Estrogen Suppresses Osteoclast Function and Cytoskeletal Remodeling via Modulation of the RhoA-Pkn3-c-Src Signaling Pathway in Ovariectomized Rats

Postmenopausal osteoporosis (PMOP) is characterized by excessive bone resorption caused by increased osteoclast activity. Estrogen remains an effective therapeutic option for preventing bone loss; however, its regulatory effects on osteoclast cytoskeletal remodeling have not been fully clarified. In this study, an ovariectomized (OVX) rat model and RANKL-induced RAW264.7 osteoclasts were used to investigate the role of the RhoA-Pkn3-c-Src signaling pathway in the anti-resorptive effects of estrogen. Bone microarchitecture was evaluated by micro-computed tomography, while osteoclast activity and pathway-related proteins were assessed using histological staining and immunofluorescence. In vitro, osteoclast differentiation, bone resorption, actin ring formation, and protein interactions were analyzed, with additional validation using a RhoA agonist. Estrogen treatment markedly alleviated OVX-induced bone loss, reduced osteoclast number and activity, and improved trabecular bone microarchitecture. These changes were accompanied by decreased expression of RhoA, Pkn3, c-Src, and Pyk2 in osteoclasts. Consistently, estrogen suppressed RANKL-induced osteoclast differentiation, bone resorption, and cytoskeletal remodeling in vitro, whereas activation of RhoA partially attenuated these effects. Collectively, these findings suggest that the protective effects of estrogen against osteoclast-mediated bone resorption are associated with modulation of the RhoA-Pkn3-c-Src signaling pathway, providing additional insight into the mechanisms underlying PMOP.