Tatsuya Sueyoshi

Comparative study of human and mouse pregnane X receptor agonistic activity in 200 pesticides using in vitro reporter gene assays

The nuclear receptor, pregnane X receptor (PXR), is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism. Recent studies have shown that PXR activation may affect energy metabolism as well as the endocrine and immune systems. In this study, we characterized and compared the agonistic activities of a variety of pesticides against human PXR (hPXR) and mouse PXR (mPXR). We tested the hPXR and mPXR agonistic activity of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 7 ureas, and 44 others) by reporter gene assays using COS-7 simian kidney cells. Of the 200 pesticides tested, 106 and 93 activated hPXR and mPXR, respectively, and a total of 111 had hPXR and/or mPXR agonistic activity with greater or lesser inter-species differences. Although all of the pyrethroids and most of the organochlorines and acid amides acted as PXR agonists, a wide range of pesticides with diverse structures also showed hPXR and/or mPXR agonistic activity. Among the 200 pesticides, pyributicarb, pretilachlor, piperophos and butamifos for hPXR, and phosalone, prochloraz, pendimethalin, and butamifos for mPXR, acted as particularly potent activators at low concentrations in the order of 10⁻⁸-10⁻⁷ M. In addition, we found that several organophosphorus oxon- and pyributicarb oxon-metabolites decreased PXR activation potency compared to their parent compounds. These results suggest that a large number of structurally diverse pesticides and their metabolites possess PXR-mediated transcriptional activity, and their ability to do so varies in a species-dependent manner in humans and mice.

The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

ABSTRACT PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as atrans-acting factor for the phenobarbital-inducibleCyp2b10 gene.

Phenobarbital-Responsive Nuclear Translocation of the Receptor CAR in Induction of the CYP2B Gene

ABSTRACT The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR. Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB responsive through an okadaic acid-sensitive nuclear translocation process.

Relative Activation of Human Pregnane X Receptor versus Constitutive Androstane Receptor Defines Distinct Classes of CYP2B6 and CYP3A4 Inducers

Both the human pregnane X receptor (hPXR) and constitutive androstane receptor (hCAR) are capable of regulating CYP3A4 and CYP2B6 gene expression. However, the majority of currently identified CYP3A4 and CYP2B6 inducers are confirmed activators of hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known to induce CYP3A4 and/or CYP2B expression were evaluated for relative activation of hPXR versus hCAR. Because of the high basal but low chemical-induced activation of hCAR in immortalized cells, alternative methods were used to evaluate hCAR activation potential. Thirteen of the 16 compounds were classified as moderate to strong hPXR activators. In contrast, carbamazepine (CMZ), efavirenz (EFV), and nevirapine (NVP) were classified as negligible or weak hPXR activators at concentrations associated with efficacious CYP2B6 reporter or endogenous gene induction in primary human hepatocytes, suggesting potential activation of hCAR. Subsequent experiments demonstrated that these three drugs efficiently induced nuclear accumulation of in vivo-transfected enhanced yellow fluorescent protein-hCAR and significantly increased expression of a CYP2B6 reporter gene when hCAR was expressed in CAR–/– mice. In addition, using a recently identified, chemically responsive splice variant of hCAR (hCAR3), the hCAR activation profiles of the 16 compounds were evaluated. By combining results from the hPXR- and hCAR3-based reporter gene assays, these inducers were classified as hPXR, hCAR, or hPXR/hCAR dual activators. Our results demonstrate that CMZ, EFV, and NVP induce CYP2B6 and CYP3A4 preferentially through hCAR and that hCAR3 represents a sensitive tool for in vitro prediction of chemical-mediated human CAR activation.

Differential Regulation of Hepatic CYP2B6 and CYP3A4 Genes by Constitutive Androstane Receptor but Not Pregnane X Receptor

Accumulated evidence suggests that cross-talk between the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) results in shared transcriptional activation of CYP2B and CYP3A genes. Although most data imply symmetrical cross-regulation of these genes by rodent PXR and CAR, the actual selectivities of the corresponding human receptors are unknown. The objective of this study was to evaluate the symmetry of human (h) PXR and hCAR cross-talk by comparing the selectivities of these receptors for CYP2B6 and CYP3A4. Human hepatocyte studies revealed nonselective induction of both CYP2B6 and CYP3A4 by hPXR activation but marked preferential induction of CYP2B6 by selective hCAR activation. Gel shift assays demonstrated that hPXR exhibited strong and relatively equal binding to all functional response elements in both CYP2B6 and CYP3A4 genes, whereas hCAR displayed significantly weak binding to the CYP3A4 proximal ER6 motif. In cell-based transfection assays, hCAR displayed greater activation of CYP2B6 reporter gene expression compared with CYP3A4 with constructs containing both proximal and distal regulatory elements. Furthermore, in agreement with binding observations, transfection assays using promoter constructs containing repeats of CYP2B6 DR4 and CYP3A4 ER6 motifs revealed an even greater difference in reporter activation by hCAR. In contrast, hPXR activation resulted in less discernible differences between CYP2B6 and CYP3A4 reporter gene expression. These results suggest asymmetrical cross-regulation of CYP2B6 and CYP3A4 by hCAR but not hPXR in that hCAR exhibits preferential induction of CYP2B6 relative to CYP3A4 because of its weak binding and functional activation of the CYP3A4 ER6.

Drug-activated nuclear receptors CAR and PXR

The metabolism and elimination of drugs is mainly mediated by cytochrome P450 (CYP) enzymes, aided by conjugative enzymes and transport proteins. An integral aspect of this elimination process is the induction of drug metabolism through activation of gene expression of metabolic and transport proteins. There is compelling evidence that induction is regulated by drug-activated nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR). This review outlines the basic properties of CAR and PXR, their ligands and target genes, and the mechanisms of the induction process. The implications of nuclear receptor-mediated induction for drug research are also discussed.

The Peptide Near the C Terminus Regulates Receptor CAR Nuclear Translocation Induced by Xenochemicals in Mouse Liver

ABSTRACT In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318–6322, 1999). To define the translocation mechanism, fluorescent protein-tagged human CAR (hCAR) was expressed in the mouse livers using the in situ DNA injection and gene delivery systems. As in the wild-type hCAR, the truncated receptor lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to the nucleus, indicating that the PB-inducible translocation is AF2 independent. Deletion of the 30 C-terminal residues abolished the receptor translocation, and subsequent site-directed mutagenesis delineated the PB-inducible translocation activity of the receptor to the peptide L313GLL316AEL319. Ala mutations of Leu313, Leu316, or Leu319 abrogated the translocation of CAR in the livers, while those of Leu312 or Leu315 did not affect the nuclear translocation. The leucine-rich peptide dictates the nuclear translocation of hCAR in response to various PB-type inducers and appears to be conserved in the mouse and rat receptors.

Nuclear Pregnane X Receptor Cross-talk with FoxA2 to Mediate Drug-induced Regulation of Lipid Metabolism in Fasting Mouse Liver

Upon drug activation, the nuclear pregnane X receptor (PXR) regulates not only hepatic drug but also energy metabolism. Using Pxr–/– mice, we have now investigated the PXR-mediated repression of lipid metabolism in the fasting livers. Treatment with PXR activator pregnenolone 16α-carbonitrile (PCN) down-regulated the mRNA levels of carnitine palmitoyltransferase 1A (in β-oxidation) and mitochondrial 3-hydroxy-3-methylglutarate-CoA synthase 2 (in ketogenesis) in wild-type (Pxr+/+) mice only. In contrast, the stearoyl-CoA desaturase 1 (in lipogenesis) mRNA was up-regulated in the PCN-treated Pxr+/+ mice. Reflecting these up- and down-regulations and consistent with decreased energy metabolism, the levels of hepatic triglycerides and of serum 3-hydroxybutylate were increased and decreased, respectively, in the PCN-treated Pxr+/+ mice. Using gel shift, glutathione S-transferase pull-down and cell-based reporter assays, we then examined whether PXR could cross-talk with the insulin response forkhead factor FoxA2 to repress the transcription of the Cpt1a and Hmgcs2 genes, because FoxA2 activates these genes in fasting liver. PXR directly bound to FoxA2 and repressed its activation of the Cpt1a and Hmgcs2 promoters. Moreover, ChIP assays showed that PCN treatment attenuated the binding of FoxA2 to these promoters in fasting Pxr+/+ but not Pxr–/– mice. These results are consistent with the conclusion that PCN-activated PXR represses FoxA2-mediated transcription of Ctp1a and Hmgcs2 genes in fasting liver.

Dephosphorylation of Threonine 38 Is Required for Nuclear Translocation and Activation of Human Xenobiotic Receptor CAR (NR1I3)

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the α-helix spanning from residues 29–42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.

Phenobarbital Indirectly Activates the Constitutive Active Androstane Receptor (CAR) by Inhibition of Epidermal Growth Factor Receptor Signaling

The epidermal growth factor receptor is an unexpected target of the barbiturate phenobarbital. Antagonistic Activation Phenobarbital stimulates the transcription of genes in the liver that encode drug metabolism enzymes by indirectly stimulating the constitutive active androstane receptor (CAR). Mutoh et al. identified epidermal growth factor receptor (EGFR) as a cell surface binding target of phenobarbital. Phenobarbital bound to EGFR and blocked the binding of the ligand EGF, thereby preventing the activation of EGFR. This inhibition of EGFR promoted the activation of CAR. Molecular simulation predicted that phenobarbital and EGF share binding sites on EGFR. Together, the findings indicate that phenobarbital stimulates the nuclear activity of CAR by inhibiting the activity of EGFR at the cell surface. Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr52, which then promoted the dephosphorylation of CAR at Thr38 by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.