Tanimoto Ayuko, Yamaguchi Yu, Kadowaki Tomoko, Sakai Eiko, Oyakawa Shun, Ono Yusuke, Yoshida Noriaki, Tsukuba Takayuki
Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
J Cell Biochem. 2023 Oct;124(10):1486-1502. doi: 10.1002/jcb.30457. Epub 2023 Aug 11.
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.
骨骼肌由单核成肌细胞融合形成的多核肌管组成。骨骼肌分化，即肌生成，已通过小鼠骨骼肌成肌细胞系C2C12进行了研究。据报道，几种“小”Rab蛋白，即主要的膜运输调节因子，可能调节C2C12细胞中的膜蛋白运输；然而，Rab蛋白在肌生成中的作用仍未得到探索。Rab44是“大”Rab GTP酶家族的成员，最近被确定为破骨细胞分化的负调节因子。在本研究中，我们使用C2C12细胞发现，在成肌细胞分化为肌管的过程中，Rab44的表达上调。敲低Rab44可增强成肌细胞分化和肌管形成。与这些结果一致，敲低成肌细胞中的Rab44可增加几种肌生成标记基因的表达水平。敲低Rab44增加了肌生成素和肌融合素的表面积累，这是多核化所需的两种融合蛋白，意味着细胞融合增强。相反，Rab44过表达抑制成肌细胞分化和管形成，并伴有一些肌生成标记物的表达降低。此外，发现Rab44主要定位于溶酶体，Rab44过表达改变了溶酶体的数量和大小。考虑到潜在的分子机制，Rab44过表达损害了C2C12细胞中雷帕霉素复合物1（mTORC1）的信号通路。也就是说，Rab44过表达细胞中mTORC1及其下游mTORC1底物（如S6和P70-S6K）的磷酸化水平明显低于对照细胞。这些结果表明，Rab44通过控制融合蛋白运输和mTORC1信号通路负调节成肌细胞向肌管的分化。
