Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes
Graduate School of Biomedical Sciences; Department of Internal Medicine III; Department of Cell Biology
Medical Subject Headings
Animals; Antibodies; Base Sequence; Binding Sites; Binding, Competitive; Humans; Mice; Nucleic Acid Conformation; Osteocalcin; Osteopontin; Osteosarcoma; Point Mutation; Protein Conformation; Rats; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoid X Receptors; Sialoglycoproteins; Steroids; Transcription Factors; Tumor Cells, Cultured; Vitamin D
Life Sciences | Medicine and Health Sciences
The 1 alpha,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 homo- and 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-alpha (RXR). The OC- and OP-VDR/RXR alpha 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/RXR alpha complexes may reflect specialized requirements for VD3 regulation of OC and OP gene expression in response to physiological cues mediating osteoblast differentiation.
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Citation: Mol Endocrinol. 1996 Nov;10(11):1444-56.
Molecular endocrinology (Baltimore, Md.)
Staal, Ada; Van Wijnen, Andre J.; Birkenhager, Jan C.; Pols, Huibert A. P.; Prahl, Jean; DeLuca, Hector F.; Gaub, Marie-Pierre; Lian, Jane B.; Stein, Gary S.; van Leeuwen, Johannes P. T. M.; and Stein, Janet L., "Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes" (1996). GSBS Student Publications. 1156.