Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C108
Yap is a novel regulator of C2C12 myogenesis
K. I. Watt1, R. Judson1, P. Medlow3, K. Reid1, T. B. Kurth2, J. G. Burniston4, A. Ratkevicius1, C. De Bari2, H. Wackerhage1
1. Molecular Exercise Physiology, University of Aberdeen, Aberdeen, United Kingdom. 2. Regenerative Medicine, University of Aberdeen, Aberdeen, United Kingdom. 3. Sports and Exercise Science Research Institute, University of Ulster, County Antrim, United Kingdom. 4. Research Institute for Sports and Exercise Science, Liverpool John Moores University, Liverpool, United Kingdom.
The mammalian Hippo pathway is a regulator of proliferation, apoptosis and organ size that acts through the transcriptional co-activator Yes-associated protein (Yap) (1). Recent evidence in epithelial and neural progenitor cells suggests that this pathway can also regulate terminal differentiation by modulating the phosphorylation of Yap at a conserved serine residue at position 127 (2,3). To date, the expression, regulation and function of the mammalian Hippo pathway in skeletal muscle are largely unknown. The aim of this study was to test the hypothesis that core members of the mammalian Hippo pathway are expressed in mouse skeletal muscles and that Yap regulates C2C12 myogenesis. First, we performed semi quantitative RT-PCR and Western blotting to assess the expression of Hippo pathway members in skeletal muscles of 4 month old C57BL/6 male mice (n=3). We found that all of the core components of the mammalian Hippo pathway are expressed in mouse gastrocnemius, soleus and tibialis anterior muscles, C2C12 myoblasts and myotubes. To investigate the role of Yap during C2C12 myogenesis, we cultured C2C12 myoblasts until confluent then differentiated myoblasts for 96 h. Protein was extracted or cells were fixed every 24 h throughout differentiation (n=3). Western blotting and immunocytochemistry experiments showed that Yap Ser127 phosphorylation was low and Yap localised to specific loci in the nucleus of C2C12 myoblasts. After 96 h of differentiation, Yap Ser127 phosphorylation was increased by ≈28-fold (p<0.05) and Yap translocated from the nucleus to the cytosol. To test whether the observed increase of Yap Ser127 phosphorylation is required for myoblast differentiation, we transfected C2C12 myoblasts to over express hYAP1 S127A, a mutant that can not be phosphorylated at Ser127, and differentiated cells for up to 96 h (n=3). We found that the over expression of hYAP S127A prevented myotube formation, whereas the over expression of wild type hYAP1 or an empty vector had no effect. Consistent with this, hYAP1 S127A over expressing cells were negative for total myosin heavy chain and the expression of dystrophin, muscle creatine kinase and dysferlin was significantly lower than in wild type hYAP1 and empty vector controls (n=3, p<0.05). We also found that ≈3 fold more hYAP1 S127A over expressing cells were within the S phase of the cell cycle, as assessed by incorporation of IdU, after 48 h of differentiation (p<0.05) and that the expression of Myf5 and cyclinD1 was sustained, while the expression of myogenin, Mef2c and p21 were significantly reduced in hYAP1 S127A cells after 24 and 48 h of differentiation (n=3, p<0.05). All statistical comparisons were made by ANOVA with Bonferroni post hoc analysis. This data suggests that the phosphorylation of Yap at Ser127 is required for C2C12 differentiation and highlights for the first time a role for the mammalian Hippo pathway in skeletal muscle.
Where applicable, experiments conform with Society ethical requirements