Transcription of the bone sialoprotein gene is stimulated by v-Src acting through an inverted CCAAT box

Bone sialoprotein (BSP) is an early marker of differentiated osteoblasts that has been implicated in the nucleation of hydroxyapatite crystal formation during de novo bone formation. Although essentially specific to mineralizing connective tissues, BSP is also expressed ectopically by carcinomas that exhibit microcalcification and which metastasize to bone with high frequency. However, it is not known how BSP is regulated in transformed cells. Because the v-src oncogene induces expression of a number of genes that are involved in tumor growth and metastasis, including osteopontin, we have studied the effects of v-Src on transcription of the BSP gene. Transfection of mouse src-/- cells with a v-src expression vector increased the transcriptional activity of rat BSP promoter/luciferase chimeric constructs approximately 5-fold. Deletion analysis revealed that the v-Src activity was targeted to an inverted CCAAT box located immediately upstream from an inverted TATA box in the BSP promoter. Although mutation of the CCAAT box diminished the basal transcription activity of the BSP promoter, the Src-induced stimulation was completely abolished. Gel mobility shift analysis identified four nuclear factors that bound to this region of the BSP promoter, two of which required an intact CCAAT sequence. Monoclonal antibodies identified nuclear factor-Y (NF-Y) as the principal nuclear factor that bound to the CCAAT box; the second factor (beta) showing strong binding only in short constructs containing the CCAAT sequence. Transcription analyses with a dominant negative NF-Y expression vector confirmed that NF-Y mediated the action of v-Src. These studies indicate that BSP gene expression in transformed cells can be up-regulated by Src kinase activity through a mechanism mediated by the NF-Y transcription factor, which targets an inverted CCAAT box in the BSP gene promoter.