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Originally published In Press as doi:10.1074/jbc.M011033200 on February 5, 2001

J. Biol. Chem., Vol. 276, Issue 19, 15741-15746, May 11, 2001
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Cross-talk between 1,25-Dihydroxyvitamin D3 and Transforming Growth Factor-beta Signaling Requires Binding of VDR and Smad3 Proteins to Their Cognate DNA Recognition Elements*

Nanthakumar SubramaniamDagger , Gary M. Leong, Terrie-Anne Cock, Judith L. Flanagan, Colette Fong, John A. Eisman, and Alexander P. Kouzmenko

From the Bone and Mineral Research Program, The Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia

1,25-Dihydroxyvitamin D3 (vitamin D) and transforming growth factor-beta (TGF-beta ) regulate diverse biological processes including cell proliferation and differentiation through modulation of the expression of target genes. Members of the Smad family of proteins function as effectors of TGF-beta signaling pathways whereas the vitamin D receptor (VDR) confers vitamin D signaling. We investigated the molecular mechanisms by which TGF-beta and vitamin D signaling pathways interact in the regulation of the human osteocalcin promoter. Synergistic activation of the osteocalcin gene promoter by TGF-beta and vitamin D was observed in transient transfection experiments. However, in contrast to a previous report by Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science, 283, 1317-1321, synergistic activation was not detectable when the osteocalcin vitamin D response element (VDRE) alone was linked to a heterologous promoter. Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restored synergistic activation. Furthermore, this synergy was dependent on the spacing between VDRE and SBEs. The Smad3-Smad4 heterodimer was found to bind in gel shift assay to two distinct DNA segments of the osteocalcin promoter: -1030 to -989 (SBE3) and -418 to -349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, but not the distal SBE3 in this promoter reporter abolished TGF-beta responsiveness and eliminated synergistic co-activation with vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate cross-talk between the TGF-beta and vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Muscle Development Unit, Children's Medical Research Institute, Wentworthville NSW 2145, Australia. Tel.: 61-2-9687-2800l; Fax: 61-2-9687-2120; E-mail: nsubramaniam@cmri.usyd.edu.au.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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