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
Authors
Staal, AdaVan 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.
Stein, Janet L.
UMass Chan Affiliations
Department of Cell BiologyDepartment of Internal Medicine III
Graduate School of Biomedical Sciences
Document Type
Journal ArticlePublication Date
1996-11-01Keywords
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 DLife Sciences
Medicine and Health Sciences
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Show full item recordAbstract
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.Source
Mol Endocrinol. 1996 Nov;10(11):1444-56.
DOI
10.1210/mend.10.11.8923469Permanent Link to this Item
http://hdl.handle.net/20.500.14038/32593PubMed ID
8923469Related Resources
ae974a485f413a2113503eed53cd6c53
10.1210/mend.10.11.8923469
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