Functional interference between AP-1 and the vitamin D receptor on osteocalcin gene expression in human osteosarcoma cells

Eur J Biochem. 1994 Aug 15;224(1):11-20. doi: 10.1111/j.1432-1033.1994.tb19989.x.


The binding of transcription factor AP-1 and vitamin D receptor (VDR) to the composite AP-1 plus vitamin-D-responsive promoter region (AP-1 + VDRE) of the human osteocalcin gene was characterized in osteocalcin-producing (MG-63) and non-producing (U2-Os, SaOs-2) human osteosarcoma cell lines. In mobility-shift assays with AP-1 + VDRE, AP-1, and VDRE probes and nuclear extracts from these cells, one AP-1-specific and two VDR-specific (fast and slow mobility) interactions were observed. Characterization of the complexes indicated that AP-1 and VDR do not bind simultaneously to the AP-1 + VDRE oligonucleotide. Intensity of the complexes was greatly influenced by cell density: in MG-63 and SaOs-2 cells, AP-1 binding was strong during the proliferative period disappearing at confluency whereas, in U2-Os cells, AP-1 binding was prominent also at the confluent stage. Furthermore, MG-63 cells possessed the faster migrating VDR complex at all stages of confluency whereas, in U2-Os and SaOs-2 cells, it was very weak or absent. There were no detectable differences in the levels of VDR protein between these cell lines. In U2-Os cells, the level of c-jun mRNA was higher than in the other two cell lines, whereas none of these cell lines exhibited detectable levels of c-fos mRNA at the confluent stage. Exogenous c-Jun protein effectively blocked the VDR-DNA interaction. Further, all these cell lines expressed mRNA for retinoid X receptor alpha (RXR alpha), the factor suggested to be required for the VDR-DNA interaction. The presence of an accessory factor in the VDR-DNA complexes was indirectly shown by treatment of the cells with 9-cis retinoic acid and by cycloheximide. Both treatments reduced VDR binding without affecting the VDR protein level. These results suggest that AP-1 interferes with VDR binding to the AP-1 + VDRE element and that the vitamin D responsiveness of the osteocalcin gene correlates with weak AP-1 binding and strong binding of the faster migrating VDR complex.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence
  • Blotting, Northern
  • Blotting, Western
  • Cycloheximide / pharmacology
  • DNA / genetics
  • DNA / metabolism
  • Gene Expression*
  • Humans
  • Molecular Sequence Data
  • Osteocalcin / biosynthesis
  • Osteocalcin / genetics*
  • Osteosarcoma / metabolism*
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic*
  • Proto-Oncogene Proteins c-fos / genetics
  • Proto-Oncogene Proteins c-fos / metabolism
  • Proto-Oncogene Proteins c-jun / genetics
  • Proto-Oncogene Proteins c-jun / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Receptors, Calcitriol / genetics
  • Receptors, Calcitriol / metabolism*
  • Transcription, Genetic / genetics
  • Tretinoin / pharmacology
  • Tumor Cells, Cultured


  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • RNA, Messenger
  • Receptors, Calcitriol
  • Osteocalcin
  • Tretinoin
  • DNA
  • Cycloheximide