TY - JOUR
T1 - 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
AU - Staal, A.
AU - Van Wijnen, A. J.
AU - Birkenhäger, J. C.
AU - Pols, H. A.P.
AU - Prahl, J.
AU - DeLuca, H.
AU - Gaub, Marie Pierre
AU - Lian, J. B.
AU - Stein, G. S.
AU - Van Leeuwen, J. P.T.M.
AU - Stein, J. L.
PY - 1996/11/1
Y1 - 1996/11/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030445914&partnerID=8YFLogxK
U2 - 10.1210/mend.10.11.8923469
DO - 10.1210/mend.10.11.8923469
M3 - Article
C2 - 8923469
AN - SCOPUS:0030445914
SN - 0888-8809
VL - 10
SP - 1444
EP - 1456
JO - Molecular Endocrinology
JF - Molecular Endocrinology
IS - 11
ER -