Abstract
Myogenic cell proliferation and differentiation are regulated by a fibroblast growth factor (FGF) signal transduction cascade mediated by a high-affinity fibroblast growth factor receptor (FGFR). Exogenous FGF added to myogenic cultures has a mitogenic effect promoting myoblast proliferation while repressing differentiation. We have examined the regulation of the FGFR-1 gene (cek-1) in avian myogenic cultures by immunocytochemistry and Northern blot analysis. FGFR-1 protein was readily detected in undifferentiated myoblast cultures and was significantly reduced in differentiated muscle fiber cultures. Similarly, FGFR-1 mRNA was 2.5-fold more abundant in myoblast cultures than in differentiated cultures. To define the molecular mechanism regulating FGFR-1 gene expression in proliferating myoblasts and post-mitotic muscle fibers, we have isolated and partially characterized the avian FGFR-1 gene promoter. Transfection of FGFR-1 promoter-chloramphenicol acetyltransferase gene constructs into myogenic cultures identified two regions regulating expression of this gene in myoblasts. A distal region of 2226 bp conferred a high level of expression in myoblasts. This region functioned in an orientation-dependent manner and interacted with a promoter element(s) in a proximal 1058 bp promoter region to direct transcription. Deletion analysis revealed a 78 bp region that confers a high level of cek1 promoter activity in myoblasts. This DNA segment also contains Spl binding sites and interacts with a component in myoblast nuclear protein extracts. The proximal promoter region alone demonstrated no activity in directing transcription in either myoblasts or muscle fibers. Using the full-length promoter, gene expression was significantly decreased in differentiated muscle fibers relative to undifferentiated myoblasts indicating that the promoter-reporter gene constructs contain elements regulating expression of the endogenous FGFR-1 gene in both myoblasts and muscle fibers.
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