Complementary roles of farnesoid X receptor,pregnane X receptor,and constitutive androstane receptor in protection against bile acid toxicity

The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis. Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet. Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet. To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), followed by the CA diet. A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase alpha), following pretreatment with phenobarbital or TCPOBOP. In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo. Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.     

Evolutionary selection across the nuclear hormone receptor superfamily with a focus on the NR1I subfamily (vitamin D,pregnane X,and constitutive androstane receptors)

Background

The 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.

Results

The 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 ω ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated ω ratio greater than 1. CAR was also unusual in showing high relative ω 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.

Conclusion

NR 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.

     

The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity

A double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli. Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions. As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin. Comparisons of the single and double knockouts reveal specific roles for the two receptors. Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA). As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C. M., Nelson, M. C., Ong, E. S., Waxman, D. J., and Evans, R. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity. Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2. These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose. Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic. These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.     

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors

The NR1I subfamily of nuclear receptors contains a phylogenetically diverse array of receptors related to the mammalian pregnane X receptor (PXR) (NR1I2) and constitutive androstane receptor (CAR) (NR1I3). We have carried out an extensive comparative analysis of this subgroup with representatives from fish, birds, amphibians, and mammals. Four novel receptors were isolated from fish, dog, pig, and monkey for this study and combined with a previously reported set of related receptors including human PXR, rabbit PXR, mouse PXR, chicken CXR, frog benzoate X receptors (BXRalpha, BXRbeta), and human and mouse CAR. A broad range of xenobiotics, steroids, and bile acids were tested for their ability to activate the ligand binding domain of each receptor. Three distinct groups of receptors were identified based on their pharmacological profiles: 1) the PXRs were activated by a broad range of xenobiotics and, along with the mammalian PXRs, included the chicken and fish receptors; 2) the CARs were less promiscuous, had high basal activities, and were generally repressed rather than activated by those compounds that modulated their activity; and 3) the BXRs were selectively activated by a subset of benzoate analogs and are likely to be specialized receptors for this chemical class of ligands. The PXRs are differentiated from the other NR1I receptors by a stretch of amino acids between helices 1 and 3, which we designate the H1-3 insert. This insert was present in the mammalian, chicken, and fish PXRs but absent in the CARs and BXRs. Modeling studies suggest that the H1-3 insert contributes to the promiscuity of the PXRs by facilitating the unwinding of helices-6 and -7, thereby expanding the ligand binding pocket.     

Regulation of hepatic Insig-2 by the farnesoid X receptor

Activation of the farnesoid X receptor (FXRalpha) affects genes controlling many pathways, including those involved in bile acid and glucose homeostasis. Here we report that a critical gene involved in cholesterol homeostasis, Insig-2, was induced when mice or cultured cells were treated with FXRalpha agonists or infected with constitutively active FXRalpha. No such induction was observed in agonist-treated FXRalpha-/- mice. Further analysis, which included EMSAs, reporter gene activation, and chromatin immunoprecipitation, identified two functional FXRalpha response elements within intron 2 of the mouse Insig-2 gene. In addition to increasing hepatic Insig-2 protein levels in wild-type mice, FXRalpha activation also reduced lanosterol 14alpha-demethylase mRNA levels and 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein levels. Together, these changes likely account for the decrease in cholesterol synthesis observed after activation of FXR in primary hepatocytes. In conclusion, the current study links hepatic FXRalpha activation to regulation of genes involved in cholesterol synthesis.     

Farnesoid X receptor suppresses constitutive androstane receptor activity at the multidrug resistance protein-4 promoter

Multidrug resistance protein-4 (MRP4) is a member of the multidrug resistance associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive up-regulation in response to cholestatic injury or bile acid feeding. In this study we demonstrate that farnesoid X receptor (FXR) regulates MRP4 in vivo and in vitro. In vivo deletion of FXR induces MRP4 gene expression. In vitro treatment of HepG2 cells with FXR ligands, chenodeoxycholic acid (CDCA), cholic acid (CA) and the synthetic ligand GW-4064 suppresses basal mRNA level of the MRP4 gene as well as the co-treatment with CDCA and 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), an activator of constitutive androstane receptor (CAR). We found in the human MRP4 promoter a CAR responsive element (CARE) embedded within an FXR responsive element (FXRE). We cloned this region and found that FXR suppresses CAR activity in luciferase assay. Finally, we demonstrated that FXR competes with CAR for binding to this overlapping binding site. Our results support the view that FXR activation in obstructive cholestasis might worsen liver injury by hijacking a protective mechanism regulated by CAR and provides a new molecular explanation to the pathophysiology of cholestasis.     

Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor

The nuclear pregnane X receptor (PXR; NR1I2) is an integral component of the body's defense mechanism against chemical insult (chemoprotection). PXR is activated by a diverse array of lipophilic chemicals, including xenobiotics and endogenous substances, and regulates the expression of cytochromes P450, conjugating enzymes, and transporters involved in the metabolism and elimination of these potentially harmful chemicals from the body. Among the chemicals that bind and activate PXR is the toxic bile acid lithocholic acid; activation of PXR, in turn, protects against the severe liver damage caused by this bile acid.Thus, PXR serves as a physiological sensor of lithocholic acid and perhaps other bile acids and coordinately regulates genes involved in their detoxification. Interestingly, both the antibiotic rifampicin and the herbal antidepressant St. John's wort activate PXR and have anticholestatic properties, which suggests that more potent, selective PXR agonists may be useful in the treatment of biliary cholestasis or other diseases characterized by the accumulation of bile acids or other toxins in the liver.     

Characterization of DNA complexes formed by the nuclear receptor constitutive androstane receptor

The nuclear receptor constitutive androstane receptor (CAR) acts as a xenobiotic sensor and regulates the expression of enzymes, such as several cytochromes P450s and the UDP-glucuronosyltransferase (UGT) type 1A1. CAR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs). Clusters of CAR REs, referred to as phenobarbital response enhancer modules (PBREMs), have been identified in several CAR target genes. In this study we confirm that REs formed by direct repeats of two AGTTCA hexamers with 4 spacing nucleotides are optimal for the binding of CAR-RXR heterodimers. In addition, we found that the heterodimers also form complexes on everted repeat-type arrangements with 8 spacing nucleotides. We also observed that CAR is able to bind DNA as a monomer and to interact in this form with different coregulators even in the presence of RXR. Systematic variation of the nucleotides 5'-flanking to both AGTTCA hexamers showed that the dinucleotide sequence modulates the DNA complex formation of CAR monomers and CAR-RXR heterodimer by a factor of up to 20. The highest preference was found for the sequence AG and lowest for CC. The increased DNA affinity of CAR is mediated by the positively charged arginines 90 and 91 located in the carboxyl-terminal extension of the DNA-binding domain of the receptor. Furthermore, we show that one of the three CAR REs of the human UGT1A1 PBREM is exclusively bound by CAR monomers and this is regulated by ligands that bind to this nuclear receptor. This points to a physiological role for CAR monomers. Therefore, both CAR-RXR heterodimers and CAR monomers can contribute to the gene activating function of PBREMs in CAR target genes.     

Transactivation of ABCG2 through a novel cis-element in the distal promoter by constitutive androstane receptor but not pregnane X receptor in human hepatocytes

A previous report demonstrated that treatment of human hepatocytes with phenobarbital, an activator of nuclear receptor constitutive androstane receptor (CAR), increases mRNA levels of an efflux transporter ABCG2, which is involved in the excretion of xenobiotics in liver and intestine. The results suggest that human CAR (hCAR) transactivates human ABCG2 (hABCG2) expression. In this study, we confirmed increase in ABCG2 mRNA levels in human hepatocytes after adenoviral expression of hCAR and treatment with its activator. Reporter assays suggested the existence of an hCAR-responsive element between -8000 and -7485 of hABCG2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays identified a DR5 motif (direct repeat separated by five nucleotides) within the region as a binding motif of hCAR/human retinoid X receptor α heterodimer. The introduction of mutations into the DR5 motif resulted in the complete loss of the hCAR-mediated transactivation. Interestingly, human pregnane X receptor, belonging to the same NR1I subfamily as CAR, did not activate any reporter gene containing the DR5 motif. Taken together, our present findings suggest that hCAR transactivates hABCG2 through the DR5 motif located in its distal promoter in human hepatocytes and that the motif prefers hCAR to pregnane X receptor.     

Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats

The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (~80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.     

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25 pM with the linearity of the standard curve spanned to 2500 pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.     

The multidrug resistance-associated protein (MRP/ABCC) subfamily of ATP-binding cassette transporters in plants

In many different plant species, genes belonging to the multidrug resistance-associated protein (MRP, ABCC) subfamily of ABC transporters have been identified. Following the discovery of vacuolar transport systems for xenobiotic or plant-produced conjugated organic anions, plant MRPs were originally proposed to be primarily involved in the vacuolar sequestration of potentially toxic metabolites. Indeed, heterologous expression of different Arabidopsis MRPs in yeast demonstrates their activity as ATP-driven pumps for structurally diverse substrates. Recent analysis of protein-protein interactions and the characterization of knockout mutants in Arabidopsis suggests that apart from transport functions plant MRPs play additional roles including the control of plant transpiration through the stomata. Here, we review and discuss the diverse functions of plant MRP-type ABC transporters and present an organ-related and developmental analysis of the expression of Arabidopsis MRPs using the publicly available full-genome chip data.