Rab44 negatively regulates myoblast differentiation by controlling fusogenic protein transport and mTORC1 signaling.

ABSTRACT

Skeletal muscle is composed of multinucleated myotubes formed by the fusion of mononucleated myoblasts. Skeletal muscle differentiation, termed as myogenesis, have been investigated using the mouse skeletal myoblast cell line C2C12. It has been reported that several "small" Rab proteins, major membrane-trafficking regulators, possibly regulate membrane protein transport in C2C12 cells; however, the role of Rab proteins in myogenesis remains unexplored. Rab44, a member of "large" Rab GTPases, has recently been identified as a negative regulator of osteoclast differentiation. In this study, using C2C12 cells, we found that Rab44 expression was upregulated during myoblast differentiation into myotubes. Knockdown of Rab44 enhanced myoblast differentiation and myotube formation. Consistent with these results, Rab44 knockdown in myoblasts increased expression levels of several myogenic marker genes. Rab44 knockdown increased the surface accumulation of myomaker and myomixer, two fusogenic proteins required for multinucleation, implying enhanced cell fusion. Conversely, Rab44 overexpression inhibited myoblast differentiation and tube formation, accompanied by decreased expression of some myogenic markers. Furthermore, Rab44 was found to be predominantly localized in lysosomes, and Rab44 overexpression altered the number and size of lysosomes. Considering the underlying molecular mechanism, Rab44 overexpression impaired the signaling pathway of the mechanistic target of rapamycin complex1 (mTORC1) in C2C12 cells. Namely, phosphorylation levels of mTORC1 and downstream mTORC1 substrates, such as S6 and P70-S6K, were notably lower in Rab44 overexpressing cells than those in control cells. These results indicate that Rab44 negatively regulates myoblast differentiation into myotubes by controlling fusogenic protein transport and mTORC1 signaling.