CAR and PXR: the xenobiotic-sensing receptors

The xenobiotic receptors CAR and PXR constitute two important members of the NR1I nuclear receptor family. They function as sensors of toxic byproducts derived from endogenous metabolism and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. In contrast, the steroid receptors, exemplified by the estrogen receptor (ER) and glucocorticoid receptor (GR), are the sensors that tightly monitor and respond to changes in circulating steroid hormone levels to maintain body homeostasis. This divergence of the chemical- and steroid-sensing functions has evolved to ensure the fidelity of the steroid hormone endocrine regulation while allowing development of metabolic elimination pathways for xenobiotics. The development of the xenobiotic receptors CAR and PXR also reflect the increasing complexity of metabolism in higher organisms, which necessitate novel mechanisms for handling and eliminating metabolic by-products and foreign compounds from the body. The purpose of this review is to discuss similarities and differences between the xenobiotic receptors CAR and PXR with the prototypical steroid hormone receptors ER and GR. Interesting differences in structure explain in part the divergence in function and activation mechanisms of CAR/PXR from ER/GR. In addition, the physiological roles of CAR and PXR will be reviewed, with discussion of interactions of CAR and PXR with endocrine signaling pathways.     

Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes

The orphan nuclear constitutive androstane receptor (CAR) is proposed to play a central role in the response to xenochemical stress. Identification of CAR target genes in humans has been limited by the lack of a selective CAR agonist. We report the identification of 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel human CAR agonist with the following characteristics: (a) potent activity in an in vitro fluorescence-based CAR activation assay; (b) selectivity for CAR over other nuclear receptors, including the xenobiotic pregnane X receptor (PXR); (c) the ability to induce human CAR nuclear translocation; and (d) the ability to induce the prototypical CAR target gene CYP2B6 in primary human hepatocytes. Using primary cultures of human hepatocytes, the effects of CITCO on gene expression were compared with those of the PXR ligand rifampicin. The relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.     

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.     

The xenobiotic receptors PXR and CAR in liver physiology,an update

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are two nuclear receptors that are well-known for their roles in xenobiotic detoxification by regulating the expression of drug-metabolizing enzymes and transporters. In addition to metabolizing drugs and other xenobiotics, the same enzymes and transporters are also responsible for the production and elimination of numerous endogenous chemicals, or endobiotics. Moreover, both PXR and CAR are highly expressed in the liver. As such, it is conceivable that PXR and CAR have major potentials to affect the pathophysiology of the liver by regulating the homeostasis of endobiotics. In recent years, the physiological functions of PXR and CAR in the liver have been extensively studied. Emerging evidence has suggested the roles of PXR and CAR in energy metabolism, bile acid homeostasis, cell proliferation, to name a few. This review summarizes the recent progress in our understanding of the roles of PXR and CAR in liver physiology.     

The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals

BACKGROUND: Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. RESULTS: To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. CONCLUSION: Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.     

Inflammation and drug metabolism: NF-kappB and the CAR and PXR xeno-receptors

Decreased drug metabolism, hyperbilirubinemia and intrahepatic cholestasis are frequently observed during inflammation. Additionally, it has long been appreciated that exposure to drug metabolism-inducing xenobiotics can impair immune function. The nuclear receptor CAR (constitutive androstane receptor or NR1I3) and PXR (pregnane X receptor, NR1I2) control phase I (cytochrome P450 2B and 3A), phase II (GSTA, UGT1A1), and transporter (MDR1, SLC21A6, MRP2) genes involved in drugs metabolism, bile acids and bilirubin clearance in response to xenobiotics. It is well known that inflammation, through the activation of NF-kappaB pathway, leads to a decrease of CAR, PXR and RXRalpha expression and the expression of their target genes. In addition, a new study reveals the mutual repression between PXR and NF-kappaB signaling pathways, providing a molecular mechanism linking xenobiotic metabolism and inflammation.     

ALAS1 gene expression is down-regulated by Akt-mediated phosphorylation and nuclear exclusion of FOXO1 by vanadate in diabetic mice

Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic ALAS1 (δ-aminolaevulinate synthase 1), the first and rate-limiting enzyme of the haem pathway. Acute porphyria attacks are usually treated by the administration of glucose; its effect is apparently related to its ability to inhibit ALAS1 by modulating insulin plasma levels. It has been shown that insulin blunts hepatocyte ALAS1 induction, by disrupting the interaction of FOXO1 (forkhead box O1) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α). We evaluated the expression of ALAS1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadate on the enzyme regulation to evaluate its potential for the treatment of acute porphyria attacks. Both ALAS1 mRNA and protein content were induced in diabetic animals, accompanied by decreased Akt phosphorylation and increased nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. Vanadate reversed ALAS1 induction, with a concomitant PI3K (phosphoinositide 3-kinase)/Akt pathway activation and subsequent reduction of nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. These findings support the notion that the FOXO1-PGC-1α complex is involved in the control of ALAS1 expression and suggest further that a vanadate-based therapy could be beneficial for the treatment of acute porphyria attacks.     

Structural and Functional Similarity of Amphibian Constitutive Androstane Receptor with Mammalian Pregnane X Receptor

The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARα and β) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARα being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARα ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARα possesses a flexible helix 11’. Modest increase in the recruitment of coactivator PGC-1α may contribute to the enhanced basal activity of three gain-of-function xlCARα mutants humanizing key LBD amino acid residues. xlCARα and PXR appear to constitute an example of convergent evolution.     

Molecular interactions of dioxins and DLCs with the xenosensors (PXR and CAR): An in silico risk assessment approach

Dioxins and dioxin‐like compounds (DLCs) are known to cause endocrine disruption in humans and animals. Being lipophilic xenobiotic chemicals, they can be easily absorbed into the biological system from the surrounding environments, thereby causing various health dysfunctions. In the present study, a total of 100 dioxins and DLCs were taken, and their binding pattern was assessed with the xenosensors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in comparison with the corresponding known inhibitors and a well‐studied endocrine disrupting xenobiotic, bisphenol A (BPA). The nuclear receptors CAR and PXR are known to play a significant role in handling potential toxins by coordinating cellular transport and metabolic functions of the same. Among different endocrine‐disrupting chemicals used in the present study, DLCs (PCDFs and PCBs) elicited better interactions in comparison with the parent dioxin (polychlorinated dibenzodioxins) compounds. On comparing D scores of all the compounds against both the receptors, PCDF 8‐hydroxy‐3,4‐dichlorodibenzofuran (8‐OH‐DCDF) and PCB tetrachlorobenzyltoluene (TCBT) exhibited significant molecular interactions against PXR (−7.633 kcal mol⁻¹) and CAR (−8.389 kcal mol⁻¹), respectively. Predominant interactions were found to be H‐bonding, π‐π stacking, hydrophobic, polar, and van der Waals. By contrast, BPA and some natural ligands tested in this study showed lower binding affinities with these receptors than certain DLCs reported herein, ie, certain DLCs might be more toxic than the proven toxic agent, BPA. Such studies play a pivotal role in the risk assessment of exposure to dioxins and DLCs on human health.     

孕烷X受体的翻译后修饰及其功能调控作用研究进展

孕烷X 受体（PXR）是一类配体依赖性的核受体亚家族,可感受外源物质,被多种药物激活。PXR 可转录调控多种与药物代谢 相关的药物代谢酶和药物转运体的表达,参与药物代谢调控。PXR 转录活性的变化可改变药物在体内的代谢过程,继而诱发潜在药物不 良反应,与药物药代动力学研究和临床药物治疗密切相关,并有潜力成为防治药物介导的肝损伤和逆转化疗药物耐药的新型药物靶标。 综述了目前已发现的PXR 翻译后修饰及其对PXR 功能调控机制的研究进展。     

Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells

Efficient handling of oxidative stress is critical for the survival of organisms. The orphan nuclear receptor pregnane X receptor (PXR) is important in xenobiotic detoxification through its regulation of phase I and phase II drug-metabolizing/detoxifying enzymes and transporters. In this study we unexpectedly found that the expression of an activated human PXR in transgenic female mice resulted in a heightened sensitivity to paraquat, an oxidative xenobiotic toxicant. Heightened paraquat sensitivity was also seen in wild-type mice treated with the mouse PXR agonist pregnenolone-16alpha-carbonitrile. The PXR-induced paraquat sensitivity was associated with decreased activities of superoxide dismutase and catalase, enzymes that scavenge superoxide and hydrogen peroxide, respectively. Paradoxically, the general expression and activity of glutathione S-transferases, a family of phase II enzymes that detoxify electrophilic and cytotoxic substrates, was also induced in the transgenic mice. PXR regulates glutathione S-transferase expression in an isozyme-, tissue-, and sex-specific manner, and this regulation is independent of the nuclear factor-erythroid 2 p45-related factor 2/Kelch-like Ech-associated protein 1 pathway. In cell cultures, expression of activated human PXR sensitizes the cancerous colon and liver cells to the cytotoxic effect of paraquat, which is associated with an increased production of the reactive oxygen species. The current study reveals a novel function of PXR in the mammalian oxidative stress response, and this regulatory pathway may be implicated in carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage.     

Xenobiotic-Induced Hepatocyte Proliferation Associated with Constitutive Active/Androstane Receptor (CAR) or Peroxisome Proliferator-Activated Receptor α (PPARα) Is Enhanced by Pregnane X Receptor (PXR) Activation in Mice

Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN) alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.