Distinct Conformations of Vitamin D Receptor/Retinoid X Receptor-α Heterodimers Are Specified by Dinucleotide Differences in the Vitamin D-Responsive Elements of the Osteocalcin and Osteopontin Genes

A. Staal, A. J. Van Wijnen, J. C. Birkenhäger, H. A.P. Pols, J. Prahl, H. DeLuca, Marie Pierre Gaub, J. B. Lian, G. S. Stein, J. P.T.M. Van Leeuwen, J. L. Stein

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71 Scopus citations

Abstract

The 1α,25-dihydroxyvitamin D3 (VD3)-dependent stimulation of osteocalcin (OC) and osteopontin (OP) gene transcription in bone tissue is mediated by interactions of trans-activating factors with distinct VD3-responsive elements (VDREs). Sequence variation between the OC- and OP-VDRE steroid hormone half-elements provides the potential for recognition by distinct hormone receptor homoand heterodimers. However, the exact composition of endogenous VD3- induced complexes recognizing the OC- and OP-VDREs in osteoblasts has not been definitively established. To determine the identity of these complexes, we performed gel shift immunoassays with nuclear proteins from ROS 17/ 2.8 osteoblastic cells using a panel of monoclonal antibodies. We show that VD3- inducible complexes interacting with the OC- and OP-VDREs represent two distinct heterodimeric complexes, each composed of the vitamin D receptor (VDR) and the retinoid X receptor-α (RXR). The OC- and OP-VDR/RXRα heterodimers are immunoreactive with RXR antibodies and several antibodies directed against the ligand-binding domain of the VDR. However, while the OC-VDRE complex is also efficiently recognized by specific monoclonal antibodies contacting epitopes in or near the VDR DNA-binding domain (DBD) (between amino acids 57-164), the OP-VDRE complex is not efficiently recognized by these antibodies. By systematically introducing a series of point-mutations in the OC-VDRE, we find that two internal nucleotides of the proximal OC-VDRE half-site (nucleotide -449 and -448; 5′-AGGACA) determine differences in VDR immunoreactivity. These results are consistent with the well established polarity of RXR heterodimer binding to bipartite hormone response elements, with the VDR recognizing the 3′-half-element. Furthermore, our data suggest that the DBD of the VDR adopts different protein conformations when contacting distinct VDREs. Distinctions between the OC- and OP-VDR/RXRα complexes may reflect specialized requirements for VD3 regulation of OC and OP gene expression in response to physiological cues mediating osteoblast differentiation.

Original languageEnglish (US)
Pages (from-to)1444-1456
Number of pages13
JournalMolecular Endocrinology
Volume10
Issue number11
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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    Staal, A., Van Wijnen, A. J., Birkenhäger, J. C., Pols, H. A. P., Prahl, J., DeLuca, H., Gaub, M. P., Lian, J. B., Stein, G. S., Van Leeuwen, J. P. T. M., & Stein, J. L. (1996). Distinct Conformations of Vitamin D Receptor/Retinoid X Receptor-α Heterodimers Are Specified by Dinucleotide Differences in the Vitamin D-Responsive Elements of the Osteocalcin and Osteopontin Genes. Molecular Endocrinology, 10(11), 1444-1456. https://doi.org/10.1210/mend.10.11.8923469