Transforming growth factor-β1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1 binding site

Osteocalcin (OC), a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix, is down-regulated upon treatment with transforming growth factor (TGF)-β1. To address the potential role of OC gene expression in relation to TGF-β1 regulation of bone formation and resorption, we examined the transcriptional activity of the rat OC promoter after TGF-β1 treatment. 5' deletion analysis of rat OC promoter- chloramphenicol acetyltransferase constructs demonstrated that TGF-β1 treatment repressed chloramphenicol acetyltransferase activity by 2.4-fold in transient transfections of ROS 17/2.8 cells. A 29-bp region between -162 and -134 identified as the TGF-β1 response domain, conferred TGF-β1 responsiveness to the -108 to +24 rat OC basal promoter in an orientation dependent manner. Mutation of an activator protein-1/cAMP-response element- like motif (-146 to -139) abolished TGF-β1 responsiveness of the construct. In vitro gel-mobility shift and competition assays using wild-type and mutated oligonucleotides and antibodies indicate that Fra-2, a Fos related transcription factor, binds to this motif. We show that Fra-2 is an activator of the OC promoter, and TGF-β1 inhibits this activation. Our results demonstrate that Fra-2 is hyperphosphorylated upon TGF-β1 treatment of ROS 17/2.8 cells. Additionally, treatment of cells with a staurosporine protein kinase C inhibitor abrogates TGF-β1 mediated down-regulation of the OC promoter activity. Together, these results demonstrate that TGF-β1 responsiveness of the rat osteocalcin gene in ROS 17/2.8 cells is mediated through an activator protein-1 like cis-acting element that interacts with Fra-2. Furthermore, our results are consistent with a critical role for TGF- β1 induced phosphorylation of Fra-2 in the repression of OC gene transcription.