P0174FHL3 Differentially Regulates Expression of Four MyHC Isoforms through Interaction with MyoD and CREB
Muscle fiber type composition plays an important role in animal production and human health. Four adult myosin heavy chain (MyHC) isoforms, including MyHC1 /slow, MyHC 2a, MyHC 2x and MyHC 2b are considered as molecular markers to distinguish different muscle fiber types. Therefore, it is of great significance to discover genes regulating the MyHC expression and elucidate its molecular mechanism. Four and a half LIM domain portein 3 (FHL3), a member of LIM protein superfamily, regulates myoblast differentiation, and gene expression through interaction with proteins such as MyoD, Sox15 and CREB. However, how FHL3 regulates MyHC expression is currently not clear.
Results
Real-time PCR, western blotting and immunofluorescence staining were used to detect the expression of MyHC isoforms after FHL3 overexpression and knockdown in C2C12 myoblasts. FHL3 down-regulated the expression of MyHC1/ slow, and up-regulated the expression of MyHC 2a and MyHC 2b, while no significant effects on the MyHC 2x expression. FHL3 inhibited the expression of MyHC1/slow by its interaction with MyoD, and promoted the expression of MyHC 2a mainly through association with phosphoryled cAMP response element binding protein (pCREB) to bind the cyclic AMP-responsive elements (CRE) in its promoter. FHL3 could interact with CREB , pCREB and p300 in vitro. EMSA and ChIP assay showed that pCREB had stronger interaction with the CRE sites of MyHC 2a promoter than that of unphosphoryled CREB, and FHL3 could affect the binding ability of pCREB with the CRE sites.
Conclusion
We established the model that FHL3 promotes the expression of MyHC 2a through CREB-mediated transcription and inhibits MyHC 1/slow by inhibiting MyoD during C2C12 myoblast differentiation, suggesting that FHL3 plays important roles in the regulation of muscle fiber type composition.