Influences Of Osteoprotegerin On The Differentiation Of Osteoclast And The Rho Signalling Pathways

To clarify the mechanism of osteoprotegerin (OPG) inhibits osteoclasts (OCs) differentiation and the role of Rho signaling pathway in this mechanism, mouse monocytic RAW264.7 cells were incubated with 25 ng/mL M-CSF and 30 ng/mL RANKL for osteoclastogenesis. In the cultivation of the cells,0,20,40, and 80ng/mL OPG were for 24 hours. The morphological changes of OCs were observed by TRAP staining. The bone resorption effect of OCs were detected by bone resorption lacunae assay. In addtion. the changes of F-actin rings were observed by immunofluorescent. The morphologic changes of pseudopod were observed by scanning electron microscopy. Furthermore, the transcription levels of bone resorption related genes, such as TRAP, Cathepsin K, Cdc42V1, Cdc42V2, Rac1, Rac2, Rac3, were examined using real-time PCR. Key signaling proteins involved in the Rho signaling pathway, such as TRAP, Cathepsin K, PAK1, PAK2, PAK3, LIMK and cofilin were monitored via Western blot.The results were as follows:①OC differentiation and bone resorption related genes or proteins, such as TRAP. Cathepsin K, were all significantly enhancedin M-CSF and RANKL-induced RAW264.7 cells (P<0.05 or P<0.01). ②After 3 days of culture, OPG were added for 24h. Results displayed that the number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly decreased compared to the control group. OC differentiation and bone resorption related proteins showed the similar trend (P<0.01). ③OPG can inhibited OCs differentiation and down-regulated the formation of F-actin rings in osteoclast. Pseudopodia corpuscle aggregation and reduction of arrangement ability were occurred in OPG-treated groups. Simultaneously, the number of F-actin rings,filopodia and lamellipodia were decreased.④OPG could significantly reduced the transcription levels of Rho related genes, such as Cdc42V1, Cdc42V2, Rac2, Rac3 (P<.0.05 or P<0.01). Furthermore, OPG significantly down-regulated the amount of phosphorylated sites in Ser144 and Thr423 of PAK1, Ser141 and Thr402 of PAK2, Thr508 of LIMK1, Thr505 of LIMK2 (P<0.05 or P<0.01), while increased phosphorylation of cofilin in a concentration-dependent manner (P<0.05 or P<0.01).In summary, these findings suggested that OPG can inhibited the differentiation and bone resorption of osteoclast and the Rho signalling pathway were involved in this mechanism. The data demonstrated that OPG can reduced the transcription levels of Cdc42V1、Cdc42V2、Rac2、 Rac3,resulted in down-regulated the phosphorylation of PAK and LIMK. Consequently, the phosphorylation of cofilin were up-regulated and then the differentiation of osteoclast were inhibited.