Intracellular Distribution and Involvement of GPR30 in the Actions of E2 on C2C12 Cells

Abstract

G-protein-coupled receptor 30 (GPR30) is an estrogen receptor that initiates several rapid, non-genomic signaling events triggered by E2.GPR30 has recently been identified in C2C12 cells; however, little is known about the intracelular distribution and its role in C2C12 myoblastsand myotubes. By western blotting and immunohistochemistry, we evidenced expression of GPR30. While in C2C12 myoblasts, the receptorwas present in nucleus, mitochondria, and endoplasmic reticulum, in C2C12 myotubes, it was additionally found in cytoplasm. Using trypanblue uptake assay to determine cellular death and fluorescent microscopy to evaluate picnotic nuclei and mitochondrial distribution, wedemonstated that treatment of C2C12 myoblasts with G1 (GPR30 agonist) did not protect the cells against apoptosis induced by H2O2 as E2.However, when G15 (GPR30 antagonist) was used, E2 could not prevent the damage caused by the oxidative stress. Further, some of themolecular mechanisms involved were investigated by wertern blot assays. Thus, E2 was able to induce AKT phosphorylation in apoptoticconditions and ERK phosphorylation in proliferating C2C12 cells but not when the cultures were incubated with G15. Additionally, using G15antagonist we have found that GPR30 participates in the myogenin expression and creatine kinase activity stimulated by E2 in the first steps ofC2C12 differentiation. Althogether these findings provide evidences showing that GPR30 is expressed in diverse intracellular compartments inundifferentiated and differentiated C2C12 cells and mediates E2 actions.