Transcriptional regulation of the human Runx2/Cbfa1 gene promoter by bone morphogenetic protein-7.

It is well established that core binding factor Runx2/Cbfa1 is required for osteoblast recruitment and differentiation from mesenchymal stem cells. Transcriptional regulation of the Runx2/Cbfa1 gene by osteogenic factors such as bone morphogenetic proteins (BMPs) plays an important role in the stimulation of bone formation by these cytokines. BMP7 (also termed OP-1) is a member of the transforming growth factor beta (TGF-beta) superfamily and induces osteoblast differentiation from mesenchymal precursor stem cells in vitro as well as bone formation in vivo. This study examines the effects of BMP7 on markers of osteoblast differentiation and specifically on human Runx2/Cbfa1 gene transcription in a mouse C2C12 myoblast cell line where it induces expression of both alkaline phosphatase (ALP) and endogenous Runx2/Cbfa1. To further understand the mechanisms of human Runx2/Cbfa1 transcriptional regulation by BMP7, we cloned 3.0 kb of the human Runx2/Cbfa1 gene 5'-upstream flanking region and created a series of promoter deletions cloned into luciferase-based reporter vectors (Runx2/Cbfa1/Luc). Sequence data revealed six copies of the osteoblastic cis-acting element (OSE2) in the proximal promoter region. In C2C12 cells transiently transfected with Runx2/Cbfa1/Luc deletion constructs, transcriptional activity of Runx2/Cbfa1 was upregulated up to 2-fold after 24 h of BMP7 treatment. Mutational analysis demonstrated that the minimal responsive promoter region for BMP7-regulated transcription maps to a proximal -74 OSE2 site. Electromobility shift assays with C2C12 cellular extracts indicate that BMP7 increases binding of OSE2 promoter sequences, and supershift assays with anti-Runx2/Cbfa1 antibodies demonstrate that Runx2/Cbfa1 is part of the nucleoprotein complex binding OSE2. Together, these data indicate BMP7 can upregulate Runx2/Cbfa1 gene expression in C2C12 myoblast cells, and suggest that Runx2/Cbfa1 may bind to OSE2 elements within its own promoter to autoregulate gene transcription in differentiating osteoblasts.