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Transforming growth factor-beta 1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1
C Banerjee1, J L Stein, A J Van Wijnen
1Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655, USA.
Abstract:
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.
Insights
Transforming growth factor-beta 1 (TGF-β1) down-regulates osteocalcin (OC) gene expression by inhibiting Fra-2 transcription factor activity. This involves TGF-β1-induced hyperphosphorylation of Fra-2, impacting bone formation regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Osteocalcin (OC) is a key protein in bone mineralization.
- Transforming growth factor-beta 1 (TGF-β1) is known to down-regulate OC expression.
- The precise molecular mechanisms of TGF-β1's regulation of OC gene expression are not fully understood.
Purpose of the Study:
- To investigate the role of the rat osteocalcin (OC) gene promoter in response to TGF-β1.
- To identify the specific DNA elements and transcription factors involved in TGF-β1-mediated regulation of OC gene expression.
- To elucidate the signaling pathway by which TGF-β1 represses OC transcription.
Main Methods:
- 5' deletion analysis of rat OC promoter-chloramphenicol acetyltransferase constructs.
- Transient transfections in ROS 17/2.8 cells.
- In vitro gel-mobility shift and competition assays.
- Mutation analysis of cis-acting elements.
- Western blot analysis for protein phosphorylation.
Main Results:
- TGF-β1 treatment repressed OC promoter activity by 2.4-fold.
- A 29-bp region (-162 to -134) was identified as the TGF-β1 response domain.
- Mutation of an AP-1/CRE-like motif (-146 to -139) abolished TGF-β1 responsiveness.
- Fra-2, a Fos-related transcription factor, binds to this motif and activates the OC promoter.
- TGF-β1 treatment led to hyperphosphorylation of Fra-2, inhibiting its activity.
- Inhibition of protein kinase C abrogated TGF-β1-mediated repression of OC promoter activity.
Conclusions:
- TGF-β1 responsiveness of the rat OC gene is mediated by an AP-1-like element binding Fra-2.
- TGF-β1-induced hyperphosphorylation of Fra-2 plays a critical role in repressing OC gene transcription.
- These findings provide insights into the regulation of bone formation and resorption by TGF-β1.