Transcriptional regulation of osteopontin gene in vivo by PEBP2αA/CBFA1 and ETS1 in the skeletal tissues

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2 alpha A/core binding factor (CBFA) 1 have been suggested to play important roles in ossification, The overlapping localization of opn and PEBP2 alpha A/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2 alpha A knockout mice, indicated that the transcription of opn gene was controlled by PEBP2 alpha A. In the present study, we determined the direct regulation of PEBP2 alpha A on the opn promoter activity, Opn promoter activity was markedly enhanced by PEBP2 alpha A and ETS1 in a synergistic manner, The synergistic effect was diminished when either the PEBP2 alpha A or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2 alpha A and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2 alpha A and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2 alpha A to MC3T3/E1 cells expressing endogenous PEBP2 alpha A, ETS1 and opn, These findings suggest that PEBP2 alpha A and ETS1 cooperate iii vivo to regulate expression of the opn gene in the skeletal tissue, Cell type-specific regulation of Opn gene expression will also be discussed.