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.     

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.     

Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR

The constitutive androstane receptor (CAR, NR1I3) is a key regulator of xenobiotic and endobiotic metabolism. The ligand-binding domains of murine (m) and human (h) CAR are divergent relative to other nuclear hormone receptors, resulting in species-specific differences in xenobiotic responses. Here we identify the widely used antiemetic meclizine (Antivert; Bonine) as both an agonist ligand for mCAR and an inverse agonist for hCAR. Meclizine increases mCAR transactivation in a dose-dependent manner. Like the mCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, meclizine stimulates binding of steroid receptor coactivator 1 to the murine receptor in vitro. Meclizine administration to mice increases expression of CAR target genes in a CAR-dependent manner. In contrast, meclizine suppresses hCAR transactivation and inhibits the phenobarbital-induced expression of the CAR target genes, cytochrome p450 monooxygenase (CYP)2B10, CYP3A11, and CYP1A2, in primary hepatocytes derived from mice expressing hCAR, but not mCAR. The inhibitory effect of meclizine also suppresses acetaminophen-induced liver toxicity in humanized CAR mice. These results demonstrate that a single compound can induce opposite xenobiotic responses via orthologous receptors in rodents and humans.     

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.     

Altered glucocorticoid metabolism represents a feature of macroph‐aging

The aging process is characterized by a chronic, low‐grade inflammatory state, termed “inflammaging.” It has been suggested that macrophage activation plays a key role in the induction and maintenance of this state. In the present study, we aimed to elucidate the mechanisms responsible for aging‐associated changes in the myeloid compartment of mice. The aging phenotype, characterized by elevated cytokine production, was associated with a dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis and diminished serum corticosteroid levels. In particular, the concentration of corticosterone, the major active glucocorticoid in rodents, was decreased. This could be explained by an impaired expression and activity of 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1), an enzyme that determines the extent of cellular glucocorticoid responses by reducing the corticosteroids cortisone/11‐dehydrocorticosterone to their active forms cortisol/corticosterone, in aged macrophages and peripheral leukocytes. These changes were accompanied by a downregulation of the glucocorticoid receptor target gene glucocorticoid‐induced leucine zipper (GILZ) in vitro and in vivo. Since GILZ plays a central role in macrophage activation, we hypothesized that the loss of GILZ contributed to the process of macroph‐aging. The phenotype of macrophages from aged mice was indeed mimicked in young GILZ knockout mice. In summary, the current study provides insight into the role of glucocorticoid metabolism and GILZ regulation during aging.     

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.     

Steroid hormone biotransformation and xenobiotic induction of hepatic steroid metabolizing enzymes

Normal reproductive development depends on the interplay of steroid hormones with their receptors at specific tissue sites. The concentrations of hormone ligands in the circulation and at target sites are maintained through coordinated regulation on steroid biosynthesis and degradation. Changed bioavailability of steroids, through alteration of steroidogenesis or biotransformation rates, leads to changes in endocrine function. Steroid hormones lose their receptor reactivity in most cases when they are bound to binding proteins, while metabolic conversion can result in either active or inactive metabolites. Hydroxylation by cytochrome P450 (CYP) enzymes and conjugation with glucuronide and sulfate are among the major hepatic pathways of steroid inactivation. The expression of these biotransformation enzymes can be induced by many xenobiotics. The barbiturate phenobarbital and the environmental toxicant 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) are among the well characterized inducers for the CYP 2B and 3A enzymes and selected conjugation enzymes. The induction of the steroid biotransformation enzymes is partly mediated through the activation of a group of nuclear receptors including the glucocorticoid receptor, the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), and the peroxisome proliferator activated receptors (PPAR). Drug or chemical-induced increases in hepatic enzyme activities are often a basis for drug-drug interactions that lead to enhanced elimination and reduced therapeutic efficacy of steroidal drugs. The effects of enzyme induction on endogenous steroid clearance, along with its possible consequence, are less well understood. While enzyme induction by xenobiotics may increase clearance of the endogenous steroid, regulatory mechanisms for steroid homeostasis may adapt and compensate for altered clearance.     

Rat pregnane X receptor: molecular cloning, tissue distribution, and xenobiotic regulation.

An orphan nuclear receptor, termed the pregnane X receptor (PXR), has recently been cloned from mouse and human and defines a novel steroid signaling pathway (Cell 92, 73-82, 1998; Proc. Natl. Acad. Sci. USA 95, 12208-122313, 1998). Transient cotransfection experiments demonstrate that the PXR responds to structurally dissimilar compounds and confers the induction of cytochrome P4503A (CYP3A), a subfamily of enzymes that involve the metabolism of two-thirds of drugs and other xenobiotics. In this report, we describe the molecular cloning, tissue distribution, and xenobiotic regulation of a rat PXR designated rPXR-1. rPXR-1 exhibits a 95% sequence identity with the mouse PXR, but only 79% identity with the human PXR, providing the molecular basis that rats and mice have a similar CYP3A induction profile but differ from humans. rPXR-1 gene was expressed abundantly in liver, intestine, and, to a lesser extent, kidney, lung, and stomach. The tissue distribution and the relative abundance of rPXR-1 mRNA among these tissues resemble those of CYP3A, suggesting that PXR is important not only for induction but also for constitutive expression of these enzymes. Xenobiotics known to induce liver microsomal enzymes showed differential effects on the rPXR-1 expression as determined by Northern blot analysis. Dexamethasone, for example, increased the accumulation of rPXR-1 mRNA, whereas troleandomycin slightly suppressed it. Compounds that increase PXR expression (inducers) and compounds that interact with PXR (ligands) likely have synergistic effects on CYP3A induction, which provides a novel molecular explanation for drug-drug interactions.     

The antiapoptotic role of pregnane X receptor in human colon cancer cells

The orphan nuclear receptor pregnane X receptor (PXR) plays an important role in the detoxification of foreign and endogenous chemicals, including bile acids. PXR promotes bile acid elimination by activating bile acid-detoxifying enzymes and transporters. Certain bile acids are known to promote colonic carcinogenesis by inducing colon cancer cell apoptosis. However, whether and how PXR plays a role in colon cancer apoptosis has not been reported. In this study, we showed that activation of PXR by genetic (using a constitutively activated PXR) or pharmacological (using PXR agonist rifampicin) means protected the PXR-overexpressing colon cancer HCT116 cells from deoxycholic acid-induced apoptosis. Interestingly, activation of PXR also protected HCT116 cells from adriamycin-induced cell death, suggesting that the antiapoptotic effect of PXR was not bile acid specific. Moreover, the antiapoptotic effect of PXR in HCT116 cells appeared to be independent of xenobiotic enzyme regulation, because these cells had little basal and inducible expression of bile acid-detoxifying enzymes. Instead, SuperArray analysis showed that PXR-mediated deoxycholic acid resistance was associated with up-regulation of multiple antiapoptotic genes, including BAG3, BIRC2, and MCL-1, and down-regulation of proapoptotic genes, such as BAK1 and TP53/p53. Treatment with rifampicin in colon cancer LS180 cells, a cell line known to express endogenous PXR, also inhibited apoptosis. Activation of PXR in transgenic mice inhibited bile acid-induced colonic epithelial apoptosis and sensitized mice to dimethylhydrazine-induced colonic carcinogenesis, suggesting that the antiapoptotic effect of PXR is conserved in normal colon epithelium. In summary, our results have established the antiapoptotic role of PXR in both human colon cancer cells and normal mouse colon epithelium.