Interaction of two novel 14-epivitamin D-3 analogs with vitamin D-3 receptor-retinoid X receptor heterodimers on vitamin D-3 responsive elements

This study provides a detailed and exact evaluation of the interactions bet ween vitamin D-3 receptor (VDR), retinoid X receptor (RXR), and vitamin D-3 responsive elements (VDREs) mediated by two novel 14-epianalogs of 1,25-di hydroxyvitamin D [1,25(OH)(2)D-3], 19-nor-14-epi-23-yne-1,25(OH)(2)D-3 (TX 522) and 19-nor-14,20bisepi-23-yne-1,25(OH)(2)D-3 (TX 527), Both analogs we re more potent (14- and 75-fold, respectively) than 1,25(OH),D, in inhibiti ng cell proliferation and inducing cell differentiation. However, DNA-indep endent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limi ted protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA-dependent assays su ch as gel shift experiments revealed that those analogs were slightly more potent (3-7 times) than 1,25(OH),D, in enhancing binding of VDR-RXR heterod imers to a direct repeat spaced by three nucleotides (DR3) type VDRE, The f unctional consequences of the ligand-VDR-RXR-VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14-epia nalogs enhanced transcription of VDRE containing reporter constructs more e fficiently than 1,25(OH)(2)D-3 in COS-1 and MCF-7 cells regardless of the p resence of ketoconazole. Transactivation activity is suggested to be a cell -specific process because maximal transcriptional induction and the half-ma ximal transactivation concentration for each reporter construct were differ ent in both cell lines. The superagonistic transactivation activity closely resembled the biological potency of these analogs on the inhibition of MCF -7 cell proliferation. These data clearly indicate that superagonistic acti vity starts beyond the binding of the ligand-heterodimer (VDR-RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.