Interaction of two novel 14-epivitamin D3 analogs with vitamin D3 receptor-retinoid X receptor heterodimers on vitamin D3 responsive elements.

This study provides a detailed and exact evaluation of the interactions between 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-dihydroxyvitamin 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 were more potent (14- and 75-fold, respectively) than 1,25(OH),D, in inhibiting cell proliferation and inducing cell differentiation. However, DNA-independent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limited protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA-dependent assays such 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 heterodimers to a direct repeat spaced by three nucleotides (DR3) type VDRE, The functional consequences of the ligand-VDR-RXR-VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14-epianalogs enhanced transcription of VDRE containing reporter constructs more efficiently than 1,25(OH)(2)D-3 in COS-1 and MCF-7 cells regardless of the presence of ketoconazole. Transactivation activity is suggested to be a cell-specific process because maximal transcriptional induction and the half-maximal transactivation concentration for each reporter construct were different 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 activity starts beyond the binding of the ligand-heterodimer (VDR-RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.