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Functional evolution of the vitamin D and pregnane X receptors.

ABSTRACT: The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the Ciona VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (Petromyzon marinus), zebrafish (Danio rerio), and African clawed frog (Xenopus laevis).Human, mouse, frog, zebrafish, and lamprey VDRs were found to have similar ligand selectivities for vitamin D derivatives. In contrast, using cultured primary hepatocytes, only zebrafish showed evidence of PXR-mediated induction of enzyme expression, with increases in testosterone 6beta-hydroxylation activity (a measure of cytochrome P450 3A activity in other species) and flurbiprofen 4-hydroxylation activity (measure of cytochrome P450 2C activity) following exposure to known PXR activators. A separate assay in vivo using zebrafish demonstrated increased hepatic transcription of another PXR target, multidrug resistance gene (ABCB5), following injection of the major zebrafish bile salt, 5alpha-cyprinol 27-sulfate. The PXR target function, testosterone hydroxylation, was detected in frog and sea lamprey primary hepatocytes, but was not inducible in these two species by a wide range of PXR activators in other animals. Analysis of the sea lamprey draft genome also did not show evidence of a PXR gene.Our results show tight conservation of ligand selectivity of VDRs across vertebrate species from Agnatha to mammals. Using a functional approach, we demonstrate classic PXR-mediated effects in zebrafish, but not in sea lamprey or African clawed frog liver cells. Using a genomic approach, we failed to find evidence of a PXR gene in lamprey, suggesting that VDR may be the original NR1I gene.

SUBMITTER: Reschly EJ

PROVIDER: S-EPMC2263054 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Functional evolution of the vitamin D and pregnane X receptors.

Reschly Erica J EJ Bainy Afonso Celso Dias AC Mattos Jaco Joaquim JJ Hagey Lee R LR Bahary Nathan N Mada Sripal R SR Ou Junhai J Venkataramanan Raman R Krasowski Matthew D MD

BMC evolutionary biology 20071112

<h4>Background</h4>The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate Ciona intestinalis (sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in ca ...[more]

PMID: 17997857

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Project description: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 (Petromyzon marinus) and zebrafish (Danio rerio) 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 (Tetraodon nigriviridis), 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 Tetraodon receptors.Tetraodon 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. Tetraodon 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.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.

Project description:BackgroundThe nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domains of NRs, the DNA-binding domain (DBD) and LBD, in addition to the full-length sequences. Additional functional data is also reported for activation of PXR and the vitamin D receptor (VDR; NR1I1) to gain further insight into the evolution of the NR1I subfamily.ResultsThe NR genes appear to be subject to strong purifying selection, particularly in the DBDs. Estimates of the ratio of the non-synonymous to synonymous nucleotide substitution rates (the omega ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated omega ratio greater than 1. CAR was also unusual in showing high relative omega ratios in both the DBD and LBD, a finding that may relate to the recent appearance of the CAR gene (presumably by duplication of a pre-mammalian PXR gene) just prior to the evolution of mammals. Functional analyses of the NR1I subfamily show that human and zebrafish PXRs show similar activation by steroid hormones and early bile salts, properties not shared by sea lamprey, mouse, or human VDRs, or by Xenopus laevis PXRs.ConclusionNR genes generally show strong sequence conservation and little evidence for positive selection. The main exceptions are PXR and CAR, genes that may have adapted to cross-species differences in toxic compound exposure.

Project description:BackgroundThe pregnane X receptor (PXR) shows the highest degree of cross-species sequence diversity of any of the vertebrate nuclear hormone receptors. In this study, we determined the pharmacophores for activation of human, mouse, rat, rabbit, chicken, and zebrafish PXRs, using a common set of sixteen ligands. In addition, we compared in detail the selectivity of human and zebrafish PXRs for steroidal compounds and xenobiotics. The ligand activation properties of the Western clawed frog (Xenopus tropicalis) PXR and that of a putative vitamin D receptor (VDR)/PXR cloned in this study from the chordate invertebrate sea squirt (Ciona intestinalis) were also investigated.ResultsUsing a common set of ligands, human, mouse, and rat PXRs share structurally similar pharmacophores consisting of hydrophobic features and widely spaced excluded volumes indicative of large binding pockets. Zebrafish PXR has the most sterically constrained pharmacophore of the PXRs analyzed, suggesting a smaller ligand-binding pocket than the other PXRs. Chicken PXR possesses a symmetrical pharmacophore with four hydrophobes, a hydrogen bond acceptor, as well as excluded volumes. Comparison of human and zebrafish PXRs for a wide range of possible activators revealed that zebrafish PXR is activated by a subset of human PXR agonists. The Ciona VDR/PXR showed low sequence identity to vertebrate VDRs and PXRs in the ligand-binding domain and was preferentially activated by planar xenobiotics including 6-formylindolo-[3,2-b]carbazole. Lastly, the Western clawed frog (Xenopus tropicalis) PXR was insensitive to vitamins and steroidal compounds and was activated only by benzoates.ConclusionIn contrast to other nuclear hormone receptors, PXRs show significant differences in ligand specificity across species. By pharmacophore analysis, certain PXRs share similar features such as human, mouse, and rat PXRs, suggesting overlap of function and perhaps common evolutionary forces. The Western clawed frog PXR, like that described for African clawed frog PXRs, has diverged considerably in ligand selectivity from fish, bird, and mammalian PXRs.

Project description:Postsynaptic N-methyl-d-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action.Significance statementSynaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

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Functional evolution of the vitamin D and pregnane X receptors.

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Functional evolution of the vitamin D and pregnane X receptors.

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