GPR30 Research Articles

Stimulatory Anticancer Effect of Resveratrol Mediated by G Protein-Coupled Estrogen Receptor in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most high-risk cancers; however, it has been suggested that estrogen signaling in CRC could have a protective effect. Therefore, we focused on the function of the G protein-coupled estrogen receptor (GPER) among the estrogen receptors in CRC. In this study, we investigated the therapeutic effect of resveratrol via GPER in CRC (RKO and WiDr) cells, CRC cell-derived xenograft models, and organoids (30T and 33T). Resveratrol significantly suppressed cell viability and proliferation in highly GPER-expressing RKO cells compared to that in low GPER-expressing WiDr cells. In xenograft models, resveratrol also delayed tumor growth and exhibited a high survival rate depending on GPER expression in RKO-derived tumors. Furthermore, resveratrol significantly inhibited the viability of organoids with high GPER expression. Additionally, the anticancer effect of resveratrol on CRC showed that resveratrol rapidly responded to GPER, while increasing the expression of p-ERK and Bax and cleaving PARP proteins.

Expression of functional mineralocorticoid receptor (MR) and G-protein coupled estrogen receptor (GPER) in human T lymphocytes

Aldosterone plays a key role in controlling blood pressure (BP) values by maintaining body salt, water, and fluid homeostasis. Excess aldosterone production is associated with arterial hypertension, cardiovascular and metabolic diseases, partly via generation of an inflammatory state followed by fibrotic changes in the organs that are target of hypertension. Aldosterone exerts genomic effects that are known to involve activation of the mineralocorticoid receptor (MR). Other aldosterone effects, including those usually defined as ‘rapid’ or ‘non genomic’, involve additional receptors as the G-protein coupled estrogen receptor (GPER). To date, the receptor(s) implicated in the inflammatory action of aldosterone in cells of the innate and adaptive immunity are unknown. Considering the potential role of T-lymphocytes in adaptive immunity in arterial hypertension and related hypertension-mediated organ damage (HMOD), we herein investigated and quantified the expression of the MR and GPER in human CD4+ and CD8+ T-cells. Results provided compelling evidence for the presence at the mRNA and protein level and suggest a functional role of these receptors in the two T-lymphocyte subtypes, thus indicating that they can represent a potential target for modulation of steroid hormone-induced inflammation and ensuing HMOD.

GPER: An Estrogen Receptor Key in Metastasis and Tumoral Microenvironments.

Estrogens and their role in cancer are well-studied, and some cancer types are classified in terms of their response to them. In recent years, a G protein-coupled estrogen receptor (GPER) has been described with relevance in cancer. GPER is a pleiotropic receptor with tissue-specific activity; in normal tissues, its activation is related to correct development and homeostasis, while in cancer cells, it can be pro- or anti-tumorigenic. Also, GPER replaces estrogen responsiveness in estrogen receptor alpha (ERα)-lacking cancer cell lines. One of the most outstanding activities of GPER is its role in epithelial-mesenchymal transition (EMT), which is relevant for metastasis development. In addition, the presence of this receptor in tumor microenvironment cells contributes to the phenotypic plasticity required for the dissemination and maintenance of tumors. These characteristics suggest that GPER could be a promising therapeutic target for regulating cancer development. This review focuses on the role of GPER in EMT in tumorigenic and associated cells, highlighting its role in relation to the main hallmarks of cancer and possible therapeutic options.

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

Prunetin in a GPR30-dependent manner mitigates renal ischemia/reperfusion injury in rats via interrupting indoxyl sulfate/TLR4/TRIF, RIPK1/RIPK3/MLKL, and RIPK3/PGAM5/DRP-1 crosstalk

The potential health benefits of phytochemicals in preventing and treating diseases have gained increasing attention. Here, we proved that the methylated isoflavone prunetin possesses a reno-therapeutic effect against renal ischemia/reperfusion (I/R) insult by activating G protein-coupled receptor 30 (GPR30). After choosing the therapeutic dose of prunetin against renal I/R injury in the pilot study, male Sprague Dawley rats were allocated into 5 groups; viz., sham-operated (SO), SO injected with 1 mg/kg prunetin intraperitoneally for three successive days, untreated I/R, I/R treated with prunetin, and I/R treated with G-15, the selective GPR30 blocker, followed by prunetin. Treatment with prunetin reversed the I/R renal injury effect and majorly restored normal renal function and architecture. Mechanistically, prunetin restored the I/R-induced depletion of renal GPR30, an impact that was canceled by the pre-administration of G-15. Additionally, post-administration of prunetin normalized the boosted inflammatory markers indoxyl sulfate, TLR4, and TRIF and abrogated renal cell demise by suppressing necroptotic signaling, verified by the inactivation of p-RIPK1, p-RIPK3, and p-MLKL while normalizing the inhibited caspase-8. Besides, prunetin reversed the I/R-mediated mitochondrial fission by inhibiting the protein expression of PGMA5 and p-DRP-1. All these favorable impacts of prunetin were nullified by G-15. To sum up, prunetin exhibited a significant reno-therapeutic effect evidenced by the enhancement of renal morphology and function, the suppression of the inflammatory cascade indoxyl sulfate/TLR4/TRIF, which turns off the activated/phosphorylated necroptotic trajectory RIPK1/RIPK3/MLKL, while enhancing caspase-8. Additionally, prunetin opposed the mitochondrial fission pathway RIPK3/PGMA5/DRP-1, effects that are mediated via the activation of GPR30.

The Mechanism of Bushen Zhuyun Prescription in Regulating Luteal Phase Deficiency via Intervention of the Kiss1/GPR54 System in the Hypothalamus

Luteal phase deficiency (LPD) is a significant contributor to infertility and miscarriage. Bushen Zhuyun Prescription (BSZY) is a traditional Chinese medicine formula that has potential to treat LPD. To investigate the effect of BSZY in treating LPD, SD rats with complete estrous cycles were divided into blank, model, dydrogesterone (DYD), high-dose BSZY (BSZY-HD), and low-dose BSZY (BSZY-LD) groups. All the groups were received mifepristone gavage except for the blank group. The pathological changes were observed in the hypothalamus, and the analysis of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), gonadotropin-releasing hormone (GnRH), and cyclic adenosine monophosphate (cAMP) levels in serum. Additionally, the mRNA and their protein expression levels of estrogen receptor α (ERα), ERβ, G protein-coupled receptor 30 (GPR30), kisspeptin 1 (Kiss1), and GPR54 were assessed; and the key molecules expression in cAMP/protein kinase A (PKA) pathway were analyzed. The results revealed that mifepristone treatment caused a decrease in FSH and cAMP levels and an increase in E2 and LH levels, which were normalised in the BSZY treatment groups. The expression of ERα and ERβ mRNA and protein in the hypothalamus was higher in the BSZY groups compared to the blank group. BSZY also resulted in increased expression of Kiss-1, GPR54, and PKA, as well as decreased expression of cAMP-response element binding protein (CREB), c-Fos, phospho-CREB (p-CREB), and p-c-Fos, compared to model group. No significant difference observed in GPR30 expression between the mifepristone and BSZY groups. The mRNA and protein of Kiss1 and GPR54 were decreased in model group and increased after BSZY treatment, while the mRNA and protein expression of PKA, CREB, c-Fos, p-CREB, and p-c-Fos were also decreased significantly. In conclusion, the findings demonstrate that BSZY could effectively regulate hypothalamic ER, interfere with the Kiss1/GPR54 signal transduction system, activate the cAMP/PKA pathway, and regulate key transduction molecules expression of PKA, CREB, c-Fos, there by effecting the level of reproductive hormones and has the potential to treat LPD-related infertility.

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.

Predictive value of first-trimester GPR120 levels in gestational diabetes mellitus.

Early diagnosis of gestational diabetes mellitus (GDM) reduces the risk of unfavorable perinatal and maternal consequences. Currently, there are no recognized biomarkers or clinical prediction models for use in clinical practice to diagnosing GDM during early pregnancy. The purpose of this research is to detect the serum G-protein coupled receptor 120 (GPR120) levels during early pregnancy and construct a model for predicting GDM. This prospective cohort study was implemented at the Women's Hospital of Jiangnan University between November 2019 and November 2022. All clinical indicators were assessed at the Hospital Laboratory. GPR120 expression was measured in white blood cells through quantitative PCR. Thereafter, the least absolute shrinkage and selection operator (LASSO) regression analysis technique was employed for optimizing the selection of the variables, while the multivariate logistic regression technique was implemented for constructing the nomogram model to anticipate the risk of GDM. The calibration curve analysis, area under the receiver operating characteristic curve (AUC) analysis, and the decision curve analysis (DCA) were conducted for assessing the performance of the constructed nomogram. Herein, we included a total of 250 pregnant women (125 with GDM). The results showed that the GDM group showed significantly higher GPR120 expression levels in their first trimester compared to the normal pregnancy group (p < 0.05). LASSO and multivariate regression analyses were carried out to construct a GDM nomogram during the first trimester. The indicators used in the nomogram included fasting plasma glucose, total cholesterol, lipoproteins, and GPR120 levels. The nomogram exhibited good performance in the training (AUC 0.996, 95% confidence interval [CI] = 0.989-0.999) and validation sets (AUC=0.992) for predicting GDM. The Akaike Information Criterion of the nomogram was 37.961. The nomogram showed a cutoff value of 0.714 (sensitivity = 0.989; specificity = 0.977). The nomogram displayed good calibration and discrimination, while the DCA was conducted for validating the clinical applicability of the nomogram. The patients in the GDM group showed a high GPR120 expression level during the first trimester. Therefore, GPR120 expression could be used as an effective biomarker for predicting the onset of GDM. The nomogram incorporating GPR120 levels in early pregnancy showed good predictive ability for the onset of GDM.

GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway

The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

GPER1 contributes to T3-induced osteogenesis by mediating glycolysis in osteoblast precursors.

Triiodothyronine (T3) is critical to osteogenesis, which is the key factor in bone growth. Our transcriptomic and metabolomic analysis results indicated that T3 leads to enhanced expression of G protein-coupled estrogen receptor 1 (GPER1) as well as increases in glycolysis metabolite levels. Accordingly, our study aimed to explore the role of GPER1-mediated glycolysis in T3-regulated osteogenesis. The MC3T3-E1 cell line was used as an osteoblast precursor model. After treatment with T3, a GPER1-specific antagonist (G15) and inhibitor of glycolysis (3PO) were used to explore the roles of GPER1 and glycolysis in T3-regulated osteogenesis, as measured by ALP activity, Alizarin red staining intensity and osteogenic molecule expression. Our results showed that T3 promoted osteogenesis-related activity, which was reversed by treatment with G15. In addition, T3 enhanced the glycolytic potential and production of lactic acid (LD) in MC3T3-E1 cells, and treatment with G15 restored the aforementioned effects of T3. Ultimately, the pharmacological inhibition of glycolysis with 3PO blocked the ability of T3 to enhance osteogenic activities. In conclusion, GPER1 mediates glycolysis in osteoblast precursors, which is critical for T3-promoted osteogenesis.

Icariin inhibits hypertrophy by regulation of GPER1 and CaMKII/HDAC4/MEF2C signaling crosstalk in ovariectomized mice

Icariin (ICA), a flavonoid phytoestrogen, was isolated from traditional Chinese medicine Yin Yang Huo (Epimedium brevicornu Maxim.). Previous studies reporting the cardioprotective effects of ICA are available; however, little is known about the impact of ICA on cardioprotection under conditions of reduced estrogen levels. This study aimed to provide detailed information regarding the antihypertrophic effects of ICA in ovariectomized female mice. Female mice were subjected to ovariectomy (OVX) and transverse aortic constriction and then orally treated with ICA at doses of 30, 60 or 120 mg/kg/day for 4 weeks. Morphological assessments, echocardiographic parameters, histological analyses, and immunofluorescence were performed to evaluate cardiac hypertrophy. Cardiomyocytes from mice or rats were stimulated using phenylephrine, and cell surface and hypertrophy markers were tested using immunofluorescence and qPCR. Western blotting, qPCR, and luciferase reporter gene assays were used to assess the expression of proteins and mRNA and further investigate the proteins related to the G-protein coupled estrogen receptor (GPER1) and CaMKII/HDAC4/MEF2C signaling pathways in vivo and in vitro. ICA blocks cardiac hypertrophy induced by pressure overload in OVX mice. Additionally, we demonstrated that ICA activated GPER1 and inhibited the nuclear export or promoted the nuclear import of histone deacetylase 4 (HDAC4) through regulation of phosphorylation of calmodulin-dependent protein kinase II (CaMKII) and further improved the repression of myocyte enhancer factor-2C (MEF2C). ICA ameliorated cardiac hypertrophy in OVX mice by activating GPER1 and inhibiting the CaMKII/HDAC4/MEF2 signaling pathway.

GPER involvement in inflammatory pain

Chronic pain is a worldwide refractory health disease that causes major financial and emotional burdens and that is devastating for individuals and society. One primary source of pain is inflammation. Current treatments for inflammatory pain are weakly effective, although they usually replace analgesics, such as opioids and non-steroidal anti-inflammatory drugs, which display serious side effects. Emerging evidence indicates that the membrane G protein-coupled estrogen receptor (GPER) may play an important role in the regulation of inflammation and pain. Herein, we focus on the consequences of pharmacological and genetic GPER modulation in different animal models of inflammatory pain. We also provide a brief overview of the putative mechanisms including the direct action of GPER on pain transmission and inflammation.

The Role of G Protein-Coupled Estrogen Receptor (GPER) in Vascular Pathology and Physiology.

Estrogen is indispensable in health and disease and mainly functions through its receptors. The protection of the cardiovascular system by estrogen and its receptors has been recognized for decades. Numerous studies with a focus on estrogen and its receptor system have been conducted to elucidate the underlying mechanism. Although nuclear estrogen receptors, including estrogen receptor-α and estrogen receptor-β, have been shown to be classical receptors that mediate genomic effects, studies now show that GPER mainly mediates rapid signaling events as well as transcriptional regulation via binding to estrogen as a membrane receptor. With the discovery of selective synthetic ligands for GPER and the utilization of GPER knockout mice, significant progress has been made in understanding the function of GPER. In this review, the tissue and cellular localizations, endogenous and exogenous ligands, and signaling pathways of GPER are systematically summarized in diverse physiological and diseased conditions. This article further emphasizes the role of GPER in vascular pathology and physiology, focusing on the latest research progress and evidence of GPER as a promising therapeutic target in hypertension, pulmonary hypertension, and atherosclerosis. Thus, selective regulation of GPER by its agonists and antagonists have the potential to be used in clinical practice for treating such diseases.

PJA1 mediates the effects of astrocytic GPR30 on learning and memory in female mice.

Hormone replacement therapy (HRT) is not recommended for treating learning and memory decline in menopausal women because it exerts adverse effects by activating classic estrogen receptors ERα and ERβ. The membrane estrogen receptor G protein-coupled receptor 30 (GPR30) has been reported to be involved in memory modulation; however, the underlying mechanisms are poorly understood. Here, we found that GPR30 deletion in astrocytes, but not in neurons, impaired learning and memory in female mice. Astrocytic GPR30 depletion induced A1 phenotype transition, impairing neuronal function. Further exploration revealed that Praja1 (PJA1), a RING ubiquitin ligase, mediated the effects of astrocytic GPR30 on learning and memory by binding to Serpina3n, which is a molecular marker of neuroinflammation in astrocytes. GPR30 positively modulated PJA1 expression through the CREB signaling pathway in cultured murine and human astrocytes. Additionally, the mRNA levels of GPR30 and PJA1 were reduced in exosomes isolated from postmenopausal women while Serpina3n levels were increased in the plasma. Together, our findings suggest a key role for astrocytic GPR30 in the learning and memory abilities of female mice and identify GPR30/PJA1/Serpina3n as potential therapeutic targets for learning and memory loss in peri- and postmenopausal women.

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.

Differential G Protein-Coupled Estrogen Receptor-1 Regulation of Counter-Regulatory Transmitter Marker and 5′-AMP-Activated Protein Kinase Expression in Ventrolateral versus Dorsomedial Ventromedial Hypothalamic Nucleus

Introduction: The ventromedial hypothalamic nucleus (VMN) is an estrogen receptor (ER)-rich structure that regulates glucostasis. The role of nuclear but not membrane G protein-coupled ER-1 (GPER) in that function has been studied. Methods: Gene silencing and laser-catapult microdissection/immunoblot tools were used to examine whether GPER regulates transmitter and energy sensor function in dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN counter-regulatory nitrergic and γ-Aminobutyric acid (GABA) neurons. Results: Intra-VMN GPER siRNA administration to euglycemic animals did not affect VMNdm or -vl nitrergic neuron nitric oxide synthase (nNOS), but upregulated (VMNdm) or lacked influence on (VMNvl) GABA nerve cell glutamate decarboxylase_65/67 (GAD) protein. Insulin-induced hypoglycemia (IIH) caused GPER knockdown-reversible augmentation of nNOS, 5′-AMP-activated protein kinase (AMPK), and phospho-AMPK proteins in nitrergic neurons in both divisions. IIH had dissimilar effects on VMNvl (unchanged) versus VMNdm (increased) GABAergic neuron GAD levels, yet GPER knockdown affected these profiles. GPER siRNA prevented hypoglycemic upregulation of VMNvl and -dm GABA neuron AMPK without altering pAMPK expression. Conclusions: Outcomes infer that GPER exerts differential control of VMNdm versus -vl GABA transmission during glucostasis and is required for hypoglycemic upregulated nitrergic (VMNdm and -vl) and GABA (VMNdm) signaling. Glycogen metabolism is reported to regulate VMN nNOS and GAD proteins. Data show that GPER limits VMNvl glycogen phosphorylase (GP) protein expression and glycogen buildup during euglycemia but mediates hypoglycemic augmentation of VMNvl GP protein and glycogen content; VMNdm glycogen mass is refractory to GPER control. GPER regulation of VMNvl glycogen metabolism infers that this receptor may govern local counter-regulatory transmission in part by astrocyte metabolic coupling.

2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 μM (vs. 6.79 μM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of β-tubulin proteins in LTED cells than in MCF-7 cells. Among the β-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

GPER1 Activation Exerts Anti-Tumor Activity in Multiple Myeloma.

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

Endocannabinoid System Receptors at the Hip and Stifle Joints of Middle-Aged Dogs: A Novel Target for the Therapeutic Use of Cannabis sativa Extract in Canine Arthropathies.

The endocannabinoid system (ECS) has emerged as a potential therapeutic target in veterinary medicine due to its involvement in a wide range of physiological processes including pain, inflammation, immune function, and neurological function. Modulation of the ECS receptors has been shown to have anti-inflammatory, analgesic, and immunomodulatory effects in various animal models of disease, including dogs with osteoarthritis. The goal of this study was to identify and compare the cellular expression and distribution of cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related G protein-coupled receptor 55 (GPR55) on the synovial cells of hip and stifle joints of seven dogs of different breeds without overt signs of osteoarthritis (OA). The synovial membranes of seven hips and seven stifle joints were harvested post mortem. The expression of the CB1R, CB2R, and GPR55 present in the synovial tissues was investigated using qualitative and quantitative immunofluorescence and Western blot (Wb) analysis. Synoviocytes of the stifle and hip joints expressed CB1R, CB2R, and GPR55 immunoreactivity (IR); no significant differences were observed for each different joint. Cannabinoid receptor 2- and GPR55-IR were also expressed by macrophages, neutrophils, and vascular cells. The ECS receptors were widely expressed by the synovial elements of dogs without overt signs of OA. It suggests that the ECS could be a target for the therapeutic use of Cannabis sativa extract in canine arthropathies.

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.