Glycogen Synthase Kinase Mediated Control of Skeletal Myogenesis in Myoblasts and Alveolar Rhabdomyosarcoma

Myogenic differentiation is a critical and highly regulated process that occurs during embryogenesis. Many studies have investigated the role of Muscle Regulatory Factor (MRF) and Myocyte Enhancer Factor 2 (MEF2) proteins in orchestrating myotube formation. More recently phosphorylation of these transcription factors has been shown to influence their ability to regulate muscle gene expression and hence control myogenic differentiation. One kinase that has been shown to prevent myogenic differentiation is GSK3β, although the precise mechanism by which this occurs is poorly understood. Alveolar rhabdomyosarcoma (ARMS) is a highly malignant mesenchymal tumor that originates from immature striated muscle tissue that does not properly differentiate despite the expression of MRF and MEF2 factors. The overall purpose of my research was to better understand how GSK3β regulates skeletal myogenesis and to explore the molecular basis for the lack of myogenic differentiation in ARMS. To achieve this objective, a variety of loss and gain of function studies were conducted in several tissue culture models, to assess the effect of GSK3β on MEF2 and MRF transcriptional activity as well as two MEF2 target genes: KLF6 and Myogenin, which are involved in myoblast proliferation and cell fusion (and hence differentiation) respectively. Although an in vitro kinase assay revealed that MEF2A was not a GSK3β substrate, inhibition of GSK3 resulted in enhanced MEF2 activity and that regulation of MEF2 by GSK3β was indirect, through p38 MAPK: a potent activator of MEF2. This first study documented that cross-talk between p38 MAPK and GSK3β signaling converges on MEF2 activity in skeletal and cardiac muscle both in vitro and in vivo. A paradoxical feature of ARMS is the expression of Myogenin, a MRF that regulates cell fusion and hence terminal differentiation. Upon confirming that GSK3β was predominantly un-phosphorylated and hence de-repressed in PAX3-FOXO1 expressing ARMS cells, the second study revealed that Myogenin was also a GSK3β substrate in vitro. Mutation of the S160, S164 GSK3 phosphoacceptor sites resulted in (i) a decrease in a phosphorylated form of Myogenin by western blot analysis, (ii) de-repression of Myogenin transcriptional activity, and (iii) reduced the ability of ARMS-derived Rh30 cells to proliferate and form colonies in a colony formation assay. The final study identified a novel, proliferative role for MEF2D target gene and GSK3β substrate, KLF6 in skeletal muscle. Further analysis revealed that TGFβ signaling and not MEF2 protein expression is required for KLF6 expression and induction of myoblast proliferation, in a Smad3-dependent manner. The data revealed that TGFβ signaling regulates myogenesis through two distinct pathways: (i) cell proliferation in a Smad3/KLF6 dependent manner and (ii) inhibition of myotube formation in an ERK1/2 MAPK dependent manner.