Dysregulated osteoclast function contributes to skeletal diseases. However, the specific ubiquitination regulators of the osteoclastogenesis repressor MafB, particularly at the post-translational level, remain undefined. This study aims to identify ubiquitin-specific proteases (USPs) that deubiquitinate MafB and enhance its stability.

We constructed a MafB-conjugated luciferase and overexpressed 40 individual USPs, measuring changes in luciferase activity. The identified USP was overexpressed in human CD14⁺ peripheral blood mononuclear cells (PBMCs) to evaluate its effect. Osteoclast differentiation was assessed through osteoclast marker Integrin alpha-V (CD51) staining and Western blot analysis. Co-immunoprecipitation (co-IP) was performed to assess the interplay. The influence on MafB ubiquitination and degradation was evaluated via immunoprecipitation and Western blot. Finally, MafB was knocked down in the USP-overexpressing PBMCs to analyze its effect on osteoclast differentiation.

Overexpression of ubiquitin-specific protease 29 (USP29) significantly increased MafB expression by approximately 75% (p < 0.0001). Elevated USP29 levels strongly inhibited osteoclastic differentiation in CD14⁺ PBMCs (p < 0.0001). USP29 was found to interact with MafB, markedly reducing its ubiquitination and subsequent degradation in PBMCs (p < 0.001). Knocking down MafB in USP29-overexpressing PBMCs alleviated the inhibitory effect of USP29 on osteoclastogenesis.

USP29 acts as a potent stabilizer of MafB, inhibiting osteoclastogenesis in human CD14⁺ PBMCs, at least in part, by enhancing MafB stability. These findings expand our understanding of USP29’s role and the post-translational regulation of MafB. Furthermore, USP29 serves as a vital factor that controls osteoclast differentiation, and its regulatory function is at least partially mediated by deubiquitinating and stabilizing MafB.