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伊藤 佐生智イトウ サオトモ

所属部署薬学研究科衛生化学分野
職名准教授
メールアドレスs-itohphar.nagoya-cu.ac.jp
ホームページURLhttp://www.phar.nagoya-cu.ac.jp/hp/esk/index.html
生年月日
Last Updated :2019/05/24

研究者基本情報

学歴

  • 名古屋市立大学

学位

  • 名古屋市立大学/博士(薬学)

研究活動情報

研究キーワード

    細菌毒素, 黄色ブドウ球菌, 免疫回避

論文

  • Staphylococcal superantigen-like 12 activates murine bone marrow derived mast cells., Kobayashi M, Kitano T, Nishiyama S, Sanjo H, Onozaki K, Taki S, Itoh S, Hida S, Biochemical and biophysical research communications,   2019年02月, 査読有り
  • Staphylococcal α-hemolysin does not induce cell damage in murine mast cells but it augments the degranulation induced by FcεRI cross-linking and ionomycin., Hayashi K, Itoh S, Morikawa A, Onozaki K, Taki S, Tsuji T, Hida S, Biochemical and biophysical research communications, 508, (1) 263 - 269,   2019年01月, 査読有り
  • Identification of the Regions Responsible for Binding to Human Immunoglobulin G in Staphylococcal Superantigen-Like Protein 10, Taichi Nishimura , Saotomo Itoh* , Kikuo Onozaki , Tsutomu Tsuji , Shigeaki Hida , BPB reports, 1, (2) 35 - 39,   2018年12月, 査読有り, Staphylococcal superantigen-like 10 (SSL10) is one of the immunoglobulin G (IgG) binding proteins produced by Staphylococcus aureus (S. aureus). SSL10 is reported to bind to Fc region of human IgG and interfere its effector functions. As SSL10 shows no homology with other staphylococcal IgG binding proteins, the mechanism of interaction between SSL10 and IgG remains to clear. In this study we attempted to identify the regions of SSL10 that are responsible for binding to human IgG (hIgG) by analyzing the binding ability of chimeras between SSL10 and its paralog, SSL7. The chimeras that retained either β1-β3 or β10-β12 of SSL10 bound to immobilized hIgG. On the other hand, chimeras that lacked both of these regions did not show binding activity to hIgG. In far western analysis, biotinylated hIgG interacted with SSL10 and chimera that retained β1-β3 and β10-β12 of SSL10. Collectively, SSL10 has two responsible regions for binding to hIgG, one is located in N-terminal half of oligonucleotide/oligosaccharide-binding (OB)-fold domain and the other is in C-terminal half of β-grasp domain. These findings would contribute to understand the mechanism of immune evasion of S. aureus and also to develop vaccines and drugs against S. aureus.
  • Identification of matrix metalloproteinase 9-interacting sequences in staphylococcal superantigen-like protein 5, Katsuhiro Kohno, Saotomo Itoh, Akari Hanai, Takemasa Takii, Toshinobu Fujiwara, Kikuo Onozaki, Tsutomu Tsuji, Shigeaki Hida, Biochemical and Biophysical Research Communications, 497, 713 - 718,   2018年03月04日, © 2018 Elsevier Inc. Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, β1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4β9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved β1-3 but not α4β9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted β1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored β1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the β1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.
  • Role of sialic acid-containing glycans of matrix metalloproteinase-9 (MMP-9) in the interaction between MMP-9 and staphylococcal superantigen-like protein 5, Chisato Kurisaka, Teruaki Oku, Saotomo Itoh, Saotomo Itoh, Tsutomu Tsuji, Microbiology and Immunology, 62, 168 - 175,   2018年03月01日, © 2018 The Societies and John Wiley & Sons Australia, Ltd Staphylococcal superantigen-like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase (MMP)-9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid-containing glycans of MMP-9, but the roles of sialic acids in the interaction between SSL5 and MMP-9 are still controversial. In the present study, we prepared recombinant glutathione S-transferase-tagged SSL5 (GST-SSL5) and analyzed its binding capacity to MMP-9 by pull-down assay after various modifications of its carbohydrate moieties. We observed that GST-SSL5 specifically bound to MMP-9 from a human monocytic leukemia cell line (THP-1 cells) and inhibited its enzymatic activity in a concentration-dependent manner. After MMP-9 was treated with neuraminidase, its binding activity towards GST-SSL5 was markedly decreased. Furthermore, recombinant MMP-9 produced by sialic acid-deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild-type CHO-K1 cells. Treatment of MMP-9 with PNGase F to remove N-glycan resulted in no significant change in the GST-SSL5/MMP-9 interaction. In contrast, the binding of GST-SSL5 to MMP-9 secreted from THP-1 cells cultured in the presence of an inhibitor for the biosynthesis of O-glycan (benzyl-GalNAc) was weaker than the binding of GST-SSL5 to MMP-9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid-containing O-glycans of MMP-9 for the interaction of MMP-9 with GST-SSL5.
  • 5-Hydroxy-2-methylpyridine isolated from cigarette smoke condensate aggravates collagen-induced arthritis in Mice, Masafumi Takeno, Shinya Kitagawa, Junpei Yamanaka, Mayumi Teramoto, Haruka Tomita, Naohiro Shirai, Saotomo Itoh, Shigeaki Hida, Kazuichi Hayakawa, Kikuo Onozaki, Takemasa Takii, Takemasa Takii, Biological and Pharmaceutical Bulletin, 41, 877 - 884,   2018年01月01日, © 2018 The Pharmaceutical Society of Japan. The risk of rheumatoid arthritis (RA) is linked to environmental and genetic factors. Cigarette smoking is an established environmental risk factor for the disease that contributes to its development and severity. Previously, we found that cigarette smoke condensate (CSC), both mainstream and sidestream, aggravates collagen type II-induced arthritis (CIA), which was observed following either intraperitoneal inoculation or nasal exposure. In the present study, we aimed to identify the compound in CSC, which aggravates CIA. By sequential fractionation and analysis, extraction with water/ether in different pH values, silica gel column chromatography, TLC, octadecyl silica (ODS) HPLC, GC/MS, and NMR, the active compound was identified as 5-hydroxy-2-methylpyridine (5H2MP). Its isomer 2-hydroxy-3-methylpyridine, but not 3-hydroxy-2-meth-ylpyridine, was also active. 5H2MP was not mutagenic, and did not exhibit aryl hydrocarbon receptor-dependent activity. Our data help clarify the mechanism underlying the pathogenic effects of cigarette smoking on RA.
  • Identification of the blood coagulation factor interacting sequences in staphylococcal superantigen-like protein 10, Saotomo Itoh, Saotomo Itoh, Takemasa Takii, Takemasa Takii, Kikuo Onozaki, Tsutomu Tsuji, Shigeaki Hida, Biochemical and Biophysical Research Communications, 485, 201 - 208,   2017年03月25日, © 2017 Elsevier Inc. Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10β1-β3 (23MEMKN ISALK HGKNN LRFKF RGIKI QVL60) bound to immobilized prothrombin, and mutants that contained SSL10β10-β12 (174SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK207) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.
  • Staphylococcal superantigen-like proteins, immune-disturbing protein family of S. Aureus, Saotomo Itoh, Seikagaku, 89, 861 - 865,   2017年01月01日
  • Early-shared Mycobacterium bovis bacillus Calmette-Guérin sub-strains induce Th1 cytokine production in vivo, Keiichi Taniguchi, Yuuji Miyatake, Daisuke Hayashi, Atsuro Takami, Saotomo Itoh, Saburo Yamamoto, Shigeaki Hida, Kikuo Onozaki, Takemasa Takii, Microbiology and Immunology, 59, 684 - 689,   2015年11月01日, © 2015 The Societies and Wiley Publishing Asia Pty Ltd. Interleukin-12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL-2 and IFN-γ. We have previously reported greater IL-12 production from macrophages infected with early-shared BCG sub-strains (ex. BCG-Japan, -Sweden) than from those infected with late-shared BCG (ex. BCG-Pasteur and -Connaught). In this study, we investigated the Th1 cytokine-inducing activity of splenocytes co-cultured with BCG-infected DCs. Early-shared BCG-infected DCs produced IL-12 and TNF-α. Furthermore, when they were co-cultured with purified protein derivative-stimulated DCs, the splenocytes of mice immunized with BCG-Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG-Connaught. In conclusion, early-shared BCG sub-strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.
  • Development of liposomal nanoconstructs targeting P-selectin (CD62P)-expressing cells by using a sulfated derivative of sialic acid, Saotomo Itoh, Kumi Kawano, Kana Takeshita, Yoshie Maitani, Tsutomu Tsuji, Pharmaceutical Research, 31, 2868 - 2875,   2014年05月, © 2014 Springer Science+Business Media New York. Purpose: NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets.Methods: The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy.Results: The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry.Conclusions: These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets.
  • Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain, Saotomo Itoh, Saotomo Itoh, Ryosuke Yokoyama, Go Kamoshida, Toshinobu Fujiwara, Hiromi Okada, Takemasa Takii, Tsutomu Tsuji, Satoshi Fujii, Hideki Hashizume, Kikuo Onozaki, Journal of Biological Chemistry, 288, 21569 - 21580,   2013年07月26日, Background: Staphylococcal superantigen-like proteins (SSLs) share structural similarity with superantigens but no superantigenic activity. Their functions remained unclear. Results: SSL10 binds to prothrombin and factor Xa via the γ-carboxyglutamic acid domain. SSL10 inhibits the penultimate step of plasma clotting. SSL10 slightly inhibits clotting by coagulase. Conclusion: SSL10 inhibits blood coagulation. Significance: This work presents a novel function of SSLs, disturbing blood coagulation. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Reactivation of immune responses against Mycobacterium tuberculosis by boosting with the CpG oligomer in aged mice primarily vaccinated with Mycobacterium bovis BCG, Keiichi Taniguchi, Takemasa Takii, Saburo Yamamoto, Jun ichi Maeyama, Sumiko Iho, Mitsuo Maruyama, Narushi Iizuka, Yuriko Ozeki, Yuriko Ozeki, Sohkichi Matsumoto, Tomohiro Hasegawa, Yuuji Miyatake, Saotomo Itoh, Kikuo Onozaki, Immunity and Ageing, 10,   2013年06月22日, Background: Mycobacterium bovis bacillus Calmette Guérin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation.Findings: The booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-γ, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H37Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50's in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4+CD44highCD62Lhigh, central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H37Rv.Conclusions: Th1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming. © 2013 Taniguchi et al.; licensee BioMed Central Ltd.
  • Staphylococcal superantigen-like protein 8 (SSL8) binds to tenascin C and inhibits tenascin C-fibronectin interaction and cell motility of keratinocytes.., Itoh S, Yamaoka N, Kamoshida G, Takii T, Tsuji T, Hayashi H, Onozaki K., Biochem Biophys Res Commun, in press,   2013年
  • Cigarette Smoke Condensate Extracts Induce IL-1-Beta Production from Rheumatoid Arthritis Patient-Derived Synoviocytes, But Not Osteoarthritis Patient-Derived Synoviocytes, Through Aryl Hydrocarbon Receptor-Dependent NF-Kappa-B Activation and Novel NF-Kap, Adachi M, Okamoto S, Chujyo S, Arakawa T, Yokoyama M, Yamada K, Hayashi A, Akita K,Takeno M, Itoh S, Takii T, Waguri-Nagaya Y, Otsuka T, Hayakawa K, Miyazawa K, Onozaki K., J Interferon Cytokine Res, in press,   2013年
  • Cytokine secretion from human monocytes potentiated by p-selectin-mediated cell adhesion., Suzuki J, Hamada E, Shodai T, Kamoshida G, Kudo S, Itoh S, Koike J, Nagata K, Irimura T, Tsuji T., Int Arch Allergy Immunol, 160, 152 - 160,   2013年
  • Staphylococcal superantigen-like protein 10 binds to phosphatidylserine and apoptotic cells, Saotomo Itoh, Saotomo Itoh, Ryosuke Yokoyama, Chizuko Murase, Takemasa Takii, Tsutomu Tsuji, Kikuo Onozaki, Microbiology and Immunology, 56, 363 - 371,   2012年06月01日, Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins that have structural similarities to staphylococcal superantigens. Although SSLs do not have superantigenic activity, some of them have been reported to bind to host immune related molecules and they have been implicated in immune evasion by S. aureus. In this study, we showed that SSL10 is capable of binding to phospholipids. SSL10 bound to phosphatidylserine (PS) containing liposome, but not to phosphatidylcholine liposome. SSL10, but not SSL7, bound to PS containing liposome, suggesting that SSL10 specifically binds to PS. Analysis of PS binding ability among recombinant truncated SSL10 fragments revealed that the β-barrel in the N-terminal oligonucleotide/oligosaccharide-binding (OB)-fold domain contributes to PS binding capacity. Fluorescein isothiocyanate labeled OB-fold of SSL10 stained hydrogen peroxide treated Jurkat cells. Annexin V is widely utilized for detection of apoptosis. Unlike annexin V, the OB-fold domain of SSL10 also bound to apoptotic cells in the presence of EDTA, suggesting that the OB-fold of SSL10 recognizes PS and apoptotic cells in a Ca 2+ independent manner. These findings suggest SSL10 and its derived peptides may be a novel detection tool for apoptotic cells. © 2012 The Societies and Blackwell Publishing Asia Pty Ltd.
  • Constitutive turnover of phosphorylation at Thr-412 of human p57/coronin-1 regulates the interaction with actin., Oku T, Nakano M, Kaneko Y, Ando Y, Kenmotsu H, Itoh S, Tsuiji M, Seyama Y, Toyoshima S, Tsuji T, J Biol Chem., 287, 42910 - 42920,   2012年
  • Involvement of transcription factor Ets-1 in the expression of the α3 integrin subunit gene., Kamoshida G, Matsuda A, Katabami K, Kato T, Mizuno H, Sekine W, Oku T, Itoh S, Tsuiji M, Hattori Y, Maitani Y, Tsuji T., FEBS J, 279, 4535 - 4546,   2012年
  • Staphylococcal superantigen-like protein 3 binds to the Toll-like receptor 2 extracellular domain and inhibits cytokine production induced by Staphylococcus aureus, cell wall component, or lipopeptides in murine macrophages., Yokoyama R, Itoh S, Kamoshida G, Takii T, Fujii S, Tsuji T, Onozaki K., Infect Immun., 80, 2816 - 2825,   2012年
  • Monocyte differentiation induced by co-culture with tumor cells involves RGD-dependent cell adhesion to extracellular matrix., Kamoshida G, Matsuda A, Sekine W, Mizuno H, Oku T, Itoh S, Irimura T, Tsutomu Tsuji T., Cancer Letter, 315, 145 - 152,   2012年
  • Etiological role of cigarette smoking in rheumatoid arthritis: Nasal exposure to cigarette smoke condensate extracts augments the development of collagen-induced arthritis in mice., Okamoto S, Adachi M, Chujo S, Yamada K, Akita K, Itoh S, Takii T, Hayakawa K, Onozaki K., Biochem Biophys Res Commun, 404, 1088 - 1092,   2011年
  • Staphylococcal superantigen-like protein 5 inhibits matrix metalloproteinase 9 from human neutrophils, Saotomo Itoh, Eri Hamada, Go Kamoshida, Kana Takeshita, Teruaki Oku, Tsutomu Tsuji, Infection and Immunity, 78, 3298 - 3305,   2010年07月01日, Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass finger-printing analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [KD] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (Ki = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl- phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
  • IL-1-induced ERK1/2 activation up-regulates p21Waf1/Cip1 protein by inhibition of degradation via ubiquitin independent pathway in human melanoma cells A375.Biochemical and biophysical research communications., Arakawa T, Hayashi H, Itoh S, Takii T, Onozaki K., Biochem Biophys Res Commun, 392, 369 - 372,   2010年
  • Staphylococcal superantigen-like protein 10 (SSL10) binds to human immunoglobulin G (IgG) and inhibits complement activation via the classical pathway., Itoh S, Hamada E, Kamoshida G, Yokoyama R, Takii T, Onozaki K, Tsuji T., Molecular Immunology., 47, 932 - 938,   2010年
  • Preventive effect of α-tocopherol and glycyrrhizin against platelet-neutrophil complex formation induced by hemodialysis membranes, Kana Takeshita, Chie Susuki, Saotomo Itoh, Tsutomu Tsuji, International Journal of Artificial Organs, 32, 282 - 290,   2009年01月01日, Background: The intradialytic activation of leukocytes is a major cause of hemodialysis (HD)-associated complications. Contact between blood and HD membranes frequently induces the formation of microaggregates composed of activated platelets and leukocytes, causing leukocyte activation that includes the generation of reactive oxygen species (ROS). This complex formation is mediated primarily by the interaction between P-selectin on activated platelets and its counter-ligands on leukocytes. Objective: We examined the preventive effects of α-tocopherol and glycyrrhizin in vitro against platelet-neutrophil microaggregate formation and neutrophil ROS production induced by HD membranes. Methods and Results: Microaggregate formation induced by the incubation of heparinized whole blood with polysulfone (PS) HD membranes was effectively inhibited by α-tocopherol and glycyrrhizin. α-Tocopherol, but not glycyrrhizin, was found to inhibit PS membrane-induced P-selectin expression on the platelet surface; however, glycyrrhizin did inhibit both the formation of neutrophil-platelet microaggregates induced by adenosine diphosphate (ADP) and the adhesion of HL60 leukemic cells to P-selectin-expressing Chinese hamster ovary (CHO) cells, suggesting that glycyrrhizin acts as a competitive inhibitor of P-selectin-mediated cell adhesion. Finally, these compounds almost completely abrogated PS membrane-induced and platelet-dependent ROS production by neutrophils. Conclusions: These results suggest that α-tocopherol and glycyrrhizin may function as preventive agents of HD-associated leukocyte activation though the modulation of platelet-leukocyte interaction. © Wichtig Editore, 2009.
  • Redistribution of P-selectin ligands on neutrophil cell membranes and the formation of platelet-neutrophil complex induced by hemodialysis membranes, Saotomo Itoh, Kana Takeshita, Chie Susuki, Kazunori Shige-eda, Tsutomu Tsuji, Biomaterials, 29, 3084 - 3090,   2008年07月01日, The formation of platelet-neutrophil microaggregates and successive activation of neutrophils are closely related to hemodialysis-associated complications. The microaggregate is mediated primarily by the interaction between P-selectin (CD62P) expressed on activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1, CD162) expressed on neutrophils. We previously reported that the clustered distribution of PSGL-1 on the cell membranes of chemokine-treated neutrophils caused upregulation of the microaggregate formation. In this study, we found that neutrophils treated with human plasma that had been incubated with hemodialysis membranes greatly enhanced the microaggregate formation. The membrane-treated plasma also induced PSGL-1 to form a cap-like cluster on the neutrophil surface. Analysis of several hemodialysis membranes with different materials indicated that the inducibility for the cap-like cluster formation of PSGL-1 parallels their ability to activate the complement system. Both the enhancement of microaggregate formation and the redistribution of PSGL-1 induced by the hemodialysis membrane-treated plasma were almost completely abrogated in the presence of a specific antagonist for the complement component C5a receptor, W-54011. These results strongly suggest that the generation of anaphylatoxin C5a through complement activation induced by hemodialysis membranes is responsible for the clustered redistribution of PSGL-1 in neutrophils leading to the increase in the platelet-neutrophil microaggregate formation. The present study indicates the importance of synergistic exacerbation of complement activation and platelet-neutrophil microaggregate formation in developing hemodialysis-associated complications. © 2008 Elsevier Ltd. All rights reserved.
  • Exclusion of actin-binding protein p57/coronin-1 from bacteria-containing phagosomes in macrophages infected with Legionella, Tsuyoshi Hayashi, Masaki Miyake, Takashi Fukui, Noriko Sugaya, Takashi Daimon, Saotomo Itoh, Teruaki Oku, Tsutomu Tsuji, Satoshi Toyoshima, Yasuyuki Imai, Biological and Pharmaceutical Bulletin, 31, 861 - 865,   2008年05月01日, Legionella pneumophila, the causative agent of Legionnaires' disease, is a human pathogen that multiplies within alveolar macrophages. L. pneumophila establishes specialized phagosomes in which it evades the host defense through largely unknown mechanisms. Here we analyzed the role of an actin-binding protein, p57/coronin-1, a member of the coronin protein family, during Legionella infection. On fluorescence microscopy, p57/coronin-1 and F-actin were found to be co-localized at the sites on the plasma membrane where L. pneumophila adhered to U937 human macrophage-like cells. The localization of p57/coronin-1 at the sites of bacterial adherence was inhibited by treatment with cytochalasin D (an inhibitor of actin polymerization), suggesting that p57/coronin-1 is involved in the actin-dependent uptake of L. pneumophila into U937 cells. In addition, we showed that p57/coronin-1 was excluded from phagosomes containing live L. pneumophila throughout the infection, whereas transient accumulation of p57/coronin-1 was observed on phagosomes containing Texas-Red-labeled opsonized zymosan (TROpZ) or heat-killed L. pneumophila at an early stage of phagocytosis. The exclusion of p57/coronin-1 from phagosomes containing live another Legionella species Legionella gratiana at an early stage of infection was also observed. Taken together, these results suggest that the endocytic pathways of live Legionella species are distinct from general phagocytic pathways, which lead to lysosomal degradation. © 2008 Pharmaceutical Society of Japan.
  • Changes in adhesive and migratory characteristics of hepatocellular carcinoma (HCC) cells induced by expression of α3β1 integrin, Hiromi Mizuno, Masaharu Ogura, Yuta Saito, Wakana Sekine, Rikio Sano, Toshie Gotou, Teruaki Oku, Saotomo Itoh, Kouji Katabami, Tsutomu Tsuji, Biochimica et Biophysica Acta - General Subjects, 1780, 564 - 570,   2008年03月01日, The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of α3β1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for α3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of α3 integrin (α3A and α3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the α3A and α3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an α3 integrin-dependent manner, indicating that transfected α3Aβ1 and α3Bβ1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of α3β1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis. © 2007 Elsevier B.V. All rights reserved.
  • Erratum: Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation (Journal of Cell Science vol. 120 (2796-2806)), Ala Eddine Deghmane, Hafid Soualhine, Horacio Bach, Khalid Sendide, Saotomo Itoh, Andrea Tam, Sanaa Noubir, Amina Talal, Raymond Lo, Satoshi Toyoshima, Yossef Av-Gay, Zakaria Hmama, Journal of Cell Science, 120,   2007年10月01日
  • Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation, Ala Eddine Deghmane, Hafid Soulhine, Horacio Bach, Khalid Sendide, Saotomo Itoh, Andrea Tam, Sanaa Noubir, Amina Talal, Raymond Lo, Satoshi Toyoshima, Yossef Av-Gay, Zakaria Hmama, Journal of Cell Science, 120, 2796 - 2806,   2007年08月15日, Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite intense studies of the mycobacterial phagosome, the mechanism of mycobacterial persistence dependent on prolonged phagosomal retention of the coat protein coronin-1 is still unclear. The present study demonstrated that several mycobacterial proteins traffic intracellularly in M. bovis BCG-infected cells and that one of them, with an apparent subunit size of Mr 50,000, actively retains coronin-1 on the phagosomal membrane. This protein was initially termed coronin-interacting protein (CIP)50 and was shown to be also expressed by M. tuberculosis but not by the non-pathogenic species M. smegmatis. Cell-free system experiments using a GST-coronin-1 construct showed that binding of CIP50 to coronin-1 required cholesterol. Thereafter, mass spectrometry sequencing identified mycobacterial lipoamide dehydrogenase C (LpdC) as a coronin-1 binding protein. M. smegmatis over-expressing Mtb LpdC protein acquired the capacity to maintain coronin-1 on the phagosomal membrane and this prolonged its survival within the macrophage. Importantly, IFNγ-induced phagolysosome fusion in cells infected with BCG resulted in the dissociation of the LpdC-coronin-1 complex by a mechanism dependent, at least in part, on IFNγ-induced LRG-47 expression. These findings provide further support for the relevance of the LpdC-coronin-1 interaction in phagosome maturation arrest.
  • Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: A role of lipid microdomains, Saotomo Itoh, Chie Susuki, Kana Takeshita, Kisaburo Nagata, Tsutomu Tsuji, Tsutomu Tsuji, Journal of Leukocyte Biology, 81, 1414 - 1421,   2007年06月01日, P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet-leukocyte and endothelium-leukocyte interactions. The treatment of neutrophils with a low concentration of IL-8 induced the redistribution of PSGL-1 to one end of the cell to form a cap-like structure. We investigated the role of lipid microdomains in the redistribution of PSGL-1 and its effect on the adhesive characteristics of IL-8-treated neutrophils. The redistribution of PSGL-1 induced by IL-8 was inhibited by cholesterol-perturbing agents such as methyl-β-cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL-1 was enriched in a low-density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X-100 was used for the solubilization, PSGL-1 was no longer recovered in the low-density fraction, although GM1 ganglioside remained in the low-density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL-1 differed from that of GM1 ganglioside, suggesting that PSGL-1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL-8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross-linking of PSGL-1 with antibodies. These results suggest that the association of PSGL-1 with lipid microdomains is essential for its redistribution induced by IL-8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P-selectin. © Society for Leukocyte Biology.
  • Platelet activation through interaction with hemodialysis membranes induces neutrophils to produce reactive oxygen species, Saotomo Itoh, Chie Susuki, Tsutomu Tsuji, Journal of Biomedical Materials Research - Part A, 77, 294 - 303,   2006年05月01日, The intradialytic activation of leukocytes is one of the major causes of hemodialysis-associated complications. During hemodialysis, the formation of microaggregates consisting of platelets and neutrophils has been observed to accompany the production of reactive oxygen species (ROS) by leukocytes. In this study, we investigated the interaction of platelets and neutrophils with hemodialysis membranes in vitro to elucidate the mechanism underlying microaggregate formation and its relevance to leukocyte activation. The production of ROS in neutrophils was induced by the coincubation of neutrophils with polysulfone (PS) membranes, and was increased when platelets were present in the neutrophil suspension. Neutrophils that were incubated with polymethylmethacrylate (PMMA) membranes in the presence of platelets also produced significant levels of ROS, suggesting that the presence of platelets augmented ROS production in neutrophils. Platelets adhered more firmly to hydrophobic membranes such as PS and PMMA membranes than to hydrophilic membranes, such as those composed of regenerated cellulose (RC) or ethylene vinylalcohol copolymer (EVAL). The adhesion of platelets to dialysis membranes composed of different materials was correlated with those membranes' ability to induce platelet activation as assessed by the cell surface expression of P-selectin. Moreover, coincubation of neutrophils with platelets that had been treated with hydrophobic membranes induced a higher level of superoxide anion relative to those treated with hydrophilic membranes in association with the P-selectin-mediated microaggregate formation. These results suggest that platelets activated through interaction with hemodialysis membranes stimulate neutrophils to produce ROS via P-selectin-mediated adhesion, and that this property of adhesion to platelets is critical for the biocompatibility of hemodialysis membranes. © 2006 Wiley Periodicals, Inc.
  • Characterization of the promoter for the α3 integrin gene in various tumor cell lines: Roles of the Ets- and Sp-family of transcription factors, Kouji Katabami, Takumi Kato, Rikio Sano, Masaharu Ogura, Hiromi Mizuno, Saotomo Itoh, Tsutomu Tsuji, Tsutomu Tsuji, Journal of Cellular Biochemistry, 97, 530 - 543,   2006年02月15日, The α3β1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for α3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5′-flanking region of the mouse α3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cel lines. However, we found that these eel lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for α3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of α3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate α3 integrin gene expression and that the contribution of each element depends on the type of tumor cells. © 2005 Wiley-Liss, Inc.
  • Transforming growth factor-β1 upregulates transcription of α3 integrin gene in hepatocellular carcinoma cells via Ets-transcription factor-binding motif in the promoter region, Kouji Katabami, Hiromi Mizuno, Rikio Sano, Yuta Saito, Masaharu Ogura, Saotomo Itoh, Tsutomu Tsuji, Tsutomu Tsuji, Clinical and Experimental Metastasis, 22, 539 - 548,   2005年11月01日, The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of α3β1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-β1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of α3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of α3β1 integrin by TGF-β1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-β1 induced the expression of α3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5′-flanking region of the mouse α3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-β1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-β1 stimulation. The nuclear proteins extracted from TGF-β1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-β1 stimulates HepG2 cells to express a higher level of α3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype. © Springer 2006.
  • Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region, Teruaki Oku, Saotomo Itoh, Rie Ishii, Kensuke Suzuki, William M. Nauseef, Satoshi Toyoshima, Tsutomu Tsuji, Biochemical Journal, 387, 325 - 331,   2005年04月15日, The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell. © 2005 Biochemical Society.
  • Acetylcholine-induced translocation of RhoA in freshly isolated single smooth muscle cells of rat bronchi, Yoshihiko Chiba, Tetsuro Uchida, Hiroyasu Sakai, Teruaki Oku, Saotomo Itoh, Tsutomu Tsuji, Miwa Misawa, Journal of Pharmacological Sciences, 95, 479 - 482,   2004年08月01日, By using immunofluorostaining and confocal laser microscopy, acetylcholine-induced translocation of RhoA was visualized in freshly isolated bronchial smooth muscle cells of the rat. The cellular distribution of RhoA at rest was observed uniformly in the cytosolic space with no staining in the nucleus, whereas acetylcholine stimulation induced a relocalization of RhoA to the cell periphery. From the results of line scans and surface plots, the peripheral to cytosolic ratio of RhoA was significantly increased by acetylcholine stimulation. Thus, the present study clearly demonstrated an acetylcholine-induced translocation of RhoA to the plasma membrane in single bronchial smooth muscle cells of the rat.
  • Inhibition of P-selectin-mediated cell adhesion by a sulfated derivative of sialic acid, Tomonori Shodai, Junsuke Suzuki, Sanae Kudo, Saotomo Itoh, Masaki Terada, Shuji Fujita, Hajime Shimazu, Tsutomu Tsuji, Biochemical and Biophysical Research Communications, 312, 787 - 793,   2003年12月19日, P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-α production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation. © 2003 Elsevier Inc. All rights reserved.
  • Heterogeneity of RNA polymerase gene (rpoB) sequences of Mycobacterium gordonae clinical isolates identified with a DNA probe kit and by conventional methods, Saotomo Itoh, Yuko Kazumi, Chiyoji Abe, Mitsuyoshi Takahashi, Mitsuyoshi Takahashi, Journal of Clinical Microbiology, 41, 1656 - 1663,   2003年04月01日, In a previous study, we have evaluated genetic identification by using the rpoB gene, which was recently introduced by Kim et al. (J. Clin. Microbiol. 39:2102-2109, 2001; J. Clin. Microbiol. 37:1714-1720, 1999). In this process, we examined the rpoB gene heterogeneity of clinical isolates identified as Mycobacterium gordonae with the conventional biological and biochemical tests and/or a commercially available DNA probe kit. Sequencing of the rpoB gene of 34 clinical isolates revealed that M. gordonae clinical isolates were classified into four major clusters (A, B, C, and D). Interestingly, organisms belonging to cluster D (15 isolates) did not hybridize with M. gordonae ATCC 14470 and specifically possessed urease activity. Therefore, it could be considered to be a novel mycobacterium. The identification of M. gordonae is known to have ambiguous results sometimes. On the other hand, identification of clinical isolates seems to be inconvenient and unsuitable because of a more than 99% 16S rRNA gene similarity value between clusters. These findings suggest that the existence of M. gordonae-like mycobacteria that share similar biochemical and biological characteristics with the 16S rRNA gene of an M. gordonae type strain but less similarity at the genomic DNA level may have complicated the identification of M. gordonae in many laboratories. Furthermore, compared with hsp65 PCR restriction analysis (PRA), rpoB PRA would have the advantage of producing no ambiguous results because of the intracluster homogeneity of the rpoB gene. In this case, rpoB would provide clearer results than hsp65, even if PRA analysis was used. We demonstrated that these M. gordonae-like mycobacteria were easily distinguished by PRA of the rpoB sequence. Additionally, the significance of this M. gordonae-like cluster may help to establish the comparison between the M. gordonae isolates from a clinical specimen and an infectious process in a given patient and to determine the true incidence of infection with this microorganism.
  • Two regions responsible for the actin binding of p57, a mammalian coronin family actin-binding protein, Teruaki Oku, Saotomo Itoh, Masamitsu Okano, Akiko Suzuki, Kensuke Suzuki, Shizuo Nakajin, Tsutomu Tsuji, William Michael Nauseef, Satoshi Toyoshima, Biological and Pharmaceutical Bulletin, 26, 409 - 416,   2003年01月01日, The actin-binding protein p57, a member of the coronin protein family, is expressed in a variety of immune cells. It has five WD repeats and a coiled-coil motif containing a leucine zipper, both of which are known to mediate protein-protein interactions. In order to identify the precise actin-binding regions in p57, and to assess the contribution of these structural motifs, we prepared various truncated p57 as fusion proteins with glutathione S-transferase (GST) and examined their actin-binding activity. A co-sedimentation assay demonstrated that p571-371(C-terminal truncated p57) had the ability to bind F-actin, but p57372-461(a fragment containing the coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p571-34) was found to bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that p571-34was co-localized with F-actin in COS-1 cells after the transfection with the p571-34construct. Deletion of10KFRHVF15, a sequence conserved among coronin-related proteins, from p571-34abolished its actin-binding activity, suggesting that this sequence with basic and hydrophobic amino acids is crucial for p57 to bind to F-actin. However, the N-terminal deletion mutant p5763-461retained the binding ability to F-actin. This result suggests the presence of a second actin-binding region. Further deletion analysis revealed that p57111-204, which includes the second and third WD repeats, also exhibited weak actin-binding activity in the co-sedimentation assay. Taken together, these data strongly suggest that at least two regions within Met-1 to Asp-34 and Ile-111 to Glu-204 of p57 are responsible for its binding to the actin cytoskeleton.
  • The role of protein kinase C in the transient association of p57, a coronin family actin-binding protein, with phagosomes, Saotomo Itoh, Kensuke Suzuki, Jun Nishihata, Mitsusada Iwasa, Teruaki Oku, Shizuo Nakajin, William Michael Nauseef, Satoshi Toyoshima, Biological and Pharmaceutical Bulletin, 25, 837 - 844,   2002年07月01日, Phagocytosis of opsonized zymosan (OpZ) particles by differentiated cells of the human leukemic cell line HL-60 induced transient periphagosomal association of p57, a coronin family actin-binding protein, and F-actin with dissociation from the phagosomes after ingestion was completed. Coincident with OpZ ingestion, p57 phosphorylation increased transiently and peaked with its dissociation from phagosomes. Since p57 contains several putative sites for protein kinase C (PKC) phosphorylation, we examined the effect of PKC on p57 phosphorylation and association with the phagosome. Purified p57 was phosphorylated in vitro by PKC isoforms α and δ, and PMA, an activator of PKC, induced p57 phosphorylation in HL-60 cells. Furthermore, chelerythrine, a specific PKC inhibitor, blocked p57 phosphorylation and the dissociation of p57 and F-actin from phagosomes, whereas wortmannin, genistein, and H-89 did not. Chelerythrine also inhibited the translocation of LAMP-1, a marker protein of lysosomes, to the OpZ-containing phagosomes, indicating that PKC-mediated phosphorylation is required for phagosome-lysosome fusion. Taken together, these data suggest that PKC-mediated phosphorylation of p57 triggers its dissociation from phagosomes, an event that may be necessary for the fusion of phagosomes with lysosomes.
  • Evidence of autophosphorylation in Txk: Y91 is an autophosphorylation site, Jun Ichi Kashiwakura, Noboru Suzuki, Mitsuhiro Takeno, Saotomo Itoh, Teruaki Oku, Tsuyoshi Sakane, Shizuo Nakajin, Satoshi Toyoshima, Biological and Pharmaceutical Bulletin, 25, 718 - 721,   2002年06月01日, We have previously shown that Txk, a member of Tec family tyrosine kinase, is expressed in Th1 and Th0 cells and directly contributes to gene transcription of Th1-related proteins, including interferon (IFN)-γ, through nuclear translocation in response to mitogenic stimuli. Btk, another member of Tec family tyrosine kinase, has been shown to have a Src family tyrosine kinase-dependent transphosphorylation site and an autophosphorylation site. However, little is known about the phosphorylation mechanism of Txk, except that 420 tyrosine residue was identified as the transphosphorylation site. In this study, we found that Txk autophosphorylated itself by using an in vitro kinase assay. To elucidate the role of phosphorylation in Txk function, we studied IFN-γ secretion by Jurkat T cells expressing mutant Txk proteins. While transfection with the wild-type Txk resulted in increased IFN-γ production, the function was abrogated by disruption of the ATP biding site, which is presumably involved in the autophosphorylation mechanism. The results suggest that phosphorylated Txk is an active form to promote IFN-γ synthesis. The 91 tyrosine residue of Txk is deduced to be an autophosphorylation site by comparing its structure with Btk. In Jurkat cells transfected with Txk Y91A, IFN-γ production was decreased in comparison with the wild-type Txk transfected Jurkat cells. These data suggest that phosphorylation of the 91 tyrosine residue in Txk plays a positive regulatory role in Txk function.
  • CHOP, a basic leucine zipper transcriptional factor, contributes to the antiproliferative effect of IL-1 on A375 human melanoma cells through augmenting transcription of IL-6, T. Hattori, S. Itoh, H. Hayashi, T. Chiba, T. Takii, K. Yoshizaki, K. Onozaki, Journal of Interferon and Cytokine Research, 21, 323 - 332,   2001年08月20日, Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.
  • Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375, Saotomo Itoh, Takayuki Hattori, Hidetoshi Hayashi, Yukiko Mizutani, Makoto Todo, Takemasa Takii, De Yang, John C. Lee, Senya Matsufuji, Yasuko Murakami, Taku Chiba, Kikuo Onozaki, Kikuo Onozaki, Journal of Immunology, 162, 7434 - 7440,   1999年06月15日, The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375- C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up- regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down- regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.
  • Acquired resistance to the anti-proliferative effect of interleukin-1 and interleukin-6 is a recessive phenotype in A375 human melanoma cells, S. Itoh, H. Hayashi, D. Yang, T. Takii, K. Onozaki, Melanoma Research, 7, 455 - 462,   1997年12月01日, The proliferation of human melanoma cell line A375-6 is inhibited by several cytokines, including interleukin-1 (IL-1) and interleukin-6 (IL-6). However, during a long period of culture, the cells progressively acquire resistance to IL-1 irrespective of functional IL-1 receptor expression. These cells constitutively produce IL-1α and IL-6, and also acquire resistance to IL-6. In order to investigate the mechanism of the acquired resistance to these cytokines, we performed somatic cell hybridization experiments. Parental cells for the construction of hybrid cells were rendered G418- or hygromycin B-resistant by transfection with expression vectors containing drug-resistant genes. Hybridization was conducted using IL-1-resistant subclones A375-R8 and R19 and an IL-1 highly sensitive clone C 2-1, which was originally resistant but became sensitive to IL-1 upon transfection with a human type I IL-1 receptor (IL-1R) expression plasmid. Cells produced by hybridization of resistant cells and C 2-1 cells appeared to be sensitive to IL-1 and IL-6. In contrast, production of IL-1 was augmented in the hybrid cells. These results suggest that resistance to IL-1 and IL-6 is a recessive phenotype, while production of IL-1 is dominant in melanoma cells.
  • Resistance to IL-1 anti-proliferative effect, accompanied by characteristics of advanced melanoma, permits invasion of human melanoma cells in vitro, but not metastasis in the nude mouse, Hidetoshi Hayashi, Reiko Shimizu, Kaori Fujii, Saotomo Itoh, De Yang, Kikuo Onozaki, International Journal of Cancer, 71, 416 - 421,   1997年06月09日, We reported earlier that IL-1 inhibits the growth of human melanoma cells (A375-6), and that these cells become resistant to IL-1 after prolonged periods of culture. The resistant cells constitutively produce IL-α and IL- 6 with IL-6 production was induced by endogenous IL-1 in an autocrine manner. The cells are also resistant to IL-6 anti-proliferative effects. In the present study, we show that the resistant clones exhibited up-regulated expression of intercellular-adhesion molecule I (ICAM-1) and vitronectin receptor (integrin α(v)β 3) when compared with the IL-1-sensitive clone, A375-6. Moreover, these IL-1-resistant clones exhibited many other metastatic characteristics, such as expression of IL-8 mRNA, production of matrix metalloproteinases (MMP-2 and MMP-9), and augmented invasion activity. However, contrary to our expectations, the IL-1-resistant cells did not exhibit experimental metastasis in a nude-mouse model, similarly to the IL- 1-sensitive parental A375-6 cell line. In contrast, the highly metastatic clone A375-SM exhibited α(v)β 3 expression at a level comparable to that of the IL-1-resistant cells, but expressed low or no ICAM-1, metalloproteinase and displayed little in vitro invasion activity. These results show that the metastatic characteristics of IL-1-resistant cells are not sufficient to produce metastasis in vivo and suggest that these resistant clones may provide a good model system for characterizing the molecular mechanisms of metastasis.
  • Interleukin 1 (IL-1) production is not essential for acquired resistance of human A375 melanoma cells to anti-proliferative effect of IL-1, Saotomo Itoh, Hidetoshi Hayashi, Naoko Watanabe, Yoshiro Kobayashi, Takemasa Takii, Kikuo Onozaki, International Journal of Cancer, 65, 805 - 811,   1996年03月15日, The proliferation of human melanoma cell line A375-6 is inhibited by interteukin I (IL-1). However, the cells acquired resistance to IL-1 after a long period of culture. We have reported that 2 resistant subclones, A375-R8 and -R19, produced IL-1α constitutively and that IL-1 induced IL-6 production in an autocrine manner. Therefore, we supposed that IL-1α production renders the cells resistant to IL-1. To investigate the relationship between IL-1α production and IL-1 resistance, we transfected the IL-1α expression plasmid to the IL-1-sensitive clone, A375-6, and the anti-sense mRNA expression plasmid to IL-1-resistant cells, A375-R8 and -R19. A37S-6MS, a transfectant of mature IL-1α expression plasmid, expressed IL-1α mRNA and produced IL-1 activity at a level comparable to the resistant cells. The transfectant also produced IL-6 and exhibited augmented expression of Mn-SOD mRNA. However, IL-1 sensitivity of this transfectant was not affected. With respect to sensitivity to anti-proliferative effects of other cytokines, such as IL-6 and TNFα, there was no difference between the transfectant and parent cells. Although A375-R8PH10 and -R19PH10, transfectants of IL-1α anti-sense mRNA expression plasmid, exhibited a decrease in the level of IL-1 production, their IL-1 sensitivity did not differ from parent cells. These results, therefore, suggest that IL-1α duction is not essential or sufficient for the acquisition of resistance to the anti-proliferative effect of IL-1. © 1996 Wiley-Liss, Inc.
  • Molecular cloning of human antizyme cDNA, De Yang, Takemasa Takii, Hidetoshi Hayashi, Saotomo Itoh, Masaru Hayashi, Kikuo Onozaki, Biochemistry and Molecular Biology International, 38, 957 - 964,   1996年01月01日, We have cloned the cDNA encoding the human ornithine decarboxylase antizyme from a 5'-stretch cDNA library of human B-cell lymphoma Daudi. The cloned human antizyme cDNA fragment consists of 1063 bp, has 80% homology to the rat antizyme cDNA, but shows almost no homology to the E. coli antizyme gene. Northern hybridization analysis shows that this gene is expressed in a number of human cell lines with an estimated mRNA transcript size of about 1.1 kb. The size of the mRNA suggests that the cloned cDNA fragment probably represents the full length of humam antizyme mRNA transcript. Comparison of the human and rat antizymes demonstrates that they are highly conserved at both nucleotide and peptide levels.

特許

  • ワクチンアジュバント及び免疫増強剤, 瀧井 猛将, 小野嵜 菊夫, 山中 淳平, 伊藤 佐生智, 竹野 聖史, 北川 慎也, 特願2014-140033, 特開2016-017044
  • 抗凝固薬及びその用途, 伊藤 佐生智, 瀧井 猛将, 小野嵜 菊夫, 横山 領介, 辻 勉, 鴨志田 剛, 特願2011-090473, 特開2012-224549

競争的資金

  • 黄色ブドウ球菌毒素SSLの免疫かく乱作用に着目した感染予防・治療法の確立と創薬, 伊藤 佐生智
  • 創薬に指向した黄色ブドウ球菌免疫かく乱タンパク質ファミリーSSLの機能解析, 星薬科大学→名古屋市立大学, 伊藤 佐生智
  • がんの浸潤・転移の素過程におけるインテグリン-マトリックス相互作用の解析, 星薬科大学, 辻 勉
  • 血液細胞間クロストークに着目した長期透析合併症の予防法の確立, 星薬科大学, 伊藤 佐生智
  • 結核菌の細胞内寄生性を標的にした新規結核治療法の開発, 星薬科大学, 伊藤 佐生智


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