Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells

Abstract

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 (U2-0s, 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 U2-0s 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 U2-0s 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 U2-0s 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 a (RXRa), 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.

Steroid hormone receptor expression and action in bone

“…These results were later confirmed by Jaaskelainen et al [236]. mRNAs encoding RXRα and RXRβ have been demonstrated in rat osteosarcoma cells [232], rat bone [328] and MC3T3-E1 cells [329].…”

A conserved lysine in the estrogen receptor DNA binding domain regulates ligand activation profiles at AP-1 sites, possibly by controlling interactions with a modulating repressor

“…7A) abolished unliganded TRα activity, and actually allowed unliganded TRα to suppress AP-1 activity below basal, just as the ERα.K206A mutation abolished anti-estrogen activation and allowed anti-estrogens to suppress AP-1 activity below basal. Thus, the K72A mutation allows TRα to enhance AP-1 dependent transcription in the presence of T 3 , just as ERα.K206A allows ERα to activate AP-1 activity in response to estradiol, and equivalent K > A mutations allow other nuclear receptors to enhance AP-1 activity in response to their cognate ligands [10,11,17]. …”

Steroid hormone receptor expression and action in bone

“…VDR mRNA has also been demonstrated in human, rat and mouse osteoblastic cells [75,76,130,232,233] and localized to rat and human osteoblasts by in situ hybridization [234] and in situ RT-PCR [235]. VDR protein has also been detected in rat and human osteosarcoma cells [236,237] and has been localized to the osteoblasts in rat bone by immunocytochemistry [238][239][240]. Johnson et al [240] specifically studied the expression of the VDR in rats throughout skeletal development.…”