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喜井 勲  キイ イサオ

教員組織学術研究院(農学系)電話番号
教育組織農学部 農学生命科学科 生命機能科学コースFAX番号
職名教授メールアドレス
住所〒399-4598 長野県上伊那郡南箕輪村8304ホームページURL

プロフィール

研究分野
常態系口腔科学
薬理学
腫瘍診断、治療学
神経科学一般
生体材料学
生体医工学
分子生物学
薬系衛生、生物化学
ケミカルバイオロジー
キーワード:リン酸化酵素 , フォールディング , 神経新生 , DYRK1A , 分子シャペロン , 品質管理 , 特異的阻害剤 , ケミカルバイオロジー , 化合物スクリーニング , ハイスループットアッセイ
現在の研究課題
リン酸化酵素フォールディング中間体を標的とした創薬研究
キーワード:リン酸化酵素 , フォールディング , 阻害剤 , 神経疾患 , 糖尿病
所属学会
所属学会
国際ケミカルバイオロジー学会
日本メカノバイオロジー学会
日本ケミカルバイオロジー学会
日本分子生物学会
日本薬理学会
メカノセンシング研究会
学歴
出身大学院
2005 , 東京工業大学大学院生命理工学研究科 博士後期課程 , Graduate School of Bioscience and Biotechnology
2002 , 東京工業大学大学院生命理工学研究科
2002 , 東京工業大学大学院生命理工学研究科 修士課程 , Graduate School of Bioscience and Biotechnology
2000 , 東京工業大学 生命理工学部 , School of Bioscience and Biotechnology
1996 , 愛媛県立今治西高等学校

出身学校・専攻等(大学院を除く)
2000 , 東京工業大学 生命理工学部
1996 , 愛媛県立今治西高等学校

取得学位
博士(理学) , 東京工業大学
受賞学術賞
2021 , 認定ベンチャーキャピタル(VC)賞
2018 , エーザイ賞・帝人ファーマ賞(共同受賞)
2018 , BRAVE賞 コスモ・バイオ賞(共同受賞)
2009 , Travel Fellowship
2004 , 優秀演題賞
2004 , Young Investigator Award
研究職歴等
研究職歴
2020- , 信州大学 農学部/大学院農学専攻 教授
2019-2020 , 信州大学 農学部/大学院農学専攻 准教授(独立)
2018-2019 , 理化学研究所 科技ハブ産連本部 ユニットリーダー
2017-2018 , 理化学研究所 科学技術ハブ推進本部 ユニットリーダー
2016-2016 , 理化学研究所  ライフサイエンス技術基盤研究センター  イメージング応用研究グループ 健康・病態科学研究チーム 上級研究員
2015-2016 , 理化学研究所  ライフサイエンス技術基盤研究センター  イメージング応用研究グループ 健康・病態科学研究チーム 研究員
2010-2015 , 京都大学大学院 医学研究科 形態形成機構学 特定助教
2010-2010 , 東京医科歯科大学大学院 疾患生命科学研究部  形質発現制御学 特任助教
2005-2010 , 東京工業大学大学院 生命理工学研究科  生命情報専攻 助教

研究活動業績

研究業績(著書・
発表論文等)
書籍等出版物
リン酸化酵素に対する選択的阻害剤の開発, CSJカレントレビュー 生物活性分子のケミカルバイオロジー : 標的同定と作用機構 = Chemical biology of bioactive molecules, 134-139
日本化学会 編 化学同人 2015
Author:喜井 勲; 萩原 正敏


間葉系幹細胞と軟骨・骨・筋肉, 再生医学の基礎, 161-196
名古屋大学出版会 2003
Author:工藤 明; 喜井 勲


論文
Structure-activity relationship for the folding intermediate-selective inhibition of DYRK1A.
European journal of medicinal chemistry,227:113948-113948 2022(Jan. 05)
Author:Yuka Miyazaki; Masaki Kikuchi; Koji Umezawa; Aurelie Descamps; Daichi Nakamura; Gaku Furuie; Tomoe Sumida; Kanako Saito; Ninako Kimura; Takashi Niwa; Yuto Sumida; Takashi Umehara; Takamitsu Hosoya; Isao Kii
Abstract:DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.


The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated.
Pathogens (Basel, Switzerland),11(1) 2021(Dec. 26)
Author:Takashi Nishiyama; Koji Umezawa; Kentaro Yamada; Masaharu Takahashi; Satoshi Kunita; Mulyanto; Isao Kii; Hiroaki Okamoto
Abstract:The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.


S1PR3-G12-biased agonist ALESIA targets cancer metabolism and promotes glucose starvation.
Cell chemical biology 2021(Feb. 02)
Author:Masayasu Toyomoto; Asuka Inoue; Kei Iida; Masatsugu Denawa; Isao Kii; Francois Marie; Ngako Kadji; Takayuki Kishi; Dohyun Im; Tatsuro Shimamura; Hiroshi Onogi; Suguru Yoshida; So Iwata; Junken Aoki; Takamitsu Hosoya; Masatoshi Hagiwara
Abstract:Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G12-biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy.


Assembly of four modules onto a tetraazide platform by consecutive 1,2,3-triazole formations.
Chemical communications (Cambridge, England),57(7):899-902 2021(Jan. 28)
Author:Suguru Yoshida; Yuki Sakata; Yoshihiro Misawa; Takamoto Morita; Tomoko Kuribara; Harumi Ito; Yuka Koike; Isao Kii; Takamitsu Hosoya
Abstract:Efficient consecutive 1,2,3-triazole formations using multiazide platforms are disclosed. On the basis of unique clickability of the 1-adamantyl azido group, a four-step synthesis of tetrakis(triazole)s was achieved from a tetraazide platform molecule. This method was applied to a convergent synthesis of tetrafunctionalized probes in a modular synthetic manner.


Druggable Transient Pockets in Protein Kinases.
Molecules (Basel, Switzerland),26(3) 2021(Jan. 27)
Author:Koji Umezawa; Isao Kii
Abstract:Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of "cryptic inhibitor-binding sites." These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.


Identification of synthetic inhibitors for the DNA binding of intrinsically disordered circadian clock transcription factors.
Chemical communications (Cambridge, England),56(76):11203-11206 2020(Sep. 24)
Author:Yusuke Hosoya; Wataru Nojo; Isao Kii; Takanori Suzuki; Miki Imanishi; Junko Ohkanda
Abstract:Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescent in vitro binding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.


Indolizines Enabling Rapid Uncaging of Alcohols and Carboxylic Acids by Red Light-Induced Photooxidation.
Organic letters,22(14):5434-5438 2020(Jul. 17)
Author:Kenji Watanabe; Nodoka Terao; Isao Kii; Reiko Nakagawa; Takashi Niwa; Takamitsu Hosoya
Abstract:The irradiation of red light-emitting-diode light (λ = 660 nm) to 3-acyl-2-methoxyindolizines in the presence of a catalytic amount of methylene blue triggered the photooxidation of the indolizine ring, resulting in a nearly quantitative release of alcohols or carboxylic acids within a few minutes. The method was applicable for photouncaging various functional molecules such as a carboxylic anticancer drug and a phenolic fluorescent dye from the corresponding indolizine conjugates, including an insulin-indolizine-dye conjugate.


HaloTag-based conjugation of proteins to barcoding-oligonucleotides.
Nucleic acids research,48(2):e8 2020(Jan. 24)
Author:Junshi Yazaki; Yusuke Kawashima; Taisaku Ogawa; Atsuo Kobayashi; Mayu Okoshi; Takashi Watanabe; Suguru Yoshida; Isao Kii; Shohei Egami; Masayuki Amagai; Takamitsu Hosoya; Katsuyuki Shiroguchi; Osamu Ohara
Abstract:Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.


Screening of novel drugs for inhibiting hepatitis E virus replication.
Journal of virological methods,270:1-11 2019(Aug.)
Author:Nishiyama T; Kobayashi T; Jirintai S; Kii I; Nagashima S; Prathiwi Primadharsini P; Nishizawa T; Okamoto H
Abstract:Hepatitis E, which is caused by hepatitis E virus (HEV), is generally a self-limiting, acute, and rarely fatal disease. It is sometimes fulminant and lethal, especially during pregnancy. Indeed, it occasionally takes a chronic course in immunocompromised individuals. To cure hepatitis E patients, the broad-spectrum antivirals (ribavirin and pegylated interferon α) are used. However, this treatment is insufficient and unsafe in some patients due to embryoteratogenic effects, leukopenia, and thrombocytopenia. In this study, we constructed an HEV replication reporter system with Gaussia luciferase for comprehensively screening anti-HEV drug candidates, and developed a cell-culture system using cells robustly producing HEV to validate the efficacy of anti-HEV drug candidates. We screened anti-HEV drug candidates from United States Food and Drug Administration-approved drugs using the established HEV replication reporter system, and investigated the selected candidates and type III interferons (interferon λ1-3) using the cell-culture system. In conclusion, we constructed an HEV replicon system for anti-HEV drug screening and a novel cell-culture system to strictly evaluate the replication-inhibitory activities of the obtained anti-HEV candidates. Our findings suggested that interferon λ1-3 might be effective for treating hepatitis E.


A facile preparation of functional cycloalkynes via an azide-to-cycloalkyne switching approach.
Chemical communications (Cambridge, England),55(24):3556-3559 2019(Mar.)
Author:Yoshida S; Kuribara T; Ito H; Meguro T; Nishiyama Y; Karaki F; Hatakeyama Y; Koike Y; Kii I; Hosoya T
Abstract:A facile method for preparing various functional cycloalkynes, including proteins incorporated with a cycloalkyne moiety, from the corresponding azides is developed. Treatment of diynes bearing strained and terminal alkyne moieties with a copper salt enabled terminal alkyne-selective click conjugation with azides, whereas a more azidophilic strained alkyne moiety was transiently protected from the click reaction via complexation with copper.


Alzheimer Aβ Assemblies Accumulate in Excitatory Neurons upon Proteasome Inhibition and Kill Nearby NAKα3 Neurons by Secretion.
iScience,13:452-477 2019(Mar.)
Author:Komura H; Kakio S; Sasahara T; Arai Y; Takino N; Sato M; Satomura K; Ohnishi T; Nabeshima YI; Muramatsu SI; Kii I; Hoshi M
Abstract:We identified ∼30-mer amyloid-β protein (Aβ) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aβ levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.


Practical Application of Periostin as a Biomarker for Pathological Conditions.
Advances in experimental medicine and biology,1132:195-204 2019
Author:Isao Kii
Abstract:In physiological condition, periostin is expressed in limited tissues such as periodontal ligament, periosteum, and heart valves. Periostin protein is mainly localized on extracellular collagen bundles and in matricellular space. On the other hand, in pathological condition, expression of periostin is induced in disordered tissues of human patients. In tumor development and progression, periostin is elevated mainly in its microenvironment and stromal tissue rich in extracellular matrix. Tumor stromal fibroblasts highly express periostin and organize the tumor-surrounding extracellular matrix architecture. In fibrosis in lung, liver, and kidney, proliferating activated fibroblasts express periostin and replace normal functional tissues with dense connective tissues. In inflammation and allergy, inflammatory cytokines such as IL-4 and IL-13 induce expression of periostin that plays important roles in pathogenesis of these diseases. The elevated levels of periostin in human patients could be detected not only in tissue biopsy samples but also in peripheral bloods using specific antibodies against periostin, because periostin secreted from the disordered tissues is transported into blood vessels and circulates in the cardiovascular system. In this chapter, I introduce the elevated expression of periostin in pathological conditions, and discuss how periostin could be utilized as a biomarker in disease diagnosis.


Periostin Functions as a Scaffold for Assembly of Extracellular Proteins.
Advances in experimental medicine and biology,1132:23-32 2019
Author:Isao Kii
Abstract:Periostin is a secretory matricellular protein with a multi-domain structure that is composed of an amino-terminal EMI domain, a tandem repeat of four FAS 1 domains, and a carboxyl-terminal domain (CTD). Periostin has been suggested to function as a scaffold for assembly of several extracellular matrix proteins as well as its accessory proteins (Fig. 3.1, Table 3.1), which underlies highly sophisticated extracellular architectures. This scaffold function is likely due to periostin's multi-domain structure, in which the adjacent domains in periostin interact with different kinds of proteins, put these interacting proteins in close proximity, and promote intermolecular interactions between these proteins, leading to their assembly into a large complex. In this chapter, I introduce the proteins that interact with each of the adjacent domains in periostin, and discuss how the multi-domain structure of periostin functions as a scaffold for the assembly of the interacting proteins, and how it underlies construction of highly sophisticated extracellular architectures.


Quantification of receptor activation by oxytocin and vasopressin in endocytosis-coupled bioluminescence reduction assay using nanoKAZ.
Analytical biochemistry,549(15):174-183 2018(May 15)
Author:Isao Kii; Shino Hirahara-Owada; Masataka Yamaguchi; Takashi Niwa; Yuka Koike; Rie Sonamoto; Harumi Ito; Kayo Takahashi; Chihiro Yokoyama; Takuya Hayashi; Takamitsu Hosoya; Yasuyoshi Watanabe
Abstract:Oxytocin (OXT) and arginine vasopressin (AVP) are structurally similar neuropeptide hormones that function as neurotransmitters in the brain, and have opposite key roles in social behaviors. These peptides bind to their G protein-coupled receptors (OXTR and AVPRs), inducing calcium ion-dependent signaling pathways and endocytosis of these receptors. Because selective agonists and antagonists for these receptors have been developed as therapeutic and diagnostic agents for diseases such as psychiatric disorders, facile methods are in demand for the evaluation of selectivity between these receptors. In this study, we developed a quantitative assay for OXT- and AVP-induced endocytosis of their receptors. The mutated Oplophorus luciferase, nanoKAZ, was fused to OXTR and AVPRs to enable rapid quantification of agonist-induced endocytosis by bioluminescence reduction. Agonist stimulation significantly decreases bioluminescence of nanoKAZ-fused receptors in living cells. Using this system, we evaluated clinically used OXTR antagonist atosiban and a reported pyrazinyltriazole derivative, hereby designated as PF13. Atosiban acted as an antagonist of AVPR1a, as well as an agonist for AVPR1b, whereas PF13 antagonized OXTR more selectively than atosiban, as reported previously. This paper shows a strategy for quantification of agonist-induced endocytosis of OXTR and AVPRs, and confirms its potent utility in the evaluation of agonists and antagonists.


Staudinger reaction using 2,6-dichlorophenyl azide derivatives for robust aza-ylide formation applicable to bioconjugation in living cells.
Chemical communications (Cambridge, England),54(57):7904-7907 2018(May)
Author:Meguro T; Terashima N; Ito H; Koike Y; Kii I; Yoshida S; Hosoya T
Abstract:Efficient formation of water- and air-stable aza-ylides has been achieved using the Staudinger reaction between electron-deficient aromatic azides such as 2,6-dichlorophenyl azide and triarylphosphines. The reaction proceeds rapidly and has been successfully applied to chemical modification of proteins in living cells.


Periostin function in communication with extracellular matrices.
Journal of cell communication and signaling,12(1):301-308 2018(Mar.)
Author:Akira Kudo; Isao Kii
Abstract:Periostin is a secretory protein with a multi-domain structure, comprising an amino-terminal cysteine-rich EMI domain, four internal FAS 1 domains, and a carboxyl-terminal hydrophilic domain. These adjacent domains bind to extracellular matrix proteins (type I collagen, fibronectin, tenascin-C, and laminin γ2), and BMP-1 that catalyzes crosslinking of type I collagen, and proteoglycans, which play a role in cell adhesion. The binding sites on periostin have been demonstrated to contribute to the mechanical strength of connective tissues, enhancing intermolecular interactions in close proximity and their assembly into extracellular matrix architectures, where periostin plays further essential roles in physiological maintenance and pathological progression. Furthermore, periostin also binds to Notch 1 and CCN3, which have functions in maintenance of stemness, thus opening up a new field of periostin action.


Convergent synthesis of trifunctional molecules by three sequential azido-type-selective cycloadditions
Chemical Communications,54(30):3705-3708 2018
Author:Suguru Yoshida; Kimiyuki Kanno; Isao Kii; Yoshihiro Misawa; Masatoshi Hagiwara; Takamitsu Hosoya
Abstract:A facile strategy for the synthesis of trifunctional molecules involving three sequential selective triazole-forming reactions is proposed. This method exploits three kinds of mechanistically different azido-type-selective cycloadditions. Three different azidophiles could be efficiently connected to a triazido platform molecule with three types of azido groups in a consecutive manner, which rendered a practical trifunctional molecule readily available.


DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain
Scientific Reports,7(1):6420 2017(Dec. 01)
Author:Samira Abu Jhaisha; Esti W. Widowati; Isao Kii; Rie Sonamoto; Stefan Knapp; Chrisovalantis Papadopoulos; Walter Becker
Abstract:Two missense mutations of the DYRK1B gene have recently been found to co-segregate with a rare autosomal-dominant form of metabolic syndrome. This gene encodes a member of the DYRK family of protein kinases, which depend on tyrosine autophosphorylation to acquire the catalytically active conformation. The mutations (H90P and R102C) affect a structural element named DYRK homology (DH) box and did not directly interfere with the conformation of the catalytic domain in a structural model of DYRK1B. Cellular assays showed that the mutations did not alter the specific activity of mature kinase molecules. However, a significant part of the mutant DYRK1B protein accumulated in detergent-insoluble cytoplasmic aggregates and was underphosphorylated on tyrosine. The mutant DYRK1B variants were more vulnerable to the HSP90 inhibitor ganetespib and showed enhanced binding to the co-chaperone CDC37 as compared to wild type DYRK1B. These results support the hypothesis that the mutations in the DH box interfere with the maturation of DYRK1B by tyrosine autophosphorylation and compromise the conformational stability of the catalytic domain, which renders the kinase susceptible to misfolding.


Periostin and its interacting proteins in the construction of extracellular architectures.
Cellular and molecular life sciences : CMLS,74(23):4269-4277 2017(Dec.)
Author:Isao Kii; Harumi Ito
Abstract:Periostin is a matricellular protein that is composed of a multi-domain structure with an amino-terminal EMI domain, a tandem repeat of four FAS 1 domains, and a carboxyl-terminal domain. These distinct domains have been demonstrated to bind to many proteins including extracellular matrix proteins (Collagen type I and V, fibronectin, tenascin, and laminin), matricellular proteins (CCN3 and βig-h3), and enzymes that catalyze covalent crosslinking between extracellular matrix proteins (lysyl oxidase and BMP-1). Adjacent binding sites on periostin have been suggested to put the interacting proteins in close proximity, promoting intermolecular interactions between each protein, and leading to their assembly into extracellular architectures. These extracellular architectures determine the mechanochemical properties of connective tissues, in which periostin plays an important role in physiological homeostasis and disease progression. In this review, we introduce the proteins that interact with periostin, and discuss how the multi-domain structure of periostin functions as a scaffold for the assembly of interacting proteins, and how it underlies construction of highly sophisticated extracellular architectures.


Prenatal neurogenesis induction therapy normalizes brain structure and function in Down syndrome mice
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA,114(38):10268-10273 2017(Sep.)
Author:Akiko Nakano-Kobayashi; Tomonari Awaya; Isao Kii; Yuto Sumida; Yukiko Okuno; Suguru Yoshida; Tomoe Sumida; Haruhisa Inoue; Takamitsu Hosoya; Masatoshi Hagiwara
Abstract:Down syndrome (DS) caused by trisomy of chromosome 21 is the most common genetic cause of intellectual disability. Although the prenatal diagnosis of DS has become feasible, there are no therapies available for the rescue of DS-related neurocognitive impairment. A growth inducer newly identified in our screen of neural stem cells (NSCs) has potent inhibitory activity against dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) and was found to rescue proliferative deficits in Ts65Dn-derived neurospheres and human NSCs derived from individuals with DS. The oral administration of this compound, named ALGERNON (altered generation of neurons), restored NSC proliferation in murine models of DS and increased the number of newborn neurons. Moreover, administration of ALGERNON to pregnant dams rescued aberrant cortical formation in DS mouse embryos and prevented the development of abnormal behaviors in DS offspring. These data suggest that the neurogenic phenotype of DS can be prevented by ALGERNON prenatal therapy.


Downregulation of neuropilin-1 on macrophages modulates antibody-mediated tumoricidal activity
CANCER IMMUNOLOGY IMMUNOTHERAPY,66(9):1131-1142 2017(Sep.)
Author:Kosuke Kawaguchi; Eiji Suzuki; Mariko Nishie; Isao Kii; Tatsuki R. Kataoka; Masahiro Hirata; Masashi Inoue; Fengling Pu; Keiko Iwaisako; Moe Tsuda; Ayane Yamaguchi; Hironori Haga; Masatoshi Hagiwara; Masakazu Toi
Abstract:Neuropilin-1 (NRP-1)-expressing macrophages are engaged in antitumor immune functions via various mechanisms. In this study, we investigated the role of NRP-1 on macrophages in antibody-mediated tumoricidal activity. Treatment of macrophages with NRP-1 knockdown or an anti-NRP-1-neutralizing antibody significantly suppressed antibody-dependent cellular cytotoxicity and modulated cytokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model bearing human epidermal growth factor receptor-2 (HER2)-positive breast cancer xenografts showed that antibody-mediated antitumor activity and tumor infiltration of CD4(+) T lymphocytes were significantly downregulated when peripheral blood mononuclear cells in which NRP-1 was knocked down were co-administered with an anti-HER2 antibody. These results revealed that NRP-1 expressed on macrophages plays an important role in antibody-mediated antitumor immunity. Taken together, the induction of NRP-1 on macrophages may be a therapeutic indicator for antibody treatments that exert antibody-dependent cellular cytotoxicity activity, although further studies are needed in order to support this hypothesis.


Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells
SCIENTIFIC REPORTS,7(1):8097 2017(Aug.)
Author:Naoki Ito; Isao Kii; Noriaki Shimizu; Hirotoshi Tanaka; Takeda Shin'ichi
Abstract:Satellite cells comprise a functionally heterogeneous population of stem cells in skeletal muscle. Separation of an undifferentiated subpopulation and elucidation of its molecular background are necessary to identify the reprogramming factors to induce skeletal muscle progenitor cells. In this study, we found that intracellular esterase activity distinguishes a subpopulation of cultured satellite cells with high stemness using esterase-sensitive cell staining reagent, calcein-AM. Gene expression analysis of this subpopulation revealed that defined combinations of transcription factors (Pax3, Mef2b, and Pitx1 or Pax7, Mef2b, and Pitx1 in embryonic fibroblasts, and Pax7, Mef2b and MyoD in adult fibroblasts) reprogrammed fibroblasts into skeletal muscle progenitor cells. These reprogrammed cells formed Dystrophin-positive mature muscle fibers when transplanted into a mouse model of Duchenne muscular dystrophy. These results highlight the new marker for heterogenous population of cultured satellite cells, potential therapeutic approaches and cell sources for degenerative muscle diseases.


Three-Dimensional Localization of an Individual Fluorescent Molecule with Angstrom Precision
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,139(26):8990-8994 2017(Jul.)
Author:Taku Furubayashi; Kazuya Motohashi; Keisuke Wakao; Tsuyoshi Matsuda; Isao Kii; Takamitsu Hosoya; Nobuhiro Hayashi; Mahito Sadaie; Fuyuki Ishikawa; Michio Matsushita; Satoru Fujiyoshi
Abstract:Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic-reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 10(6) fluorescence photons from the molecule were accumulated in 5 min.


Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy
SCIENTIFIC REPORTS,7:46126 2017(May)
Author:Yukiya Sako; Kensuke Ninomiya; Yukiko Okuno; Masayasu Toyomoto; Atsushi Nishida; Yuka Koike; Kenji Ohe; Isao Kii; Suguru Yoshida; Naohiro Hashimoto; Takamitsu Hosoya; Masafumi Matsuo; Masatoshi Hagiwara
Abstract:Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease. Various attempts are underway to convert severe DMD to a milder phenotype by modulating the splicing of the dystrophin gene and restoring its expression. In our previous study, we reported TG003, an inhibitor of CDC2-like kinase 1 (CLK1), as a splice-modifying compound for exon-skipping therapy; however, its metabolically unstable feature hinders clinical application. Here, we show an orally available inhibitor of CLK1, named TG693, which promoted the skipping of the endogenous mutated exon 31 in DMD patient-derived cells and increased the production of the functional exon 31-skipped dystrophin protein. Oral administration of TG693 to mice inhibited the phosphorylation of serine/arginine-rich proteins, which are the substrates of CLK1, and modulated pre-mRNA splicing in the skeletal muscle. Thus, TG693 is a splicing modulator for the mutated exon 31 of the dystrophin gene in vivo, possibly possessing therapeutic potential for DMD patients.


Periostin is required for matricellular localization of CCN3 in periodontal ligament of mice
JOURNAL OF CELL COMMUNICATION AND SIGNALING,11(1):5-13 2017(Mar.)
Author:Issei Takayama; Hideyuki Tanabe; Takashi Nishiyama; Harumi Ito; Norio Amizuka; Minqi Li; Ken-ichi Katsube; Isao Kii; Akira Kudo
Abstract:CCN3 is a matricellular protein that belongs to the CCN family. CCN3 consists of 4 domains: insulin-like growth factor-binding protein-like domain (IGFBP), von Willebrand type C-like domain (VWC), thrombospondin type 1-like domain (TSP1), and the C-terminal domain (CT) having a cysteine knot motif. Periostin is a secretory protein that binds to extracellular matrix proteins such as fibronectin and collagen. In this study, we found that CCN3 interacted with periostin. Immunoprecipitation analysis revealed that the TSP1-CT interacted with the 4 repeats of the Fas 1 domain of periostin. Immunofluorescence analysis showed co-localization of CCN3 and periostin in the periodontal ligament of mice. In addition, targeted disruption of the periostin gene in mice decreased the matricellular localization of CCN3 in the periodontal ligament. Thus, these results indicate that periostin was required for the matricellular localization of CCN3 in the periodontal ligament, suggesting that periostin mediated an interaction between CCN3 and the extracellular matrix.


Periostin Deficiency Causes Severe and Lethal Lung Injury in Mice With Bleomycin Administration
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY,64(7):441-453 2016(Jul.)
Author:Hirofumi Kondoh; Takashi Nishiyama; Yoshinao Kikuchi; Masashi Fukayama; Mitsuru Saito; Isao Kii; Akira Kudo
Abstract:Pulmonary capillary leakage followed by influx of blood fluid into the air space of lung alveoli is a crucial step in the progression of acute lung injury (ALI). This influx is due to increased permeability of the alveolar-capillary barrier. The extracellular matrix (ECM) between the capillary and the epithelium would be expected to be involved in prevention of the influx; however, the role of the ECM remains to be addressed. Here, we show that the ECM architecture organized by periostin, a matricellular protein, plays a pivotal role in the survival of bleomycin-exposed mice. Periostin was localized in the alveolar walls. Although periostin-null mice displayed no significant difference in lung histology and air-blood permeability, they exhibited early lethality in a model of bleomycin-induced lung injury, compared with their wild-type counterparts. This early lethality may have been due to increased pulmonary leakage of blood fluid into the air space in the bleomycin-exposed periostin-null mice. These results suggest that periostin in the ECM architecture prevents pulmonary leakage of blood fluid, thus increasing the survival rate in mice with ALI. Thus, this study provides an evidence for the protective role of the ECM architecture in the lung alveoli.


Selective inhibition of the kinase DYRK1A by targeting its folding process
NATURE COMMUNICATIONS,7:11391 2016(Apr.)
Author:Isao Kii; Yuto Sumida; Toshiyasu Goto; Rie Sonamoto; Yukiko Okuno; Suguru Yoshida; Tomoe Kato-Sumida; Yuka Koike; Minako Abe; Yosuke Nonaka; Teikichi Ikura; Nobutoshi Ito; Hiroshi Shibuya; Takamitsu Hosoya; Masatoshi Hagiwara
Abstract:Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.


Periostin promotes secretion of fibronectin from the endoplasmic reticulum
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,470(4):888-893 2016(Feb.)
Author:Isao Kii; Takashi Nishiyama; Akira Kudo
Abstract:Extracellular matrix (ECM) proteins are synthesized in the endoplasmic reticulum (ER), transported to the extracellular milieu through the secretory pathway, and assembled into an extracellular architecture. A previous study of ours showed that periostin, a secretory protein, interacts with fibronectin and is involved in ECM remodeling. Here we show that periostin played a role in fibronectin secretion from the ER. Co-immunoprecipitation and in situ proximity ligation assays revealed an interaction between periostin and fibronectin in the ER. Although accumulation of fibronectin was detected in the ER of fibroblastic C3H10T1/2 cells, forced expression of periostin in those cells decreased the accumulation of fibronectin in the ER, suggesting that periostin promoted the secretion of fibronectin. A substitution mutant of tryptophan at the position 65 to alanine in the EMI domain of periostin, which caused periostin to lose its ability to interact with fibronectin, did not decrease the accumulation. Furthermore, targeted disruption of periostin in mice caused the non-fibrillar and ectopic deposition of fibronectin in the periodontal ligament. Thus, these results demonstrate a subcellular role of periostin in promotion of fibronectin secretion from the ER. (C) 2016 Elsevier Inc. All rights reserved.


Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ
SCIENTIFIC REPORTS,5:12728 2015(Aug.)
Author:Rie Sonamoto; Isao Kii; Yuka Koike; Yuto Sumida; Tomoe Kato-Sumida; Yukiko Okuno; Takamitsu Hosoya; Masatoshi Hagiwara
Abstract:The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.


Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly.
Proceedings of the National Academy of Sciences of the United States of America,112(32):E4465-E4474 2015(Aug.)
Author:Ohnishi T; Yanazawa M; Sasahara T; Kitamura Y; Hiroaki H; Fukazawa Y; Kii I; Nishiyama T; Kakita A; Takeda H; Takeuchi A; Arai Y; Ito A; Komura H; Hirao H; Satomura K; Inoue M; Muramatsu S; Matsui K; Tada M; Sato M; Saijo E; Shigemitsu Y; Sakai S; Umetsu Y; Goda N; Takino N; Takahashi H; Hagiwara M; Sawasaki T; Iwasaki G; Nakamura Y; Nabeshima Y; Teplow DB; Hoshi M
Abstract:Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid beta-protein (A beta) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical A beta oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na+/K+-ATPase alpha 3 subunit (NAK alpha 3). ASPD-binding to NAK alpha 3 impaired NAK alpha 3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-A beta-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAK alpha 3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAK alpha 3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAK alpha 3 interaction.


Design and synthesis of a potent inhibitor of class 1 DYRK kinases as a suppressor of adipogenesis
BIOORGANIC & MEDICINAL CHEMISTRY,23(15):4434-4441 2015(Aug.)
Author:So Masaki; Isao Kii; Yuto Sumida; Tomoe Kato-Sumida; Yasushi Ogawa; Nobutoshi Ito; Mitsuhiro Nakamura; Rie Sonamoto; Naoyuki Kataoka; Takamitsu Hosoya; Masatoshi Hagiwara
Abstract:Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer's disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARc and C/EBPa. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome. (C) 2015 Elsevier Ltd. All rights reserved.


Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice
MOLECULAR PHARMACOLOGY,88(2):316-325 2015(Aug.)
Author:Satoshi Morooka; Mitsuteru Hoshina; Isao Kii; Takayoshi Okabe; Hirotatsu Kojima; Naoko Inoue; Yukiko Okuno; Masatsugu Denawa; Suguru Yoshida; Junichi Fukuhara; Kensuke Ninomiya; Teikichi Ikura; Toshio Furuya; Tetsuo Nagano; Kousuke Noda; Susumu Ishida; Takamitsu Hosoya; Nobutoshi Ito; Nagahisa Yoshimura; Masatoshi Hagiwara
Abstract:Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.


CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses
JOURNAL OF CLINICAL INVESTIGATION,124(8):3479-3488 2014(Aug.)
Author:Makoto Yamamoto; Hiroshi Onogi; Isao Kii; Suguru Yoshida; Kei Iida; Hiroyuki Sakai; Minako Abe; Toshiaki Tsubota; Nobutoshi Ito; Takamitsu Hosoya; Masatoshi Hagiwara
Abstract:A wide range of antiviral drugs is currently available; however, drug-resistant viruses have begun to emerge and represent a potential public health risk. Here, we explored the use of compounds that inhibit or interfere with the action of essential host factors to prevent virus replication. In particular, we focused on the cyclin-dependent kinase 9 (CDK9) inhibitor, FIT-039, which suppressed replication of a broad spectrum of DNA viruses through inhibition of mRNA transcription. Specifically, FIT-039 inhibited replication of herpes simplex virus 1 (HSV-1), HSV-2, human adenovirus, and human cytomegalovirus in cultured cells, and topical application of FIT-039 ointment suppressed skin legion formation in a murine HSV-1 infection model. FIT-039 did not affect cell cycle progression or cellular proliferation in host cells. Compared with the general CDK inhibitor flavopiridol, transcriptome analyses of FIT-039-treated cells revealed that FIT-039 specifically inhibited CDK9. Given at concentrations above the inhibitory concentration, FIT-039 did not have a cytotoxic effect on mammalian cells. Importantly, administration of FIT-039 ameliorated the severity of skin lesion formation in mice infected with an acyclovir-resistant HSV-1, without noticeable adverse effects. Together, these data indicate that FIT-039 has potential as an antiviral agent for clinical therapeutics.


Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors
MOLECULAR PAIN,10:17 2014(Mar.)
Author:Takashi Kurihara; Eri Sakurai; Masayasu Toyomoto; Isao Kii; Daisuke Kawamoto; Toshihide Asada; Tsutomu Tanabe; Megumu Yoshimura; Masatoshi Hagiwara; Atsuro Miyata
Abstract:Background: The phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund's adjuvant (CFA) models. Results: We have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1-100 pmol) or IC261 (0.1-1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 mu M) or IC261 (1 mu M) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models. Conclusions: These results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.


The Niche Component Periostin Is Produced by Cancer-Associated Fibroblasts, Supporting Growth of Gastric Cancer through ERK Activation
AMERICAN JOURNAL OF PATHOLOGY,184(3):859-870 2014(Mar.)
Author:Yoshinao Kikuchi; Akiko Kunita; Caname Iwata; Daisuke Komura; Takashi Nishiyama; Kazuhiro Shimazu; Kimiko Takeshita; Junji Shibahara; Isao Kii; Yasuyuki Morishita; Masakazu Yashiro; Kosei Hirakawa; Kohei Miyazono; Akira Kudo; Masashi Fukayama; Takeshi G. Kashima
Abstract:Overexpression of periostin (POSTN), an extraceaular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II-IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising Linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn(-/-) mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn(-/-) mice. These findings suggest that POSTN produced by cancerassociated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer.


Cell-based chemical screen identifies a novel type of kinase inhibitor that targets autophosphorylation of the kinase DYRK1A
JOURNAL OF PHARMACOLOGICAL SCIENCES,118:112P-112P 2012
Author:Isao Kii; Yukiko Okuno; Yuto Sumida; Takamitsu Hosoya; Masatoshi Hagiwara


Double click reaction: A novel method for chemical modification of biomolecules
Seikagaku,84(4):306-311 2012
Author:Isao Kii; Suguru Yoshida; Takamitsu Hosoya


Remodeling of Actin Cytoskeleton in Mouse Periosteal Cells under Mechanical Loading Induces Periosteal Cell Proliferation during Bone Formation
PLOS ONE,6(9):e24847 2011(Sep.)
Author:Daisuke Sakai; Isao Kii; Kazuki Nakagawa; Hiroko N. Matsumoto; Masateru Takahashi; Suguru Yoshida; Takamitsu Hosoya; Kazuo Takakuda; Akira Kudo
Abstract:Background: The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo. Principal Findings: We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells. Conclusions: These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.


Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma
TUMOR BIOLOGY,32(3):611-622 2011(Jun.)
Author:Masahiko Fujiwara; Takeshi G. Kashima; Akiko Kunita; Isao Kii; Daisuke Komura; Agamemnon E. Grigoriadis; Akira Kudo; Hiroyuki Aburatani; Masashi Fukayama
Abstract:S100A4, a 10-12 kDa calcium-binding protein, plays functional roles in tumor progression and metastasis. The present study aimed to investigate the function of S100A4 in osteosarcoma (OS) metastasis, using a loss-of-function approach. Our previous expression profiling analysis revealed that S100a4 was preferentially expressed in the highly metastatic mouse OS cell line, LM8. Introducing a short hairpin ribonucleic acid (shRNA) targeting S100a4 using a newly established vector containing insulators and transposons, we established stable LM8 subclones with almost 100% silencing of endogenous S100a4 protein. These transfectants showed a significant suppression of cell migration in vitro as well as a marked reduction in their ability to colonize the lung and form pulmonary metastases in vivo following intravenous inoculation, whereas there was no significant change in cell proliferation or cell attachment to fibronectin, laminin, and type I collagen. Expression and phosphorylation of ezrin, an emerging OS metastasis-associated factor, and expression of MMPs, remained the same in S100a4-shRNA clones. In 61 human OS, immunohistochemical analysis showed that lesional cells in 85.2% samples expressed S100A4 protein, and the immunoreactivity was primarily cytoplasmic, but it also showed occasional nuclear localization. Chondroblastic and osteoblastic OS subtypes expressed more S100A4 than fibroblastic subtypes. The causative role of S100A4 in OS lung metastasis shown in the murine xenograft model, together with the high proportion of primary human OS expressing S100A4, suggest that S100A4 protein represents an important potential target for future OS therapy.


Delayed Re-Epithelialization in Periostin-Deficient Mice during Cutaneous Wound Healing
PLOS ONE,6(4):e18410 2011(Apr.)
Author:Takashi Nishiyama; Isao Kii; Takeshi G. Kashima; Yoshinao Kikuchi; Atsushi Ohazama; Masashi Shimazaki; Masashi Fukayama; Akira Kudo
Abstract:Background: Matricellular proteins, including periostin, are important for tissue regeneration. Methods and Findings: Presently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient (-/-) mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin gamma 2. Periostin was associated with laminin gamma 2, and this association enhanced the proteolytic cleavage of the laminin gamma 2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin-/- mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin-/- mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-kappa B. Conclusion: These results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing.


Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A
NATURE COMMUNICATIONS,1:86 2010(Oct.)
Author:Yasushi Ogawa; Yosuke Nonaka; Toshiyasu Goto; Eriko Ohnishi; Toshiyuki Hiramatsu; Isao Kii; Miyo Yoshida; Teikichi Ikura; Hiroshi Onogi; Hiroshi Shibuya; Takamitsu Hosoya; Nobutoshi Ito; Masatoshi Hagiwara
Abstract:Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, INDY, a benzothiazole derivative showing a potent ATP-competitive inhibitory effect with IC50 and K-i values of 0.24 and 0.18 mu M, respectively. X-ray crystallography of the Dyrk1A/INDY complex revealed the binding of INDY in the ATP pocket of the enzyme. INDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed NFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proINDY, a prodrug of INDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proINDY in vivo.


Periostin Associates with Notch1 Precursor to Maintain Notch1 Expression under a Stress Condition in Mouse Cells
PLOS ONE,5(8):e12234 2010(Aug.)
Author:Hideyuki Tanabe; Issei Takayama; Takashi Nishiyama; Masashi Shimazaki; Isao Kii; Minqi Li; Norio Amizuka; Ken-ichi Katsube; Akira Kudo
Abstract:Background: Matricellular proteins, including periostin, modulate cell-matrix interactions and cell functions by acting outside of cells. Methods and Findings: In this study, however, we reported that periostin physically associates with the Notch1 precursor at its EGF repeats in the inside of cells. Moreover, by using the periodontal ligament of molar from periostin-deficient adult mice (Pn(-/-) molar PDL), which is a constitutively mechanically stressed tissue, we found that periostin maintained the site-1 cleaved 120-kDa transmembrane domain of Notch1 (N1 (TM)) level without regulating Notch1 mRNA expression. N1 (TM) maintenance in vitro was also observed under such a stress condition as heat and H(2)O(2) treatment in periostin overexpressed cells. Furthermore, we found that the expression of a downstream effector of Notch signaling, Bcl-xL was decreased in the Pn(-)(-/) molar PDL, and in the molar movement, cell death was enhanced in the pressure side of Pn(-/-) molar PDL. Conclusion: These results suggest the possibility that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition, which is caused by its physical association with the Notch1 precursor.


Interaction between periostin and BMP-1 promotes proteolytic activation of lysyl oxidase.
The Journal of biological chemistry,285(17):13294-303 2010(Apr. 23)
Author:Takumi Maruhashi; Isao Kii; Mitsuru Saito; Akira Kudo
Abstract:Intra- and intermolecular covalent cross-linking between collagen fibrils, catalyzed by lysyl oxidase (LOX), determines the mechanical properties of connective tissues; however, mechanisms that regulate the collagen cross-linking according to tissue specificity are not well understood. Here we show that periostin, a secretory protein in the dense connective tissues, promotes the activation of LOX. Previous studies showed that periostin null mice exhibit reduced collagen cross-linking in their femurs, periosteum, infarcted myocardium, and tendons. Presently, we showed that active LOX protein, formed by cleavage of its propeptide by bone morphogenetic protein-1 (BMP-1), was decreased in calvarial osteoblast cells derived from periostin null mice. Overexpression of periostin promoted the proteolytic cleavage of the propeptide, which increased the amount of active LOX protein. The results of co-immunoprecipitation and solid phase binding assays revealed that periostin interacted with BMP-1. Furthermore, this interaction probably resulted in enhanced deposition of BMP-1 on the extracellular matrix, suggesting that this enhanced deposition would lead to cleavage of the propeptide of LOX. Thus, we demonstrated that periostin supported BMP-1-mediated proteolytic activation of LOX on the extracellular matrix, which promoted collagen cross-linking.


Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture
JOURNAL OF BIOLOGICAL CHEMISTRY,285(3):2028-2039 2010(Jan.)
Author:Isao Kii; Takashi Nishiyama; Minqi Li; Ken-ichi Matsumoto; Mitsuru Saito; Norio Amizuka; Akira Kudo
Abstract:Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.


Strain-promoted double-click reaction for chemical modification of azido-biomolecules
ORGANIC & BIOMOLECULAR CHEMISTRY,8(18):4051-4055 2010
Author:Isao Kii; Akira Shiraishi; Toshiyuki Hiramatsu; Takeshi Matsushita; Hidehiro Uekusa; Suguru Yoshida; Makoto Yamamoto; Akira Kudo; Masatoshi Hagiwara; Takamitsu Hosoya
Abstract:The strain-promoted "double-click" (SPDC) reaction using Sondheimer diyne, a novel convergent method conjugating three molecules spontaneously, has enabled us to readily modify an azido-biomolecule with a small reporter azido-molecule.


Expression, Purification and Characterization of Soluble Recombinant Periostin Protein Produced by Escherichia coli
JOURNAL OF BIOCHEMISTRY,146(5):713-723 2009(Nov.)
Author:Issei Takayama; Isao Kii; Akira Kudo
Abstract:Periostin is a matricellular protein participating in the tissue remodelling of damaged cardiac tissue after acute myocardial infarction and of the periodontal ligament in mice. However, further studies on the periostin protein have been limited by the intrinsic difficulty of purifying this protein produced in Escherichia coli due to its insolubility. Here, we demonstrate the expression of recombinant periostin protein with high solubility and monodispersity in E. coli. Periostin is composed of an amino-terminal EMI domain, a tandem repeat of 4 fas1 domains (RD1-4), and a carboxyl-terminal region (CTR). We expressed the RD4-CTR region tagged with GST at amino-terminal and 6 Histidine at carboxyl-terminal end in E. coli. The recombinant protein was purified by using GSH-Sepharose and nickel chelation affinity chromatography, followed by gel filtration chromatography. The RD4-CTR protein exhibited high solubility and monodispersity. On average, 9.1 mg of purified RD4-CTR was routinely obtained from 1 L of culture media. Furthermore, the RD4-CTR was biochemically active, because it bound to the RD1-4, the same as intact periostin protein that had been purified from mammalian cells. Our results should enable us to produce the periostin recombinant protein in large quantities and facilitate future studies on functional and structural analyses of periostin.


Periostin, a novel marker of intramembranous ossification, is expressed in fibrous dysplasia and in c-Fos-overexpressing bone lesions
HUMAN PATHOLOGY,40(2):226-237 2009(Feb.)
Author:Takeshi G. Kashimaa; Takashi Nishiyama; Kazuhiro Shimau; Masashi Shimazaki; Isao Kii; Agamemnon E. Grigoriadis; Masashi Fukayama; Akira Kudo
Abstract:Fibrous dysplasia is a benign bone disease caused by a mutation in the gene for the stimulatory guanine nucleotide-binding protein Gs alpha, leading to high cyclic adenosine monophosphate levels. Histologically, fibrous dysplasia is characterized by the production of fibrous tissue accompanied by the deposition of ectopic type I collagen and other bone-associated extracellular matrix proteins, as well as by irregular woven intramembranous bone onto which type I collagen-containing Sharpey fibers are often attached. Fibrous dysplasia is also characterized by high expression of c-Fos/c-Jun, known targets for cyclic adenosine monophosphate signaling. In this study, we examined the expression of the bone-related extracellular matrix protein, periostin, and its known receptor, integrin alpha v beta 3 (CD51/61), in normal bones as well as in fibrous dysplasia. Immunohistochemistry and in Situ hybridization studies revealed that periostin was expressed in the extracellular matrix during intramembranous but not endochondral ossification, as well as in the fibrous component of fibrous dysplasia; and all cells adjacent to periostin-positive regions expressed CD51/61. Importantly, periostin was abundantly localized to Sharpey fibers. To investigate the contribution of c-Fos, we examined transgenic mice overexpressing c-fos, which develop sclerotic lesions closely resembling those found in fibrous dysplasia. In all lesions, transformed osteoblasts expressed high levels of periostin, whereas normal osteoblasts did not. Our results show that periostin is a novel marker for intramembranous ossification, and is a good candidate as a diagnostic tool and/or a therapeutic target in fibrous dysplasia. Moreover, the Gs alpha-cyclic adenosine monophosphate-c-Fos pathway might represent one mechanism of periostin up-regulation in fibrous dysplasia, resulting in altered collagen fibrillogenesis characteristic of this disease. (C) 2009 Elsevier Inc. All rights reserved.


Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY,56(8):753-764 2008(Aug.)
Author:Yoshinao Kikuchi; Takeshi G. Kashima; Takashi Nishiyama; Kazuhiro Shimazu; Yasuyuki Morishita; Masashi Shimazaki; Isao Kii; Hisanaga Horie; Hideo Nagai; Akira Kudo; Masashi Fukayama
Abstract:Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immuno-electron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin(-/-) mice (similar to 0.6-fold) compared with periostin(+/+) mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger (similar to 1.5-fold) when cultured with fibroblasts derived from periostin(+/+) mice or periostin-transfected NIH3T3 cells than with those from periostin(-/-) mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.


Periostin is essential for cardiac healing after acute myocardial infarction
JOURNAL OF EXPERIMENTAL MEDICINE,205(2):295-303 2008(Feb.)
Author:Masashi Shimazaki; Kazuto Nakamura; Isao Kii; Takeshi Kashima; Norio Amizuka; Minqi Li; Mitsuru Saito; Keiichi Fukuda; Takashi Nishiyama; Satoshi Kitajima; Yumiko Saga; Masashi Fukayama; Masataka Sata; Akira Kudo
Abstract:Acute myocardial infarction (AMI) is a common and lethal heart disease, and the recruitment of fibroblastic cells to the infarct region is essential for the cardiac healing process. Although stiffness of the extracellular matrix in the infarct myocardium is associated with cardiac healing, the molecular mechanism of cardiac healing is not fully understood. We show that periostin, which is a matricellular protein, is important for the cardiac healing process after AMI. The expression of periostin protein was abundant in the infarct border of human and mouse hearts with AMI. We generated periostin(-/-) mice and found no morphologically abnormal cardiomyocyte phenotypes; however, after AMI, cardiac healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness caused by a reduced number of alpha smooth muscle actin-positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes were rescued by gene transfer of a spliced form of periostin. Moreover, the inhibition of FAK or alpha v-integrin, which blocked the periostin-promoted cell migration, revealed that alpha v-integrin, FAK, and Akt are involved in periostin signaling. Our novel findings show the effects of periostin on recruitment of activated fibroblasts through FAK-integrin signaling and on their collagen fibril formation specific to healing after AMI.


GFP transgenic mice reveal active canonical Wnt signal in neonatal brain and in adult liver and spleen
GENESIS,45(2):90-100 2007(Feb.)
Author:Akemi Moriyama; Isad Kii; Takehiko Sunabori; Suguru Kurihara; Issei Takayama; Masashi Shimazaki; Hideyuki Tanabe; Masayuki Oginuma; Masashi Fukayama; Yumi Matsuzaki; Yumiko Saga; Akira Kudo
Abstract:In the past decades, the function of the Writ canonical pathway during embryogenesis has been intensively investigated; however, little survey of neonatal and adult tissues has been made, and the role of this pathway remains largely unknown. To investigate its role in mature tissues, we generated two new reporter trans genic mouse lines, ins-TOPEGFP and ins-TOPGAL, that drive EGFP and beta-galactosidase expression under TCF/ beta-catenin, respectively. To obtain the accurate expres sion pattern, we flanked these transgenes with the HS4 insulator to reduce chromosomal positional effects. Analysis of embryos showed that the reporter genes were activated in regions where canonical Writ activity has been implicated. Furthermore, their expression patterns were consistent in both lines, indicating the accuracy of the reporter signal. In the neonatal brain, the reporter signal was detected in the mesencephalon and hippocampus. In the adult mice, the reporter signal was found in the mature pericenteral hepatocytes in the normal liver. Furthermore, during inflammation the number of T cells expressing the reporter gene increased in the adult spleen. Thus, in this research, we identified two organs, i.e., the liver and spleen, as novel organs in which the Writ canonical signal is in motion in the adult. These transgenic lines will provide us broader opportunities to investigate the function of the Writ canonical pathway in vivo.


Periostin is an extracellular matrix protein required for eruption of incisors in mice
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,342(3):766-772 2006(Apr.)
Author:Kii, I; N Amizuka; L Minqi; S Kitajima; Y Saga; A Kudo
Abstract:A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin(-/-) mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin(-/-) mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone. (c) 2006 Elsevier Inc, All rights reserved.


Identification of cysteine residues critically involved in homodimer formation and protein expression of human ATP-binding cassette transporter ABCG2: A new approach using the Flp recombinase system
Journal of Experimental Therapeutics and Oncology,5(3):205-222 2006
Author:Kanako Wakabayashi; Hiroshi Nakagawa; Tatsuhiko Adachi; Isao Kii; Eiry Kobatake; Akira Kudo; Toshihisa Ishikawa
Abstract:Since ABCG2 is a half-transporter in the ATP-binding cassette (ABC) transporter family, it has been suspected that ABCG2 functions as a homodimer. In the present study, we have investigated the molecular mechanism underlying homodimer formation of ABCG2. Based on the amino acid sequence of ABCG2, three cysteine residues (Cys592, Cys603, and Cys608) are expected to exist in the extracellular loop. To identify a cysteine residue(s) required for homodimer formation, we have substituted those cysteine residues to glycine by site-directed mutagenesis and stably expressed the resulting variants in Flp-In™-293 cells. Substitution of the amino acid at position 603 from cysteine to glycine (C603G) completely diminished homodimer formation, whereas substitution of both Cys592 and Cys608 to glycine residues (C592G/C608G) had no effect on homodimer formation. These results strongly suggest that Cys603 is prerequisite for homodimer formation of ABCG2 via a disulfide bond. On the other hand, immunohistochemistry experiments revealed that the C592G/C608G variant is mainly located in intracellular compartments. The C592G/C608G variant exhibited lower activity of ATP-dependent methotrexate (MTX) transport, and its expression did not confer Flp-In-293 cells resistance to SN-38 or mitoxantrone. Cys592 and Cys608 appear to be important for intracellular sorting of the de novo synthesized ABCG2 protein to the plasma membrane. Taken together, cysteine residues in the extra-cellular loop are considered to play pivotal roles in homodimer formation and plasma membrane localization of ABCG2. © 2006 Old City Publishing, Inc.


Immunohistochemical localization of periostin in tooth and its surrounding tissues in mouse mandibles during development.
The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology,281(2):1264-1275 2004(Dec.)
Author:Suzuki H; Amizuka N; Kii I; Kawano Y; Nozawa-Inoue K; Suzuki A; Yoshie H; Kudo A; Maeda T
Abstract:Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption.


Cell-cell interaction mediated by cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage
JOURNAL OF BONE AND MINERAL RESEARCH,19(11):1840-1849 2004(Nov.)
Author:Kii, I; N Amizuka; J Shimomura; Y Saga; A Kudo
Abstract:We studied cadherin-11 function in the differentiation of mesenchymal cells. Teratomas harboring the cadherin-11 gene generated bone and cartilage preferentially. Cadherin-11 transfectants of C2C12 cells and cadherin-11 and/or N-cadherin transfectants of L cells showed that cadherin-11 together with N-cadherin-induced expression of ALP and FGF receptor 2. These results suggest that cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage in a different manner from N-cadherin.


Inactivation of Rho/ROCK signaling is crucial for the nuclear accumulation of FKHR and myoblast fusion
JOURNAL OF BIOLOGICAL CHEMISTRY,279(45):47311-47319 2004(Nov.)
Author:T Nishiyama; Kii, I; A Kudo
Abstract:Myoblast fusion is a critical process for the terminal differentiation of skeletal muscle. To elucidate the intracellular mechanisms regulating myoblast fusion, we studied the roles of signaling through the small GTPase Rho and its effector, the Rho-associated kinase ROCK, in myoblast fusion of mouse C2C12 cells. We found that Rho activity, which was high in proliferating myoblasts, decreased during myogenesis. Expression of a constitutively active form of Rho blocked myoblast fusion, but not the earlier steps of differentiation. Consistently, ROCK activity was also decreased in differentiating C2C12 cells, and an active ROCK mutant prevented their fusion. Furthermore, inactivation of ROCK by the specific inhibitor Y-27632 enhanced myoblast fusion, even in cells expressing the active Rho mutant. Thus, the down-regulation of Rho/ROCK signaling is required for myoblast fusion. We also found that Rho/ROCK signaling was required for retaining FKHR, a transcription factor implicated in myoblast fusion, in the cytoplasm and that inactivation of ROCK was essential for the nuclear accumulation of FKHR that took place just before the onset of myoblast fusion. Moreover, ROCK directly phosphorylated FKHR in vitro. We conclude that the inactivation of Rho/ROCK signaling is a prerequisite for FKHR nuclear translocation and myoblast fusion in C2C12 cells, providing evidence for a novel regulatory role of Rho/ROCK signaling in myogenic differentiation.


A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport
BIOCHEMICAL JOURNAL,373(Pt 3):767-774 2003(Aug.)
Author:H Mitomo; R Kato; A Ito; S Kasamatsu; Y Ikegami; Kii, I; A Kudo; E Kobatake; Y Sumino; T Ishikawa
Abstract:Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482) and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK293 cells. [Arg(482)]ABCG2 transports [H-3]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482).]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S-octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg 412 is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.


Targeted disruption of cadherin-11 leads to a reduction in bone density in calvaria and long bone metaphyses
JOURNAL OF BONE AND MINERAL RESEARCH,16(7):1265-1271 2001(Jul.)
Author:J Kawaguchi; Y Azuma; K Hoshi; Kii, I; S Takeshita; T Ohta; H Ozawa; M Takeichi; O Chisaka; A Kudo
Abstract:The migration and adhesion of osteoblasts requires several classical cadherins, Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-ll in osteoblastogenesis, cadherin-ll null mutant mice were investigated, Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploe of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area-of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-ll leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-ll plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.


The transition of cadherin expression in osteoblast differentiation from mesenchymal cells: Consistent expression of cadherin-11 in osteoblast lineage
JOURNAL OF BONE AND MINERAL RESEARCH,16(2):260-269 2001(Feb.)
Author:J Kawaguchi; Kii, I; Y Sugiyama; S Takeshita; A Kudo
Abstract:Osteoblasts are derived originally from pluripotent mesenchymal stem cells on migration into the bone matrix, To elucidate the contribution of classical cadherins in this differentiation pathway, we developed a new protocol for their analysis and studied their specific expressions in various cell lines of the mesenchymal lineage, including osteoblasts. N-cadherin was expressed constitutively in all cell lines examined except an osteocyte-like cell line whereas cadherin-11 was expressed selectively in preosteoblast and preadipocyte cell lines. P-cadherin also was expressed in primary cultures of calvarial cells and mature osteoblasts at a relatively low level compared with N-cadherin and cadherin-11. M-cadherin was expressed only in a premyoblast cell line. We observed the transition of cadherin expression from M-cadherin to cadherin-11 in the premyoblast cell line when osteogenic differentiation was induced by treatment with bone morphogenetic protein 2 (BMP-2), while the expression of N-cadherin remained unchanged. In contrast, when a preadipocyte cell line, which shows a similar pattern of cadherin expression to osteoblasts, was induced to undergo adipogenic differentiation, the expression of N-cadherin and cadherin-11 was decreased. These observations characterize the cadherin expression profile of mesenchymal lineage cells, especially osteoblasts, which regularly express cadherin-11. Cadherin-11 may affect cell sorting, alignment, and separation through differentiation.


講演・口頭発表等
An alternative strategy to develop a selective kinase inhibitor
WPI-NanoLSI Special Seminar, Kanazawa University 2020(Jan. 30)
Presenter:喜井 勲


リン酸化酵素のフォールディング中間体を標的とした創薬研究
新学術領域「分子夾雑の生命化学」第2回 関東地区シンポジウム(東京大学) 2019(Oct. 16)
Presenter:喜井 勲


リン酸化酵素のフォールディング中間体を標的とした創薬研究
第8回生命科学阿波おどりシンポジウム(徳島大学) 2019(Aug. 16)
Presenter:喜井 勲


リン酸化酵素フォールディング中間体を標的とした創薬研究
新学術領域「中分子戦略」「分子夾雑化学」ジョイントシンポジウム・第21回生命化学研究会(大阪大学) 2018(Sep. 08)
Presenter:喜井 勲


リン酸化酵素の活性・阻害に影響を与える細胞内分子夾雑環境の理解と創薬応用
新学術領域研究「分子夾雑の生命化学」領域会議 2018(May 22)
Presenter:喜井 勲


タンデムHaloTagによる高感度蛍光イメージング技術の開発
新学術領域「新生鎖の生物学」第5回若手ワークショップ 2018(May 16)
Presenter:喜井 勲


Cotranslational ubiquitinationとmRNA安定性の関連性についての仮説
第6回CCR4-NOT研究会 2018(May 13)
Presenter:喜井 勲


細胞外マトリックス形成の蛍光追跡イメージング技術の開発
平成29年度生体医歯工学共同研究拠点成果報告会 2018(Mar. 09)
Presenter:喜井 勲


ナトリウム代謝イメージングによる腎臓機能画像診断
Kobe×BRAVE Acceleration programピッチ大会 2018(Mar. 04)
Presenter:本村 信治; 喜井 勲; 羽場 宏光; 薬師寺 秀樹


MetalloDiagnosis GREIによる生体微量元素動態診断
BRAVE Acceleration program最終ピッチ大会 2017(Dec. 18)
Presenter:本村 信治; 喜井 勲; 羽場 宏光; 薬師寺 秀樹


Staudinger反応を利用した安定なアザイリドの形成に基づく分子連結法の開発
第44回有機典型元素化学討論会 2017(Dec. 07)
Presenter:寺嶌紀和; 目黒友啓; 小池悠華; 伊藤晴海; 伊藤晴海; 喜井勲; 吉田優; 細谷孝充; 細谷孝充


神経新生促進によるダウン症モデルにおける脳発達障害の是正
2017年度生命科学系学会合同年次大会 2017(Dec. 06)
Presenter:小林亜希子; 粟屋智就; 喜井勲; 隅田有人; 奥野友紀子; 和根崎圭子; 吉田優; 隅田ともえ; 井上治久; 細谷孝充; 萩原正敏


がん細胞の代謝がもたらす組織微小環境を標的にしたがん治療
2017年度生命科学系学会合同年次大会 2017(Dec. 06)
Presenter:豊本雅靖; 井上飛鳥; 飯田慶; 出縄政嗣; 喜井勲; 林佳子; 岸貴之; 小野木博; 吉田優; 細谷孝充; 青木淳賢; 萩原正敏


Duchenne型筋ジストロフィーに対して変異ジストロフィンエキソンのスキッピング誘導活性を有する経口投与可能な新規CLK1阻害剤の開発
2017年度生命科学系学会合同年次大会 2017(Dec. 06)
Presenter:佐古有季哉; 二宮賢介; 奥野友紀子; 豊本雅靖; 西田篤史; 小池悠華; 大江賢治; 喜井勲; 吉田優; 橋本有弘; 細谷孝充; 松尾雅文; 萩原正敏


マルチカラーパルスチェイスによる細胞外マトリックスの継時的変化の可視化
2017年度生命科学系学会合同年次大会 2017(Dec. 06)
Presenter:伊藤 晴海; 小池 悠華; 三好 洋美; 吉田 優; 渡辺 恭良; 細谷 孝充; 喜井 勲


Sequential Multicolor Pulse-Chase Imaging of Protein Dynamics in Extracellular Matrix in Living Fibroblasts
The 4th RIKEN/Karolinska Institute/SciLifeLab Joint Symposium 2017(Nov. 16)
Presenter:Harumi Ito; Yuka Koike; Hiromi Miyoshi; Suguru Yoshida; Yasuyoshi Watanabe; Takamitsu Hosoya; Isao Kii


リン酸化酵素の新生鎖における品質管理機構の解明
新学術領域「新生鎖の生物学」領域会議 2017(Nov. 08)
Presenter:喜井 勲


Selective inhibition of the kinase DYRK1A by targeting its folding process
新学術領域「新生鎖の生物学」第4回若手ワークショップ 2017(Aug. 29)
Presenter:喜井 勲


リン酸化酵素DYRK1Aのフォールディング中間体阻害メカニズムの解析
日本ケミカルバイオロジー学会第12回年会 2017(Jun. 07)
Presenter:喜井 勲; 小池 悠華


環状アルキンの銅錯体形成を利用した生体分子の化学修飾法の開発
日本ケミカルバイオロジー学会第12回年会 2017(Jun. 07)
Presenter:吉田 優; 栗原ともこ; 伊藤晴海; 喜井 勲; 畠山泰朋; 唐木文霞; 渡辺恭良; 細谷孝充


Quality control mechanism of DYRK1A folding intermediate
International Symposium on Protein Quality Control 2017(Jun. 04)
Presenter:喜井 勲


Selective inhibition of the kinase DYRK1A by targeting its folding process
RIKEN SAKURA Symposium 2017 2017(Mar. 29)
Presenter:喜井 勲


環状アルキン‐銅錯体を利用する生体分子の化学修飾法の開発
日本化学会第97春季年会 2017(Mar. 16)
Presenter:吉田優; 栗原とも子; 伊藤晴海; 喜井勲; 畠山泰朋; 唐木文霞; 渡辺恭良; 細谷孝充


Selective inhibition of the kinase DYRK1A by targeting its folding process
第2回AMED次世代がん若手研究者ワークショップ 2016(Nov. 29)
Presenter:喜井 勲; 祖納元 りえ; 隅田 有人; 小池 悠華; 細谷 孝充; 萩原 正敏


タウ蛋白質分解誘導による認知症治療を目指した新規化合物の発見
第89回日本生化学会大会 2016(Sep. 25)
Presenter:豊本 雅靖; 笹ヶ迫 智紀; 喜井 勲; 小池 悠華; 林 佳子; 石田 憲太郎; 隅田 有人; 吉田 優; 細谷 孝充; 萩原 正敏


クライオ1分子蛍光イメージングによる高精度三次元位置決定
分子科学討論会 2016(Sep. 13)
Presenter:本橋和也; 古林琢; 若尾佳祐; 松下道雄; 石川冬木; 喜井勲; 藤芳暁


低分子化合物FINDYによるリン酸化酵素DYRK1Aのフォールディング阻害メカニズムの解析
日本ケミカルバイオロジー学会第11回年会 2016(Jun. 15)
Presenter:喜井 勲; 祖納元 りえ; 隅田 有人; 小池 悠華; 隅田 ともえ; 細谷 孝充; 萩原 正敏


ダウン症モデルでの神経新生の低下を改善するDYRK1A阻害剤の創製
日本ケミカルバイオロジー学会第11回年会 2016(Jun. 15)
Presenter:小林 亜希子; 喜井 勲; 隅田 有人; 吉田 優; 細谷 孝充; 萩原 正敏


多機能性分子プローブ開発を指向したマルチアジド化合物を用いる機能性分子集積法の開発
日本ケミカルバイオロジー学会第11回年会 2016(Jun. 15)
Presenter:吉田 優; 三澤 善大; 栗原 ともこ; 森田 隆太; 喜井 勲; 渡辺 恭良; 細谷 孝充


Duchenne型筋ジストロフィーに対するエキソンスキッピング治療薬候補化合物である新規CLK1特異的阻害剤
日本ケミカルバイオロジー学会第11回年会 2016(Jun. 15)
Presenter:佐古 有季哉; 二宮 賢介; 奥野 友紀子; 豊本 雅靖; 西田 篤史; 小池 悠華; 大江 賢治; 喜井 勲; 吉田 優; 細谷 孝充; 松尾 雅文; 萩原 正敏


タウ蛋白質分解誘導によるタウオパチー治療を目指した新規化合物の発見
日本ケミカルバイオロジー学会第11回年会 2016(Jun. 15)
Presenter:豊本 雅靖; 笹ヶ迫 智紀; 喜井 勲; 小池 悠華; 林 佳子; 石田 憲太郎; 隅田 有人; 吉田 優; 細谷 孝充; 萩原 正敏


新しいマルチアジド化合物をプラットフォーム分子として用いる機能性分子集積法の開発
日本化学会第96春季年会 2016(Mar. 24)
Presenter:吉田優; 三澤善大; 栗原ともこ; 森田隆太; 喜井勲; 渡辺恭良; 細谷孝充


DYRK1Aはダウン症モデルにおける神経新生低下を制御する
第38回日本神経科学大会 2015(Jul. 28)
Presenter:小林 亜希子; 喜井 勲; 隅田 有人; 隅田 ともえ; 細谷 孝充; 萩原 正敏


変異ジストロフィン遺伝子に対するエクソンスキップ誘導活性を有する化合物の探索
第17回日本RNA学会年会 2015(Jul. 17)
Presenter:二宮賢介; 佐古有季哉; 喜井勲; 細谷孝充; 萩原正敏


Neuropollin-1陽性免疫細胞はトラスツズマブによる免疫応答を惹起し抗腫瘍活性を誘導する
第23回日本乳癌学会総会 2015(Jul. 02)
Presenter:河口 浩介; 鈴木 栄治; 喜井 勲; 片岡 竜貴; 平田 勝啓; 羽賀 博典; 萩原 正敏; 戸井 雅和


SRPK1およびCK2阻害活性を有する新規血管新生阻害薬の開発
日本眼科学会雑誌 2015(Mar. 18)
Presenter:諸岡諭; 喜井勲; 奥野友紀子; 吉田優; 福原淳一; 野田航介; 伊藤暢聡; 石田晋; 細谷孝充; 萩原正敏; 吉村長久


SRPK1をターゲットとした化合物スクリーニングによる新規血管新生阻害薬の開発
血管 2015(Jan. 31)
Presenter:諸岡諭; 保科光輝; 喜井勲; 奥野友紀子; 吉田優; 伊藤暢聡; 細谷孝允; 吉村長久; 萩原正敏


極低温共焦点蛍光顕微鏡の開発による細胞の多色イメージング
分子科学討論会 2015
Presenter:本橋和也; 若尾圭祐; 稲川博敬; 古林琢; 喜井勲; 定家真人; 石川冬木; 松下道雄; 藤芳暁


リン酸化酵素DYRK1Aのフォールディング中間体を標的とした新規阻害剤の開発
日本薬理学会近畿部会 2014(May)
Presenter:喜井勲; 奥野友紀子; 隅田有人; 後藤利保; 吉田優; 澁谷浩司; 安倍美奈子; 伊藤暢聡; 細谷孝充; 萩原正敏


リン酸化酵素とシャペロンの結合を指標とした阻害剤評価系の構築
日本分子生物学会年会 2014
Presenter:祖納元りえ; 喜井勲; 小池悠華; 萩原正敏


A Novel type of kinase inhibitor that targets the folding intermediate of DYRK1A
The 2nd Annual Conference of the International Chemical Biology Society 2013(Oct. 07)
Presenter:Isao Kii; Yuto Sumida; Toshiyasu Goto; Yukiko Okuno; Yosuke Nonaka; Minako Abe; Suguru Yoshida; Tomoe Kato; Teikichi Ikura; Nobutoshi Ito; Hiroshi Shibuya; Takamitsu Hosoya; Masatoshi Hagiwara


異種アジド基の特性にもとづく逐次マルチクリック反応の開発
複素環化学討論会 2013(Oct. 01)
Presenter:細谷孝充; 吉田優; 菅野貴美幸; 喜井勲; 森田隆太; 田中淳子; 松下武司; 萩原正敏


多機能性分子プローブ創製に有用な異種アジド選択的逐次クリック反応の開発
有機合成シンポジウム 2013(May 27)
Presenter:吉田優; 菅野貴美幸; 喜井勲; 松下武司; 萩原正敏; 細谷孝充


異種アジド選択的逐次クリック反応:機能性分子集積への展開
日本化学会 2013(Mar. 08)
Presenter:菅野貴美幸; 吉田優; 喜井勲; 松下武司; 萩原正敏; 細谷孝充


炎症性疼痛モデルマウスに対する新規カゼインキナーゼ阻害薬の抗侵害効果
日本薬理学雑誌 2013(Mar.)
Presenter:川元 大輔; 喜井 勲; 豊本 雅靖; 朝田 俊秀; 吉村 惠; 萩原 正敏; 宮田 篤郎; 栗原 崇


温度数Kの色素分子の3次元イメージング技術の設計と実現
分子科学討論会 2013
Presenter:虎谷泰靖; 丸尾美奈子; 稲川博敬; 喜井勲; 林宣広; 細谷孝充; 松下道雄; 藤芳暁


PUF60は筋分化特異的な選択的スプライシングの調節因子であり,筋発生に必要である
日本RNA学会年会 2012(Jul. 18)
Presenter:片岡直行; 野島孝之; 大城(井手上; 杜子; 武内章英; 喜井勲; 萩原正敏


セルベース・スクリーニングを活用した神経変性疾患治療薬の探索
解剖学雑誌 2012(Jun. 01)
Presenter:笹ケ迫知紀; 喜井勲; 石田憲太郎; 細谷孝充; 萩原正敏


炎症性疼痛モデルマウスに対する新規カゼインキナーゼ阻害薬の抗侵害効果
日本薬理学会西南部会 2012
Presenter:川元大輔; 喜井勲; 豊本雅靖; 朝田俊秀; 吉村惠; 萩原正敏; 宮田篤郎; 栗原崇


ダブルクリック反応の開発と生体分子の化学修飾法への応用
有機合成化学セミナー 2011(Aug. 31)
Presenter:吉田優; 白石旭; 喜井勲; 平松俊行; 松下武司; 植草秀裕; 山本誠; 工藤明; 萩原正敏; 細谷孝充


Targeted inhibition of autophosphorylation of Down syndrome associated kinase DYRK1A leads to its proteasome-mediated degradation
US-JAPAN CONFERENCE ON INFLAMMATION, DIABETES AND CANCER 2011(Aug. 04)
Presenter:Isao Kii; Yukiko Okuno; Yuto Sumida; Takamitsu Hosoya; Masatoshi Hagiwara


細胞メカノセンシングの新展開 網目状の細胞外マトリックス形成を介した結合組織の力学環境適応(Recent Advances in Cell Mechanosensing Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture)
日本細胞生物学会大会 2011(May)
Presenter:喜井 勲


ダブルクリック反応:生体分子の新しい化学修飾法
日本化学会 2011(Mar. 11)
Presenter:細谷孝充; 喜井勲; 白石旭; 平松俊行; 松下武司; 植草秀裕; 吉田優; 山本誠; 工藤明; 萩原正敏


ミリストイル基を介したHIV Nefの免疫細胞のシグナル伝達系への介入
日本分子生物学会年会 2011
Presenter:細谷悟史; 戸田収; 遠藤毅; 根岸瑠美; 福島慶子; 伊藤由馬; 十川久美子; PAXTON Thanai; 喜井勲; 朴明宣; 大野敏; 竹内真粧美; 横川隆志; 西川一八; 山下克子; 徳永万喜洋; 細谷孝充; 林宣宏


神経細胞における生体膜ラフトの動作メカニズムの解明
日本分子生物学会年会 2011
Presenter:戸田収; 細谷悟史; 遠藤毅; 根岸瑠美; 福島慶子; 伊藤由馬; 十川久美子; PAXTON Thanai; 喜井勲; 朴明宣; 大野敏; 竹内真粧美; 横川隆志; 西川一八; 山下克子; 徳永万喜洋; 細谷孝充; 林宣宏


骨膜における骨芽細胞のアクチン細胞骨格は、メカニカルストレス依存的に再形成され骨膜肥厚・骨形成に関与する
日本骨代謝学会学術集会 2010(Jul.)
Presenter:酒井 大輔; 喜井 勲; 中川 一樹; 松本 裕子; 細谷 孝充; 高久田 和夫; 工藤 明


periostinは歯根膜においてNotch1の安定性を調節することで細胞生存を制御している
日本骨代謝学会学術集会 2010(Jul.)
Presenter:田辺 英幸; 高山 一成; 西山 尚志; 島崎 雅司; 喜井 勲; 李 敏啓; 網塚 憲生; 勝部 憲一; 工藤 明


non‐RI光親和性標識法による新規Wntシグナル調節因子の探索
日本薬理学会関東部会 2009(Jul. 11)
Presenter:細谷孝充; 井上敦; 平松俊行; 喜井勲; 工藤明


ペリオスチンはBMP1と結合することでリジン酸化酵素(LOX)を活性化し、コラーゲン架橋を促進する
日本骨代謝学会学術集会 2009(Jul.)
Presenter:丸橋 拓海; 喜井 勲; 工藤 明


ペリオスチンは網目状細胞外マトリックス構築のスキャフォールドとして機能することで、脛骨過労性骨膜炎の発症を予防する
日本骨代謝学会学術集会 2009(Jul.)
Presenter:喜井 勲; 工藤 明


Bone Whole Imagingによるメカニカルストレス依存的アクチン細胞骨格の分子動態解析
日本骨代謝学会学術集会 2008(Oct.)
Presenter:酒井 大輔; 喜井 勲; 中川 一樹; 松本 裕子; 高久田 和夫; 工藤 明


ペリオスチンは、ゴルジ体においてフィブロネクチンとテネイシン-Cの結合の足場として機能し、脛骨過労性骨膜炎の発症を抑制する
日本骨代謝学会学術集会 2008(Oct.)
Presenter:喜井 勲; 西山 尚志; 原田 伊知郎; 李 敏啓; 松本 健一; 斉藤 充; 松本 裕子; 市川 知宏; 高山 一成; 田辺 英幸; 高久田 和夫; 網塚 憲生; 工藤 明


胃癌におけるペリオスチン 癌微小環境において間質線維芽細胞がペリオスチンを介して癌細胞の増殖を促進する(Periostin in gastric cancer: Stromal fibroblasts support growth of cancer cell via periostin in cancer microenvironment)
日本癌学会総会 2008(Sep.)
Presenter:菊地 良直; 岩田 要; 西山 尚志; 鹿島 健司; 狩野 光伸; 森下 保幸; 喜井 勲; 八代 正和; 新谷 裕加子; 平川 弘聖; 宮園 浩平; 工藤 明; 深山 正久


歯周組織の恒常性維持に必須な細胞外マトリックスタンパク質,ペリオスチンの分子機能解析
生化学 2008
Presenter:高山一成; 喜井勲; 工藤明


靭帯様コラーゲンゲル培養法を用いた細胞外マトリクスの繊維化形成モデルの解析
日本バイオマテリアル学会大会 2007(Nov. 26)
Presenter:市川知広; 原田伊知郎; 喜井勲; 赤池敏宏


ヒトペリオスチンは線維性骨異形成症に発現し、そのコラーゲン線維形成に関与する
日本骨代謝学会学術集会 2007(Jun.)
Presenter:西山 尚志; 鹿島 健司; 島津 和弘; 大狭 淳; 島崎 雅司; 喜井 勲; Grigoriadis Agamemnon E; 深山 正久; 工藤 明


ペリオスチンによって安定化されるフィブロネクチンマトリックスは、メカニカルストレス感知機構を担う
日本骨代謝学会学術集会 2007(Jun.)
Presenter:喜井 勲; 網塚 憲生; 李 敏啓; 中川 一樹; 高山 一成; 島崎 雅司; 田辺 英幸; 西山 尚志; 松本 裕子; 高久田 和夫; 市川 知広; 原田 伊知郎; 赤池 敏宏; 工藤 明


マウスの頭蓋顔面骨格形成時におけるFGF受容体1のカドヘリン-11による安定化(Cadherin-11 stabilizes FGF Receptor 1 during craniofacial skeletal development in mice)
日本発生生物学会・日本細胞生物学会合同大会 2007(May)
Presenter:喜井 勲; 徐 方; 下村 淳子; 網塚 憲生; 常 智杰; 工藤 明


創傷治癒時に誘導される筋繊維芽細胞に発現するペリオスチン機能解析
再生医療 2007(Feb. 19)
Presenter:西山尚志; 島崎雅司; 喜井勲; 深山正久; 鹿島健司; 工藤明


ペリオスチンは力学的負荷依存的骨形成に必須である
生化学 2007
Presenter:中川一樹; 喜井勲; 松本裕子; 西山尚志; LI Minqi; 網塚憲生; 高久田和夫; 工藤明


periostinとCCN3によるNotchシグナルの制御
日本骨代謝学会学術集会 2006(Jul.)
Presenter:田辺 英幸; 喜井 勲; 森山 明美; 李 敏啓; 網塚 憲生; 勝部 憲一; 工藤 明


ペリオスチンは力学的負荷依存的骨形成に必須である
日本骨代謝学会学術集会 2006(Jul.)
Presenter:喜井 勲; 松本 裕子; Li Minqi; 田辺 英幸; 中川 一樹; 高山 一成; 東 由明; 高久田 和夫; 網塚 憲生; 工藤 明


ヒトABCG2タンパク質の翻訳後修飾‐Cys603を介した二量体形成‐
日本分子生物学会年会 2005(Nov. 25)
Presenter:若林香菜子; 中川大; 足立達彦; 喜井勲; 工藤明; 小畠英理; 石川智久


骨肉腫におけるペリオスチンの発現および機能解析
日本癌学会学術総会 2005(Aug. 15)
Presenter:鹿島健司; 島津和弘; 西山尚志; 島崎雅司; 喜井勲; 深山正久; 工藤明


ヒト癌組織におけるペリオスチン発現の検討
日本癌学会学術総会 2005(Aug. 15)
Presenter:菊地良直; 島津和弘; 鹿島健司; 島崎雅司; 喜井勲; 柴原純二; 仁木利郎; 深山正久; 工藤明


歯の移動実験におけるペリオスチン遺伝子欠損マウスの歯根膜の組織学的変化
日本骨代謝学会学術集会 2005(Jun. 20)
Presenter:李敏啓; 網塚憲生; 喜井勲; 工藤明; 前田健康


Mechanical Stress Dependent Remodeling of the Periodontal Ligament Is Defective In Periostin Deficient Mice; Mechanotransduction through Periostin Protein
The 26 th Annual Meeting of the American Society for Bone and Mineral Research 2004(Oct. 01)
Presenter:Isao Kii; Norio Amizuka; Li Minqi; Satoshi Kitajima; Yumiko Saga; Akira Kudo


骨芽細胞におけるperiostinとCCN3の会合とNotch signalの制御
日本骨代謝学会学術集会 2004(Aug.)
Presenter:田辺 英幸; 喜井 勲; 網塚 憲生; 勝部 憲一; 工藤 明


ペリオスチンノックアウトマウスではメカニカルストレスによる歯根膜リモデリングが破綻している
日本骨代謝学会学術集会 2004(Aug.)
Presenter:喜井 勲; 網塚 憲生; 李 敏啓; 竹内 亀一; 前田 健康; 北嶋 聡; 菅野 純; 井上 達; 相賀 裕美子; 工藤 明


低分子量GTPase Rhoとそのエフェクター分子による骨格筋分化制御
日本分子生物学会年会 2003(Nov. 25)
Presenter:西山朋子; 喜井勲; 工藤明


細胞間接着分子カドヘリンによる骨芽細胞分化誘導
日本骨代謝学会 2001(Jul. 19)
Presenter:喜井勲; 工藤明


カドヘリンによる骨芽細胞分化制御
日本分子生物学会年会 2000(Nov. 25)
Presenter:喜井勲; 川口実太郎; 工藤明


カドヘリンによる骨芽細胞分化制御
日本細胞生物学会大会 2000(Aug.)
Presenter:喜井 勲; 川口 実太郎; 工藤 明


MISC
非オピオイド鎮痛薬の創製
鎮痛薬・オピオイドペプチドシンポジウムプログラム・抄録集,40th 2021
Author:豊本雅靖; 豊本雅靖; 栗原崇; 中川貴之; 井上飛鳥; 木村亮; 喜井勲; 喜井勲; 澤田照夫; 澤田照夫; 荻原孝史; 荻原孝史; 永安一樹; 岸貴之; 小野木博; 小野木博; 田口純平; 吉田優; 青木淳賢; 青木淳賢; 細谷孝充; 萩原正敏


タンデムタグ含有14-3-3プロテオミクス解析による抗がん活性フシコクシン誘導体の作用機序の解明
日本化学会春季年会講演予稿集(Web),101st 2021
Author:増田遼馬; 伊賀上祥汰; 喜井勲; 大神田淳子


天然変性概日時計転写因子を標的とする阻害剤の開発
日本化学会春季年会講演予稿集(Web),101st 2021
Author:細谷侑佑; 能條航; 喜井勲; 鈴木孝紀; 今西未来; 大神田淳子


赤色光によるアンケージングが可能な3-アシルインドリジンの簡便合成
反応と合成の進歩シンポジウム講演要旨集 2020
Author:渡邊賢司; 寺尾和花; 喜井勲; 中川れい子; 丹羽節; 細谷孝充; 細谷孝充


半導体コンプトンカメラGREIによるNa+/K+の同時イメージング
JSMI Report,12(2):170 2019(May)
Author:本村 信治; 喜井 勲; 羽場 宏光; 薬師寺 秀樹; 渡辺 恭良; 榎本 秀一


リン酸化酵素のフォールディング阻害技術を基盤とした創薬研究
東京生化学研究会助成研究報告集,33 2019
Author:喜井勲


細胞外マトリックス形成の蛍光追跡イメージング技術の開発
生体医歯工学共同研究拠点成果報告書,平成29年度:50 2018(Apr.)
Author:喜井 勲; 伊藤 晴海; 細谷 孝充; 吉田 優


神経新生促進によるダウン症モデルにおける脳発達障害の是正
生命科学系学会合同年次大会,2017年度:[2P 2017(Dec.)
Author:小林 亜希子; 粟屋 智就; 喜井 勲; 隅田 有人; 奥野 友紀子; 和根崎 圭子; 吉田 優; 隅田 ともえ; 井上 治久; 細谷 孝充; 萩原 正敏


がん細胞の代謝がもたらす組織微小環境を標的にしたがん治療
生命科学系学会合同年次大会,2017年度:[3AT25 2017(Dec.)
Author:豊本 雅靖; 井上 飛鳥; 飯田 慶; 出縄 政嗣; 喜井 勲; 林 佳子; 岸 貴之; 小野木 博; 吉田 優; 細谷 孝充; 青木 淳賢; 萩原 正敏


Duchenne型筋ジストロフィーに対して変異ジストロフィンエキソンのスキッピング誘導活性を有する経口投与可能な新規CLK1阻害剤の開発
生命科学系学会合同年次大会,2017年度:[2P 2017(Dec.)
Author:佐古 有季哉; 二宮 賢介; 奥野 友紀子; 豊本 雅靖; 西田 篤史; 小池 悠華; 大江 賢治; 喜井 勲; 吉田 優; 橋本 有弘; 細谷 孝充; 松尾 雅文; 萩原 正敏


神経新生促進によるダウン症モデルにおける脳発達障害の是正
生命科学系学会合同年次大会,2017年度:[2AT26 2017(Dec.)
Author:小林 亜希子; 粟屋 智就; 喜井 勲; 隅田 有人; 奥野 友紀子; 和根崎 圭子; 吉田 優; 隅田 ともえ; 井上 治久; 細谷 孝充; 萩原 正敏


マルチカラーパルスチェイスによる細胞外マトリックスの継時的変化の可視化
生命科学系学会合同年次大会,2017年度:[1P 2017(Dec.)
Author:伊藤 晴海; 小池 悠華; 三好 洋美; 吉田 優; 渡辺 恭良; 細谷 孝充; 喜井 勲


Staudinger反応を利用した安定なアザイリドの形成に基づく分子連結法の開発
有機典型元素化学討論会講演要旨集,44th 2017
Author:寺嶌紀和; 目黒友啓; 小池悠華; 伊藤晴海; 伊藤晴海; 喜井勲; 吉田優; 細谷孝充; 細谷孝充


環状アルキン-銅錯体を利用する生体分子の化学修飾法の開発
日本化学会春季年会講演予稿集(CD-ROM),97th 2017
Author:吉田優; 栗原とも子; 伊藤晴海; 喜井勲; 畠山泰朋; 唐木文霞; 渡辺恭良; 細谷孝充


タウ蛋白質分解誘導による認知症治療を目指した新規化合物の発見
日本生化学会大会プログラム・講演要旨集,89回:[3P 2016(Sep.)
Author:豊本 雅靖; 笹ヶ迫 智紀; 喜井 勲; 小池 悠華; 林 佳子; 石田 憲太郎; 隅田 有人; 吉田 優; 細谷 孝充; 萩原 正敏


Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer
CANCER RESEARCH,76 2016(Jul.)
Author:Kosuke Kawaguchi; Eiji SUzuki; Masashi Inoue; Isao Kii; Tatsuki R. Karaoke; Masahiro Hiram; Keiko Iwaisako; Pu Fengling; Mariko Niche; Ayane Yamaguchi; Hironori Haga; Masatoshi Hagiwara; Masakazu Toi


2種のアジド分子を効率的に連結する手法の開発
有機合成化学協会誌,74(5):453 2016(May)
Author:吉田優; 喜井勲; 細谷孝充; 細谷孝充
Abstract:"Click reaction", epitomized by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is an emerging method for conjugating molecules efficiently in broad fields, including materials chemistry and chemical biology. A copper-free variant, strain-promoted azide-alkyne cycloaddition (SPAAC), using functionalized cyclooctyne derivatives that spontaneously react with azides, have enabled the chemical modification of azido-incorporated biomolecules in cultured cells and in living animals, greatly expanding the utility of click chemistry. A variety of cyclooctyne derivatives bearing functional moieties, such as fluorescent or biotinyl groups, have been developed and some of them have become commercially available. However, on-demand synthesis is required for those with a new functionality, which is not always easy because of the high reactivity of the strained alkyne moiety. To conjugate two azide molecules easily, we have developed strain promoted "double-click" reaction using Sondheimer diyne that bears two strained alkyne moieties. This novel convergent method capable of conjugating three molecules spontaneously has allowed for facile modification of an azido-biomolecule with a small reporter azido-molecule. We have also developed a transient protection method of cyclooctynes from cycloaddition with an azide via 1 1 complexation with a cationic copper (I) salt. Protection of a cyclooctyne bearing a terminal alkyne with a copper salt enabled the selective copper-catalyzed click conjugation with an azide at the terminal alkyne moiety, facilitating easy preparation of cyclooctyne derivatives.


Knockdown of neuropilin-1 in monocytes impaired lymphocyte migration and anti-tumor activity in a humanized mouse model
CANCER RESEARCH,75 2015(Aug.)
Author:Kosuke Kawaguchi; Eiji Suzuki; Isao Kii; Tatsuki R. Kataoka; Masahiro Hirata; Hironori Haga; Masatoshi Hagiwara; Masakazu Toi


Neuropollin-1陽性免疫細胞はトラスツズマブによる免疫応答を惹起し抗腫瘍活性を誘導する
日本乳癌学会総会プログラム抄録集,23回:352 2015(Jul.)
Author:河口 浩介; 鈴木 栄治; 喜井 勲; 片岡 竜貴; 平田 勝啓; 羽賀 博典; 萩原 正敏; 戸井 雅和


Identification of a dual inhibitor of SRPK1 and CK2 that attenuates pathological angiogenesis of macular degeneration in mice
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE,56(7) 2015(Jun.)
Author:Satoshi Morooka; Isao Kii; Yukiko Okuno; Nobutoshi Ito; Masatoshi Hagiwara; Nagahisa Yoshimura


リガンド構造展開と活性制御の実際 リン酸化酵素に対する選択的阻害剤の開発
CSJ Current Review,(19):134-139,1(4) 2015(Apr.)
Author:喜井勲; 萩原正敏


SRPK1およびCK2阻害活性を有する新規血管新生阻害薬の開発
日本眼科学会雑誌,119(臨増):178 2015(Mar.)
Author:諸岡 諭; 喜井 勲; 奥野 友紀子; 吉田 優; 福原 淳一; 野田 航介; 伊藤 暢聡; 石田 晋; 細谷 孝充; 萩原 正敏; 吉村 長久


SRPK1をターゲットとした化合物スクリーニングによる新規血管新生阻害薬の開発
血管,38(1):33 2015(Jan.)
Author:諸岡 諭; 保科 光輝; 喜井 勲; 奥野 友紀子; 吉田 優; 伊藤 暢聡; 細谷 孝允; 吉村 長久; 萩原 正敏


京都大学臨床研究ハイウェイを活用した難治疾患・がん等の新規治療法の開発 新規Clk阻害剤の開発,新規DYRK1A阻害薬の開発
京都大学臨床研究ハイウェイを活用した難治疾患・がん等の新規治療法の開発 平成26年度 総括・分担研究報告書,:4‐9 2015
Author:萩原正敏; 喜井勲; 高橋良輔


変異ジストロフィン遺伝子に対するエクソンスキップ誘導活性を有する化合物の探索
日本RNA学会年会要旨集,17th 2015
Author:二宮賢介; 佐古有季哉; 喜井勲; 細谷孝充; 萩原正敏


リン酸化酵素DYRK1Aのフォールディング中間体を標的とした新規阻害剤の開発
日本薬理学会近畿部会プログラム・要旨集,125th:87 2014(May)
Author:喜井勲; 奥野友紀子; 隅田有人; 後藤利保; 吉田優; 澁谷浩司; 安倍美奈子; 伊藤暢聡; 細谷孝充; 萩原正敏


【研究成果を薬につなげるアカデミア創薬の戦略と実例】(第2章)アカデミア創薬研究の今を知る 《注目の標的からの創薬展開》 キナーゼの多彩な立体構造を標的とした創薬
実験医学,32(2):279 2014(Feb.)
Author:喜井 勲; 萩原 正敏
Abstract:キナーゼは細胞内において多彩な立体構造をとっている。創薬研究で活用されている精製キナーゼの立体構造情報は、キナーゼの1つの側面を表したに過ぎない。そのため、精製キナーゼを用いた化合物スクリーニングでは、多彩な立体構造を標的とする阻害剤は見落とされてきた。このような新しいタイプの阻害剤は、細胞を用いた評価系においてのみヒットすると考えられる。本稿では、細胞内のキナーゼを標的とした創薬研究について概説する。後半では、われわれが細胞を用いた化合物スクリーニングによって見出したキナーゼのフォールディング中間体を特異的に阻害する化合物について紹介する。(著者抄録)


京都大学臨床研究ハイウェイを活用した難治疾患・がん等の新規治療法の開発 新規Clk阻害剤の開発,新規DYRK1A阻害薬の開発
京都大学臨床研究ハイウェイを活用した難治疾患・がん等の新規治療法の開発 平成25年度 総括・分担研究報告書,:3‐7 2014
Author:萩原正敏; 喜井勲; 高橋良輔


リン酸化酵素とシャペロンの結合を指標とした阻害剤評価系の構築
日本分子生物学会年会プログラム・要旨集(Web),37th 2014
Author:祖納元りえ; 祖納元りえ; 喜井勲; 小池悠華; 萩原正敏


炎症性疼痛モデルマウスに対する新規カゼインキナーゼ阻害薬の抗侵害効果
日本薬理学雑誌,141(3):38P 2013(Mar.)
Author:川元 大輔; 喜井 勲; 豊本 雅靖; 朝田 俊秀; 吉村 惠; 萩原 正敏; 宮田 篤郎; 栗原 崇


京都大学臨床研究ハイウェイを活用した難治疾患・がん等の新規治療法の開発 新規DYRK1A阻害薬の開発,新規Clk阻害剤の開発
京都大学臨床研究ハイウェイを活用した難治疾患・がん等の新規治療法の開発 平成24年度 総括研究報告書,:3‐8 2013
Author:萩原正敏; 喜井勲; 高橋良輔


異種アジド選択的逐次クリック反応:機能性分子集積への展開
日本化学会講演予稿集,93rd(4) 2013
Author:菅野貴美幸; 吉田優; 喜井勲; 松下武司; 萩原正敏; 細谷孝充


セルベース・スクリーニングを活用した神経変性疾患治療薬の探索
解剖学雑誌,87(2):38 2012(Jun.)
Author:笹ヶ迫 知紀; 喜井 勲; 石田 憲太郎; 細谷 孝充; 萩原 正敏


ダブルクリック反応:生体分子の新しい化学修飾法
生化学,84(4):306 2012(Apr.)
Author:喜井勲; 吉田優; 細谷孝充


PUF60は筋分化特異的な選択的スプライシングの調節因子であり,筋発生に必要である
日本RNA学会年会要旨集,14th 2012
Author:片岡直行; 野島孝之; 大城(井手上)杜子; 武内章英; 喜井勲; 萩原正敏


細胞メカノセンシングの新展開 網目状の細胞外マトリックス形成を介した結合組織の力学環境適応(Recent Advances in Cell Mechanosensing Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture)
日本細胞生物学会大会講演要旨集,63回:90 2011(May)
Author:喜井 勲


ミリストイル基を介したHIV Nefの免疫細胞のシグナル伝達系への介入
日本分子生物学会年会プログラム・要旨集(Web),34th 2011
Author:細谷悟史; 戸田収; 遠藤毅; 根岸瑠美; 福島慶子; 伊藤由馬; 伊藤由馬; 十川久美子; 十川久美子; PAXTON Thanai; 喜井勲; 朴明宣; 大野敏; 竹内真粧美; 横川隆志; 西川一八; 山下克子; 徳永万喜洋; 徳永万喜洋; 細谷孝充; 林宣宏


神経細胞における生体膜ラフトの動作メカニズムの解明
日本分子生物学会年会プログラム・要旨集(Web),34th 2011
Author:戸田収; 細谷悟史; 遠藤毅; 根岸瑠美; 福島慶子; 伊藤由馬; 伊藤由馬; 十川久美子; 十川久美子; PAXTON Thanai; 喜井勲; 朴明宣; 大野敏; 竹内真粧美; 横川隆志; 西川一八; 山下克子; 徳永万喜洋; 徳永万喜洋; 細谷孝充; 林宣宏


periostinは歯根膜においてNotch1の安定性を調節することで細胞生存を制御している
日本骨代謝学会学術集会プログラム抄録集,28回:221 2010(Jul.)
Author:田辺 英幸; 高山 一成; 西山 尚志; 島崎 雅司; 喜井 勲; 李 敏啓; 網塚 憲生; 勝部 憲一; 工藤 明


骨膜における骨芽細胞のアクチン細胞骨格は、メカニカルストレス依存的に再形成され骨膜肥厚・骨形成に関与する
日本骨代謝学会学術集会プログラム抄録集,28回:223 2010(Jul.)
Author:酒井 大輔; 喜井 勲; 中川 一樹; 松本 裕子; 細谷 孝充; 高久田 和夫; 工藤 明


ペリオスチンはBMP1と結合することでリジン酸化酵素(LOX)を活性化し、コラーゲン架橋を促進する
日本骨代謝学会学術集会プログラム抄録集,27回:177 2009(Jul.)
Author:丸橋 拓海; 喜井 勲; 工藤 明


ペリオスチンは網目状細胞外マトリックス構築のスキャフォールドとして機能することで、脛骨過労性骨膜炎の発症を予防する
日本骨代謝学会学術集会プログラム抄録集,27回:176 2009(Jul.)
Author:喜井 勲; 工藤 明


ペリオスチンは、ゴルジ体においてフィブロネクチンとテネイシン-Cの結合の足場として機能し、脛骨過労性骨膜炎の発症を抑制する
日本骨代謝学会学術集会プログラム抄録集,26回:172 2008(Oct.)
Author:喜井 勲; 西山 尚志; 原田 伊知郎; 李 敏啓; 松本 健一; 斉藤 充; 松本 裕子; 市川 知宏; 高山 一成; 田辺 英幸; 高久田 和夫; 網塚 憲生; 工藤 明


Bone Whole Imagingによるメカニカルストレス依存的アクチン細胞骨格の分子動態解析
日本骨代謝学会学術集会プログラム抄録集,26回:172 2008(Oct.)
Author:酒井 大輔; 喜井 勲; 中川 一樹; 松本 裕子; 高久田 和夫; 工藤 明


胃癌におけるペリオスチン 癌微小環境において間質線維芽細胞がペリオスチンを介して癌細胞の増殖を促進する(Periostin in gastric cancer: Stromal fibroblasts support growth of cancer cell via periostin in cancer microenvironment)
日本癌学会総会記事,67回:417 2008(Sep.)
Author:菊地 良直; 岩田 要; 西山 尚志; 鹿島 健司; 狩野 光伸; 森下 保幸; 喜井 勲; 八代 正和; 新谷 裕加子; 平川 弘聖; 宮園 浩平; 工藤 明; 深山 正久


靱帯様コラーゲンゲル培養法を用いた細胞外マトリクスの繊維化形成モデルの解析
日本バイオマテリアル学会大会予稿集,29回:344 2007(Nov.)
Author:市川 知広; 原田 伊知郎; 喜井 勲; 赤池 敏宏


ペリオスチンによって安定化されるフィブロネクチンマトリックスは、メカニカルストレス感知機構を担う
日本骨代謝学会学術集会プログラム抄録集,25回:238 2007(Jun.)
Author:喜井 勲; 網塚 憲生; 李 敏啓; 中川 一樹; 高山 一成; 島崎 雅司; 田辺 英幸; 西山 尚志; 松本 裕子; 高久田 和夫; 市川 知広; 原田 伊知郎; 赤池 敏宏; 工藤 明


ヒトペリオスチンは線維性骨異形成症に発現し、そのコラーゲン線維形成に関与する
日本骨代謝学会学術集会プログラム抄録集,25回:282 2007(Jun.)
Author:西山 尚志; 鹿島 健司; 島津 和弘; 大狭 淳; 島崎 雅司; 喜井 勲; Grigoriadis Agamemnon E.; 深山 正久; 工藤 明


マウスの頭蓋顔面骨格形成時におけるFGF受容体1のカドヘリン-11による安定化(Cadherin-11 stabilizes FGF Receptor 1 during craniofacial skeletal development in mice)
日本発生生物学会・日本細胞生物学会合同大会要旨集,40回・59回:169 2007(May)
Author:喜井 勲; 徐 方; 下村 淳子; 網塚 憲生; 常 智杰; 工藤 明


【歯科と骨粗鬆症 骨生物学と歯科医学の融合点】ペリオスチンと歯根膜・骨外膜
Clinical Calcium,17(2):202 2007(Jan.)
Author:喜井 勲; 工藤 明
Abstract:歯と骨には、歯根膜と骨外膜という共通性の高い組織が存在する。共通点の一つは強靱なコラーゲン線維を備え、物理的な力(メカニカルストレス)に対して抗う組織であることである。もう一つは、双方ともメカニカルストレスを感知して自らの組織改変を誘導するといった高いメカニカルストレス応答性を備えている点である。本稿では、歯根膜と骨外膜におけるこれらの共通点を繋ぐ分子としてペリオスチンを紹介する。ペリオスチンは、歯・骨組織では歯根膜と骨外膜に特異的に発現する細胞外マトリックスタンパク質である。我々は、ペリオスチンが骨代謝と歯科学の融合に貢献すると期待している。(著者抄録)


ペリオスチンは力学的負荷依存的骨形成に必須である
生化学 2007
Author:中川一樹; 喜井勲; 松本裕子; 西山尚志; LI Minqi; 網塚憲生; 高久田和夫; 工藤明


創傷治癒時に誘導される筋繊維芽細胞に発現するペリオスチン機能解析
再生医療,6 2007
Author:西山尚志; 島崎雅司; 喜井勲; 深山正久; 鹿島健司; 工藤明


periostinとCCN3によるNotchシグナルの制御
日本骨代謝学会学術集会プログラム抄録集,24回:171 2006(Jul.)
Author:田辺 英幸; 喜井 勲; 森山 明美; 李 敏啓; 網塚 憲生; 勝部 憲一; 工藤 明


ペリオスチンは力学的負荷依存的骨形成に必須である
日本骨代謝学会学術集会プログラム抄録集,24回:170 2006(Jul.)
Author:喜井 勲; 松本 裕子; Li Minqi; 田辺 英幸; 中川 一樹; 高山 一成; 東 由明; 高久田 和夫; 網塚 憲生; 工藤 明


肝細胞癌におけるペリオスチン発現の検討 肝細胞癌のマーカーになりうるか?
日本病理学会会誌,95(1):362 2006(Apr.)
Author:菊地 良直; 柴原 純二; 鹿島 健司; 西山 尚志; 島津 和弘; 喜井 勲; 工藤 明; 深山 正久


ファシクリンIファミリータンパク質の働き―脊椎動物に存在するペリオスチンの機能解析―
生化学,78(1):16 2006(Jan.)
Author:喜井勲; 工藤明


ヒト癌組織におけるペリオスチン発現の検討
日本癌学会総会記事,64回:380 2005(Sep.)
Author:菊地 良直; 島津 和弘; 鹿島 健司; 島崎 雅司; 喜井 勳; 柴原 純二; 仁木 利郎; 深山 正久; 工藤 明


Expression of periostin in human normal tissue and fibrous dysplasia.
JOURNAL OF BONE AND MINERAL RESEARCH,20(9):S140 2005(Sep.)
Author:T Kashima; K Shimazu; T Nishiyama; M Shimazaki; Kii, I; M Fukayama; AE Grigoriadis; A Kudo


歯の移動実験におけるペリオスチン遺伝子欠損マウスの歯根膜の組織学的変化
日本骨代謝学会学術集会プログラム抄録集,23回:229 2005(Jun.)
Author:李 敏啓; 網塚 憲生; 喜井 勲; 工藤 明; 前田 健康


骨肉腫におけるペリオスチンの発現および機能解析
日本癌学会学術総会記事,64th 2005
Author:鹿島健司; 鹿島健司; 島津和弘; 島津和弘; 西山尚志; 西山尚志; 島崎雅司; 喜井勲; 深山正久; 工藤明


ヒトABCG2タンパク質の翻訳後修飾-Cys603を介した二量体形成-
日本分子生物学会年会講演要旨集,28th 2005
Author:若林香菜子; 中川大; 足立達彦; 喜井勲; 工藤明; 小畠英理; 石川智久


Immunohistochemical localization of periostin in tooth and its surrounding tissues in mouse mandibles during development
ANATOMICAL RECORD PART A-DISCOVERIES IN MOLECULAR CELLULAR AND EVOLUTIONARY BIOLOGY,281A(2):1264 2004(Dec.)
Author:H Suzuki; N Amizuka; Kii, I; Y Kawano; K Nozawa-Inoue; A Suzuki; H Yoshie; A Kudo; T Maeda
Abstract:Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption. (C) 2004 Wiley-Liss, Inc.


Mechanical stress dependent remodeling of the periodontal ligament is defective in periostin deficient mice; Mechanotransduction through periostin protein.
JOURNAL OF BONE AND MINERAL RESEARCH,19:S21 2004(Oct.)
Author:Kii, I; N Amizuka; S Kitajima; M Li; K Takeuchi; T Maeda; J Kanno; T Inoue; Y Saga; A Kudo


Notch signaling is activated by binding of periostin or CCN3 to Notch1 in osteoblasts
JOURNAL OF BONE AND MINERAL RESEARCH,19:S275 2004(Oct.)
Author:H Tanabe; Kii, I; N Amizuka; K Katsube; A Kudo


ペリオスチンノックアウトマウスではメカニカルストレスによる歯根膜リモデリングが破綻している
日本骨代謝学会学術集会プログラム抄録集,22回:98 2004(Aug.)
Author:喜井 勲; 網塚 憲生; 李 敏啓; 竹内 亀一; 前田 健康; 北嶋 聡; 菅野 純; 井上 達; 相賀 裕美子; 工藤 明


骨芽細胞におけるperiostinとCCN3の会合とNotch signalの制御
日本骨代謝学会学術集会プログラム抄録集,22回:163 2004(Aug.)
Author:田辺 英幸; 喜井 勲; 網塚 憲生; 勝部 憲一; 工藤 明


【骨粗鬆症学 基礎・臨床研究の新しいパラダイム】骨代謝調節系 骨芽細胞の機能・骨形成メカニズム 骨形成促進因子 細胞間接着分子カドヘリン
日本臨床,62(増刊2 骨粗鬆症学):85 2004(Feb.)
Author:喜井 勲; 工藤 明


Cell-cell interaction mediated by cadherin-11 selectively enhances the mesenchymal tissue formation.
JOURNAL OF BONE AND MINERAL RESEARCH,17:S338 2002(Sep.)
Author:Kii, I; A Kudo


細胞間接着分子カドヘリンによる骨芽細胞分化誘導
日本骨代謝学会雑誌,19(2):26 2001(Jul.)
Author:喜井 勲; 工藤 明


Targeted disruption of cadherin-11 leads to an osteomalacia-like phenotype owing to a reduction in bone density.
JOURNAL OF BONE AND MINERAL RESEARCH,15:S197 2000(Sep.)
Author:J Kawaguchi; Y Azuma; K Hoshi; Kii, I; S Takeshita; T Ohta; H Ozawa; M Takeichi; O Chisaka; A Kudo


カドヘリンによる骨芽細胞分化制御
日本細胞生物学会大会講演要旨集,53回:81 2000(Aug.)
Author:喜井 勲; 川口 実太郎; 工藤 明

特許等知的財産
登録済特許
環状化合物、環状化合物の製造方法および生体分子の修飾法
環状化合物、環状化合物の製造方法および生体分子の修飾法
精神神経疾患又は悪性腫瘍に関する化合物及び医薬組成物
精神神経疾患又は悪性腫瘍に関する化合物及び医薬組成物
スクリーニング方法、タンパク質の不安定性及び/又は安定性を誘導する物質、及び、タンパク質の活性評価
AMPKのリン酸化を亢進する化合物を有効成分とする組成物