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.     

Evolution of pharmacologic specificity in the pregnane X receptor

Background  

The 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.     

Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation

The arylamide 2-acetylaminofluorene (AAF) is a powerful carcinogen displaying a marked promoting activity, also known to regulate expression of liver detoxifying proteins. In this study we identified CYP3A23, a major inducible cytochrome P-450 (CYP) isoform, as an AAF target in hepatocytes. Indeed, exposure to AAF of primary rat hepatocytes resulted in a marked up-regulation of CYP3A23 expression at both mRNA and protein levels. Using CYP3A23 reporter gene constructs, we further demonstrated that AAF activated the CYP3A23 Direct Repeat 3 (DR3) promoter element interacting with the nuclear pregnane X receptor (PXR). Moreover, the PXR antagonist ecteinascidin-743 fully suppressed AAF-related CYP3A23 induction. Low doses of AAF inhibiting DNA synthesis in hepatocytes however failed to trigger PXR-related CYP3A23 induction and PXR-negative epithelial liver cells remained sensitive to the mito-inhibitory effects of AAF. Such data indicate that AAF up-regulates CYP3A23 through PXR activation but does not require PXR for exerting its carcinogenic promoting properties based on inhibition of cell growth.     

Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide

Cross-talk between nuclear receptors involved in the control of drug metabolism is being increasingly recognised as a source of drug side effects. Omeprazole is a well known activator of the aryl hydrocarbon receptor (AhR). We investigated the regulation of AhR by omeprazole-sulphide, a degradation metabolite of omeprazole, using CYP1A mRNA induction, reporter gene assay, receptor DNA binding, ligand binding, nuclear translocation, trypsin digests, and drug metabolism analysis in mouse Hepa-1c1c7, human HepG2 cells and primary human hepatocytes. Omeprazole-sulphide is a pure antagonist of AhR in Hepa-1c1c7 and HepG2 hepatoma cell lines. In Hepa-1c1c7 cells, omeprazole-sulphide is a ligand of AhR, inhibits AhR activation to a DNA-binding form, induces a specific pattern of AhR trypsin digestion and inhibits AhR nuclear translocation and subsequent degradation in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, in highly differentiated primary human hepatocytes treated with rifampicin an agonist of the pregnane X receptor (PXR), omeprazole-sulphide behaves as an agonist of AhR. Inhibition of drug metabolizing enzymes by ketoconazole restores the antagonist effect of omeprazole-sulphide. Metabolic LC/MS analysis reveals that omeprazole-sulphide (AhR antagonist) is efficiently converted to omeprazole (AhR activator) by cytochrome P450 CYP3A4, a target gene of PXR, in primary human hepatocytes but not in hepatoma cells in which PXR is not expressed. This report provides the first evidence for a cross-talk between PXR/CYP3A4 and AhR. In addition, it clearly shows that conclusions drawn from experiments carried out in cell lines may lead to erroneous in vivo predictions in man.     

CYP3A4 and pregnane X receptor humanized mice

Marked species differences exist in P450 expression and activities. In order to produce mouse models that can be used to more accurately predict human drug and carcinogen metabolism, P450- and xenobiotic receptor humanized mice are being prepared using bacterial artificial chromosomes (BAC) and P1 phage artificial chromosomes (PAC) genomic clones. In some cases, transgenic mice carrying the human genes are bred with null-mice to produce fully humanized mice. Mice expressing human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 were generated and characterized. Studies with the CYP3A4-humanized (hCYP3A4) mouse line revealed new information on the physiological function of this P450 and its role in drug metabolism in vivo. With this mouse line, CYP3A4, under certain circumstances, was found to alter the serum levels of estrogen resulting in deficient lactation and low pup survival as a result of underdeveloped mammary glands. This hCYP3A4 mouse established the importance of intestinal CYP3A4 in the pharmacokinetics of orally administered drugs. The hCYP3A4 mice were also used to establish the mechanisms of potential gender differences in CYP3A4 expression (adult female > adult male) that could account for human gender differences in drug metabolism and response. The pregnane X receptor (PXR) is also involved in induction of drug metabolism through its target genes including CYP3A4. Since species differences exist in ligand specificity between human and mice, a PXR-humanized mouse (hPXR) was produced that responds to human PXR activators such as rifampicin but does not respond to the rodent activator pregnenalone 16alpha-carbonitrile.     

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.     

PXR-mediated transcriptional activation of CYP3A4 by cryptotanshinone and tanshinone IIA

Danshen (Radix Salvia miltiorrhiza) is a famous Traditional Chinese Medicine used widely for the treatment of coronary heart disease and cerebrovascular disease. Diterpenoid tanshinones including tanshinone I, tanshinone IIA and cryptotanshinone are the major bioactive components from Danshen herb. Previous reports have demonstrated that Danshen extracts could induce the expression of CYP3A in rodents, however, the constituents responsible for Danshen-mediated CYP3A induction and the underlying molecular mechanisms remain unknown. The discovery of a family of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR) and glucocorticoid receptor (GR) gives insight into the molecular explanation of CYP3A induction by xenobiotics. In the present study, interactions between Danshen constituents and human PXR were evaluated using a reporter gene assay. Our observations showed that Danshen ethanol extract could activate human PXR and induce the CYP3A4 reporter construct in HepG2 cells. Tanshinone IIA and cryptotanshinone were identified as efficacious PXR agonists, and cryptotanshinone activated the CYP3A4 promoter more strongly than tanshinone IIA. Furthermore, CAR and GR were also involved in the induction of CYP3A4 expression by tanshinones, though their roles seemed not as important as PXR. Treatment of LS174T cells with cryptotanshinone or tanshinone IIA resulted in a significant increase of CYP3A4 mRNA, which was consistent with the results from the reporter gene assay. Collectively, activation of PXR and the resultant CYP3A4 induction mediated by cryptotanshinone and tanshinone IIA provide a molecular mechanism for previously observed CYP3A induction by Danshen extracts, and our findings also suggest that caution should be taken when Danshen products are used in combination with therapeutic drugs metabolized by CYP3A4.     

Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor.

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm-100 microm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low-dexamethasone component (nmol concentrations) of low amplitude (factor of 3-4) followed by a high-dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15-30). We show that the low-dexamethasone component results from the glucocorticoid receptor-mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic-independent manner. At supramicromolar concentrations (>10 microm), dexamethasone binds to and activates pregnane X receptor thus producing the high-dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).