RETINOID-X RECEPTORS STIMULATE AND 9-CIS RETINOIC ACID INHIBITS 1,25-DIHYDROXYVITAMIN D3-ACTIVATED EXPRESSION OF THE RAT OSTEOCALCIN GENE

The vitamin D receptor (VDR) binds the vitamin D-responsive element (V DRE) as a heterodimer with an unidentified receptor auxiliary factor ( RAF) present in mammalian cell nuclear extracts. VDR also interacts wi th the retinoid X receptors (RXRs), implying that RAF may be related t o the RXRs. Here we demonstrate that highly purified HeLa cell RAF con tained RXRbeta immunoreactivity and that both activities copurified an d precisely coeluted in high-resolution hydroxylapatite chromatography . Furthermore, an RXRbeta-specific antibody disrupted VDR-RAF-VDRE com plexes in mobility shift assays. These data strongly indicate that HeL a RAF is highly related to or is identical to RXRbeta. Consequently, t he effect of the 9-cis retinoic acid ligand for RXRs was examined in 1 ,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression system s. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 muM) ma rkedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also inte rfered with vitamin D responsiveness, but it was consistently less pot ent than the 9-cis isomer. Transient transfection studies revealed tha t attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Further more, overexpression of both RXRbeta and RXRalpha augmented 1,25(OH)2D 3 responsiveness in transient expression studies. Direct analysis of V DRE binding in mobility shift assays demonstrated that heteromeric int eractions between VDR and RXR were enhanced by 1,25(OH)2D3 and were no t affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a reg ulatory mechanism for osteocalcin gene expression that involves 1,25(O H)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis reti noic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs a way from VDR-mediated transcription and towards other RXR-dependent tr anscriptional pathways.