Autophagy Regulator Rufy 4 Promotes Osteoclastic Bone Resorption by Orchestrating Cytoskeletal Organization via Its RUN Domain

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the <i>Rufy4</i> gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing <i>Rufy4</i> enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type <i>Rufy4</i> in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a <i>Rufy4</i> variant lacking the RUN domain mimics the effects of <i>Rufy4</i> knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4's potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.