Advance

MASUDA Seiji

    Department of Food Science and Nutrition　Professor
Last Updated :2024/04/25

Researcher Information

Degree

  • Doctor of Agriculture（Kyoto University）

URL

Research funding number

  • 20260614

ORCID ID

J-Global ID

Research Interests

  • 食品分子生物学   食品生化学   mRNA核外輸送   選択的mRNAスプライシング   mRNA品質管理   processing   タンパク質生産   細胞工学   mRNA   Molecular Biology   

Research Areas

  • Life sciences / Applied biochemistry
  • Life sciences / Applied molecular and cellular biology
  • Life sciences / Cell biology
  • Life sciences / Molecular biology

Academic & Professional Experience

  • 2021/04 - Today  Kindai UniversityFaculty of Agriculture Department of Food Science and Nutrition教授
  • 2007/04 - 2021/03  京都大学大学院生命科学研究科准教授
  • 1999/04 - 2007/03  京都大学大学院生命科学研究科助教授
  • 2001/07 - 2003/09  Harvard Universityメディカルスクールポスドク研究員
  • 1998/04 - 1999/03  京都大学大学院農学研究科助手
  • 1993/10 - 1998/03  Kyoto UniversityFaculty of Agriculture助手
  • 1993/04 - 1993/09  日本学術振興会特別研究員

Education

  • 1993/03 - Today  京都大学博士（農学）
  • 1990/04 - 1993/03  Kyoto University  Graduate School of Agriculture  食品工学科 博士後期課程
  • 1988/04 - 1990/03  Kyoto University  Graduate School of Agriculture  食品工学専攻 博士前期課程
  • 1984/04 - 1988/03  Kyoto University  Faculty of Agriculture  食品工学科

Association Memberships

  • 日本栄養食糧学会   日本動物細胞工学会   日本分子生物学会   日本農芸化学会   日本生化学会   

Published Papers

  • Ken-ichi Fujita; Tomohiro Yamazaki; Akila Mayeda; Seiji Masuda
    Biochemical and Biophysical Research Communications Elsevier BV 703 149682 - 149682 0006-291X 2024/02 [Refereed]
     
    UAP56 and URH49 are closely related RNA helicases that function in selective mRNA processing and export pathways to fine-tune gene expression through distinct complex formations. The complex formation of UAP56 and URH49 is believed to play a crucial role in regulating target mRNAs. However, the mechanisms underlying this complex formation have not been fully elucidated. Here we identified the regions essential for the complex formation of both helicases. The terminal regions of UAP56 and the C-terminal region of URH49 were indispensable for their respective complex formation. Further analysis revealed that a specific amino acid at the C-terminus of UAP56 is critical for its complex formation. Alanine substitution of this amino acid impairs its complex formation and subsequently affected its mRNA processing and export activity. Our study provides a deeper understanding of the basis for the complex formation between UAP56 and URH49.
  • Seiji Masuda; Ken-ichi Fujita; Misa Ito; Midori Irie; Kotaro Harada; Naoko Fujiwara; Yuya Ikeda; Hanae Yoshioka; Tomohiro Yamazaki; Masaki Kojima; Bunzo Mikami; Akila Mayeda
    Nature communications Research Square Platform LLC 15 (1) 455 - 455 2024/01 [Refereed]
     
    Abstract Messenger RNA export is a regulated pathway that control gene expression and specific physiological events. In humans, closely related RNA helicases, UAP56 and URH49 shape selective mRNA export pathways through distinct apo-complex formation and remodeling to similar ATP-complex to achieve precise gene regulation. The difference in apo-complex is the key to functional divergence. However, the profile of the apo-complex formed by URH49 (named apo-AREX complex) and why UAP56 and URH49 exhibit distinct complex formation remain unknown. Here, we investigated unidentified apo-AREX components. RUVBL1, RUVBL2, ILF2, ILF3, and HNRNPM were physically and functionally associated with URH49, indicating the key components of the apo-AREX complex. Integrating analysis of crystal structure and complex formation of UAP56/URH49 chimera mutants demonstrated that their structural features contribute to respective complex formation. This study provides insights into the specific function of two helicases and into how two helicases diverged from a single ancestral gene, Sub2.
  • Shuntaro Miyake; Seiji Masuda
    RNA Biology Informa UK Limited 19 (1) 1244 - 1255 1547-6286 2022/11 [Refereed]
  • Naoko Fujiwara; Maki Shigemoto; Mizuki Hirayama; Ken-ichi Fujita; Shigeto Seno; Hideo Matsuda; Masami Nagahama; Seiji Masuda
    Nucleic Acids Research Oxford University Press (OUP) 50 (15) 8779 - 8806 0305-1048 2022/07 [Refereed]
     
    Abstract Recent in vitro reconstitution analyses have proven that the physical interaction between the exosome core and MTR4 helicase, which promotes the exosome activity, is maintained by either MPP6 or RRP6. However, knowledge regarding the function of MPP6 with respect to in vivo exosome activity remains scarce. Here, we demonstrate a facilitative function of MPP6 that composes a specific part of MTR4-dependent substrate decay by the human exosome. Using RNA polymerase II-transcribed poly(A)+ substrate accumulation as an indicator of a perturbed exosome, we found functional redundancy between RRP6 and MPP6 in the decay of these poly(A)+ transcripts. MTR4 binding to the exosome core via MPP6 was essential for MPP6 to exert its redundancy with RRP6. However, at least for the decay of our identified exosome substrates, MTR4 recruitment by MPP6 was not functionally equivalent to recruitment by RRP6. Genome-wide classification of substrates based on their sensitivity to each exosome component revealed that MPP6 deals with a specific range of substrates and highlights the importance of MTR4 for their decay. Considering recent findings of competitive binding to the exosome between auxiliary complexes, our results suggest that the MPP6-incorporated MTR4-exosome complex is one of the multiple alternative complexes rather than the prevailing one.
  • Yasutaka Yamanaka; Takaki Ishizuka; Ken-ichi Fujita; Naoko Fujiwara; Masashi Kurata; Seiji Masuda
    International Journal of Molecular Sciences MDPI AG 23 (5) 2555  2022/02 [Refereed]
     
    Calcium homeostasis endoplasmic reticulum protein (CHERP) is colocalized with the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or perinuclear region, and has been involved in intracellular calcium signaling. Structurally, CHERP carries the nuclear localization signal and arginine/serine-dipeptide repeats, like domain, and interacts with the spliceosome. However, the exact function of CHERP in the nucleus remains unknown. Here, we showed that poly(A)+ RNAs accumulated in the nucleus of CHERP-depleted U2OS cells. Our global analysis revealed that CHERP regulated alternative mRNA splicing events by interaction with U2 small nuclear ribonucleoproteins (U2 snRNPs) and U2 snRNP-related proteins. Among the five alternative splicing patterns analyzed, intron retention was the most frequently observed event. This was in accordance with the accumulation of poly(A)+ RNAs in the nucleus. Furthermore, intron retention and cassette exon choices were influenced by the strength of the 5′ or 3′ splice site, the branch point site, GC content, and intron length. In addition, CHERP depletion induced anomalies in the cell cycle progression into the M phase, and abnormal cell division. These results suggested that CHERP is involved in the regulation of alternative splicing.
  • Analyses of putative anti-cancer potential of three STAT3 signaling inhibitory compounds derived from Salvia officinalis.
    Maho Yanagimichi; Katsutoshi Nishino; Akiho Sakamoto; Ryusei Kurodai; Kenji Kojima; Nozomu Eto; Hiroko Isoda; Riadh Ksouri; Kazuhiro Irie; Taiho Kambe; Seiji Masuda; Toru Akita; Kazuhiro Maejima; Masaya Nagao
    Biochemistry and Biophysics Reports 2020/12 [Refereed]
  • Ken-Ichi Fujita; Takaki Ishizuka; Mizuki Mitsukawa; Masashi Kurata; Seiji Masuda
    International journal of molecular sciences 21 (6) 2020/03 [Refereed]
     
    Human transcriptomes are more divergent than genes and contribute to the sophistication of life. This divergence is derived from various isoforms arising from alternative splicing. In addition, alternative splicing regulated by spliceosomal factors and RNA structures, such as the RNA G-quadruplex, is important not only for isoform diversity but also for regulating gene expression. Therefore, abnormal splicing leads to serious diseases such as cancer and neurodegenerative disorders. In the first part of this review, we describe the regulation of divergent transcriptomes using alternative mRNA splicing. In the second part, we present the relationship between the disruption of splicing and diseases. Recently, various compounds with splicing inhibitor activity were established. These splicing inhibitors are recognized as a biological tool to investigate the molecular mechanism of splicing and as a potential therapeutic agent for cancer treatment. Food-derived compounds with similar functions were found and are expected to exhibit anticancer effects. In the final part, we describe the compounds that modulate the messenger RNA (mRNA) splicing process and their availability for basic research and future clinical potential.
  • Ken-Ichi Fujita; Tomohiro Yamazaki; Kotaro Harada; Shigeto Seno; Hideo Matsuda; Seiji Masuda
    Biochimica et biophysica acta. Gene regulatory mechanisms in press (2) 194480 - 194480 2020/01 [Refereed]
     
    The TREX complex integrates information from nuclear mRNA processing events to ensure the timely export of mRNA to the cytoplasm. In humans, UAP56 and its paralog URH49 form distinct complexes, the TREX complex and the AREX complex, respectively, which cooperatively regulate the expression of a specific set of mRNA species on a genome wide scale. The difference in the complex formation between UAP56 and URH49 are thought to play a critical role in the regulation of target mRNAs. To date, the underlying mechanism remains poorly understood. Here we characterize the formation of the TREX complex and the AREX complex. In the ATP depleted condition, UAP56 formed an Apo-TREX complex containing the THO subcomplex but not ALYREF and CIP29. URH49 formed an Apo-AREX complex containing CIP29 but not ALYREF and the THO subcomplex. However, with the addition of ATP, both the Apo-TREX complex and the Apo-AREX complex were remodeled to highly similar ATP-TREX complex containing the THO subcomplex, ALYREF and CIP29. The knockdown of URH49 caused a reduction in its target mRNAs and a cytokinesis failure. Similarly, cytokinesis abnormality was observed in CIP29 knockdown cells, suggesting that CIP29 belongs to the URH49 regulated mRNA export pathway. Lastly, we confirmed that the export of mRNA in URH49-dependent pathway is achieved by NXF1, which is also observed in UAP56-dependent pathway. Our studies propose an mRNA export model that the mRNA selectivity depends on the Apo-form TREX/AREX complex, which is remodeled to the highly similar ATP-form complex upon ATP loading, and integrated to NXF1.
  • Masashi Kurata; Naoko Fujiwara; Ken-Ichi Fujita; Yasutaka Yamanaka; Shigeto Seno; Hisato Kobayashi; Yusaku Miyamae; Nobuyuki Takahashi; Yasuyuki Shibuya; Seiji Masuda
    iScience 22 336 - 352 2019/12 [Refereed]
     
    Cancer cells often exhibit extreme sensitivity to splicing inhibitors. We identified food-derived flavonoids, apigenin and luteolin, as compounds that modulate mRNA splicing at the genome-wide level, followed by proliferation inhibition. They bind to mRNA splicing-related proteins to induce a widespread change of splicing patterns in treated cells. Their inhibitory activity on splicing is relatively moderate, and introns with weak splice sites tend to be sensitive to them. Such introns remain unspliced, and the resulting intron-containing mRNAs are retained in the nucleus, resulting in the nuclear accumulation of poly(A)+ RNAs in these flavonoid-treated cells. Tumorigenic cells are more susceptible to these flavonoids than nontumorigenic cells, both for the nuclear poly(A)+ RNA-accumulating phenotype and cell viability. This study illustrates the possible mechanism of these flavonoids to suppress tumor progression in vivo that were demonstrated by previous studies and provides the potential of daily intake of moderate splicing inhibitors to prevent cancer development.
  • Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1.
    MASUDA SeijiNishino K; Uesugi H; Hirasawa A; Ohtera A; Miyamae Y; Neffati M; Isoda H; Kambe T; Masuda S; Irie K; Nagao M
    Biochem Biophys Res Commun. S0006-291X (doi: 10.1016/j.bbrc.2019.11.03) 32157-6 - Epub ahead of print 2019/11 [Refereed]
  • Masumi Okamura; Yasutaka Yamanaka; Maki Shigemoto; Yuya Kitadani; Yuhko Kobayashi; Taiho Kambe; Masaya Nagao; Issei Kobayashi; Katsuzumi Okumura; Seiji Masuda
    PLoS ONE 14 (7) 2019/07 [Refereed]
     
    © 2019 Okamura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. There are errors in Fig 1, “Nuclear accumulation of poly (A)+ RNA and deficiency of cell growth by knock-down of DBP5, GLE1 and IPPK,” panel E and caption. Please see the correct Fig 1 and correct caption here. (Figure presented).
  • Masumi Okamura; Yasutaka Yamanaka; Maki Shigemoto; Yuya Kitadani; Yuhko Kobayashi; Taiho Kambe; Masaya Nagao; Issei Kobayashi; Katsuzumi Okumura; Seiji Masuda
    PLoS ONE Public Library of Science 13 (5) e0197165  1932-6203 2018/05 [Refereed]
     
    DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knockdown of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism.
  • Y. Mori; F. Okazaki; C. Inuo; Y. Yamaguchi; S. Masuda; S. Sugiura; T. Fukuie; M. Nagao; I. Tsuge; T. Yosikawa; A. Yagami; K. Matsunaga; T. Fujisawa; K. Ito; H. Narita; Y. Kondo; Fruits Allergy Component Study Group, Japan
    Allergologia et Immunopathologia Elsevier Doyma 46 482 - 490 1578-1267 2018 [Refereed]
     
    Background: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. Methods: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). Results: Peach-allergic patients (n = 27) were diagnosed and categorized into oral reaction (n = 10) or systemic reaction (n = 17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P < 0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P < 0.05). Conclusions: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.
  • Inhibition of mRNA processing activity from ginger-, clove- and cinnamon-extract, and by two ginger constituents, 6-gingerol and 6-shogaol.
    Morimoto M; Mitsukawa M; Fujiwara C; Kawamura Y; Masuda S
    Bioscience. Biotechnology. Biochem. 83 498 - 501 2018 [Refereed]
  • Masashi Kurata; Yuki Murata; Keiko Momma; Intisar Fouad Ali Mursi; Masakazu Takahashi; Yusaku Miyamae; Taiho Kambe; Masaya Nagao; Hiroshi Narita; Yasuyuki Shibuya; Seiji Masuda
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 81 (3) 551 - 554 0916-8451 2017/03 [Refereed]
     
    Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.
  • Inhibition of mRNA Maturation by Compounds Which Have a Flavonoid Skeleton,
    Kurata M; Morimoto M; Kawamura Y; Mursi I FA; Momma K; Takahashi M; Miyamae Y; Kambe T; Nagao N; Narita H; Shibuya Y; Masuda, S
    Biochem. Mol. Biol 2 46 - 53 2017 [Refereed]
  • Shigeyuki Fujimoto; Tokuji Tsuji; Takashi Fujiwara; Taka-aki Takeda; Chengfeng Merriman; Ayako Fukunaka; Yukina Nishito; Dax Fu; Eitan Hoch; Israel Sekler; Kazuhisa Fukue; Yusaku Miyamae; Seiji Masuda; Masaya Nagao; Taiho Kambe
    BIOCHEMICAL JOURNAL PORTLAND PRESS LTD 473 2611 - 2621 0264-6021 2016/09 [Refereed]
     
    Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.
  • Yusaku Miyamae; Yukina Nishito; Naomi Nakai; Yoko Nagumo; Takeo Usui; Seiji Masuda; Taiho Kambe; Masaya Nagao
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 477 (1) 40 - 46 0006-291X 2016/08 [Refereed]
     
    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. (C) 2016 Elsevier Inc. All rights reserved.
  • Anna Ohtera; Yusaku Miyamae; Kotaro Yoshida; Kazuhiro Maejima; Toru Akita; Akira Kakizuka; Kazuhiro Irie; Seiji Masuda; Taiho Kambe; Masaya Nagao
    ACS CHEMICAL BIOLOGY AMER CHEMICAL SOC 10 (12) 2794 - 2804 1554-8929 2015/12 [Refereed]
     
    Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a ligand-activated transcription factor that plays an important role in adipogenesis and glucose metabolism. The ligand-binding pocket (LBP) of PPAR gamma has a large Y-shaped cavity with multiple subpockets where multiple ligands can simultaneously bind and cooperatively activate PPAR gamma. Focusing no, this unique property of the PPAR gamma LBP, we describe e novel two-step cell-based strategy to develop PPAR gamma ligands, first, a combination of ligands that cooperatively. activates PPAR gamma was identified using a luciferase reporter assay. Second, hybrid ligands were designed and synthesized. For proof of concept, we focused on covalent agonists, which activate PPAR gamma through a unique activation mechanism regulated by a covalent linkage with the Cys285 residue in the PPAR gamma LBP. Despite their biological significance and pharmacological potential, few covalent PPAR gamma,agonists are known except for endogenous fatty acid metabolites, With our strategy, we determined that plant-derived cinnamic acid derivatives cooperatively activated PPAR gamma by combining with GW9662, an irreversible antagonist, GW9662 covalently reacts with the Cys285 residue. A docking study predicted that a cinnamic acid derivative can bind to the open cavity in GW9052-bound PPAR gamma LBP., On the basis of the putative binding mode; strut-totes of both ligands were linked successfully to create a potent PPAR gamma agonist; which enhanced the, transactivation potential, of PPAR gamma at submicromolar levels through covalent modification, Of Cys285. Out approach could lead to the discovery of novel high-potency PPAR gamma agonists.
  • Ayako Hashimoto; Katsuma Ohkura; Masakazu Takahashi; Kumiko Kizu; Hiroshi Narita; Shuichi Enomoto; Yusaku Miyamae; Seiji Masuda; Masaya Nagao; Kazuhiro Irie; Hajime Ohigashi; Glen K. Andrews; Taiho Kambe
    BIOCHEMICAL JOURNAL PORTLAND PRESS LTD 472 183 - 193 0264-6021 2015/12 [Refereed]
     
    Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.
  • Yoshiko Yasuda; Mitsugu Fujita; Eiji Koike; Koshiro Obata; Mitsuru Shiota; Yasushi Kotani; Terunaga Musha; Sachiyo Tsuji-Kawahara; Takao Satou; Seiji Masuda; Junko Okano; Harufumi Yamasaki; Katsumi Okumoto; Tadao Uesugi; Shinichi Nakao; Hiroshi Hoshiai; Masaki Mandai
    PLOS ONE PUBLIC LIBRARY SCIENCE 10 (4) e0122458  1932-6203 2015/04 [Refereed]
     
    The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed epsilon,gamma, and a globins as well as myoglobin (Mb) to produce tetrameric alpha 2 epsilon 2 and alpha 2 gamma 2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1 alpha expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire e,. and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.
  • Masumi Okamura; Haruko Inose; Seiji Masuda
    GENES MDPI AG 6 (1) 124 - 149 2073-4425 2015/03 [Refereed]
     
    In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC.
  • Gene Regulation through mRNA Expression.
    Inose H; Mukai K; Ito M; Masuda S
    Adv. Biol. Chem 5 45 - 57 2015 [Refereed]
  • Identification of 6-Octadecynoic acid from Marrubium vulgare L. as a PPARγ agonist
    Anna,Ohtera; Yusaku,Miyamae; Naomi,Nakai; Atsushi,Kawauchi; Kawata,Kiyokazu; Junkyu,Hann; Hiroko,Isoda; Mohamed,Neffati; Kazuhiro,Maejima; Toru,Akita; Naoki,Mori; Kazuhiro,Irie; Taiho,Kambe; Seiji,Masuda; Masaya,Nagao
    Book of proceedings Tunisian-Japanese Symposium on Society, Science and Technology (TJASSST 2013) Session I: Life Science, Food and Agriculture Ibaraki Insatsu Co., Ltd 90 - 93 2014/03
  • Shigeyuki Fujimoto; Naoya Itsumura; Tokuji Tsuji; Yasumi Anan; Natsuko Tsuji; Yasumitsu Ogra; Tomoki Kimura; Yusaku Miyamae; Seiji Masuda; Masaya Nagao; Taiho Kambe
    PLoS ONE 8 (10) e77445  1932-6203 2013/10 [Refereed]
     
    The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1-/-MT-/-ZnT4-/- cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1-/-MT-/-ZnT4-/- cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1-/-MT-/-ZnT4-/- cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled. © 2013 Fujimoto et al.
  • Anna Ohtera; Yusaku Miyamae; Naomi Nakai; Atsushi Kawachi; Kiyokazu Kawada; Junkyu Han; Hiroko Isoda; Mohamed Neffati; Toru Akita; Kazuhiro Maejima; Seiji Masuda; Taiho Kambe; Naoki Mori; Kazuhiro Irie; Masaya Nagao
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 440 (2) 204 - 209 0006-291X 2013/10 [Refereed]
     
    6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor gamma (PPAR gamma). Fibrogenesis caused by hepatic stellate cells is inhibited by PPAR gamma whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L, were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPAR gamma agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPAR gamma agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA.), which both have a triple bond but in different positions, activated PPAR gamma in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPAR gamma-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPAR gamma agonists. (C) 2013 Elsevier Inc. All rights reserved.
  • Abdalla Akef; Hui Zhang; Seiji Masuda; Alexander F. Palazzo
    NUCLEUS-AUSTIN LANDES BIOSCIENCE 4 (4) 326 - 340 1949-1034 2013/07 [Refereed]
     
    In vertebrates, the majority of mRNAs that encode secreted, membrane-bound or mitochondrial proteins contain RNA elements that activate an alternative m RNA nuclear export (ALR.EX) pathway. Here we demonstrate that mRNAs containing ALREX-promoting elements are trafficked through nuclear speckles. Although ALREX-promoting elements enhance nuclear speckle localization, additional features within the m RNA largely drive this process. Depletion of two TREX-associated RNA helicases, UAP56 and its paralog URH49, or inhibition of the TREX-associated nuclear transport factor, TAP, not only inhibits ALREX but also appears to trap these mRNAs in nuclear speckles. mRNAs that contain ALREX- promoting elements associate with UAP56 in vivo. Finally, we demonstrate that mRNAs lacking a poly(A)-tail are not efficiently exported by the ALREX pathway and show enhanced association with nuclear speckles. Our data suggest that within the speckle, ALREX-promoting elements, in conjunction with the poly(A)-tail, likely stimulate UAP56/URH49 and TAP dependent steps that lead to the eventual egress of the export-competent rriFINP from these structures.
  • Ken-ichi Fujita; Masumi Okamura; Sachiko Nishimoto; Tomoya Kurihara; Keiko Momma; Yusaku Miyamae; Taiho Kambe; Masaya Nagao; Hiroshi Narita; Seiji Masuda
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 76 (6) 1248 - 1251 0916-8451 2012/06 [Refereed]
     
    A number of proteins complete mRNA processing in the nucleus, thus, inhibitor of mRNA processing is worth finding to analyze the mechanism of mRNA maturation in detail. Here, we established a monitoring system for mRNA processing using a test compound, spliceostatin A (SSA), which inhibits mRNA splicing. This system should serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing.
  • 山崎 智弘; 増田 誠司
    化学と生物 Japan Society for Bioscience, Biotechnology, and Agrochemistry 50 (1) 9 - 11 0453-073X 2012/01
  • Fukunaka Ayako; Kurokawa Yayoi; Teranishi Fumie; Sekler Israel; Oda Kimimitsu, Ackla; M Leigh; Faundez Victor; Hiromura Makoto; Masuda Seiji; Nagao Masaya; Enomoto Shuichi; Kambe Taiho
    J Biol Chem 286 (18) 16363 - 16373 1083-351X 2011/05 
    A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein sta
  • Yuki Aihara; Naoko Fujiwara; Tomohiro Yamazaki; Taiho Kambe; Masay Nagao; Yutaka Hirose; Seiji Masuda
    JOURNAL OF BIOTECHNOLOGY ELSEVIER SCIENCE BV 153 (3-4) 86 - 91 0168-1656 2011/05 [Refereed]
     
    Recent research into mRNA maturation processes in the nucleus has identified a number of proteins involved in mRNA transcription, capping, splicing, end processing and export. Among them, the Tap-p15 heterodimer acts as an mRNA export receptor. Tap-p15 is recruited onto fully processed mRNA in the nucleus, which is ready for export to the cytoplasm, through associating with Aly or SR proteins on mRNA, or by directly associating with a constitutive transport element (CTE), an RNA element derived from type D retroviruses. mRNA containing a CTE is exported to the cytoplasm by directly associating with Tap-p15, even in the absence of Tap-recruiting proteins such as Aly or SR proteins on the mRNA. Here, we showed that the use of a CTE enhanced the expression of recombinant protein in human cell lines. The co-expression of reporter proteins and Tap-p15 also enhanced recombinant protein expression. Moreover, the use of a CTE and Tap-p15 synergistically further enhanced the recombinant protein expression. In addition to Tap-p15, several Tap-p15-recruiting proteins, including Aly and SR proteins, enhanced recombinant protein expression, albeit independently of the CTE. The incorporation of a CTE and Tap-p15-recruiting proteins into protein expression system is useful to increase recombinant protein yield in human cells. (C) 2011 Elsevier B.V. All rights reserved.
  • Ayako Fukunaka; Yayoi Kurokawa; Fumie Teranishi; Israel Sekler; Kimimitsu Oda; M. Leigh Ackland; Victor Faundez; Makoto Hiromura; Seiji Masuda; Masaya Nagao; Shuichi Enomoto; Taiho Kambe
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 286 (18) 16363 - 16373 0021-9258 2011/05 [Refereed]
     
    A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase ( TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo- form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.
  • Satoshi Abe; Ryuzo Sasaki; Seiji Masuda
    CYTOTECHNOLOGY SPRINGER 63 (2) 101 - 109 0920-9069 2011/03 [Refereed]
     
    Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.
  • Constitutively active soluble form of erythropoietin receptor suppresses growth and angiogenesis of xenografts of transfected cancer cell lines.
    Yasuda, Y; Maeda, Y; Hara, S; Tanaka, M; Koike, E; Watanabe, Y; Masuda, S; Yamasaki, H; Konishi, H; Horiuchi, Y; Hoshiai, H
    J. Cancer Therapy 2, 40-53 2 40 - 53 2011 [Refereed]
  • Tomohiro Yamazaki; Naoko Fujiwara; Hiroko Yukinaga; Miki Ebisuya; Takuya Shiki; Tomoya Kurihara; Noriyuki Kioka; Taiho Kambe; Masaya Nagao; Eisuke Nishida; Seiji Masuda
    MOLECULAR BIOLOGY OF THE CELL AMER SOC CELL BIOLOGY 21 (16) 2953 - 2965 1059-1524 2010/08 [Refereed]
     
    Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49-CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.
  • Naoko Fujiwara; Mayu Yoshikawa; Tomohiro Yamazaki; Taiho Kambe; Masaya Nagao; Seiji Masuda
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 74 (7) 1512 - 1516 0916-8451 2010/07 [Refereed]
     
    Screening of mRNA export factors in Saccharomyces cerevisiae and Drosophila melanogaster has identified a number of mRNA processing factors involved in multiple mRNA processing steps. However, only limited information is available on human cells. Here we established a screening system searching for mRNA processing factors in human cells by combining the luciferase reporter system and fluorescence in situ hybridization, which evaluates the nuclear/cytoplasmic distribution of bulk poly(A)(+) RNA. This system makes it possible to search for the compounds affecting mRNA processing from the various resources.
  • Yoshiko Yasuda; Yasuhiro Maeda; Eiji Koike; Yoh Watanabe; Seiji Masuda; Harufumi Yamasaki; Katsumi Okumoto; Yoshitaka Horiuchi; Hiroshi Hoshiai
    RECENT ADVANCES IN CLINICAL MEDICINE WORLD SCIENTIFIC AND ENGINEERING ACAD AND SOC 337 - + 1790-5125 2010 [Refereed]
     
    Erythropoietin (Epo) therapy for combatting anemia or fatigue in cancer patients has become a controversial issue. We have previously reported our study of 24 malignant human cell lines which express Epo and its receptor (EpoR) mRNAs and secrete Epo protein; blockade of Epo-signal destroyed the xenografts of female malignancies [1] and cancer cell lines [2]. We speculated that the conflicting clinical outcomes are due to the individual responsiveness to Epo of the various cancers. We measured the expression levels of Epo and EpoR mRNAs and the amount of Epo protein secreted and demonstrated the presence of EpoR protein in these 24 cell lines, some of which had anoxia-inducible Epo and/or EpoR mRNA. Additionally in seven selected cell lines with known amounts of Epo and EpoR expression, rhEpo triggered and the EpoR antagonist (EMP9) inhibited tyrosine phosphorylation of STAT5. They showed a rhEpo-induced growth that corresponded generally to the level of the constitutive activation of tyrosine phosphorylation of STAT5. Further, EMP9-suppressed growth depended inversely on the amount of Epo secretion. These data justify our speculation that the growth of very many cancers is promoted by their own Epo-signal which may or may not be accelerated by exogenous rhEpo.
  • Ayako Fukunaka; Tomoyuki Suzuki; Yayoi Kurokawa; Tomohiro Yamazaki; Naoko Fujiwara; Kaori Ishihara; Hitoshi Migaki; Katsuzumi Okumura; Seiji Masuda; Yuko Yamaguchi-Iwai; Masaya Nagao; Taiho Kambe
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 284 (45) 30798 - 30806 0021-9258 2009/11 [Refereed]
     
    The majority of CDF/ZnT zinc transporters form homooligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.
  • DEAD-box型RNAヘリカーゼUAP56およびURH49による遺伝子発現制御を介した染色体分配機構における異なる役割
    山崎 智弘; 藤原 奈央子; 栗原 朋也; 幸長 弘子; 戎家 美紀; 西田 栄介; 木岡 紀幸; 神戸 大朋; 永尾 雅哉; 増田 誠司
    日本生化学会大会プログラム・講演要旨集 (公社)日本生化学会 82回 3T18a - 2 2009/09
  • Wataru Matsuura; Tomohiro Yamazaki; Yuko Yamaguchi-Iwai; Seiji Masuda; Masaya Nagao; Glen K. Andrews; Taiho Kambe
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 73 (5) 1142 - 1148 0916-8451 2009/05 [Refereed]
     
    The SLC39A family of zinc transporters can be divided into four subfamilies (I, II, LIV-1, and gufA) in vertebrates, but studies of their functions have been restricted exclusively to members of subfamilies 11 and LIV-1. In this study, we characterized SLC39A9 (ZIP9), the only member of subfamily I in vertebrates. Confocal microscopy demonstrated that transiently expressed, HA-tagged human ZIP9 (hZIP9-HA) was localized to the trans-Golgi network regardless of zinc status. Disruption of the ZIP9 gene in DT40 cells did not change the growth rate, sensitivity to high zinc and manganese concentrations during long-term culture, or cellular zinc status after short-term incubation with zinc. The alkaline phosphatase activity of ZIP9(-/-) cells did not change in cells cultured in medium containing normal zinc levels. In contrast, the activity of this enzyme decreased in wild-type cells cultured in zinc deficient medium but less so in ZIP9(-/-) cells under these conditions. Stable over-expression of hZIP9-HA moderately decreased alkaline phophatase activity. These results suggest that ZIP9 functions to regulate zinc homeostasis in the secretory pathway without significantly altering cytosolic zinc homeostasis.
  • Fumito Tani; Michiko Ohno; Yuichi Furukawa; Masami Sakamoto; Seiji Masuda; Naofumi Kitabatake
    MOLECULAR IMMUNOLOGY PERGAMON-ELSEVIER SCIENCE LTD 46 (7) 1326 - 1339 0161-5890 2009/04 [Refereed]
     
    Surface expression of Hsp70 members has been previously reported on human tumor cell lines. Here we examined how the inducible mouse Hsp72 can be expressed on the surface of two types of murine tumor cell lines in response to non-lethal heat shock. Exposure to 42 degrees C for 2 h led to the intracellular production of Hsp72 for both murine LL/2 lung carcinoma and B16 melanoma cells. Flow cytometric analyses showed that living LL/2 carcinoma, but not B16 melanoma, transported a fraction of inducible Hsp72 to the cell-surface membrane. Induction of the surface expression of Hsp72 occurred upon non-lethal heat shock only when Hsp72 expression was forced to be elevated in B16 transfectants. Hsp72 expressed on the LL/2 cell surface was detected by the monoclonal antibody that recognized the epitope of 504-617 amino acid residues, but not by another antibody with the epitope of 122-264 residues. When we analyzed the binding of recombinant full-length Hsp72 to mouse splenocytes, significant binding was observed for innate immune cells such as CD11b(+)-, CD11c(+)-, or NK1.1(+)-cells. The recombinant variants obtained by truncation of the C-terminal helical region of Hsp72 exhibited more robust binding to these innate immune cells in a similar fashion, however, further deletion offered less binding to those immunocytes. Two fragment variants lacking the N-terminal nucleotide-binding domain were found to extensively bind to peritoneal macrophages. Taken together with these results, it thus follows that the sentinels in an innate immune system, macrophages, dendritic cells and NK cells, can be involved in the surveillance of functionally aberrant cells through the recognition of a specific C-terminal structure of Hsp70 as a danger signal in living cells. (C) 2008 Elsevier Ltd. All rights reserved.
  • Fukunaka A; Suzuki T; Kurokawa Y; Yamazaki T; Fujiwara N; Ishihara K; Migaki H; Okumura K; Masuda S; Yamaguchi-Iwai Y; Nagao M; Kambe T
    J. Biol. Chem. 284 30798 - 30806 2009 [Refereed]
  • D Watanabe; K Suzuma; S Matsui; M Kurimoto; J Kiryu; M Kita; Suzuma, I; H Ohashi; T Ojima; T Murakami; T Kobayashi; S Masuda; M Nagao; N Yoshimura; H Takagi
    NEW ENGLAND JOURNAL OF MEDICINE MASSACHUSETTS MEDICAL SOC 353 (8) 782 - 792 0028-4793 2005/08 [Refereed]
     
    Background: Although vascular endothelial growth factor (VEGF) is a primary mediator of retinal angiogenesis, VEGF inhibition alone is insufficient to prevent retinal neovascularization. Hence, it is postulated that there are other potent ischemia-induced angiogenic factors. Erythropoietin possesses angiogenic activity, but its potential role in ocular angiogenesis is not established. Methods: We measured both erythropoietin and VEGF levels in the vitreous fluid of 144 patients with the use of radioimmunoassay and enzyme-linked immunosorbent assay. Vitreous proliferative potential was measured according to the growth of retinal endothelial cells in vitro and with soluble erythropoietin receptor. In addition, a murine model of ischemia-induced retinal neovascularization was used to evaluate erythropoietin expression and regulation in vivo. Results: The median vitreous erythropoietin level in 73 patients with proliferative diabetic retinopathy was significantly higher than that in 71 patients without diabetes (464.0 vs. 36.5 mIU per milliliter, P<0.001). The median VEGF level in patients with retinopathy was also significantly higher than that in patients without diabetes (345.0 vs. 3.9 pg per milliliter, P<0.001). Multivariate logistic-regression analyses indicated that erythropoietin and VEGF were independently associated with proliferative diabetic retinopathy and that erythropoietin was more strongly associated with the presence of proliferative diabetic retinopathy than was VEGF. Erythropoietin and VEGF gene-expression levels are up-regulated in the murine ischemic retina, and the blockade of erythropoietin inhibits retinal neovascularization in vivo and endothelial-cell proliferation in the vitreous of patients with diabetic retinopathy in vitro. Conclusions: Our data suggest that erythropoietin is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy.
  • S Masuda; R Das; H Cheng; E Hurt; N Dorman; R Reed
    GENES & DEVELOPMENT COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT 19 (13) 1512 - 1517 0890-9369 2005/07 [Refereed]
     
    In yeast, the TREX complex contains the THO transcription elongation complex, which functions in direct co-transcriptional recruitment of the mRNA export proteins Sub2 and Yra1 to nascent transcripts. Here we report the identification of the human THO complex and show that it associates with spliced mRNA, but not with unspliced pre-mRNA in vitro. Transcription is not required for this recruitment. We also show that the human THO complex colocalizes with splicing factors in nuclear speckle domains in vivo. Considering that splicing occurs cotranscriptionally in humans, our data indicate that recruitment of the human TREX complex to spliced mRNA is not directly coupled to transcription, but is instead coupled to transcription indirectly through splicing.
  • M Maruyama; M Kishimoto; K Ishida; Y Watanabe; M Nishikawa; S Masuda; R Sasaki; Y Takakura
    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS ELSEVIER SCIENCE INC 438 (2) 174 - 181 0003-9861 2005/06 [Refereed]
     
    It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts. (c) 2005 Elsevier Inc. All rights reserved.
  • A Ogawa; S Terada; N Sakuragawa; S Masuda; M Nagao; M Miki
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING SOC BIOSCIENCE BIOENGINEERING JAPAN 96 (5) 448 - 453 1389-1723 2003/11 [Refereed]
     
    Human amniotic epithelial (HAE) cells have great potential for successful use in cell therapy, since they do not cause acute rejection upon allotransplantation. However, to date, HAE cells have not well been studied. We previously reported that HAE cells produce erythropoietin (EPO), which is known to be a regulator of hematopoiesis, and that the induction mechanism of HAE cells is unknown, although EPO production from HAE cells is not increased by hypoxia which induces several cell types to produce EPO. In this study, we determined whether female sex hormones, including progesterone and 17beta-estradiol, affect the EPO production of HAE cells. Bioactive measurement of EPO activity in the culture supernatants of HAE-SV40 cells, which were immortalized by transfection with a simian virus 40 large T antigen, revealed that EPO bioactivity was significantly increased by treatment with progesterone, but not 17beta-estradiol. Treatment of HAE-SV40 cells with progesterone transiently increased the EPO mRNA level by fivefold, while there was no change in response to 17beta-estradiol. Furthermore, the progesterone receptor (PR)-B was detected in both HAE cells and HAE-SV40 cells by Western blotting. These results suggest that EPO synthesis in HAE-SV40 cells is stimulated by progesterone, but not by 17beta-estradiol, and thus it is highly likely that the EPO synthesis of HAE cells is also regulated by progesterone.
  • Y Yasuda; Y Fujita; S Masuda; T Musha; K Ueda; H Tanaka; H Fujita; T Matsuo; M Nagao; R Sasaki; Y Nakamura
    CARCINOGENESIS OXFORD UNIV PRESS 23 (11) 1797 - 1805 0143-3334 2002/11 [Refereed]
     
    The accumulating evidence that erythropoietin and erythropoietin receptor are expressed in various non-haematopoietic organs suggests that erythropoietin signalling might be involved in the growth of tumours, but this possibility has never been examined. We found that mRNAs for erythropoietin and erythropoietin receptor are expressed in malignant tumours of female reproductive organs, where erythropoietin levels are higher than in normal tissues. Furthermore, tumour cells and capillary endothelium showed erythropoietin receptor immunoreactivity. To investigate the role of the erythropoietin/erythropoietin receptor pathway in these tumours, we injected mouse monoclonal antibody against erythropoietin or the soluble form of erythropoietin receptor into blocks of tumour specimens and cultured the blocks. After 12 h of injections, these blocks were examined and compared with control blocks injected with mouse monoclonal antibody, heat denatured soluble form of erythropoietin receptor, mouse serum or saline. Tumour cells and capillaries were markedly decreased in a dose-dependent manner after either injection. A marked increase of the cells containing fragmented DNA and the histopathological characteristics of these cells suggest that the decrease in tumour cells and capillary endothelial cells was due to apoptotic cell death. The co-existence of JAK2 and phosphorylated-JAK2, and STAT5 and phosphorylated STAT5, all of which are involved in the mitogenic signalling of erythropoietin, was found frequently in tumour cells and capillary endothelial cells in the untreated blocks. In contrast, most of the phosphorylated-JAK2- or phosphorylated-STAT5-positive cells had disappeared in the experimental blocks. Moreover, reduced tyrosine phosphorylation of STAT5 in the experimental blocks was confirmed by western blotting analysis. The results strongly indicate that erythropoietin signalling contributes to the growth and/or survival of both transformed cells and capillary endothelial cells in these tumours. Thus, deprivation of erythropoietin signalling may be a useful therapy for erythropoietin-producing malignant tumours.
  • Y. Yasuda; M. Okano; M. Nagao; S. Masuda; Y. Fujita; R. Sasaki
    Anatomical science international / Japanese Association of Anatomists Springer Science and Business Media LLC 77 (1) 58 - 63 0022-7722 2002 [Refereed]
  • S Masuda; SK Moon; T Kambe; M Nagao; R Sasaki
    ANIMAL CELL TECHNOLOGY : BASIC & APPLIED ASPECTS, VOL 12 SPRINGER 139 - 143 2002 [Refereed]
     
    Oxygen supply is one of the major problems in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of human therapeutic recombinant proteins can be maintained or enhanced under low oxygen concentrations. To overcome the reduced productivity of recombinant protein in a low oxygen concentration, we have developed a new recombinant protein production system using hypoxia-response enhancer. We have prepared a permanent CHO cell lines producing recombinant human Epo under control of LDHA promoter/HRE. Epo production was highly hypoxia-inducible. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells cultured in 21% and 2% oxygen. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even under low oxygen concentrations.
  • M Nagao; S Masuda; M Chikuma-Esaki; T Kobayashi; R Sasaki
    ANIMAL CELL TECHNOLOGY : BASIC & APPLIED ASPECTS, VOL 12 SPRINGER 325 - 329 2002 [Refereed]
     
    Erythropoietin (Epo) produced by the kidney regulates erythropoiesis, brain EPO prevents neuron death, and uterine Epo stimulates estrogen (E-2)-dependent uterine angiogenesis. To characterize the tissue-specific regulation of Epo gene expression, ovariectomized mice were given E-2 and/or exposed to hypoxia and the temporal patterns of stumuli-induced changes in Epo mRNA levels were examined. Epo mRNA levels in the kidney, cerebrum, and cerebellum were elevated markedly within 4 hr after exposure to hypoxia. While the elevated level of Epo mRNA in the kidney decreased markedly within 8 hr despite continuous hypoxia, the high level in the cerebrum was sustained, indicating that down-regulation operates in the kidney but not in the brain. E-2 transiently induced Epo mRNA in the uterus but not in the kidney, cerebrum and cerebellum. Interestingly, the uterine Epo mRNA was hypoxia-inducible only in the presence of E-2. Thus Epo expression appears to be regulated in a tissue-specific manner, endorsing the tissue-specific functions of Epo.
  • Masuda S; Sträßer K; Mason P; Pfannstiel J; Oppizzi M; Rodriguez-Navarro S; Rondón AG; Aguilera A; Struhl K; Reed R; Hurt E (The; first three authors contributed equally to this work
    Nature 417 304 - 308 2002 [Refereed]
  • MASUDA Seiji
    Nippon Nōgeikagaku Kaishi Japan Society for Bioscience, Biotechnology, and Agrochemistry 75 (10) 1067 - 1075 0002-1407 2001/10
  • R Sasaki; S Masuda; M Nagao
    NEWS IN PHYSIOLOGICAL SCIENCES NEWS IN PHYSIOLOGICAL SCIENCES 16 (3) 110 - 113 0886-1714 2001/06 [Refereed]
     
    Erythropoietin (EPO) is produced in the brain, uterus, and oviduct. Brain EPO plays a neuroprotective role, and uterine EPO is likely involved in estrogen-dependent angiogenesis. Hypoxic induction of brain EPO markedly differs from that in the kidney. EPO in the uterus and oviduct is estrogen inducible.
  • Y. Yasuda; T. Musha; H. Tanaka; Y. Fujita; H. Fujita; H. Utsumi; T. Matsuo; S. Masuda; M. Nagao; R. Sasaki; Y. Nakamura
    British Journal of Cancer 84 (6) 836 - 843 0007-0920 2001/03 [Refereed]
     
    We have recently shown that malignant tumours from the ovary and uterus expressed erythropoietin (Epo) and its receptor (EpoR), and that deprivation of Epo signal in tumour blocks induced death of malignant cells and capillary endothelial cells in vitro (Yasuda et al, submitted). These in vitro results prompted us to examine the effect of Epo-signal withdrawal on tumours in vivo. RT-PCR analysis demonstrated the expression of mRNAs for Epo and EpoR in the transplants of uterine and ovarian tumours in nude mice. Then we injected locally anti-Epo antibody or soluble form of EpoR into the transplants. At 12 h, 1, 7 or 14 days after the injection, all transplants were resected and examined macro- and microscopically. Tumour size was reduced in Epo signal-deprived transplants. Immunohistochemical examinations revealed destruction of Epo-responding malignant and capillary endothelial cells through apoptotic death. The degree of tumour regression correlated well with the dose and frequency of the injections. Control xenografts with saline injection or needle insertion showed well-developed tumour masses. This Epo response pathway will have profound implications for our understanding of the development and progression of malignant tumours and for the use of Epo-signal deprivation as an effective therapy. © 2001 Cancer Research Campaign.
  • GP Chen; Y Ito; S Masuda; R Sasaki
    CYTOTECHNOLOGY KLUWER ACADEMIC PUBL 35 (1) 3 - 8 0920-9069 2001/01 [Refereed]
     
    Chinese hamster ovary (CHO) cells were transfected with both genes encoding erythropoietin (Epo) and epidermal growth factor receptor (EGFR). The transfection of the Epo gene was confirmed by an enzyme-linked immunoassay. Overexpression of EGFR was confirmed by Western blotting of EGFR. The transfected CHO cells were cultured in serum-free medium in the presence of soluble epidermal growth factor (EGF) or immobilized EGF. The CHO cells overexpressing EGFR grew in the presence of less EGF than the cells not overexpressing EGFR. In addition, the growth of EGFR-overexpressing CHO cells was enhanced in the presence of immobilized EGF more efficiently than in the presence of soluble EGF. The amount of Epo secreted from the cells increased linearly with the increase of growth rate. Consequently, culture of CHO cells coexpressing Epo and EGFR on EGF-immobilized matrix was the most efficient for Epo production.
  • M Chikuma; S Masuda; T Kobayashi; M Nagao; R Sasaki
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM AMER PHYSIOLOGICAL SOC 279 (6) E1242 - E1248 0193-1849 2000/12 [Refereed]
     
    Erythropoietin (Epo) produced by the kidney regulates erythropoiesis. Recent evidence suggests that Epo in the cerebrum prevents neuron death and Epo in the uterus induces estrogen (E-2)-dependent uterine angiogenesis. To elucidate how Epo expression is regulated in these tissues, ovariectomized mice were given E-2 and/or exposed to hypoxia, and the temporal patterns of Epo mRNA levels were examined. Epo mRNA levels in the kidney and cerebrum were elevated markedly within 4 h after exposure to hypoxia. Although the elevated level of Epo mRNA in the kidney decreased markedly within 8 h despite continuous hypoxia, the high level in the cerebrum was sustained for greater than or equal to 24 h, indicating that downregulation operates in the kidney but not in the brain. E-2 transiently induced Epo mRNA in the uterus but not in the kidney and cerebrum. Interestingly, the uterine Epo mRNA was hypoxia inducible only in the presence of E-2. Thus Epo expression appears to be regulated in a tissue-specific manner, endorsing the tissue-specific functions of Epo.
  • R Sasaki; S Masuda; M Nagao
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 64 (9) 1775 - 1793 0916-8451 2000/09 [Refereed]
     
    Erythropoietin (Epo), which is produced by the kidney in the adult and by the liver in the fetus, increases red blood cells by supporting the survival of erythroid progenitor cells and stimulating their differentiation and proliferation via binding to Epo receptor (EpoR). The main signal in the control of Epo production is oxygen; hypoxia stimulates Epo production through activation of Epo gene transcription. Tremendous progress in our understanding of molecular mechanisms of Epo action on erythroid cells and regulation of the Epo production has been made by manipulation of cDNAs and genes of Epo and EpoR. Studies on hypoxic induction of Epo gene transcription led to the identification of hypoxia-inductible factor (HIF-1), a transcriptional factor, that functions as a global regulator of hypoxic gene expression. Paracrine Epo/EpoR systems that are independent of the endocrine erythropoietic system (kidney/bone marrow) have been found in the central nervous system and uterus. Novel functions of Epo at these local sites and tissue-specific regulation of Epo production including a newly found potent regulator (estrogen) have been proposed. The tissue-specific regulation rationalizes the specific functions of Epo produced by individual tissues.
  • S Masuda; T Kobayashi; M Chikuma; M Nagao; R Sasaki
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM AMER PHYSIOLOGICAL SOC 278 (6) E1038 - E1044 0193-1849 2000/06 [Refereed]
     
    Previously, we showed that erythropoietin (Epo) is produced in the mouse uterus, where Epo is indispensable for estrogen (E-2)-dependent angiogenesis. Expression of uterine Epo mRNA is stimulated by E-2 and hypoxia. The hypoxic induction requires the presence of E-2. In the present study, we examined other female reproductive organs in the mouse with respect to Epo mRNA expression and its stimuli (E-2 and hypoxia)-induced changes. Although Epo mRNA expression was seen in the ovary and oviduct, the E-2-induced stimulation of Epo mRNA was found only in the oviduct. The E-2-induced stimulation in the oviduct was transient and rapidly downregulated. Epo mRNA expression in the oviduct was hypoxia inducible, in both the presence and the absence of E-2. E-2-dependent production of Epo and its mRNA expression were also found by use of cultured oviducts. The E-2 action is probably mediated through the E-2 receptor, and de novo protein synthesis is not required for E-2 induction of Epo mRNA. In the oviduct, the ampulla and isthmus regions produce Epo.
  • K Fujisawa; K Yagasaki; R Funabiki; S Masuda; R Sasaki
    NUTRITION RESEARCH PERGAMON-ELSEVIER SCIENCE LTD 20 (5) 685 - 693 0271-5317 2000/05 [Refereed]
     
    Erythropoietin (EPO) is a glycoprotein that stimulates red blood cell production and the kidney is the major source of EPO production. We examined the effects on serum EPO concentration of a low (8.5%) casein diet (8.5C), nephritis induction, and supplementation of the limiting amino acids, 0.3% L-methionine and 0.36% L-threonine, to the low casein diet (8.5CMT), using a 20% casein diet (20C) as the control. Nephritis was induced in rats by an intravenous injection of anti-rat kidney glomerular basement membrane rabbit antiserum. Serum EPO concentration decreased when normal rats were fed the 8.5C, and this decrease was quickly restored when refed the 20C or 8.5CMT. Serum EPO concentration was significantly lower in 20C-fed nephritic rats than in 20C-fed normal rats. In the nephritic state as well as the normal state, serum EPO concentration was significantly decreased in the 8.5C-fed animals compared with the 20C-fed ones. This fall in serum EPO was restored to the concentration of 20C-fed rats by 8.5CMT feeding. The 8.5CMT also reduced proteinuria and hypercholesterolemia in nephritis without the severe growth retardation. These results suggest that the dietary manipulation of 8.5CMT has a diversity of beneficial effects on nephritis. (C) 2000 Elsevier Science Inc.
  • S Masuda; SK Moon; T Kambe; M Nagao; R Sasaki
    BIOTECHNOLOGY AND BIOENGINEERING JOHN WILEY & SONS INC 67 (2) 157 - 164 0006-3592 2000/01 [Refereed]
     
    Oxygen supply is one of the major problems in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a-high-productivity of human therapeutic recombinant proteins can be maintained or enhanced under law oxygen concentrations. A number of hypoxia-inducible genes have been found in animal cells and the induction in most: cases is due to hypoxic activation of the gene transcription. A consensus sequence (HRE = hypoxia-response enhancer) responsible for the hypoxic activation exists in these genes and the binding of a protein,which is widely distributed in animal cells, to this sequence responding to hypoxia activates the promoter activity. The promoter of lactate dehydrogenase A gene is: active in Chinese hamster ovary (CHO) cells and the vicinal HRE stimulates the promoter activity efficiently in hypoxia; We have prepared a number of permanent CHO cell lines producing recombinant human erythropoietin (Epo) under control of this promoter/HRE. Epo production was highly hypoxia-inducible when the wild-type of HRE was used but uninducible when the mutant HRE was used. There was little difference in the in vitro and in vivo activities,:and glycosylation between Epo produced by the cells cultured in 21% and 2% oxygen. Furthermore, forced expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel:System:wh ich guarantees a high productivity even uner low oxygen concentrations. (C) 2000 John Wiley & Sons, Inc.
  • Y Nakamura; H Ohigashi; S Masuda; A Murakami; Y Morimitsu; Y Kawamoto; T Osawa; M Imagawa; K Uchida
    CANCER RESEARCH AMER ASSOC CANCER RESEARCH 60 (2) 219 - 225 0008-5472 2000/01 [Refereed]
     
    Here we report the molecular mechanism underlying the induction of glutathione S-transferase (GST) in rat liver epithelial RL34 cells treated with a cancer chemopreventive isothiocyanate compound, benzylisothiocyanate (BITC), BITC was found to significantly induce GST activity in RL34 cells. Northern and Western blot analyses demonstrated that BITC specifically enhanced the production of the class pi GST isozyme (GSTP1), Our studies demonstrated for the first time that the addition of BITC to the cells resulted in an immediate increase in the reactive oxygen intermediates (ROIs) detected by a fluorescence probe, 2',7'-dichlorofluorescin diacetate. The level of the ROIs in the cells treated with BITC (10 mu M) was similar to 50-fold higher than those in the control cells. Furthermore, glutathione depletion by diethyl maleate significantly enhanced BITC-induced ROI production and accelerated the BITC-induced elevation of the GST activity, whereas pretreatment of the cells with glutathione inhibited both the ROI production and GST induction. The structure-activity relationship of the isothiocyanates also indicated that the ROI-producing activities closely correlated with their GST-inducing potencies. Moreover, the GSTP1 enhancer I-containing region was found to be essential for induction of the GSTP1 gene by intracellular ROI inducers such as BITC and diethyl maleate. These data suggest the involvement of the redox regulation on the induction of GSTP1 by BITC.
  • S. Masuda; M. Nagao; R. Sasaki
    International Journal of Hematology 70 (1) 1 - 6 0925-5710 1999/07 [Refereed]
  • S Masuda; M Nagao; R Sasaki
    ANIMAL CELL TECHNOLOGY: CHALLENGES FOR THE 21ST CENTURY SPRINGER 249 - 253 1999 
    Erythropoietin (EPO) has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report EPO protects neurons against ischemia-induced cell death and the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from nitric oxide-induced death.
  • S Masuda; E Kada; M Nagao; R Sasaki
    CYTOTECHNOLOGY SPRINGER 29 (3) 207 - 213 0920-9069 1999 [Refereed]
     
    In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in some other properties between Epo produced by these astrocyte cell lines and that by CHO cells. Digestion of Epo with glycosidases indicated that there was a little difference in glycosylation of Epo produced by two types of the cells.
  • T Iguchi; S Sogo; H Hisha; S Taketani; Y Adachi; R Miyazaki; H Ogata; S Masuda; R Sasaki; M Ito; S Fukuhara; S Ikehara
    STEM CELLS ALPHAMED PRESS 17 (2) 82 - 91 1066-5099 1999 [Refereed]
     
    Hepatocyte growth factor (HGF) is a multifunctional cytokine with early hematopoiesis-stimulatory activity. Here, we focus on its erythropoiesis-stimulatory effect on highly purified human hematopoietic progenitor cells (CD34(+)/CD45(+) cells) derived from the cord blood. In immunoblot analyses, c-met protein (a receptor of HGF) was detected in the CD34(+)/CD45(+) cells, although the expression levels were different among samples. The c-met expression was facilitated by incubation of the cells with stem cell factor (SCF) or interleukin 3 (IL-3), even if the expression level had been low, IL-6, G-CSF, or erythropoietin (EPO) did not show such a stimulatory effect on the c-met expression of the cells. When HGF was added to the CD34(+)/CD45(+) cells in the presence of SCF, the numbers of CD36(+)/CD11b(-) cells (very early erythroid lineage cells) and BFU-E increased. EPO-dependent tyrosine phosphorylation of Stat 5 also increased, but the EPO receptor (EPO-R) expression remained unchanged in the CD34(+)/CD45(+) cells treated with SCF + HGF, Our present study suggests that stimulation of the HGF/c-met signal is concomitant with induction of c-met protein by SCF, The subsequent enhancement of signal transduction via the activation of Stat 5 from the EPO-R plays a crucial role in the commitment of hematopoietic stem cells into erythroid lineage cells.
  • S Masuda; E Kada; M Nagao; R Sasaki
    CYTOTECHNOLOGY SPRINGER 31 (1-2) 177 - 182 0920-9069 1999 [Refereed]
     
    In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in some other properties between Epo produced by these astrocyte cell lines and that by CHO cells.
  • HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells.
    Iguchi T; Sogo S; Hisha H; Taketani S; Adachi Y; Miyazaki R; Ogata H; Masuda S; Sasaki R; Ito M; Fukuhara S; Ikehara S
    Stem Cells 17 82 - 91 1999 [Refereed]
  • Y Sadamoto; K Igase; M Sakanaka; K Sato; H Otsuka; S Sakaki; S Masuda; R Sasaki
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC 253 (1) 26 - 32 0006-291X 1998/12 [Refereed]
     
    Erythropoietin (EPO) prevents the ischemia-induced delayed neuronal death in the hippocampal CAI field in gerbils. EPO receptor (EPOR) is also expressed in the cerebral cortex but its function is not known. To examine whether EPO has a neuroprotective action in the cortex, EPO was infused into the cerebroventricles of stroke-prone spontaneously hypertensive rats with permanent occlusion of the left middle cerebral artery. Morris water maze test indicated that EPO infusion alleviated the ischemia-induced place navigation disability. The left (ischemic)-to-right (contralateral nonischemic) (L/R) ratio of cerebrocortical area in the EPO-infused ischemic group was larger than that in the vehicle-infused ischemic group. The occlusion caused secondary thalamic degeneration but infusion of EPO prevented the decrease in the L/R ratio of thalamic area and supported neuron survival in the ventroposterior thalamic nucleus. In situ hybridization indicated that EPOR mRNA was upregulated in the periphery (ischemic penumbra) of a cerebrocortical infarct after occlusion of the middle cerebral artery, suggesting that an increased number of EPOR in neurons facilitates the EPO signal transmission, thereby preventing the damaged area from enlarging. (C) 1998 Academic Press.
  • Y Yasuda; S Masuda; M Chikuma; K Inoue; M Nagao; R Sasaki
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 273 (39) 25381 - 25387 0021-9258 1998/09 [Refereed]
     
    Although erythropoietin (Epo) has been shown to possess in vitro angiogenic activity, its physiological significance has not been demonstrated. Normally angiogenesis does not occur actively in adults but an exception is the female reproductive organ. In the uterine endometrium, angiogenesis takes place actively for supporting the endometrial growth that occurs during transition from the diestrus to estrous stage. This transition is under control of 17 beta-estradiol (E-2), an ovarian hormone, and can be mimicked by injection of E-2 to ovariectomized (OVX) mouse. Thus, the uterus is a pertinent site to examine the Epo function in angiogenesis. We found that Epo protein and its mRNA were produced in an E-2-dependent manner, when the uterus from OVX mouse was cultured in vitro. The de novo protein synthesis was not needed for E-2 induction of Epo mRNA. Administration of E-2 to OVX mouse induced a rapid and transient increase in Epo mRNA in the uterus. Injection of Epo into the OVX mouse uterine cavity promoted blood vessel formation in the endometrium. Furthermore, injection of the soluble Epo receptor capable of binding with Epo into the uterine cavity of non-OVX mouse in diestrus stale inhibited the endometrial transition to proestrus stage, whereas heat-inactivated soluble Epo receptor allowed the transition to occur. These results, combined with our finding that the endothelial cells in uterine endometrium express Epo receptor, strongly suggest that Epo is an important factor for the E-2-dependent cyclical angiogenesis in uterus.
  • M Sakanaka; TC Wen; S Matsuda; S Masuda; E Morishita; M Nagao; R Sasaki
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 95 (8) 4635 - 4640 0027-8424 1998/04 [Refereed]
     
    Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity, Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.
  • T Kambe; J Tada; M Chikuma; S Masuda; M Nagao; T Tsuchiya; PJ Ratcliffe; R Sasaki
    BLOOD W B SAUNDERS CO 91 (4) 1185 - 1195 0006-4971 1998/02 [Refereed]
     
    Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in pig and Hep3B cells. The Epo gene promoter and 3' enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of pig Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in pig and Hep3B cells, and mutation of a hypoxia-inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in pig cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. pig cells may lack an unidentified regulalor(s) required for interaction of the Epo enhancer with Epo and LDHA promoters. (C) 1998 by The American Society of Hematology.
  • S Masuda; M Chikuma; E Morishita; M Nagao; R Sasaki
    ANIMAL CELL TECHNOLOGY: BASIC & APPLIED ASPECTS, VOL 9 SPRINGER 23 - 27 1998 
    It has been thought that erythropoietin exclusively acts on erythroid precursor cells in vivo. However, we describe that erythropoietin is produced by astrocytes and its receptor is expressed in neurons. Erythropoietin production by astrocytes is dependent on oxygen tension and activated by insulin and insulin-like growth factors. Furthermore, erythropoietin protected the cultured hippocampal and cerebral cortical neurons from glutamate neurotoxicity. These results indicate that, in the central nervous system, erythropoietin acts on neurons in a paracrine fashion, and erythropoietin and its receptor system is independent of that for erythropoiesis.
  • M Nagao; K Inoue; SK Moon; S Masuda; H Takagi; S Udaka; R Sasaki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 61 (4) 670 - 674 0916-8451 1997/04 [Refereed]
     
    Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants mere affinity purified using a monoclonal antibody-fixed gel for EPO and an EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities, Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.
  • S Masuda; M Chikuma; R Sasaki
    BRAIN RESEARCH ELSEVIER SCIENCE BV 746 (1-2) 63 - 70 0006-8993 1997/01 [Refereed]
     
    Erythropoietin (EPO) is established as a major regulator of erythropoiesis. However, we and others have shown that neurons express erythropoietin receptor (EPO-R), that astrocytes produce EPO and that EPO may act as a neurotrophic factor in the CNS. We also found that EPO production is activated by insulin and insulin-like growth factors (IGFs) in astrocytes in a dose-dependent manner and that IGF-I was the most potent activator. The concentrations required for half-maximal activation were 3 nM IGF-I, 10 nM IGF-II and 100 nM insulin. The oxygen concentration regulates EPO production; hypoxia stimulates EPO production in astrocytes. The stimulatory effect of IGFs and insulin on EPO production in astrocytes was not affected by the oxygen concentration of astrocyte culture. Insulin and IGFs did nor increase the total protein synthesis of astrocytes but increased EPO mRNA levels, indicating that EPO production is stimulated at the mRNA level. It appeared that the growth factor-induced accumulation of EPO mRNA in astrocytes was caused by activation of the tyrosine kinase-signal transduction pathway, because tyrosine phosphorylation of receptors for IGF-I and insulin was activated when astrocytes were stimulated by these growth factors.
  • E Morishita; S Masuda; M Nagao; Y Yasuda; R Sasaki
    NEUROSCIENCE PERGAMON-ELSEVIER SCIENCE LTD 76 (1) 105 - 116 0306-4522 1997/01 [Refereed]
     
    Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity. Copyright (C) 1996 IBRO.
  • K Ikura; K Takahata; R Shinagawa; S Masuda; R Sasaki
    CYTOTECHNOLOGY KLUWER ACADEMIC PUBL 23 (1-3) 77 - 85 0920-9069 1997 [Refereed]
     
    Transglutaminase catalyzes the intermolecular cross-linking of peptides between Gin and Lys residues, forming an epsilon-(gamma-glutamyl) lysine bond. Amyloid beta-peptide, a major constituent of the deposits in Alzheimer disease, contains Lys16, Lys28, and Gln15 which may act as substrates of transglutaminase. Transglutaminase treatment of amyloid beta-peptide (1-28) and amyloid beta-peptide (1-40) yielded cross-linked oligomers. Transglutaminase-treated A beta retarded neurite extension of PC12 cells, and rat cultured neurons of hippocampus and septum, brain areas severely affected by Alzheimer disease, and subsequently caused cell death, whereas the transglutaminase-untreated counterparts did not show harmful effects. The transglutaminase-catalyzed oligomers of amyloid beta-peptide and their neurotoxicity may be involved in two characteristics in Alzheimer disease, neuronal degeneration and formation of the insoluble deposits.
  • SK Moon; S Takeuchi; T Kambe; T Tsuchiya; S Masuda; M Nagao; R Sasaki
    CYTOTECHNOLOGY KLUWER ACADEMIC PUBL 25 (1-3) 79 - 88 0920-9069 1997 [Refereed]
     
    Oxygen is a limiting nutrient in animal cell culture and its supply is still worthy of improvement for production of useful proteins with a high efficiency. From a different point of view, development of the system by which a high productivity can be maintained even under hypoxic condition as well as under normoxic condition may be important. A number of hypoxia-inducible genes have been found in eucaryotic cells and the induction in most cases, if not all, is due to hypoxic activation of the gene transcription. Transcription of erythropoietin gene is highly hypoxia-inducible and the induction is achieved by binding of a protein, which is widely distributed in animal cells, to a short DNA stretch (erythropoietin enhancer) in the 3' Ranking region of erythropoietin gene. Using a hepatoma cell line (Hep3B) that produces the endogenous erythropoietin in an oxygen-dependent manner and Chinese hamster ovary cells that have been widely used for production of recombinant proteins, we show that, under hypoxic condition, the erythropoietin enhancer can activate not only the promoter of erythropoietin gene but also promoters of cytomegalovirus early genes and eucaryotic polypeptide chain elongation factor gene, both of which are very active in animal cells under normoxic condition.
  • Y Shirai; M Inagaki; M Yamaguchi; T Kambe; M Nagao; S Masuda; R Sasaki
    CYTOTECHNOLOGY KLUWER ACADEMIC PUBL 25 (1-3) 71 - 77 0920-9069 1997 [Refereed]
     
    Expression of specific genes is a strategy of animal cells for adaptation to oxygen deficiency and the mechanism underlying the hypoxic activation of gene expression may be useful for efficient production of recombinant proteins by animal cells, because oxygen is a limiting factor in animal cell cultures. We prepared an animal cell line harboring the plasmid in which expression of a reporter gene, beta-galactosidase, is controlled by an enhancer responsible for the hypoxic activation of gene transcription. The purpose of this paper is to understand this hypoxic production of recombinant proteins quantitatively by a mathematical model originally developed based on the following hypotheses; 1 lacZ (the reporter gene) is transcribed after HIF-1 protein complex is bound to the hypoxic enhancer, 2. beta-galactosidase synthesis rate is limited at the transcription of lacZ, 3. HIF-1 is an inactive form under a normal oxygen concentration, 4. Oxygen works as a repressor in the synthesis of HIF-1 protein, 5. Both beta-galactosidase and HIF-1 are decomposed according to the first order reaction. The effects of hypoxic duration as well as oxygen concentration on the beta-galactosidase production were successfully predicated by the model.
  • Nagao M; Inoue K; Moon SK; Masuda S; Takagi H; Udaka S; Sasaki R
    Biosci. Biotech. Biochem. 61 670 - 674 1997 [Refereed]
  • Shirai Y; Inagaki M; Yamaguchi M; Kambe T; Nagao M; Masuda S; Sasaki R
    Cytotechnology 25 71 - 77 1997 [Refereed]
  • E. Morishita; S. Masuda; M. Nagao; Y. Yasuda; R. Sasaki
    Neuroscience 76 (1) 105 - 116 0306-4522 1996/12 [Refereed]
     
    Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin-receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.
  • 永尾雅哉; 増田誠司; 神戸大朋; 佐々木隆造
    蛋白質 核酸 酵素 共立出版 41 (16) 2522 - 2531 0039-9450 1996/12
  • Y Yasuda; M Okano; M Nagao; S Masuda; H Konishi; K Ueda; T Matsuo; K Tsujiguchi; S Tajima; R Sasaki; T Tanimura
    DEVELOPMENTAL DYNAMICS WILEY-LISS 207 (2) 184 - 194 1058-8388 1996/10 [Refereed]
     
    Erythropoiesis begins first in the visceral yolk sec (VYS) of the embryo; however, the involvement of erythropoietin (EPO) in yolk-sac erythropoiesis has not been studied adequately. This study reports the expression of EPO in normal and hypoxic VYSs and alterations in yolk sac components induced by retinoic acid (RA) in mice. Gravid mice (plug day = day 0 of gestation) were given one oral dose of 60 mg/kg of RA in olive oil on days 6, 6.5, 7, 7.5, or 8 of gestation and were sacrificed 2.5, 3, or 3.5 days later. Control mice received olive oil without RA. None of the dams developed anemia, but more than 80% of the embryos of the dams that received RA on day 6, 6.5, or 7 of gestation had avascular yolk sacs (AVYs) and anemia. In these AVYs, the adenosine triphosphate (ATP) level was as low as 18-59% of that in the control VYSs. Reverse transcription-polymerase chain reaction and Southern analysis of products demonstrated that mRNA for EPO receptor (EPR) was expressed in both VYSs and AVYs on days 9-11 of gestation, and EPO mRNA was present in VYSs and AVYs on days 9 and 10 of gestation and in vehicle-exposed VYSs on day 11 of gestation. Furthermore, enzyme immunoassay of EPO indicated that AVYs contained more EPO protein than control VYSs. Light microscopy revealed that, in AVYs, in addition to the defective hemopoietic cells, the endodermal layer was exclusively altered: The presence of focal proliferated regions and the separation from the mesenchyme led to a single layer from which some immature cells seemed to be migrating. Immunolocalization of EPO showed its presence in all components of VYSs with a characteristic distribution pattern: In the endodermal layer, cells with positive EPO staining decreased as gestation advanced, and erythroid precursor cells showed positive staining. In AVYs, the proliferated endodermal cells had EPO in abundance; in the separated regions, the distinction between positive and negative EPO staining became clearer than that in the control VYSs, and the immature cells in the lumens also had EPO. EPR was seen on the cell surface of the corresponding cells that reacted to EPO. These findings suggest that VYSs not only produce EPO temporarily but also respond to the oxygen content in situ. EPO and EPR appear to be synthesized in the endodermal cells of the VYSs that are likely to respond to the circumstances induced by RA. (C) 1996 Wiley-Liss, Inc.
  • E Morishita; H Narita; M Nishida; N Kawashima; K Yamagishi; S Masuda; M Nagao; H Hatta; R Sasaki
    BLOOD W B SAUNDERS CO 88 (2) 465 - 471 0006-4971 1996/07 [Refereed]
     
    A hybridoma cell line producing the monoclonal antibody against erythropoietin receptor (EpoR) was established using the soluble ectodomain of mouse erythropoietin receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR, Epitope mapping with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources, Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons. (C) 1996 by The American Society of Hematology.
  • Regulation of gene expression by oxygen
    M. Nagao; S. Masuda; T. Kambe; R. Sasaki
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 41 (16) 2522 - 2531 1996 [Refereed]
  • Yasuda Y; Okano M; Nagao M; Masuda S; Konishi H; Ueda K; Matsuo T; Tsujiguchi K; Tajima S; Sasaki R; Tanimura T
    Develop. Dyn 207 184 - 194 1996 [Refereed]
  • 増田誠司; 永尾雅哉; 佐々木隆造
    化学と生物 Japan Society for Bioscience, Biotechnology, and Agrochemistry 33 (7) 437 - 446 0453-073X 1995/07
  • M Nagao; S Masuda; M Ueda; R Sasaki
    CYTOTECHNOLOGY KLUWER ACADEMIC PUBL 18 (1-2) 83 - 91 0920-9069 1995 [Refereed]
     
    We describe possible functions of carbohydrates attached to growth factors and strategies to examine the functions, concentrating on erythropoietin, a major regulator of erythropoiesis. Erythropoietin in erythropoiesis functions as an endocrine hormone; it is produced by kidney cells and transferred into the circulation to hemopoietic sites. In the brain, erythropoietin acts on neurons in a paracrine fashion. Comparison of glycosylation has been made between kidney and brain erythropoietins.
  • M Nagao; S Masuda; M Ueda; R Sasaki
    ANIMAL CELL TECHNOLOGY: DEVELOPMENTS TOWARDS THE 21ST CENTURY KLUWER ACADEMIC PUBL 427 - 430 1995 [Refereed]
  • M Nagao; S Masuda; M Ueda; R Sasaki
    ANIMAL CELL TECHNOLOGY: DEVELOPMENTS TOWARDS THE 21ST CENTURY KLUWER ACADEMIC PUBL 431 - 441 1995 [Refereed]
  • M Nagao; S Masuda; M Ueda; R Sasaki
    CYTOTECHNOLOGY KLUWER ACADEMIC PUBL 18 (1-2) 83 - 91 0920-9069 1995 [Refereed]
     
    We describe possible functions of carbohydrates attached to growth factors and strategies to examine the functions, concentrating on erythropoietin, a major regulator of erythropoiesis. Erythropoietin in erythropoiesis functions as an endocrine hormone; it is produced by kidney cells and transferred into the circulation to hemopoietic sites. In the brain, erythropoietin acts on neurons in a paracrine fashion. Comparison of glycosylation has been made between kidney and brain erythropoietins.
  • M OKANO; S MASUDA; H NARITA; S MASUSHIGE; S KATO; S IMAGAWA; R SASAKI
    FEBS LETTERS ELSEVIER SCIENCE BV 349 (2) 229 - 233 0014-5793 1994/08 [Refereed]
     
    Retinoic acid (RA) stimulated the production of erythropoietin (Epo) in a human hepatoma cell line, HepG2 cells. The stimulation was due to the accumulation of Epo mRNA. The Epo production in HepG2 cells was also dependent on O-2 tension for cell culture but the enhancement of Epo production by RA was independent of O-2 tension, indicating that RA exerts its effect through a pathway different from O-2. Oral administration of RA to the vitamin A-depleted rats elevated the concentration of Epo in serum. These results suggest that RA up-regulates EPO production in vivo as well as in vitro.
  • S MASUDA; M OKANO; K YAMAGISHI; M NAGAO; M UEDA; R SASAKI
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 269 (30) 19488 - 19493 0021-9258 1994/07 [Refereed]
     
    It has been shown that neurons express erythropoietin (Epo) receptor, but the production of Epo protein in neural tissues has not been demonstrated. Cerebral cells of rat fetuses were cultured, and Epo in the spent medium was measured with an enzyme-linked immunoassay. Production of the immunoreactive Epo was dependent on O-2 tension for cell culture; hypoxia enhanced the production. The immunoreactive Epo purified from the spent medium stimulated the growth of Epo-dependent myeloid cells and formation of fetal liver erythroid colonies. These biological activities were completely inhibited by the anti-Epo antiserum and the extracellular domain of the Epo receptor capable of binding with Epo. When brain Epo was compared with serum Epo, brain Epo was smaller in size and more active in vitro at low ligand concentrations. These differences appear to be caused by the different extent of sialylation. Analyses with the reverse transcription-polymerase chain reaction method indicated that the regulation of Epo production by oxygen operates at the level of its mRNA. Immunochemical staining of the immortalized clonal cells revealed that astrocytes produced brain Epo. These results provide a novel site of Epo production and suggest that Epo acts on neurons in a paracrine fashion.
  • Characterization of Erythropoietin Receptor on two Neuronal Cell Line (共著)
    増田 誠司
    Animal Cell Technology : Basic & Applied Aspects,6/,567-574 1994
  • Okano M; Masuda S; Narita H; Masushige S; Kato; Imagawa S; Sasaki R
    FEBS Lett. 349 229 - 233 1994 [Refereed]
  • Y YASUDA; M NAGAO; M OKANO; S MASUDA; R SASAKI; H KONISHI; T TANIMURA
    DEVELOPMENT GROWTH & DIFFERENTIATION WILEY-BLACKWELL 35 (6) 711 - 722 0012-1592 1993/12 [Refereed]
  • M OKANO; H SUGA; S MASUDA; M NAGAO; H NARITA; K IKURA; R SASAKI
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY TAYLOR & FRANCIS LTD 57 (11) 1882 - 1885 0916-8451 1993/11 [Refereed]
     
    We isolated erythropoietin (Epo) from anemic-rat serum with 1.3 x 10(6)-fold purification and 38% recovery using immunoaffinity chromatography. The isolated Epo migrated in SDS polyacrylamide gel with a molecular size of 37 kDa. Biological properties of rat Epo were compared with those of human Epo using target cells of primate and murine origins. When murine cells were used as target cells for assaying Epo, rat Epo stimulated proliferation of the cells with a 50% lower potency than did human Epo. The activity of rat Epo on human cells was only 25% of that of human Epo. Studies of Epo binding to the receptor indicated that rat and human Epos were not distinguishable in binding to murine cells; however, rat Epo bound to the receptor on human cells with an affinity much lower than that of human Epo. Rat Epo was digested with N-glycanase. Complete removal of N-linked sugars converted the native Epo to the deglycosylated form with 18 kDa. The in vitro activity of deglycosylated Epo was 2.5-fold higher than that of the native Epo.
  • S MASUDA; M NAGAO; K TAKAHATA; Y KONISHI; F GALLYAS; T TABIRA; R SASAKI
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 268 (15) 11208 - 11216 0021-9258 1993/05 [Refereed]
     
    Radioiodinated erythropoietin (Epo) was bound specifically to the cells of two non-erythroid clonal lines, PC12 and SN6, which expressed neuronal characteristics. The binding was time-, cell number-, and dose-dependent and was reversible. Although the cloned Epo receptor from PC12 cells (derived from rat adrenal medulla) was identical to that from rat erythroid cells, significant differences in the ligand binding properties between two cell lineages were found; 1) PC12 cells had a single class of binding sites with very low affinity (K(d) = 16 nM), whereas erythroid cells had two classes of binding sites with different affinities (K(d) = 95 pM for high affinity sites and 1.9 nM for low affinity sites), and 2) cross-linking experiments revealed one cross-linked product of 105 kDa for PC12 cells and two products of 140 and 120 kDa for erythroid cells. Taken together with additional results, the presence of a putative accessory protein(s) that may alter the ligand binding affinity through interaction with Epo receptor is discussed. The binding of Epo to PC12 cells caused a rapid increase in the cytosolic concentration of free calcium. The presence of EGTA had no effect on the Epo binding but completely inhibited the calcium increase, indicating that Epo stimulated the calcium influx from outside of the cells. The addition of Epo to the culture media of PC12 cells elevated the intracellular concentrations of monoamines.
  • M NAGAO; S MATSUMOTO; S MASUDA; R SASAKI
    BLOOD W B SAUNDERS CO 81 (10) 2503 - 2510 0006-4971 1993/05 [Refereed]
  • Production and Characterization of Recombinant Soluble Form Erythropoietin Receptor (共著)
    増田 誠司
    Animal Cell Technology : Basic & Applied Aspects,5,71-79 1993
  • M NAGAO; H SUGA; M OKANO; S MASUDA; H NARITA; K IKURA; R SASAKI
    BIOCHIMICA ET BIOPHYSICA ACTA ELSEVIER SCIENCE BV 1171 (1) 99 - 102 0006-3002 1992/11 [Refereed]
     
    The cDNA for the rat erythropoietin (EPO) has been cloned and sequenced. The deduced amino acid sequence consists of 166 amino acid residues, which has a 79% and 95% homology with human and mouse EPOs, respectively. Many short stretches, highly conserved in primate and rodent EPOs, are found in the 3'-noncoding region when insertions and deletions are taken into consideration.
  • M NAGAO; S MASUDA; S ABE; M UEDA; R SASAKI
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS 188 (2) 888 - 897 0006-291X 1992/10 [Refereed]
  • S MASUDA; Y HISADA; R SASAKI
    FEBS LETTERS ELSEVIER SCIENCE BV 298 (2-3) 169 - 172 0014-5793 1992/02 [Refereed]
     
    Erythropoietin (EPO) stimulates proliferation and differentiation of late erythroid precursor cells (CFU-E) and thereby determines the rate of erythropoiesis. Liver is the major erythropoietic site in a fetus. We dealt with developmental changes in CFU-E and EPO receptor (EPO-R) of fetal mouse liver. The affinity of the EPO-R to EPO was unchanged during fetal development. The population size of CFU-E, the number of EPO-R per liver cell, and EPO-R mRNA decreased as gestation proceeded, in a pattern indicating that the expression of EPO-R on erythroid precursor cells in fetal mouse liver is governed mostly by the process of mRNA production.
  • S MASUDA; E TSUDA; K YAMAGUCHI; K AKAI; G KAWANISHI; M UEDA; R SASAKI
    ANIMAL CELL TECHNOLOGY : BASIC & APPLIED ASPECTS, VOL 4 KLUWER ACADEMIC PUBL 499 - 507 1992
  • K YAMAGUCHI; K AKAI; G KAWANISHI; M UEDA; S MASUDA; R SASAKI
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 266 (30) 20434 - 20439 0021-9258 1991/10 [Refereed]
     
    Erythropoietin (Epo) has three N-linked sugar chains. Codons for asparagine at N-glycosylation sites in genomic human Epo DNA were replaced with those for glutamine. The wild-type Epo gene and seven mutants that lacked N-glycosylation sites in every possible combination were introduced into baby hamster-kidney cells. To study the role of the N-linked sugars in Epo biosynthesis, Epo protein expressed transiently was measured by an enzyme-linked immunoassay. The elimination of all three N-glycosylation sites decreased Epo production to 10% of that of the wild-type Epo. Wild-type and mutant Epos produced by stably transfected cells were partially purified to investigate their properties. Removal of N-glycosylation sites changed affinity of Epo to the receptor. The in vitro activity of Epo that lost all N-glycosylation sites was comparable with that of the wild-type Epo, while the in vivo activity severely decreased. These results indicate that N-linked sugars of Epo have two major functions; N-linked sugars are important for 1) proper biosynthesis and/or secretion and 2) expression of the in vivo activity probably by enhancing survival in the circulation. N-Linked sugars of Epo affect binding affinity of the ligand to the receptor but do not play a key role in expression of the in vitro activity.
  • H KIMATA; A YOSHIDA; C ISHIOKA; S MASUDA; R SASAKI; H MIKAWA
    CLINICAL AND EXPERIMENTAL IMMUNOLOGY BLACKWELL SCIENCE LTD 85 (1) 151 - 156 0009-9104 1991/07 [Refereed]
     
    The effect of human recombinant erythropoietin (Epo) on B cell responses was studied in a serum-free medium. Epo enhanced IgM production and thymidine uptake by a human IgM-producing lymphoblastoid cell line, CBL. This effect was specific to Epo since enhancement was blocked by anti-Epo antibody but not by control antibody. Among the various cytokines, interleukin-4 (IL-4) enhanced IgM production and thymidine uptake while IL-6 enhanced IgM production without affecting thymidine uptake. In contrast, other cytokines including IL-1-beta, IL-2, IL-5, interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), or granulocyte/macrophage colony-stimulating factor (GM-CSF) were without effect. However, the enhancing effect of Epo is different from that of IL-4 or IL-6, since Epo effect was not blocked by anti-IL-4 antibody or anti-IL-6 antibody. Moreover, specific binding of Epo was detected on CBL cells. Epo also enhanced immunoglobulin (IgG, IgM and IgA) production and thymidine uptake by purified tonsil small resting B cells stimulated by Staphylococcus aureus Cowan strain I (SAC) or by large activated B cells. In contrast, Epo had no effect on unstimulated small resting B cells These results indicate that Epo could directly stimulate activated and differentiated B cells and could enhance B cell immunoglobulin production and proliferation.
  • Erythropoietin Receptor
    増田 誠司
    SEIKAGAKU,63,28-32 1991
  • S MASUDA; Y HISADA; M UEDA; R SASAKI
    ANIMAL CELL CULTURE AND PRODUCTION OF BIOLOGICALS KLUWER ACADEMIC PUBL 345 - 352 1991
  • J HIRATA; H SATO; H TAKAHIRA; S SHIOKAWA; T ENDO; J NISHIMURA; M KATSUNO; S MASUDA; R SASAKI; Y FUKUMAKI; H NAWATA; H OKANO
    LEUKEMIA STOCKTON PRESS 4 (5) 365 - 372 0887-6924 1990/05 [Refereed]
  • E TSUDA; G KAWANISHI; M UEDA; S MASUDA; R SASAKI
    EUROPEAN JOURNAL OF BIOCHEMISTRY SPRINGER VERLAG 188 (2) 405 - 411 0014-2956 1990/03 [Refereed]
  • R SASAKI; K HITOMI; S MASUDA; M UEDA; G KAWANISHI
    TRENDS IN ANIMAL CELL CULTURE TECHNOLOGY V C H PUBLISHERS 155 - 160 1990
  • K HITOMI; S MASUDA; K ITO; M UEDA; R SASAKI
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS 160 (3) 1140 - 1148 0006-291X 1989/05 [Refereed]

Books etc

  • 技術情報協会 (ContributormRNA転写・プロセシング・核外輸送・品質管理における相互連携の重要性 (p279-288) 、藤田賢一、堀史人、鵜飼生望、前田明、増田誠司)技術情報協会 2023/02 9784861049378 411p
  • 動物細胞におけるmRNAスプライシングをコントロールする生理活性物質およびタンパク質生産性の向上
    増田 誠司 (ContributorP89-99)CMC出版 2021/11
  • ８章「遺伝情報の流れ」
    藤田尚志; 増田誠司 (Contributor京大発!フロンティア生命科学」p120-138)講談社 2018/03
  • Applied RNA Bioscience
    Masuda S; Izawa S (Joint editor)Springer 2018/02
  • The production of recombinant proteins from mammalian cells using RNA element
    Mursi IFA; Masuda S (Joint workChapter 9)Springer 2018
  • 効率的なmRNA核外輸送系を用いたタンパク質生産法
    岡村真純; 平山瑞季; 猪瀬春子; 増田誠司 (Joint work「動物細胞の培養を成功させる条件設定集」p447-450)技術情報協会 2014
  • mRNA biogenesis in the nucleus and its export to the cytoplasm. “Current Frontiers and Perspectives in Cell Biology”, eds by Najman,
    Fujiwara, N; Shiki, T; Masuda InTech - Open Access Publisher, ISBN 978-953-51-0544-2, pp131-152 2012
  • mRNA輸送体の多様化とその生物学的意義
    山崎智弘; 増田誠司 (Joint work化学と生物, 50, 9-11)2012
  • mRNA核外輸送の効率化による動物細胞タンパク質生産
    松村嘉員; 増田誠司 (Joint workバイオサイエンスとインダストリー, 70, 373-375,)2012
  • mRNA biogenesis in the nucleus and its export to the cytoplasm
    Fujiwara N; Shiki T; Masuda S (Joint workp131-152)InTech 2012
  • 基礎生物学テキストシリーズ「分子生物学」 深見泰夫編、3章 遺伝情報の発現・ 転写・プロセシング、4章 遺伝情報の輸送・翻訳
    増田誠司 化学同人 2011
  • mRNAの一生 - mRNAプロセシング・核外輸送・品質管理ネットワーク
    福中彩子; 山崎智弘; 藤原奈央子; 増田誠司 (Joint work化学と生物、46, 90-100)2008
  • mRNAのスプライシングと核外輸送について
    増田誠司 (Single work化学と生物、43, 387-390)2005
  • mRNAの転写・スプライシング・核外輸送の共役ならびに成熟mRNAの選択的認識機構
    増田誠司 (Single work生化学、75, 613-616)2003
  • エリスロポエチンの組織特異的発現調節の発見と応用生化学的研究
    増田誠司 (Single work日本農芸化学会誌, 75, 1067-1075)2001
  • Production of erythropoietin in female reproductive organs and its angiogenic function in the uterus.
    Nagao M; Masuda S; Chikuma-Esaki M; Kobayashi T; Yasuda Y; Sasaki R (Reprod. Biotechnol. Relating Physiol., 147-152,2001)2001
  • Erythropoietin: Multiple Physiological Functions and Regulation of Biosynthesis.
    Sasaki R; Masuda S; Nagao M (Biosci. Biotechnol. Biochem., 64, 1775-1793)2000
  • "Erythropoietin" in The Encyclopedia of Bioprocess Technology : Fermentation, Biocatalysis, & Biosaparation edited by Flickinger, MC. and Drew, SW. (共著)
    増田 誠司 Wiley publishers,/,1113-1122 1999
  • エリスロポエチンと神経細胞
    増田誠司; 佐々木隆造 (Joint work血液フロンティア、9, 64-67)1999
  • 赤血球造血因子（エリスロポエチン）の生理作用と生合成の調節
    増田誠司; 佐々木隆造 (Joint work健康と栄養のライフサイエンス、4、17-22,)1999
  • Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production.
    Masuda S; Nagao M; Sasaki R (Joint workInt. J. Hematol., 70, 1-6,)1999
  • Erythropoietin in The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, & Biosaparation
    Masuda S; Sasaki R (Joint workp1113-1122)Wiley publishers 1999
  • エリスロポエチン遺伝子の発現制御
    増田誠司; 佐々木隆造 (Joint workBIO Clinica, 13, 1169-1173,)1998
  • 酸素による遺伝子発現の制御
    永尾雅哉; 増田誠司; 神戸大朋; 佐々木隆造 (Single translation蛋白質 核酸 酵素、41, 2522-2531)1996
  • Erythropoietin processing in erythropoietic system and central nervous system (共著)
    増田 誠司 Animal Cell Technology : Developments towards the 21st Century,/,431-441 1995
  • 神経系に作用するサイトカインとそのレセプター
    増田誠司; 永尾雅哉; 佐々木隆造 (化学と生物、33、437-446)1995
  • エリスロポエチン
    佐々木隆造; 増田誠司 (廣川タンパク質化学、第8巻、成長因子II、林恭三、古川昭栄編集、p175-182)廣川書店 1992
  • エリトロポエチンレセプター
    増田誠司; 佐々木隆造 (生化学、63、28−32)1991

Conference Activities & Talks

  • カツオ動脈球由来エラスチンペプチドの高血圧性腎血管障害抑制作用の解明
    竹森久美子; 竹森久美子; 中村優希; 増田誠司; 増田誠司
    日本動脈硬化学会総会・学術集会プログラム・抄録集(Web)  2023

MISC

Industrial Property Rights

  • 特願2022-104280:細胞培養に適した温度応答性ヒドロゲル  2022年/06/29
    上高原浩, 櫻井優佳, 増田誠司, 新延信吾
  • 特願2019-061856:mRNA成熟阻害剤  2019年/03/27
    増田誠司, 森本茉莉, 光川瑞葵, 藤井繁佳, 金谷華帆, 黒澤真一郎
  • 特願2016-011389:mRNA成熟阻害剤  2016年/01/25
    増田誠司, 藤井繁佳, 黒澤真一郎
  • 特許特許6099075号:mRNA輸送を介した効率的タンパク質生産法  
    増田誠司
  • 特許特許平06-038787:可溶性エリスロポエチン受容体蛋白質、およびそのアミノ酸配列をコードする遺伝子    1994/02/15
    佐々木隆造, 永尾雅哉, 増田誠司, 上田正次

Awards & Honors

  • 2017 科学研究費 審査委員表彰
     
    受賞者: 増田 誠司
  • 2012 日本農芸化学研究企画賞
     
    受賞者: 増田 誠司
  • 2011 長瀬研究振興賞
     
    受賞者: 増田 誠司
  • 2001 日本農芸化学会奨励賞
     JPN

Research Grants & Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2022/04 -2025/03 
    Author : 竹森 久美子; 増田 誠司
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2022/04 -2025/03 
    Author : 増田 誠司; 藤田 賢一
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2021/04 -2025/03 
    Author : 加藤 伸一郎; 宮本 大模; 増田 誠司; 尾関 哲也; 青木 尚史; 長尾 徹; 渋谷 恭之
     
    口腔内には様々な病変が存在するが，その中で前がん病変として広く知られているものが口腔内潜在性悪性疾患(OPMDs : Oral Potentially Malignant Disorders)である。OPMDsの一つである口腔白板症は治療法に薬物療法や慎重な経過観察、外科的切除などが提案されているが、有効性は信頼できるものではなく、予防や治療に関する統一された管理方法はない状態である。本研究では、過去に報告した活性フラボノイドによる抗腫瘍効果を元に、実際に製薬へつなげることである。製薬形態に関してはディスカッションの結果、フィルム製剤とした。白板症が慢性的な刺激によって生じることがあることから、患部を刺激から保護し、薬効を長時間患部に届かせる狙いがある。将来的な臨床応用を考え、患者ごとに内容量や形の調整ができるよう3Dプリンタを用いた製薬を行う。今回の実験に関しては、内容量や形は一律で設定している。まずは4NQO溶解水を用いた前がん病変モデルラットに対して製薬したフィルム製剤を塗布し、解析を行う。最終的に可能であれば製剤の安全性を確認した後、ヒトを対象とする医学系研究に関する倫理指針に基づき、倫理審査委員会に研究計画書を提出し承認を得てから臨床研究を実施する。ヒトへの臨床応用へ展開するために、探索的にまず第Ⅰ相試験を白板症患者で行う。先の動物実験から安全性の高い低用量より開始し、第Ⅱ相試験に移行するための推奨容量を決定する。 将来的な展望：本研究の将来的なエンドポイントは、新規口腔用貼付製剤の有効性を明らかにすることであり、最終的には多施設共同での臨床試験へと展開する予定である。具体的には名古屋市立大学口腔外科学と愛知学院大学顎顔面外科学講座を中心とした関連研修施設を登録施設として、口腔潜在的悪性疾患を有する100名規模の患者を対象に臨床試験を実施する。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2021/07 -2024/03 
    Author : 増田 誠司; 後藤 剛
     
    食品の機能性に関する研究は年々盛んになっており、特定保健用食品(トクホ)や機能性表示食品が登場している。このように超高齢社会を迎えた日本では食品成分に高い機能性を求める社会状況が定着しており、今後これまでにない機能性を持つ食品が期待されている。ただ食品成分からの機能性の研究は、時間のかかる古典的な手法が今でも主流であり、迅速な解析手法の開発が望まれている。本研究は、食品中の活性化合物をリード化合物として活性化合物を迅速に探索する試みである。そのために幅広い強さの活性化合物について構造と活性に関する情報ライブラリを整備する。その情報に基づき候補化合物を推定する。最後に候補化合物の機能性を評価するリバーススクリーニング手法を用いて探索を効率化する。食品分野において、構造活性相関を用いて効果的な活性化合物を系統的に類縁体から探索する試みは広がっておらず、本研究は食品分野に新たな概念を導入することが期待できる。 これまでに見出した活性化合物から共通構造としてカテコール構造に着目した。いくつかの類縁体で解析すると、比較的弱いながらも活性が存在していた。この結果から既知の食品成分由来活性化合物をリード化合物とし、活性発現に必要な構造を推定し、実際の活性評価とを繰り返す手法の有効性をある程度まで示すことができ、第一段階としてまずますの結果と判断している。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2020/04 -2024/03 
    Author : 上高原 浩; 増田 誠司; 吉永 新; 田中 義正
     
    バイオマスの高機能化を最終目的に、細胞壁構成全成分の分子特性に関する統合的研究を遂行した。 バイオマスの直接エーテル化による三成分分離に関する基礎研究として、カラマツ鋸屑のエーテル化の炭素数 (Cx, x=1, 2) の違いが三成分分離に与える影響を検討した。アルカリ処理時間、エーテル化反応時間がエーテル化反応及び細胞壁構成成分の分離に与える影響を検討し、これまで得られているメチル化（C1）と同様に、エチル化（C2）の場合でもエーテル化反応は良好に進行し木質成分を分離可能であることがわかった。 次いで、その理由を明らかにするため、カラマツ鋸屑からMilled Wood Lignin (MWL), Glucomannan, xylanを抽出し、それらの基礎的な反応性を調べた。さらに、MWL抽出残渣のメチル化物ついて、その生成物を分析した。現在、それらの反応性の違いを詳細に検討中である。 加えて、反応溶媒であるtetrabutylammonium fluoride (TBAF)/dimethylsulfoxide (DMSO)がエーテル化反応に与える影響を詳細に検討するため、前述の溶媒系に加え異なる溶媒系が反応に与える影響も検討した。結果として、TBAF/DMSO系の優れた反応性が再確認されたものの、現時点でTBAF/DMSOに優る溶媒系の発掘には至っていない。 また、木質細胞壁成分由来ブロックコポリマーの高機能化を目指し、温度応答性バイオマスヒドロゲルの合成を行なった
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Masuda Seiji
     
    The nuclear export of mRNA is carried out by mRNA exporters. So far, our research group has identified TREX complex and AREX complex as mRNA exporters in humans, and compared the mRNAs selectively exported by TREX complex and AREX complex, and the structures of UAP56 and URH49, which are central components of these complexes. In this study, our research group isolated and identified new AREX complex components and analyzed their functions. The results revealed that mRNA exporters are also involved in mRNA splicing, contrary to the previously accepted concept This study provides a new insight into the molecular mechanism of selective mRNA export by diversified mRNA exporters in humans.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/06 -2021/03 
    Author : Masuda Seiji
     
    Apigenin and luteolin (active flavonoids) have strong inhibitory activity of mRNA splicing. To servey affected introns by active flavonoid treatment, we performed NGS analysis and detected significant changes in the alternative mRNA splicing of various genes. The characteristics of the target introns had shorter length, higher GC content, and lower splice site score. The reporter-gene was then generated to verify the above observations. The results showed that active flavonoids caused intron retention and resulted in splicing inhibition, for introns with weak splice sites.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2017/06 -2019/03 
    Author : Masuda Seiji
     
    Calcium signaling is well known as a second messenger in the cell. Part of the signal is delivered into the nucleus and evoked the specific set of gene transcription. However, there is no information whether calcium signaling affect the regulation of alternative mRNA splicing at the genome wide revel. Alternative mRNA splicing produces more proteins than genes encoding proteins. The use of alternative mRNA splicing becomes frequent when living organisms acquire a complexity in their body. In this study, the regulation of alternative mRNA splicing was examined when calcium signaling was induced.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2019/03 
    Author : Kamitakahara Hiroshi; Hayakawa Kazuhisa
     
    For the sake of the effective utilization of biomass resources, we developed derivatization-triggered fractionation of biomass, followed by the reconjugation of fractions to afford 3 biomass-based functional materials. Cellulose part was converted into thermoresponsive supramolecular hydrogels, biocompatible biomass-based plastics, and molecules for antibody drugs as medical materials. Hemicellulose part was converted to biomass-based surfactant. Lignin part was converted into amphiphilic nanoparticles.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : Shibuya Yasuyuki
     
    Currently, there are no drugs effective for treating oral leukoplakia, which is an oral precancerous lesion. Accordingly, we have screened the oral leukoplakia preventive active compounds of dietary origin in the present study. To find out the active compounds, we have established a monitoring system which can serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing. AS a result, we found that the isoflavone fraction extracted from soybean possessed mRNA processing inhibitory activity. Moreover, we found that Apigenin, Luteolin and Chrysin which were flavonoids have the strong mRNA maturation inhibitory activity. Using a rat model of tongue precancerous lesion with 4-nitroquinoline 1-oxide (4NQO), it has been also demonstrated that apigenin improved the precancerous lesion.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2017/03 
    Author : Masuda Seiji
     
    Alternative mRNA splicing is a fundamental mechanism to produce plenty of protein more than those encoded in its genome. The understanding of alternative splicing will make us to develop the therapy strategy for diseases based on the inadequate miss-splicing. CHERP is first identified as Ca2+ signaling related protein in the endoplasmic reticulum. Then, CHERP is also localized in the nucleus, suggesting CHERP has another function. Here, I examined the CHERP regulating genes and alternative splicing. To analyze the genome wide expression change of each mRNA and exon, the exon array analysis was performed. To know the function of CHERP, then, GO term analysis was also carried out. The depletion of CHERP caused the decreased expression of cell cycle, mitosis, cytokinesis, meiosis, DNA repair and DNA replication GO term and increased the induction of apoptosis GO term, suggesting that CHERP has a role for cell proliferation and survival by regulating mRNA splicing.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2017/03 
    Author : Masada Seiji
     
    UAP56, a DEAD box RNA helicase, functions to mRNA export from the nucleus to the cytoplasm. In higher eukaryote, there is a paralog of UAP56, named URH49. Although their amino acid homology is more than 90%, they form different complex and export specific set of mRNAs. In this study, I analyzed the region to regulate the complex formation using molecular biology technique and the structural basis using structural biology method. The results indicate that the region regulating the complex formation is within N domain of UAP56 and URH49 and that their structures are similar but the coordination of N and C domains were different between UAP56 and URH49 and the structure of linker region between N and C domains was also different.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/04 -2015/03 
    Author : MASUDA Seiji
     
    The proteins for the clinical use are produced in mammalian cells because they needs post translational modification such as glycosylation, formation of adequate disulfide-bond, acylation and phosphorylation for their activity in human body. The protein production in mammalian cells, however, takes much cost than that in bacteria. The author found that the mRNA export process becomes the limited step than transcription or translation process. In the present study, mRNA export receptor is created to establish a novel protein production system in mammalian cells by modifying the sequence of mRNA export receptor. The mutant receptor produce more reporter protein than the wild type export receptor.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/04 -2014/03 
    Author : MASUDA Seiji; OKUMURA Katsuzumi
     
    Messenger RNA exporter regulates the mRNA processing. In human, mRNA exporter is diversified. Here, I am focusing on the mechanism of the complex formation of TREX and AREX as functional mRNA exporters. The analysis using chimera mutants and point-mutated proteins, I have shown that TREX and AREX complex formation by UAP56 and URH49 are regulated by single amino acid exchange. This indicates that UAP56 and URH49 were diversified from the common ancestor and obtained the specific function through forming specific complex.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2011/04 -2013/03 
    Author : 増田 誠司
     
    真核生物においてmRNAは核内で転写された後、様々なmRNAプロセシングを受けて成熟mRNAとなる。mRNAプロセシング間の共役は、共役因子と呼ばれるタンパク質複合体が制御している。TREX複合体はmRNAのスプライシングと核外輸送を共役する機能を持つ共役因子であり、その構成因子 UAP56は進化的に保存されている。UAP56には、ヒトやマウスなどのほ乳類では90%の相同性を持つURH49が存在する。我々は、UAP56と URH49がそれぞれ異なる複合体TREXならびにAREXを形成すること、それぞれの複合体を通して異なる生理機能を担っていることを明らかにしてきた。しかし非常に相同性の高い両者が形成する複合体の構築原理は全く分かっておらず、両者の機能の違い、複合体形成の違いを決定する領域を同定し、複合体形成の違いに起因する機能の違いを明らかにすることを目的としている。 まずUAP56とURH49で比較的異なる末端配列を欠損させた欠損変異体と、末端配列を入れ替えたキメラ変異体を作成した。加えてコア領域についても、両者で異なるアミノ酸を1残基ずつ入れ替えたキメラ変異体を作成した。これらの変異体に対して免疫沈降法、mRNA輸送能を検証し、UAP56とURH49の違いを決定する領域の同定を試みた。 末端欠損変異体の解析から、UAP56とURH49のいずれの欠損変異体においても、TREXあるいはAREX複合体形成能が低下することを観察した。一方で末端のキメラ変異体は、いずれも元のUAP56あるいはURH49と同じ複合体形成能を示した。細胞内機能についても同様の結果であった。ついでコア領域を入れ替えたキメラ変異体について機能を検証した結果、１アミノ酸を変異したURH49変異体がUAP56と同様の機能を持つことを複合体形成能とmRNA輸送能の解析から明らかにした。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2012 
    Author : MASUDA Seiji; OKUMURA Katsuzumi
     
    The mRNA quality control in the nucleus remains largely unknown. Here, to analyze the molecular function of mRNA quality control factors in the nucleus, whole transcriptome analysis of the mRNA dynamics in the nucleus was exploited. And the importance of the mRNA quality control was also investigated using the conventional biochemical and molecular biology methods. The whole transcriptome was analyzed from the cells treated with siRNA of RRP45, a core component of exosome, or MTR4, a subcomponent of exosome. Whole reads were mapped on the human genome and the changes of the number of reads mapped on the specific region were compared between siRNA treated- and control cells. The genes whose expressions were markedly increased wereselected by the treatment of siRNA of RRP45 or MTR4. Next, the identification and localization of human TRAMP (PAPD5-ZCCHC7-MTR4) complex were analyzed using conventional methods like immunoprecipitation and immunostaining. Exosome and MTR4 were localized in the nucleolus. PAPD5 and ZCCHC7 were localized in the nucleus. The candidate molecules of human TRAMP complex were associated in the nuclear extract.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008 -2010 
    Author : MASUDA Seiji; MOMMA Keiko
     
    The TREX complex has an essential function in mRNA biogenesis. Here I have studied the function of UAP56 and URH49, whish are the component of the TREX complex, in view of the relationship between mRNA biogenesis and genome stability. Fragmentation from chromosome DNA was observed when URH49 was knocked down rather than that UAP56 was. This phenotype was rescued by the forced expression of URH49, RNaseH, SF2 or RNPS1. These results suggest that the TREX complex functions to regulate the interaction of nascent RNA with genomic DNA to prevent the genome instability.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2005 
    Author : MASUDA Seiji
     
    mRNA, used as a template for protein synthesis in eukaryotes, is synthesized by RNA polymerase II complex in nucleus and undergoes a multiple process for maturation including capping, splicing and poly-adenylation. Recent lines of evidence indicate that these mRNA maturation process form a extensive network by a number of coupling factors. We found THO complex as a one of these coupling factor. Furthermore, we named the TREX complex as transcription and export coupling complex by combined with THO complex, Sub2 (UAP56 in human) and Yra1 (Aly in human), first one is mainly supposed to act as a transcription elongation and middle and last ones act as export factors. Previous studies indicate that THO complex in yeast directly recruited into transcribing RNA polymerase II elongation complex and THO complex, in turn, recruits Sub2 and Yra1, and thereby couples transcription and export. In this study, we have investigated the characterization and functional analysis of human THO complex. We found, first, human THO complex is composed of six protein components, second, does not affect the mRNA transcription and splicing in contrast to yeast case, and third, is recruited on mRNA during splicing. We, therefore, hypothesized that THO complex recruitment onto mRNA is regulated by the intron in general. This idea is also supported by the finding that intron in yeast gene inhibits the recruitment of THO complex to transcribing pre-mRNA.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2000 -2001 
    Author : SASAKI Ryuzo; KAMBE Taiho
     
    Erythropoietin (EPO) is a glycoprotein that is produced by the kidney in adults. It has been belived for long time that stimulation of erythropoiesis is a sole physiological function of EPO but we have demonstrated that EPO is produced in brain and protects neurons from ischemic damage. This finding has been supported by a number of papers subsequently publised and recombinant human EPO is going to be examined as a therapeutic of various brain diseases. Expression of EPO gene is markedly enhanced by hypoxia. Expression of EPO gene in both kidney and brain is hypoxia-inducible but the expression patterns are quite different. When animals are exposed to hypoxia (7 % oxygen), EPO mRNA in both organs increases 40-80-fold at 4 hr after exposure. EPO mRNA is kept at a high level as far as animals are exposed to hypoxia. Although the mechanism remains unknown, this finding implicates the physiological importance that (1) the continuous high level of the kidney EPO mRNA causes erthrocytosis, leading to various vascular disorders and therefore EPO mRNA in the kidney must be decreased despite of hypoxia and that (2) EPO in the brain is required for neuroprotection under hyoxia and therefore a high level continues during hypoxia. We have shown that EPO is also produced in the uterus where EPO acts as an angiogenic factor for periodical growth of uterine endometrial layer. EPO production in the uterus is also hypoxia-inducible but estrogen is required for hypoxic induction of EPO.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2001 
    Author : NAGAO Masaya; SASAKI Ryuzo; MASUDA Seiji
     
    Erythropoietic stimulation has been believed to be the sole physiological function of erythropoietin (Epo). Recently, however, the central nervous system and female reproductive organs have been found to produce Epo. To elucidate how Epo expression is regulated in these tissues, ovariectomized mice were given estrogen (E_2) and/or exposed to hypoxia, and the temporal patterns of Epo mRNA levels were examined. E_2 transiently induced Epo mRNA in the uterus but not in the kidney and cerebrum. Interestingly, the uterine Epo mRNA was hypoxia inducible only in the presence of E_2. Epo mRNA level in the kidney and cerebrum were elevated markedly within 4 h after exposure to hypoxia. Although the elevated level of Epo mRNA in the kidney decreased markedly within 8 h despite continuous hypoxia, the high level in the cerebrum was sustained for 【greater than or equal】 24 h, indicating that down regulation operates in the kidney but not in the brain. Thus Epo expression appears to be regulated in a tissue-specific manner, endorsing the tissue-specific function of Epo. In the next study, we examined other female reproductive organs in the mouse with respect to Epo mRNA expression and its stimuli (E_2 and hypoxia)-induced changes. Although Epo mRNA expression was seen in the ovary and oviduct, the E_2-induced stimulation of Epo mRNA was found only in the oviduct. The E_2-induced stimulation in the oviduct was transient and rapidly downregulated. Epo mRNA expression in the oviduct was hypoxia inducible, in both the presence and the absence of E2. We found that Epo is also produced in the male reproductive organs. Cultured epididymis produces Epo. Expression of epididymal Epo mRNA was drastically increased in the course of sexual maturation. Expression of Epo mRNA by the interstitial cells between the ductus epididymidis was found by in situ hybridization technique.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1997 -1999 
    Author : NAGAO Masaya; MASUDA Seiji
     
    Erythropoietin (Epo) is a glycoprotein, and its sugar chains are important for its in vivo biological activity. So, this year we examined the biological activities of recombinant Epo (rEpo) produced by cultured Chinese hamster ovary (CHO) cells in normoxia or hypoxia and analyzed the structure of its sugar chains. For this purpose we used the promoter of lactate dehydrogenase A gene, which is active in CHO cells and its vicinal hypoxia-response enhancer (HRE) stimulates the promoteractivity efficiently in hypoxia. We have prepared a number of permanent CHO cell lines producing rEPO under control of this promoter/HRE. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells in 21% and 2% oxygen. Further more, forced expression of hypoxia-inducible factor-1α (HIF-1α) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even under low oxygen concentration. HIF-1 is involved in hypoxia response. AKT kinase thought to be involved in the signal transudation of hypoxia response. So we co-transfected a constitutive active AKT kinase expression plasmid and a luciferase reporter plasmid under control of LDH A promoter/HRE into Cos1 cells. Expression of constitutive active AKT kinase alone could not stimulate the expression of the reporter in normoxic condition, but it could potentiate the expression of the reporter in hypoxia. It was reported that dominant-negative AKT could depress the hypoxic induction. These results suggest that AKT kinase is necessary but not sufficient for hypoxic induction.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1997 -1999 
    Author : SASAKI Ryuzo; YASUDA Yoshiko; MASUDA Seiji; NAGAO Masaya
     
    Erythropoietin (EPO) is a glycoprotein produced by the kidney in an oxygen-dependent manner; hypoxia activates EPO production dramatically. EPO has been believed to act exclusively on erythroid precursor cells, stimulating red blood cell formation. However, we found that EPO is produced in other sites where EPO production is regulated in tissue-specific manner and that EPO manifests unexpected new physiological functions in these sites as summarized below. 1.EPO is produced in the central nervous system. The brain EPO acts on EPO receptor expressed in neurons, preventing the neuronal damage from ischemic insult. EPO production is hypoxia-inducible as it is in the kidney. 2.EPO is also produced in female reproductive organs (uterus and oviduct). The uterine EPO stimulates uterine angiogenesis that occurs periodically in the estrous cycle but the oviductal function of EPO remains to be studied. Estrogen is a major inducer of EPO in both organs, although oxygen also contributes the regulation of EPO production. Interestingly, the hypoxic activation of the EPO gene expression in the uterus requires the presence of estrogen. 3.Oxygen supply is one of the major problems in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of recombinant proteins can be maintained or enhanced under low oxygen concentrations. A number of hypoxia-inducible genes have been found in animal cells. A consensus sequence (HRE=hypoxia-response enhancer) responsible for the hypoxic activation exists in these genes. We provided a novel system which guarantees a high productivity even under low oxygen concentrations by the use of the biological strategy based on the hypoxic induction of gene transcription.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1998 -1998 
    Author : 佐々木 隆造; 増田 誠司; 永尾 雅哉
     
    本研究は、ガン組織が常に低酸素状態にあることを利用して、低酸素エンハンサーを用いて特異的に腫瘍細胞にのみ細胞死を誘発する方法論を開発することを目的とした。 プロモーターには、全ての細胞で低酸素エンハンサーと相互作用しうる乳酸脱水素酵素(LDH)のプロモーターを使用し、アンチセンスRNAを発現させる因子は、解糖系の律速酵素であるLDHおよび多薬耐性の原因遺伝子の1つであるMDRを対象とした。これらをHep3B細胞に遺伝子導入し、アンチセンスRNAを発現した細胞を各々50株取得した。これらの細胞株の増殖を、in vitroにおいて目的とする性質(低酸素或いは薬剤投与による死滅)が獲得されているかについて検証した。LDHのアンチセンスRNAを発現した細胞と親株を低酸素分圧に導入し、細胞増殖活性を測定した。しかし親株に比べて有為に増殖活性の低下したクローンはなかった。LDHプロモーターの活性は十分強いが、LDH遺伝子の発現も多いために発現したアンチセンスRNAではLDH mRNAを十分には阻害できなかったと考えられた。一方MDRのアンチセンスRNAを発現する細胞株については低酸素にした後、抗ガン剤を投与した。その結果、薬剤に対する抵抗性がある程度低下した。しかし今回導入したHep3B細胞には抗ガン活性を持つ因子としてMDR以外にMRPも発現しているらしく期待したほどの効果は得られなかった。以上低酸素に応答するアンチセンスRNA発現系はガン細胞の増殖を抑制する系として有効であるが、今後より有効性を確認するためには発現させるアンチセンスRNAの種類を検討する必要があると考えられた。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1997 -1998 
    Author : 増田 誠治
     
    従来造血ホルモンとして知られていたエリスロポエチン(EPO)の脳・神経系における作用を明らかにすることを目的とした。 神経伝達物質としてグルタミン酸は必須の因子である。しかし脳虚血の際には過剰のグルタミン酸が放出される結果、ある種のニューロンはグルタミン酸の興奮毒性により死滅する。これまでにEPOは中枢神経系ではアストロサイトが生産すること、EPOはin vtroにおいてグルタミン酸の毒性によるニューロン死を効果的に防止することを明らかにしてきた。本研究では砂ネズミを用いてin vivoにおいてもEPOがニューロン死を防止するかについて検討した。 EPO又はEPO可溶型受容体を砂ネズミの脳内に投与し、脳虚血に伴うニューロン死への影響を精査した。砂ネズミは、虚血による神経細胞死を解析するのに最適の動物である。砂ネズミに一定の虚血を施すと、1週間後には海馬CA1領域のニューロンの半分は遅発性神経細胞死を起こし選択的に死ぬ。海馬は学習および記憶に重要な組織であり、虚血により海馬の神経細胞が死んだ動物の学習記憶能力は著しく低下する。砂ネズミの学習記憶能力はステップダウン型回避学習実験により判定し、その後海馬領域の神経細胞密度を測定することによりニューロン死保護効果の総合評価を行った。EPOの投与はニューロン死を低滅し、逆に可溶型EPO受容体の投与は内因性のEPOの作用を阻害することによりニューロン死を促進した。これよりアストログリアの生産する内因性のEPO自体が中枢神経系に生理作用を持つことを証明した。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1996 -1996 
    Author : 増田 誠司
     
    従来造血ホルモンとして知られていたエリスロポエチン(EPO)の脳・神経系における作用を明らかにするために、本研究では遺伝子改変動物の作製、初代培養系を用いたEPOの作用メカニズムに関する研究を行った。脳におけるEPO産生細胞はアストロサイトであり、その産生は低酸素により顕著に誘導される。脳虚血傷害において海馬領域は特に脆弱であるため、EPOは脳内における低酸素ストレスによるニューロン死に関与するのではないかと考えた。 (1)神経系特異的にEPOの作用を阻害する遺伝子改変動物の作製 γ-エノラーゼのプロモーターの下流にEPO受容体cDNAの細胞外領域を接続し、神経系特異的に可溶性EPO受容体が発現する発現ベクターを作成した。これをマイクロインジェクション法によりマウス受精卵に導入し、トランスジェニックマウスを作成した。得られた産仔の中からゲノムに組み込まれた遺伝子が安定して子孫に伝えられる系統を選出した。 虚血による神経細胞死のin vitroモデルにおけるEPOの作用 脳虚血によるニューロン死は、中枢神経系の主要な神経伝達物質であるグルタミン酸の細胞外異常蓄積による。事実、初代培養ニューロンをグルタミン酸に暴露することによりニューロン死が観察され、虚血による神経細胞死のin vitroモデルとされている。ラット胎児の海馬及び大脳皮質領域より初代培養ニューロンを調製し、グルタミン酸暴露に先立ち培養液にEPOを添加し、EPO存在下でニューロンを培養しておくと、グルタミン酸によるニューロン死がほぼ完全に抑制された。EPOがグルタミン酸によるニューロン死を抑制するためには、EPOによる細胞外からのカルシウムイオンの流入が必要であること、新規の蛋白合成が必要であることを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1995 -1995 
    Author : 増田 誠司
     
    エリスロポエチン(EPO)は、赤血球系前駆細胞の赤血球への分化・増殖に必須の糖タンパク質であり、胎児では肝臓、成体では肝臓で生産される。申請者は神経細胞に対してもEPOは作用することを示し、中枢神経系ではアストロサイトがEPOを生産し、ニューロンにEPO受容体を介して作用することを明らかにした。これによりEPOは脳・肝臓・腎臓において組織特異的発現をしていることが判明した。本研究はEPOの脳・肝臓・腎臓特異的に発現するために必須の領域を決定し、そこに結合する転写因子を同定することによりEPOの組織特異的発現調節機構を解明することを目的として研究を行った。 EPO遺伝子の組織特異的発現に必要な転写調節領域の決定するために、様々な領域のヒトEPO構造遺伝子の上流および下流領域をルシフェラーゼ遺伝子の両端に接続した。これをEPOを生産する初代培養アストロサイト細胞、肝ガン細胞Hep3Bに導入し、トランジエントのルシフェラーゼ活性を指標として、脳・肝臓におけるEPO産生に必要な領域の決定を試みた。その結果、肝臓での発現にはEPOの上流0.2Kbおよび下流の酸素応答配列が存在すればよいことがわかった。いっぽう脳での発現は、上流3.5Kbと下流の酸素応答配列をルシフェラーゼに接続したものでは、酸素応答配列だけを接続したものに比べて転写の活性化が見られた。しかし上流8.5Kbを接続すると転写は抑制された。従ってこの領域は脳での特異的発現に関する部分ではないと推察された。この知見から脳での特異的発現には、されに上流あるいは下流の配列が必要であることが明らかとなった。 脳でのEPO受容体の発現部位を明らかにするために、18日目のラットの免疫組織化学的染色を行い、海馬および大脳皮質に陽性のニューロンが存在することを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1994 -1994 
    Author : 増田 誠司
     
    エリスロポエチン(EPO)は、赤血球前駆細胞に特異的に作用して、赤血球への分化と増殖を促進すると信じられてきたが、神経細胞の性質を持つPC12やSN6細胞に機能的なEPO受容体が発現していることを見出した。さらに18日目のラット胎児脳の初代培養を低酸素分圧下で行うことにより、EPOが分泌されることを明らかにした。そこで中枢神経系を構成する細胞群のうちどの細胞がEPOを生産するのかを調べた。その結果、抗グリア繊維性酸性タンパク質抗体に対して陽性を示す細胞(アストロサイト)がEPOを生産していた。これより神経細胞がEPO受容体を発現し、神経細胞をとりまくアストロサイトがEPOを生産していることが判明した。ついで腎臓で生産され血中を流れるEPO(血清EPO)とアストロサイトの生産するEPO(脳EPO)について、性状比較を行った。その結果、脳EPOの方が2倍活性が高く、またレクチンに対する結合性実験より、脳EPOは糖鎖末端のシアル酸含量が血清EPOに比べて少ないことが判明した。シアル酸は、EPOのin vitroの活性発現に阻害的に働くことから、脳EPOは、シアル産をつけないことで活性を高め、パラクライン様式に適応していると考えられた。 次にEPO産生脳細胞培養系を用いてEPO産生を増強する因子の検索を行った。材料はきのこの抽出液や緑茶の各成分の抽出液および生体材料などを用いた。しかし食品由来のEPO産生を増強する因子は発見できなかった。ところが驚いたことにインスリンが、EPOの産生を3倍に増強することが判明した。インスリンの主な作用は、食品の摂取によって上昇したグルコースをグリコーゲンに変換することであり、EPO産生にも作用していることは、食品とEPOについて考える上で大変興味深い現象である。一方肝ガン細胞由来のEPO産生細胞HepG2はインスリンによる効果は見られなかった。よってインスリンの効果は脳特異的であると考えられた。
  • Studies on function of erythropoietin
  • Regulation of gene expression by hypoxia
  • 核内mRNA品質管理機構の制御
    Author : 増田 誠司
  • mRNAプロセシングを制御する食品因子
    Author : 増田 誠司
  • 動物細胞におけるタンパク質生産法
    Author : 増田 誠司
  • エリスロポエチン受容体の発現制御
    Author : 増田 誠司
  • エリスロポエチンの生理機能
    Author : 増田 誠司
  • mRNA核外輸送の制御機構
    Author : 増田 誠司
  • mRNAのプロセシング制御機構
    Author : 増田 誠司

Teaching Experience

  • 食品機能学実験 I・II食品機能学実験 I・II
  • 食品機能学演習食品機能学演習 Kindai University
  • 基礎ゼミ基礎ゼミ Kindai University
  • 特別講義 I - IV特別講義 I - IV Kindai University
  • 専門英語I・II専門英語I・II Kindai University
  • 栄養学実験栄養学実験 Kindai University
  • 食品学食品学 Kindai University
  • 化学実験化学実験 Kindai University
  • 分析化学分析化学 Kindai University
  • 自校学習自校学習 Kindai University
  • 総合演習総合演習 Kindai University

Others

  • 2019/03 -2019/03  指導学生・院生の受賞等 
    光川瑞葵 Outstanding Poster presentation 
International student seminar
  • 2018/03 -2018/03  指導学生・院生の受賞等 
    原田光太郎 Outstanding Poster presentation International student seminar
  • 2017/04 -2017/04  指導学生・院生の受賞等 
    倉田雅志 若手優秀ポスター賞 日本口腔科学会
  • 2017/02 -2017/02  指導学生・院生の受賞等 
    倉田雅志 賛助会員特別賞 日本農芸化学会関西支部
  • 2015/06 -2015/06  指導学生・院生の受賞等 
    藤田賢一 ベストプレゼンテーション賞優秀賞 日本RNA学会

Other link

researchmap


Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.