| Fibroblast growth factor receptors (FGFRs) represent a family of tyrosine kinase receptors which mediate the diverse biological functions of fibroblast growth factors (FGFs). Previous studies have shown that FGFRs downregulate in response to cell differentiation. However, a universally accepted rationale or mechanism for this action remains unclear. We evaluated the regulation of FGFR1 gene expression in chicken skeletal muscle myoblasts versus differentiated myofibers utilizing immunohistochemistry, northern blot analysis, and transient transfections of primary cell cultures. Immunohistochemistry of undifferentiated cultured myoblasts treated with an anti-FGFR1 antibody exhibited prominent receptor staining. On the other hand, cultures of differentiated myotubes demonstrated less staining. RNA northern blot analysis, utilizing a 476 bp fragment from the FGFR1 cDNA, revealed that FGFR1 was downregulated after muscle differentiation. To identify the elements responsible for FGFR-1 gene regulation in proliferating myoblasts and post-mitotic muscle fibers, we have isolated and partially characterized the avian FGFR1 gene (cek1) promoter. A fragment of 3.2 kilobases of the FGFR1 promoter linked to the chloramphenicol acetyltransferase (CAT) gene drove reporter gene activity in undifferentiated myoblasts and identified two regions--a distal 2226 bp fragment and a 1058 bp proximal fragment--regulating expression of this gene in myoblasts. The distal region acted in an orientation specific manner and conferred a high level of expression of reporter gene activity in myoblasts. Conversely, reporter gene activity in differentiated cultures, transfected with the same construct, was significantly decreased. In addition, the proximal 1058 bp of the FGFR1 promoter failed to elicit reporter gene activity in either undifferentiated or differentiated cultures. These results indicate that the distal 2226 bp promoter fragment guides transcription of the avian FGFR1 gene in a differentiation-specific manner; and the 3.2 FGFR1CAT transgene effectively mimics the expression of the endogenous FGFR1 gene. |
