Molecular Mechanism of the Vitamin D Antagonistic Actions of (23S)-25-Dehydro-1α-Hydroxyvitamin D3-26,23-Lactone Depends on the Primary Structure of the Carboxyl-Terminal Region of the Vitamin D Receptor

Abstract

We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.

On the Mechanism Underlying (23S)-25-Dehydro-1α(OH)-vitamin D3-26,23-lactone Antagonism of hVDRwt Gene Activation and Its Switch to a Superagonist

“…The flexible docking results also provide a rationale for how MK can function as a partial agonist (7,10) or superagonist (Fig. 4, F and G).…”

“…Therefore, MK makes vdW interactions with the helix-3/helix-12 interface residues in Region 1 and explains why MK is not a complete antagonist in hVDRwt (Figs. 2 and 4A) (7,10,16).…”

“…1); however, the mode of antagonism is different for these two molecules (7,10). The ZK side-chain molecular volume is significantly enhanced relative to 1,25D; therefore, ZK acts as a traditional NR antagonist by blocking VDR helix-12 closure (5).…”

“…1) undergoes a 1,4-Michael addition (i.e. formation of a covalent MK-VDR adduct) with Cys-403 and/or Cys-410 of the human VDR (hVDR), neither of which are present in the rVDR primary sequence (10). Yet another hypothesis proposes that the energetic stability of the rVDR closed helix-12 conformer is enhanced relative to hVDR (7).…”

“…Therefore, to gain a molecular understanding to MK antagonism of 1,25D-hVDR gene activation and species specificity (7,10,(13)(14)(15)(16)(17) we applied traditional structure-function assays, molecular modeling, and a vitamin D sterol-VDR conformational ensemble model (18,19). Our comprehensive structure-function studies demonstrate that MK antagonism hinges on its reduced side-chain molecular volume allowing it to bind favorably to two distinct regions within the hVDRwt LBP.…”