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

The 1 alpha,25-dihydroxyvitamin D-3 (VD3)-dependent stimulation of ost eocalcin (OC) and osteopontin (OF) gene transcription in bone tissue i s mediated by interactions of trans-activating factors with distinct V D3-responsive elements (VDREs). Sequence variation between the OC- and OP-VDRE steroid hormone half-elements provides the potential for reco gnition 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 establi shed. To determine the identity of these complexes, we performed gel s hift immunoassays with nuclear proteins from ROS 17/2.8 osteoblastic c ells using a panel of monoclonal antibodies. We show that VD3- inducib le complexes interacting with the OC- and OP-VDREs represent two disti nct heterodimeric complexes, each composed of the vitamin D receptor ( VDR) and the retinoid X receptor-alpha (RXR). The OC- and OP-VDR/RXR a lpha heterodimers are immunoreactive with RXR antibodies and several a ntibodies directed against the ligand-binding domain of the VDR. Howev er, while the OC-VDRE complex is also efficiently recognized by specif ic monoclonal antibodies contacting epitopes in or near the VDR DNA-bi nding domain (DBD) (between amino acids 57-164), the OP-VDRE complex i s not efficiently recognized by these antibodies. By systematically in troducing a series of point-mutations in the OC-VDRE, we find that two internal nucleotides of the proximal OC-VDRE half-site (nucleotide -4 49 and -448; 5'-AGGACA) determine differences in VDR immunoreactivity. These results are consistent with the well established polarity of RX R heterodimer binding to bipartite hormone response elements, with the VDR recognizing the 3'-half-element. Furthermore, our data suggest th at the DBD of the VDR adopts different protein conformations when cont acting distinct VDREs. Distinctions between the OC- and OP-VDR/RXR alp ha complexes may reflect specialized requirements for VD3 regulation o f OC and OP gene expression in response to physiological cues mediatin g osteoblast differentiation.