G Protein-coupled Receptor 30 Expression Research Articles

Joint Inflammation Correlates with Joint GPR30 Expression in Males and Hippocampal GPR30 Expression in Females in a Rat Model of Rheumatoid Arthritis.

It is not entirely clear how the interaction between joint inflammation and the central nervous system (CNS) response in rheumatoid arthritis (RA) works, and what pathophysiology underlies the sex differences in coexisting neuropsychiatric comorbidities. It is known that estrogen hormones reduce inflammation in RA and that this occurs mainly via the stimulation of G protein-coupled receptor-30 (GPR30), also known as G protein-coupled estrogen receptor (GPER) 1. However, changes in GPR30 expression and sex differences induced by local and systemic inflammation in RA are not yet known. Our aim was to reveal sex differences in the expression and association of joint GPR30 with local and systemic inflammation, clinical course and furthermore with hippocampal GPR30 expression during pristane-induced arthritis (PIA) in Dark Agouti (DA) rats, an animal model of RA. Furthermore, we demonstrated sex-specific differences in the association between joint and systemic inflammation and hippocampal microglia during PIA. Our results suggest sex-specific differences not only in the clinical course and serum levels of pro-inflammatory cytokines but also in the expression of GPR30. Female rats show greater synovial inflammation and greater damage to the articular cartilage compared to males during PIA attack. Male rats express higher levels of synovial and cartilaginous GPR30 than females during PIA, which correlates with a less severe clinical course. The correlation between synovial and cartilaginous GPR30 and joint inflammation scores (Krenn and Mankin) in male rats suggests that the more severe the joint inflammation, the higher the GPR30 expression. At the same time, there is no particular upregulation of hippocampal GPR30 in males. On the other hand, female rats express higher levels of neuroprotective GPR30 in the hippocampus than male rats at the basic level and during PIA attack. In addition, females have a higher number of Iba-1+ cells in the hippocampus during PIA attack that strongly correlates with the clinical score, serum levels of IL-17A, and Krenn and Mankin scores. These results suggest that male rats are better protected from inflammation in the joints and female rats are better protected from the inflammation in the hippocampus during a PIA attack, independently of microglia proliferation. However, in the remission phase, synovial GPR30 expression suddenly increases in female rats, as does hippocampal GPR30 expression in males. Further experiments with a longer remission period are needed to investigate the molecular background of these sex differences, as well as microglia phenotype profiling.

G protein-coupled estrogen receptor (GPER)/GPR30 forms a complex with the β1-adrenergic receptor, a membrane-associated guanylate kinase (MAGUK) scaffold protein, and protein kinase A anchoring protein (AKAP) 5 in MCF7 breast cancer cells

G protein-coupled receptor 30 (GPR30), also named G protein-coupled estrogen receptor (GPER), and the β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCR) that are implicated in breast cancer progression. Both receptors contain PSD-95/Discs-large/ZO-1 homology (PDZ) motifs in their C-terminal tails through which they interact in the plasma membrane with membrane-associated guanylate kinase (MAGUK) scaffold proteins, and in turn protein kinase A anchoring protein (AKAP) 5. GPR30 constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. We hypothesized that this inhibition is a consequence of a plasma membrane complex of these receptors. Using co-immunoprecipitation, confocal immunofluorescence microscopy, and bioluminescence resonance energy transfer (BRET), we show that GPR30 and β1AR reside in close proximity in a plasma membrane complex when transiently expressed in HEK293. Deleting the GPR30 C-terminal PDZ motif (-SSAV) does not interfere with the receptor complex, indicating that the complex is not PDZ-dependent. MCF7 breast cancer cells express GPR30, β1AR, MAGUKs, and AKAP5 in the plasma membrane, and co-immunoprecipitation revealed that these proteins exist in close proximity also under native conditions. Furthermore, expression of GPR30 in MCF7 cells constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. AKAP5 also inhibits β1AR-mediated cAMP production, which is not additive with GPR30-promoted inhibition. These results argue that GPR30 and β1AR form a PDZ-independent complex in MCF7 cells through which GPR30 constitutively and PDZ-dependently inhibits β1AR signaling via receptor interaction with MAGUKs and AKAP5.

EQUOL, A SOYBEAN METABOLITE WITH ESTROGEN-LIKE FUNCTIONS, DECREASES LIPOPOLYSACCHARIDE-INDUCED HUMAN NEUTROPHIL EXTRACELLULAR TRAPS IN VITRO.

Objective: Neutrophil extracellular traps (NETs) defend against acute infections. However, their overexpression causes organ failure during sepsis. Control of NET formation may improve the outcomes of patients with sepsis. Equol, a soybean isoflavone, is a female hormone analog, which prevents inflammation. We evaluated the effects of equol on NET formation in human neutrophils during inflammatory stimulation in vitro . Methods: Healthy volunteers provided blood samples. An enzyme-linked immunosorbent assay assessed serum equol concentrations. Neutrophil extracellular trap formation in neutrophils was induced by lipopolysaccharide treatment. Enzyme-linked immunosorbent assay quantified DNA-binding elastase, and immunostaining assessed NET formation. Reverse-transcription quantitative polymerase chain reaction and Western blotting detected G-protein-coupled receptor 30 (GPR30) or peptidyl arginine deiminase 4 (PAD4) expression. Flow cytometry assessed neutrophil phagocytic ability with inactivated Escherichia coli . Results: In neutrophils derived from males with low-serum equol levels (low-serum equol group), equol significantly decreased DNA-binding elastase levels and NET formation. Equol did not decrease NETs in neutrophils from males with high-serum equol levels. GPR30 expression of neutrophils was higher in the low-serum than in the high-serum equol group. PAD4 mRNA levels and nuclear PAD4 protein expression also decreased more than the vehicle control in the low-serum equol group. Equol did not alter the phagocytic ability of neutrophils. In neutrophils from young females, equol had no inhibitory effect on NET formation. Conclusions: Equol decreases lipopolysaccharide-induced NET formation in neutrophils from males via inhibition of PAD4 expression. Our findings provide a rationale for investigating a new therapeutic approach using equol to control neutrophil activity during sepsis.

Sexual dimorphic distribution of G protein-coupled receptor 30 in pain-related regions of the mouse brain.

Sex differences in pain sensitivity have contributed to the fact that medications for curing chronic pain are unsatisfactory. However, the underlying mechanism remains to be elucidated. Brain-derived estrogen participates in modulation of sex differences in pain and related emotion. G protein-coupled receptor 30 (GPR30), identified as a novel estrogen receptor with a different distribution than traditional receptors, has been proved to play a vital role in regulating pain affected by estrogen. However, the contribution of its distribution to sexually dimorphic pain-related behaviors has not been fully explored. In the current study, immunofluorescence assays were applied to mark the neurons expressing GPR30 in male and female mice (in metestrus and proestrus phase) in pain-related brain regions. The neurons that express CaMKIIα or VGAT were also labeled to observe overlap with GPR30. We found that females had more GPR30-positive (GPR30+ ) neurons in the primary somatosensory (S1) and insular cortex (IC) than males. In the lateral habenula (LHb) and the nucleus tractus solitarius (NTS), males had more GPR30+ neurons than females. Moreover, within the LHb, the expression of GPR30 varied with estrous cycle phase; females in metestrus had fewer GPR30+ neurons than those in proestrus. In addition, females had more GPR30+ neurons, which co-expressed CaMKIIα in the medial preoptic nucleus (mPOA) than males, while males had more than females in the basolateral complex of the amygdala (BLA). These findings may partly explain the different modulatory effects of GPR30 in pain and related emotional phenotypes between sexes and provide a basis for comprehension of sexual dimorphism in pain related to estrogen and GPR30, and finally provide new targets for exploiting new treatments of sex-specific pain.

Inhibition of GPR30 sensitized gefitinib to NSCLC cells via regulation of epithelial-mesenchymal transition.

Introduction: G-protein coupled receptor 30 (GPR30) is associated with cell metastasis and drug resistance in many different cancer cells. The present study aimed to reveal the sensitivity of GPR30 to gefitinib in non-small cell lung cancer (NSCLC) cells.Methods: Cell viability and proliferation were detected using cell counting kit 8 and 5-ethynyl-2'-deoxyuridine assays, respectively. Western blotting and quantitative real-time reverse transcription PCR were used to detect GPR30 or epithelial-mesenchyme transition (EMT)-related mRNA and protein expression.Results: The results showed that GPR30 expression is associated with gefitinib sensitivity. G15, as a GPR30 antagonist, reduced GPR30 expression. We chose the maximum concentration of G15 with minimal cytotoxicity to detect cell viability after combined treatment with gefitinib in NSCLC cells, which indicated that G15 could increase sensitivity to gefitinib. However, the effect of G15 on gefitinib sensitivity disappeared after treatment with a small interfering RNA targeting GPR30. Further research showed that G15 or GPR30 siRNA treatment could upregulate E-cadherin and downregulate vimentin levels.Conclusion: Taken together, these data suggested that G15 could enhance NSCLC sensitivity to gefitinib by inhibition of GPR30 and EMT.

Protective role of estrogen through G-protein coupled receptor 30 in a colitis mouse model.

Estrogen and its receptors are involved in the pathogenesis of gastrointestinal diseases such as colitis. However, the role of the membrane estrogen receptor G-protein-coupled receptor 30 (GPR30) in colitis is poorly understood. We therefore investigated the effect of estrogen in dextran sulfate sodium (DSS)-induced colitis. Male C57BL/6 mice were administered 1.5% DSS for 5days and treated with 17β-estradiol (E2), GPR30 agonist (G1), or GPR30 antagonist (G15) for 8days. Inflammation grade was evaluated by disease activity index (DAI) and histomorphological score. Colon tissues were immunohistochemically analyzed and revealed high expression of membrane GPR30, histone 3 lysine 36 dimethylation, and lysine 79 trimethylation in normal mouse colon epithelial cells but significantly decreased expression in DSS-treated mice, whereas the expression was partially preserved after treatment with E2 or G1. Colon shortening and DAI were significantly lower in E2- and G1-treated mice compared to DSS-treated mice. Caudal type homeobox 2 (CDX2) expression and cell proliferation differed in normal colon epithelial cells but overlapped in those of DSS-treated mice. Administration of E2 and G1 reduced CDX2 expression and cell proliferation. Altered expression of claudin-2 and occludin were observed in the colonic epithelium of DSS-treated mice, and these changes were significantly lower in the colon of E2- and G1-treated mice. These results indicate that estrogen regulates histone modification, cell proliferation, and CDX2 expression through GPR30, which affects intestinal epithelial barrier function. We conclude that estrogen protects against intestinal epithelial damage through GPR30 by enhancing intestinal epithelial barrier function in DSS-induced colitis in mice.

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.

Environmental-relevant bisphenol A exposure promotes ovarian cancer stemness by regulating microRNA biogenesis.

Bisphenol A (BPA) is a ubiquitous environmental xenobiotic impacting millions of people worldwide. BPA has long been proposed to promote ovarian carcinogenesis, but the detrimental mechanistic target remains unclear. Cancer stem cells (CSCs) are considered as the trigger of tumour initiation and progression. Here, we show for the first time that nanomolar (environmentally relevant) concentration of BPA can markedly increase the formation and expansion of ovarian CSCs concomitant. This effect is observed in both oestrogen receptor (ER)-positive and ER-defective ovarian cancer cells, suggesting that is independent of the classical ERs. Rather, the signal is mediated through alternative ER G-protein-coupled receptor 30 (GPR30), but not oestrogen-related receptor α and γ. Moreover, we report a novel role of BPA in the regulation of Exportin-5 that led to dysregulation of microRNA biogenesis through miR-21. The use of GPR30 siRNA or antagonist to inhibit GPR30 expression or activity, respectively, resulted in significant inhibition of ovarian CSCs. Similarly, the CSCs phenotype can be reversed by expression of Exportin-5 siRNA. These results identify for the first time non-classical ER and microRNA dysregulation as novel mediators of low, physiological levels of BPA function in CSCs that may underlie its significant tumour-promoting properties in ovarian cancer.

Transcription factor Krüppel-like factor 4 upregulated G protein-coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia-reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury is a common clinical event occurring during multiple clinical pathological processes. Here, we designed this paper to discuss the role of G protein-coupled receptor 30 (GPR30) playing in intestinal I/R injury. An oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate the pathological process of I/R injury. With the application of enzyme-linked immunosorbent assay, TUNEL, and transepithelial electrical resistance (TEER) assays, the levels of inflammatory cytokines, cell apoptosis, and intestinal integrity were estimated. The corresponding proteins were estimated by applying western blot. Immunofluorescence was conducted to examine N-terminal Gasdermin D (GSDMD-N) expression. The interplay between KLF4 and GPR30 was demonstrated by dual-luciferase reporter assay and chromatin immunoprecipitation. The results showed that GPR30 was downregulated in Caco-2 cells exposed to OGD/R. GPR30 overexpression reduced the production of TNF-α, IL-6, IL-1β, and IL-18, the TUNEL-positive cells, as well as the contents of p-p65, Cox-2, Inos, Bax, and cleaved-PARP, but elevated the expression of Bcl-2 in OGD/R-induced Caco-2 cells. In addition, OGD/R-induced the reduction of TEER value and reduced expression of tight junction proteins in Caco-2 cells, which was partially restored by GPR30 overexpression. Furthermore, GPR30 suppressed nod-like receptor pyrin 3inflammasome and GSDMD-N expression. It was evidenced that Krüppel-like factor 4 (KLF4) could directly bind to GPR30 promoter and positively regulate GPR30 expression. The regulation of GPR30 overexpression above was weakened by KLF4 knockdown. Collectively, our findings suggested that KLF4 could transcriptionally upregulate GPR30, and GPR30 prevented intestine I/R injury by inhibiting inflammation and apoptosis, and maintaining intestinal integrity that provides potential targets for mitigating the I/R injury.

GPR30 knockdown weakens the capacity of CAF in promoting prostate cancer cell invasion via reducing macrophage infiltration and M2 polarization.

Cancer-associated fibroblasts (CAFs) can promote the development and metastasis of prostate cancerpartly by mediating tumor-associated inflammation. An increasing amount of studies have focused on the functional interactions between CAFs and immune cells in the tumor microenvironment (TME). We previously reported that G protein-coupled receptor 30 (GPR30) was highly expressed in prostate CAFs and plays a crucial rolein prostate stromal cell activation. However, the effect and underlying mechanism of GPR30 expression in prostate CAFs affecting the interaction between CAFs and tumor-associated macrophages (TAMs) need further elucidation. Here, we found that, compared with CAF-shControl, CAF-shGPR30 inhibited macrophagemigration through transwell migration assays, which should be attributed to the decreased expression of C-X-C motif chemokine ligand 12 (CXCL12). In addition, macrophages treated with a culture medium of CAF-shGPR30 exhibited attenuated M2 polarization with downregulated M2-like markers expression. Moreover, macrophages stimulated with a culture medium of CAF-shGPR30 were less efficient in promoting activation of fibroblast cells and invasion of PCa cells. Finally, cocultured CAF-shGPR30 and macrophages suppressed PCa cell invasion compared to cocultured CAF-shControl and macrophages by decreasing interleukin-6 (IL-6) secretion, and this effect could be abrogated with rescue expression of IL-6. Our results pinpoint the function of GPR30 in prostate CAFs on regulating the CAF-TAM interaction in the TME and provide new insights into PCa therapies via regulating TME.

Downregulated GPR30 expression in the epileptogenic foci of female patients with focal cortical dysplasia type IIb and tuberous sclerosis complex is correlated with 18 F-FDG PET-CT values.

Focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) are typical causes of developmental delay and refractory epilepsy. G‐protein‐coupled receptor 30 (GPR30) is a specific estrogen receptor that is critical in neurodevelopment, neuroinflammation, and neuronal excitability, suggesting that it plays a potential role in the epilepsy of patients with FCDIIb and TSC. Therefore, we investigated the role of GPR30 in patients with FCDIIb and TSC. We found that the expression of GPR30 and its downstream protein kinase A (PKA) pathway were decreased and negatively correlated with seizure frequency in female patients with FCDIIb and TSC, but not in male patients. GPR30 was widely distributed in neurons, astrocytes, and microglia, and its downregulation was especially notable in microglia. The GPR30 agonist G‐1 increased the expression of PKA and p‐PKA in cultured cortical neurons, and the GPR30 antagonist G‐15 exhibited the opposite effects of G‐1. The NF‐κB signaling pathway was also activated in the specimens of female patients with FCDIIb and TSC, and was regulated by G‐1 and G‐15 in cultured cortical neurons. We also found that GPR30 regulated cortical neuronal excitability by altering the frequency of spontaneous excitatory postsynaptic currents and the expression of NR2A/B. Further, the relationship between GPR30 and glycometabolism was evaluated by analyzing the correlations between GPR30 and 18F‐FDG PET‐CT values (standardized uptake values, SUVs). Positive correlations between GPR30 and SUVs were found in female patients, but not in male patients. Intriguingly, GPR30 expression and SUVs were significantly decreased in the epileptogenic tubers of female TSC patients, and ROC curves indicated that SUVs could predict the localization of epileptogenic tubers. Taken together, our results suggest a potential protective effect of GPR30 in the epileptogenesis of female patients with FCDIIb and TSC.

G protein-coupled receptor 30 activation protects hepatic ischemia-reperfusion injury of liver tissue through inhibiting NLRP3 in the rat model

ABSTRACT One of the most prominent characteristics of hepatic ischemia-reperfusion injury (HI/R) is an intense inflammatory reaction, which plays a key role in inflammatory injury induced by ischemia-reperfusion. Nucleotide-binding oligomerization domain-containing protein (NOD-), leucine-rich repeat (LRR), and pyrin domains-containing protein 3 (NLRP3) are involved in the inflammatory injury of ischemia-reperfusion as an important pattern recognition receptor for innate immunity. G protein-coupled receptor 30 (GPR30) is a newly identified as 7-transmembrane G protein-coupled receptor and can be activated by many stimulations including estrogen. The current study aims to explore whether GPR30 agonist (G1) can alleviate hepatic ischemia-reperfusion injury HI/R by inhibiting NLRP3. An induced HI/R rat model was generated, blood and liver samples were gathered and subjected to histological examination, biochemical assays, Western blot assays, and qRT-PCR. Our results indicated GPR30 agonist (G1) pretreatment or NLRP3 silencing significantly decreased the serum levels of Interleukin 1β (IL-1β), alanine aminotransferase (ALT) and aspartate aminotransferase, improved histological alterations and hepatocyte apoptosis. Moreover, G1 pretreatment or NLRP3 silencing downregulated the protein level of Caspase-1 and pro-Interleukin 1β (pro-IL-1β) while G1 pretreatment upregulated the expression of GPR30 (p < 0.05). In conclusion, the salutary effects of GPR30 agonists on HI/R are mediated at least in part through downregulating NLRP3 expression. GPR30 may be used as a therapy target of HI/R.

Daidzein stimulates fatty acid-induced fat deposition in C2C12 myoblast cells via the G protein-coupled receptor 30 pathway

Fat deposition in skeletal muscle is an important aspect of improving meat quality. Isoflavones can promote animal anabolism, but whether and how they regulate muscle fat deposition remain largely unclear. In this study, we explored the role and corresponding molecular mechanism of one of the major isoflavones, daidzein, in fat deposition in C2C12 myoblast cells. In the absence of fatty acids (FAs), daidzein did not promote triglyceride synthesis and lipid droplet formation in cells but increased sterol regulatory element-binding protein 1c (SREBP-1c) expression and maturation. In the presence of FAs, daidzein enhanced FAs-induced fat deposition and the SREBP-1c signaling. Daidzein promoted FAs-induced nuclear factor κB1 (NFκB1) phosphorylation and activated the SREBP-1c signaling in a PI3K-dependent manner. G protein-coupled receptor 30 (GPR30) knockdown but not estrogen receptor α (ERα) knockdown blocked the stimulation of daidzein on the PI3K-NFκB1-SREBP-1c signaling pathway, while both knockdown did not affect the stimulation of FAs on this signaling. qRT-PCR and ChIP-qPCR further detected that daidzein stimulated NFκB1-targeted SREBP-1c transcription. Daidzein did not affect ERα expression in cells, but it stimulated GPR30 expression and cytoplasmic localization. These results reveal that daidzein promotes FAs-induced fat deposition through the GPR30 signaling in C2C12 myoblast cells.

GPR30 Promotes the Phenotypic Switching of Vascular Smooth Muscle Cells via Activating the AKT and ERK Pathways.

BackgroundLower extremity varicose veins (LEVVs) are a common venous disorder of venous dilation and tortuosity. The functional integrity of vascular smooth muscle cells (VSMCs), the majority of the cells in venous tissues, and their phenotypic differences play important roles in the occurrence and development of LEVV. However, the underlying mechanism remains unclear.MethodsThe expression of estrogen receptors ERα and ERβ and G-protein-coupled receptor 30 (GPR30) in LEVV tissues and the role of GPR30 in VSMC phenotypic switching were examined by Western blotting and quantitative real-time PCR. Finally, the related mechanisms underlying LEVVs were explored by Western blotting.ResultsThe serum estradiol content was increased in LEVV patients compared with normal control patients, but the mRNA levels of ERα and ERβ were not significantly different. GPR30 was overexpressed in LEVVs, and high expression of GPR30 promoted the maintenance of a synthetic phenotype in which OPN, MMP-1 and MMP-9 were highly expressed and α-SMA was poorly expressed in VSMCs. Finally, the mechanism by which GPR30 promotes the phenotypic switching of VSMCs is dependent on the ERK1/2 and AKT pathways.ConclusionGPR30 may contribute to the pathogenesis of LEVVs by promoting the maintenance of a synthetic phenotype in VSMCs by activating the ERK1/2 and AKT pathways, and GPR30 might be a novel therapeutic target for clinical LEVV treatment.

Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer.

BackgroundG protein-coupled estrogen receptor (GPER), or G protein-coupled receptor 30 (GPR30), is reported to mediate non-genomic estrogen signaling. GPR30 associates with breast cancer (BC) outcome and may contribute to tamoxifen resistance. We investigated the expression and prognostic significance of GPR30 in metachronous contralateral breast cancer (CBC) as a model of tamoxifen resistance.MethodsTotal GPR30 expression (GPR30TOT) and plasma membrane-localized GPR30 expression (GPR30PM) were analyzed by immunohistochemistry in primary (BC1; nBC1 = 559) and contralateral BC (BC2; nBC2 = 595), and in lymph node metastases (LGL; nLGL1 = 213; nLGL2 = 196). Death from BC (BCD), including BC death or death after documented distant metastasis, was used as primary end-point.ResultsGPR30PM in BC2 and LGL2 were associated with increased risk of BCD (HRBC2 = 1.7, p = 0.03; HRLGL2 = 2.0; p = 0.02). In BC1 and BC2, GPR30PM associated with estrogen receptor (ER)-negativity (pBC1<0.0001; pBC2<0.0001) and progesterone receptor (PR)-negativity (pBC1 = 0.0007; pBC2<0.0001). The highest GPR30TOT and GPR30PM were observed in triple-negative BC. GPR30PM associated with high Ki67 staining in BC1 (p<0.0001) and BC2 (p<0.0001). GPR30TOT in BC2 did not associate with tamoxifen treatment for BC1. However, BC2 that were diagnosed during tamoxifen treatment were more likely to express GPR30PM than BC2 diagnosed after treatment completion (p = 0.01). Furthermore, a trend was observed that patients with GPR30PM in an ER-positive BC2 had greater benefit from tamoxifen treatment.ConclusionPM-localized GPR30 staining is associated with increased risk of BC death when expressed in BC2 and LGL2. Additionally, PM-localized GPR30 correlates with prognostic markers of worse outcome, such as high Ki67 and a triple-negative subtype. Therefore, PM-localized GPR30 may be an interesting new target for therapeutic exploitation. We found no clear evidence that total GPR30 expression is affected by tamoxifen exposure during development of metachronous CBC, or that GPR30 contributes to tamoxifen resistance.

GPR30 expression and function in breast cancer cells are induced through a cis‑acting element targeted by ETS factors.

The capacity that G protein‑coupled receptor30 (GPR30) has demonstrated for triggering estrogen‑dependent signaling pathways has attracted the interest of breast cancer researchers; however, the reported expression profiles and functions of GPR30 in breast cancer are inconsistent. The main purpose of the present investigation was to identify transcriptional mechanisms underlying the expression of GPR30 that allow a better understanding of its role in breast cancer progression. In the cell lines used as different polarity models in the present study, it was determined immunologically that GPR30 is expressed in normal mammary gland cells and that this expression decreased considerably during breast cancer development, where cell identity is lost. However, it was also determined that, in spite of low GPR30 expression levels in breast cancer cells with little differentiation, this membrane estrogen receptor (ER) is able to increase cell viability and suppress migration in cells that have acquired metastatic capacity. In addition, through transient expression assays in breast cancer cells, it was revealed that a transcriptional mechanism dependent on protein kinase A and susceptible to retinoic acid in ER‑positive cells induces GPR30 expression through a cis‑regulatory element for E26 transformation‑specific transcription factors, located between ‑631 and ‑625bp from the GPR30 translation start codon. Overall, these results suggested that invitro transcriptional regulation of GPR30 expression in breast cancer cells may serve a relevant role in the conservation of an epithelial phenotype, and also may be important to avoid the transition to metastasis.

G Protein-Coupled Receptor 30 (GPR30) Expression Pattern in Inflammatory Bowel Disease Patients Suggests its Key Role in the Inflammatory Process. A Preliminary Study.

G protein-coupled receptor 30 (GPR30) is a recently de-orphanized estrogen receptor that mediates the effects of estrogens on different cells. It has been postulated that in inflammatory bowel diseases (IBD) activation of GPR30 blocks the pathways dependent on pro-inflammatory cytokines. The aim of our study was to investigate GPR30 expression in patients with IBD and its potential implication in future therapies. Fifty-seven patients were enrolled in our study: 20 subjects with Crohn's disease (CD), 22 with ulcerative colitis (UC) and 15 controls. In each subject, biopsies were taken from various left-colonic locations. Gene and protein expression of GPR30 was quantified using real time RT-PCR or Western blot. GPR30 mRNA and protein expression were detected in all tested colonic tissues. No significant differences in GPR30 gene expression were observed. In non-inflamed areas, GPR30 protein was strongly increased in CD patients, but moderately in UC patients (p= 0.014 and p=0.143, respectively, vs. controls). In CD patients, a significantly lower GPR30 protein content in inflamed than in non-inflamed tissue was observed (p=0.039). The change was independent of patient gender. Our observations indicate that GPR30 may play a role in the development and progression of inflammatory lesions in IBD, thus affecting disease severity, and consequently IBD treatment. Therefore, GPR30 may become an attractive target for novel anti-IBD drugs, particularly in CD.

Androgen receptor, aromatase, oestrogen receptor α/β and G protein-coupled receptor 30 expression in the testes and epididymides of adult sheep.

The androgen receptor (AR) plays a key role in reproduction, and aromatase (P450arom), nuclear oestrogen receptors (ERs) α and β, and G protein-coupled receptor 30 (GPR30) are important for testicular and epididymal cell proliferation and development. In the study, we have investigated the expression and localization of AR, P450arom, ERα, ERβ and GPR30 in testes and epididymides of sexually mature sheep by quantitative reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. The results demonstrate that the AR, P450arom and ERα levels in the caput and corpus epididymis were significantly lower than those in the testis and cauda epididymis (p<.05), the ERβ level in the testis was significantly higher than in the caput, corpus and cauda epididymis (p<.05), and the GPR30 level in the caput epididymis was significantly lower than in the testis and corpus and cauda epididymis (p<.05). These receptors were mainly detected in epididymal epithelial, basal, smoothmuscle, Sertoli and Leydig cells, as well as in spermatozoa. Taken together, the results suggest that sheep epididymides and testes have the potential for estradiol synthesis and are the targets of both androgens and estradiol. These results provide a foundation for further studies on the mechanisms of androgens and estradiol signalling in the testes and epididymides of sheep.

Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target.

BackgroundBRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1.MethodsExpression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays.ResultsThe expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G2/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression.ConclusionsLack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.

Postnatal Expression Patterns of Estrogen Receptor Subtypes and Choline Acetyltransferase in Different Regions of the Papez Circuit

The Papez circuit is crucial for several brain functions, including long-term memory and emotion. Estradiol modulates cognitive functions based on the expression pattern of its receptor subtypes including estrogen receptor (ER) α, β, and G protein-coupled receptor 30 (GPR30). Similarly, the activity in the cholinergic system correlates with several brain functions, such as learning and memory. In this study, we used immunofluorescence to examine the expression patterns of ERβ and Western blotting to analyze GPR30 and choline acetyltransferase (ChAT) expression, in different regions of the Papez circuit, including the prefrontal cortex, hippocampus, hypothalamus, anterior nucleus of the thalamus, and cingulum in female rats at postnatal days (PND) 1, 10, and 56. Our main finding was that the highest expression of ERβ and GPR30 was noted in each brain area of the Papez circuit in the PND1 rats, whereas the expression of ChAT was the highest in PND10 rats. These results provide vital information on the postnatal expression patterns of ER subtypes and ChAT in different regions of the Papez circuit.