Stimulation of mouse osteopontin promoter by v-Src is mediated by a CCAAT box-binding factor.

Osteopontin is an arginine-glycine-aspartic acid-containing cell adhesion protein, which is frequently expressed in transformed cells and is thought to play a role in tumorigenesis. v-Src is a transforming viral oncogene product encoded by Rous sarcoma virus (RSV). We report that v-Src expression in HT1080 fibrosarcoma cells significantly stimulates mouse osteopontin promoter activity. We also determined the v-Src response element in the osteopontin promoter as an inverted CCAAT box located at -53 to -49 from the transcription start site. Mutations of the CCAAT box disrupts protein-DNA interaction and diminishes both v-Src stimulation and basal promoter activity. A CCAAT box-containing fragment corresponding to -155 to -122 of RSV long terminal repeat competed with the -72 to -38 fragment of mouse osteopontin promoter for specific protein binding in the gel shift assay. A polyclonal antibody against CBF, a CCAAT box-binding factor, supershifted in gel shift assays the protein-DNA complex formed by nuclear extract of HT1080 with either the RSV CCAAT box fragment or with the osteopontin -72 to -38 fragment. Moreover, both osteopontin mRNA levels and enhancer activity of CCAAT box-containing -72 to -38 fragment were significantly elevated in v-src-transformed NIH 3T3 cells relative to parental cells. These findings suggest that the elevated osteopontin expression in transformed cells could be due, at least in part, to v-Src stimulation of the osteopontin promoter and that this effect is mediated by a CBF-like factor.