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 adhes ion protein, which is frequently expressed in transformed cells and is thought to play a role in tumorigenesis. v-Src is a transforming vira l oncogene product encoded by Rous sarcoma virus (RSV). We report that v-Src expression in HT1080 fibrosarcoma cells significantly stimulate s mouse osteopontin promoter activity. We also determined the v-Src re sponse element in the osteopontin promoter as an inverted CCAAT box lo cated at -53 to -49 from the transcription start site. Mutations of th e CCAAT box disrupts protein-DNA interaction and diminishes both v-Src stimulation and basal promoter activity. A CCAAT box-containing fragm ent corresponding to -155 to -122 of RSV long terminal repeat competed with the -72 to -38 fragment of mouse osteopontin promoter for specif ic protein binding in the gel shift assay. A polyclonal antibody again st CEF, a CCAAT box-binding factor, supershifted in gel shift assays t he protein-DNA complex formed by nuclear extract of HT1080 with either the RSV CCAAT box fragment or with the osteopontin -72 to -38 fragmen t. Moreover, both osteopontin mRNA levels and enhancer activity of CCA AT box-containing -72 to -38 fragment were significantly elevated in v -src-transformed NIH 3T3 cells relative to parental cells. These findi ngs suggest that the elevated osteopontin expression in transformed ce lls could be due, at least in part, to v-Src stimulation of the osteop ontin promoter and that this effect is mediated by a CBF-like factor.