Osteogenic transcription factors and proto-oncogene regulate bone sialoprotein gene transcription

Runt homeodomain protein 2 (Runx2), distalless 5 (Dlx5) and Smad1 are transcription factors that play critical roles in controlling the differentiation of osteoblasts and mineralization of bone. Proto-oncogene tyrosine-protein kinase, Src, is an enzyme encoded by the Src gene. The normal cellular gene is called cellular-Src (c-Src). Bone sialoprotein (BSP), a protein implicated in the initial mineralization of newly formed bone, is an early phenotypic marker of differentiated osteoblasts. In this study, we used overexpression plasmids with Runx2, Dlx5, Smad1 or c-Src inserts to search for the effects of these transcription factors and proto-oncogene on BSP gene expression using rat osteoblast-like ROS 17/2.8. When we used Runx2, Dlx5 or c-Src overexpression plasmids for the transfection, BSP and Runx2 mRNA levels were increased in ROS 17/2.8 cells. However, overexpression of Smad1 did not induce BSP and Runx2 mRNA. Transient transfection analyses were performed using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. Transfection of ROS 17/2.8 cells with Runx2, Dlx5 or c-Src overexpression plasmid increased the luciferase activities of the constructs, pLUC3 (-116 to +60), pLUC4 (-425 to +60) and pLUC5 (-801 to +60). However, Smad1 overexpression had no effect on the luciferase activities. These results demonstrate that overexpression of Runx2, Dlx5 or c-Src stimulates BSP transcription, and suggest that Runx2, Dlx5 and c-Src might be crucial transcriptional regulators of mineralization and bone formation.