TRANSCRIPTIONAL ACTIVITY OF A FLUORINATED VITAMIN-D ANALOG ON VDR-RXR-MEDIATED GENE-EXPRESSION

The transcriptional activity of the hexafluorinated derivative of 1,25 -dihydroxyvitamin D-3 [1,25-(OH)(2)D-3], 26,26,26,27,27,27-hexafluoro- 1,25-dihydroxyvitamin D-3 [F-6-1,25-(OH)(2)D-3], was examined in cultu red cells by a transient expression assay (CAT assay) using expression vectors for the rat nuclear vitamin D-3 receptor (VDR) and the rat 9- cis-retinoic acid receptor (RXR beta), and a reporter plasmid containi ng a consensus vitamin D-3 response element (VDRE) consisting of two d irectly repeated AGGTCA motifs spaced by 3 bp (DR3). At physiological concentrations, the transcriptional activity of F-6-1,25-(OH)(2)D-3 wa s 2-4 times more potent than that of 1,25-(OH)(2)D-3 in both nontarget (HeLa) and target (UMR106) cells for 1,25-(OH)(2)D-3. The transcripti onal activity of F-6-1,25-(OH)(2)D-3 was also higher when the endogeno us target gene (osteopontin), which has a VDRE related to the DR3 in i ts promoter, was induced. A gel-shift assay using DR3 as a probe and i n vitro synthesized receptors showed that the ligand-induced DNA bindi ng of VDR required RXR to form a heterodimer. Moreover, in this assay we found that F-6-1,25-(OH)(2)D-3 induced the receptor-DNA complex at a 10-fold lower concentration than 1,25-(OH)(2)D-3 without influencing the dissociation kinetics. However, the binding affinity of F-6-1,25- (OH)(2)D-3 for VDR was slightly lower than that of 1,25-(OH)(2)D-3. Th e increased DNA binding of ligand-bound VDR by introducing hexafluorin es into 1,25-(OH)(2)D-3 may potentiate the transcriptional activity. T hus, the higher biological activity of F-6-1,25-(OH)(2)D-3 may be exer ted at least in part by enhanced transcriptional activity.