FUNCTIONAL INTERFERENCE BETWEEN AP-1 AND THE VITAMIN-D RECEPTOR ON OSTEOCALCIN GENE-EXPRESSION IN HUMAN OSTEOSARCOMA CELLS

The binding of transcription factor AP-1 and vitamin D receptor (VDR) to the composite AP-I plus vitamin-D-responsive promoter region (AP-1VDRE) of the human osteocalcin gene was characterized in osteocalcin-p roducing (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 pr obes and nuclear extracts from these cells, one AP-1-specific and two VDR-specific (fast and slow mobility) interactions were observed. Char acterization of the complexes indicated that AP-1 and VDR do not bind simultaneously to the AP-1+VDRE oligonucleotide. Intensity of the comp lexes was greatly influenced by cell density: in MG-63 and SaOs-2 cell s, AP-1 binding was strong during the proliferative period disappearin g at confluency whereas, in U2-Os cells, AP-1 binding was prominent al so at the confluent stage. Furthermore, MG-63 cells possessed the fast er 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 detectabl e differences in the levels of VDR protein between these cell lines. I n U2-Os cells, the level of c-jun mRNA was higher than in the other tw o cell lines, whereas none of these cell lines exhibited detectable le vels of c-fos mRNA at the confluent stage. Exogenous c-Jun protein eff ectively blocked the VDR-DNA interaction. Further, all these cell line s 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 ost eocalcin gene correlates with weak AP-1 binding and strong binding of the faster migrating VDR complex.