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 th at has been implicated in the nucleation of hydroxyapatite crystal formatio n during de novo bone formation. Although essentially specific to mineraliz ing connective tissues, BSP is also expressed ectopically by carcinomas tha t exhibit microcalcification and which metastasize to bone with high freque ncy. However, it is not known how BSP is regulated in transformed cells. Be cause the v-src oncogene induces expression of a number of genes that are i nvolved in tumor growth and metastasis, including osteopontin, we have stud ied the effects of v-Src on transcription of the BSP gene. Transfection of mouse src-/- cells with a v-src expression vector increased the transcripti onal activity of rat BSP promoter/ luciferase chimeric constructs approxima tely 5-fold. Deletion analysis revealed that the v-Src activity was targete d to an inverted CCAAT box located immediately upstream from an inverted TA TA box in the BSP promoter. Although mutation of the CCAAT box diminished t he basal transcription activity of the BSP promoter, the Src-induced stimul ation was completely abolished. Gel mobility shift analysis identified four nuclear factors that bound to this region of the BSP promoter, two of whic h required an intact CCAAT sequence. Monoclonal antibodies identified nucle ar 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 construc ts containing the CCAAT sequence, Transcription analyses with a dominant ne gative NF-Y expression vector confirmed that NF-Y mediated the action of v- Src. These studies indicate that BSP gene expression in transformed cells c an be up-regulated by Src kinase activity through a mechanism mediated by t he NF-Y transcription factor, which targets an inverted CCAAT box in the BS P gene promoter.