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 myoblasts and myotubes. By western blotting and immunohistochemistry, we evidenced expression of GPR30. While in C2C12 myoblasts, the receptor was present in nucleus, mitochondria, and endoplasmic reticulum, in C2C12 myotubes, it was additionally found in cytoplasm. Using trypan blue uptake assay to determine cellular death and fluorescent microscopy to evaluate picnotic nuclei and mitochondrial distribution, we demonstated 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 the molecular mechanisms involved were investigated by wertern blot assays. Thus, E2 was able to induce AKT phosphorylation in apoptotic conditions and ERK phosphorylation in proliferating C2C12 cells but not when the cultures were incubated with G15. Additionally, using G15 antagonist we have found that GPR30 participates in the myogenin expression and creatine kinase activity stimulated by E2 in the first steps of C2C12 differentiation. Althogether these findings provide evidences showing that GPR30 is expressed in diverse intracellular compartments in undifferentiated and differentiated C2C12 cells and mediates E2 actions.

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

“…Elevated expression of Erβ and Gpr30 was independent of E2 treatment and was limited to cultures with fully formed myotubes in both Con-Ve and E2 treated cells. While the precise role of Erβ in fully formed myotubes is unknown, an increase in GPR30 during late myogenesis is attributed to its protective role from oxidative stress through activation of creatine kinase [46]. We observed a 1.3-fold increase in Erα expression at 1 h only in E2 treated cells followed by a downregulation.…”

“…E2 also interacts with a membrane G-protein coupled receptor 30 (GPR30) [32]. Both the intracellular and membrane receptors have been associated with in-vitro myoblast differentiation through signal transduction pathways [13,23,46]. Elevated expression of Erβ and Gpr30 was independent of E2 treatment and was limited to cultures with fully formed myotubes in both Con-Ve and E2 treated cells.…”

17β-Estradiol on the Expression of G-Protein Coupled Estrogen Receptor (GPER/GPR30) Mitophagy, and the PI3K/Akt Signaling Pathway in ATDC5 Chondrocytes In Vitro

“…ERα and ERβ are localized mainly in the cell nucleus but are also found in the mitochondria [ 11 ]. The estrogen receptor GPER/GPR30 is mainly localized in the endoplasmic reticulum but is also found in the mitochondria [ 12 ].…”

17β-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes

“…MYH is the dominant myofibrillar protein in differentiated muscle and a downregulation would occur during an inhibitory process. On the contrary, other authors described that E2 promotes differentiation of rat MB cell line and induces changes of some differentiation markers such as myogenin and creatine kinase (Galluzzo et al, 2009; Ronda & Boland, 2016). Unfortunately, a large heterogeneity exists in literature which includes the use of different cell lines and the choice of different time points for treatment and evaluation, making difficult to take a definitive conclusion about E2 effect on muscular differentiation.…”