Hiroki Tojima

Ligand dependent hepatic gene expression profiles of nuclear receptors CAR and PXR

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are key regulators of drug-metabolizing enzymes and transporters. These two receptors are closely associated with each other and also have overlapping functions. This study investigated the overall hepatic gene expression profiles and the regulatory roles of these nuclear receptors using CAR/PXR single and double knockout mice. Basal and ligand-stimulated gene expression profiles were obtained in each mouse using cDNA microarrays and a reverse transcriptase-polymerase chain reaction. Enzymes such as Cyp2b10, Cyp3a11, Cdc20 and Cdk1 displayed both CAR- and PXR-dependent induction. Inversely, enzymes such as Cyp4a10, Fos and Mme displayed both CAR- and PXR-dependent repression. Enzymes such as Cyp1a1, Cyp1a2 and c-Myc represented the group of genes only induced by CAR. Enzymes such as Aacs represented the group of genes induced only by the PXR. CAR and PXR are closely associated and have diverse roles, both as positive and negative regulators of hepatic genes including xenobiotic metabolism, apoptosis, cholesterol biosynthesis, lipid metabolism, and cytokine signaling pathways.

Nuclear receptors CAR and PXR; therapeutic targets for cholestatic liver disease.

Cholestasis results in the intrahepatic retention of cytotoxic bile acids (BA) and it can thus lead to liver injury. Hydrophilic BA ursodeoxycholic acid (UDCA) is currently used to treat cholestasis but its efficacy is limited. Nuclear receptors are key regulators of various processes including metabolism of xeno- and endobiotics such as BA and drugs. Recent studies have made significant progress in elucidating the mechanisms which regulate the BA metabolism by nuclear receptors. The nuclear receptor FXR plays the role of master regulator of BA homeostasis and is a promising drug target for cholestatic liver disease. In addition to FXR, the nuclear receptors CAR and PXR function as sensors of toxic byproducts and regulator of BA homeostasis. Ligands for both receptors including phenobarbital have been used to treat cholestatic liver diseases before the mechanisms of these receptors were revealed. Novel compounds targeting CAR and PXR with specific effects and fewer side effects will therefore be useful for the treatment of cholestatic liver diseases. This article will review the current knowledge of the xenobiotic-sensing nuclear receptors CAR and PXR, while also discussing their potential role in the treatment of cholestatic liver diseases.

Constitutive active/androstane receptor promotes hepatocarcinogenesis in a mouse model of non-alcoholic steatohepatitis.

The nuclear receptor constitutive active/androstane receptor (CAR) acts as a sensor of toxic byproducts derived from the endogenous metabolism and exogenous chemicals. We previously reported that CAR is responsible for exacerbating hepatic injury and fibrosis in a dietary model of non-alcoholic steatohepatitis (NASH) via upregulation of lipid peroxidation. In this study, we investigated the pathological roles of the CAR in the development of hepatocellular carcinoma in NASH model. CAR+/+ and CAR-/- mice were fed methionine- and choline-deficient (MCD) diet after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine (DEN) at 2 weeks of age. Interestingly, the MCD diet dramatically promoted DEN-induced hepatocarcinogenesis in CAR+/+ mice. However, the deletion of CAR leads to a significantly lower tumor incidence and smaller tumor diameter. Hepatocytes of MCD-treated-CAR+/+ mice showed a significantly higher staining frequency of Ki-67, a marker of cell proliferation, and exhibited a higher expression of c-Myc and FoxM1 transcripts compared with MCD-treated CAR-/- mice. Immunohistochemistry revealed the nuclear translocation of CAR thus suggesting that the activation of CAR signaling increased in the hepatocytes of CAR+/+ mice fed MCD diet. In addition, in vitro experiments using the CAR stably expressed cell line with TCPOBOP have suggested that CAR activation directly leads to cell proliferation. Survival was significantly lower in the CAR+/+ mice fed the MCD diet in comparison with the CAR-/- mice. Taken together, these results suggest that CAR may therefore play a critical role in the hepatocarcinogenesis of the murine NASH model via the upregulation of cell proliferation.

Expression of amino acid transporters (LAT1, ASCT2 and xCT) as clinical significance in hepatocellular carcinoma.

Amino acid transporters play an important role in tumor progression and survival of cancer cells. However, the prognostic significance of L‐type amino acid transporter 1 (LAT1), system ASC amino acid transporter‐2 (ASCT2) and xCT expression in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of this study is to investigate the clinicopathological significance of these amino acid transporters in patients with HCC.

Xenobiotic-sensing nuclear receptors CAR and PXR as drug targets in cholestatic liver disease.

Cholestasis results in the intrahepatic retention of cytotoxic bile acid and it can thus lead to liver injury and/or liver fibrosis. Cholestatic liver damage is counteracted by a variety of intrinsic hepatoprotective mechanisms including a complex network of drug metabolizing enzymes and transporters. During the last decade, much progress has been made in dissecting the mechanisms which regulate the hepatic xeno- and endobiotic metabolism by nuclear receptors. The xenobiotic receptors CAR and PXR are two important members of the NR1I nuclear receptor family. They function as sensors of toxic byproducts derived from the endogenous metabolism and of exogenous chemicals, in order to enhance their elimination. Ligands for both receptors, including phenobarbital, have already been used to treat cholestatic liver diseases before the mechanisms of these receptors were revealed. Furthermore, Yin Zhi Huang, a traditional Chinese herbal medicine, which has been used to prevent and treat neonatal jaundice, was identified to be a CAR ligand which also accelerates bilirubin clearance. Therefore, CAR and PXR have a protective effect on cholestasis by activating both detoxification enzymes and transporters. As a result, novel compounds targeting CAR and PXR with specific effects and fewer side effects will therefore be useful for the treatment of cholestatic liver diseases. This article will review the current knowledge on xenobiotic-sensing nuclear receptors CAR and PXR, while also discussing their potential role in the treatment of cholestatic liver diseases.

Histone Deacetylase Inhibitors Induce Cytochrome P450 2B by Activating Nuclear Receptor Constitutive Androstane Receptor

Valproic acid, a histone deacetylase (HDAC) inhibitor, induces the cytochrome P450 2B subfamily. However, the effects of HDAC inhibitors on CYP2B induction are still not fully understood. Nuclear receptor constitutive androstane receptor (CAR) is a key regulator of CYP2B induction. In this study, we investigated the effect of HDAC inhibitors on CAR-mediated CYP2B induction. The expression of CYP2B6 mRNA was induced in HepG2 cells stably expressing mouse CAR (Ym17) by HDAC inhibitors including valproic acid, phenylbutyrate, and trichostatin A. HDAC inhibitors activated the phenobarbital-responsive enhancer module of the CYP2B6 promoter in transient transfection reporter assays with Ym17 cells. Furthermore, HDAC inhibitors synergistically augmented the effect of the CAR ligand, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, in the transactivation of CYP2B6 mRNA and the promoter assay in Ym17 cells. Intraperitoneal injection of HDAC inhibitors induced Cyp2b10 mRNA in wild-type mice. However, such induction was not observed in CAR(−/−) mice. Immunoprecipitation demonstrated that CAR formed a complex with HDACs. HDAC inhibitors diminished the binding between CAR and HDAC1 and augmented the binding of steroid receptor coactivator-1 (SRC-1) to CAR. Furthermore, small interfering RNA knockdown of HDAC1 increased CYP2B6 mRNA expression. These results provide novel insight into the mechanism by which HDAC inhibitors affect gene expression of CYP2B6. HDAC inhibitors have the potential to up-regulate CYP2B6 through the dissociation of HDAC1 and recruitment of SRC-1 to receptor CAR.

Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice

Abstract Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20 mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.

Factors predicting the overall response and overall survival in hepatocellular carcinoma patients undergoing balloon-occluded transcatheter arterial chemoembolization (B-TACE): A retrospective cohort study

This study aimed to investigate the factors predicting overall response and overall survival in hepatocellular carcinoma patients undergoing balloon‐occluded transcatheter arterial chemoembolization (B‐TACE).

Constitutive androstane receptor and pregnane X receptor cooperatively ameliorate DSS-induced colitis

BACKGROUND Nuclear receptor pregnane X receptor (PXR) was shown to be protective in case of dextran sulfate sodium (DSS)-induced colitis. Constitutive androstane receptor (CAR) belongs to the same nuclear receptor subfamily with PXR. The roles of both receptors in DSS-induced colitis were evaluated. METHODS Wild-type, Car-null, Pxr-null, and Car/Pxr-null mice were treated with a CAR/PXR agonist or vehicle and administered 2.5% DSS in the drinking water. The typical clinical symptoms, histological scoring, proinflammatory cytokine, and apoptosis were analyzed. RESULTS Mice treated with the PXR agonist pregnenolone-16α-carbonitrile (PCN) were protected from DSS-induced colitis, as in a previous study. Mice treated with the CAR agonist, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were also protected from DSS-induced colitis. Interestingly, the protective effects of PCN in the Car-null mice and those of TCPOBOP in the Pxr-null mice both decreased. PCN or TCPOBOP pretreatment significantly decreased the macrophage and monocyte infiltration in DSS-induced colitis. PXR and CAR agonists reduced the mRNA expression of several proinflammatory cytokines in a PXR- and CAR-dependent manner, respectively. CAR inhibited apoptosis by inducing Gadd45b. PXR inhibited TNF-α and IL-1b and CAR induced Gadd45b in in vitro cell analyses. CONCLUSIONS We showed that CAR and PXR cooperatively ameliorate DSS-induced colitis. PXR and CAR protected against DSS-induced colitis by inhibiting proinflammatory cytokines and apoptosis, respectively.

Faecal lactoferrin is a useful biomarker for mucosal healing in patients with ulcerative colitis during granulocyte and monocyte adsorptive apheresis therapy.

The study investigated the value of faecal lactoferrin as a follow‐up biomarker for mucosal healing of ulcerative colitis during granulocyte and monocyte adsorptive apheresis (GMA) therapy.