G Protein-coupled Receptor 30 mRNA Research Articles

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.

Downregulation of G‑protein‑coupled receptor 30 in the hippocampus attenuates the neuroprotection of estrogen in the critical period hypothesis.

The aim of the present study was to investigate the role of G-protein-coupled receptor 30 (GPR30) in long-term 17β-estradiol (E2) deprivation (LTED) in a rat model with global cerebral ischemia (GCI), and its therapeutic target for ischemic stroke in the clinical setting. Following bilateral ovariectomy, GCI was induced in rats 1 or 10 weeks post-surgery. To determine the protein and mRNA expression levels of GPR30 in the hippocampal CA1 region of LTED rats, short-term E2 deprivation (STED) rats and naturally aging rats, western blot analysis and reverse transcription-quantitative polymerase chain reaction were performed. The results of the present study demonstrated that E2 treatment revealed significant neuroprotection post-GCI in STED rats, but not in LTED rats, as well as a decrease in the expression levels of GPR30 in the hippocampal CA1 region. In LTED rats,. Notably, no effects were observed on the ubiquitination of GPR30 following investigation in STED or LTED rats. While the protein and mRNA expression levels of GPR30 were also decreased in the hippocampal CA1 region of female 24-month-old rats compared with 3-month-old rats. E2 treatment initiated for the entire ovariectomy period elevated GPR30 mRNA and protein expression levels, and attenuated the loss of hippocampal neurons in the GCI-induced CA1 region, indicating that E2 treatment exerted robust neuroprotection within LTED rats. However, the neuroprotective effect of E2 may be blocked by G15. The results of the present study revealed that downregulation of GPR30 expression may attenuate the neuroprotection of E2 within LTED conditions in rats post-ovariectomy by leading to neuronal insensitivity to E2 neuroprotection following cerebral ischemia. These results provide evidence that GPR30 may have potential as a novel therapeutic target for the treatment of clinical ischemic stroke.

Relative abundance of G protein-coupled receptor 30 and localization in testis and epididymis of sheep at different developmental stages

The G protein-coupled receptor 30 (GPR30) is a transmembrane estrogen receptor that binds to estrogen, and has been confirmed to have an important role in testicular cell proliferation and development. The main objective of this study was to examine GPR30 gene expression and localization in the testis and epididymis of sheep at different developmental stages. Testes, including the epididymis, were collected from Polled Dorset x Mongolian cross rams at one (n=4; wt), three (n=4; wt), six (n=4; wt), nine (n=4; wt) and 12 (n=4; wt) months of age. The 12-month-old hybrid crossbred sheep were exsanguinated via puncture of the jugular vein. Relative abundance of GPR30 mRNA was detected by quantitative PCR, and localization of the protein was examined by immunohistochemistry. Semi-quantitative analysis of GPR30 protein was performed by western blotting. The relative abundance of GPR30 mRNA was similar in the epididymis tail for rams at 6, 9, and 12mo of age. Further, relative abundance of GPR30 mRNA in the testes and caput epididymis of 1-, 3-, 6-, 9-, and 12-month-old crossbred rams did not increase with age. The GPR30 mRNA was detected in epididymal interstitial and principal cells, and in the epididymal cavity, spermatocytes, spermatogonial stem cells, Sertoli and Leydig cells, and spermatozoon of ram testes. Western blotting indicated the GPR30 protein was present in 9- and 12-month-old crossbred sheep corpus, cauda epididymis (P<0.05). The results suggest that relative abundance of GPR30 mRNA is time-dependent and localization-specific.

Expression of Estrogen Receptors Alpha (ER-α), Beta (ER-β), and G Protein-Coupled Receptor 30 (GPR30) in Testicular Tissue of Men with Klinefelter Syndrome.

Men with Klinefelter syndrome (KS) present severe hormonal dysregulation, particularly elevated serum estradiol concentration. Estrogens act through specific receptors and regulate testes development and spermatogenesis. Herein, we evaluated GPR30, ERα, and ERβ mRNA expression in testis of KS men and men with 46XY karyotype by reverse transcriptase and quantitative PCR. ERβ transcripts are the most abundant in testicular tissue of 46XY men. Notably, testicular GPR30 transcription in KS men was approximately 12 times higher. Since GPR30 is essential to mediate estrogen effects over steroidogenesis, our data illustrate that GPR30 may underpin the testicular alterations observed in KS men.

Chaihu-Shugan-San Administration Ameliorates Perimenopausal Anxiety and Depression in Rats

Chaihu-Shugan-San (CSS) is a traditional Chinese herbal formula that is widely used for treating perimenopausal symptoms in China; however, its mechanisms remain unknown. The present study was designed to investigate potential CSS mechanisms in rats with unpredicted chronic mild stress (UCMS) and normally aging rats (52 weeks of age). We performed the sucrose consumption test along with the forced swimming test to confirm depression-like behavior and the open field test (OFT) to confirm anxiety-like behavior in the animals. In addition, we used an enzyme-linked immunosorbent assay to measure serum and hippocampal estradiol (E2) levels and a quantitative real-time polymerase chain reaction to assess hippocampal mRNA levels of estrogen receptors (ERs) α and β as well as G protein-coupled receptor 30 (GPR30). We found that CSS administration resulted in a significant increase in the ratio of hippocampal ERα and ERβ mRNA (ERα/ERβ ratio) in UCMS rats (p<0.001). However, no significant changes were observed in E2 levels, ERα mRNA expression, and GPR30 mRNA expression. In contrast, changes in ERα/ERβ mRNA ratio were sensitively associated with changes in mood states in the animal models. These findings suggest that enhancement of ERα/ERβ ratio may play a role in the pharmacological mechanisms of CSS. Furthermore, this ratio can be employed as a potential index for evaluating mood states in animal models and can be considered as a therapeutic target for perimenopausal anxiety and depression in the future.

Expression of estradiol receptor, GPR30, in bovine anterior pituitary and effects of GPR30 agonist on GnRH-induced LH secretion

G-protein – coupled receptor 30 (GPR30) is an estradiol receptor located on the plasma membrane, and it initiates several rapid, non-genomic signaling events. GPR30 has recently been identified in rat anterior pituitary (AP); however, little is known about the role of GPR30 in controlling luteinizing hormone (LH) secretion from gonadotropes in animals. To fill this research gap, we hypothesized that GPR30 is expressed in bovine AP and mediates estradiol inhibition of gonadotropin-releasing hormone (GnRH)-induced LH release. We confirmed the expressions of GPR30 mRNA and protein by RT-PCR, western blotting, and immunohistochemistry. We cultured bovine AP cells (n=8) for 3 days in steroid-free conditions and then treated them with increasing concentrations (0.001nM, 0.01nM, 0.1nM, 1nM, and 10nM) of estradiol or a GPR30-specific agonist, G1, for 5min before GnRH stimulation. As expected, estradiol at 0.001–0.1nM inhibited the GnRH-stimulated LH secretion. However, we found also that G1 at 0.001nM was able to inhibit this secretion (P<0.05). In contrast, both estradiol and G1 at higher doses were less efficient in suppressing the GnRH-stimulated LH secretion. Neither estradiol nor G1 suppressed GnRH-stimulated follicle-stimulating hormone secretion. In separate experiments, fluorescent immunohistochemistry and immunocytochemistry revealed that approximately 50% of GPR30-positive cells express LH, and about 30% of LH-positive cells express GPR30. In conclusion, GPR30 is expressed in bovine gonadotropes and other AP cells and may partially contribute to rapid negative estradiol feedback of GnRH-induced LH secretion.

Insulinotropic and Antidiabetic Effects of 17β-Estradiol and the GPR30 Agonist G-1 on Human Pancreatic Islets

We have recently shown that 17β-estradiol (E2) and the synthetic G protein-coupled receptor 30 (GPR30) ligand G-1 have antiapoptotic actions in mouse pancreatic islets, raising the prospect that they might exert beneficial effects also in human islets. The objective of the present study was to identify the expression of GPR30 in human islets and clarify the role of GPR30 in islet hormone secretion and β-cell survival. GPR30 expression was analyzed by confocal microscopy, Western blot, and quantitative PCR in islets from female and male donors. Hormone secretion, phosphatidylinositol hydrolysis, cAMP content, and caspase-3 activity in female islets were determined with conventional methods and apoptosis with the annexin-V method. Confocal microscopy revealed GPR30 expression in islet insulin, glucagon, and somatostatin cells. GPR30 mRNA and protein expression was markedly higher in female vs. male islets. An amplifying effect of G-1 or E2 on cAMP content and insulin secretion from isolated female islets was not influenced by the E2 genomic receptor (ERα and ERβ) antagonists ICI 182,780 and EM-652. Cytokine-induced (IL-1β plus TNFα plus interferon-γ) apoptosis in islets cultured for 24 h at 5 mmol/liter glucose was almost abolished by G-1 or E2 treatment and was not affected by the nuclear estrogen receptor antagonists. Concentration-response studies on female islets from healthy controls and type 2 diabetic subjects showed that both E2 and G-1 displayed important antidiabetic actions by improving glucose-stimulated insulin release while suppressing glucagon and somatostatin secretion. In view of these findings, we propose that small molecules activating GPR30 could be promising in the therapy of diabetes mellitus.

G-1-Activated Membrane Estrogen Receptors Mediate Increased Contractility of the Human Myometrium

Estrogens are key mediators of increased uterine contractility at labor. We sought to determine whether membrane-associated estrogen receptors, such as the recently described seven-transmembrane receptor G protein-coupled receptor 30 (GPR30), mediated some of this effect. Using human myometrium obtained at term cesarean section before or after the onset of labor, we demonstrated the presence of GPR30 mRNA and protein using quantitative RT-PCR and Western blotting. GPR30 receptor was localized to the cell membrane and often colocalized with calveolin-1. Using the specific estrogen membrane receptor agonist G-1 and myometrial explants, we showed that membrane receptor activation led to phosphorylation of MAPK and the actin-modifying small heat shock protein 27. Using myometrial strips incubated with G-1 or vehicle we demonstrated that estrogen membrane receptor activation increased the myometrial contractile response to oxytocin. These data suggest that activation of the plasma membrane estrogen receptor GPR30 likely participates in the physiology of the human myometrium during pregnancy and identifies it as a potential target to modify uterine activity.

Involvement of estradiol-17β and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20β-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17β, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary

Estradiol-17beta (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation.

Expression of G Protein-Coupled Receptor 30 in the Hamster Ovary: Differential Regulation by Gonadotropins and Steroid Hormones

The nongenomic actions of estradiol-17beta are mediated by transmembrane estrogen receptors. Recently, G protein-coupled receptor 30 (GPR30) has been suggested to be a transmembrane estrogen receptor that can mediate rapid and transcription-independent estradiol-17beta signaling in different cell types. However, the expression, regulation, or biological relevance of GPR30 in the ovary remains unknown. We examined the expression and hormonal regulation of GPR30 mRNA and protein in hamster ovarian cells during the estrous cycle and after hypophysectomy and hormone replacement. GPR30 protein expression was high in the theca, appreciable in the granulosa, but low in luteal cells. GPR30 protein levels in granulosa and theca cells increased steadily with the development of preantral and antral follicles, respectively. GPR30 mRNA and protein levels increased significantly on diestrous (d 3 of the estrous cycle), but decreased on d 4 at 1600 h after the LH surge. GPR30 mRNA levels increased significantly after hypophysectomy. Although steroid treatment failed to alter ovarian GPR30 mRNA levels, either FSH or LH effectively reduced the levels. Interestingly, the decrease in GPR30 mRNA corresponded to a marked increase in the receptor protein levels. FSH treatment, either alone or together with LH, resulted in a marked increase in GPR30 immunostaining in granulosa cells. LH alone significantly increased immunostaining in theca cells. These results suggest that GPR30 is expressed in the membrane of hamster granulosa and theca cells, and the expression is regulated by gonadotropins. The unique pattern of GPR30 expression suggests that gonadotropin-regulated follicular cell functions may involve GPR30 activity.

The Interactions Between GPR30 and the Major Biomarkers in Infiltrating Ductal Carcinoma of the Breast in an Asian Population

G-protein-coupled receptor 30 (GPR30) has been reported to be a novel estrogen receptor alpha (ERalpha) in vitro. Therefore, the interactions among GPR30, ERalpha, progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2/neu), and their prognostic utilities in the infiltrating ductal carcinoma (IDC) of the breast were evaluated. Messenger RNA (mRNA) levels of GPR30, ERalpha, PR and HER-2/neu in the tumor samples of 118 Taiwanese IDC patients and 27 non-tumor mammary tissues were measured via quantitative polymerase chain reaction analyses. The correlations of GPR30 mRNA levels with clinical parameters, i.e. tumor/non-tumor, ERalpha, PR, HER-2/neu, age, lymph node metastasis, lymph-vascular invasion, grade, stage and patient survival, were assessed by using appropriate statistical analyses. GPR30 expression was observed to be lower in IDC (p < 0.001) than in non-tumor mammary tissues. Importantly, GPR30 mRNA level was positively correlated with that of ERalpha (p = 0.001) and PR (p = 0.001) but not correlated with that of HER-2/neu when they were analyzed as continuous variables. However, lower GPR30 was noticed in tumors with HER-2/neu protein overexpression. GPR30 expression was not correlated with age, lymph node metastasis, lymph-vascular invasion, grade and stage in IDC. GPR30 expression was not an independent prognostic factor for patient survival. GPR30 expression is downregulated in IDC. GPR30 is preferentially co-expressed with ER and/or PR but is lowly expressed in HER-2/neu(+) tumors. The correlation of GPR30 expression with clinical parameters, including patient survival, was not evident in this cohort.

Cloning of novel LERGU mRNAs in GPR30 3′ untranslated region and detection of 2 bp‐deletion polymorphism in gastric cancer

The improved IGCR (In-Gel Competitive Reassociation) method was applied to the analysis of human gastric cancer genomic DNA to identify its alterations, and it appeared that the IGCR library contained a fragment of 3'-untranslated region (3' UTR) of G-protein coupled receptor 30 (GPR30) mRNA. When we searched genomic DNA pairs of gastric cancer patients with this IGCR clone, we found the deletion polymorphism with or without 2 bp (Cytosine and Thymine; CT). We confirmed the existence of a novel mRNA in GPR30 3'UTR by northern blotting, cloned this novel mRNA and named it Leucine Rich Protein in GPR30 3'UTR (LERGU). The EST database search gave one alternative splicing form in this 3' UTR, which was named as LERGU-1. A novel alternative splicing form of this mRNA was also identified from the stomach total RNA, which was named LERGU-2. The LERGU mRNA was also detected in eight gastric cancer cell lines, but GPR30 mRNA scarcely existed. Furthermore, we detected the 2 bp-deletion form in genomic DNAs and mRNAs derived from gastric cancers, but not in other type cancers. Since the 2 bp-deletion position on LERGU corresponds to its alternative splicing site, this deletion may produce a frame-shifted protein. Overall, our findings suggest that a mutation or disappearance of the normal LERGU protein may have a function in the development of gastric cancer.

G protein-coupled receptor 30 is critical for a progestin-induced growth inhibition in MCF-7 breast cancer cells.

The issue of how progesterone affects mammary gland growth is controversial, and the mechanism governing the effects of the hormone remains mostly unknown. We have previously shown that G protein-coupled receptor 30 (GPR30) is a progestin target gene whose expression correlates with progestin-induced growth inhibition in breast cancer cells. In this study, we investigate the role of GPR30 in regulating cell proliferation and mediating progestin-induced growth inhibition. When progestin failed to inhibit the growth of MCF-7 cells and instead stimulated growth, GPR30 was down-regulated. In this way, the inhibitory or stimulatory affects that progestin has on proliferation correlated with the level of expression of GPR30. Transient expression of GPR30 resulted in a marked inhibition of cell proliferation independent of estrogen treatment. GPR30 antisense was used to evaluate the role of GPR30 expression in progestin-induced growth inhibition. A diminished GPR30 mRNA expression by the antisense stimulated growth. Interestingly, GPR30 antisense abrogated the growth inhibitory effect of progestin and progesterone. Indeed, progestin induced 1) a reduction in cell proliferation, 2) G1-phase arrest, and 3) down-regulation of cyclin D1 was diminished. These data suggest that the orphan receptor, GPR30, is important for the inhibitory effect of progestin on growth.

Progestin upregulates G-protein-coupled receptor 30 in breast cancer cells.

A differential display method was used to study genes the expression of which is altered during growth inhibition induced by medroxyprogesterone acetate (MPA). A transcript of G-protein-coupled receptor 30 (GPR30) was upregulated by MPA in estrogen-treated MCF-7 breast cancer cells. Northern-blot analysis showed a progestin-specific primary target gene, which was enhanced by progesterone and different progestins, but not by dihydrotestosterone or dexamethasone, and which was abrogated by antiprogestin RU486. The dose-dependent and time-dependent increase in GPR30 mRNA expression correlated with MPA-induced growth inhibition in MCF-7 cells. Additionally, GPR30 upregulation by progestin correlated with growth inhibition when a comparison was made between different breast cancer cell lines. The ERK1/ERK2 pathway is capable of inducing progesterone receptor-dependent and ligand-dependent transcription. Thus we sought to establish whether different MAPK pathway inhibitors affect progestin-induced GPR30 mRNA regulation. The regulation of GPR30 was independent of ERK pathway activation, but the p38 pathway inhibitor induced GPR30 expression, which suggested a potential gene regulation pathway. These data demonstrate a new progestin target gene, the expression of which correlates with growth inhibition.