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Molecular and Cellular Biology, October 2004, p. 8847-8861, Vol. 24, No. 20
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.20.8847-8861.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bone-Specific Transcription Factor Runx2 Interacts with the 1,25-Dihydroxyvitamin D₃ Receptor To Up-Regulate Rat Osteocalcin Gene Expression in Osteoblastic Cells

Roberto Paredes,^1, Gloria Arriagada,^1, Fernando Cruzat,¹ Alejandro Villagra,¹ Juan Olate,¹ Kaleem Zaidi,² Andre van Wijnen,² Jane B. Lian,² Gary S. Stein,² Janet L. Stein,² and Martin Montecino^1*

Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Concepcion, Chile,¹ Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts²

Received 10 December 2003/ Returned for modification 12 January 2004/ Accepted 19 July 2004

Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1,25-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 1,25-dihydroxyvitamin D₃-responsive element (VDRE), abolishes 1,25-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 1,25-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 1,25-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 1,25-dihydroxyvitamin D₃, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.

R.P. and G.A. equally contributed to this work.

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