Distinct conformational changes induced by 20-epi analogues of 1 alpha,25-dihydroxyvitamin D3 are associated with enhanced activation of the vitamin D receptor.

The relative affinities of the 1 alpha,25-dihydroxyvitamin D3 (1,25-D3) analogues 20-epi-1 alpha,25-dihydroxyvitamin D3 (IE) and 20-epi-22-oxa-24a,26a,27a-tri-homo-1 alpha,25-dihydroxyvitamin D3 (ID) to the nuclear vitamin D receptor (VDR) are similar to that of 1,25-D3, but their antiproliferative action is 1000-fold greater. We tested whether the greater antiproliferative effect of these analogues is due to a differential activation of the VDR. In ROS 17/2.8 cells, the effective doses required to produce 50% maximal stimulation (ED50) of transfected reporter genes driven by either the osteocalcin or the osteopontin vitamin D-response elements (VDRE) were 5 x 10(-9) M, 10(-10) M, and 10(-11) M for 1,25-D3, ID, and IE, respectively. Similar results were obtained when recombinant human VDR was cotransfected into CV-1 cells with an osteocalcin VDRE-reporter plasmid. We found that in vitro the sensitivity of 1,25-D3-induced and analogue-induced receptors to proteases was different. The ED50 for binding to VDRE, as determined by electrophoretic mobility shift assays, was significantly higher for 1,25-D3-induced than for analogue-induced VDR. The concentration of retinoid X receptor (RXR) was significantly lower in 1,25-D3-induced than analogue-induced VDR complexes with VDRE. We therefore conclude that IE and ID augment transcriptional activity of VDR more than 1,25-D3 does, by producing conformational changes that enhance dimerization of VDR with RXR. We suggest that these conformational changes are due to differences in the contact sites of the 20-epi analogues and 1,25-D3 with the VDR.