Raf signaling stimulates and represses the human collagen X promoter through distinguishable elements

Endochondral bone growth is regulated through the rates of proliferation and differentiation of growth plate chondrocytes. While little is known about the intracellular events controlling these processes, the protein kinase c-Raf, a central component of the cellular signal transduction machinery, has recently been shown to be expressed only by differentiated, hypertrophic chondrocytes. The involvement of c-Raf in the transcriptional regulation of the hypertrophic chondrocyte-specific collagen X gene was investigated using cotransfections of collagen X reporter plasmids and expression vectors for mutant c-Raf proteins. Both activated and dominant-negative forms of c-Raf reduced the activity of the collagen X promoter to approximately 30%. The element mediating the repressing effect of activated c-Raf was located between nucleotides -2864 and -2410 of the promoter, whereas the effect of the dominant-negative form of c-Raf was conferred by the 462 nucleotides immediately upstream of the transcription start site. Inhibition of MEK1/2 and ERK1/2, downstream components of Raf-signaling, also caused repression of basal collagen X promoter activity. These data suggest that c-Raf regulates collagen X promoter activity positively and negatively through different cis-acting elements and represent the first evidence of c-Raf activity described in hypertrophic chondrocytes.