G Protein-coupled Estrogen Receptor Antagonist Research Articles

17-β-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-β-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2’,7’-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

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

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

Genistein Alleviates Dextran Sulfate Sodium-induced Ulcerative Colitis in Mice by Regulating Th17/Treg Cell Balance: Implication for the G Protein-coupled Estrogen Receptor

Objectives: This study aimed to evaluate the therapeutic effects and mechanism of genistein on dextran sulfate sodium-induced mouse model of ulcerative colitis (UC). Materials and Methods: A DSS-induced mouse model for UC was used in this study. Mice were then treated with genistein or a combination of genistein and the G protein-coupled estrogen receptor (GPER) antagonist G15. Colon length, disease activity index (DAI), spleen index (SI), histopathological alterations, and integrity of the colonic barrier were evaluated. The expression of inflammatory cytokines and antioxidant capacity were detected. Populations of T helper 17 cells (Th17) and Regulatory T cells (Treg) in the mesenteric lymph nodes (MLN) were analyzed. The expression of a transcription factor for Th17 and Treg in the colonic tissues was detected. Results: Genistein treatment significantly alleviated DSS-induced colitis, summarized as increased colonic length, decreased DAI, SI, improved histopathological alterations, and colonic barrier integrity. Genistein treatment restrained pro-inflammatory cytokines and oxidative enzyme release while promoting anti-inflammatory and anti-oxidative enzyme release. Flow cytometry indicated that genistein significantly reduced the Th17 population while boosting Treg populations. Furthermore, genistein inhibited the expression of transcription factors associated with Th17 and promoted the expression of transcription factors associated with Treg in the colonic tissue. Intriguingly, these observed effects of genistein were abolished when UC mice were treated with a combination of genistein and GPER antagonist G15. Conclusion: This study suggests that genistein is potent in protecting against DSS-induced colonic injuries by rebalancing Th17/Treg and that GPER may be a vital target for genistein-mediated immunomodulatory effects in UC.

FRI126 Autoreactive T Cells To Angiotensin II Type 1 Receptor In Salt-dependent Hypertension Due To Primary Aldosteronism

Abstract Disclosure: R. Scarpa: None. M. Piazza: None. B. Caroccia: None. S. Carraro: None. F. Vianello: None. F. Cinetto: None. C. Agostini: None. T.M. Seccia: None. G. Rossi: None. Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but the pathogenic role of this acquired immunity is still unknown. Primary Aldosteronism, the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary (‘essential’) hypertension and by high titer of autoantibodies against the type 1 angiotensin receptor (AT1R). As T cells are broadly recognised as HMOD-mediators, we aim to investigate: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of know aldosterone receptors on T lymphocytes. The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number by droplet digital PCR and immunoblotting from healthy donors (HD) subjects and PA patients. Peripheral blood mononuclear cells (PBMCs) from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFNɣ release with flow cytometry. PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR>MR>>GPER. Aldosterone significantly increased IFNɣ release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-γ production by CD8+ T cells by acting via the MR, as it was prevented (p < 0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p < 0.003), but also by G36 (p < 0.02), and mimicked by G1. These results showed the presence of AT1R-autoreactive T cells in patients with PA. Moreover, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs. Presentation: Friday, June 16, 2023

MiR-206 inhibits estrogen signaling and ovarian cancer cell migration without affecting GPER

AimsEstrogen-regulated pathways are involved in the etiology and progression of epithelial ovarian cancer (EOC), but the relative contribution of estrogen receptor isoforms is unclear. Only a subset of patients responds to antiestrogens including tamoxifen. Based on our previous evidence that miR-206 behaves as an oncosuppressor in EOC, we hypothesized that miR-206 would interfere with G protein-coupled estrogen receptor (GPER)-mediated signaling and cell motility. Main methodsPFKFB3 and FAK proteins from OC cells challenged with selective estrogen receptor agonist and antagonist were measured by Western blotting. Cell proliferation and motility were analyzed by MTT and Boyden chamber, respectively. Estrogen-dependent cells were transfected with miR-206 mimic or control using Lipofectamine. Key findingsThe migration of SKOV3 and OVCAR5 cells significantly increased following treatment with 17β-estradiol (E2) and the selective GPER agonist G1. However, tamoxifen failed to inhibit E2 effect and even promoted SKOV3 cell migration. Estrogen receptor ligands did not affect SKOV3 proliferation. The GPER antagonist G15 significantly prevented E2-mediated upregulation of PFKFB3 expression, while G1 concentration-dependently upregulated PFKFB3 levels. Consistent with the functional link between PFKFB3 and FAK activation, E2 and G1 increased FAK phosphorylation at Tyr397. Transfection with miR-206 abolished estrogen-induced EOC migration and down-regulated PFKFB3 protein levels. Notably, miR-206 transfection reduced ERα protein abundance, whereas GPER amount was unchanged. SignificanceBy blocking estrogen signaling and G1-induced EOC cell invasiveness with no direct interference with GPER levels, miR-206 mimics have the potential to act as pathway-selective antagonists and deserve further testing as RNA therapeutics in estrogen-dependent EOC.

Different Expression Pattern of G Protein-Coupled Estrogen Receptor GPER1 in Esophageal Squamous Cell Carcinoma and Adenocarcinoma.

Esophageal carcinoma is a male-dominant malignancy worldwide, and esophageal adenocarcinoma (EAC) shows more significant sex bias than esophageal squamous cell carcinoma (ESCC) in morbidity and mortality. The G protein-coupled estrogen receptor 1 (GPER1) is involved in several sex-related cancers; however, its expression level in esophageal carcinoma has been poorly investigated and its role is not precisely defined, depending on histological types. In the present study, the mRNA levels of GPER1 in esophageal carcinoma were collected from GEPIA and Oncomine databases for meta-analyses. The protein expression levels of GPER1 were detected by immunohistochemistry in the tissue microarray of EAC and ESCC. The GPER1 selective agonist G1, antagonist G15, and siRNA were applied in vitro to investigate their impacts on esophageal cell lines. Analysis of the RNA levels from the databases showed a decreased expression of GPER1 in overall esophageal carcinoma, and low expression levels of GPER1 were found to be associated with low survival of tumor patients. However, in the subgroup of EAC and its precancerous lesion, Barrett's esophagus, overexpression of GPER1 RNA was increased when compared with the normal tissues. The average staining scores of GPER1 protein in the tissue microarray of EAC were significantly higher than normal esophageal samples, and the rate of positive staining increased with the grade of poor tumor differentiation. The scores of GPER1 protein in ESCC tissues were lower than those in the normal tissues. The results from cell line experiments in vitro showed that the GPER1 agonist G1 inhibited proliferation and promoted apoptosis of ESCC cells EC109 with positive expression of GPER1. G1 had no obvious effect on normal esophageal NE2 cells with weak expression of GPER1. In addition, GPER1 RNA knockdown and application of antagonist G15 reversed the effects of G1 on EC109. The results of this study indicate that the expression levels of GPER1 are higher in EAC than in ESCC, which might be correlated with the dimorphic estrogen signaling pathway in different types of esophageal carcinoma.

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

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

Neuroprotective Potential of Raloxifene via G-Protein-Coupled Estrogen Receptors in Aβ-Oligomer-Induced Neuronal Injury.

Amyloid-β (Aβ) is one of the causes of Alzheimer's disease (AD), damaging nerve membranes and inducing neurotoxicity. AD is more prevalent in female patients than in male patients, and women are more susceptible to developing AD due to the decline in estrogen levels around menopause. Raloxifene, a selective estrogen receptor modulator, exhibits protective effects by activating the transmembrane G-protein-coupled estrogen receptor (GPER). Additionally, raloxifene prevents mild cognitive impairment and restores cognition. However, the influence of raloxifene via GPER on highly toxic Aβ-oligomers (Aβo)-induced neurotoxicity remains uncertain. In this study, we investigated the GPER-mediated neuroprotective effects of raloxifene against the neurotoxicity caused by Aβo-induced cytotoxicity. The impact of raloxifene on Aβo-induced cell damage was evaluated using measures such as cell viability, production of reactive oxygen species (ROS) and mitochondrial ROS, peroxidation of cell-membrane phospholipids, and changes in intracellular calcium ion concentration ([Ca2+]i) levels. Raloxifene hindered Aβo-induced oxidative stress and reduced excessive [Ca2+]i, resulting in improved cell viability. Furthermore, these effects of raloxifene were inhibited with pretreatment with a GPER antagonist. Our findings suggest that raloxifene safeguards against Aβo-induced neurotoxicity by modifying oxidative parameters and maintaining [Ca2+]i homeostasis. Raloxifene may prove effective in preventing and inhibiting the progression of AD.

AUTOREACTIVE T CELLS TO ANGIOTENSIN II TYPE 1 RECEPTOR IN SALT-DEPENDENT HYPERTENSION DUE TO PRIMARY ALDOSTERONISM

Objective: Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but its pathogenic role is still unknown. Primary Aldosteronism (PA), the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary hypertension and by high titer of autoantibodies against the angiotensin type-1 receptor (AT1R). As T cells are broadly recognised as HMOD-mediators we investigated: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of aldosterone receptors on T lymphocytes. Design and Methods: The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number and immunoblotting from healthy donors and PA patients. PBMCs from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFN? release with flow cytometry. Results: PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR>MR>>GPER. Aldosterone significantly increased IFN? release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-? production by CD8+ T cells by acting via the MR, as it was prevented (p<0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p<0.003), but also by G36 (p<0.02), and mimicked by G1. Conclusion: These results showed the presence of AT1R-autoreactive T cells in patients with PA and demonstrates that, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs.

Aldosterone activates ERK phosphorylation in the nucleus tractus solitarius

Sodium intake effect of aldosterone has attracted much attention. In our recent study, aldosterone can play a nongenomic regulatory role on rapid sodium intake in the NTS (nucleus tractus solitarius) by activating G protein-coupled estrogen receptor (GPER), and it exhibited an obvious time-dependent and concentration-dependent regulation. However, the molecular mechanism how aldosterone regulated sodium intake rapidly, is unclear. To determine the molecular mechanism of rapid sodium intake regulation of aldosterone, rats with a stainless-steel cannula in the NTS were used (n = 6 each subgroup), and were injected different concentrations of aldosterone/G1 (GPER agonist)/G15 (GPER antagonist) at different time points, then detected ERK1/2 protein expression. The results showed that aldosterone/G1 increased the ERK1/2 protein phosphorylation, and presented a time-dependent and concentration-dependent similar to sodium intake; Meanwhile, G15 partially blocked this effect at least. Taken together, we postulate that ERK1/2 protein may influence nongenomic sodium intake regulated by aldosterone at nucleus tractus solitarius level.

Regulation of human endothelial cell migration by oral contraceptive estrogen receptor ligands

Ethinylestradiol (EE) and estetrol (E4) are the two main estrogenic agents used in combined oral contraceptives. These compounds have different binding affinity to and efficacy on estrogen receptors (ER) subtypes. We previously reported that treatment with estrogenic agents enhances angiogenesis via nongenomic, G protein-coupled estrogen receptor (GPER)-dependent mechanisms. However, the impact of EE and E4 on human endothelial function has been little investigated. EE and E4 (10−9– 10−7 M) significantly enhanced migration of human umbilical vein endothelial cells (HUVECs) using scratch and Boyden chamber assays. Mechanistically, both agents increased accumulation of phosphorylated protein tyrosine kinase 2 on tyrosine 397 (FAK Y397), a key player in endothelial cell motility, after 30-min treatment. Treatment with increasing concentrations of EE, but not E4, enhanced accumulation of the glycolysis activator PFKFB3. Of note, effects of EE and E4 on endothelial migration and signalling proteins were abolished by addition of the GPER antagonist G36 (10−6 M). Thus, EE and E4 induced comparable endothelial responses in vitro, suggesting no apparent alterations of vascular remodelling and regeneration capacity by oral contraceptives containing these agents.

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

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

G protein estrogen receptor as a potential therapeutic target in Raynaud's phenomenon.

Exaggerated cold-induced vasoconstriction can precipitate a pathogenesis called Raynaud's phenomenon (RP). Interestingly, RP is significantly more prevalent in females than age-matched men, highlighting the potential implication of 17β-estradiol (E2) in the etio-pathogenesis of this disease. Indeed, we have previously reported that E2 stimulates the expression of vascular alpha 2C-adrenoceptors (α2C-AR), the sole mediator of cold-induced constriction of cutaneous arterioles. This induced expression occurs through the cyclic adenosine monophosphate → exchange protein activated by cAMP→ Ras-related protein 1→ c-Jun N-terminal kinase→ activator protein-1 (cAMP/Epac/Rap/JNK/AP-1 pathway). On the basis that estrogen-induced rapid cAMP accumulation and JNK activation occurs so rapidly we hypothesized that a non-classic, plasma membrane estrogen receptor was the mediator. We then showed that an impermeable form of E2, namely E2:BSA, mimics E2 effects suggesting a role for the membranous G-protein coupled estrogen receptor (GPER) in E2-induced α2C-AR expression. Our current working hypothesis and unpublished observations further cement this finding, as G1, a GPER agonist, mimics while G15, a GPER antagonist, abrogates estrogen's effect on the expression of vascular α2C-AR. These, and other observations, highlight the potential of GPER as a tractable target in the management of RP, particularly in pre-menopausal women.

Estradiol Regulates the Expression and Secretion of Antimicrobial Peptide S100A7 via the ERK1/2-Signaling Pathway in Goat Mammary Epithelial Cells.

S100A7 has received extensive attention in the prevention and treatment of mastitis across a broad spectrum, yet there is a little information about its mechanism, especially in the immunomodulatory effects of estrogen. In the present study, based on the milk bacteriological culture (BC) of 30 dairy goats, the concentration of both estrogen and S100A7 in the BC-positive samples was not significantly different than in the BC-negative samples; the estrogen abundance in subclinical and clinical mastitis samples also showed only a limited difference; compared with healthy samples, the S100A7 abundance in subclinical mastitis samples differed little, while it was significantly decreased in clinical mastitis samples. Moreover, the relationship between estrogen and S100A7 was positive, and the regression equation was y = 0.3206x + 23.459. The goat mammary epithelial cells (gMECs) were isolated and treated with 1, 10, 100 nM E2 and/or 5 μg/mL lipopolysaccharide (LPS), respectively, for 6 h. Compared with control samples, 5 μg/mL LPS, 10 nM E2 and 100 nM E2 markedly induced S100A7 expression and secretion. More than separated treatment, the cooperation of LPS and E2 also significantly increased S100A7 expression, rather than S100A7 secretion. The p-ERK was up-regulated markedly with 100 nM E2 treatment, while the expression of p-JNK, p-p38 and p-Akt had little effect. The G protein-coupled estrogen receptor 1(GPER1) agonist G1 markedly induced S100A7 expression and secretion in gMECs, and the estrogen nuclear receptor antagonist ICI and GPER1 antagonist G15 significantly repressed this process. In conclusion, E2 binds to nuclear and membrane receptors to regulate the expression and secretion of S100A7 via the ERK1/2-signaling pathway in gMECs.

Epimedin B exerts neuroprotective effect against MPTP-induced mouse model of Parkinson's disease: GPER as a potential target

Mitochondrial dysfunction and oxidative stress play important roles in the neuropathogenesis of Parkinson’s disease (PD). Epimedin B, the second highest active ingredient in the flavonoids of Herba Epimedii, has been proven effective in treating osteoporosis and oxaliplatin-induced peripheral neuropathy. The present study aims to investigate the neuroprotective effects of Epimedin B in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP)-induced mouse model of PD, and the involvement of G protein-coupled estrogen receptor (GPER)-mediated anti-apoptosis as well as anti-endoplasmic reticulum stress. Molecular docking revealed that Epimedin B could directly bind to GPER at the same site as GPER agonist G1 and the binding energy was − 7.3 kcal/mol. Epimedin B treatment ameliorated MPTP-induced motor dysfunction and alleviated the decreased contents of DA with its metabolites in the striatum and the loss of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantial nigra pars compacta (SNpc). Epimedin B treatment markedly prevented MPTP-induced changes in apoptosis-related protein Bcl-2 and Bax as well as endoplasmic reticulum stress-related protein glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). Pharmacological blockade with GPER antagonist G15 could antagonize these neuroprotective effects of Epimedin B on the nigrostriatal system. Moreover, the anti-apoptosis and anti-endoplasmic reticulum stress effects of Epimedin B against MPTP toxicity were significantly reduced in GPER knockout (GPER-/-) mice. The present study provides the first evidence that Epimedin B can protect against MPTP-induced PD mice model. GPER may be a potential target for the neuroprotective effect of Epimedin B against PD.

Bisphenol F blocks Leydig cell maturation and steroidogenesis in pubertal male rats through suppressing androgen receptor signaling and activating G-protein coupled estrogen receptor 1 (GPER1) signaling

Bisphenol F (BPF) is a new analog of bisphenol A (BPA). BPA has deleterious effects on the male reproductive system, but the effect of BPF has not been studied in detail. In this study we focus on the effect of BPF on Leydig cell maturation. Male Sprague-Dawley rats were gavaged with 0, 1, 10, or 100 mg/kg BPF from postnatal days 35–56. BPF significantly reduced serum testosterone levels and sperm count in cauda epididymis at dose ≥1 mg/kg. It significantly down-regulated the expression of steroidogenic enzymes, while increasing FSHR and SOX9 levels at 10 and 100 mg/kg. Further studies showed that BPF reduced NR3C4 expression in Leydig and Sertoli cells without affecting its levels in peritubular myoid cells. BPF markedly increased GPER1 in Leydig cells at 100 mg/kg, and it significantly reduced SIRT1 and PGC1α levels in the testes at 100 mg/kg. BPF significantly inhibited testosterone production by immature Leydig cells at 50 μM after 24 h of treatment, which was completely reversed by NR3C4 agonist 7α-methyl-19-nortestosterone and partially reversed by GPER1 antagonist G15 not by ESR1 antagonist ICI 182,780. In conclusion, BPF negatively affects Leydig cell maturation in pubertal male rats through NR3C4 antagonism and GPER1 agonism.

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

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

Genistein accelerates glucose catabolism via activation the GPER-mediated cAMP/PKA-AMPK signaling pathway in broiler chickens

Genistein, the most abundance of phytoestrogens in soybeans, has beneficial effects in regulating metabolism-related disease; however, there is few available literatures about whether genistein regulates glucose metabolism that in turn affects the lipid accumulation in animals or humans. The current study showed that genistein promoted glucose uptake by enhancing glucose transporter-2 (GLUT2) protein level; and it also increased the activity of phosphofructokinase-1 (PFK) and pyruvate dehydrogenase (PDH), and the mRNA level of succinate dehydrogenase (SDH) both in broiler chickens or hepatocytes. Moreover, genistein obviously increased the p-LKB1 and p-AMPKα protein levels both in vivo and in vitro. Furthermore, the enhancement of genistein on glucose uptake and catabolism were reversed in hepatocytes pre-treated with AMPK inhibitor Compound C, and the increasing of genistein on the p-LKB1 and p-AMPKα protein levels were also reversed in hepatocytes pre-treated with PKA inhibitor H89. Importantly, the results showed that genistein simultaneously increased the estrogen receptor β (ERβ) and G protein-coupled estrogen receptor (GPER) protein levels, but the elevation effect of genistein on cAMP content was completely reversed in hepatocytes pre-treated with GPER antagonist G15, rather than ERβ inhibitor PHTPP. Meanwhile, the increasing of p-LKB1 and p-AMPKα protein levels induced by genistein were also reversed in hepatocytes pre-treated with G15. Collectively, our data demonstrated that genistein improves glucose metabolism via activating the GPER-mediated cAMP/PKA-AMPK signaling pathway. These findings provide theoretical basis for genistein as a promising nutritional supplemental to alleviate metabolism disorders and related diseases in animals or even humans.

PD16-09 THE ROLE OF GPER SIGNALING IN DEVELOPMENT OF PROSTATIC ENLARGEMENT IN HIGH FAT DIET INDUCED OBESITY RAT MODEL

You have accessJournal of UrologyCME1 May 2022PD16-09 THE ROLE OF GPER SIGNALING IN DEVELOPMENT OF PROSTATIC ENLARGEMENT IN HIGH FAT DIET INDUCED OBESITY RAT MODEL Shinsuke Mizoguchi, Kenichi Mori, Toshitaka Shin, and Hiromitsu Mimata Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Kenichi MoriKenichi Mori More articles by this author , Toshitaka ShinToshitaka Shin More articles by this author , and Hiromitsu MimataHiromitsu Mimata More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002548.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although there has been increasing evidence that indicates positive relationship between metabolic syndrome and emergence in benign prostatic hyperplasia (BPH), the detailed pathophysiological mechanism is still unknown. Estrogen, which is increased in obese patients, is considered to be one of the key factors in development of BPH and G protein-coupled estrogen receptor (GPER) has been reported to be implicated in symptomatic BPH. To elucidate a role of GPER signaling in development of BPH, we evaluated changes in prostatic weight and inflammatory profiles in the prostate by GPER inhibition using high fat diet induced obesity rat model. METHODS: Male Wistar rats (8 weeks old) were divided into three groups; rats with normal diet group (ND, n=6), rats with high fat diet induced obesity group (HFD, n=6), and HFD treated by G15 (G15, n=6) which is GPER antagonist. The high fat diet contains 32% fat. These rats were maintained for twelve weeks followed by two weeks treatment by oral administration of placebo or G15 (0.1mg/kg) daily. Lateral lobes of prostate were harvested to assess prostatic weight, histological change and mRNA expression of GPER, IL1β, TGFβ, ERK1/2 by qPCR. RESULTS: HFD showed significantly increased body weight, visceral fat and prostatic weight as well as significant upregulation of gene expressions in GPER, IL1β, TGFβ, and ERK1/2 compared to ND. Hematoxylin and eosin (HE) staining showed inflammatory cell infiltration in prostatic tissue section from HFD. In contrast, HFD treated by G15 demonstrated significantly decreased prostatic weight in association with downregulation of IL1β, TGFβ and ERK1/2 compared to HFD with placebo. mRNA expression of GPER was not significantly changed between HFD and G15 group. CONCLUSIONS: We demonstrated improvement of prostatic enlargement and inflammation by GPER inhibition, which suggest GPER signaling has an important role in development of BPH in obese patients. Therefore, GPER inhibition could be an therapeutic target of BPH with metabolic syndrome. Source of Funding: KAKENHI 20K18094 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e272 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Kenichi Mori More articles by this author Toshitaka Shin More articles by this author Hiromitsu Mimata More articles by this author Expand All Advertisement PDF DownloadLoading ...

G protein-coupled estrogen receptor 1 mediates proliferation and adipogenic differentiation of goat adipose-derived stem cells through ERK1/2-NF-κB signaling pathway.

Adipose tissue formation and moderate fat deposition are important for the production performance and eating quality of livestock meats. The self-renewal and adipogenic differentiation of adipose-derived stem cells are responsible for the formation and development of adipose tissue. In addition, estrogen targeting G protein-coupled estrogen receptor 1 (GPER1) has been reported to modulate cell proliferation and differentiation during tissue and organ development. However, the potential correlation among estrogen, GPER1, proliferation, and adipogenic differentiation in goat adipose-derived stem cells (gADSCs) is still unclear. Herein, we demonstrated that 17β-estradiol enhances the proliferative ability of gADSCs, indicated by the increased cell number and cell viability, accompanied by up-regulated expressions of cyclin D1 and PCNA. Meanwhile, the adipogenic differentiation is promoted by 17β-estradiol, supported by higher ccumulation of intracellular lipids and increased expressions of PPARγ, ACC, and FABP4. Notably, these activities are all obviously reduced by administration with GPER1 antagonist G15, but GPER1 agonist G1 enhances cell proliferation and adipogenic differentiation. Moreover, GPER1 silencing diminishes cell proliferation and adipogenic differentiation. In parallel, 17β-estradiol elevates the protein level of nuclear p-p65. Furthermore, the phosphorylation of p65 is enhanced by G1 but inhibited by G15 and GPER1 silencing. In addition, the phosphorylation of p65 is mediated by ERK1/2, suggesting that estrogen targeting GPER1 regulates cell proliferation and adipogenic differentiation of gADSCs through the ERK1/2-NF-κB signaling pathway. This study may provide a strong theoretical basis for improving meat quality, flavor, and cold resistance of livestock.