Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin

Intestinal P-glycoprotein, which is encoded by the MDR1 gene, plays an important role in the absorption and presystemic elimination of many xenobiotics. Hence, an understanding of the factors regulating its expression and function is of substantial interest. In addition to genetic factors, exposure to drugs such as rifampin can profoundly affect its expression. So far, the mechanisms by which rifampin induces MDR1 expression are poorly understood. Recent studies demonstrate that the nuclear receptor PXR (pregnane X receptor) is involved in xenobiotic induction of CYP3A4. Because CYP3A4 and MDR1 are often co-induced, we investigated whether a similar mechanism is also involved in MDR1 induction. The human colon carcinoma cell line LS174T was used as an intestinal model to study induction because in these cells the endogenous MDR1 gene is highly inducible by rifampin. The 5'-upstream region of human MDR1 was examined for the presence of potential PXR response elements. Several binding sites were identified that form a complex regulatory cluster at about -8 kilobase pairs. Only one DR4 motif within this cluster is necessary for induction by rifampin. We conclude that induction of MDR1 is mediated by a DR4 motif in the upstream enhancer at about -8 kilobase pairs, to which PXR binds.     

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.     

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.     

Induction of cytochrome P4502B: Role of regulatory elements and nuclear receptors

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.     

Functionally conserved xenobiotic responsive enhancer in cytochrome P450 3A7

Nuclear receptors CAR and PXR play a key role in cytochrome P450 gene induction by xenobiotics. Human cytochrome P450 3A7 (CYP3A7) is expressed from early in gestation until the perinatal period, when there is a switch in expression to CYP3A4. Here we demonstrate that a PXR and CAR responsive enhancer is located approximately 8 kb upstream of the proximal CYP3A7 promoter. This distal xenobiotic responsive enhancer module (XREM) is conserved with the XREM of CYP3A4. Interestingly, not only the XREM, but also the entire promoters exhibit 90% sequence identity up to -8.8 kb, indicating a close evolutionary distance. We propose that the promoters have coevolved to functionally conserve P450 gene induction in response to xenobiotics through CAR and PXR. Thus, nuclear receptors for xenobiotics may not only play a role to provide a survival advantage during adulthood, but also to protect the embryo against endogenous and exogenous toxins.     

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.     

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.     

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.     

Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, lipoteichoic acid

Expression of hepatic drug metabolizing enzymes (DMEs) is altered in infection and inflammation. However, the role of Gram+ve bacterial components and their receptor, Toll-like receptor (TLR) 2 in regulation of hepatic DMEs is unknown. Gene expression of DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRα). The TLR2 ligand, lipoteichoic acid (LTA) reduced RNA levels of CAR and its target genes, Cyp2b10, Cyp2a4 and Sultn in mouse liver (∼60-80% reduction). Hepatic genes regulated by PXR and CAR, Cyp3a11 and Mrp2 were moderately reduced by LTA, along with ∼50% reduction of PXR RNA and nuclear protein levels of RXRα. The effects of LTA were significantly attenuated by pre-treatment with the Kupffer cell inhibitor, gadolinium chloride, indicating that Kupffer cells contribute to LTA-mediated down-regulation of hepatic genes. These results indicate that treatment with Gram+ve bacterial components preferentially down-regulate CAR and its target genes in the liver.     

P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR.

The biochemistry of foreign compound metabolism and the roles played by individual cytochrome P450 (CYP) enzymes in drug metabolism and in the toxification and detoxification of xenochemicals prevalent in the environment are important areas of molecular pharmacology and toxicology that have been widely studied over the past decade. Important advances in our understanding of the mechanisms through which foreign chemicals impact on these P450-dependent metabolic processes have been made during the past 2 years with several key discoveries relating to the mechanisms through which xenochemicals induce the expression of hepatic P450 enzymes. Roles for three "orphan" nuclear receptor superfamily members, designated CAR, PXR, and PPAR, in respectively mediating the induction of hepatic P450s belonging to families CYP2, CYP3, and CYP4 in response to the prototypical inducers phenobarbital (CAR), pregnenolone 16alpha-carbonitrile and rifampicin (PXR), and clofibric acid (PPAR) have now been established. Two other nuclear receptors, designated LXR and FXR, which are respectively activated by oxysterols and bile acids, also play a role in liver P450 expression, in this case regulation of P450 cholesterol 7alpha-hydroxylase, a key enzyme of bile acid biosynthesis. All five P450-regulatory nuclear receptors belong to the same nuclear receptor gene family (family NR1), share a common heterodimerization partner, retinoid X-receptor (RXR), and are subject to cross-talk interactions with other nuclear receptors and with a broad range of other intracellular signaling pathways, including those activated by certain cytokines and growth factors. Endogenous ligands of each of those nuclear receptors have been identified and physiological receptor functions are emerging, leading to the proposal that these receptors may primarily serve to modulate hepatic P450 activity in response to endogenous dietary or hormonal stimuli. Accordingly, P450 induction by xenobiotics may in some cases lead to a perturbation of endogenous regulatory circuits with associated pathophysiological consequences.