Sp1- and Sp3-mediated Transcriptional Regulation of the Fibroblast Growth Factor Receptor 1 Gene in Chicken Skeletal Muscle Cells

Abstract

Expression of the fibroblast growth factor receptor 1 (FGFR1) gene in skeletal muscle is positively regulated in proliferating myoblasts and declines during differentiation. We have characterized the cis-regulatory elements in the proximal region of the FGFR1 promoter which render positive transcriptional activity. Multiple elements between ؊69 and ؊14 activate the FGFR1 promoter. Myoblast transfections revealed that potential Sp transcription factor binding sites are required for promoter activity. Electromobility shift assays indicated that myoblast nuclear proteins specifically bind to these cis-elements and that differentiated myotube nuclear extracts do not form these same complexes. In addition, Southwestern blot analysis detected binding of the most proximal Sp motif to a Sp1-like protein present in myoblast nuclear extracts but not in myotubes. In corroboration, Sp1 and Sp3 proteins were detected only in myoblasts and not in differentiated myotubes. Finally, transfection of Drosophila SL2 cells showed that Sp1 is a positive regulator of FGFR1 promoter activity and that Sp3 is a coactivator via the proximal Sp binding sites. These studies demonstrate that the FGFR1 promoter is activated by Sp transcription factors in proliferating myoblasts and demonstrate at least part of the mechanism by which FGFR1 gene expression is downregulated in differentiated muscle fibers.During vertebrate myogenesis, mesodermally derived cells within myogenic lineages proliferate as mononucleated myoblasts before differentiation into postmitotic, multinucleated muscle fibers. Both the sustained proliferation of myoblasts and subsequent withdrawal from the cell cycle as a part of differentiation are regulated by signal transduction cascades initiated by environmental signals including growth factors. Members of the fibroblast growth factor (FGF) 1 family of signaling molecules are capable of sustaining myoblast proliferation and delaying differentiation. The cellular effects of FGF signaling are mediated through a small family of fibroblast growth factor receptors (FGFRs). FGF1 and FGF2 possess well documented mitogenic activity for skeletal myoblasts, and these factors bind to FGFR1 in the cell surface of proliferating myoblasts. In addition to skeletal muscle myoblasts, FGFR1 is also expressed during development of the brain, skin, bones, and cardiac muscle (1). It has recently been shown that FGFR1 can be translocated to the nucleus via importin B and that it plays an important role in the regulation of the cell cycle by inducing nuclear target genes (2). However, FGF signaling declines during myoblast differentiation. This decline is the result of loss of cell surface receptor and a coordinate decrease in FGFR1 mRNA (3-5).The significance of the developmentally regulated expression of FGFR1 in relation to muscle growth and patterning in vivo has been partially examined. Myoblast differentiation and muscle fiber formation were delayed in chick limb musculature overexpressing wild type FGFR1. Conversely, premature differentiat...

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