The evolution of farnesoid X, vitamin D, and pregnane X receptors: insights from the green-spotted pufferfish (Tetraodon nigriviridis) and other non-mammalian species

<p>Abstract</p> <p>Background</p> <p>The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (<it>Petromyzon marinus</it>) and zebrafish (<it>Danio rerio</it>) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (<it>Tetraodon nigriviridis</it>), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the <it>Tetraodon </it>receptors.</p> <p>Results</p> <p><it>Tetraodon </it>FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. <it>Tetraodon </it>PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not.</p> <p>Conclusions</p> <p>Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for zebrafish and sterically bent bile acids for the pufferfish) and receptor selectivity that matches these differences in endogenous ligands. Our observations to date present an integrated picture of the co-evolution of bile salt structure and changes in the binding pockets of three nuclear hormone receptors across the species studied.</p

Thyroid receptor antagonism as a contributory mechanism for adipogenesis induced by environmental mixtures in 3T3-L1 cells

We previously demonstrated that indoor house dust extracts could induce adipogenesis in pre-adipocytes, suggesting a potential role for indoor contaminant mixtures in metabolic health. Herein, we investigated the potential role of thyroid receptor beta (TRβ) antagonism in adipogenic effects (dust-induced triglyceride accumulation and pre-adipocyte proliferation) following exposure to environmental mixtures (indoor house dust extracts). Concentrations of specific flame retardants were measured in extracts, and metabolic health information was collected from residents (n = 137). 90% of dust extracts exhibited significant adipogenic activity, &gt;60% via triglyceride accumulation, and &gt;70% via pre-adipocyte proliferation. Triglyceride accumulation was positively correlated with concentrations of each of twelve flame retardants, despite most being independently inactive; this suggests a putative role for co-exposures or mixtures. We further reported a positive correlation between dust-induced triglyceride accumulation and serum thyroid stimulating hormone concentrations, negative correlations with serum free triiodothyronine and thyroxine concentrations, and a positive and significant association between dust-induced triglyceride accumulation and residents' body mass index (BMI). We hypothesized that inhibition of TR antagonism might counteract these effects, and both addition of a TR agonist and siRNA knock-down of TR resulted in decreased dust-induced triglyceride accumulation in a subset of samples, bolstering this as a contributory mechanism. These results highlight a contributory role of environmental TR antagonism as a putative factor in metabolic health, suggesting that further research should evaluate this mechanism and determine whether in vitro adipogenic activity could have utility as a biomarker for metabolic health in residents

Chemical Mixtures Isolated from House Dust Disrupt Thyroid Receptor β Signaling

House dust is a source of exposure to chemicals that can impact hormone regulation. This study was designed to evaluate the potential of house dust mixtures ( n = 137) to disrupt thyroid hormone nuclear receptor signaling in a cell-based reporter assay and to examine associations with thyroid hormones (TH) measured in residents of the homes. Approximately 41% of the extracts (ranging from 10.5 to 4.097 μg of dust/mL) significantly antagonized thyroid receptor β (TRβ) signaling by 20-67% relative to the hormone control. The concentrations of 12 flame retardants (FRs) quantified in the mixtures were significantly correlated with TRβ antagonism; however, they were inactive when tested individually. We hypothesize that the observed antagonism is due to mixture effects or unidentified compounds that co-occur with FRs. Dust extract potency was significantly associated with free thyroxine (FT4, rs = -0.64, p &lt; 0.001), suggesting that more potent dust samples are associated with higher FT4 levels in residents. Overall, these results suggest that house dust is a significant source of exposure to TH-disrupting chemicals, and TRβ may have a role in mediating effects of exposure on TH levels. Additional studies are needed to identify the chemical(s) driving the observed effects on TRβ and to determine if these changes lead to any adverse outcomes

Molecular cloning, functional characterization, and evolutionary analysis of vitamin D receptors isolated from basal vertebrates.

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators

Molecular cloning, functional characterization, and evolutionary analysis of vitamin D receptors isolated from basal vertebrates.

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators

Evolutionary and Functional Diversification of the Vitamin D Receptor-Lithocholic Acid Partnership

<div><p>The evolution, molecular behavior, and physiological function of nuclear receptors are of particular interest given their diverse roles in regulating essential biological processes. The vitamin D receptor (VDR) is well known for its canonical roles in calcium homeostasis and skeletal maintenance. Additionally, VDR has received an increased amount of attention due to the discovery of numerous non-calcemic functions, including the detoxification of lithocholic acid. Lithocholic acid is a toxic metabolite of chenodeoxycholic acid, a primary bile acid. The partnership between the VDR and lithocholic acid has been hypothesized to be a recent adaptation that evolved to mediate the detoxification and elimination of lithocholic acid from the gut. This partnership is speculated to be limited to higher vertebrates (birds and mammals), as lower vertebrates do not synthesize the parent compound of lithocholic acid. However, the molecular functions associated with the observed insensitivity of basal VDRs to lithocholic acid have not been explored. Here we characterize canonical nuclear receptor functions of VDRs from select species representing key nodes in vertebrate evolution and span a range of bile salt phenotypes. Competitive ligand binding assays revealed that the receptor’s affinity for lithocholic acid is highly conserved across species, suggesting that lithocholic acid affinity is an ancient and non-adaptive trait. However, transient transactivation assays revealed that lithocholic acid-mediated VDR activation might have evolved more recently, as the non-mammalian receptors did not respond to lithocholic acid unless exogenous coactivator proteins were co-expressed. Subsequent functional assays indicated that differential lithocholic acid-mediated receptor activation is potentially driven by differential protein-protein interactions between VDR and nuclear receptor coregulator proteins. We hypothesize that the vitamin D receptor-lithocholic acid partnership evolved as a by-product of natural selection on the ligand-receptor partnership between the vitamin D receptor and the native VDR ligand: 1α,25-dihydroxyvitamin D₃, the biologically active metabolite of vitamin D₃.</p></div

Mammalian 2-hybrid analysis of VDR interaction with SRC/p160 coactivators in response to 1, 25D₃.

<p>The above graphs depict (A) lamprey VDR, (B) skate VDR, (C) bichir VDR, and (D) human VDR. Assays were conducted both in the presence (+) and absence (-) of pCDNA-RXR_WT. HepG2 cells were transiently transfected with pVP16-VDR as prey and pM-SRC1 (orange bars), pM-GRIP1 (blue bars), or pM-ACTR (yellow bars) as bait, along with the 5XGal4-TATA-Luc reporter and pRL-CMV as an internal luciferase control. Cells were exposed to 120 nM 1, 25D₃ in media for 24 hours. Protein-protein interaction was measured via dual-luciferase assays as described in the Materials and Methods. Data are represented as the mean fold interaction ± SEM (n = 4). Data are normalized to VDR + empty pM vector (no coactivators). Asterisks above bars represent a significant interaction between VDR and the SRC/p160 coactivators: *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Asterisks above brackets indicate the addition of RXR_WT significantly enhanced VDR-SRC/p160 interaction. The interaction between pVP16-skate VDR and pM-SRC1 was tested with an unpaired t-test: t₆ = 16.56, p < 0.0001 (†).</p

Analysis of LCA-mediated VDR transactivation with the SRC/p160 family of nuclear receptor coactivators.

<p>(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 RXR_WT (#) or the indicated SRC/p160 coactivator (Δ) had a significantly greater effect on VDR transactivation than either the SRC/p160 coactivator or RXR_WT alone. Data are represented as the average fold activation normalized to VDR alone ± SEM (n = 3).</p

Chemical structures of vertebrate bile alcohols, bile acids, and vitamin D receptor ligands.

<p>(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.</p