研究者詳細 - 児玉　進

論文 - 児玉　進

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Structural characterization of 1,3-bis-tert-butyl monocyclic benzene derivatives with agonistic activity towards retinoid X receptor alpha 査読国際誌

Susumu Kodama, Shuzo Matsumoto, Yuta Takamura, Michiko Fujihara, Masaki Watanabe, Atsushi Ono, Hiroki Kakuta

Toxicology Letters 373 76 - 83 2022年11月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier BV

Retinoid X receptor alpha (RXRα) plays pivotal roles in multiple biological processes, but limited information is available on the structural features of chemicals that show low affinity for RXRα, but nevertheless cause significant activation, though these may represent a human health hazard. We recently discovered that several industrial chemicals having 1,3-bis-tert-butylbenzene as a common chemical structure exhibit agonistic activity towards rat RXRα. In this study, we explored the structure-activity relationship of 1,3-bis-tert-butyl monocyclic benzene derivatives for RXRα activation by means of in vitro and in silico analyses. The results indicate that a bulky substituent at the 5-position is favorable for agonistic activity towards human RXRα. Since 1,3-bis-tert-butyl monocyclic benzene derivatives with bulky hydrophobic moieties differ structurally from known RXRα ligands such as 9-cis-retinoic acid and bexarotene, our findings may be helpful for the development of structural alerts in the safety evaluation of industrial chemicals for RXRα-based toxicity to living organisms.

DOI： 10.1016/j.toxlet.2022.11.003

Association between in vitro nuclear receptor-activating profiles of chemical compounds and their in vivo hepatotoxicity in rats 査読

Susumu Kodama, Nao Yoshii, Akihiro Ota, Jun-ichi Takeshita, Kouichi Yoshinari, Atsushi Ono

The Journal of Toxicological Sciences 46 ( 12 ) 569 - 587 2021年12月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Toxicology

The liver plays critical roles to maintain homeostasis of living organisms and is also a major target organ of chemical toxicity. Meanwhile, nuclear receptors (NRs) are known to regulate major liver functions and also as a critical target for hepatotoxic compounds. In this study, we established mammalian one-hybrid assay systems for five rat-derived NRs, namely PXR, PPARα, LXRα, FXR and RXRα, and evaluated a total of 326 compounds for their NR-activating profiles. Then, we assessed the association between their NR-activating profile and hepatotoxic endpoints in repeated-dose toxicity data of male rats from Hazard Evaluation Support System. In the in vitro cell-based assays, 68, 38, 20, 17 and 17 compounds were identified as positives for PXR, PPARα, LXRα, FXR and RXRα, respectively. The association analyses demonstrated that the PXR-positive compounds showed high frequency of endpoints related to liver hypertrophy, such as centrilobular hepatocellular hypertrophy, suggesting that PXR activation is involved in chemical-induced liver hypertrophy in rats. It is intriguing to note that the PXR-positive compounds also showed statistically significant associations with both prolonged activated partial thromboplastin time and prolonged prothrombin time, suggesting a possible involvement of PXR in the regulation of blood clotting factors. Collectively, our approach may be useful for discovering new functions of NRs as well as understanding the complex mechanism for hepatotoxicity caused by chemical compounds.

DOI： 10.2131/jts.46.569

PXR Functionally Interacts with NF-κB and AP-1 to Downregulate the Inflammation-Induced Expression of Chemokine CXCL2 in Mice. 査読国際誌

Maya Okamura, Ryota Shizu, Taiki Abe, Susumu Kodama, Takuomi Hosaka, Takamitsu Sasaki, Kouichi Yoshinari

Cells 9 ( 10 ) 2020年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Pregnane X receptor (PXR) is a liver-enriched xenobiotic-responsive transcription factor. Although recent studies suggest that PXR shows anti-inflammatory effects by suppressing nuclear factor kappa B (NF-κB), the detailed mechanism remains unclear. In this study, we aimed to elucidate this mechanism. Mice were treated intraperitoneally with the PXR agonist pregnenolone 16α-carbonitrile (PCN) and/or carbon tetrachloride (CCl4). Liver injury was evaluated, and hepatic mRNA levels were determined via quantitative reverse transcription polymerase chain reaction. Reporter assays with wild-type and mutated mouse Cxcl2 promoter-containing reporter plasmids were conducted in 293T cells. Results showed that the hepatic expression of inflammation-related genes was upregulated in CCl4-treated mice, and PCN treatment repressed the induced expression of chemokine-encoding Ccl2 and Cxcl2 among the genes investigated. Consistently, PCN treatment suppressed the increased plasma transaminase activity and neutrophil infiltration in the liver. In reporter assays, tumor necrosis factor-α-induced Cxcl2 expression was suppressed by PXR. Although an NF-κB inhibitor or the mutation of an NF-κB-binding motif partly reduced PXR-dependent suppression, the mutation of both NF-κB and activator protein 1 (AP-1) sites abolished it. Consistently, AP-1-dependent gene transcription was suppressed by PXR with a construct containing AP-1 binding motifs. In conclusion, the present results suggest that PXR exerts anti-inflammatory effects by suppressing both NF-κB- and AP-1-dependent chemokine expression in mouse liver.

DOI： 10.3390/cells9102296

Functional interaction between PXR and YAP in xenobiotic-dependent liver hypertrophy and drug metabolism. 査読

Abe T, Shizu R, Sasaki T, Shimizu Y, Hosaka T, Kodama S, Matsuzawa A, Yoshinari K

The Journal of pharmacology and experimental therapeutics 371 ( 3 ) 590 - 601 2019年9月

Role of YAP activation in nuclear receptor CAR-mediated proliferation of mouse hepatocytes 査読国際誌

Taiki Abe, Yuto Amaike, Ryota Shizu, Miki Takahashi, Makoto Kano, Takuomi Hosaka, Takamitsu Sasaki, Susumu Kodama, Atsushi Matsuzawa, Kouichi Yoshinari

Toxicological Sciences 165 ( 2 ) 408 - 419 2018年10月

Points-to-consider documents: Scientific information on the evaluation of genetic polymorphisms during non-clinical studies and phase I clinical trials in the Japanese population. 査読国際誌

Masahiro Hiratsuka, Noriyasu Hirasawa, Yoshiteru Oshima, Susumu Kodama, Toshio Miyata, Takashi Dan, Hiroyuki Takatoku, Hideaki Kuribayashi, Ryosuke Nakamura, Yoshiro Saito

Drug metabolism and pharmacokinetics 33 ( 3 ) 141 - 149 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Pharmacotherapy shows striking individual differences in pharmacokinetics and pharmacodynamics, involving drug efficacy and adverse reactions. Recent genetic research has revealed that genetic polymorphisms are important intrinsic factors for these inter-individual differences. This pharmacogenomic information could help develop safer and more effective precision pharmacotherapies and thus, regulatory guidance/guidelines were developed in this area, especially in the EU and US. The Project for the Promotion of Progressive Medicine, Medical Devices, and Regenerative Medicine by the Ministry of Health, Labour and Welfare, performed by Tohoku University, reported scientific information on the evaluation of genetic polymorphisms, mainly on drug metabolizing enzymes and transporters, during non-clinical studies and phase I clinical trials in Japanese subjects/patients. We anticipate that this paper will be helpful in drug development for the regulatory usage of pharmacogenomic information, most notably pharmacokinetics.

DOI： 10.1016/j.dmpk.2018.01.005

Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acid 査読

Taiki Abe, Mirei Takahashi, Makoto Kano, Yuto Amaike, Chizuru Ishii, Kazuhiro Maeda, Yuki Kudoh, Toru Morishita, Takuomi Hosaka, Takamitsu Sasaki, Susumu Kodama, Atsushi Matsuzawa, Hiroyuki Kojima, Kouichi Yoshinari

ARCHIVES OF TOXICOLOGY 91 ( 6 ) 2365 - 2374 2017年6月

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 HEIDELBERG

Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor alpha is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.

DOI： 10.1007/s00204-016-1888-3

Pregnenolone 16α-carbonitrile ameliorates concanavalin A-induced liver injury in mice independent of the nuclear receptor PXR activation 査読

Susumu Kodama, Takuto Shimura, Hideaki Kuribayashi, Taiki Abe, Kouichi Yoshinari

Toxicology Letters 271 58 - 65 2017年4月

PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor 査読

Ryota Shizu, Taiki Abe, Satoshi Benoki, Miki Takahashi, Susumu Kodama, Masaaki Miayata, Atsushi Matsuzawa, Kouichi Yoshinari

BIOCHEMICAL JOURNAL 473 ( 3 ) 257 - 266 2016年2月

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

Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnaneXreceptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16 alpha-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression.

DOI： 10.1042/BJ20150734

Pregnane X Receptor Represses HNF4α Gene to Induce Insulin-Like Growth Factor-Binding Protein IGFBP1 that Alters Morphology of and Migrates HepG2 Cells 査読

Susumu Kodama, Yuichi Yamazaki, Masahiko Negishi

Molecular Pharmacology 88 ( 4 ) 746 - 757 2015年10月

Predictive Genomic Markers for Severe Adverse Drug Reactions 招待査読

Yoshiro Saito, Susumu Kodama, Emiko Sugiyama, Ryosuke Nakamura

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 135 ( 4 ) 589 - 595 2015年4月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：PHARMACEUTICAL SOC JAPAN

Severe adverse drug reactions are an important issue to be considered during proper drug usage in postmarketing period. Most severe adverse reactions are idiosyncratic and unrelated to their pharmacological actions via primary targets. Although these reactions were not predictable, recent developments in the field of genomics have revealed closely associated markers responsible for some severe adverse reactions, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). This review demonstrates genomic biomarkers for SJS/TEN and drug-induced liver injury (DILI) that were found mainly in Japanese patients and reveal ethnic differences. We and other groups have found the following associations of SJS/TEN with susceptible drugs: 1) HLA-B*58:01 for allopurinol-related cases; 2) HLA-B*15:11 and HLA-A*31:01 for carbamazepine-related cases; 3) HLA-B*51:01 for phenobarbital-related cases; 4) HLA-A*02:07 for zonisamide-related cases; 5) CYP2C9*3 for phenytoin-related cases; and 6) HLA-A*02:06 for cold medicine-related cases. The allele frequencies of these related HLA types vary among Asian populations. In addition, direct (non-covalent) binding of carbamazepine or an allopurinol metabolite, oxypurinol, to the associated HLA-type proteins was suggested. Associated genomic biomarkers are also summarized for DILI in Japanese and Caucasian populations. The application of these genomic biomarkers to prevent the onset of a reaction has been utilized in a few countries. However, in Japan, the package inserts only contain precautions that cite the research findings. To overcome this limitation, the following points should be addressed: 1) factors responsible for the development of SJS/TEN should be identified in addition to the above-mentioned HLA alleles; and 2) an inexpensive genotyping strategy and assay methods should be developed to provide a pharmacoeconomical viewpoint. Further research on severe adverse reactions is warranted.

DOI： 10.1248/yakushi.14-00249-3

特異体質性薬物毒性の研究最前線重篤副作用に関する予測ゲノムマーカー

斎藤嘉朗, 児玉進, 杉山永見子, 中村亮介

薬学雑誌 135 ( 4 ) 589 - 595 2015年4月

記述言語：日本語出版者・発行元：(公社)日本薬学会

国立医薬品食品衛生研究所では、収集した重症薬疹を対象に、被疑薬別にゲノムバイオマーカー探索を行っている。全ゲノムにわたる網羅的な遺伝子多型解析も行っているが、これまでに見出されたマーカーとしては、ヒト白血球抗原型が多い。特に、HLAクラスI蛋白質は、皮膚を含む全身の細胞に発現しており、表皮細胞壊死との関連が注目されている。重症薬疹の発症報告数と被疑薬・バイオマーカー研究・分子論的機構、薬剤性肝障害発症と関連するヒト白血球抗原型について述べた。

その他リンク： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2015&ichushi_jid=J01459&link_issn=&doc_id=20150407300006&doc_link_id=130005060821&url=https%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F130005060821&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_3.gif

Sulfotransferase genes: Regulation by nuclear receptors in response to xeno/endo-biotics 招待査読

Susumu Kodama, Masahiko Negishi

DRUG METABOLISM REVIEWS 45 ( 4 ) 441 - 449 2013年11月

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：INFORMA HEALTHCARE

Pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR), members of the nuclear receptor superfamily, are two major xeno-sensing transcription factors. They can be activated by a broad range of lipophilic xenobiotics including therapeutics drugs. In addition to xenobiotics, endogenous compounds such as steroid hormones and bile acids can also activate PXR and/or CAR. These nuclear receptors regulate genes that encode enzymes and transporters that metabolize and excrete both xenobiotics and endobiotics. Sulfotransferases (SULTs) are a group of these enzymes and sulfate xenobiotics for detoxification. In general, inactivation by sulfation constitutes the mechanism to maintain homeostasis of endobiotics. Thus, deciphering the molecular mechanism by which PXR and CAR regulate SULT genes is critical for understanding the roles of SULTs in the alterations of physiological and pathophysiological processes caused by drug treatment or environmental exposures.

DOI： 10.3109/03602532.2013.835630

PXR cross-talks with internal and external signals in physiological and pathophysiological responses 査読

Susumu Kodama, Masahiko Negishi

DRUG METABOLISM REVIEWS 45 ( 3 ) 300 - 310 2013年8月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INFORMA HEALTHCARE

Pregnane X receptor (PXR), an orphan member of the nuclear receptor superfamily, is a major xeno-sensing transcription factor. In response to xenobiotic exposure, PXR regulates genes involved in the metabolism and transport of xenobiotics to protect the body from their harmful effects. Recent progress has revealed that PXR responds not only to such external signals but also to internal signals to help the body adapt to changes in the internal environment, including dysregulation of the immune system. PXR responds to external and internal signals by up-or down-regulating certain metabolic pathways and cellular signals through gene regulation. PXR is a potential therapeutic target for inflammatory as well as metabolic diseases, although its activation may also have unfavorable effects on human health. This review will discuss the recent progress in the understanding of the physiological and pathophysiological roles of PXR and their implications in human diseases and drug therapy by elucidating the molecular mechanisms underlying PXR-mediated gene regulation.

DOI： 10.3109/03602532.2013.795585

Xenobiotic-induced hepatocyte proliferation associated with constitutive active/androstane receptor (CAR) or peroxisome proliferator-activated receptor α (PPARα) is enhanced by pregnane X receptor (PXR) activation in mice 査読

Ryota Shizu, Satoshi Benoki, Yuki Numakura, Susumu Kodama, Masaaki Miyata, Yasushi Yamazoe, Kouichi Yoshinari

PloS One 8 ( 4 ) e61802 2013年4月

医療品評価をめぐる最近の話題市販後安全性確保に係るバイオマーカーと診断

斎藤嘉朗, 前川京子, 田島陽子, 児玉進, 黒瀬光一

レギュラトリーサイエンス学会誌 3 ( 1 ) 43 - 55 2013年1月

記述言語：日本語出版者・発行元：(一社)レギュラトリーサイエンス学会

発生頻度や患者背景の多様性の点から、市販後に明らかになる副作用は比較的多く、特に重篤な副作用は医薬品の適正使用の観点から問題となっている。バイオマーカーは副作用の予測および早期診断に有用と期待され、ゲノムを中心に解析が進展している。例えば、カルバマゼピン、アロプリノール等による重症薬疹に関しては、その発症と強く関連するヒト白血球抗原(HLA)型が、日本人を含む多くの人種で見いだされている。また薬物性肝障害に関しても、予測ゲノムバイオマーカーが明らかになってきている。一方で、環境的要因を反映しうるタンパク質および体内代謝物マーカーについても期待が寄せられており、少数であるが報告例もある。本項では、著者らが行っている研究を含め、これらの最新の知見を、民族差を含めて紹介する。バイオマーカー研究がさらに進み、医薬品のより安全な使用に応用されることを期待したい。一方で、これらを実臨床に応用するためには、前向き研究により診断の有用性を示すことが極めて重要であり、その進展が期待される。(著者抄録)

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated .BETA.-glucuronidase reporter gene expression system 査読

INUI Hideyuki, GION Keiko, UTANI Yasushi, WAKAI Taketo, KODAMA Susumu, EUN Heesoo, KIM Yun‐Seok, OHKAWA Hideo

Journal of Environmental Science and Health. Part B. Pesticides, Food Contaminants, and Agricultural Wastes 47 ( 4 ) 233 - 239 2012年4月

Assays of PCB congeners and organochlorine insecticides with the transgenic Arabidopsis and tobacco plants carrying recombinant guinea pig AhR and GUS reporter genes 査読

Keiko Gion, Hideyuki Inui, Hideaki Sasaki, Yasushi Utani, Susumu Kodama, Hideo Ohkawa

JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART B-PESTICIDES FOOD CONTAMINANTS AND AGRICULTURAL WASTES 47 ( 7 ) 599 - 607 2012年

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

Certain congeners of polychlorinated biphenyls (PCBs) and organochlorine insecticides are ligands of aryl hydrocarbon receptors (AhRs) in animals. A recombinant guinea pig (g) AhR, XgDV, was constructed by fusing the ligand-binding domain of gAhR, the DNA-binding domain of LexA, and the transactivating domain of VP16. Then, the expression unit of beta-glucuronidase (GUS) reporter gene regulated by XgDV was introduced into Arabidopsis and tobacco plants. When the transgenic Arabidopsis XgDV plants were cultured on Murashige-Skoog (MS) medium containing PCB congeners, the GUS activity in the plants increased toxic equivalent (TEQ)-dependently. The GUS activity in the transgenic Arabidopsis XgDV plants cultured on MS medium containing the organochlorine insecticide dieldrin was also induced. On the other hand, in the case of DDT, the GUS activity induced by 3-methylcholanthere in the plants decreased. The transgenic Arabidopsis XgDV plants detected 1000 ng g(-1) PCB126 in 1 g of soils. Thus the XgDV plants seemed to be useful for convenient assays of PCB congeners and organochlorine insecticides, without any extraction and purification steps.

DOI： 10.1080/03601234.2012.668453

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system 査読

Hideyuki Inui, Keiko Gion, Yasushi Utani, Taketo Wakai, Susumu Kodama, Heesoo Eun, Yun-Seok Kim, Hideo Ohkawa

Journal of Environmental Science and Health. Part. B 47 ( 1 ) 59 - 65 2012年

Liganded pregnane X receptor represses the human sulfotransferase SULT1E1 promoter through disrupting its chromatin structure 査読

Susumu Kodama, Fardin Hosseinpour, Joyce A. Goldstein, Masahiko Negishi

NUCLEIC ACIDS RESEARCH 39 ( 19 ) 8392 - 8403 2011年10月

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：OXFORD UNIV PRESS

Pregnane X receptor (PXR), acting as a xenobiotic-activated transcription factor, regulates the hepatic metabolism of therapeutics as well as endobiotics such as steroid hormones. Given our finding that PXR activation by rifampicin (RIF) represses the estrogen sulfotransferase (SULT1E1) gene in human primary hepatocytes and hepatocellular carcinoma Huh7 cells, here we have investigated the molecular mechanism of this repression. First the PXR-responsive enhancer was delineated to a 100 bp sequence (-1000/-901), which contains three half sites that constitute the overlapping direct repeat 1 (DR1) and direct repeat 2 (DR2) motifs and two forkhead factor binding sites. siRNA knockdown, chromatin immunoprecipitation and chromatin conformation capture assays were employed to demonstrate that hepatocyte nuclear factor 4 alpha (HNF4 alpha) bound to the PXR-responsive enhancer, and activated the enhancer by looping its position close to the proximal promoter. Upon activation by RIF, PXR indirectly interacted with the enhancer, decreasing the interaction with HNF4 alpha and dissolving the looped SULT1E1 promoter with deacetylation of histone 3. Removal of the DR sites from the enhancer hampers the ability of HNF4 alpha to loop the promoter and that of PXR to repress the promoter activity. Thus, PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen.

DOI： 10.1093/nar/gkr458

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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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