Transforming growth factor-beta 1 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)-beta 1. To address the potential role of OC gene expression in relation to TGF-beta 1 regulation of bone formation and resorption, we examined the transcriptional activity of the rat OC promoter after TGF-beta 1 treatment. 5' deletion analysis of rat OC promoter-chloramphenicol acetyltransferase constructs demonstrated that TGF-beta 1 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-beta 1 response domain, conferred TGF-beta 1 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-beta 1 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-beta 1 inhibits this activation. Our results demonstrate that Fra-2 is hyperphosphorylated upon TGF-beta 1 treatment of ROS 17/2.8 cells. Additionally, treatment of cells with a staurosporine protein kinase C inhibitor abrogates TGF-beta 1 mediated down-regulation of the OC promoter activity. Together, these results demonstrate that TGF-beta 1 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-beta 1 induced phosphorylation of Fra-2 in the repression of OC gene transcription.
Banerjee
C
C
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655, USA.
Stein
J L
JL
Van Wijnen
A J
AJ
Frenkel
B
B
Lian
J B
JB
Stein
G S
GS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051921:Fos-Related Antigen-2; D005786:Gene Expression Regulation; D008745:Methylation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015675:Osteocalcin / Q000235:genetics*; D012516:Osteosarcoma; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051381:Rats; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; C497874:Fosl2 protein, rat; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D015675:Osteocalcin