Fig 1.
Chemical structures of vertebrate bile alcohols, bile acids, and vitamin D receptor ligands.
(A) The structure of cholesterol, from which all bile acids and alcohols are derived. The left panel (B-D) depicts representative bile alcohols: (B) 5α-petromyzonol, a C24 bile alcohol that is a unique and minor component of the lamprey bile alcohol pool, (C) 5β-scymnol, the dominant bile alcohol of cartilaginous fish, and (D) 5α-cyprinol, the bile alcohol of zebrafish and other Cypriniformes. The middle panel (E-G) depicts representative bile acids: (E) the C27 trihydroxy bile acid that is the major bile acid of the Japanese medaka, and the two dominant C24 bile acids of vertebrates: (F) cholic acid (CA), and (G) chenodexocycholic acid (CDCA), the parent compound of LCA. The right panel depicts the two VDR ligands in this study: (H) lithocholic acid (LCA), the toxic metabolite of CDCA, and (I) 1α,25-dihydroxyvitamin D3 (1,25D3), the biologically active form of vitamin D3.
Fig 2.
VDR competitive ligand binding assays for LCA in the presence of 1,25D3.
Transfected cell lysates containing expressed VDR and human RXRWT were incubated with a saturating concentration of 1,25D3 (4 nM) and a range of LCA concentration (0–1 mM). Reactions were incubated at 4°C for 18 hours. Bound and free ligands were separated as described in the Materials and Methods. The affinity of LCA for each VDR ortholog was determined by calculating the dissociation constant (Ki) using the Cheng-Prusoff equation. The above graphs represent the combined specific binding data from three separate experiments (mean ± SEM).
Table 1.
VDR Transactivation mediated by 100 μM LCA
Fig 3.
Analysis of VDR-RXR interactions in response to LCA.
The top panel (A) illustrates the effect of exogenous human RXR on VDR transactivation in response to 100 μM LCA in transient transactivation assays. Human RXR constructs included wild-type RXR (RXRWT) and the RXR mutant lacking the c-terminal AF2 region (RXRAF2). VDR activation was measured via dual-luciferase assays and data were analyzed via 2-way ANOVA followed by Bonferroni’s multiple comparisons test. Asterisks over bars indicate a significant increase in VDR transactivation compared to the VDR only control (black bars), and asterisks over brackets indicate a significant difference in VDR activation in the presence of RXRWT (blue bars) vs. RXRAF2 (orange bars): *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Data are represented as the average fold activation normalized to VDR alone ± SEM (n = 3). The bottom panel (B and C) illustrates results from mammalian 2-hybrid assays that analyzed VDR-RXR heterodimerization in response to 100 μM LCA. VDR-RXR protein-protein interaction was measured via dual-luciferase assays, and analyzed via 2-way ANOVA followed by Bonferroni’s multiple comparisons test. Asterisks above bars represent a significant interaction compared to the VDR only control (black bars): *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Asterisks over the brackets indicate that the addition of the SRC1 coactivator significantly increased VDR-RXR interaction (striped bars) compared to VDR-RXR interaction in the absence of SRC1 (solid bars). Data are represented as the average fold interaction ± SEM (n = 3).
Fig 4.
Analysis of LCA-mediated VDR transactivation with the SRC/p160 family of nuclear receptor coactivators.
(A) and (B) illustrate the effects of exogenous human SRC/p160 nuclear receptor coactivators on VDR transactivation in response to 100 μM LCA in transient transactivation assays. The effect of the SRC/p160 coactivators on VDR transactivation was analyzed via 2-way ANOVA followed by Bonferroni’s multiple comparisons test. Asterisks represent a significant difference in transactivation compared to VDR in the absence of coactivators (black bars): *** = p < 0.001, ** = p < 0.01, * = p < 0.05. The Δ and # symbols indicate that the co-transfection of RXRWT (#) or the indicated SRC/p160 coactivator (Δ) had a significantly greater effect on VDR transactivation than either the SRC/p160 coactivator or RXRWT alone. Data are represented as the average fold activation normalized to VDR alone ± SEM (n = 3).
Fig 5.
Analysis of protein-protein interactions between VDR and SRC/p160 nuclear receptor coactivators.
(A) and (B) depict the results of mammalian 2-hybrid assays that assessed LCA-mediated protein-protein interactions between VDR and members of the SRC/p160 family of nuclear receptor coactivators. Significant VDR-SRC protein-protein interactions were analyzed via 2-way ANOVA followed by Bonferroni’s multiple comparisons test. Asterisks represent significant VDR-SRC interaction: *** = p < 0.001, ** = p < 0.01, * = p < 0.05. The Δ symbol indicates that the cotransfection of RXRWT significantly increased VDR-SRC/p160 interaction compared to the absence of RXRWT. Data are represented as the average fold activation normalized to VDR alone ± SEM (n = 3).
Fig 6.
Heatmap depicting the results of the bioinformatic summary analysis.
Analysis included mammalian 2-hybrid (M2H) and transient transactivation (TT) data for lamprey (lVDR), skate (sVDR), bichir (bVDR), human (hVDR), zebrafish (zfVDRa, zfVDRb) and medaka (mVDRa, mVDRb) in response to LCA and 1,25D3. The Picket Plot to the right of the heat map indicates the presence (black box) or absence (no box) of coregulators in each assay. Data were normalized for each assay (rows) across all species (columns) to account for magnitudinal response differences. The resulting matrix was subjected to unsupervised, hierarchical clustering using Manhattan distance and complete linkage. Bootstrap resampling was performed over the assays according to presence/absence of each of the coregulators to identify drivers of the overall cluster pattern as well as subclusters. For each of 10,000 bootstrap samples of assays, the accuracy was measured by counting the number of times the overall cluster pattern and species subclusters [lVDR, bVDR, sVDR, mVDRb and zfVDRb] and [mVDRa, zfVDRa, hVDR] were identical compared to the raw data.