Gingipains promote RANKL-induced osteoclastogenesis through the enhancement of integrin β3 in RAW264.7 cells

As a crucial virulence factor of Porphyromonas gingivalis, gingipains play an important role in periodontal destruction. This study aimed to investigate the effect of gingipains on osteoclastogenesis. We used RAW264.7 cells as osteoclast precursors in our study. In experimental groups, cells were treated with gingipains and/or receptor activator of nuclear factor-κB ligand (RANKL). Tartrate-resistant Acid Phosphatase (TRAP) activity staining assay showed osteoclast precursors and RANKL-induced mature osteoclasts were increased in a gingipains dose-dependent manner. Real-time reverse transcription polymerase chain reaction analysis demonstrated that gingipains upregulated osteoclastic genes including the protease Cathepsin K (Ctsk), matrix metalloprotein 9 (Mmp9), nuclear factor of activated T cells 1 (Nfatc1) and Acid Phosphatase 5, tartrate resistant (ACP5) in a time-dependent manner. Western blotting assays presented upregulated expressions of TNF receptor-activating factor 6 (TRAF6) and Integrin β3 induced by gingipains and RANKL compared to RANKL alone. Enhanced integrin-related signaling was also demonstrated by elevated phosphorylations of FAK and paxillin compared to control. Moreover, the pit resorption assays showed that gingipains augmented bone resorptive function of osteoclasts induced by RANKL. When we used Cilengitide to block Integrin αvβ3, gingipains reversed the reduction of formation and resorptive function in RANKL-induced osteoclasts, as they enhanced Integrin αvβ3 levels more than RANKL treatment alone. In conclusion, our data suggest that gingipains augmented the differentiation and function of mature osteoclasts induced by RANKL through the increase in Integrin αvβ3.