Carboxylic ester antagonists of 1α,25-dihydroxyvitamin D3 show cell-specific actions Original Research Article

bstract Background: The nuclear hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) acts through the transcription factor vitamin D receptor (1α,25(OH)2D3 receptor, VDR) via combined contact with the retinoid X receptor (RXR), coactivator proteins and specific DNA binding sites (1α,25(OH)2D3 response elements, VDREs). Ligand-mediated conformational changes of the VDR are the basis of the molecular mechanisms of nuclear 1α,25(OH)2D3 signaling. Cell-specific VDR antagonists would allow to dissect and fine regulate the pleiotropic 1α,25(OH)2D3 endocrine system affecting the regulation of calcium homeostasis, bone mineralization and other cellular functions. Results: Two carboxylic ester analogues of 1α,25(OH)2D3, ZK159222 and ZK168281, which have additional cyclopropyl rings and allylic alcohol substructures in their side chain, were characterized in different 1α,25(OH)2D3 target tissues as functional antagonists of 1α,25(OH)2D3 signaling. In all tested systems, ZK168281 showed lower residual agonistic activity and higher antagonistic effects than ZK159222, but the strength of these effects was cell-specific. Both antagonists were shown to act via the same mechanisms: they selectively stabilize an antagonistic conformation of the ligand-binding domain of the VDR within VDR–RXR–VDRE complexes, which then inhibits the interaction of the VDR with coactivator proteins and an induction of transactivation. Interestingly, cells that have been treated with antagonists were found to contain VDR–RXR heterodimers in a different conformation than cells that were stimulated with an agonist. Moreover, the strength of the functional antagonism of ZK159222 and ZK168281 appears to depend on the VDR/RXR expression ratio and high RXR levels were found to reduce the antagonistic effect of both compounds. In support of this observation, the overexpression of an transactivation function 2 (AF-2) deletion mutant of RXR resulted for both ZK159222 and ZK168281 in a reduced agonistic activity and an increased antagonistic effect. Conclusions: A novel, more potent VDR antagonist, ZK168281, was identified, which stabilizes VDR–RXR heterodimers in living cells in a different conformation than agonists. In addition, the VDR/RXR ratio was found as the major discriminating factor for understanding cell-specific effects of VDR antagonists. Article Outline