title: 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.
abstract: 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.
authors.affiliation: Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Staal
authors.forename: A J
authors.initials: AJ
authors.lastname: van Wijnen
authors.forename: J C
authors.initials: JC
authors.lastname: Birkenhäger
authors.forename: H A
authors.initials: HA
authors.lastname: Pols
authors.forename: J
authors.initials: J
authors.lastname: Prahl
authors.forename: H
authors.initials: H
authors.lastname: DeLuca
authors.forename: M P
authors.initials: MP
authors.lastname: Gaub
authors.forename: J B
authors.initials: JB
authors.lastname: Lian
authors.forename: G S
authors.initials: GS
authors.lastname: Stein
authors.forename: J P
authors.initials: JP
authors.lastname: van Leeuwen
authors.forename: J L
authors.initials: JL
authors.lastname: Stein
mesh_terms: D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D051379:Mice; D009690:Nucleic Acid Conformation; D015675:Osteocalcin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053495:Osteopontin; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D047488:Retinoid X Receptors; D012795:Sialoglycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013256:Steroids / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014807:Vitamin D / Q000378:metabolism*
chemical_list: D000906:Antibodies; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; C506897:Spp1 protein, rat; D013256:Steroids; D014157:Transcription Factors; D015675:Osteocalcin; D053495:Osteopontin; D014807:Vitamin D