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-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.
Jääskeläinen
T
T
Department of Biochemistry and Biotechnology and A. I. Virtanen Institute, University of Kuopio, Finland.
Pirskanen
A
A
Ryhänen
S
S
Palvimo
J J
JJ
Deluca
H F
HF
Mäenpää
P H
PH
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics*; D012516:Osteosarcoma / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D015675:Osteocalcin; D014212:Tretinoin; D004247:DNA; D003513:Cycloheximide