PEX5 acts as a negative regulator of RANKL-induced osteoclastogenesis in vitro and inflammatory calvarial bone...

Peroxisomal biogenesis factor 5 (PEX5), a peroxisomal import receptor, is well recognized for its role in protein trafficking and oxidative stress regulation. However, its function in bone metabolism remains unclear. Given the established impact of oxidative stress on osteoclast differentiation, this study explores the previously uncharacterized role of PEX5 in osteoclastogenesis and bone resorption. Using bone marrow-derived macrophages, we examined the effects of PEX5 knockdown (siPEX5) and recombinant PEX5 protein (rpPEX5) on osteoclast differentiation. Osteoclast activity was evaluated through TRAP staining, F-actin ring formation, and bone resorption assays. qRT-PCR and Western blot analyses assessed gene and protein expression, while an lipopolysaccharide (LPS)-induced calvarial bone loss model provided in vivo validation. PEX5 expression declined during osteoclast differentiation, and its suppression promoted osteoclastogenesis by increasing c-Fos, NFATc1, and osteoclast-specific gene expression. Loss of PEX5 also enhanced receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced activation of Akt, MAPK, IκB, and calcium-dependent pathways, accelerating osteoclast maturation. In contrast, rpPEX5 treatment effectively inhibited osteoclast differentiation and bone resorption by downregulating NFATc1 and dampening RANKL-mediated signaling. In vivo, rpPEX5 administration mitigated LPS-induced bone loss by preserving bone structure and reducing osteoclast activity. These findings reveal a novel function of PEX5 as a regulator of osteoclast differentiation, independent of its peroxisomal role. The extracellular activity of PEX5 suggests a broader regulatory mechanism in bone metabolism, with potential therapeutic implications for osteolytic diseases.