Cross-talk between 1,25-dihydroxyvitamin D-3 and transforming growth factor-beta signaling requires binding of VDR and Smad3 proteins to their cognate DNA recognition elements

1,25-Dihydroxyvitamin D-3 (vitamin D) and transforming growth factor-beta ( TGF-beta) regulate diverse biological processes including cell proliferatio n and differentiation through modulation of the expression of target genes. Members of the Smad family of proteins function as effecters of TGF-beta s ignaling pathways whereas the vitamin D receptor (VDR) confers vitamin D si gnaling. We investigated the molecular mechanisms by which TGF-beta and vit amin D signaling pathways interact in the regulation of the human osteocalc in promoter. Synergistic activation of the osteocalcin gene promoter by TGF -beta and vitamin D was observed in transient transfection experiments. How ever, in contrast to a previous report by Yanagisawa, J., Yanagi, Y., Masuh iro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Rawabata, M ., Miyazono, K., and Kato, S. (1999) Science, 283, 1317-1321, synergistic a ctivation was not detectable when the osteocalcin vitamin D response elemen t (VDRE) alone was linked to a heterologous promoter. Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restore d synergistic activation. Furthermore, this synergy was dependent on the sp acing 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, brit not the distal SBE3 in this promoter reporter ab olished TGF-beta responsiveness and eliminated synergistic co-activation wi th vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate c ross-talk between the TGF-beta acid vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter.