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

Mol Endocrinol. 1996 Nov;10(11):1444-56. doi: 10.1210/mend.10.11.8923469.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • Humans
  • Mice
  • Nucleic Acid Conformation
  • Osteocalcin / chemistry
  • Osteocalcin / genetics*
  • Osteocalcin / metabolism
  • Osteopontin
  • Osteosarcoma / genetics
  • Osteosarcoma / metabolism
  • Osteosarcoma / pathology
  • Point Mutation
  • Protein Conformation
  • Rats
  • Receptors, Calcitriol / chemistry*
  • Receptors, Calcitriol / immunology
  • Receptors, Calcitriol / metabolism
  • Receptors, Retinoic Acid / chemistry*
  • Receptors, Retinoic Acid / immunology
  • Receptors, Retinoic Acid / metabolism
  • Retinoid X Receptors
  • Sialoglycoproteins / chemistry
  • Sialoglycoproteins / genetics*
  • Sialoglycoproteins / metabolism
  • Steroids / metabolism
  • Transcription Factors / chemistry*
  • Transcription Factors / immunology
  • Transcription Factors / metabolism
  • Tumor Cells, Cultured
  • Vitamin D / metabolism*

Substances

  • Antibodies
  • Receptors, Calcitriol
  • Receptors, Retinoic Acid
  • Retinoid X Receptors
  • SPP1 protein, human
  • Sialoglycoproteins
  • Spp1 protein, mouse
  • Spp1 protein, rat
  • Steroids
  • Transcription Factors
  • Osteocalcin
  • Osteopontin
  • Vitamin D