Abstract

The vitamin D receptor (VDR) mediates vitamin D signaling in numerous physiological and pharmacological processes, including bone and calcium metabolism, cellular growth and differentiation, immunity, and cardiovascular function. Although transcriptional regulation by VDR has been investigated intensively, an understanding of ligand-selective dynamic VDR conformations remains elusive. Here, we examined ligand-dependent dynamic interactions of VDR with retinoid X receptor (RXR), steroid receptor coactivator 1 (SRC-1), and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) in cells using fluorescence resonance energy transfer (FRET) and chromatin immunoprecipitation (ChIP) assays. We compared the effects of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], lithocholic acid (LCA), and (25R)-25-adamantyl-1α,25-dihydroxy-2-methylene-22,23-didehydro-19,26,27-trinor-20-epivitamin D₃ (ADTT), a partial agonist/antagonist vitamin D derivative. In the absence of ligand, VDR homodimers were preferred to RXR heterodimers and were associated with SMRT. 1,25(OH)₂D₃ induced heterodimerization with RXR, dissociation of SMRT, and association of SRC-1. LCA and ADTT induced those effects to a lesser extent at concentrations that did not induce expression of the VDR target gene CYP24A1 in human embryonic kidney (HEK) 293 cells. Unlike in HEK293 cells, ADTT increased CYP24A1 expression in HCT116 cells and increased the association of VDR and SMRT on the CYP24A1 promoter. The results indicate that ligand-selective conformation may lead to unique cofactor complex formation in a cell context-dependent manner. The combination of FRET and ChIP assays is a powerful tool useful in understanding ligand-selective dynamic VDR conformations and the development of selective VDR modulators.