The Effects And Mechanisms Of Estrogen Receptor GPR30in Human Leiomyoma And Vascular Smooth Muscle Cells

Part One The Effects and Mechanisms of estrogen receptor GPR30in human leiomyomaObjective:Uterine leiomyoma, characterized proliferation of smooth muscle cells (SMCs), is the most common benign tumor in women and occur in women at reproductive age. They are the primary indication for hysterectomy, and are the cause of significant morbidity from profuse menstrual bleeding, pelvic discomfort and so on. Estrogen plays a critical role in the pathogenesis of human uterine leiomyomas. However, the novel ER G-protein-coupled estrogen receptor (GPR30) mediates the proliferative effects induced by17β-estradiol has not been investigated in human uterine leiomyomas and their matched myometrial tissues. Our study was designed to determine differential expression of G-protein-coupled receptor30(GPR30) in uterine leiomyoma and its matched myometrium and investigate the effect and mechanism of GPR30in pathogenesis of leiomyomas.Methods and Results:GPR30expression examined in both tissues, which were obtained from patients (35to50years old) undergoing hysterectomies at the Tianjin Central Hospital for Obstetrics and Gynecology and cultured cells, devried from leiomyoma tissues and its matched myometrium tissues. Using Western blot and real-time quantitative polymerase chain reaction analyses, we found that GPR30was highly expressed in uterine leiomyomas compared with their matched myometrium. In only three out of nine patients examined was GPR30protein detectable by Western blot analysis in myometrial tissues, but at statistically significantly lower levels than in their leiomyomas. Confocal microscopy revealed the nuclear localization of GPR30in leiomyoma tissues and cultured leiomyoma smooth muscle cells (SMCs). Treatment with0.1mM17β-estradiol increased mRNA expression of GPR30in leiomyoma SMCs but decreased expression in myometrial SMCs. Treatment with G-1, a GPR30agonist, stimulated phosphorylation of p44/42mitogen-activated protein kinase (MAPK) in both SMC types. PD98059, the MEK inhibitor, completely inhibited G-1-induced phosphorylation of p44/42in myometrium SMCs, but not in SMCs from leiomyoma.Conclusion(s):1. GPR30is abundantly expressed in uterine leiomyomas tissues and the cultured uterine leiomyomas smooth muscle cells. Immunofluorescence and confocal microscopy revealed the nuclear localization of GPR30in leiomyoma tissues and cultured leiomyoma smooth muscle cells (SMCs).2. Estradiol stimulation of GPR30mRNA expression in cultured smooth muscle cells derived from uterine leiomymas.3.G-1, the selective agonist of GPR30, activates the p44/42MAPK pathway in cultured leiomyoma and myometrium SMCs. Part Two The Effects and Mechanisms of estrogen receptor GPR30in Vascular Smooth Muscle CellsObjective:Atherosclerosis is the primary cause of heart disease and stroke. The proliferation of vascular smooth muscle cells is the cytopathology of the incidence cardiovascular diseases, such as atherosclerosis. However, estrogen still has inhibitory effects on neointima formation in mice without both ERs, suggesting the presence of a mechanism independent on ER-a and-β. Therefore, we speculated that activation of a novel estrogen receptor called G-protein coupled receptor30, GPR30, might result in inhibition of neointima formation through the inhibitory effect on smooth muscle cells proliferation. Our study was designed to test this hypothesis and investigate the the inhibitory effect and mechanism of GPR30on vascular smooth muscle cells proliferation.Methods and Results:We took advantage of the neointimal formation model through ligation of mouse carotid arteries and examined the effect of G-1, a specific ligand for GPR30. As expected, G-1treatment significantly inhibited the development of neointimal lesions resulting from ligation. To further investigate the underlying mechanism, we examined G-1effects on cultured human vascular smooth muscle cells (SMCs). Notably, G-1treatment induced phosphorylation and activation of epidermal growth factor (EGF) receptor and subsequently p44/42mitogen activated protein kinase (MAPK) in human vascular SMCs. Taken together, our results show that G-1inhibits neointimal formation through inhibition of SMC proliferation involving a mechanism independent of EGF receptor and p44/42MAPK pathway.Conclusions:1. In vivo, G-1inhibits the injury-induced-neointima formation in mouse injured carotid arteries.2. G-1, GPR30agonist, inhibits the proliferation of human vascular smooth muscle cells.3. Treatment with G-1, activates p44/42MAPK pathway through EGFR to inhibit the proliferation of human vascular smooth muscle cells.