17β-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells

Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E ) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E elevated [Ca ] and increased Ca oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E induces the non-genomic responses Ca release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E responses. © 2012 Springer Science+Business Media, LLC.