Selective recognition of vitamin D receptor conformations mediates promoter selectivity of vitamin D analogs

The transcription factor VDR is the nuclear receptor for 1 alpha,25-dihydro xyvitamin D-3 (VD) and the mediator of all genomic actions of the nuclear h ormone and its synthetic analogs. The sharp biological profile of the model VD analog 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1 '(E),3'(E )-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) (i.e., its h igh antiproliferative effect combined with low calcemic actions) has been c orrelated with the selectivity of EB1089 to activate heterodimeric complexe s of VDR with its partner retinoid X receptor (RXR) on VD response elements (VDREs). These VDREs are formed by an inverted palindromic arrangement of two hexameric core binding motifs spaced by nine nucleotides (IP9) rather t han VDREs that are formed by direct repeats with three intervening nucleoti des (DR3). In this report, ligand-dependent gel-shift assays were used for a comparison of the ability of VD and EB1089 to stabilize VDR-RXR heterodim ers on these two VDRE types. The gel-shift assays revealed EB1089 to be mor e sensitive for complexes on IP9-type VDREs than on DR3-type VDREs. In addi tion, a gel-shift clipping method was established to identify and compare c omplexes of ligand-stabilized VDR-RXR heterodimers on different VDREs. On e ach VDRE, two complexes could be discriminated that seemed to contain diffe rent functional conformations of the VDR and allowed a more differential vi ew on DNA-complexed VDR-RXR heterodimers. The VDR-RXR conformation (which w as more ligand-sensitive) gained through EB1089 a higher affinity (7-fold) for DNA binding and a more sensitive (9-fold) activation of an IP9-type VDR E than of a DR3-type VDRE, whereas with the natural hormone VD, no VDRE-typ e preference could be observed. This indicates that promoter selectivity of VDR ligands is based on their property to selectively increase affinity fo r VDREs and very sensitively stabilize VDR conformations in VDR-RXR-VDRE co mplexes.