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

Abstract

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2αA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2αA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2αA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2αA. In the present study, we determined the direct regulation of PEBP2αA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2αA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2αA 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 PEBP2αA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2αA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2αA to MC3T3/E1 cells expressing endogenous PEBP2αA, ETS1 and opn. These findings suggest that PEBP2αA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.