G Protein-Coupled estrogen receptor negatively regulates cell rigidity and osteogenic differentiation in bone marrow-derived mesenchymal stem cells.

Estrogen plays pivotal roles in regulating bone formation and mineralization via estrogen receptors. Although the expression of G protein-coupled estrogen receptor-1 (GPER-1) is widespread in eukaryotic cells like bone marrow-derived mesenchymal stem cells (BMSCs), the precise mechanism by which this membrane receptor dictates BMSC differentiation has not been thoroughly established. In this study, we investigated whether GPER-1 regulates BMSC osteogenesis by modulating cytoskeletal dynamics and cellular rigidity via the Ras homolog family member A (RhoA) signaling pathway. Murine BMSCs were treated with the GPER-1 agonist G1, antagonist G15, or GPER-1-targeted siRNA, and changes in cell morphology, F-actin organization, focal adhesion, and stiffness were assessed using confocal and atomic force microscopy. RhoA activity was measured using pull-down assays, and osteogenic differentiation was evaluated based on Alizarin Red S (ARS) staining and osteogenic gene expression. Treatment with G1 significantly suppressed RhoA activation in BMSCs, reduced the thickness of actin filaments and number of focal adhesions, and diminished cellular tension. Importantly, GPER-1 activation reduced mineralization and osteogenic gene expression. Furthermore, treatment with G15 attenuated the inhibitory effects of G1. These findings indicate that GPER-1 negatively regulates the osteogenic differentiation of murine BMSCs by inhibiting RhoA-mediated cytoskeletal remodeling, associated with the RhoA/actin/cell rigidity axis. Collectively, our findings in this study reveal a previously unrecognized regulatory role of GPER-1 in bone biology and identify this receptor as a potential therapeutic target for modulating stem cell differentiation during skeletal regeneration and osteoporosis.