Abstract
Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1α,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1α,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1α,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1α,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1α,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a “master” transcriptional regulator of osteoblast differentiation, and 1α,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
We thank Jose Gutierrez and Soraya Gutierrez for helpful discussions and critical reading of the manuscript.
This work was supported by grants from FONDECYT (1030749 to M.M. and 2010103 to R.P.), NIH-FIRCA grant TW00990 (to G.S. and M.M.), DIUC grant 201.031.090-1.4 (to M.M.), and NIH grants AR48818 and AR39588 (to G.S.) and DE12528 (to J.L.). F.C. has been supported by a fellowship from MECESUP UCH9903, and R.P. has been supported by a scholarship from Fundacion Andes.
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