Vitamin D₃ Derivatives with Adamantane or Lactone Ring Side Chains are Cell Type-Selective Vitamin D Receptor Modulators

Abstract

The vitamin D receptor (VDR) mediates the biological actions of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], the active form of vitamin D, which regulates calcium homeostasis, immunity, cellular differentiation, and other physiological processes. We investigated the effects of three 1,25(OH) 2 D 3 derivatives on VDR function. AD47 has an adamantane ring and LAC67a and LAC67b have lactone ring substituents at the side chain position. These vitamin D derivatives bind to VDR but do not stabilize an active cofactor conformation. In a VDR transfection assay, AD47 and LAC67b act as partial agonists and all three compounds inhibit VDR activation by 1,25(OH) 2 D 3 . The derivatives enhanced the heterodimerization of VDR with the retinoid X receptor, an effect unrelated to agonist/antagonist activity. AD47 and LAC67b weakly induced recruitment of the SRC-1 cofactor to VDR, and all three derivatives inhibited the recruitment of p160 family cofactors to VDR induced by 1,25(OH) 2 D 3 . It is noteworthy that AD47 induced DRIP205 recruitment as effectively as 1,25(OH) 2 D 3 , whereas LAC67a and LAC67b were not effective. We examined the expression of endogenous VDR target genes and the nuclear protein levels of VDR and cofactors in several cell lines, including cells derived from intestine, bone, and monocytes, and found that the vitamin D 3 derivatives act as cell type-selective VDR modulators. The data indicate that side chain modification is useful in the development of VDR antagonists and tissue-selective modulators. Further elucidation of the molecular mechanisms of action of selective VDR modulators will be essential for their clinical application.The vitamin D receptor (VDR; NR1I1) is a member of the nuclear receptor superfamily that regulates physiological processes, including cell growth and differentiation, embryonic development, and metabolic homeostasis (Makishima, 2005). Nuclear receptor transcriptional activity is modulated by ligands such as steroids, retinoids, and other lipid-soluble compounds. Upon ligand binding, nuclear receptors undergo a conformational change in the cofactor binding site and activation function 2 (AF2) helices, which results in dynamic exchange of cofactor complexes, allowing nuclear receptors to modulate the transcription of specific target genes (Rosenfeld et al., 2006). In the absence of ligand, corepressors bind to the AF2 surface composed of portions of helix (H) 3, loop 3-4, H4/5, and H11. Ligand binding reduces the receptor affinity for corepressors and recruits coactivators to the altered AF2 surface formed by repositioning of H12. Recent studies suggest that DNA sequence-specific effects of transcription factor activity are associated with site-specific interaction with cofactor complexes (Rosenfeld et al., 2006). Chemical modification of ligands may induce AF2 conformations and cofactor interactions distinct from those of natural ligands and can result in cell type-selective modulation of target gene expression.The active form of vitamin D 3 , 1,25-dihydroxyvitamin D...

Hairless Modulates Ligand-Dependent Activation of the Vitamin D Receptor-Retinoid X Receptor Heterodimer

“…pCMX-FLAG-VDR, pCMX-VP16-VDR, pCMX-GAL4-SRC-1, pC-MX-GAL4-N-CoR, pCMX-GAL4-SMRT, Spp×3-tk-LUC, hCYP3A4-ER6×3-tk-LUC, and MH100 (UAS)×4-tk-LUC were previously reported. 3,6,16) Mutations were introduced into pCMX-FLAG-VDR using a QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, U.S.A.) and pCMX-FLAG-VDR mutants were sequenced before use to verify DNA sequence fidelity.…”

“…Transfections in HEK293 cells were performed by the calcium phosphate coprecipitation method. 16) Transfection experiments used 15 ng of each expression plasmid in combination with 50 ng of reporter plasmid and 10 ng of pCMX-β-galactosidase in each well of a 96-well plate. Eight hours after transfection, compounds were added.…”

Lithocholic acid derivatives act as selective vitamin D receptor modulators without inducing hypercalcemia

“…Transfections in HEK293 cells were performed by the calcium phosphate coprecipitation assay as described previously (10). Eight hours after transfection, compounds were added.…”

“…IR1x3-tk-LUC, hCYP3A4-ER6x3-tk-LUC, Som-LUC, and GAL4-responsive MH100(UAS)x4-tk-LUC reporters were used to evaluate the activities of FXR, PXR, GPBAR1, and GAL4-VDR, respectively (13,17). The ligand binding domain from VDR was inserted into pGEX vector (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) to generate pGEX-VDR (10). All plasmids were sequenced before use to verify DNA sequence fidelity.…”

“…The ligand binding domains of human VDR (GenBank accession number NM_000376) was inserted into the pCMX-GAL4 vector to make pCMX-GAL4-VDR (10). Fragments of human farnesoid X receptor (FXR; GenBank accession number NM_005123), pregnane X receptor (PXR; GenBank accession number NM_022002), and G protein-coupled bile acid receptor 1 (GPBAR1; GenBank accession number NM_170699) were inserted into the pCMX vector to make pCMX-FXR, pCMX-PXR, and pCMXGPBAR1, respectively (13,16).…”