Ubiquitin-Specific Protease 4 Antagonizes Osteoblast Differentiation Through Dishevelled.

The canonical Wnt/β-catenin signaling pathway plays a pivotal role and is essentially required for the osteoblast differentiation and bone formation. In this study, we found ubiquitin-specific peptidase 4 (USP4) to strongly inhibit the Wnt/β-catenin signaling by removing Lysine-63 linked poly-ubiquitin chain from Dishevelled (Dvl). Ectopic expression of USP4 promoted β-catenin poly-ubiquitination and thus inhibited Wnt-induced accumulation of cytosolic β-catenin and counteracted Wnt-induced transcriptional activity. Moreover, USP4 knockdown or USP4 knockout led to an increase in the active β-catenin levels and in activation of Wnt/β-catenin-induced transcription. Functional studies in C2C12 myoblasts and KS483 osteoprogenitor cells showed that ectopic expression of USP4 resulted in impaired activation of endogenous Wnt3a-induced genes and decreased osteoblast differentiation and mineralization, whereas USP4 depletion showed the opposite effect. These results identify USP4 as a novel regulator of Dvl in Wnt/β-catenin signal and show its involvement in Wnt3a-induced osteoblast differentiation.