduced cardiac atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) induced by Ang II. Ang Ⅱ treatment of myocardial fibrosis in mice was suppressed after administration of G1. GPR30 deficiency produced the opposite results. Oxidative stress and apoptosis were enhanced in the mice heart induced by Ang II, which were suppressed by G1 administration, but were further exacerbated after GPR30 deficiency. The outcomes demonstrated that GPR30 participated in the regulation of cardiac hypertrophy and fibrosis. Activation of GPR30 ameliorated cardiac hypertrophy and fibrosis by reducing oxidative stress and apoptosis.

In vivo arterial stiffness, but not isolated artery endothelial function, varies with the mouse estrus cycle

Due to the increasing inclusion of female mice in research, it is important to understand the influence of the estrus cycle on vascular function. In humans, endothelial function and arterial stiffness vary throughout the menstrual cycle; however, these functional changes have yet to be investigated for the mouse estrus cycle. During the estrus cycle, circulating estrogen is highest during the proestrus and estrus phases and lowest during diestrus, while progesterone peaks during estrus. Estrogens bind to estrogen receptor α (ERα), estrogen receptor β (ERβ), and estrogen G‐protein coupled receptor (GPR30) in vascular cells. Activation of ERα and GPR30 increases nitric oxide (NO) bioavailability. The purpose of this study was to determine the effect of the estrus cycle phase on arterial stiffness, vascular endothelial function, and estrogen receptor expression.In young female C57Bl/6J mice (n=23; 5.96 ± 0.04 months), we determined the estrus cycle via vaginal cytology and plasma hormone concentrations. We assessed large artery stiffness via aortic pulse wave velocity (PWV). Endothelial function was assessed by dose responses to acetylcholine (ACh) and insulin in pressurized mesenteric arteries (MAs) and posterior cerebral arteries (PCAs). NO‐mediated dilation was determined by the difference in vasodilation to ACh in the presence vs. absence of the NO synthase inhibitor L‐NAME. Endothelium independent dilation was assessed by the dose responses to sodium nitroprusside. We quantified gene expression of ERα (Esr1), ERβ (Esr2), and GPR30 (Gper1) in MAs and PCAs.Plasma progesterone concentrations were higher for mice in estrus (5.7 ± 1.1 ng/mL) compared to proestrus (2.5 ± 0.6 ng/mL; p=0.04) and tended to be higher in diestrus (3.9 ± 1.1 ng/mL, p=0.14), verifying the cytology classifications. Plasma 17β‐estradiol and estrone were below detection in most mice. There was a higher plasma testosterone concentration during diestrus (0.08 ± 0.02 ng/mL) compared to other stages (estrus 0.02 ± 0.00 ng/mL, p=0.006; proestrus 0.01 ± 0.00 ng/mL, p=0.001).Aortic PWV was lower for mice in estrus compared with mice in diestrus (3.0 ± 0.1 vs. 3.4 ± 0.1 m/s, p=0.005). Aortic PWV for mice in proestrus did not differ from the other stages (3.2 ± 0.1 m/s, p>0.05). The MA and PCA responses to ACh, insulin and sodium nitroprusside, as well as NO‐mediated dilation, were not different between estrus cycle phases (p>0.05). PCA Esr2 gene expression for mice in estrus was 57% lower than diestrus (p=0.008) and 54% lower than proestrus (p=0.04). Gene expression did not differ between estrus cycle phases for Esr1 and Gper1 in either the MA or PCA (p>0.05), and MA Esr2 was not detectable.In summary, we found that the estrus phase in mice is associated with lower in vivo large artery stiffness. In contrast, there were no differences between estrus cycle phase in ex vivo resistance artery endothelial function. Changes to estrogen receptor gene expression during the estrus cycle are limited to ERβ in the cerebral arteries. These results suggest that estrus phase should be considered when measuring in vivo arterial stiffness in young female mice.

Biochanin A Regulates Key Steps of Inflammation Resolution in a Model of Antigen-Induced Arthritis via GPR30/PKA-Dependent Mechanism.

Biochanin A (BCA) is a natural organic compound of the class of phytochemicals known as flavonoids and isoflavone subclass predominantly found in red clover (Trifolium pratense). It has anti-inflammatory activity and some pro-resolving actions, such as neutrophil apoptosis. However, the effect of BCA in the resolution of inflammation is still poorly understood. In this study, we investigated the effects of BCA on the neutrophilic inflammatory response and its resolution in a model of antigen-induced arthritis. Male wild-type BALB/c mice were treated with BCA at the peak of the inflammatory process (12 h). BCA decreased the accumulation of migrated neutrophils, and this effect was associated with reduction of myeloperoxidase activity, IL-1β and CXCL1 levels, and the histological score in periarticular tissues. Joint dysfunction, as seen by mechanical hypernociception, was improved by treatment with BCA. The resolution interval (Ri) was also quantified, defining profiles of acute inflammatory parameters that include the amplitude and duration of the inflammatory response monitored by the neutrophil infiltration. BCA treatment shortened Ri from ∼23 h observed in vehicle-treated mice to ∼5.5 h, associated with an increase in apoptotic events and efferocytosis, both key steps for the resolution of inflammation. These effects of BCA were prevented by H89, an inhibitor of protein kinase A (PKA) and G15, a selective G protein–coupled receptor 30 (GPR30) antagonist. In line with the in vivo data, BCA also increased the efferocytic ability of murine bone marrow–derived macrophages. Collectively, these data indicate for the first time that BCA resolves neutrophilic inflammation acting in key steps of the resolution of inflammation, requiring activation of GPR30 and via stimulation of cAMP-dependent signaling.

Activation of GPR30 with G1 inhibits oscillatory shear stress-induced adhesion of THP-1 monocytes to HAECs by increasing KLF2.

Atherosclerosis is a chronic inflammatory disease known to be mediated by numerous factors, among which endothelial dysfunction plays a critical role. Oscillatory shear stress induces endothelial cells to lose their anti-atherosclerotic properties and downregulates the expression of the innate protective transcription factor, Krüppel-like factor 2 (KLF2), which is typically upregulated in vascular endothelial cells in response to harmful stimuli. Oxidative stress and inflammation impair endothelial function and damage their survival. Oscillatory shear stress also promotes generation of reactive oxygen species and production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby further promoting endothelial dysfunction and formation of atherosclerotic plaque. A major event in the development of atherosclerotic plaque is rolling and adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules including vascular cellular adhesion molecule 1 and endothelial-selectin. Expression of these molecules is also upregulated by oscillatory shear stress. Estrogen has long been recognized as a protective agent against atherosclerosis, but the mechanisms through which estrogen receptors prevent atherogenesis remain unclear. In the present study, we investigated the role of the G-coupled protein estrogen receptor (GPR30) in oscillatory shear stress- induced endothelial dysfunction. We show that agonism of GPR30 by its specific agonist G1 prevented oscillatory shear stress -induced oxidative stress markers and production of inflammatory cytokines and adhesion molecules. As a result, GPR30 activation suppresses monocytes adhesion to endothelial cells. Furthermore, we demonstrate that GPR30 prevents oscillatory shear stress- induced downregulation of KLF2 via ERK5 pathway. These findings suggest that endothelial GPR30 is potential target to suppress oscillatory shear stress mediated atherogenesis.

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERβ and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERβ agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERβ antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERβ but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs.

Protective mechanism of GPR30 agonist G1 against ultraviolet B-induced injury in epidermal stem cells

The regeneration of the skin is vital to our wound healing and skin repair abilities. Adult epidermal stem cells (ESCs) have been shown to have the potential to renew old and dead skin cells, and ESCs have been implemented in stem cell-based therapies. GPR30 is a G protein-coupled membrane receptor for oestrogen, which has been shown to regulate cell proliferation and programmed cell death. Here, we examined the biological function of GPR30 in isolated adult murine ESCs. We show that GPR30 is fairly expressed in ESCs and is repressed upon ultraviolet B (UV-B) treatment in a dose-dependent manner. The activation of GPR30 by its agonist G1 ameliorates UV-B induced cellular oxidative stress and induction of IL-6 and IL-8. Furthermore, G1 protects against UV-B-induced cell death and improves the viability of ESCs. G1 also suppresses UV-B-induced HMGB-1 expression and protects the capacity of ESCs from the harm by UV-B radiation. Mechanistically, we show that co-treatment with G1 rescues UV-B-induced reduced Wnt1, cyclin D1 and β-catenin production, indicating the involvement of conical Wnt/β-catenin. Collectively, our data indicate that the activation of GPR30 has a protective role in ESCs, and GPR30 agonist G1-mediated ESC protection has potential implications in stem cell-based therapies for skin diseases.

Issue Highlights

IUBMB LifeVolume 71, Issue 11 p. 1660-1660 Issue HighlightsFree Access Issue Highlights First published: 12 October 2019 https://doi.org/10.1002/iub.2183AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 1661 Immunological implications of epidermal growth factor receptor signaling in persistent infections Tanushree Mukherjee, Kithiganahalli Narayanaswamy Balaji Epidermal Growth Factor Receptor (EGFR) is classically known for its implications in the etiology of diverse cancers by regulating cell division, differentiation and death. However, the ability of the oncogenic receptor to interact with several cellular intermediates, modulate signaling events and alter gene expression is also tapped by infectious agents to drive their pathogenesis. There is a wide array of nodes where pathogens intersect with EGFR signaling ranging from host cell adhesion/entry to phagosomal maturation and inflammation. Therefore, the understanding of these mechanisms would offer effective EGFR-directed therapeutic/vaccination strategies against the rising incidence of antibiotic-resistant infections. 1685 Leishmania species-dependent functional duality of toll-like receptor 2 Abdollah Jafarzadeh, Maryam Nemati, Iraj Sharifi, Arathi Nair, Divanshu Shukla, Prashant Chauhan, Hossain Khorramdelazad, Arup Sarkar, Bhaskar Saha TLR2-mediated host-protective or disease-exacerbating immune responses are dependent on the infecting Leishmania species. A) An overview of TLR2/2 or TLR1/2, TLR2/6-mediated elimination or persistence of Leishmania in different immune cells. B) Signaling through TLR2/2 in NK-cells, macrophages, DCs and TH1-cells leads to elimination, whereas signaling through TLR1/2 and TLR2/6 in macrophages, TH1-cells and Treg-cells results in persistence of L. major. Signaling through TLR2/2 in DCs, Treg-cells, TH1-cells and CD8+T-cells leads to L. donovani elimination, whereas through TLR2/2, TLR1/2 and TLR2/6 in L. donovani persistence. In L. mexicana, and L. infantum infections TLR2-mediated host-protective responses and L. amazonensis and L. braziliensis infections disease-exacerbating response are triggered 1751 Activation of GPR30 promotes osteogenic differentiation of MC3T3-E1 cells: An implication in osteoporosis Xiaozong Lin, Li Li, Shuliang Wu, Jun Tian, Weizhuo Zheng Expression of GPR30 is upregulated during osteoblast differentiation and that agonism of GPR30 significantly increases some key markers of mineralization including alkaline phosphatase, osteocalcin, osterix, and type I collagen. GPR30 agonism upregulates expression of Runx2, which is recognized as an essential transcription factor involved in bone formation. Additionally, through a series of adenosine monophosphate-activated protein kinase (AMPK) -inhibition experiments using compound C, we show that the positive effects of GPR30 on mineralization and differentiation of pre-osteoblasts are mediated through the AMPK /anti-acetyl-CoA carboxylase (ACC) pathway. Our findings demonstrate the potential of GPR30 as a novel target for the treatment and prevention of osteoporosis. REFERENCE 1Mukherjee Tanushree, Balaji Kithiganahalli Narayanaswamy. Immunological implications of epidermal growth factor receptor signaling in persistent infections. IUBMB Life. 2019, 71, 1661– 1671. 2Jafarzadeh Abdollah, Nemati Maryam, Sharifi Iraj, Nair Arathi, Shukla Divanshu, Chauhan Prashant, Khorramdelazad Hossain, Sarkar Arup, Saha Bhaskar. Leishmania species-dependent functional duality of tolllike receptor 2. IUBMB Life. 2019, 71, 1685– 1700. 3Lin Xiaozong, Li Li, Wu Shuliang, Tian Jun, Zheng Weizhuo. Activation of GPR30 promotes osteogenic differentiation of MC3T3-E1 cells: An implication in osteoporosis. IUBMB Life. 2019, 71, 1751– 1759. Volume71, Issue11November 2019Pages 1660-1660 ReferencesRelatedInformation

Activation of GPR30 promotes osteogenic differentiation of MC3T3-E1 cells: An implication in osteoporosis.

Osteoporosis is an age-related disease characterized by reduced bone volume and disturbed bone metabolism. Novel therapies to rescue or prevent reduced bone mass by guiding the differentiation of pluripotent bone marrow stromal cells away from adipocyte differentiation and toward osteoblastic differentiation may serve as a valuable treatment option against osteoporosis. Estrogen has long been recognized as a key effector of bone formation and mineralization, but the exact mechanisms involved remain poorly understood. In the present study, we investigated the role of the estrogen-specific G protein-coupled receptor 30 (GPR30/GPER) using its specific agonist G1 in MC3T3-E1 preosteoblast cells. Our findings demonstrate that expression of GPR30 is upregulated during osteoblast differentiation and that agonism of GPR30 significantly increases some key markers of mineralization including alkaline phosphatase, osteocalcin, osterix, and type I collagen. We also demonstrate that GPR30 agonism upregulates expression of Runx2, which is recognized as an essential transcription factor involved in bone formation. Additionally, through a series of adenosine monophosphate-activated protein kinase (AMPK)-inhibition experiments using compound C, we show that the positive effects of GPR30 on mineralization and differentiation of preosteoblasts are mediated through the AMPK/anti-acetyl-CoA carboxylase (ACC) pathway. Taken together, the findings of the present study demonstrate the potential of GPR30 as a novel target for the treatment and prevention of osteoporosis.

Estrogen regulation of cardiac cAMP-L-type Ca2+ channel pathway modulates sex differences in basal contraction and responses to \u03b22AR-mediated stress in left ventricular apical myocytes

Backgrounds/AimMale and female hearts have many structural and functional differences. Here, we investigated the role of estrogen (E2) in the mechanisms of sex differences in contraction through the cAMP-L-type Ca2+channel pathway in adult mice left ventricular (LV) apical myocytes at basal and stress state.MethodsIsolated LV apical myocytes from male, female (Sham) and ovariectomised mice (OVX) were used to investigate contractility, Ca2+ transients and L-type Ca2+ channel (LTCC) function. The levels of β2AR, intracellular cAMP, phosphodiesterase (PDE 3 and PDE 4), RyR2, PLB, SLN, and SERCA2a were compared among the experimental groups.ResultsWe found that (1) intracellular cAMP, ICaL density, contraction and Ca2+ transient amplitudes were larger in Sham and OVX + E2 myocytes compared to male and OVX. (2) The mRNA expression of PDE 3 and 4 were lower in Sham and OVX + E2 groups compared with male and OVX groups. Treatment of myocytes with IBMX (100 μM) increased contraction and Ca2+ transient amplitude in both sexes and canceled differences between them. (3) β2AR-mediated stress decreased cAMP concentration and peak contraction and Ca2+ transient amplitude only in male and OVX groups but not in Sham or OVX + E2 groups suggesting a cardioprotective role of E2 in female mice. (4) Pretreatment of OVX myocytes with GPR30 antagonist G15 (100 nM) abolished the effects of E2, but ERα and ERβ antagonist ICI 182,780 (1 μM) did not. Moreover, activation of GPR30 with G1 (100 nM) replicated the effects of E2 on cAMP, contraction and Ca2+ transient amplitudes suggesting that the acute effects of E2 were mediated by GPR30 via non-genomic signaling. (5) mRNA expression of RyR2 was higher in myocytes from Sham than those of male while PLB and SLN were higher in male than Sham but no sex differences were observed in the mRNA of SERCA2a.ConclusionCollectively, these results demonstrate that E2 modulates the expression of genes related to the cAMP-LTCC pathway and contributes to sex differences in cardiac contraction and responses to stress. We also show that estrogen confers cardioprotection against cardiac stress by non-genomic acute signaling via GPR30.

Low concentration of BPA induces mice spermatocytes apoptosis via GPR30.

Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human testicular function remains to be identified. GPR30 is a novel membrane estrogen receptor with high-affinity and low-capacity binding to estrogens. We demonstrated that estrogen receptor α (ERα), estrogen receptor β (ERβ) as well as GPR30 are expressed in mouse spermatocyte-derived GC-2 cells using Real-time PCR. We treated the cells with different doses of BPA and found that even low doses of BPA can inhibit GC-2 cell growth using MTT assay. To make sure which receptor is responsible for the biological function of BPA, we used ER down-regulator ICI and indicated that BPA could bind to GPR30. We also observed that BPA was able to induce Erk1/2 phosphorylation in GC-2 cells and proved that this process was mediated by GPR30–related EGFR-MAPK pathway using western blot. By Real-time PCR, we found that the expression of c-Fos was up-regulated and Cyclin D1 gene was down-regulated, in the presence of BPA and ICI. The results of MTT assay, comet assay and flow cytometry indicated that the activation of GPR30 induced by BPA inhibited the cell growth and induced cell apoptosis and ICI, GPR30 siRNA, EGFR inhibitor (AG), and MAPK (PD) inhibitor could partially reverse this effect. Immunohistochemistry on the testis of BPA –damaged mice showed that BPA induced spermatocyte apoptosis without affecting the seminiferous tubules and spermatocyte. In conclusion, BPA triggered spermatocyte apoptosis via GPR30.

Activation of GPR30 stimulates GTP-binding of Gαi1 protein to sustain activation of Erk1/2 in inhibition of prostate cancer cell growth and modulates metastatic properties

Previously, we reported that GPR30 activation by the receptor-specific, non-estrogenic ligand G-1 inhibited in vitro and in vivo growth of prostate cancer (PCa) cells via sustained Erk1/2 activation. Mechanism underlying the sustained Erk1/2 activation for PCa cell growth inhibition remains unclear. Here we report that G-1, through GPR30, activated Gαi1 proteins to sustain Erk1/2 activation but failed to activate adenylyl cyclase (AC) for cAMP production in PCa cells. The chemical-induced activation of AC-cAMP-PKA signaling attenuated Erk1/2 activity and blocked the cell growth inhibitory effects of G-1. Furthermore, PCa predominantly expressed Gαi1 proteins. Silencing of Gαi1 expression blocked the inhibitory effects of G-1 on PCa cell growth. By gene expression profiling, GPR30 activation by G-1 interfered expression of cell cycle regulators and machinery elements to modulate PCa cell growth and the RACGAP1 interactome to control metastatic properties. In this regard, we demonstrated that G-1 inhibited PCa cell migration and invasion with reduced formations of filopodia and stress fibers through a GPR30-dependent pathway. Taken together, our findings revealed the underlying mechanism for sustaining Erk1/2 activation upon GPR30 activation by G-1 in PCa cells and the GPR30-mediated pathways in controlling PCa cell growth and metastatic properties.

GPR30 regulates diet-induced adiposity in female mice and adipogenesis in vitro.

Recent studies showed that GPR30, a seven-transmembrane G-protein-coupled receptor, is a novel estrogen receptor (ER) that mediates some biological events elicited by estrogen in several types of cancer cells. However, its physiological or pathological role in vivo is unclear. Here, we show that GPR30 knockout (GPRKO) female mice were protected from high-fat diet (HFD)-induced obesity, blood glucose intolerance, and insulin resistance. The decreased body weight gain in GPRKO female mice is due to the reduction in body fat mass. These effects occurred in the absence of significant changes in food intake, intestinal fat absorption, triglyceride metabolism, or energy expenditure. However, GPR30 had no significant metabolic effects in male mice fed the HFD and both sexes of mice fed a chow diet. Further, GPR30 expression levels in fat tissues of WT obese female mice were greatly increased, whereas ERα and β expression was not altered. Deletion of GPR30 reduced adipogenic differentiation of adipose tissue-derived stromal cells. Conversely, activation of GPR30 enhanced adipogenic differentiation of 3T3-L1 preadipocytes. These findings provide evidence for the first time that GPR30 promotes adipogenesis and therefore the development of obesity in female mice exposed to excess fat energy.

Importance of Estrogenic Signaling and Its Mediated Receptors in Prostate Cancer.

Prostate cancer (PCa) treatment was first established by Huggins and Hodges in 1941, primarily described as androgen deprivation via interference of testicular androgen production. The disease remains incurable with relapse of hormone-refractory cancer after treatments. Epidemiological and clinical studies disclosed the importance of estrogens in PCa. Discovery of estrogen receptor ERβ prompted direct estrogenic actions, in conjunction with ERα, on PCa cells. Mechanistically, ERs upon ligand binding transactivate target genes at consensus genomic sites via interactions with various transcriptional co-regulators to mold estrogenic signaling. With animal models, Noble revealed estrogen dependencies of PCa, providing insight into potential uses of antiestrogens in the treatment. Subsequently, various clinical trials were conducted and molecular and functional consequences of antiestrogen treatment in PCa were delineated. Besides, estrogens can also trigger rapid non-genomic signaling responses initiated at the plasma membrane, at least partially via an orphan G-protein-coupled receptor GPR30. Activation of GPR30 significantly inhibited in vitro and in vivo PCa cell growth and the underlying mechanism was elucidated. Currently, molecular networks of estrogenic and antiestrogenic signaling via ERα, ERβ and GPR30 in PCa have not been fully deciphered. This crucial information could be beneficial to further developments of effective estrogen- and antiestrogen-based therapy for PCa patients.

GPR30 decreases with vascular aging and promotes vascular smooth muscle cells maintaining differentiated phenotype and suppressing migration via activation of ERK1/2.

Estrogen receptors, including classic nuclear receptors ERα, ERβ, and membrane receptor GPR30, are expressed in vascular tissues and exert protective actions in vascular diseases. But the expression pattern and functional roles of GPR30 in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we found that ERα, ERβ, and GPR30 were decreased with VSMCs passaging in vitro or growing in vivo and activation of GPR30 promoted ERα expression. Then, we validated that activation of GPR30 significantly decreased migratory capability of VSMCs and suppressed ERα, whereas PDGF-BB (20 ng/mL) treatment caused increase of migration. And activation of GPR30 led to reduction of osteopontin and cellular retinol binding protein 1, enhancement of calponin and 3F8, and upregulation of total and phosphorylated ERK1/2 expression in VSMCs knocked down by GPR30, ERα, and ERβ or treated with PDGF-BB. These data suggest that GPR30 promotes VSMCs reducing migration and maintaining differentiated phenotype via activation of ERK1/2 pathway. Our findings provide novel mechanisms of GPR30 protection of VSMCs as well as a new target for prevention of vascular aging.

Activation of GPR30 improves exercise capacity and skeletal muscle strength in senescent female Fischer344 × Brown Norway rats

The molecular mechanisms of muscle weakness and sarcopenia in postmenopausal women are largely unknown. To determine the effect of a new estrogen receptor, GPR30, in the maintenance of exercise capacity and skeletal muscle function in females, the selective GPR30 agonist, G1 (100 μg/kg/day), or vehicle (V, soybean oil) was administered subcutaneously daily (n = 7 per group) to ovariectomized (OVX) 27-month-old Fischer 344 × Brown Norway (F344BN) female rats. Following 8 weeks of treatment, the exercise capacity (treadmill walk time to exhaustion) was reduced in OVX vs. sham rats (5.1 ± 1.4 vs. 11.0 ± 0.9 min, P < 0.05), and chronic G1 restored exercise capacity (12.9 ± 1.2 min; P < 0.05 vs. OVX-V). Similarly, the peak twitch of electrically stimulated soleus muscles was decreased by 22% in OVX vs. sham rats (P < 0.05), and G1 attenuated this decline (P < 0.05). Western blot analysis showed that chronic G1 treatment attenuated OVX-associated decreases in heat shock protein (HSP) 90, HSP70, and HSP27 expressions. In vitro studies using the L6 myoblast cell line demonstrated that G1 increased mRNA levels of HSPs in cultured cells. Collectively, these data demonstrate that the activation of GPR30 mitigates the adverse effects of estrogen loss on exercise capacity and skeletal muscle contractile function in old F344BN rats. The protective effects of GPR30 might be through its upregulation of heat shock proteins in skeletal muscle.

Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30

Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3−/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3−; control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.

Activation of GPR30 inhibits cardiac fibroblast proliferation.

The incidence of left ventricular diastolic dysfunction significantly increases in postmenopausal women suggesting the association between estrogen loss and diastolic dysfunction. The in vivo activation of G protein-coupled estrogen receptor (GPR30) attenuates the adverse effects of estrogen loss on cardiac fibrosis and diastolic dysfunction in mRen2.Lewis rats. This study was designed to address the effects of GPR30 on cardiac fibroblast proliferation in rats. The expression of GPR30 in cardiac fibroblasts isolated from adult Sprague-Dawley rats was confirmed by RT-PCR, Western blot analysis, and immunofluorescence staining. Results from BrdU incorporation assays, cell counting, carboxyfluorescein diacetate succinimidyl ester labeling in conjunction with flow cytometry, and Ki-67 staining showed that treatment with G1, a specific agonist of GPR30, inhibited cardiac fibroblast proliferation in a dose-dependent manner, which was associated with decreases in CDK1 and cyclin B1 protein expressions. In the GPR30-KO cells, BrdU incorporation, and CDK1 and cyclin B1 expressions significantly increased when compared to GPR30-intact cells. G1 had no effect on BrdU incorporation, CDK1 and cyclin B1 mRNA levels in GPR30-KO cells. In vivo studies showed increases in CDK1 and cyclin B1 mRNA levels, Ki-67-positive cells, and the immunohistochemistry staining of vimentin, a fibroblast marker, in the left ventricles from ovariectomized mRen2.Lewis rats versus hearts from ovary-intact littermates; 2 weeks of G1 treatment attenuated these adverse effects of estrogen loss. This study demonstrates that GPR30 is expressed in rat cardiac fibroblasts, and activation of GPR30 limits proliferation of these cells likely via suppression of the cell cycle proteins, cyclin B1, and CDK1.

EGFR participates downstream of ERα in estradiol-17β-D-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets.

Estradiol-17β-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner.

Role of GPR30 in mediating estradiol effects on acetylcholine release in the hippocampus

We have hypothesized that estradiol enhances basal forebrain cholinergic function and cognitive performance, at least in part, via activation of the novel estrogen receptor GPR30. Here we evaluated the effects of estradiol, G-1 (a selective GPR30 agonist), and tamoxifen (TAM; an ERα/ERβ antagonist that also acts as a GPR30 agonist), on acetylcholine (ACh) release in the hippocampus, as well as the ability to block the effects of 17β-estradiol (E) or TAM with the GPR30 antagonist G-15. Note that G-1 was included to evaluate the effects of selectively activating GPR30, whereas TAM was included to differentiate effects of E associated with activation of GPR30 vs. ERα or ERβ. The study was designed to test effects on potassium-stimulated release, as well as on ACh release stimulated by feeding. Effects of feeding were included because the tasks we used previously to demonstrate beneficial effects of E on cognitive performance were motivated by food reward, and we hypothesized that E may enhance performance by increasing ACh release in association with that reward. Ovariectomized rats were treated for 1week, and ACh release was evaluated using in vivo microdialysis. In addition, rats were fed at the same time daily for several days and were fasted overnight prior to microdialysis. For each rat, ACh release was evaluated under basal conditions, in response to feeding, and in response to elevated potassium. Both feeding and elevated potassium increased ACh release in the hippocampus. In response to feeding, E, G-1, and TAM all significantly increased the percent change in release. The effects of E and TAM were blocked by G-15, and the effects of combining E+TAM did not differ significantly from the effects of E or TAM alone. In response to elevated potassium, E, and TAM significantly increased the percent change in ACh release. G-1 produced a slightly lesser effect. The effect of TAM was reduced by G-15, but the effect of E was not. These findings suggest that activation of GPR30 is both necessary and sufficient to account for the effects of E on ACh release associated with feeding. In contrast, activation of GPR30 appears to be sufficient, but may not be necessary for increased release associated with elevated potassium. The changes associated with feeding are consistent with the effects of E, G-1 and G-15 on acquisition of a spatial learning task previously described. These data confirm and extend previous reports, and support a hypothesis wherein E treatment can improve learning on specific tasks by activating GPR30 and enhancing ACh release in association with food reward.