G Protein-coupled Receptor 30 Signaling Research Articles

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.

The activation of G protein-coupled receptor 30 increases pro-opiomelanocortin gene expression through cAMP/PKA/NR4A pathway in mouse pituitary corticotroph AtT-20 cells

G protein-coupled receptor 30 (GPR30) signaling plays an important role in many regulatory pathways, such as gene expression, cell proliferation and migration. However, whether GPR30 is involved in transcription of the pro-opiomelanocortin (Pomc) gene in pituitary corticotroph cells is currently unknown. Here, we report that GPR30 signaling, activated by the GPR30 specific agonist G-1, increases Pomc expression in the mouse corticotroph cell line AtT-20. G-1 also increased nuclear receptor subfamily 4 group A member 1- and 2-dependent transcription activity and phosphorylation of cyclic adenosine monophosphate response element binding protein. Furthermore, protein kinase A inhibitors strongly attenuated G-1-mediated transactivation. The findings suggest that G-1 stimulates GPR30-mediated mechanisms via cyclic adenosine monophosphate/protein kinase A/nuclear receptor subfamily 4 group A members activity in the regulation of Pomc in corticotroph cells.

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.

GPR30 signaling to regulate epithelial-mesenchymal transition and predict survival in ovarian cancer.

e17041 Background: G protein-coupled receptor 30 (GPR30) is a 7-transmembrane estrogen receptor that functions alongside traditional estrogen receptors to regulate the cellular responses to estrogen. Recent studies suggest that the high expression of GPR30 is associated with a poor prognosis in breast cancer or endometrial cancer. Although the role of GPR30 in ovarian cancer was unclear, we revealed that GPR30 is associated with poor prognosis in ovarian cancer. On the other hand, Epithelial-to-Mesenchymal Transition (EMT) is involved in cancer metastasis. The purpose of this study is to reveal how GPR30 was associated with poor prognosis and whether associated with EMT in ovarian cancer. Methods: We examined whether GPR30 signaling activates the EGFR-Akt pathway in an ovarian cancer cell line (Caov-3) by a Western blotting analysis. We also examined the effect of GPR30 on EMT were evaluated in Caov-3, which were cultured both in two-dimensional (2D) culture and three-dimensional (3D) culture model. GPR30 agonist, G1, was used to confirm the regulatory effects of GPR30 on the change of phenotypic modulation and EMT markers expression. Results: The phosphorylation of the EGFR and Akt could be significantly enhanced by G1 (p < 0.05) and inhibited by a Src family kinase inhibitor. In 3D culture, the stimulation of GPR30 leads the floating and sphere formation in Caov-3. G1-induced EMT was observed with related regulation of EMT markers expression at both mRNA and protein level. G1 induced the decrease of E-cadherin level and the increase of Snail and Vimentin in RT-PCR and Western blotting. Knockdown of GPR30, using siRNA, blocked G1-induced EMT. Conclusions: GPR30 increases the phosphorylation of Akt via the EGFR in ovarian cancer cells and changes ovarian cancer cells to the EMT state.GPR30 might be an important molecule related to metastasis process in ovarian cancer.

Estrogen stimulated migration and invasion of estrogen receptor-negative breast cancer cells involves an ezrin-dependent crosstalk between G protein-coupled receptor 30 and estrogen receptor beta signaling

Estrogen mediates important cellular activities in estrogen receptor negative (ER-) breast cancer cells via membrane associated G protein-coupled receptor 30 (GPR30). However, the biological role and mechanism of estrogen action on cell motility and invasion in this aggressive kind of tumors remains poorly understood. We showed here that treatment with 17β-estradiol (E2) in ER-negative cancer cells resulted in ezrin-dependent cytoskeleton rearrangement and elicited a stimulatory effect on cell migration and invasion. Mechanistically, E2 induced ezrin activation was mediated by distinct mechanisms in different cell contexts. In SK-BR-3 cells with a high GPR30/ERβ ratio, silencing of GPR30 was able to abolish E2 induced ERK1/2, AKT phosphorylation and ezrin activation, whereas in MDA-MB-231 cells with low GPR30/ERβ ratio, E2 stimulated ezrin activation was mediated by the ERβ/PI3K/AKT signaling pathway. Importantly, we showed that activation of GPR30 signaling significantly prevents ERβ activation induced ezrin phosphorylation, cell migration and invasion, indicating an antagonist effect between GPR30 and ERβ signaling in MDA-MB-231 cells. These findings highlight the important interplay between different estrogen receptors in estrogen induced cell motility and invasiveness in ER-negative breast cancer cells.

Estrogen receptor GPR30 exerts anxiolytic effects by maintaining the balance between GABAergic and glutamatergic transmission in the basolateral amygdala of ovariectomized mice after stress

G-protein-coupled receptor 30 (GPR30)/G-protein-coupled estrogen receptor is a novel estrogen membrane receptor that localizes to the cell membrane and endoplasmic reticulum. GPR30 is widely distributed and has numerous physiological functions in the central nervous system. We found that GPR30 is highly expressed in the basolateral amygdala (BLA). Additionally, GPR30 expression in the amygdala of ovariectomized (OVX) mice significantly increased after acute stress and was accompanied by anxiety-like behaviors. These effects, however, were reversed by local infusion of the GPR30 agonist (G1) in the BLA. Protein assessments revealed that G1 attenuated the up-regulation of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and NR2A-containing N-methyl-d-aspartate receptors (NMDARs) in the BLA of OVX mice using an acute stress paradigm. In the same model, we found that the agonist also blocked the down-regulation of γ-aminobutyric acid A (GABAA) receptors and NR2B-containing NMDARs. Electrophysiological recording showed that the activation of GPR30 increased the inhibitory synaptic transmission in the BLA. Overall, our results indicate that estradiol reduces anxiety-like behaviors induced by acute stress at least partially through GPR30 signaling, maintaining the balance between GABAergic and glutamatergic transmission in the BLA of OVX-stressed mice.

G protein‐coupled receptor 30 contributes to improved remyelination after cuprizone‐induced demyelination

Estrogen exerts neuroprotective and promyelinating actions. The therapeutic effect has been shown in animal models of multiple sclerosis, in which the myelin sheath is specifically destroyed in the central nervous system. However, it remains unproven whether estrogen is directly involved in remyelination via the myelin producing cells, oligodendrocytes, or which estrogen receptors are involved. In this study, we found that the membrane-associated estrogen receptor, the G protein-coupled receptor 30 (GPR30), also known as GPER, was expressed in oligodendrocytes in rat spinal cord and corpus callosum. Moreover, GPR30 was expressed throughout oligodendrocyte differentiation and promyelinating stages in primary oligodendrocyte cultures derived from rat spinal cords and brains. To evaluate the role of signaling via GPR30 in promyelination, a specific agonist for GPR30, G1, was administered to a rat model of demyelination induced by cuprizone treatment. Histological examination of the corpus callosum with oligodendrocyte differentiation stage-specific markers showed that G1 enhanced oligodendrocyte maturation in corpus callosum of cuprizone-treated animals. It also enhanced oligodendrocyte ensheathment of dorsal root ganglion (DRG) neurons in co-culture and myelination in cuprizone-treated animals. This study is the first evidence that GPR30 signaling promotes remyelination by oligodendrocytes after demyelination. GPR30 ligands may provide a novel therapy for the treatment of multiple sclerosis.

G Protein-Coupled Receptor 30 Mediates Estrogen-Induced Proliferation of Primordial Germ Cells Via EGFR/Akt/β-Catenin Signaling Pathway

In vertebrates, estrogens are required for the normal development and function of postnatal gonads. However, it remains unclear whether estrogens are able to modulate development of the fetal germ cells. Here, we show that, unexpectedly, chicken primordial germ cells (PGC) lacking estrogen receptor α/β still proliferate in response to 17β-estradiol (E(2)). This is due to the capacity of G protein-coupled receptor 30 (GPR30), existing on PGC, to directly bind E(2). Knockdown experiments suggest that GPR30 is required for E(2)-stimulated PGC proliferation. Furthermore, this estrogen-induced activation of GPR30 is revealed to occur through the Gβγ-subunit protein-dependent and through the matrix metalloproteinase-dependent transactivation of the epidermal growth factor receptor. Epidermal growth factor receptor activation results in a series of intracellular events, including activation of the phosphatidylinositol 3-kinase/serine-threonine kinase/β-catenin pathway, which are followed by the induction of c-fos, c-myc, cyclin D1/E, and B-cell lymphoma 2 expression, and the inhibition of B-cell lymphoma 2-associated X protein expression and caspase3/9 activity. This eventually leads to decreased apoptosis and increased PGC proliferation. Collectively, these findings offer novel insights into the dynamic mechanism of estrogen action on PGC proliferation and suggest that E(2)/GPR30 signaling might play an important role in regulating fetal germ cell development, particularly at the stage before sexual differentiation.

Regulation of HRG-β1-induced proliferation, migration and invasion of MCF-7 cells by upregulation of GPR30 expression

The cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There is evidence of crosstalk between the G protein-coupled receptor 30 (GPR30), the newly discovered estrogen receptor (ER), and the ErbB family. Heregulin (HRG)-β1, the ligand for ErbB3 and ErbB4, upregulates GPR30 expression in MCF-7, T-47D and BT-474 breast cancer cell lines that express ERα. In the present study, recombinant human HRG-β1 was used to investigate the upregulation of GPR30 expression by HRGs in MCF-7 breast cancer cells which were ERα-positive. In MCF-7 cells, the ErbB2 inhibitor, AG825, the MAPK inhibitor, PD98059, and the MEK1/2 inhibitor, U0126, blocked the HRG-β1-induced GPR30 expression. 17-β-estradiol (E2) boosted the HRG-β1-induced proliferation, migration and invasion of MCF-7 cells. Similar to E2, the specific GPR30 agonist, G-1, promoted HRG-β1-induced migration and invasion, but inhibited growth. Using the specific GPR30 antagonist, G-15, or the small interfering RNA for GPR30, the functions of GPR30 after treatment with HRG-β1 were further investegated. The results from our study indicate that the interruption between GPR30 signaling and the ErbB family system may serve as a promising therapeutic strategy for breast cancer.

Heregulin-β1-induced GPR30 upregulation promotes the migration and invasion potential of SkBr3 breast cancer cells via ErbB2/ErbB3–MAPK/ERK pathway

Estrogen receptor (ER)-negative breast cancer cells are probably more aggressive with larger metastatic potential than ER-positive cells. Loss of ER in recurrent breast cancer is associated with poor response to endocrine therapy. G protein-coupled receptor 30 (GPR30) is expressed in half of ER-negative breast cancers. Tumor cell-derived heregulin-β1 (HRG-β1) is also found mainly in ER-negative cancer. In SkBr3 breast cancer cells that lack ER but express GPR30, HRG-β1 upregulates mRNA and protein levels of GPR30 by promoting ErbB2–ErbB3 heterodimerization and activating the downstream MAPK–ERK signaling pathway. Moreover, GPR30 boosts HRG-β1-induced migration and invasion of SkBr3 cells after combinative treatment with E2, 4-hydroxy-tamoxifen or the specific GPR30 agonist G-1, which are blocked by the specific GPR30 antagonist G-15 or the transfection with the small interfering RNA for GPR30. The ErbB2 inhibitor AG825 and the MEK1/2 inhibitor U0126 also partly inhibit the enhanced migration and invasion. Therefore, HRG-β1-induced migration and invasion partly depend on the upregulation of GPR30 expression through activation of the ErbB2–ERK pathway in SkBr3 cells. The results of this study indicate that the crosstalk between GPR30 and HRGs signaling is important for endocrine therapy resistance and may provide a new therapeutic way to treat breast cancer.

The membrane estrogen receptor GPR30 mediates cadmium-induced proliferation of breast cancer cells

Cadmium (Cd) is a nonessential metal that is dispersed throughout the environment. It is an endocrine-disrupting element which mimics estrogen, binds to estrogen receptor alpha (ERα), and promotes cell proliferation in breast cancer cells. We have previously published that Cd promotes activation of the extracellular regulated kinases, erk-1 and -2 in both ER-positive and ER-negative human breast cancer cells, suggesting that this estrogen-like effect of Cd is not associated with the ER. Here, we have investigated whether the newly appreciated transmembrane estrogen receptor, G-protein coupled receptor 30 (GPR30), may be involved in Cd-induced cell proliferation. Towards this end, we compared the effects of Cd in ER-negative human SKBR3 breast cancer cells in which endogenous GPR30 signaling was selectively inhibited using a GPR30 interfering mutant. We found that Cd concentrations from 50 to 500 nM induced a proliferative response in control vector-transfected SKBR3 cells but not in SKBR3 cells stably expressing interfering mutant. Similarly, intracellular cAMP levels increased about 2.4-fold in the vector transfectants but not in cells in which GPR30 was inactivated within 2.5 min after treatment with 500 nM Cd. Furthermore, Cd treatment rapidly activated (within 2.5 min) raf-1, mitogen-activated protein kinase kinase, mek-1, extracellular signal regulated kinases, erk-1/2, ribosomal S6 kinase, rsk, and E-26 like protein kinase, elk, about 4-fold in vector transfectants. In contrast, the activation of these signaling molecules in SKBR3 cells expressing the GPR30 mutant was only about 1.4-fold. These results demonstrate that Cd-induced breast cancer cell proliferation occurs through GPR30-mediated activation in a manner that is similar to that achieved by estrogen in these cells.

Amelioration of collagen‐induced arthritis and immune‐associated bone loss through signaling via estrogen receptor α, and not estrogen receptor β or G protein–coupled receptor 30

The effects of estrogen may be exerted via the nuclear estrogen receptors (ERs) ERalpha or ERbeta or via the recently proposed transmembrane estrogen receptor G protein-coupled receptor 30 (GPR-30). The purpose of this study was to elucidate the ER specificity for the ameliorating effects of estrogen on arthritis and bone loss in a model of postmenopausal rheumatoid arthritis (RA). Female DBA/1 mice underwent ovariectomy or sham operation, and type II collagen-induced arthritis was induced. Mice were treated subcutaneously 5 days/week with the specific agonists propylpyrazoletriol (PPT; for ERalpha), diarylpropionitrile (DPN; for ERbeta), G1 (for GPR-30), or with a physiologic dose of estradiol. Clinical arthritis scores were determined continuously. At termination of the study, bone mineral density (BMD) was analyzed, paws were collected for histologic assessment, serum was analyzed for cytokines and markers of bone and cartilage turnover, and bone marrow was subjected to fluorescence-activated cell sorting. Treatment with PPT as well as estradiol dramatically decreased the frequency and severity of arthritis. Furthermore, estradiol and PPT treatment resulted in preservation of bone and cartilage, as demonstrated by increased BMD and decreased serum levels of bone resorption markers and cartilage degradation markers, whereas no effect was seen after DPN or G1 treatment. In a well-established model of postmenopausal RA, ERalpha, but not ERbeta or GPR-30 signaling, was shown to ameliorate the disease and the associated development of osteoporosis. Since long-term treatment with estrogen has been associated with significant side effects, increased knowledge about the mechanisms behind the beneficial effects of estrogen is useful in the search for novel treatments of postmenopausal RA.

403 Molecular analysis of eighteen most recurrent mutations in the BRCA1 gen in 59 Chilean breast cancer families

Cadmium (Cd) is a nonessential metal that is dispersed throughout the environment. It is an endocrine-disrupting element which mimics estrogen, binds to estrogen receptor alpha (ERα), and promotes cell proliferation in breast cancer cells. We have previously published that Cd promotes activation of the extracellular regulated kinases, erk-1 and -2 in both ER-positive and ER-negative human breast cancer cells, suggesting that this estrogen-like effect of Cd is not associated with the ER. Here, we have investigated whether the newly appreciated transmembrane estrogen receptor, G-protein coupled receptor 30 (GPR30), may be involved in Cd-induced cell proliferation. Towards this end, we compared the effects of Cd in ER-negative human SKBR3 breast cancer cells in which endogenous GPR30 signaling was selectively inhibited using a GPR30 interfering mutant. We found that Cd concentrations from 50 to 500 nM induced a proliferative response in control vector-transfected SKBR3 cells but not in SKBR3 cells stably expressing interfering mutant. Similarly, intracellular cAMP levels increased about 2.4-fold in the vector transfectants but not in cells in which GPR30 was inactivated within 2.5 min after treatment with 500 nM Cd. Furthermore, Cd treatment rapidly activated (within 2.5 min) raf-1, mitogen-activated protein kinase kinase, mek-1, extracellular signal regulated kinases, erk-1/2, ribosomal S6 kinase, rsk, and E-26 like protein kinase, elk, about 4-fold in vector transfectants. In contrast, the activation of these signaling molecules in SKBR3 cells expressing the GPR30 mutant was only about 1.4-fold. These results demonstrate that Cd-induced breast cancer cell proliferation occurs through GPR30-mediated activation in a manner that is similar to that achieved by estrogen in these cells.