The Pregnane X Receptor Binds to Response Elements in a Genomic Context-Dependent Manner, and PXR Activator Rifampicin Selectively Alters the Binding Among Target Genes

Song, Xiulong; Xie, Mingxing; Zhang, He; Li, Yuxin; Sachdeva, Karuna; Yan, Bingfang

doi:10.1124/dmd.32.1.35

Cited by 49 publications

(52 citation statements)

References 28 publications

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“…Although TgCYP3A4/hPXR mice contain CYP3A7 and CYP3A4, CYP3A7 mRNAs levels were considerably less than the expression of CYP3A4. CYP3A7 induction was also noted in adult TgCYP3A4/hPXR mice after RIF treatment, which might be attributed to the high homology of the PXR binding motif between CYP3A7 and CYP3A4 (Song et al, 2004). However, it is minor compared with the robust induction of CYP3A4 by RIF.…”

Section: Human Pxr and Cyp3a4 Regulate Apap Toxicitymentioning

confidence: 93%

Rifampicin-Activated Human Pregnane X Receptor and CYP3A4 Induction Enhance Acetaminophen-Induced Toxicity

Cheng¹,

Ma²,

Krausz³

et al. 2009

Drug Metab Dispos

115

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ABSTRACT:Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAPinduced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/ hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

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Section: Human Pxr and Cyp3a4 Regulate Apap Toxicitymentioning

confidence: 93%

Rifampicin-Activated Human Pregnane X Receptor and CYP3A4 Induction Enhance Acetaminophen-Induced Toxicity

Cheng¹,

Ma²,

Krausz³

et al. 2009

Drug Metab Dispos

115

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“…Huh 7 cells were plated in 24-well plates in DMEM supplemented with 10% delipidated fetal bovine serum at a density of 5 ϫ 10 4 cells per well and cultured overnight. Transient transfection was conducted by lipofection with LipofectAMINE and Plus Reagent (Invitrogen) as described previously (Song et al, 2004). For all the transfections, standard amounts of plasmid DNA used per well were 100 ng for BSEP promoter construct, 100 ng for nuclear receptor expression plasmids, and 10 ng for the null-Renilla luciferase plasmid as an internal control.…”

Section: Methodsmentioning

confidence: 99%

“…The reporter enzyme activities were assayed with the Dual-Luciferase Reporter assay system as described previously (Song et al, 2004). Treated Huh 7 cells were washed once with phosphate-buffered saline and lysed by adding 100 l of passive lysis buffer (Promega) with gentle rocking for 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting pBSEP-Luc reporter construct was sequence-verified. Expression construct of nuclear receptor pregnane X receptor (PXR) and its target reporter construct pCYP3A4DP-Luc were described previously (Song et al, 2004 Site-Directed Mutagenesis. Mutations were introduced by a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

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Oxysterol 22(R)-Hydroxycholesterol Induces the Expression of the Bile Salt Export Pump through Nuclear Receptor Farsenoid X Receptor but Not Liver X Receptor

Deng

Yang²,

Yang³

et al. 2005

J Pharmacol Exp Ther

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Oxysterols are intermediates in the synthesis of bile acids and steroid hormones from cholesterol and function as ligands for liver X receptor (LXR). Bile salt export pump (BSEP) is responsible for canalicular secretion of bile acids and is tightly regulated by its substrates bile acids through nuclear receptor farnesoid X receptor (FXR). In a microarray study using human hepatocytes, BSEP was markedly induced not only by chenodeoxycholic acid (CDCA) but also by oxysterol 22(R)-hydroxycholesterol [22(R)-OHC]. We hypothesized that the expression of BSEP was induced by oxysterols through activation of LXR. To test the hypothesis, human primary hepatocytes or hepatoma cells were treated with 22(R)-OHC, and expression of BSEP was determined. The level of BSEP mRNA was increased as much as 5-fold upon oxysterol induction. In contrast to our hypothesis, the oxysterol-induced up-regulation of BSEP is mediated through FXR but not LXR. BSEP promoter activity was markedly induced by 22(R)-OHC in the presence of FXR but not LXRs. Mutation of the FXR element IR1 in the BSEP promoter significantly reduced its ability to respond to oxysterol induction. To determine whether 22(R)-OHC and CDCA bind to similar structural features of FXR, site-directed mutagenesis was performed in the FXR ligand binding domain. Mutation of residues R331 and I352 abolished activation mediated by CDCA and 22(R)-OHC. In contrast, substitution of residues L340 and R351 differentiated CDCA-and 22(R)-OHC-mediated activation. In conclusion, oxysterol 22(R)-OHC functions as an FXR ligand to induce BSEP expression and differs in the binding with FXR from CDCA.

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“…After de-cross-linking and protease digestion, DNA fragments were recovered by QIAquick PCR purification kit (QIAGEN). Quantitative PCR was performed using specific sets of primers (Table 1) (Song et al, 2004). PCR products were also resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.…”

mentioning

confidence: 99%

Synergistically Enhanced CYP2B6 Inducibility between a Polymorphic Mutation in CYP2B6 Promoter and Pregnane X Receptor Activation

Ferguson²,

Wang³

2010

Molecular Pharmacology

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CYP2B6 is a highly inducible and polymorphic enzyme involved in the metabolism of an increasing number of clinically important drugs. Significant interindividual variability in CYP2B6 expression has been attributed to either genetic polymorphisms or chemical-mediated induction through the activation of constitutive androstane receptor and/or pregnane X receptor (PXR). It was reported that the Ϫ82T3 C substitution within the CYP2B6*22 allele creates a functional CCAAT/enhancer-binding protein (C/EBP) binding site and enhances the basal expression of the CYP2B6 gene. Here, we explored whether this polymorphic mutation could affect drug-mediated induction of CYP2B6. Cell-based promoter reporter assays demonstrated that CYP2B6 luciferase activity was synergistically enhanced in the presence of both Ϫ82T3 C mutation and rifampicin (RIF)-activated PXR. On the other hand, this synergism was attenuated by disrupting the C/EBP binding site or knocking down C/EBP␣ expression. Mechanistic studies revealed that C/EBP␣ plays an important role in such synergism by directly interacting with PXR; enhancing RIF-mediated recruitment of PXR to the Ϫ82T3 C harboring CYP2B6 promoter; and looping the PXRbound distal phenobarbital-responsive enhancer module toward the proximal C/EBP binding site. Furthermore, the genotype-phenotype association was evaluated in cultured human primary hepatocytes from 44 donors. Interestingly, RIF-mediated induction of CYP2B6 in four Ϫ82T/C carriers was higher compared with that in the reference Ϫ82T/T homozygotes. Together, our results demonstrate, for the first time, a synergistic interplay between a CYP2B6 polymorphism and PXRmediated induction, which may contribute to the large individual variations and inducibility of CYP2B6 in humans.

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The Pregnane X Receptor Binds to Response Elements in a Genomic Context-Dependent Manner, and PXR Activator Rifampicin Selectively Alters the Binding Among Target Genes

Cited by 49 publications

References 28 publications

Rifampicin-Activated Human Pregnane X Receptor and CYP3A4 Induction Enhance Acetaminophen-Induced Toxicity

Rifampicin-Activated Human Pregnane X Receptor and CYP3A4 Induction Enhance Acetaminophen-Induced Toxicity

Oxysterol 22(R)-Hydroxycholesterol Induces the Expression of the Bile Salt Export Pump through Nuclear Receptor Farsenoid X Receptor but Not Liver X Receptor

Synergistically Enhanced CYP2B6 Inducibility between a Polymorphic Mutation in CYP2B6 Promoter and Pregnane X Receptor Activation

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