論文 - 児玉　進

Pregnane X Receptor PXR Activates the GADD45 beta Gene, Eliciting the p38 MAPK Signal and Cell Migration 査読

Susumu Kodama, Masahiko Negishi

JOURNAL OF BIOLOGICAL CHEMISTRY   286 ( 5 )   3570 - 3578   2011年2月

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Pregnane X receptor (PXR) was originally characterized as a transcription factor that induces hepatic drug metabolism by activating cytochrome P450 genes. Here we have now demonstrated a novel function of PXR, that of eliciting p38 mitogen-activated protein kinase (MAPK) phosphorylation for cell migration. Upon xenobiotic activation of ectopic human PXR, human hepatocellular carcinoma HepG2 cells were found to exhibit increased phosphorylation of p38 MAPK and to subsequently change morphology and migrate. p38 MAPK was responsible for the regulation of these morphological changes and cell migration because the p38 MAPK inhibitor SB239063 repressed both. Prior to this phosphorylation, PXR directly activated the early response GADD45 beta gene by binding to a distal direct repeat 4 site of the GADD45 beta promoter. Ectopic expression of GADD45 beta increased p38 MAPK phosphorylation, whereas siRNA knockdown of GADD45 beta decreased the PXR-induced p38 MAPK phosphorylation, confirming that GADD45 beta can regulate PXR-induced p38 MAPK phosphorylation in HepG2 cells. These results indicate that PXR activates the GADD45 beta gene, increasing p38 MAPK phosphorylation, and leading HepG2 cells to change morphology and migrate. The GADD45 beta gene is a direct target for PXR, eliciting cell signals to regulate various cellular functions.

DOI： 10.1074/jbc.M110.179812

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Recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants 査読

Susumu Kodama, Kumiko Okada, Keiko Akimoto, Hideyuki Inui, Hideo Ohkawa

PLANT BIOTECHNOLOGY JOURNAL   7 ( 2 )   119 - 128   2009年2月

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

Dioxin residues widely contaminate soil and agricultural products at low concentrations and may accumulate in organisms at the top of food chains owing to their physicochemical properties. In this study, we have developed novel, dioxin-inducible, reporter gene expression systems regulated by recombinant aryl hydrocarbon receptors (AhRs). The recombinant AhRs, referred to as XDVs, consist of the DNA-binding domain of the bacterial repressor protein LexA, a 90-kDa heat shock protein- and ligand-binding regulatory domain from mouse AhR, and the transactivation domain of herpes simplex virus regulatory protein VP16. Transgenic tobacco plants carrying XDVs absorb various AhR ligands, including 3-methylcholanthrene, beta-naphthoflavone and indigo from solid medium and vermiculite, and show dose- and time-dependent expression of the beta-glucuronidase reporter gene. The results clearly suggest that XDVs are functional transcription factors that respond to AhR ligands, and that the XDV-mediated reporter gene expression system is applicable to bioassays for dioxin residues in the environment.

DOI： 10.1111/j.1467-7652.2008.00378.x

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Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto the CYP24A1 Promoter to Attenuate Vitamin D-3 Activation 査読

Yoshihiro Konno, Susumu Kodama, Rick Moore, Nobuhiro Kamiya, Masahiko Negishi

MOLECULAR PHARMACOLOGY   75 ( 2 )   265 - 271   2009年2月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We have studied the molecular mechanism by which the nuclear xenobiotic receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) regulate transcription of the vitamin D-3 24-hydroxylase (CYP24A1) gene. In the absence of vitamin D-3, PXR activates the CYP24A1 gene by directly binding to and transactivating vitamin D-response elements (VDREs) within its promoter. Vitamin D-3 activates the CYP24A1 promoter by dissociating the corepressor silencing mediator for retinoid and thyroid hormone receptors ( SMRT) from the vitamin D receptor (VDR) on those VDREs. PXR strongly represses vitamin D-3 activation of the CYP24A1 gene, in which PXR indirectly binds to and prevents vitamin D-3-dependent dissociation of SMRT from the CYP24A1 promoter. The degree of the PXR-mediated locking of SMRT depends on the relative concentration of vitamin D 3 to the human PXR activator rifampicin; SMRT increased its dissociation as this ratio increased. CAR is also found to prevent dissociation of SMRT from the CYP24A1 promoter. Thus, our present study defines the novel molecular mechanism by which PXR and CAR mediate drug interactions with vitamin D 3 to regulate the CYP24A1 gene. Pxr(+/+) and Pxr(-/-) mice were continuously treated with mouse PXR activator PCN to evaluate the hypothesis that induction of the Cyp24a1 gene is responsible for the loss of bone mineral density often observed in patients treated continuously with PXR-activating drugs. PCN-dependent loss of mineral density is observed in the metaphyseal bones of only the Pxr(+/+) mice. This loss, however, does not correlate with the expression levels of the Cyp24a1 gene in these mice.

DOI： 10.1124/mol.108.051904

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The roles of nuclear receptors CAR and PXR in hepatic energy metabolism 招待 査読

Yoshihiro Konno, Masahiko Negishi, Susumu Kodama

DRUG METABOLISM AND PHARMACOKINETICS   23 ( 1 )   8 - 13   2008年

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAP SOC STUDY XENOBIOTICS  

Nuclear receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) were originally characterized as transcription factors regulating the hepatic genes that encode drug metabolizing enzymes. Recent works have now revealed that these nuclear receptors also play the critical roles in modulating hepatic energy metabolism. While CAR and PXR directly bind to their response sequences phenobarbital-responsive enhancer module (PBREM) and xenobiotic responsive enhancer module (XREM) in the promoter of target genes to increase drug metabolism, the receptors also cross talk with various hormone responsive transcription factors such as forkhead box O1 (FoxO1), forkhead box A2 (FoxA2), cAMP-response element binding protein, and peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC 1 alpha) to decrease energy metabolism through down-regulating gluconeogenesis, fatty acid oxidation and ketogenesis and up-regulating lipogenesis. In addition, CAR modulates thyroid hormone activity by regulating type 1 deiodinase in the regenerating liver. Thus, CAR and PXR are now placed at the crossroad where both xenobiotics and endogenous stimuli co-regulate liver function.

DOI： 10.2133/dmpk.23.8

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Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants 査読

Stisumu Kodama, Kumiko Okada, Hideyuki Inui, Hideo Ohkawa

PLANTA   227 ( 1 )   37 - 45   2007年12月

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems for a beta-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter in transgenic tobacco plants. On treatment with the AhR ligands 3-methyl-cholanthrene (MC), beta-naphthoflavone (beta NF), and indigo, the transgenic tobacco plants exhibited enhanced GUS activity, presumably by inducible expression of the reporter gene. The recombinant AhR (AhRV), with the activation domain replaced by that of the Herpes simplex virus protein VP16, induced GUS activity much more than the wild-type AhR in the transgenic tobacco plants. Plants carrying AhRV expressed the GUS reporter gene in a dose- and time-dependent manner when treated with MC; GUS activity was detected at 5 nM MC on solid medium and at 12 h after soaking in 25 mu M MC. Histochemical GUS staining showed that this system was active mainly in leaf and stem. These results suggest that the AhR-mediated reporter gene expression system has potential for the bioassay of dioxins in the environment and as a novel gene expression system in plants.

DOI： 10.1007/s00425-007-0592-1

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Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene 査読

Susumu Kodama, Rick Moore, Yukio Yamamoto, Masahiko Negishi

BIOCHEMICAL JOURNAL   407 ( Pt 3 )   373 - 381   2007年11月

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

The nuclear PXR (pregnane X receptor) was originally characterized as a key transcription factor that activated hepatic genes encoding drug-metabolizing enzymes. We have now demonstrated that PXR also represses glucagon-activated transcription of the G6Pase (glucose-6-phosphatase) gene by directly binding to CREB [CRE (CAMP-response element)-binding protein]. Adenoviral-mediated expression of human PXR (hPXR) and its activation by rifampicin strongly repressed cAMP-dependent induction of the endogenous G6Pase gene in Huh7 cells. Using the - 259 by G6Pase promoter construct in cell-based transcription assays, repression by hPXR of PKA (CAMP-dependent protein kinase)-mediated promoter activation was delineated to CRE sites. GST (glutathione transferase) pull-down and immunoprecipitation assays were employed to show that PXR binds directly to CREB, while gel-shift assays were used to demonstrate that this binding prevents CREB interaction with the CRE. These results are consistent with the hypothesis that PXR represses the transcription of the G6Pase gene by inhibiting the DNA-binding ability of CREB. In support of this hypothesis, treatment with the mouse PXR activator PCN (pregnenolone 16 alpha-carbonitrile) repressed CAMP-dependent induction of the G6Pase gene in primary hepatocytes prepared from wild-type, but not from PXR-knockout, mice, and also in the liver of fasting wildtype, but not PXR-knockout, mice. Moreover, ChIP (chromatin immunoprecipitation) assays were performed to show a decreased CREB binding to the G6Pase promoter in fasting wild-type mice after PCN treatment. Thus drug activation of PXR can repress the transcriptional activity of CREB, down-regulating gluconeogenesis.

DOI： 10.1042/BJ20070481

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Phenobarbital confers its diverse effects by activating the orphan nuclear receptor car 招待 査読

S Kodama, M Negishi

DRUG METABOLISM REVIEWS   38 ( 1-2 )   75 - 87   2006年

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS INC  

In the early 1960s, phenobarbital (PB) was shown to induce hepatic drug metabolism and the induction was implicated in the molecular mechanism of drug tolerance development. Since then, it has become evident that PB not only induces drug metabolism, but also triggers pleiotropic effects on liver function., such as cell growth and communication, proliferation of the endoplasmic reticulum, tumor promotion, glucose metabolism, steroid/thyroid hormone metabolism, and bile acid synthesis. Upon activation by PB and numerous PB-type inducers, the nuclear receptor CAR mediates those pleiotropic actions by regulating various hepatic genes, utilizing multiple regulatory mechanisms.

DOI： 10.1080/03602530600569851

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Monitoring of endocrine disruptors in transgenic plants carrying aryl hydrocarbon receptor and estrogen receptor genes 査読

H Inui, H Sasaki, S Kodama, NH Chua, H Ohkawa

NEW DISCOVERIES IN AGROCHEMICALS   892   40 - 47   2005年

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Transgenic plants carrying mammalian hormone receptor genes were examined for monitoring of environmental chemicals including dioxins and endocrine disruptors. The transgenic tobacco plants expressing mouse arylhydrocarbon receptor gene detected 1ppb of the dioxin-like compound 20-methylcholanthrene in the cultured plants. In addition, the transgenic Arabidopsis plants expressing human estrogen receptor gene detected 1ppt of 17 beta-estradiol and ppb levels of the other endocrine disruptors such as bisphenol A, 4-t-octylphenol, and others in potted plants. These transgenic plants appear to be useful for on site monitoring of dioxins and estrogenic contaminants in the environment. The transgenic plants expressing AhR and ER detected 1ppb of MC under cultured conditions and 1ppt of E-2 under potted conditions, respectively. Basic technology was established for monitoring dioxins and EDs in the transgenic plants with the receptor genes. However, increase of the sensitivity towards other EDs with low affinity towards receptors is necessary. In order to overcome this problem, (1)sensitive receptors may be useful instead of mouse AhR and human ER. It is known that fish ERs were more sensitive to alkylphenols such as nonylphenol and octylphenol than mammalian ones. (2)Increase of uptake efficiency of dioxins and EDs is another way to increase sensitivity. Zucchini plants are known to have efficient translocation of polychlorinated dibenzo-p-dioxins and dibenzofurans(7). Furthermore, (3)genes synthesizing flower colors as reporter genes may be useful for visible monitoring of contaminations on site.

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Nuclear receptors CAR and PXR cross talk with FOXO1 to regulate genes that encode drug-metabolizing and gluconeogenic enzymes 査読

S Kodama, C Koike, M Negishi, Y Yamamoto

MOLECULAR AND CELLULAR BIOLOGY   24 ( 18 )   7931 - 7940   2004年9月

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

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 16alpha-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.

DOI： 10.1128/MCB.24.18.7931.7940.2004

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Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants 査読

N Shiota, S Kodama, H Inui, H Ohkawa

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   64 ( 10 )   2025 - 2033   2000年10月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1, The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv, Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did, The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.

DOI： 10.1271/bbb.64.2025

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