G protein-coupled receptor 30 mediates cell proliferation of goat mammary epithelial cells via MEK/ERK&PI3K/AKT signaling pathway

Abstract

ABSTRACT The mammary gland of mammals possesses the specific function of synthesizing, secreting, and delivering milk. Notably, mammary epithelial cells are considered to be central to control the expansion and remodeling of mammary gland into a milk-secretory organ. And the biological function of mammary gland is mainly regulated by the endocrine system, especially for estrogen. G protein-coupled receptor 30 (GPR30), an estrogen membrane receptor, mediates estrogen-induced functions of physiology and pathophysiology. However, the relationship between estrogen/GPR30 signaling and proliferation of goat mammary epithelial cells (gMECs) is still unclear. Herein, estrogen promoted cell proliferation than control, as evidence by upregulation of cell numbers, BrdU-positive cell counts, and cell viability. Of note, these activities were all obviously reduced by treatment with GPR30 antagonist G15, yet GPR30 agonist G1 increased cell proliferation than control. Further, GPR30 silencing inhibited cell proliferation than negative control. This inhibition was accompanied by a G2/M phase arrest and downregulation of cell cycle regulators. Meanwhile, estrogen increased the phosphorylation of ERK1/2 and AKT. Further, the protein level of p-ERK1/2 and p-AKT was enhanced by GPR30 agonist G1 but inhibited by GPR30 antagonist G15 and GPR30 silencing. Importantly, MEK inhibitor and PI3K inhibitor decreased the expression of cell cycle regulators, and repressed estrogen-induced and G1-driven promotion of cell proliferation, suggesting that estrogen regulated cell proliferation of gMECs through mechanisms involving cell cycle, dependent of GPR30 and MEK/ERK and PI3K/AKT signaling pathway. This may provide a strong theoretical basis for researching estrogen sustained-release drugs promoting breast development and improving lactation performance. Abbreviations: gMECs, goat mammary epithelial cells; E2, 17β-estradiol; GPR30, G protein-coupled receptor 30; shRNA, small hairpin RNA; CDK, cyclin-dependent kinase; PI3K, phosphatidylinositol 3-kinase; AKT, proteinkinase B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK1/2, extracellular signal-regulated kinase 1/2.