Masahiko Negishi

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.

The Repressed Nuclear Receptor CAR Responds to Phenobarbital in Activating the Human CYP2B6 Gene

The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. The PB induction mediated by CAR is regulated by a conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between −1733 and −1683 bp in the gene’s 5′-flanking region. An in vitro translated CAR acting as a retinoid X receptor α heterodimer binds directly to the two nuclear receptor sites NR1 and NR2 within PBREM. In a stably transfected HepG2 cell line, both PBREM and NR1 are activated by PB and PB-type compounds such as chlorinated pesticides, polychlorinated biphenyls and chlorpromazine. In addition to PBREM, CAR also transactivates the steroid/rifampicin-response element of the humanCYP3A4 gene in HepG2 cells. Thus, activation of the repressed nuclear receptor CAR appears to be a versatile mediator that regulates PB induction of the CYP2B and other genes.

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.

Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.

The Orphan Nuclear Receptor Constitutive Active/Androstane Receptor Is Essential for Liver Tumor Promotion by Phenobarbital in Mice

Hepatocellular carcinoma (HCC) is known to progress through a step often called tumor promotion. Phenobarbital (PB) is the prototype of nongenotoxic cacinogens that promote HCC in rodents. The molecular target of PB to elicit the promotion has been the subject of intense investigations over the last 30 years since it was discovered. The nuclear receptor constitutive active/androstane receptor (CAR) is activated by PB as well as by various other xenobiotics such as therapeutic drugs and environmental pollutants. CAR activation results in the transcriptional induction of numerous hepatic genes including those that encode xenobiotic-metabolizing enzymes such as a set of cytochrome P450s. In addition to PB, many CAR activators are nongenotoxic carcinogens, but the role of CAR in liver tumor promotion remains unexplored. Using Car−/− mice, we have here examined tumor promotion by chronic treatment with PB in drinking water after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine. None of the Car−/− mice developed either eosinophilic foci or advanced liver tumors, whereas all Car+/+ mice developed HCC and/or adenoma by 39 weeks. The results indicate that CAR is the molecular target of promotion by PB and that activation of this receptor is an essential requirement for liver tumor development.

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.

Nuclear Receptors CAR and PXR Cross Talk with FOXO1 To Regulate Genes That Encode Drug-Metabolizing and Gluconeogenic Enzymes

ABSTRACT The nuclear receptors CAR and PXR activate hepatic genes in response to therapeutic drugs and xenobiotics, leading to the induction of drug-metabolizing enzymes, such as cytochrome P450. Insulin inhibits the ability of FOXO1 to express genes encoding gluconeogenic enzymes. Induction by drugs is known to be decreased by insulin, whereas gluconeogenic activity is often repressed by treatment with certain drugs, such as phenobarbital (PB). Performing cell-based transfection assays with drug-responsive and insulin-responsive enhancers, glutathione S-transferase pull down, RNA interference (RNAi), and mouse primary hepatocytes, we examined the molecular mechanism by which nuclear receptors and FOXO1 could coordinately regulate both enzyme pathways. FOXO1 was found to be a coactivator to CAR- and PXR-mediated transcription. In contrast, CAR and PXR, acting as corepressors, downregulated FOXO1-mediated transcription in the presence of their activators, such as 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone 16α-carbonitrile, respectively. A constitutively active mutant of the insulin-responsive protein kinase Akt, but not the kinase-negative mutant, effectively blocked FOXO1 activity in cell-based assays. Thus, insulin could repress the receptors by activating the Akt-FOXO1 signal, whereas drugs could interfere with FOXO1-mediated transcription by activating CAR and/or PXR. Treatment with TCPOBOP or PB decreased the levels of phosphoenolpyruvate carboxykinase 1 mRNA in mice but not in Car −/− mice. We conclude that FOXO1 and the nuclear receptors reciprocally coregulate their target genes, modulating both drug metabolism and gluconeogenesis.

Crystal structure of estrogen sulphotransferase.

The structure of estrogen sulphotransferase has been solved in the presence of inactive cofactor PAP and substrate 17β-estradiol. This structure reveals structural similarities between cytosolic sulphotransf erases and nucleotide kinases.

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.

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.

Human β1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Galβ1–3Galβ1–4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 Å in the presence of the donor substrate product UDP, the catalytic Mn2+ ion, and the acceptor substrate analog Galβ1–3Galβ1–4Xyl. The enzyme is a α/β protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu227, Asp252, and Glu281 dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu281 is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp194, Asp195, Asp196) has direct interaction with the ribose of the UDP molecule as well as with the Mn2+ ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.