Arkadia represses the expression of myoblast differentiation markers through degradation of Ski and the Ski-bound Smad complex in C2C12 myoblasts

The differentiation of myoblasts is regulated by multiple extracellular and intracellular factors. Of the extracellular regulators, members of transforming growth factor-β (TGF-β) family play critical roles in the regulation of osteoblasts and myoblast differentiation. Little is known, however, about the regulation of Myostatin/TGF-β signaling during myoblast differentiation. In this study, we examined the roles of Arkadia, an E3 ubiquitin ligase, in Myostatin/TGF-β signaling and the regulation of myoblast differentiation. Knockdown of Arkadia reduced Myostatin/TGF-β signaling and enhanced the differentiation of C2C12 myoblasts. In addition, exogenous overexpression of Arkadia enhanced Myostatin/TGF-β signaling, preventing myoblast differentiation. In the absence of the activation of Myostatin/TGF-β signaling, knockdown of Arkadia enhanced myoblast differentiation via upregulation of Ski protein, an intracellular enhancer of myoblast differentiation. Arkadia likely affected the differentiation of myoblasts in a Smad-independent fashion by inducing Ski degradation. Knockdown of Arkadia increased the Myostatin-induced phosphorylation of Smad2/3 in C2C12 cells. Arkadia bound Smad2/3 via Ski to induce the ubiquitination of Smad2/3. These results suggest that Arkadia targets Ski-bound, inactive phospho-Smad2/3 to regulate positively Myostatin/TGF-β signaling. Taken together, this study indicates that Arkadia regulates myoblast differentiation through both Smad-dependent and Smad-independent pathways.