Updated on 2022/12/04

写真a

 
KATO, Takashi
 
Scopus Paper Info  
Paper Count: 0  Citation Count: 0  h-index: 13

Citation count denotes the number of citations in papers published for a particular year.

Affiliation
Faculty of Education and Integrated Arts and Sciences, School of Education
Job title
Professor

Concurrent Post

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering

  • Affiliated organization   Global Education Center

Research Institute

  • 2020
    -
    2022

    理工学術院総合研究所   兼任研究員

Education

  •  
    -
    1982

    Waseda University   Graduate School of Science and Engineering   Major in Pure and Applied Physics  

  •  
    -
    1982

    Waseda University   Graduate School of Science and Engineering   Major in Pure and Applied Physics  

  •  
    -
    1980

    Waseda University   School of Education   Department of Biology  

Degree

  • 早稲田大学   博士(理学)

  • Waseda University   D.Sc.

Research Experience

  • 2010.04
    -
     

    Shizuoka University

  • 2010.04
    -
     

    Japan Atomic Energy Agency

  • 2007.04
    -
     

    Waseda University   Faculty of Education and Integrated Arts and Sciences

  • 2006.04
    -
     

    Japan Atomic Energy Agency

  • 2005.10
    -
     

    〜2008年3月 慶応義塾大学 医学部 訪問教授

  • 2004.09
    -
     

    Waseda University   Faculty of Education and Integrated Arts and Sciences

  • 2003.04
    -
     

    Waseda University   Graduate School of Science and Engineering

  • 2002.12
    -
     

    Waseda University   Advanced Research Institute for Science and Engineering

  • 2002.04
    -
     

    Waseda University   School of Education

  • 2002.03
    -
     

    キリンビール株式会社 退職

  • 2001.11
    -
     

    キリンビール株式会社 医薬探索研究所 主幹研究員/研究推進担当部長

  • 2001.04
    -
     

    キリンビール株式会社 医薬探索研究所 主幹研究員/癌分野リーダー[専任]

  • 2000.01
    -
     

    キリンビール株式会社 医薬探索研究所 主任研究員/癌分野リーダー[専任]

  • 1999.08
    -
     

    キリンビール株式会社 医薬探索研究所 研究企画担当部長(兼務)

  • 1994.03
    -
     

    キリンビール株式会社 医薬探索研究所 主任研究員

  • 1993.08
    -
     

    キリンビール株式会社 医薬探索研究所 研究員

  • 1986.04
    -
     

    キリンビール株式会社 医薬開発研究所 研究員

  • 1982.04
    -
     

    キリンビール株式会社 開発科学研究所 研究員

  • 1982.04
    -
     

    キリンビール株式会社 総合研究所入社 技師

  •  
     
     

    (兼)

  •  
     
     

    (大学院名称変更に伴う人事所属変更)

  •  
     
     

    【大学院】 大学院 先進理工学研究科

  •  
     
     

    【学部】 教育学部

  •  
     
     

    (大学組織変更に伴う人事所属変更)

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    【大学院】 大学院 理工学研究科

  •  
     
     

    【学部】 教育学部

  •  
     
     

    生命理工学専攻生命分子機能分野 分子生理学研究

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Professional Memberships

  •  
     
     

    日本薬学会

  •  
     
     

    量子生命科学会

  •  
     
     

    日本比較免疫学会

  •  
     
     

    International Society for Experimental Hematology

  •  
     
     

    The American Society of Hematology

  •  
     
     

    The Japan Society for Comparative Endocrinology

  •  
     
     

    The Japanese Biochemical Society

  •  
     
     

    The Japanese Society of Hematology

  •  
     
     

    The Zoological Society of Japan

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Research Areas

  • Physiology

  • Cell biology

  • Chemistry and chemical methodology of biomolecules

  • Morphology and anatomical structure

  • Clinical pharmacy

Research Interests

  • Molecular Physiology, Hematology, Protein Chemistry, Cytokine, Interleukin, Hematopoiesis, Growth factor

Papers

  • MicroRNA-493-5p-mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

    Ken Yasukawa, Lee Chuen Liew, Keitaro Hagiwara, Ai Hironaka-Mitsuhashi, Xian Yang Qin, Yutaka Furutani, Yasuhito Tanaka, Hitoshi Nakagama, Soichi Kojima, Takashi Kato, Takahiro Ochiya, Luc Gailhouste

    Cancer Science   111 ( 3 ) 869 - 880  2020.03

     View Summary

    Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer-related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR-493-5p, a major tumor suppressor miRNA that inactivates miR-483-3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR-493-5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR-493-5p given its significant inhibition through 3′-UTR targeting in miR-493-5p-rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR-493-5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR-493-5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR-493-5p activity. In summary, miR-493-5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies.

    DOI PubMed

    Scopus

    17
    Citation
    (Scopus)
  • Relationship between the Induced Iron Overload Model and Hepatic Erythropoiesis in Xenopus laevis

    Kei Sato, Masamitsu Taniai, Kota Kato, Takashi Kato

    Zoological Science   37 ( 1 ) 61 - 69  2020

     View Summary

    Iron is an essential element for hemoglobin synthesis during erythropoiesis. Iron overload, in contrast, adversely affects erythropoiesis and causes organ dysfunction. Research using various animal models may help to elucidate pathophysiological mechanisms induced by excess iron. In the present study, we evaluated the relationship between iron metabolism and erythropoietic activity in the African clawed frog, Xenopus laevis. In X. laevis, both erythropoiesis and iron metabolism occur in the liver. First, we developed a method to quantify iron levels in the liver and plasma using 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl) amino] phenol (Nitroso-PSAP). We then measured iron levels and analyzed hematopoietic parameters in frogs that were orally administered sodium ferrous citrate (SFC). The hepatic iron level increased in the SFC group, but the number of erythrocytes, hematocrit, and hemoglobin concentration did not change, suggesting that the regulation of the production and release of mature erythrocytes in the liver was not directly affected by dietary iron. At four days after administration of 2 mg/kg SFC, the number of immature erythrocytes decreased in the liver. Interestingly, atypical blood cells with hyper-segmented nuclei were observed, identified by acridine orange cell staining; these atypical blood cells were hardly detectable under the steady state. Compared with previously reported results in mice, the increase in the hepatic iron levels was small, but our results indicate that SFC affects hematopoietic activity. These results establish a novel model for iron metabolism and provide new insights into the relationship between iron metabolism and erythropoiesis in vertebrates.

    DOI PubMed

    Scopus

    1
    Citation
    (Scopus)
  • MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells

    Luc Gailhouste, Lee Chuen Liew, Ken Yasukawa, Izuho Hatada, Yasuhito Tanaka, Takashi Kato, Hitoshi Nakagama, Takahiro Ochiya

    Cell Death and Disease   10 ( 8 )  2019.08

     View Summary

    Numerous studies have described the critical role played by microRNAs (miRNAs) in cancer progression and the potential of these small non-coding RNAs for diagnostic or therapeutic applications. However, the mechanisms responsible for the altered expression of miRNAs in malignant cells remain poorly understood. Herein, via epigenetic unmasking, we identified a group of miRNAs located in the imprinted delta like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3 (MEG3) locus that were repressed in hepatic tumor cells. Notably, miR-493-5p epigenetic silencing was correlated with hypermethylation of the MEG3 differentially regulated region (DMR) in liver cancer cell lines and tumor tissues from patients. Experimental rescue of miR-493-5p promoted an anti-cancer response by hindering hepatocellular carcinoma (HCC) cell growth in vitro and tumor progression in vivo. We found that miR-493-5p mediated part of its tumor-suppressor activity by abrogating overexpression of insulin-like growth factor 2 (IGF2) and the IGF2-derived intronic oncomir miR-483-3p in HCC cells characterized by IGF2 loss of imprinting (LOI). In summary, this study describes an unknown miRNA-dependent regulatory mechanism between two distinct imprinted loci and a possible therapeutic window for liver cancer patients exhibiting IGF2-miR-483 LOI and amplification.

    DOI PubMed

    Scopus

    28
    Citation
    (Scopus)
  • The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions

    Shigeki Arai, Chie Shibazaki, Motoyasu Adachi, Yoshitake Maeda, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Ryota Kuroki

    FEBS Journal   286 ( 9 ) 1717 - 1733  2019.05

     View Summary

    Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1–163 residues of hTPO (hTPO163) including the N-terminal active site domain (1–153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332). In order to clarify the structural and physicochemical properties of hTPO163, circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and −16.7% for β-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154–332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.

    DOI PubMed

    Scopus

  • Flow cytometric analysis of Xenopus laevis and X. tropicalis blood cells using acridine orange

    Kei Sato, Azusa Uehara, Sayaka Kinoshita, Ikki Nomura, Minami Yagi, Yuta Tanizaki, Yu Matsuda-shoji, Atsushi Matsubayashi, Nobuyasu Endo, Yutaka Nagai, Takashi Kato

    Scientific Reports   8 ( 1 )  2018.11

    Authorship:Corresponding author

     View Summary

    Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

    DOI PubMed

    Scopus

    5
    Citation
    (Scopus)
  • CCN3 secreted by prostaglandin E<inf>2</inf> inhibits intimal cushion formation in the rat ductus arteriosus

    Kenji Iwai, Kazumichi Nagasawa, Toru Akaike, Toshio Oshima, Takashi Kato, Susumu Minamisawa

    Biochemical and Biophysical Research Communications   503 ( 4 ) 3242 - 3247  2018.09

     View Summary

    The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E2 (PGE2)-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE2 may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE2 stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE2 in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE2 stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE2-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE2 to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE2-mediated DA remodeling.

    DOI PubMed

    Scopus

  • Multiple origins of embryonic and tadpole myeloid cells in Xenopus laevis

    Yasutaka Imai, Keisuke Ishida, Maya Nemoto, Keisuke Nakata, Takashi Kato, Mitsugu Maeno

    CELL AND TISSUE RESEARCH   369 ( 2 ) 341 - 352  2017.08  [Refereed]

     View Summary

    Rabbit anti-serum against a myeloid-cell-specific peroxidase (Mpo) of Xenopus laevis was generated to identify myeloid cells in adult and larval animals. Smears of blood samples from adult hematopoietic organs were co-stained with Mpo and with XL-2, a mouse monoclonal antibody against a leukocyte common antigen. Lymphocytes found in the thymus and spleen were XL-2(+)Mpo(-) and granulocytes found in peripheral blood cells and the spleen were XL-2(+)Mpo(+), indicating that double-staining with these two antibodies allowed classification of the leukocyte lineages. Immunohistochemical analysis of larval organs showed that XL-2(+)Mpo(-) cells were scattered throughout the liver, whereas XL-2(+)Mpo(+) cells were present mainly in the cortex region. Interestingly, a cluster of XL-2(+)Mpo(+) cells was found in the region of the larval mesonephric rudiment. The ratio of XL-2(+)Mpo(+) cells to XL-2(+) cells in the mesonephric region was approximately 80%, which was much higher than that found in other hematopoietic organs. In order to elucidate the embryonic origin of the myeloid cells in the tadpole mesonephros, grafting experiments between X. laevis and X. borealis embryos were performed to trace the X. borealis cells as donor cells. Among the embryonic tissues examined, the tailbud tissue at the early neurula stage contributed greatly to the myeloid cluster in the mesonephric region at stage 48. Therefore, at least four independent origins of the myeloid cell population can be traced in the Xenopus embryo.

    DOI

    Scopus

    3
    Citation
    (Scopus)
  • An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

    Shigeki Arai, Chie Shibazaki, Motoyasu Adachi, Eijiro Honjo, Taro Tamada, Yoshitake Maeda, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Michael Blaber, Ryota Kuroki

    PROTEIN SCIENCE   25 ( 10 ) 1786 - 1796  2016.10  [Refereed]

     View Summary

    Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 angstrom using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (Cp) measured by ITC (-1.52 +/- 0.05 kJmol(-1)K(-1)) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 approximate to 0.25 kJmol(-1)K(-1)) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

    DOI

    Scopus

    2
    Citation
    (Scopus)
  • An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

    Shigeki Arai, Chie Shibazaki, Motoyasu Adachi, Eijiro Honjo, Taro Tamada, Yoshitake Maeda, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Michael Blaber, Ryota Kuroki

    PROTEIN SCIENCE   25 ( 10 ) 1786 - 1796  2016.10  [Refereed]

     View Summary

    Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 angstrom using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (Cp) measured by ITC (-1.52 +/- 0.05 kJmol(-1)K(-1)) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 approximate to 0.25 kJmol(-1)K(-1)) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

    DOI PubMed

    Scopus

    2
    Citation
    (Scopus)
  • DIFFERENTIATION AND PROLIFERATION OF XENOPUS HEMATOPOIETIC PROGENITOR CELLS IN THE FATTY BONE MARROW

    Takaki Aiso, Yuta Tanizaki, Yoko Mochizuki, Miku Fukunaga, Takashi Kato

    EXPERIMENTAL HEMATOLOGY   44 ( 9 ) S110 - S110  2016.09  [Refereed]

  • CHARACTERIZATION OF HEMATOPOIETIC STEM/PROGENITOR CELLS EXPANDED BY XLTPO STIMULATION IN XENOPUS

    Yuta Tanizaki, Yoko Mochizuki, Takaki Aiso, Atsuko Watarai, Takashi Kato

    EXPERIMENTAL HEMATOLOGY   44 ( 9 ) S102 - S102  2016.09  [Refereed]

  • VISUALIZATION OF NUCLEATED ERYTHROCYTES AND THROMBOCYTES IN THE CIRCULATION WITH TWO-PHOTON MICROSCOPE

    Haruka Ninao, Satoshi Nishimura, Asuka Sakata, Ayaka Murase, Yuta Tanizaki, Takashi Kato

    EXPERIMENTAL HEMATOLOGY   44 ( 9 ) S92 - S93  2016.09  [Refereed]

  • THE PROPORTION OF HEPATIC HEMATOPOIETIC PROGENITOR CELLS IN XENOPUS LAEVIS DURING SYSTEMIC REMODELING AT METAMORPHOSIS

    Mike Fukunaga, Akito Hirata, Yoko Mochizuki, Takaki Aiso, Yuta Tanizaki, Sakiko Hosozawa, Kei Sato, Takashi Kato

    EXPERIMENTAL HEMATOLOGY   44 ( 9 ) S71 - S72  2016.09  [Refereed]

  • Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR

    Sumako Kameishi, Terumasa Umemoto, Yu Matsuzaki, Masako Fujita, Teruo Okano, Takashi Kato, Masayuki Yamato

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   473 ( 3 ) 704 - 709  2016.05  [Refereed]

     View Summary

    Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. (C) 2015 Elsevier Inc. All rights reserved.

    DOI

    Scopus

    8
    Citation
    (Scopus)
  • Development of Erythroid Progenitors Under Erythropoietin Stimulation in Xenopus laevis Larval Liver

    Takehito Okui, Sakiko Hosozawa, Satoka Kohama, Shingo Fujiyama, Shun Maekawa, Hiroshi Muto, Takashi Kato

    ZOOLOGICAL SCIENCE   33 ( 6 ) 575 - 582  2016  [Refereed]

     View Summary

    Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre-(NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. beta 2-globin mRNA expression peaked at day 7 after the start of culture. Larval beta 2-globin was highly expressed in NF56-derived cells, while adult beta 2-globin was detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of epor and gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

    DOI

    Scopus

    5
    Citation
    (Scopus)
  • A comparative perspective on erythropoiesis

    KATO Takashi, MAEKAWA Shun, NAGASAWA Kazumichi, OKUI Takehito, TANIZAKI Yuta

    Rinsho Ketsueki   57 ( 7 ) 925 - 932  2016

     View Summary

    <p>The acquisition of fundamental information by the use of recent technologies, including omics-based molecular analyses and total RNA sequencing, has opened the door to further advances in physiological studies on new animal models. Currently, we are endeavoring to develop a comparative hematology protocol in order to build a discovery platform. All vertebrates, with the exception of a few species, have universally peripheral erythrocytes and hemoglobin, suggesting erythropoiesis to be an evolutionary index.</p>

    CiNii

  • アフリカツメガエルにおいてトロンボポエチンは肝臓での有核栓球の産生を誘導する

    谷崎 祐太, 一杉 芽美, 大淵-下地 美也子, 石田-岩田 貴子, 田原-茂木 彩香, 目黒-石川 瑞枝, 加藤 尚志

    比較内分泌学   42 ( 157 ) 9 - 11  2016

    DOI CiNii

  • Effect of solution drift on crystalline morphology in the solution growth of off-axis 4H-SiC crystals

    Takashi Kato, Kazuhiko Kusunoki, Kazuaki Seki, Nobuhiro Okada, Kazuhito Kamei

    Materials Science Forum   858   65 - 68  2016

     View Summary

    We investigated the effect of the solution flow on crystalline morphology in off-axis 4H-SiC solution growth. In particular, we focused on the relationship between the Si solution flow and step flow directions. In step parallel flow in which the solution drifted transversely to the step flow direction of the off-axis substrate, it was possible to attain a better surface morphology than when the solution drifted in the other direction. Furthermore, the surface morphology was found to be improved as the flow velocity increased. These improvements in morphological stability are presumed to be caused by the aligning of the solute concentration fluctuation along the steps.

    DOI

  • Development of Erythroid Progenitors Under Erythropoietin Stimulation in Xenopus laevis Larval Liver

    Takehito Okui, Sakiko Hosozawa, Satoka Kohama, Shingo Fujiyama, Shun Maekawa, Hiroshi Muto, Takashi Kato

    ZOOLOGICAL SCIENCE   33 ( 6 ) 575 - 582  2016  [Refereed]

     View Summary

    Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre-(NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. beta 2-globin mRNA expression peaked at day 7 after the start of culture. Larval beta 2-globin was highly expressed in NF56-derived cells, while adult beta 2-globin was detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of epor and gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

    DOI

    Scopus

    5
    Citation
    (Scopus)
  • Thrombopoietin induces production of nucleated thrombocytes from liver cells in Xenopus laevis

    Yuta Tanizaki, Megumi Ichisugi, Miyako Obuchi-Shimoji, Takako Ishida-Iwata, Ayaka Tahara-Mogi, Mizue Meguro-Ishikawa, Takashi Kato

    SCIENTIFIC REPORTS   5  2015.12  [Refereed]

     View Summary

    The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated thrombocytes instead of platelets, the features of nucleated thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO (xlTPO). Hepatic and splenic cells were cultured in liquid suspension with recombinant xlTPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X. laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xlTPO produced mature, spindle-shaped thrombocytes that were activated by thrombin, thereby altering their morphology. XlTPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xlTPO enhanced viability of peripheral thrombocytes, indicating the xlTPO-Mpl pathway stimulates anti-apoptotic in peripheral thrombocytes. The development of thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200).

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  • The Influence of Artificially Introduced N-Glycosylation Sites on the In Vitro Activity of Xenopus laevis Erythropoietin

    Kazumichi Nagasawa, Mizue Meguro, Kei Sato, Yuta Tanizaki, Nami Nogawa-Kosaka, Takashi Kato

    PLOS ONE   10 ( 4 )  2015.04  [Refereed]

     View Summary

    Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1-3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR.

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  • Remodeling of epithelial cells and basement membranes in a corneal deficiency model with long-term follow-up

    Sumako Kameishi, Hiroaki Sugiyama, Masayuki Yamato, Yoshikazu Sado, Hideo Namiki, Takashi Kato, Teruo Okano

    Laboratory Investigation   95 ( 2 ) 168 - 179  2015.03

     View Summary

    The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.

    DOI PubMed

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  • Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies

    Yuta Tanizaki, Takako Ishida-Iwata, Miyako Obuchi-Shimoji, Takashi Kato

    Experimental Hematology   43 ( 2 ) 125 - 136  2015.02

     View Summary

    Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12+ thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12+ thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.

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  • Phylogeny and ontogeny of erythropoiesis

    Wataru Nunomura, Aurora M. Cianciarullo, Takashi Kato, Ritsuko Shimizu, Malgorzata Witeska

    BioMed Research International   2015  2015

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  • Correction: Transcription profiles of endothelial cells in the rat ductus arteriosus during a perinatal period (PLoS ONE (2013) 8, 9, (e73685) DOI: 10.1371/journal.pone.0073685)

    Norika Mengchia Liu, Tomohiro Yokota, Shun Maekawa, Ping Lü, Yun Wen Zheng, Hideki Taniguchi, Utako Yokoyama, Takashi Kato, Susumu Minamisawa

    PLoS ONE   9 ( 1 )  2014.01

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  • The therapeutic potential of a novel PSMA antibody and its IL-2 conjugate in prostate cancer

    Yoshiyuki Sugimoto, Maiko Hirota, Kazuhiro Yoshikawa, Makoto Sumitomo, Kogenta Nakamura, Ryuzo Ueda, Rinpei Niwa, Toshiyuki Suzawa, Motoo Yamasaki, Kenya Shitara, Takashi Kato, Kazuyasu Nakamura

    Anticancer Research   34 ( 1 ) 89 - 97  2014.01

     View Summary

    Prostate-specific membrane antigen (PSMA) is an attractive target for treatment of prostate cancer. Using the PSMA-recognizing mouse monoclonal antibody 2C9 obtained in our previous study, the biological activities of PSMA antibody were evaluated. Mouse-human chimeric IgG1 of 2C9 (KM2777) showed antibody-dependent cellular cytotoxicity activity against PSMA-expressing prostate cancer cells in the presence of human peripheral blood mononuclear cells (PBMCs). To increase lymphocyte-mediated cytotoxicity of KM2777, C-terminus interleukin-2 (IL-2)-fused KM2777 (KM2812) was constructed. KM2812 retained binding activity to PSMA and exhibited growth-stimulating activity equivalent to IL-2 on the IL-2-dependent T-cell line CTLL-2. Moreover, KM2812 exhibited enhanced cytotoxic activity against PSMA-expressing prostate cancer cells in the presence of PBMCs compared with KM2777. In a xenograft tumor model using PSMA-expressing prostate cancer cells, KM2812 exhibited marked antitumor activity, accompanied by complete regression of tumor in some of the KM2812-treated mice. These results suggest that KM2812 has a therapeutic potential for prostate cancer by stimulating lymphocyte-mediated antitumor cytotoxicity.

    PubMed

  • [Intestinal bleeding in patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors].

    Makoto Saito, Koh Izumiyama, Akio Mori, Tatsuro Irie, Masanori Tanaka, Masanobu Morioka, Akiyoshi Saga, Manabu Musashi, Takashi Kato, Takashi Meguro, Mishie Tanino

    [Rinshō ketsueki] The Japanese journal of clinical hematology   55 ( 1 ) 130 - 132  2014.01

     View Summary

    Tyrosine kinase inhibitors (TKIs) are highly effective in the treatment of chronic myelogenous leukemia (CML), but there have been a few adverse event reports describing gastrointestinal bleeding. We clinically analyzed two patients who developed intestinal bleeding during the administration of TKIs for CML. Platelet counts of both patients were normal. The patients showed endoscopic findings characterized by mildly hemorrhagic mucosa. The imatinib patient was diagnosed by capsule endoscopy of the small intestine, and required frequent blood transfusions. The dasatinib patient showed occult bleeding due to CD8-positive colitis. We should adequately recognize that gastrointestinal bleeding may occur during the administration of TKIs.

    PubMed

  • Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen

    Yusuke Yoshioka, Nobuyoshi Kosaka, Yuki Konishi, Hideki Ohta, Hiroyuki Okamoto, Hikaru Sonoda, Ryoji Nonaka, Hirofumi Yamamoto, Hideshi Ishii, Masaki Mori, Koh Furuta, Takeshi Nakajima, Hiroshi Hayashi, Hajime Sugisaki, Hiroko Higashimoto, Takashi Kato, Fumitaka Takeshita, Takahiro Ochiya

    Nature communications   5   3591  2014

     View Summary

    Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.

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  • Adult-Specific Systemic Over-Expression Reveals Novel In Vivo Effects of the Soluble Forms of ActRIIA, ActRIIB and BMPRII

    Kengo Yamawaki, Shinobu Ueda, Tsutomu Okada, Takeshi Oshima, Makoto Kakitani, Takashi Kato, Kazuma Tomizuka

    PLoS ONE   8 ( 10 )  2013.10

     View Summary

    Bone morphogenetic proteins (BMPs)/growth differentiation factors (GDFs), which belong to the TGF-beta superfamily, are pleiotropic factors that play a role in regulating the embryonic development and postnatal homeostasis of various organs and tissues by controlling cellular differentiation, proliferation and apoptosis. Conventional transgenic and knockout (KO) mouse approaches have provided only limited information regarding the in vivo functions of BMP signaling in adult animals due to the effects on prenatal development and the difficulty in manipulating multiligand signals simultaneously. We recently produced transgenic chimeric mice(Tg chimeras) in which the soluble IgG1-Fc fusion protein of three BMP type II receptors (ActRIIA, ActRIIB, BMPRII) was highly circulated (281-709 μg/ml), specifically in adult mouse blood. Since each BMP receptor can bind to multiple BMP ligands, these Tg chimeras should be useful to investigate the effects of trapping multiple BMP ligands. Remarkably, some phenotypes were unexpected based on previous studies, such as KO mouse analyses, presumably representing the effects of the multiple ligand trapping. These phenotypes included increased red blood cells (RBCs) and decreased viability in adults. In a further study, we focused on the phenotype of increased RBCs and found that extramedullary hematopoiesis in the spleen, not in the bone marrow, was increased using histological and flow cytometric analyses. Although it remains to be elucidated whether the transgene products affect the tissues directly or indirectly, our data provide novel and important insight into the biological functions of the soluble IgG1-Fc fusion protein of three BMP type II receptors in adults, and our approach should have broad applications to research on other ligand receptor families and studies involving mouse models. © 2013 Yamawaki et al.

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  • Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

    Kazumichi Nagasawa, Yuta Tanizaki, Takehito Okui, Atsuko Watarai, Shinobu Ueda, Takashi Kato

    Biology Open   2 ( 10 ) 1057 - 1069  2013.10

     View Summary

    The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

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  • Transcription Profiles of Endothelial Cells in the Rat Ductus Arteriosus during a Perinatal Period

    Norika Mengchia Liu, Tomohiro Yokota, Shun Maekawa, Ping Lü, Inbun Tei, Hideki Taniguchi, Utako Yokoyama, Takashi Kato, Susumu Minamisawa

    PLoS ONE   8 ( 9 )  2013.09

     View Summary

    Endothelial cells (ECs) lining the blood vessels serve a variety of functions and play a central role in the homeostasis of the circulatory system. Since the ductus arteriosus (DA) has different arterial characteristics from its connecting vessels, we hypothesized that ECs of the DA exhibited a unique gene profile involved in the regulation of DA-specific morphology and function. Using a fluorescence-activated cell sorter, we isolated ECs from pooled tissues from the DA or the descending aorta of Wistar rat fetuses at full-term of gestation (F group) or neonates 30 minutes after birth (N group). Using anti-CD31 and anti-CD45 antibodies as cell surface markers for ECs and hematopoietic derived cells, respectively, cDNAs from the CD31-positive and CD45-negative cells were hybridized to the Affymetrix GeneChip® Rat Gene 1.0 ST Array. Among 26,469 gene-level probe sets, 82 genes in the F group and 81 genes in the N group were expressed at higher levels in DA ECs than in aortic ECs (p<0.05, fold change>2.0). In addition to well-known endothelium-enriched genes such as Tgfb2 and Vegfa, novel DA endothelium-dominant genes including Slc38a1, Capn6, and Lrat were discovered. Enrichment analysis using GeneGo MetaCore software showed that DA endothelium-related biological processes were involved in morphogenesis and development. We identified many overlapping genes in each process including neural crest-related genes (Hoxa1, Hoxa4, and Hand2, etc) and the second heart field-related genes (Tbx1, Isl1, and Fgf10, etc). Moreover, we found that regulation of epithelial-to-mesenchymal transition, cell adhesion, and retinol metabolism are the active pathways involved in the network via potential interactions with many of the identified genes to form DA-specific endothelia. In conclusion, the present study uncovered several significant differences of the transcriptional profile between the DA and aortic ECs. Newly identified DA endothelium-dominant genes may play an important role in DA-specific functional and morphologic characteristics. © 2013 Liu et al.

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  • Platelet-Derived Stromal Cell-Derived Factor-1 Is Required for the Transformation of Circulating Monocytes into Multipotential Cells

    Noriyuki Seta, Yuka Okazaki, Hiroshi Miyazaki, Takashi Kato, Masataka Kuwana

    PLoS ONE   8 ( 9 )  2013.09

     View Summary

    Background:We previously described a primitive cell population derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMCs), which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s) derived from circulating CD14- cells. The present study was conducted to identify factors that induce MOMC differentiation.Methods:We cultured CD14+ monocytes on fibronectin in the presence or absence of platelets, CD14- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages.Results:The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF)-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1′s critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14+CXCR4high cell population.Conclusion:The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs. © 2013 Seta et al.

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  • Quantification and localization of erythropoietin-receptor-expressing cells in the liver of Xenopus laevis

    Takehito Okui, Yusuke Yamamoto, Shun Maekawa, Kazumichi Nagasawa, Yuka Yonezuka, Youichi Aizawa, Takashi Kato

    Cell and Tissue Research   353 ( 1 ) 153 - 164  2013.07

     View Summary

    Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2′-deoxyuridine (BrdU). We detected 0.12×108 xlEPOR+ BrdU+ cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR+/ BrdU+ cells (approximately 1.30×108 cells) in the liver. The xlEPOR+ cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia. © 2013 Springer-Verlag Berlin Heidelberg.

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  • Enhanced erythropoiesis in mice exposed to low environmental temperature

    Shun Maekawa, Hitomi Iemura, Takashi Kato

    Journal of Experimental Biology   216 ( 5 ) 901 - 908  2013.03

     View Summary

    Hematopoietic responses to environmental factors are not fully characterized. Polycythemia has been reported during exposure to low temperatures in ectothermic animals. The relationship between the causes of polycythemia and erythropoiesis during low temperature exposure is not fully understood. In this study, we exposed C57BL/6 mice to 5°C and monitored the blood cell counts and erythropoiesis. The hematocrit level increased from 45.6 to 52.2% after 14 days. Likewise, the hemoglobin concentration, initially 15.1 g dl-1, rose to 16.0 g dl-1. The reticulocyte production index significantly increased from 4 to 8% after 7 days. We examined the anatomy and cell composition of the spleens of the mice. On day5, the spleens were ̃6 mg g-1 of body mass, which was twofold greater than the spleens on day0. Flow cytometry showed fourfold more proerythroblasts on day5, compared with day0. Additionally, the number of late-stage mature erythroblasts increased on day14. Erythropoietin mRNA levels increased in the kidneys, and hypoxia-inducible genes were enhanced in the kidney. Our findings indicated that low ambient temperature is a novel erythropoietic stress, which induces polycythemia by enhanced erythropoiesis. © 2013. Published by The Company of Biologists Ltd.

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  • Comparative marker analysis of extracellular vesicles in different human cancer types

    Yusuke Yoshioka, Yuki Konishi, Nobuyoshi Kosaka, Takeshi Katsuda, Takashi Kato, Takahiro Ochiya

    Journal of Extracellular Vesicles   2 ( 1 )  2013

     View Summary

    Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived EVs play a role in cancer initiation and progression. These vesicles include both a common set of membrane and cytosolic proteins and origin-specific subsets of proteins that likely correlated to cell type-associated functions. To confirm the presence of EVs in the preparations, researchers have identified so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumour susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicates the identification of EVs but also precludes the quantitative evaluation of EV proteins. Thus, it is strongly required to explore well-conserved EV marker proteins that are present at similar levels, regardless of their tissue or cellular origin. In this study, we compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes. We found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, we propose that the detection of CD9 and/or CD81 should ensure the presence of EVs. © 2013 Yusuke Yoshioka et al.

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  • Micromanaging iron homeostasis: Hypoxia-inducible micro-RNA-210 suppresses iron homeostasis-related proteins

    Yusuke Yoshioka, Nobuyoshi Kosaka, Takahiro Ochiya, Takashi Kato

    Journal of Biological Chemistry   287 ( 41 ) 34110 - 34119  2012.10

     View Summary

    Iron is fundamental for sustaining life for living organisms, and the iron metabolism is finely regulated at different levels. In cancer cells, deregulation of the iron metabolism induces oxidative stress and drives tumor progression and metastasis; however, the molecular mechanisms of iron homeostasis are not fully understood. Here we found that iron deficiency as well as hypoxia promoted microRNA-210 (miR-210) expression. A central mediator of miR-210 transcriptional activation is the hypoxia-inducible factor (HIF)-1α, and the hypoxia-response element in the miR-210 promoter is confirmed experimentally. This is in agreement with the data from in vivo studies that have demonstrated the presence of miR-210-expressing cells at the chronic hypoxic regions of xenografted tumors. Furthermore we found two essential molecules for iron homeostasis, iron-sulfur cluster scaffold protein (ISCU) and transferrin receptor 1 (TfR), are a direct target of miR-210. Transfection ofmiR-210decreases the uptake of transferrin by inhibiting the expression of TfR. In addition, inhibition of miR-210 by anti-miR-210 up-regulates ISCU expression. These findings suggest that miR-210 works as an iron sensor and is involved in the maintenance of iron homeostasis by sustaining the TfR expression level to stimulate cell proliferation and promote cell survival in the hypoxic region within tumors. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • Hepatic confinement of newly produced erythrocytes caused by low-temperature exposure in Xenopus laevis

    Shun Maekawa, Hitomi Iemura, Yuko Kuramochi, Nami Nogawa-Kosaka, Hironori Nishikawa, Takehito Okui, Youichi Aizawa, Takashi Kato

    Journal of Experimental Biology   215 ( 17 ) 3087 - 3095  2012.09

     View Summary

    Diminished erythrocyte count and erythropoiesis have been reported during hypothermia in some ectothermic animals. In this study, the African clawed frog, Xenopus laevis, was used to investigate the cause of hypothermia-induced anemia. We developed a new model of hypothermia at 5°C and monitored blood cell count and erythropoiesis on several days. Erythrocyte count declined by 30% on the first day following cold exposure (5°C) and mRNA expression of hemeoxygenase-1 was enhanced 10-fold; accumulation of iron as a result of heme degradation was observed in the liver. One day after low-temperature exposure, erythropoietin mRNA expression was elevated in the liver and lung compared with that at normal temperature (22°C) by qRT-PCR analysis. Examination of liver sections (i.e. the erythropoietic organ) showed an increase in odianisidine-positive erythrocytes in the hepatic sinusoid 5 days after the onset of low-temperature exposure compared with normal liver. Peripheral erythrocyte count remained low, indicating that newly produced erythrocytes did not migrate from the liver to the circulation during hypothermia. In conclusion, this study reveals hypothermic anemia as being associated with hepatic erythrocyte destruction; prolonged anemia during low-temperature exposure is concomitant with newly produced erythrocytes being confined to the liver and may lead to new insights into vertebrate hematopoiesis. © 2012. Published by The Company of Biologists Ltd.

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  • Synthesis and bioassay of a boron-dipyrromethene derivative of estradiol for fluorescence imaging in vivo

    Mayumi Okamoto, Shun Kobayashi, Hiroshi Ikeuchi, Shunji Yamada, Korehito Yamanouchi, Kazumichi Nagasawa, Shun Maekawa, Takashi Kato, Isao Shimizu

    Steroids   77 ( 8-9 ) 845 - 849  2012.07

     View Summary

    C7α-substituted estradiols bind to estrogen receptors in cell nuclei, yet these derivatives remain little used in bioimaging. Here, we describe a fluorescent derivative of estradiol (E2) with a boron-dipyrromethene (BODIPY) moiety attached to C7α, synthesized by olefin metathesis reaction of 7α-allylestradiol and 9-decenyl-BODIPY. In ovariectomized rats and non-ovariectomized mice, E2-BODIPY promoted the growth of uterine tissue similar to the effect of estradiol. Twenty-four hours after subcutaneous injection of E2-BODIPY in non-ovariectomized mice, we observed fluorescence of E2-BODIPY in the nuclei of uterine epithelial cells. Our findings suggest that fluorescence microscopy can localize this derivative in E2-responsive cells during normal development and tumorigenesis in vivo. © 2012 Elsevier Inc. All rights reserved.

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    16
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  • Fibronectin binding is required for acquisition of mesenchymal/endothelial differentiation potential in human circulating monocytes

    Noriyuki Seta, Yuka Okazaki, Keisuke Izumi, Hiroshi Miyazaki, Takashi Kato, Masataka Kuwana

    Clinical and Developmental Immunology   2012  2012

     View Summary

    We previously reported monocyte-derived multipotential cells (MOMCs), which include progenitors capable of differentiating into a variety of mesenchymal cells and endothelial cells. In vitro generation of MOMCs from circulating CD14+ monocytes requires their binding to extracellular matrix (ECM) protein and exposure to soluble factor(s) derived from circulating CD14 - cells. Here, we investigated the molecular factors involved in MOMC generation by examining the binding of monocytes to ECM proteins. We found that MOMCs were obtained on the fibronectin, but not on type I collagen, laminin, or poly-L-lysine. MOMC generation was followed by changes in the expression profiles of transcription factors and was completely inhibited by either anti-α5 integrin antibody or a synthetic peptide that competed with the RGD domain for the β1-integrin binding site. These results indicate that acquisition of the multidifferentiation potential by circulating monocytes depends on their binding to the RGD domain of fibronectin via cell-surface α5β1 integrin. © 2012 Noriyuki Seta et al.

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  • Implication of Molecular Diversity and Functional Conservation of Erythropoietin Based on a Comparison of Tertiary Structures of Humans and Frogs

    Meguro Mizue, Adachi Motoyasu, Nagasawa Kazumichi, Beppu Miho, Okazaki Nobuo, Nogawa-Kosaka Nami, Tamada Taro, Kuroki Ryota, Kato Takashi

    BLOOD   118 ( 21 ) 1455  2011.11  [Refereed]

  • An integrative genomic analysis revealed the relevance of microRNA and gene expression for drug-resistance in human breast cancer cells

    Yusuke Yoshioka, Kaho Minoura, Ryou u. Takahashi, Fumitaka Takeshita, Toshiki Taya, Reiko Horii, Yayoi Fukuoka, Takashi Kato, Nobuyoshi Kosaka, Takahiro Ochiya

    Molecular Cancer   10  2011.11

     View Summary

    Background: Acquisition of drug-resistance in cancer has led to treatment failure, however, their mechanisms have not been clarified yet. Recent observations indicated that aberrant expressed microRNA (miRNA) caused by chromosomal alterations play a critical role in the initiation and progression of cancer. Here, we performed an integrated genomic analysis combined with array-based comparative hybridization, miRNA, and gene expression microarray to elucidate the mechanism of drug-resistance.Results: Through genomic approaches in MCF7-ADR; a drug-resistant breast cancer cell line, our results reflect the unique features of drug-resistance, including MDR1 overexpression via genomic amplification and miRNA-mediated TP53INP1 down-regulation. Using a gain of function study with 12 miRNAs whose expressions were down-regulated and genome regions were deleted, we show that miR-505 is a novel tumor suppressive miRNA and inhibits cell proliferation by inducing apoptosis. We also find that Akt3, correlate inversely with miR-505, modulates drug sensitivity in MCF7-ADR.Conclusion: These findings indicate that various genes and miRNAs orchestrate to temper the drug-resistance in cancer cells, and thus acquisition of drug-resistance is intricately controlled by genomic status, gene and miRNA expression changes. © 2011 Yamamoto et al; licensee BioMed Central Ltd.

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  • Identification of erythroid progenitors induced by erythropoietic activity in Xenopus laevis

    Nami Nogawa-Kosaka, Tatsuhisa Sugai, Kazumichi Nagasawa, Yuta Tanizaki, Mizue Meguro, Youichi Aizawa, Shun Maekawa, Motoyasu Adachi, Ryota Kuroki, Takashi Kato

    Journal of Experimental Biology   214 ( 6 ) 921 - 927  2011.03

     View Summary

    Oxygen is essential for the survival of animals. Red blood cells in the circulation, i.e. peripheral erythrocytes, are responsible for transporting oxygen to tissues. The regulation of erythropoiesis in vertebrates other than mammals is yet to be elucidated. Recently we identified erythropoietin, a primary regulator of erythropoiesis, in Xenopus laevis, which should enable us to identify target cells, including erythroid progenitors, and to investigate the production and development of erythroid cells in amphibians. Here, we established a semi-solid colony-forming assay in Xenopus laevis to clarify the existence of colony-forming unit-erythroid cells, the functional erythroid progenitors identified in vitro. Using this assay, we showed that recombinant xlEPO induces erythroid colony formation in vitro and detected an increased level of erythropoietin activity in blood serum during acute anemic stress. In addition, our study demonstrated the possible presence of multiple, non-x/EPO, factors in anemic serum supportive of erythroid colony formation. These results indicate that erythropoiesis mediated by erythropoietin is present in amphibian species and, furthermore, that the regulatory mechanisms controlling peripheral erythrocyte number may vary among vertebrates. © 2011. Published by The Company of Biologists Ltd.

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  • 3B1012 Activation mechanism of thrombopoietin receptor investigated by its specific ligand and neutralization antibodies(3B Membrane proteins 2,The 49th Annual Meeting of the Biophysical Society of Japan)

    Matsumoto Fumiko, Adachi Motoyasu, Shimizu Rumi, Meguro Mizue, Arai Shigeki, Tamada Taro, Kato Takashi, Kuroki Ryota

    Seibutsu Butsuri   51   S109  2011

    DOI CiNii

  • Single molecule dynamics of the thrombopoietin receptor on the plasma membrane

    Akihiko Sakamoto, Takashi Kato, Takashi Funatsu

    Seikagaku   83 ( 10 ) 912 - 919  2011

    PubMed

  • Structural and biological properties of erythropoietin in Xenopus laevis

    Nami Nogawa-Kosaka, Takayuki Hirose, Nobuyoshi Kosaka, Youichi Aizawa, Kazumichi Nagasawa, Nobuaki Uehara, Hiroshi Miyazaki, Norio Komatsu, Takashi Kato

    Experimental Hematology   38 ( 5 ) 363 - 372  2010.05

     View Summary

    Objective: Erythropoietin (EPO) and its receptor (EPOR) are key regulators of red blood cell production in mammals and fish. We aimed to investigate the structural and functional conservation of the EPO-EPOR system in amphibian erythropoiesis, using Xenopus laevis as a model. Materials and Methods: X. laevis epo (xlepo) complementary DNA was identified by referring to the Xenopus tropicalis genome database. Biological activity of recombinant xlEPO expressed in COS-1 cells was evaluated using xlEPOR-expressing murine FDC/P2 cells and human EPO-dependent UT-7/EPO cells. Expression of xlepo messenger RNA in adult X. laevis tissues in the normal state and under the condition of phenylhydrazine-induced anemia was evaluated by real-time reverse transcription polymerase chain reaction. Results: In the encoded protein, the positions of four cysteine residues were conserved; however, xlEPO had only 38% identity with human EPO. N-glycosylation sites were absent. Recombinant xlEPO induced proliferation of cell lines expressing xlEPOR and UT-7/EPO, confirming biological activity and cross-species reactivity. Despite little primary amino acid sequence similarity, the evolutionary highly conserved sequence NFLRGK was identified in the EPOR-binding site 1 region as in the human EPO protein. Strong expression of xlepo messenger RNA was detected in the lung and liver, especially in fractionated hepatocytes. No marked increase in xlepo expression was seen in the lung and liver of phenylhydrazine-induced anemic X. laevis. Conclusion: We confirmed that xlEPO is the ligand to the previously reported xlEPOR in X. laevis. xlEPO shares structural and functional similarities and differences with mammalian counterparts, and regulation of xlepo expression and its influence on the erythropoietic system appears to be unique. © 2010 ISEH - Society for Hematology and Stem Cells.

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  • Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure

    Agnieszka Banas, Takumi Teratani, Yusuke Yamamoto, Makoto Tokuhara, Fumitaka Takeshita, Mitsuhiko Osaki, Takashi Kato, Hitoshi Okochi, Takahiro Ochiya

    Journal of Gastroenterology and Hepatology (Australia)   24 ( 1 ) 70 - 77  2009.01

     View Summary

    Background and Aim: Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo. Methods: In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum. Results: We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation. Conclusions: Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo. Thus, ASC may be a superior choice for the establishment of a therapy for injured liver. © 2008 The Authors.

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  • MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma

    Yusuke Yamamoto, Nobuyoshi Kosaka, Minoru Tanaka, Fumiaki Koizumi, Yae Kanai, Takayuki Mizutani, Yoshiki Murakami, Masahiko Kuroda, Atsushi Miyajima, Takashi Kato, Takahiro Ochiya

    Biomarkers   14 ( 7 ) 529 - 538  2009

     View Summary

    We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC. © 2009 Informa UK Ltd.

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  • IFATS collection: In vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury

    Agnieszka Banas, Takumi Teratani, Yusuke Yamamoto, Makoto Tokuhara, Fumitaka Takeshita, Mitsuhiko Osaki, Masaki Kawamata, Takashi Kato, Hitoshi Okochi, Takahiro Ochiya

    Stem Cells   26 ( 10 ) 2705 - 2712  2008.10

     View Summary

    Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adipose-derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT-MSCs by their transplantation into nude mice with CCl4-caused liver injury. We observed that after transplantation, AT-MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations returned to a nearly normal level after AT-MSC transplantation. These results raised the question of how AT-MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT-MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM-MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT-MSCs secrete interleukin 1 receptor α (IL-1Rα), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BMMSCs and NHDFs. Thus, our findings suggest that ATMSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment. ©AlphaMed Press.

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  • Identification of erythropoietin-induced microRNAs in haematopoietic cells during erythroid differentiation

    Nobuyoshi Kosaka, Keiichi Sugiura, Yusuke Yamamoto, Yusuke Yoshioka, Hiroshi Miyazaki, Norio Komatsu, Takahiro Ochiya, Takashi Kato

    British Journal of Haematology   142 ( 2 ) 293 - 300  2008.07

     View Summary

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through mRNA degradation or translation inhibition. It has not yet been clearly elucidated whether miRNAs participate in haematopoietic processes such as cell differentiation, apoptosis and maintenance of adequate levels of circulating blood cells. A human miRNA microarray was used to analyze miRNA expression in the erythropoietin-dependent cell line UT-7/EPO compared to other factor-dependent UT-7 cell lines. Among 324 human miRNAs, MIRN188, MIRN362 and MIRN210 levels were significantly elevated in UT-7/EPO cells, and stimulation with EPO in UT-7 cells increased the level of these three miRNAs. Notably, knockdown of MIRN210 in UT-7/EPO cells led to apoptosis. In mouse fetal liver cells, MIRN210 expression was twofold higher in TER-119-positive cells than in TER-119-negative cells. The expression of MIRN210 was elevated during erythroid maturation in vitro. These data suggest MIRN210 to be a member a new class of regulatory miRNAs that might play an important role in erythroid maturation. © 2008 The Authors.

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  • A comparative analysis of the transcriptome and signal pathways in hepatic differentiation of human adipose mesenchymal stem cells

    Yusuke Yamamoto, Agnieszka Banas, Shigenori Murata, Madoka Ishikawa, Chun R. Lim, Takumi Teratani, Izuho Hatada, Kenichi Matsubara, Takashi Kato, Takahiro Ochiya

    FEBS Journal   275 ( 6 ) 1260 - 1273  2008.03

     View Summary

    The specific features of the plasticity of adult stem cells are largely unknown. Recently, we demonstrated the hepatic differentiation of human adipose tissue-derived mesenchymal stem cells (AT-MSCs). To identify the genes responsible for hepatic differentiation, we examined the gene expression profiles of AT-MSC-derived hepatocytes (AT-MSC-Hepa) using several microarray methods. The resulting sets of differentially expressed genes (1639 clones) were comprehensively analyzed to identify the pathways expressed in AT-MSC-Hepa. Clustering analysis revealed a striking similarity of gene clusters between AT-MSC-Hepa and the whole liver, indicating that AT-MSC-Hepa were similar to liver with regard to gene expression. Further analysis showed that enriched categories of genes and signaling pathways such as complementary activation and the blood clotting cascade in the AT-MSC-Hepa were relevant to liver-specific functions. Notably, decreases in Twist and Snail expression indicated that mesenchymal-to-epithelial transition occurred in the differentiation of AT-MSCs into hepatocytes. Our data show a similarity between AT-MSC-Hepa and the liver, suggesting that AT-MSCs are modulated by their environmental conditions, and that AT-MSC-Hepa may be useful in basic studies of liver function as well as in the development of stem cell-based therapy. © 2008 The Authors.

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  • Fluorescence labeling of a cytokine with desthiobiotin-tagged fluorescent puromycin

    Akihiko Sakamoto, Mai Yamagishi, Takafumi Watanabe, Youichi Aizawa, Takashi Kato, Takashi Funatsu

    Journal of Bioscience and Bioengineering   105 ( 3 ) 238 - 242  2008.03

     View Summary

    Fluorescence labeling of a cytokine at a specific site is required for observing cytokine-receptor interactions in living cells at the single-molecule level. Here, we demonstrated the C-terminus-specific fluorescence labeling of histidine-tagged thrombopoietin (TPO), a ligand for Mpl, with desthiobiotin-tagged fluorescent puromycin. Fluorescent TPO, purified by tandem affinity purification, stimulated the proliferation of Mpl-expressing cells. Within 10 min of its addition, fluorescent TPO was found to be diffusely distributed on the cell membranes of Mpl-expressing cells, and gradually accumulated to form fluorescent spots. This method is applicable for studying the spatial and temporal distributions of cytokines in individual living cells. © 2008 The Society for Biotechnology, Japan.

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  • Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor

    Tomoaki Nakada, Yoko Ishizuka, Takeo Iwata, Fumiyo Toyoda, Takashi Kato, J. Michael Conlon, Sakae Kikuyama

    Zoological Science   24 ( 5 ) 521 - 524  2007.05

     View Summary

    Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg 176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males. © 2007 Zoological Society of Japan.

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    4
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  • Isolation, characterization and bioactivity of a region-specific pheromone, [Val8]sodefrin from the newt Cynops pyrrhogaster

    Tomoaki Nakada, Fumiyo Toyoda, Takeo Iwata, Kazutoshi Yamamoto, J. Michael Conlon, Takashi Kato, Sakae Kikuyama

    Peptides   28 ( 4 ) 774 - 780  2007.04

     View Summary

    Previous analysis of PCR products derived from total RNA from the abdominal gland of the male newt, Cynops pyrrhogaster, inhabiting the Nara area of Japan led to the identification of a gene encoding [Val8]sodefrin, as well as the female-attracting peptide pheromone, sodefrin. In this study, purification of this sodefrin variant from the abdominal glands of male newts from the Nara area was accomplished using gel-filtration chromatography and reversed-phase HPLC. Amino acid sequence analysis and mass spectrometry confirmed that the final product was [Val8]sodefrin. A full-length cDNA encoding the biosynthetic precursor of [Val8]sodefrin was cloned and characterized. The deduced amino acid sequence of prepro[Val8]sodefrin showed 86.2% identity with that of the sodefrin precursor. The [Val8]sodefrin variant potently attracted females from the Nara area, but the variant was much less or not effective in attracting females captured in the Niigata and Chiba areas. The term aonirin ("aoni" from "aoni-yoshi", the conventional epithet of Nara) is proposed to designate this region-specific pheromone. It is speculated that the coevolution of a novel pheromone and its complementary receptor in the Nara newts may lead to reproductive isolation and eventual differentiation into a separate species. © 2006 Elsevier Inc. All rights reserved.

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  • Characterization of lobulated fibers in limb girdle muscular dystrophy type 2A by gene expression profiling

    Yoko Keira, Satoru Noguchi, Rumi Kurokawa, Masako Fujita, Narihiro Minami, Yukiko K. Hayashi, Takashi Kato, Ichizo Nishino

    Neuroscience Research   57 ( 4 ) 513 - 521  2007.04

     View Summary

    Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in CAPN3, which encodes an intracellular cysteine protease. To elucidate the fundamental molecular changes that may be responsible for the pathological features of LGMD2A, we employed cDNA microarray analysis. We divided LGMD2A muscles into two groups according to specific pathological features: an early-stage group characterized by the presence of active necrosis and a regeneration process and a later-stage group characterized by the presence of lobulated fibers. After comparing the gene expression profiles of the two groups of LGMD2A muscles with control muscles, we identified 29 genes whose mRNA expression profiles were specifically altered in muscles with lobulated fibers. Interestingly, this group included genes that encode actin filament binding and regulatory proteins, such as gelsolin, PDZ and LIM domain 3 (PDLIM3) and troponin I1. Western blot analysis confirmed the upregulation of these proteins. From these results, we propose that abnormal increased expression of actin filament binding proteins may contribute to the changes of the intra-myofiber structures, observed in lobulated fibers in LGMD2A. © 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society.

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    18
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  • マイクロRNAmiR-10a一次転写産物によるUT-7/EPO細胞の増殖能の増大

    小坂 展慶, 山本 雄介, 野川 菜美, 杉浦 圭一, 落谷 孝広, 小松 則夫, 宮崎 洋, 加藤 尚志

    臨床血液   47 ( 9 ) 1220 - 1220  2006.09  [Refereed]

  • FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation

    Nobuyoshi Kosaka, Maho Kodama, Hideo Sasaki, Yusuke Yamamoto, Fumitaka Takeshita, Yasushi Takahama, Hiromi Sakamoto, Takashi Kato, Masaaki Terada, Takahiro Ochiya

    FASEB Journal   20 ( 9 )  2006.07

     View Summary

    The FGF-4 (fibroblast growth factor 4, known as HST-1) protein is an important mitogen for a variety of cell types. However, only limited information is available concerning tissue distribution and the biological role of FGF-4 in the brain. In situ hybridization analysis revealed localization of mouse Fgf-4 mRNA in the normal postnatal mouse hippocampus, subventricular zone (SVZ), and the rostral migratory stream where new neurons generate, migrate, and become incorporated into the functional circuitry of the brain. We also investigated whether FGF-4 could promote both proliferation and differentiation of the neural progenitor cells by using an in vitro neurosphere assay. The addition of recombinant FGF-4 generated large proliferative spheres that have a multipotent differentiation ability. Furthermore, recombinant FGF-4 significantly promotes neuronal differentiation in attached clonal neurosphere culture. These findings suggest that FGF-4 has an ability to promote neural stem cell proliferation and neuronal differentiation in the postnatal brain. © FASEB.

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  • Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells

    Yusuke Yamamoto, Takumi Teratani, Hanako Yamamoto, Gary Quinn, Sigenori Murata, Rieko Ikeda, Kenji Kinoshita, Kenichi Matsubara, Takashi Kato, Takahiro Ochiya

    Hepatology   42 ( 3 ) 558 - 567  2005.09

     View Summary

    Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3β/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se. Copyright © 2005 by the American Association for the Study of Liver Diseases.

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  • Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo

    Fumitaka Takeshita, Yoshiko Minakuchi, Shunji Nagahara, Kimi Honma, Hideo Sasaki, Kotaro Hirai, Takumi Teratani, Nachi Namatame, Yusuke Yamamoto, Koji Hanai, Takashi Kato, Akihiko Sano, Takahiro Ochiya

    Proceedings of the National Academy of Sciences of the United States of America   102 ( 34 ) 12177 - 12182  2005.08

     View Summary

    Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and/or legs. We also show that the siRNA/atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3′-hydroxykinase p110-α-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-α levels was not associated with the in vivo administration of the siRNA/atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA. © 2005 by The National Academy of Sciences of the USA.

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    347
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  • Expression of erythropoietin receptor-like molecule in Xenopus laevis and erythrocytopenia upon administration of its recombinant soluble form

    Youichi Aizawa, Nami Nogawa, Nobuyoshi Kosaka, Yasutaka Maeda, Takafumi Watanabe, Hiroshi Miyazaki, Takashi Kato

    Journal of Biochemistry   138 ( 2 ) 167 - 175  2005.08

     View Summary

    The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key mechanism of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. A cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, tentatively named xlEPOR, was cloned from a cDNA library of Xenopus laevis immature erythrocytes. The comparative identities of the deduced entire amino acid sequence to mammalian EPO receptors were quite low, although functional domains indispensable for erythropoietic activities were found in the molecule. Northern analysis revealed that xlEPOR were expressed in peripheral blood cells. In the peripheral blood of phenylhydrazine-treated adult Xenopus, immature erythrocytes expressing xlEPOR were identified by in situ hybridization and immunostaining with polyclonal antibodies to xlEPOR. To confirm the biological functions of this molecule, the extracellular domain of xlEPOR (i.e., soluble xlEPOR) was administered to adult Xenopus by consecutive intracardiac injection. The peripheral erythrocyte counts were decreased gradually; meanwhile, immature erythrocytes appeared in the circulation, demonstrating that xlEPOR plays a significant physiological role in erythropoiesis in Xenopus laevis. © 2005 The Japanese Biochemical Society.

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  • Decreased prothrombotic effects of pegylated recombinant human megakaryocyte growth and development factor in thrombocytopenic state in a rat thrombosis model

    U. Nishiyama, T. Kuwaki, H. Akahori, T. Kato, Y. Ikeda, Hiroshi Miyazaki

    Journal of Thrombosis and Haemostasis   3 ( 2 ) 355 - 360  2005.02

     View Summary

    Previous in vitro studies demonstrated that thrombopoietin (TPO) acts on platelets to activate a variety of intracellular signaling pathways and to enhance platelet sensitivity to multiple agonists. Little is known, however, about whether TPO exerts prothrombotic effects in vivo. The aim of this study was to examine the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a pegylated N-terminal domain of human TPO, in a rat model of venous thrombosis. A microthrombus was photochemically induced on the vessel wall of a mesenteric venule, but the vessel was not occluded by it. A single intravenous injection of PEG-rHuMGDF (3 μg kg -1) after the thrombus generation into normal rats enhanced the thrombus size, resulting in transient thrombotic occlusion in the majority of rats. Stimulatory effects on thrombus growth were also observed following administration of glycosylated recombinant human full-length TPO (6 μg kg-1). In rats rendered thrombocytopenic by total body irradiation, however, PEG-rHuMGDF, even at 300 μ kg-1, did not induce a significant increase in thrombus size or thrombotic occlusion. Platelets from thrombocytopenic rats had decreased surface levels of c-Mp1 and decreased sensitivity to PEG-rHuMGDF in an in vitro aggregation response. Thus, decreased prothrombotic effects of PEG-rHuMGDF in thrombocytopenic rats might be the result not only of low platelet counts but also of decreased platelet reactivity to PEG-rHuMGDF. These results indicate that PEG-rHuMGDF has little effect on venous thrombus formation in thrombocytopenic states associated with high endogenous TPO levels. © 2005 International Society on Thrombosis and Haemostasis.

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  • Pegylated recombinant human megakaryocyte growth and development factor suppresses the development of megakaryoblastic leukemia in mice

    Kazunori Shibuya, Tomoaki Kuwaki, Hiromichi Akahori, Takashi Kato, Hiroshi Miyazaki

    Leukemia Research   28 ( 9 ) 941 - 946  2004.09

     View Summary

    We examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on the development of L-8057, a murine megakaryoblastic leukemia that expresses the thrombopoietin receptor c-Mpl, in mice. PEG-rHuMGDF administration prolonged survival of L-8057 leukemic mice, in which L-8057 cell growth in the spleen was decreased. L-8057 cells harvested from PEG-rHuMGDF-treated leukemic mice had decreased ability to generate leukemic colonies in vitro as well as to induce leukemia in vivo. PEG-rHuMGDF administration also resulted in prolonged survival of mice transplanted with a c-Mpl-expressing erythroleukemia, but had no effect on survival of mice transplanted with a myeloblastic leukemia that does not possess c-Mpl. Thus, PEG-rHuMGDF suppresses the development of c-Mpl-expressing leukemia in vivo in mice. © 2004 Elsevier Ltd. All rights reserved.

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  • Expression of P450 aromatase protein in developing and in sex-reversed gonads of the XX/XY type of the frog Rana rugosa

    T. Kato, K. Matsui, M. Takase, M. Kobayashi, M. Nakamura

    General and Comparative Endocrinology   137 ( 3 ) 227 - 236  2004.07

     View Summary

    Gonadal differentiation in some species of amphibians is sensitive to steroids. The phenotypic sex of XX/XY-type frogs such as Rana rugosa can be reversed from female to male by injection of testosterone into tadpoles, but little is known about the molecular mechanism of this sex reversal. To elucidate the mechanism of the sex differentiation, we examined the role of P450 aromatase (P450arom), an enzyme that converts testosterone to estrogen, during gonadal differentiation of amphibians. In this study, we first cloned a P450arom cDNA homolog of the frog R. rugosa and analyzed by RT-PCR its expression profile in developing and in female-to-male sex-reversed gonads. P450arom expression was observed in the gonad of tadpoles during ovarian differentiation and became much stronger in the developing ovary in which only immature oocytes were observed. However, its expression declined significantly in the ovary of frogs 2 months after metamorphosis, when oocytes were growing; and it was no longer seen in adult ovaries. By RT-PCR, we also examined the expression of P450arom and SF-1 (steroidogenic factor-1; the orphan nuclear receptor) in the female-to-male sex-reversed gonad. The level of P450arom mRNA was high in the ovary, but it declined rapidly after the injection of testosterone. In contrast, no change in the SF-1 (also known as Ad4BP) expression was observed. Moreover, to identify the type(s) of cells expressing P450arom protein, we performed immunostaining with an antibody against frog P450arom protein. Cells giving positive signals were observed around oocytes in the ovary of frogs 1 month after metamorphosis. They were identified as follicle cells by both light and electron microscopy. The results, taken together, indicate that P450arom protein is synthesized in follicle cells and that P450arom is very much involved in ovarian differentiation in R. rugosa. © 2004 Elsevier Inc. All rights reserved.

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  • Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment.

    Feese MD, Tamada T, Kato Y, Maeda Y, Hirose M, Matsukura Y, Shigematsu H, Muto T, Matsumoto A, Watarai H, Ogami K, Tahara T, Kato T, Miyazaki H, Kuroki R

    Proceedings of the National Academy of Sciences of the United States of America   101 ( 7 ) 1816 - 1821  2004.02  [Refereed]

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  • Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo.

    Yoshiko Minakuchi, Fumitaka Takeshita, Nobuyoshi Kosaka, Hideo Sasaki, Yusuke Yamamoto, Makiko Kouno, Kimi Honma, Shunji Nagahara, Koji Hanai, Akihiko Sano, Takashi Kato, Masaaki Terada, Takahiro Ochiya

    Nucleic acids research   32 ( 13 )  2004

     View Summary

    Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

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  • 2002〜現在の成果報告は、下記URLよりご参照ください。 http://www.f.waseda.jp/tkato/publ/publ-top.html

       2004

  • CrkL directs ASAP1 to peripheral focal adhesions

    Atsushi Oda, Ikuo Wada, Koichi Miura, Katsuya Okawa, Toshihiko Kadoya, Takashi Kato, Hiroshi Nishihara, Masae Maeda, Shinya Tanaka, Kazuo Nagashima, Chiaki Nishitani, Kazuhiko Matsuno, Masaho Ishino, Laura M. Machesky, Hiroyoshi Fujita, Paul Randazzo

    Journal of Biological Chemistry   278 ( 8 ) 6456 - 6460  2003.02

     View Summary

    Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.

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  • Marked improvement of thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura by pegylated recombinant human megakaryocyte growth and development factor

    Kazunori Shibuya, Tomoaki Kuwaki, Emiko Tahara, Chizuru Yuki, Hiromichi Akahori, Takashi Kato, Hiroshi Miyazaki

    Experimental Hematology   30 ( 10 ) 1185 - 1192  2002.10

     View Summary

    Objective. We examined the stimulatory effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production in male (NZW × BXSB) Fl (W/B F1) mice, a murine model of idiopathic thrombocytopenic purpura. Materials and Methods. A cohort of 19- to 25-week-old, severely thrombocytopenic male W/B F1 mice were given PEG-rHuMGDF at different dosing schedules. Before and at various times after therapy, platelet counts, reticulated platelets, platelet lifespan, and levels of platelet-associated immunoglobulin G were measured. Analysis of megakaryocytic cells was performed. Results. Treatment of male W/B F1 mice with PEG-rHuMGDF (30 μg/kg/day) three times per week for several weeks resulted in sustained thrombocytosis, accompanied by increased megakaryocytopoiesis in both the bone marrow and spleen. The degree of the platelet response to PEG-rHuMGDF varied between individual mice, likely reflecting the heterogeneity of the disease. Production of new platelets in response to PEG-rHuMGDF was manifested by an increase in reticulated platelets. Levels of platelet-associated immunoglobulin G decreased inversely during periods of thrombocytosis. PEG-rHuMGDF therapy also improved thrombocytopenia in male W/B F1 mice refractory to splenectomy. Platelet lifespan was not affected by PEG-rHuMGDF. Male W/B F1 mice treated with pegylated murine MGDF, a homologue of PEG-rHuMGDF, had persistent thrombocytosis for at least 7 months, suggesting that antiplatelet antibody production was not enhanced. Conclusion. PEG-rHuMGDF therapy potently stimulated platelet production, effectively ameliorating thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

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  • Thrombopoietin receptor (c-Mpl) is constitutively expressed on platelets of patients with liver cirrhosis, and correlates with its disease progression

    Toru Ishikawa, Takafumi Ichida, Satoshi Sugahara, Satoshi Yamagiwa, Yasunobu Matsuda, Kazuhiro Uehara, Takashi Kato, Hiroshi Miyazaki, Hitoshi Asakura

    Hepatology Research   23 ( 2 ) 115 - 121  2002.06

     View Summary

    Thrombocytopenia is one of the major complications of liver cirrhosis. Except for hypersplenism associated with portal hypertension, it is not known which abnormalities of thrombopoiesis cause thrombocytopenia. To evaluate thrombopoiesis in liver cirrhosis, we analyzed thrombopoietin (TPO) and its receptor c-Mp1 levels in cirrhotic patients. Expression of c-Mp1 on platelets and the serum level of TPO were investigated from 38 patients with various stages of liver cirrhosis by flow-cytometric analysis and enzyme-immuno assay. Samples obtained from 22 individuals without evidence of liver disease were used as controls. Neither platelet counts nor TPO levels correlated with disease progression defined by the Child-Pugh classification. c-Mp1 was constitutively expressed on the platelets of cirrhotic patients, and its expression level was reduced with disease progression defined by the Child-Pugh classification. In this study, serum TPO did not fluctuate according to the grade of cirrhosis. However, its receptor c-Mp1, which is expressed on platelets, was decreased significantly in severely cirrhotic patients with thrombocytopenia. Thus, a correlation between reduced c-Mp1 expression and the progression of liver cirrhosis was demonstrated. We conclude that, in addition to hypersplenism, the reduced expression of c-Mp1 may play a significant role in the thrombocytopenia observed in severe liver cirrhosis. © 2002 Elsevier Science B.V. All rights reserved.

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  • Crystallization of the functional domain of human thrombopoietin using an antigen-binding fragment derived from neutralizing monoclonal antibody.

    Kuroki R, Hirose M, Kato Y, Feese MD, Tamada T, Shigematsu H, Watarai H, Maeda Y, Tahara T, Kato T, Miyazaki H

    Acta crystallographica. Section D, Biological crystallography   58 ( Pt 5 ) 856 - 858  2002.05  [Refereed]

    DOI PubMed CiNii

  • Elevation of serum thrombopoietin precedes thrombocytosis in acute infections

    Toshikazu Shimbo, Mari Mito, Yoko Suzuki, Akira Ishiguro, Kousaku Matsubara, Hiroshi Miyazaki, Takashi Kato

    British Journal of Haematology   116 ( 3 ) 612 - 618  2002

     View Summary

    To clarify the mechanisms underlying thrombocytosis secondary to infections, we longitudinally studied serum levels of thrombopoietin (TPO) and interleukin (IL)-6 in 15 infants and young children with prominent thrombocytosis (platelets >700 × 109/l) following acute infections and 116 age-matched controls using an enzyme-linked immunosorbent assay. The subjects included nine patients with bacterial infections, three with viral infections and three with non-determined pathogens. TPO values in the controls were 2.24 ± 0.87 fmol/ml (mean ± SD) with a 95% reference interval of 0.85-4.47 fmol/ml. In the first week of infection, platelet counts were normal, but TPO values increased (∼10.73 fmol/ml). TPO levels peaked on day 4 ± 2 at 6.44 ± 2.37 fmol/ml and then fell gradually. When platelet counts peaked in the second and third weeks, TPO levels were similar to the controls. IL-6 levels in the first week rose and dropped more rapidly than TPO. Serum TPO values were significantly correlated with C-reactive protein levels (r = 0.688, P < 0.001) and IL-6 levels (r = 0.481, P = 0.027). These results suggest that TPO contributes to thrombocytosis following infections in conjunction with IL-6, arguing for additional regulatory mechanisms of blood TPO levels.

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  • Role of the thrombopoietin (TPO)/Mpl system: c-Mpl-like molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis

    Minoru Kakeda, Jun Ichi Kyuno, Takashi Kato, Mitsuo Nishikawa, Makoto Asashima

    Development Growth and Differentiation   44 ( 1 ) 63 - 75  2002

     View Summary

    Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c-Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)-4 and increased the number of both erythrocytes and leukocytes in a dose-dependent manner. Addition of anti-c-Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood-like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c-Mpl-like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal-lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c-Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c-Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.

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  • New human myelodysplastic cell line, TER-3: G-CSF specific downregulation of Ca 2+/calmodulin-dependent protein kinase IV

    Yuji Mishima, Yasuhito Terui, Yuko Mishima, Misa Katsuyama, Masaki Mori, Hiroshi Tomizuka, Toshiyuki Takizawa, Akira Miyazato, Masuzu Ueda, Muneo Yamada, Hirotoshi Hayasawa, Nobuyuki Mizunuma, Yukihito Ishizaka, Kazuma Ikeda, Takashi Kato, Keiya Ozawa, Kiyohiko Hatake

    Journal of Cellular Physiology   191 ( 2 ) 183 - 190  2002

     View Summary

    We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS. © 2002 Wiley-Liss, Inc.

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  • Involvement of protein kinase C-ε in signal transduction of thrombopoietin in enhancement of interleukin-3-dependent proliferation of primitive hematopoietic progenitors

    Noriyuki Shiroshita, Manabu Musashi, Keisuke Sakurada, Kazuhiro Kimura, Yuzo Tsuda, Shuichi Ota, Hiroshi Iwasaki, Tamotsu Miyazaki, Takashi Kato, Hiroshi Miyazaki, Akihiro Shimosaka, Masahiro Asaka

    Journal of Pharmacology and Experimental Therapeutics   297 ( 3 ) 868 - 875  2001.06

     View Summary

    We studied the effect of thrombopoietin (TPO) on interleukin-3 (IL-3)-dependent bone marrow cell colony formation of mice to clarify the role of protein kinase C (PKC) in the signal transduction of TPO for the proliferation of primitive hematopoietic progenitors. TPO alone hardly yielded colonies. However, TPO in combination with IL-3 increased colony numbers synergistically from 2- to 4-fold, compared with those supported by IL-3 alone. Serial observation of colony development showed that TPO may hasten the appearance of colonies by shortening the dormant period (Go) of primitive progenitors. Immunocytochemical studies on PKC isoforms in progenitor cells stimulated with TPO have revealed that the expression pattern of PKC-ε is changed, but not that of PKC-α, -β, -γ, -δ or -ζ. Selective PKC inhibitors, such as calphostin C and GF 109203X, and PKC-ε-specific translocation inhibitor peptide abrogated the enhancing effect of TPO on IL-3-dependent colony formation and the changes in the intracellular expression pattern of PKC-ε. These data taken together suggest that TPO has a direct effect on primitive progenitors and enhances IL-3-dependent colony formation, at least partly through the activation of PKC-ε.

    PubMed

  • An increase in circulating mast cell colony-forming cells in asthma

    H. H. Mwamtemi, K. Koike, T. Kinoshita, S. Ito, S. Ishida, Y. Nakazawa, Y. Kurokawa, K. Shinozaki, K. Sakashita, K. Takeuchi, M. Shiohara, T. Kamijo, Y. Yasui, A. Ishiguro, Y. Kawano, K. Kitano, H. Miyazaki, T. Kato, S. Sakuma, A. Komiyama

    Journal of Immunology   166 ( 7 ) 4672 - 4677  2001.04

     View Summary

    We compared a potential to generate mast cells among various sources of CD34+ peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34+ PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34+ PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34+ PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34+ PB cells between the two groups. In the presence of TPO, CD34+ PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34+ PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

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  • Platelets exposed to elevated levels of endogenous thrombopoietin in vivo have a reduced response to megakaryocyte growth and development factor in vitro

    U. Nishiyama, H. Morita, Y. Torii, T. Kuwaki, E. Shimizu, H. Akahori, T. Kato, H. Miyazaki

    Thrombosis and Haemostasis   85 ( 1 ) 152 - 159  2001

     View Summary

    Thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

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  • Early elevation of serum thrombopoietin levels and subsequent thrombocytosis in healthy preterm infants

    Kousaku Matsubara, Kunizo Baba, Hiroyuki Nigami, Hidekazu Harigaya, Akira Ishiguro, Takashi Kato, Hiroshi Miyazaki

    British Journal of Haematology   115 ( 4 ) 963 - 968  2001

     View Summary

    To verify pathophysiological mechanisms underlying thrombocytosis in low-birth-weight (LBW) preterm babies, we evaluated kinetic changes in platelet counts and thrombopoietic cytokines including thrombopoietin (TPO), interleukin 6 (IL-6) and IL-11 in 24 uncomplicated preterm infants. Platelet counts in cord blood (CB) (265 ± 64 × 109/l) were similar to adult levels, increased by d 14 (473 ± 140 × 109/l), and then remained fairly constant. Thrombocytosis (> 500 × 109/l) was observed in 9/24 (38%) subjects. Mean TPO level in CB was 5.11 ± 1.51 fmol/ml, peaked at d 2 (7.64 ± 3.28 fmol/ml), decreased at d 5 (3.93 ± 1.67 fmol/ml), and thereafter kept fairly constant during the remaining neonatal period. Compared with term infants, mean TPO levels of preterm infants in CB and at d 2 were significantly higher (P < 0.01). There was an inverse correlation between platelet counts and TPO levels (r = 0.45, P < 0.001, n = 88). Preterm neonates with thrombocytosis had significantly higher TPO values in CB than those without thrombocytosis (P < 0.05). There was no significant relationship between platelet counts and IL-6. IL-11 was not detectable. These results suggest that an early elevation of serum TPO levels is related to the subsequent thrombocytosis in LBW preterm infants.

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  • Stressful delivery influences circulating thrombopoietin (TPO) levels in newborns: Possible role for cortisol in TPO-mpl binding

    Kazuhide Ikeno, Kenichi Koike, Akihiro Takeshita, Kaori Shinjo, Tsukasa Higuchi, Tetsuo Nakabayashi, Sachiko Akanuma, Kiyoko Hizume, Akira Ishiguro, Kinya Ogami, Takashi Kato, Hiroshi Miyazaki, Ryuzo Ohno, Atsushi Komiyama

    Early Human Development   58 ( 3 ) 225 - 235  2000.06

     View Summary

    The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl+/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system. Copyright (C) 2000 Elsevier Science Ireland Ltd.

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  • Functional analysis of the c-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a 'shuttle' peptide to help secretion

    Takanori Muto, Michael D. Feese, Yoshihiro Shimada, Youko Kudou, Tomoyuki Okamoto, Tadashi Ozawa, Tomoyuki Tahara, Hideya Ohashi, Kinya Ogami, Takashi Kato, Hiroshi Miyazaki, Ryota Kuroki

    Journal of Biological Chemistry   275 ( 16 ) 12090 - 12094  2000.04

     View Summary

    Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N. linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to 1/10 that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C- terminal region was found to be important for secretion of TPO. Among six N- glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn- 234, were found to be critical for secretion, and two other locations, Ash- 319 and Ash-327, did not affect the secretion.

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  • Increased D-type cyclin expression together with decreased cdc2 activity confers megakaryocytic differentiation of a human thrombopoietin-dependent hematopoietic cell line

    Itaru Matsumura, Hirokazu Tanaka, Akira Kawasaki, Junko Odajima, Hanako Daino, Koji Hashimoto, Hiroshi Wakao, Koichi Nakajima, Takashi Kato, Hiroshi Miyazaki, Yuzuru Kanakura

    Journal of Biological Chemistry   275 ( 8 ) 5553 - 5559  2000.02

     View Summary

    At the late phase of megakaryocytopoiesis, megakaryocytes undergo endomitosis, which is characterized by DNA replication without cell division. Although a number of cell cycle regulatory molecules have been identified, the precise roles of these molecules in megakaryocytic endomitosis are largely unknown. In a human interleukin-3-dependent cell line transfected with the thrombopoietin (TPO) receptor c-mpl (F-36P-mpl), either treatment with TPO or the overexpression of activated ras (Ha-Ras(G12V)) induced megakaryocytic maturation with polyploid formation. We found that TPO stimulation or Ha-Ras(G12v) expression led to up-regulation of cyclin D1, cyclin D2, and cyclin D3 expression. In addition, expression levels of cyclin A and cyclin B were reduced during the total course of both TPO- and Ha- Ras(G12V)-induced megakaryocytic differentiation, thereby leading to decreased cdc2 kinase activity. Neither the induced expression of cyclin D1, cyclin D2, or cyclin D3 nor the expression of a dominant negative form of cdc2 alone could induce megakaryocytic differentiation of F-36P-mpl cells. In contrast, overexpression of dominant negative cdc2 together with cyclin D1, cyclin D2, or cyclin D3 facilitated megakaryocytic differentiation in the absence of TPO. These results suggest that both D-type cyclin expression and decreased cdc2 kinase activity may participate in megakaryocytic differentiation.

    DOI PubMed

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  • Expression of hepatic thrombopoietin mRNA in primary cultured hepatocytes and in rats with acute liver injury or bone marrow suppression with or without cirrhosis

    Toru Ishikawa, Takafumi Ichida, Yasunobu Matsuda, Soichi Sugitani, Motoya Sugiyama, Takashi Kato, Hiroshi Miyazaki, Hitoshi Asakura

    Journal of Gastroenterology and Hepatology (Australia)   15 ( 6 ) 647 - 653  2000

     View Summary

    Background and Aims: The main causes of thrombocytopenia in cirrhosis are thought to be platelet destruction and the reduction of thrombopoietin (TPO) expression in the liver. The mechanisms by which levels of TPO mRNA are regulated in cirrhosis have not been elucidated. In this study, we investigated some possible mechanisms. Methods: We used three experimental models: bone marrow suppression, acute liver injury and primary cultured hepatocytes. We used northern blots to assess the kinetics of TPO mRNA expression in the livers of irradiated rats (with and without cirrhosis) in acute liver injury and in primary cultured hepatocytes treated with hepatotoxin or cytokines. Results: Although the bone marrow was hypocellular, there was no apparent enhancement of TPO mRNA expression in the irradiated rats with cirrhotic livers compared with the unirradiated rats with cirrhotic livers. There were no conspicuous changes in hepatic TPO mRNA expression between the livers of the control rats and the three models of acute liver injury. There were no conspicuous changes in the levels of TPO mRNA between control hepatocytes and hepatocytes treated with hepatotoxin or cytokines. Conclusions: Our results suggest that bone marrow is not a regulator of hepatic TPO production in cirrhosis. The reduced TPO mRNA expression found in cirrhotic rats may not result merely from serious cellular damage; it may be associated with cirrhosis-specific regulatory mechanisms for the expression of the TPO gene. Further studies are needed to search for other factors that may induce reduced TPO expression. (C) 2000 Blackwell Science Asia Pty Ltd.

    DOI

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  • Strong inverse correlation between serum TPO level and platelet count in essential thrombocythemia

    Naoto Tomita, Shigeki Motomura, Rika Sakai, Katsumichi Fujimaki, Juichi Tanabe, Hitoshi Fukawa, Hiroshi Harano, Heiwa Kanamori, Kouji Ogawa, Hiroshi Mohri, Atsuo Maruta, Fumio Kodama, Yoshiaki Ishigatsubo, Tomoyuki Tahara, Takashi Kato

    American Journal of Hematology   63 ( 3 ) 131 - 135  2000

     View Summary

    Serum thrombopoietin (TPO) levels in 50 essential thrombocythemia (ET) patients were measured using a highly sensitive sandwich ELISA. In nine cases, TPO levels were measured at two points with different platelet counts. ET patients showed significantly higher serum TPO levels (n = 59, 2.70 ± 2.74 fmol/mL, P < 0.0001) than those of normal individuals (n = 29, 0.83 ± 0.36 fmol/mL). Twenty-three previously untreated ET patients also showed significantly higher serum TPO levels (1.33 ± 0.75 fmol/mL, P = 0.0066) than normal individuals. Extremely high serum TPO levels (5.46 ± 3.68 fmol/mL) were observed in ET patients with normal platelet counts. Furthermore, a strong inverse correlation was found between serum TPO levels and platelet counts in ET patients (R = -0.729, P < 0.0001). This inverse correlation also held for each of nine cases with two-point TPO measurements. In the clinical course of ET, megakaryocyte mass may parallel the platelet mass before and after chemotherapy. Although it is unknown whether overproduction of TPO exists or not in ET, total platelet and megakaryocyte mass, i.e., the total number of c-Mpl, may play a role to regulate serum TPO levels. (C) 2000 Wiley-Liss, Inc.

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  • Effect of thrombopoietin on proliferation of blasts from patients with myelodysplastic syndromes

    Shan Shun Luo, Kiyoyuki Ogata, Norio Yokose, Takashi Kato, Dan Kazuo

    Stem Cells   18 ( 2 ) 112 - 119  2000

     View Summary

    Thrombopoietin (TPO), a major cytokine involved in megakaryocytopoiesis/thrombopoiesis, may be effective for treatment of the thrombocytopenia associated with myelodysplastic syndromes (MDS). However, it has been unclear whether TPO stimulates proliferation of MDS blasts, as observed in de novo acute myeloid leukemia. This study examined this concern. When marrow cells from 37 MDS cases were cultured with or without recombinant human PEGylated TPO, TPO increased the blast number (stimulation index ≥1.5) in 9 of 16 high-risk MDS cases (refractory anemia with excess blasts [RAEB] and RAEB in transformation) and 4 of 10 cases with MDS transformed to acute leukemia (MDS-AL), but none of 11 cases with low-risk MDS (RA and RA with ringed sideroblasts). When the cell cycle of cultured cells was determined by three-color flow cytometry, TPO activated the cell cycle of MDS cells (causing a decrease in Go-phase cells) in most of the cases whose blast number increased in response to TPO. Reverse transcriptase-polymerase chain reaction analysis detected TPO receptor messenger RNA in purified blasts from all six cases examined, irrespective of the response of their blasts to TPO in culture. Analysis of the patients' characteristics identified a high-serum lactate dehydrogenase (LDH) value as being associated with blast proliferation in high-risk MDS cases (p = 0.0036). We conclude that TPO stimulates in vitro proliferation of blasts from a fraction of MDS patients. High-risk MDS patients, especially those who have a high-serum LDH value, and MDS-AL patients should be monitored with particular care in clinical trials of TPO for MDS.

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  • Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia

    N. Sawai, K. Koike, H. H. Mwamtemi, S. Ito, Y. Kurokawa, K. Sakashita, T. Kinoshita, T. Higuchi, K. Takeuchi, M. Shiohara, T. Kamijo, Y. Higuchi, H. Miyazaki, T. Kato, M. Kobayashi, M. Miyake, K. Yasui, A. Komiyama

    Journal of Leukocyte Biology   68 ( 1 ) 137 - 143  2000

     View Summary

    We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34+CD38+c-kit+ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF + SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF + TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34+CD38+c-kit+ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF + SCF. When BM CD34+CD38-c-kit+ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder.

    PubMed

  • Production of thrombopoietin by human carcinomas and its novel isoforms

    Yutaka Sasaki, Takayuki Takahashi, Hiroshi Miyazaki, Atsushi Matsumoto, Takashi Kato, Kishiko Nakamura, Sumiko Iho, Yoshiaki Okuno, Kazuwa Nakao

    Blood   94 ( 6 ) 1952 - 1960  1999.09

     View Summary

    Thrombocytosis is occasionally seen in patients with carcinomas and has been assumed to be attributable to interleukin-6 or granulocyte-macrophage colony-stimulating factor produced by carcinoma cells. In this study, we clarified whether thrombopoietin (TPO) is involved in carcinoma-associated thrombocytosis. Expression of TPO mRNA was observed in the majority of 27 carcinoma cell lines as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). There were 6 PCR products differing in size; sequence analysis showed the full-length TPO mRNA (TPO-1), 12- and 116-bp deleted variants (TPO-2 and TPO-3, respectively), and 3 novel isoforms (197- and 128- bp deleted forms and a 60-bp insert form of TPO-3; named TPO-4, TPO-5, and TPO-6, respectively). Of 27 lines, 24 expressed TPO-1 mRNA with various other isoforms. Culture supernatants of COS-1 cells transfected with TPO-5 or TPO-6 cDNA did not promote the proliferation of TPO-responsive cells, whereas Western blot analysis on the cell lysates demonstrated TPO-5 but not TPO-6 protein, suggesting poor extracellular secretion (TPO-5) or poor protein synthesis (TPO-6). TPO protein was detected in 10-fold concentrated culture supernatants of cells of these carcinoma lines, with a median concentration of 0.38 fmol/mL as evaluated by enzyme-linked immunosorbent assay. High blood TPO levels were observed with a median value of 3.46 fmol/mL (range, 0.34 to 8.67 fmol/mL) in patients with advanced carcinomas associated with thrombocytosis. These results indicate that thrombocytosis in patients with carcinomas might be caused, at least in part, by TPO produced by carcinoma cells.

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  • Thrombopoietin expression in normal and hypobaric hypoxia-induced thrombocytopenic rats

    Kuniaki Nakanishi, Fumiko Tajima, Hiroshi Osada, Takashi Kato, Hiroshi Miyazaki, Toshiaki Kawai, Chikao Torikata, Tatsuko Suga, Kunio Takishima, Takashi Aurues, Tomosumi Ikeda

    Laboratory Investigation   79 ( 6 ) 679 - 688  1999.06

     View Summary

    Thrombopoietin (TPO) is important as the physiologic regulator of platelet production. High-altitude hypoxia is a well-known cause of polycythemia and thrombocytopenia in animals. Fifty-two Wistar rats were housed for 0.5 to 21 days in a mechanical chamber in an environment equivalent to that found at 5500 m to determine (a) the cellular localization of TPO and (b) whether the decreased platelet and megakaryocyte counts in rats exposed to a hypobaric hypoxic environment (HHE) are associated with an altered TPO mRNA expression. In normal rats, there were high levels of TPO mRNA in the liver and kidney, intermediate levels in the brain and large intestine, and low levels in the skeletal muscle and small intestine. TPO mRNA and protein were expressed in Purkinje cells and neuronal cells in the brain, in proximal tubular cells and the mesangial cells of the glomeruli in the kidney, in hepatocytes and biliary duct epithelial cells, in absorptive epithelial cells in the large intestine, in the epidermis, and in the lung. The platelet count in the blood and megakaryocyte counts in the bone marrow and spleen were all decreased significantly after 5 or more days of exposure to HHE. In major producers such as the liver and kidney and in minor producers such as the brain, TPO mRNA levels, which tended to be decreased after 0.5 to 3 days of exposure to HHE, had returned to normal by about Day 5 or 7. Thus, during the HHE period with a decreased platelet count, no changes in TPO mRNA levels were detected in these three organs. In conclusion, we have demonstrated that TPO production occurs in various types of cells. In HHE, however, factors other than TPO may be involved in hypobaric hypoxia- induced thrombocytopenia in rats.

    PubMed

  • Thrombopoietin augments stem cell factor-dependent growth of human mast cells from bone marrow multipotential hematopoietic progenitors

    Nobukuni Sawai, Kenichi Koike, Hadija Hemed Mwamtemi, Tatsuya Kinoshita, Yumi Kurokawa, Kazuo Sakashita, Tsukasa Higuchi, Kouichi Takeuchi, Masaaki Shiohara, Takehiko Kamijo, Susumu Ito, Takashi Kato, Hiroshi Miyazaki, Tetsuji Yamashita, Atsushi Komiyama

    Blood   93 ( 11 ) 3703 - 3712  1999.06

     View Summary

    The effects of thrombopoietin (TPO) and/or stem cell factor (SCF) on the development of human mast cells from CD34+ bone marrow (BM) cells were investigated using a serum-deprived liquid culture system. Mast cells were identified by measurement of intracellular histamine content, immunocytochemical staining, and flow cytometric analysis. Whereas SCF alone generated only a small number of tryptase+ cells, the addition of TPO to the culture containing SCF resulted in an apparent production of mast cells from 3 weeks until at least 15 weeks. Some of the cells reacted with an antichymase monoclonal antibody as well. Based on the effects of growth factor(s) on a later phase of the mast cell growth, TPO may stimulate an early stage of mast cell development in combination with SCF, whereas subsequent growth seems to be supported by SCF alone. Single-cell culture studies indicated that the CD34+CD38-c-kit+ cells and CD34+CD38+c-kit+ cells were responsible for the SCF + TPO-dependent mast cell production. Two- step culture assays clearly showed that mast cells originated from multilineage colony-forming cells that had potential to differentiate into neutrophil/mast cell lineages, neutrophil/macrophage/mast cell lineages, or neutrophil/macrophage/mast cell/erythroid lineages. These results suggest that TPO plays an important role in the development of human mast cells from CD34+ BM cells in concert with SCF, and provide direct evidence of the differentiation into the mast cell lineage of human multipotential BM- derived progenitors.

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  • Neutrophilic cell production by combination of stem cell factor and thrombopoietin from CD34+ cord blood cells in long-term serum-deprived liquid culture

    Nobukuni Sawai, Kenichi Koike, Susumu Ito, Hadija Hemed Mwamtemi, Yumi Kurokawa, Tatsuya Kinoshita, Kazuo Sakashita, Tsukasa Higuchi, Kouichi Takeuchi, Masaaki Shiohara, Hiroshi Miyazaki, Takashi Kato, Atsushi Komiyama

    Blood   93 ( 2 ) 509 - 518  1999.01

     View Summary

    In the present study, we investigated the effects of stem cell factor (SCF) and/or thrombopoietin (TPO) on the cell production by cord blood CD34+ cells using a serum-deprived liquid culture system. Although SCF alone supported a modest production of neutrophilic cells and a remarkable generation of mast cells, the addition of TPO to the culture containing SCF caused an apparent generation of neutrophilic cells, identified by immunocytochemical staining and flow cytometric analysis. The significant production of neutrophilic cells by SCF and TPO was persistently observed from 2 weeks to 2 to 3 months of culture. The interaction between SCF and TPO on the neutrophilic cell generation was greater than the combined effects of SCF with granulocyte colony-stimulating factor (G-CSF) or granulocyte- macrophage colony-stimulating factor (GM-CSF). The addition of neutralizing antibody against G-CSF or GM-CSF did not influence the SCF + TPO-dependent neutrophilic cell production. A single-cell culture study showed that not only CD34+CD38+c-kit+ cells but also CD34+CD38-c-kit+ cells were responsible for the neutrophilic cell generation. In clonal cell cultures, GM progenitors as well as erythroid progenitors and multipotential progenitors expanded in the cultures supplemented with SCF and TPO. The neutrophilic cells grown by SCF + TPO were at myeloblast to band cell stages, and scarcely matured to segmented neutrophils. In addition, the cells generated by SCF + TPO were stained with monoclonal antibodies against myeloperoxidase, elastase, lactoferrin, and CD11b, but they had negligible levels of alkaline phosphatase (ALP) and CD35. The replating of the CD34-c-kit(-/low) CD15+ cells grown by SCF + TPO into a culture containing SCF + G-CSF permitted both the terminal maturation into segmented cells and the appearance of ALP and CD35. These results indicate the existence of a G-CSF/GM-CSF-independent system of neutrophilic cell production.

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  • Thrombopoietin-induced signal transduction and potentiation of platelet activation

    Atsushi Oda, Yoshitaka Miyakawa, Brian J. Druker, Katsutoshi Ozaki, Hideya Ohashi, Takashi Kato, Hiroshi Miyazaki, Makoto Handa, Kenji Ikebuchi, Yasuo Ikeda

    Thrombosis and Haemostasis   82 ( 2 ) 377 - 384  1999

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    16
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  • Characterization of native human thrombopoietin in the blood of normal individuals and of patients with haematologic disorders

    Atsushi Matsumoto, Tomoyuki Tahara, Haruhiko Morita, Kensuke Usuki, Hideya Ohashi, Atsuko Kokubo-Watarai, Kieko Takahashi, Eiko Shimizu, Hikaru Tsunakawa, Kinya Ogami, Hiroshi Miyazaki, Akio Urabe, Takashi Kato

    Thrombosis and Haemostasis   82 ( 1 ) 24 - 29  1999

     View Summary

    Thrombopoietin (TPO) isolated from thrombocytopenic plasma of various animal species has previously been shown to comprise only truncated forms of the molecule, presumably generated by proteolysis. Native TPO has now been partially purified from normal human plasma by immunoaffinity chromatography and was confirmed to be biologically active. Gel filtration in the presence of SDS revealed that TPO eluted in two peaks: a major peak corresponding to the elution position of fully glycosylated recombinant human TPO (rhTPO) consisting of 332 amino acid residues, and a minor peak corresponding to a smaller molecular size. Immunoblot analysis also revealed that most plasma-derived TPO migrated at the same position as fully gly- cosylated rhTPO, corresponding to molecular size of ~ 80 to 100 kDa. Furthermore, the size distribution of circulating TPO in patients with various haematologic disorders did not differ markedly from that of plasma-derived TPO from healthy individuals. These results indicate that the truncation of circulating TPO is not related to disease pathophysiology, and that the predominant form of TPO in blood is a biologically active ~ 80- to 100-kDa species. The size distribution of TPO extracted from normal platelets was similar to that of TPO in plasma; the proportion of truncated TPO was decreased by prior incubation of platelets with hirudin, indicating that the endogenous truncated TPO, at least in platelet extract, was generated by thrombin-mediated cleavage.

    DOI PubMed

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    8
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  • Serial serum thrombopoietin levels in a pregnant woman with essential thrombocythaemia

    Yosiiinobu Kanda, Sliigeru Cliiba, Yuji Tanaka, Akiyoshi Mlu'A, Alsushi Togawa, Takasiii Kato, Hiroshi Miyazaki, Ruriko Nisnino, Yosmo Yazaki, Hisamaru Hirai

    British Journal of Haematology   105 ( 1 ) 271 - 273  1999

     View Summary

    Thrombopoietin (TPO) is a ligand for c-mpl, which regulates the differentiation and maturation of megakaryocytes. Essential thrombocythaemia (ET) is a clonal myeloproliferative disorder. It has been reported that the platelet count declines during pregnancy in ET patients. We examined serial changes in the serum TPO level during the course of pregnancy in a patient with ET. The serum TPO level showed significant negative correlation with the platelet count. Although it mimicked the normal feed back system, the TPO levels were consistently higher than the normal upper limit. Accumulation of these data will be helpful in revealing the pathogenesis of ET and the decline in the platelet count during pregnancy.

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    4
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  • Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment

    Kentaro Kinjo, Masahiro Kizaki, Nobuyuki Takayama, Naohiko Michikawa, Atsushi Oda, Shin Ichiro Okamoto, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Yasuo Ikeda

    British Journal of Haematology   105 ( 2 ) 382 - 387  1999

     View Summary

    Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)-3, IL-6, IL-11, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all-trans retinoic acid (RA) treatment. During differentiation-inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10-20d of all-trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation-inducing therapy, and it would appear that TPO levels are directly regulated by all-trans RA during the first 10-20d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all-trans RA treatment, suggesting a close relationship between TPO and EPO signalling.

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    7
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  • Successful bone marrow transplantation in a child with X-linked hyper-IgM syndrome

    T. Kato, I. Tsuge, J. Inaba, K. Kato, T. Matsuyama, S. Kojima

    Bone Marrow Transplantation   23 ( 10 ) 1081 - 1083  1999

     View Summary

    We report a case of an 11-year-old boy who underwent successful bone marrow transplantation for X-linked hyper-IgM syndrome (XHIM). The donor was an HLA-matched brother. The patient was conditioned with busulfan, cyclophosphamide and anti-thymocyte globulin. He received 4.7 x 108 marrow cells per kg from the donor. Prophylaxis against graft-versus-host disease consisted of cyclosporine and short-term methotrexate. The clinical course after the bone marrow transplantation was uneventful, and 12 months after transplantation the patient was doing well with no need for therapy. We examined expression of the CD40 ligand (CD40L) on the patient's activated T lymphocytes and in vitro production of immunoglobulins by his lymphocytes. Although expression of CD40L was totally absent before the bone marrow transplant, subnormal expression appeared after the transplantation. In vitro production of IgG and IgA also was improved by the transplant. Based on our experience bone marrow transplantation appears to be a reasonable therapeutic option for patients with XHIM if HLA-matched family donors are available.

    DOI PubMed

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    24
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  • The relationship between carboplatin AUC and serum thrombopoietin kinetics in patients with lung cancer

    Mitsuru Miyazaki, Yasuhiro Fujiwara, Takeshi Isobe, Michio Yamakido, Takashi Kato, Hiroshi Miyazaki

    Anticancer Research   19 ( 1 B ) 667 - 670  1999

     View Summary

    To clarify the relationship between the carboplatin AUC and the extent of damage to thrombopoiesis, we monitored both endogenous serum TPO kinetics and carboplatin pharmacokinetics after single-agent carboplatin administration. Previously untreated 12 patients with stage IV non-small-cell lung cancer were enrolled. The actual carboplatin AUC showed a significant positive correlation with the maximum increase ratio of TPO (TPO(max)/TPO(day1) (r = 0.74, p = 0.014). Furthermore, the increase ratio of TPO at one week after administration of carboplatin (TPO(day8)/TPO(day1) showed a significant negative correlation with the following platelet nadir around day 19 (r = -0.84, p = 0.005). By monitoring the changes in endogenous TPO concentration, we could estimate the degree of thrombocytopenia and determine the indication, and thus the optimal timing of prophylactic administration of TPO before platelets are markedly reduced.

    PubMed

  • Age-related changes in thrombopoietin in children: Reference interval for serum thrombopoietin levels

    Akera Ishiguro, Tatsutoshi Nakahata, Kousaku Matsubara, Yasuhide Hayashi, Takashi Kato, Yoko Suzuki, Toshikazu Shimbo

    British Journal of Haematology   106 ( 4 ) 884 - 888  1999

     View Summary

    We studied thrombopoietin (TPO, Mpl ligand) values using a sensitive ELISA in 254 serum samples obtained from disease-free children and adult volunteers. TPO was detected in all samples, and its values ranged widely from 0.25 to 9.18 fmol/ml. When analysed by dividing the subjects into 11 age groups, the mean TPO levels from birth to 1 month of age were increased (3.73-5.92 fmol/ml). The highest values were found 2 d after birth; TPO levels then gradually decreased to adult levels (0.83 fmol/ml). The relationship between TPO values and platelet counts was not significant in all subjects (r=0.27) or in children alone (r=0.12). In children >1 month of age a 95% reference interval for serum TPO values was determined from 0.58 to 3.27 fmol/ml. A significant correlation was found between TPO values in serum and plasma; serum TPO values = -0.257 + 4.039 x plasma TPO values (r=0.951, P<0.001, n=22). This study is the first to report age-dependent changes in blood TPO levels throughout child development. Serum TPO values were significantly high up to 1 month of age and were correlated with plasma TPO levels.

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  • Liver cirrhosis with marked thrombocytopenia and highly elevated serum thrombopoietin levels

    Kiyoshi Kitano, Shigetaka Shimodaira, Toshiro Ito, Naoaki Ichikawa, Hiroshi Kodaira, Yoichi Kohara, Mayumi Ueno, Tomoyuki Tahara, Takashi Kato, Fumihiro Ishida, Kendo Kiyosawa

    International Journal of Hematology   70 ( 1 ) 52 - 55  1999

     View Summary

    Three patients with liver cirrhosis (LC) and a bleeding tendency due to marked thrombocytopenia of less than 20 × 109/1 were admitted to our hospital for further examination. Bone marrow examination revealed megakaryocytic hypoplasia in all three patients. All patients exhibited amegakaryocytic thrombocytopenic purpura, myelodysplastic syndrome, or bone marrow hypoplasia. 111In-labeled platelet kinetic studies revealed decreased platelet production in all patients. Although serum thrombopoietin (sTPO) levels are usually within the normal level in patients with LC, the sTPO levels of our patients were about 10 times higher than the levels of normal subjects (1.22 ± 0.37 fmol/ml): 13.34, 16.79, and 10.46 fmol/ml, respectively. These sTPO data supported our findings of decreased megakaryopoiesis. Our findings suggest that examination of sTPO lev els is useful in determining the etiology of marked thrombocytopenia in LC patients. © 1999 The Japanese Society of Hematology.

    PubMed

  • Molecular cloning and functional expression of feline thrombopoietin

    Haruka Matsushiro, Hirotomo Kato, Tomoyuki Tahara, Takashi Kato, Akira Iwata, Toshihiro Watari, Hajime Tsujimoto, Atsuhiko Hasegawa

    Veterinary Immunology and Immunopathology   66 ( 3-4 ) 225 - 236  1998.12

     View Summary

    Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.

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  • Thrombopoietin level is inversely related to blast count, not platelet number, in Down syndrome neonates with transient myeloproliferative disorder

    Motoki Bonno, Eiichi Azuma, Hajime Kawasaki, Xao Li Zhang, Yoshihiro Komada, Masahiro Hirayama, Masamune Higashigawa, Masakazu Umemoto, Tadashi Koike, Takashi Kato, Tomoyuki Tahara, Hiroshi Miyazaki, Minoru Sakurai

    American Journal of Hematology   58 ( 4 ) 267 - 272  1998.08

     View Summary

    Transient myeloproliferative disorder (TMD) in neonates with Down syndrome is characterized by increased megakaryoblastic cells in the peripheral blood. Despite their spontaneous regression in weeks, prognosis is not always favorable because of fatal hepatic fibrosis. In this study, blood thrombopoietin (TPO) levels were measured by ELISA in six TMD patients and the expression of c-Mpl, a ligand for TPO, was examined on the blast cells from four patients by flow cytometer. At the onset, TPO level was undetectable in one patient and significantly lower in five patients than six neonatal controls (mean 0.52 fmol/ml, range 0.30-0.93 vs. 3.70, 1.38-8.33, P < 0.001), although platelet counts were similar (mean 321 x 109/l, range 42- 1,040 vs. 253 x 109/l, 124-381). Two patients died of hepatic failure. TPO levels were measured in five patients after regression of the blast cells. With regression of blast cells, TPO levels were remarkably increased in four survived patients. In one patient with hepatic failure, TPO level was poorly elevated and relatively lower compared to the others. TPO levels were inversely correlated with blast numbers (r = -0.85, P < 0.001), but not with platelet counts (r = 0.426). Blast cells from four patients were all positive for c-Mpl. Our findings suggest that megakaryocyte mass is a major regulator of TPO levels and hepatic failure may affect the TPO level because liver is a major source of TPO production.

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  • Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: Comparison with the effects of FLT3/FLK-2 ligand and interleukin-6

    Takuya Matsunaga, Takashi Kato, Hiroshi Miyazaki, Makio Ogawa

    Blood   92 ( 2 ) 452 - 461  1998.07

     View Summary

    The effects of thrombopoietin (TPO; c-mpl ligand), FLT3/FLK-2 ligand (FL), and interleukin-6 (IL-6) on the survival of murine hematopoietic long- term reconstituting cells (LTRC) were studied by using lineage-negative, Sca- 1-positive, c-kit-positive (Lin-Sca-1+c-kit+) marrow cells from 5- fluorouracil-treated mice. We tested the ability of these cytokines to maintain the viability of LTRC by transplanting the cultured cells to lethally irradiated Ly-5 congenic mice together with compromised marrow cells. As a single agent, only TPO could maintain the LTRC. Neither IL-6 nor FL was effective by itself, but they acted synergistically to maintain the LTRC. We examined whether the maintenance of LTRC by these cytokines was due to the survival of stem cells or was the result of active cell divisions and self-renewal. To monitor cell division, we used membrane dye PKH26. Enriched cells were stained with PKH26 on day 0 and incubated in suspension culture with TPO or with IL-6 and FL for 7 days. On day 7, PKH26(low) and PKH26(high) cells were prepared by sorting and their in vivo reconstituting abilities were tested by transplantation into lethally irradiated Ly-5 congenic mice together with compromised marrow cells. PKH26(high) populations cultured with both TPO alone and the combination of IL-6 and FL showed greater reconstitution activity than that of PKH26(low) populations. These data indicate that TPO alone and the combination of IL-6 and FL can support the survival of stem cells without stimulating their active cell proliferation.

    DOI PubMed

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  • Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line

    Itaru Matsumura, Koichi Nakajima, Hiroshi Wakao, Seisuke Hattori, Koji Hashimoto, Hiroyuki Sugahara, Takashi Kato, Hiroshi Miyazaki, Toshio Hirano, Yuzuru Kanakura

    Molecular and Cellular Biology   18 ( 7 ) 4282 - 4290  1998.07

     View Summary

    Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although, TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPOinduced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPOinduced proliferation and differentiation, c-mpl was introduced into F- 36P, a human interleukin-3 (IL-3)dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate an differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F- 36P-mpl cells, and their effects on TPQ-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ~30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-ras(G12C)) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-ras(G12v)-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells-with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

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  • Quantitative analysis of thrombopoietin receptors on human megakaryocytes

    Tomoaki Kuwaki, Tetsuya Hagiwara, Chizuru Yuki, Ikuko Kodama, Takashi Kato, Hiroshi Miyazaki

    FEBS Letters   427 ( 1 ) 46 - 50  1998.05

     View Summary

    Thrombopoietin (TPO), or c-MPL ligand, is the primary regulator of megakaryocyte and platelet production. TPO receptors expressed on human megakaryocytes derived from peripheral blood (PB) and cord blood (CB) progenitors cultured in the presence of TPO have now been analyzed quantitatively. Like those on human PB platelets, TPO receptors on the cultured megakaryocytes exhibited a molecular mass of approximately 80 kDa. Various characteristics of PB- and CB-derived megakaryocytes indicated that the former were more mature than the latter. Both PB- and CB-derived megakaryocytes expressed a single class of high-affinity TPO receptors, with 1933 ± 772 (n = 3) and 184 ± 48 (n = 4) sites per cell, respectively. These data indicate that the number of TPO receptors on human megakaryocytes increases with cell maturation.

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  • Further examination of various administration protocols of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia in myelosuppressed mice

    Hiromichi Akahori, Masako Ozai, Masumi Ida, Kazunori Shibuya, Takashi Kato, Hiroshi Miyazaki

    Therapeutic Apheresis   2 ( 1 ) 58 - 64  1998.02

     View Summary

    Thrombopoietin (TPO) is the recently isolated lineage-dominant hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In vivo studies have shown that daily multiple injections of pegylated human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule related to human TPO, modified with polyethylene glycol, greatly improve thrombocytopenia and in most cases anemia and neutropenia in myelosuppressed animal models. In this study, we further examined various administration protocols of PEG-rHuMGDF on thrombocytopenia in mice treated with a combination of irradiation and carboplatin. After the myelosuppressive treatment on Day 0, mice received the same amount of PEG-rHuMGDF beginning on Day 1 by a single, 3 times (on alternate days), or 7 day daily administration. A single injection of PEG- rHuMGDF significantly reduced the severity and duration of thrombocytopenia and anemia with a concomitant accelerated recovery of megakaryocytic and erythroid progenitors in the bone marrow, similar to the 2 other administration protocols. As the start of a single injection of PEG-rHuMGDF was delayed, its therapeutic effects were attenuated. These results indicate that an administration of PEG-rHuMGDF at an earlier time after the myelosuppressive treatment is necessary to improve thrombocytopenia and anemia.

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  • Thrombopoietin in patients with hepatoblastorna

    Emiko Komura, Takafumi Matsumura, Takashi Kato, Tomoyuki Tahara, Yuko Tsunoda, Tadashi Sawada

    Stem Cells   16 ( 5 ) 329 - 333  1998

     View Summary

    Marked thrombocytosis (over 50 x 104/μl) is frequently seen in patients with hepatoblastoma. Thrombopoietin (TPO), c-mpl ligand, has recently been purified as the major physiological regulator of the thrombopoiesis and is mainly produced in the liver. Since it is possible that TPO participates in thrombocytosis and the tumor growth of this particular hepatic tumor, serum TPO levels in addition to interleukin 1β (IL-1β) and IL-6 levels were assessed in seven untreated patients by using a sandwich enzyme-linked immunosorbent assay. High serum TPO levels were observed in all of the examined patients. The level ranged from 3.15 to 11.02 (mean ± standard deviation; 6.08 ± 1.25) fmol/ml. IL-6 levels were also somewhat higher than normal. Platelet counts, however, appeared to correlate more with serum TPO levels (p = 0.1) than with IL-1β (p = 0.5) and IL-6 (p = 0.2) levels. Furthermore, using the reverse transcriptase polymerase chain reaction method, the expression of c-mpl mRNA was found in five of eight hepatoblastoma tissues as well as TPO mRNA in all eight tissues. These observations suggest that thrombocytosis in hepatoblastoma patients results from the production of cytokine members, including TPO, within tumor tissues. Additionally, it is possible that TPO might act as a type of autocrine and/or paracrine system for cellular growth in this tumor.

    DOI PubMed

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    43
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  • Native thrombopoietin: Structure and function

    Takashi Kato, Atsushi Matsumoto, Kinya Ogami, Tomoyuki Tahara, Haruhiko Morita, Hiroshi Miyazaki

    Stem Cells   16 ( 5 ) 322 - 328  1998

     View Summary

    Thrombopoietin (TPO), the c-Mpl ligand, is produced constitutively in liver and other organs, circulates in the bloodstream, and is delivered to bone marrow, where it stimulates the early development of multiple hematopoietic lineages and megakaryocytopoiesis. The concentration of TPO in blood is regulated by c-Mpl mass on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including the possible modulation of TPO mRNA abundance in bone marrow, megakaryocytopoiesis and platelet production may be regulated as a result of modulation of TPO activity by proteolytic processing that generates truncated forms of the molecule. Characterization of TPO partially purified from human plasma, however, revealed that the full-length molecule was the predominant form in the blood of both normal individuals and thrombocytopenic patients, although small amounts of truncated species were detected. Thus, truncation of TPO, at least that in the circulation examined, does not appear to contribute to the direct regulation of platelet production in response to increased demand. Given that native TPO isolated from the plasma of thrombocytopenic animals comprises truncated forms, the truncation of TPO is likely of physiological importance in the life history of this molecule.

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    43
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  • Reduced expression of thrombopoietin is involved in thrombocytopenia in human and rat liver cirrhosis

    Toru Ishikawa, Takafumi Ichida, Yasunobu Matsuda, Soichi Sugitani, Motoya Sugiyama, Takashi Kato, Hiroshi Miyazaki, Hitoshi Asakura

    Journal of Gastroenterology and Hepatology (Australia)   13 ( 9 ) 907 - 913  1998

     View Summary

    It is widely accepted that thrombocytopenia associated with liver cirrhosis is caused by increased platelet destruction in the enlarged spleen, but this issue has not yet been analysed sufficiently in terms of platelet production. Thrombopoietin is produced mainly in the liver and strongly promotes platelet production. We studied serum thrombopoietin and the levels of its mRNA in liver tissue of cirrhotic patients and also in a rat model of liver cirrhosis. Furthermore, to clarify the influence of the spleen, we investigated thrombopoietin mRNA in splenectomized rats. The serum thrombopoietin level in humans with liver cirrhosis was not significantly reduced instead of thrombocytopenia. The expression of thrombopoietin mRNA in liver tissue decreased with the progression of liver cirrhosis in both patients and the rat model and no compensatory expression was observed in other organs or nonparenchymal cells. The level of thrombopoietin mRNA did not differ significantly in splenectomized cirrhotic rats before or after administration of dimethylnitrosamine, but was lower than that in splenectomized rats without cirrhosis. We conclude that thrombocytopenia in liver cirrhosis is caused not only by platelet destruction but also by decreased platelet production, perhaps due to reduction of thrombopoietin mRNA in the liver.

    DOI

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    63
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  • Evaluation of thrombopoiesis in thrombocytopenic disorders by simultaneous measurement of reticulated platelets of whole blood and serum thrombopoietin concentrations

    Yukako Koike, Akiko Yoneyama, Jun Shirai, Tateru Ishida, Eriko Shoda, Kouji Miyazaki, Sinji Sunaga, Ryoichi Horie, Katunori Aoki, Kazuhiko Koike, Iturou Ogata, Tomoyuki Tahara, Takashi Kato, Kazuhiko Nakahara, Toshitugu Kariya, Masaaki Higashihara

    Thrombosis and Haemostasis   79 ( 6 ) 1106 - 1110  1998

     View Summary

    To evaluate thrombopoiesis in thrombocytopenic disorders, we simultaneously determined reticulated platelet counts in whole blood by FACScan flow cytometry and serum thrombopoietin (TPO) concentrations by a sensitive sandwich ELISA. The subjects were 40 healthy volunteers and 45 thrombocytopenic patients. In idiopathic thrombocytopenic purpura (ITP), the percentage of reticulated platelets was significantly elevated (5.61 ± 2.02%: mean ± SD) relative to normal controls (2.17 ± 0.90%), but serum TPO concentrations (1.91 ± 1.27 fmol/l) did not differ significantly from the normal range (1.43 ± 0.62 fmol/l). The patients with aplastic anemia (AA) had decreased reticulated platelet counts and markedly increased serum TPO concentrations (13.65 ± 10.64 fmol/l). In thrombocytopenic patients with liver cirrhosis (LC), the absolute number of reticulated platelets (1.65 ± 1.11 x 109/l) decreased similarly that in AA. However, serum TPO concentrations (1.38 ± 0.50 fmol/l) did not increase in contrast to AA. Our findings suggested a possible dual mechanism of thrombocytopenia in LC; that is, thrombocytopenia in LC results from the decreased TPO production primarily in the liver adding to an increase in platelet sequestration in the spleen.

    DOI PubMed

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    87
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  • Thrombopoietin stimulates proliferation and megakaryocytic differentiation of mouse pro-B cell line BF-TE22

    Hideya Ohashi, Haruhiko Morita, Tomoyuki Tahara, Hikaru Tsunakawa, Atsushi Matsumoto, Kinya Ogami, Takashi Kato, Hiroshi Miyazaki

    Cytotechnology   26 ( 3 ) 199 - 206  1998

     View Summary

    We have isolated and characterized a thrombopoietin (TPO)-dependent BF-TE22 cell line endogenously expressing murine Mp1, which is a subclone of murine pro-B Ba/F3 cells. TPO stimulated the proliferation of BF-TE22 cells in a dose-dependent manner, and also induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens. The results indicate that the murine Mp1 on BF-TE22 cells can transmit both proliferation and megakaryocyte lineage-specific differentiation signals to cells. Furthermore, it was shown that IL-3 inhibits the TPO-induced differentiation signals of BF-TE22 cells. These results suggest that the signals mediated by IL-3 predominate over thor of TPO in BF-TE22 cells. Thus, BF-TE22 cells will be useful for the biological and biochemical studies of the TPO-Mp1 signal transduction mechanism.

    DOI

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    1
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  • Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice

    Kazunori Shibuya, Hiromichi Akahori, Kazumi Takahashi, Emiko Tahara, Takashi Kato, Hiroshi Miyazaki

    Blood   91 ( 1 ) 37 - 45  1998.01

     View Summary

    Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG- rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 μg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit-spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG- rHuMGDF (80 μg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG- rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG- rHuMGDF, a single injection of recombinant human granulocyte colony- stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 μg/kg) treatment even when rhG-CSF doses were escalated to 1,000 μg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

    DOI PubMed

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    59
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  • Mobilization of primitive haemopoietic progenitor cells and stem cells with long-term repopulating ability into peripheral blood in mice by pegylated recombinant human megakaryocyte growth and development factor

    Y. Torii, Y. Nitta, H. Akahori, T. Tawara, T. Kuwaki, K. Ogami, T. Kato, H. Miyazaki

    British Journal of Haematology   103 ( 4 ) 1172 - 1180  1998

     View Summary

    We investigated in detail the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization in male BDF1 mice. Treatment with PEG- rHuMGDF for 5 d stimulated a striking expansion of the circulating levels of multiple types of colony-forming units in culture (CFU-c), including CFU- granulocyte-macrophage, CFU-megakaryocyte, burst-forming units-erythroid, and multipotent CFU-c, and primitive day-12 CFU-spleen. All of these progenitors were mobilized into the peripheral blood (PB) with similar kinetics; their numbers peaked after the cessation of treatment and then declined earlier than platelet numbers peaked. The maximal increase in any of the four CFU-c in the PB was attained with at least 300 μg/kg/d of PEG-rHuMGDF, whereas peripheral platelet counts plateaued at 30 μg/kg/d. Adoptive transfer with PB from PEG- rHuMGDF-treated donor mice resulted in greater survival of lethally irradiated recipients. The majority of the recipients that survived at 187 d after transplantation with PEG-rHuMGDF-mobilized PB showed significant donor engraftment at the progenitor cell level. The combined administration of appropriate doses of PEG-rHuMGDF and recombinant human granulocyte colony-stimulating factor induced a synergistic increase in the circulating levels of the four CFU-c compared to either factor alone. These results indicate that PEG-rHUMGDF as a single agent can mobilize a full spectrum of PBPCs in mice.

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    11
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  • Increased thrombopoietin levels in idiopathic thrombocytopenic purpura patients with a poor response to steroid therapy

    Y. Sakane, H. Wada, H. Kazuyo, M. Shimura, T. Nakasaki, N. Katayama, M. Nishikawa, K. Deguchi, Y. Mori, H. Shiku, T. Tahara, T. Kato

    Blood Coagulation and Fibrinolysis   9 ( 4 ) 315 - 321  1998

     View Summary

    The serum thrombopoietin (TPO) levels in 61 idiopathic thrombocytopenic purpura (ITP) patients were found to be slightly increased compared with those of 29 normal subjects. The TPO levels of the 15 ITP patients who had a poor response to steroid therapy (i.e. an unchanged platelet count) were higher than those of the 22 ITP patients who had a good response to steroid therapy (i.e. an increased platelet count) and the normal subjects. The TPO levels in the 15 ITP patients whose platelet count was higher than 10 x 104/μl after the discontinuation of steroid therapy significantly higher than those of the normal subjects. The platelet-associated immunoglobulin G (PAIgG) levels in the ITP patients who had a poor response to steroid therapy were slightly increased compared with the normal subjects and the ITP patients who had a good response to the steroid therapy and the nine ITP patients who did not undergo the steroid therapy. The serum TPO level was negatively correlated only with the megakaryocyte count in the ITP patients, and the megakaryocyte count in the ITP patients who had good responses to the steroid therapy was higher than that in those who had poor responses. These data suggest that serum TPO levels might be important for the prediction of the outcome of ITP patients who receive steroid therapy.

    DOI PubMed

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    14
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  • Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin

    Tomoyuki Tahara, Tomoaki Kuwaki, Atsushi Matsumoto, Haruhiko Morita, Hiroshi Watarai, Yoshimasa Inagaki, Hideya Ohashi, Kinya Ogami, Hiroshi Miyazaki, Takashi Kato

    Stem Cells   16 ( 1 ) 54 - 60  1998

     View Summary

    Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, c-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

    DOI PubMed

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    14
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  • Proliferative properties of human umbilical cord blood megakaryocyte progenitor cells to human thrombopoietin

    T. Hagiwara, I. Kodama, K. Horie, T. Kato, H. Miyazaki

    Experimental Hematology   26 ( 3 ) 228 - 235  1998

     View Summary

    We evaluated the effects of human thrombopoietin (TPO) alone, or in combination with other several hematopoietic factors, on megakaryocyte colony growth from human umbilical cord blood CD34+ cells in serum-depleted agar cultures. The addition of TPO alone had a concentration-dependent effect on the number of pure megakaryocyte colonies and of megakaryocytes per colony (colony size). The maximally stimulating concentration of TPO generated a greater number of megakaryocyte colonies and larger megakaryocyte colony size compared with the stimulation observed with an optimal concentration of human interleukin-3 (IL-3) or stem cell factor (SCF). At the high concentration of TPO that yielded the maximum colony numbers, a substantial proportion of megakaryocyte colonies contained 100 or more cells per colony. The combination of TPO plus SCF resulted in a synergistic enhancement of both the number and size of megakaryocyte colonies. Among the combinations of TPO plus other cytokines tested, IL-3 plus TPO had a modest effect on megakaryocyte colony numbers. The generation of megakaryocyte colonies from subpopulations of CD34+ cells was further examined. The addition of TPO alone induced a greater number of megakaryocyte colonies from CD34+CD41+ cells compared with CD34+CD41- cells, and TPO plus IL-3 exerted a synergistic effect on the number of megakaryocyte colonies only from CD34+CD41- cells. In contrast to the effects on colony numbers, TPO induced larger megakaryocyte colony sizes from CD34+CD41- cells, compared with CD34+CD41+ cells. In the case of HLA-DR expression, TPO and IL-3, administered singly or in combination, induced similar megakaryocyte colony numbers and sizes from CD34+DR+ and CD34+DR- subpopulations. Ploidy analysis revealed that the majority of megakaryocytes generated from cord blood CD34+ cells in serum-free liquid cultures containing TPO displayed 2N and 4N values, suggesting that they were immature. These results indicate that, compared with IL-3 and SCF, TPO has more potent proliferative effect on human cord blood megakaryocyte progenitors, leading to greater numbers of megakaryocyte progenitors triggered for both growth and cell division, and synergizes with SCF to enhance megakaryocyte colony growth.

    PubMed

  • Changes in serum thrombopoietin levels after splenectomy

    Naoaki Ichikawa, Kiyoshi Kitano, Shigetaka Shimodaira, Fumihiro Ishida, Toshiro Ito, Shoji Kajikawa, Tomoyuki Tahara, Takashi Kato, Kendo Kiyosawa

    Acta Haematologica   100 ( 3 ) 137 - 141  1998

     View Summary

    To clarify the role of thrombopoietin (c-Mp1 ligand, TPO) in 'hypersplenic' thrombocytopenia, we used an enzyme-linked immunosorbent assay to examine changes in serum TPO levels accompanied with splenectomy in 6 patients with liver cirrhosis, 4 patients with gastric cancer, and 2 patients with lymphoid malignancies. We also measured serum levels of other thrombopoietic cytokines such as interleukin-6 (IL-6) and erythropoietin. Platelet counts reached a maximum at day 14 after splenectomy in all subjects. In patients with liver cirrhosis, a lower elevation of platelet counts was observed compared with that in patients with gastric cancer. Serum TPO levels gradually elevated after splenectomy and reached a maximum 3.5 days after splenectomy in noncirrhotic patients, whereas peak serum TPO levels were delayed until day 7 in the cirrhosis group. IL-6 and erythropoietin showed similar kinetics between cirrhotic and noncirrhotic patients. These findings suggest that transient thrombocytosis after splenectomy may be associated with an alteration in the site of TPO catabolism by platelets from spleen to the blood and that deterioration of TPO production may play a role in thrombocytopenia in liver cirrhosis.

    DOI PubMed

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  • Plasma thrombopoietin (TPO) levels and expression of TPO receptor on platelets in patients with myelodysplastic syndromes

    H. Tamura, K. Ogata, S. Luo, K. Nakamura, N. Yokose, K. Dan, K. Tohyama, Y. Yoshida, H. Hamaguchi, H. Sakamaki, T. Kuwaki, T. Tahara, T. Kato, T. Nomura

    British Journal of Haematology   103 ( 3 ) 778 - 784  1998

     View Summary

    Data on endogenous thrombopoietin (TPO) levels and their regulation in myelodysplastic syndromes (MDS) are sparse. We examined the plasma TPO level of 85 MDS patients by a sensitive enzyme immunoassay and the platelet expression of TPO receptor (TPO-R) protein, which metabolizes endogenous TPO, in 19 MDS patients with an equilibrium binding assay using 125I-TPO. The MDS patients had higher plasma TPO levels (7.0 ± 9.3 fmol/ml) than 52 normal subjects (P<0.0001). Refractory anaemia (RA) patients (n = 39) had higher plasma TPO levels than patients (n = 28) with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-t) (P = 0.0002), irrespective of similar platelet counts in these groups. The plasma TPO level correlated inversely with the platelet count in RA patients (P = 0.0027) but not in RAEB and RAEB- t patients (P = 0.7865). These data suggest that the physiological pathway for TPO production and metabolism is conserved, at least partially, in RA, but deranged in RAEB/RAEB-t. The number of TPO-R per platelet was significantly smaller in 19 MDS patients (17.5 ± 13.3) than in normals (P = 0.0014), but similar between RA patients and patients with RAEB and RAEB-t. Further, the bone marrow megakaryocyte count, determined in 31 MDS patients, was quite similar between RA patients and patients with RAEB or RAEB-t. Thus, in addition to thrombocytopenia, a reduced platelet TPO-R number may contribute to elevated plasma TPO levels in MDS, and a regulatory pathway for circulating TPO other than platelet TPO-R and marrow megakaryocytes, such as blasts expressing TPO-R, may operate in RAEB/RAEB-t.

    DOI PubMed

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  • Thrombopoietin, steel factor and the ligand for flt3/flk2 interact to stimulate the proliferation of human hematopoietic progenitors in culture

    Masao Kobayashi, Joseph H. Laver, Stewart D. Lyman, Takashi Kato, Hiroshi Miyazaki, Makio Ogawa

    International Journal of Hematology   66 ( 4 ) 423 - 434  1997.12

     View Summary

    We have tested the effects of steel factor (SF) the ligand for flt3/flk2 (FL) and thrombopoietin (TPO, Mpl ligand), on the proliferation of primitive human bone marrow progenitors in serum-deprived culture. Varying combinations of SF, FL and TPO supported formation of only few colonies from CD34+/c- Kit(low)/CD38(neg/low) cells. However, the addition of interleukin 3 (IL-3) to the three cytokines significantly increased the number of colonies. When this population of cells was tested in suspension culture for one week for production of colony-forming cells there was synergism among SF, FL and TPO. Addition of IL-3 to the three cytokines further increased the number of erythroid colony-forming cells. The effects of these four factors on CD34+/c- Kit(low)/CD38(high) cells were merely additive. Studies of individual CD34+/c-Kit(low)/CD38(neg/low) cells demonstrated the direct effects of SF, FL and TPO. In the presence of SF, FL and TPO, approximately half of the individual CD34+/c-Kit(low)/CD38(neg/low) cells proliferated in seven day suspension culture. Addition of IL-3 to the combination of SF, FL and TPO did not increase the frequencies of proliferating clones, but increased the size of individual clones. These observations suggest that SF, FL and TPO play important roles in survival and proliferation of primitive human hematopoietic progenitors.

    DOI PubMed

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    18
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  • Serum thrombopoietin levels in patients receiving high-dose chemotherapy with support of purified peripheral blood CD34+ cells

    Yasuhiro Okamoto, Yoshifumi Kawano, Yoichi Takaue, Tsutomu Watanabe, Takashi Kato, Akihiro Shimosaka, Yasuhiro Kuroda

    Cancer Research   57 ( 22 ) 5037 - 5040  1997.11

     View Summary

    In a case control study, serum levels of thrombopoietin (TPO) were determined by a sandwich ELISA in 20 patients (median age, 7 years; range, 2- 56 years) with various malignancies who received high-dose chemotherapy and a stem cell rescue operation. The patients received two different transplant modalities: (a) 12 patients received purified autologous peripheral blood CD34+ cells; and (b) 8 patients received cells in the CD34(-) fraction, which still contains many CD34+ cells. No significant differences were observed between the two groups with regard to the duration required to achieve an absolute granulocyte count of >0.5 x 109/liter, the duration of dependence on platelet transfusion, or the number of platelet transfusions. In both groups, the serum TPO levels were inversely correlated with the circulating platelet count. Multivariate analysis demonstrated that significant determinants of the serum TPO level included the circulating platelet count (standardized regression coefficient = -0.5179), transplantation with cells in the CD34(-) fraction (0.2414), solid tumor (0.1420), and the age of the patient (-0.1236; r2 = 0.3021; P < 0.0001). These results suggest that the mode of stem cell support (i.e., the presence of accessory cells in the inoculum), age, or the type of preceding chemotherapy affects serum TPO levels after transplantation.

    PubMed

  • Markedly reduced expression of platelet c-mpl receptor in essential thrombocythemia

    Yoko Horikawa, Itaru Matsumura, Koji Hashimoto, Masamichi Shiraga, Satoru Kosugi, Seiji Tadokoro, Takashi Kato, Hiroshi Miyazaki, Yoshiaki Tomiyama, Yoshiyuki Kurata, Yuji Matsuzawa, Yuzuru Kanakura

    Blood   90 ( 10 ) 4031 - 4038  1997.11

     View Summary

    Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis through binding to the cytokine receptor c-Mpl (the product of the c-mpl proto-oncogene). In an effort to determine the pathophysiological role of TPO-c-Mpl system in essential thrombocythemia (ET), we have examined the levels of serum TPO and the expression and function of platelet c-Mpl in 17 patients with ET. In spite of extreme thrombocytosis, serum TPO levels were slightly elevated or within normal range in most, if not all, patients with ET (mean ± SD, 1.31 ± 1.64 fmol mL), as compared with normal subjects (0.76 ± 0.21 fmol/mL). Flow cytometric and Western blot analyses revealed that the expression of platelet c-Mpl was strikingly reduced in all patients with ET. Furthermore, the expression of platelet c-mpl mRNA was found to be significantly decreased in the ET patients tested. In contrast, almost identical levels of GPIIb/IIIa protein and mRNA were expressed in platelets from ET patients and normal controls. In addition to expression level, activation state of platelet c-Mpl was investigated in ET patients. Immunoblotting with anti-phosphotyrosine antibody showed that no aberrant protein-tyrosine phosphorylation was observed in platelets of ET patients before treatment with TPO, and the levels of TP-induced protein-tyrosine phosphorylation, including c-Mpl- tyrosyl phosphorylation, roughly paralleled those of c-Mpl expression, suggesting that c-Mpl-mediated signaling pathway was not constitutively activated in platelets of ET patients. These results suggested that the TPO- c-Mpl system may not be directly linked to pathogenesis of ET, and that gene(s) mutated in ET may be important in regulating the levels of c-mpl gene expression in addition to the growth and differentiation of multipotential hematopoietic stem cells.

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    170
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  • Effects and serum levels of thrombopoietin in a case of chronic thrombocytopenia with achondroplasia

    Akira Ishiguro, Tatsutoshi Nakahata, Kenji Muraoka, Tomoyuki Tahara, Hiroshi Miyazaki, Takashi Kato, Yuji Inaba, Toshikazu Shimbo

    International Journal of Hematology   66 ( 1 ) 99 - 102  1997.07

     View Summary

    Thrombopoietin (TPO), produced mainly in the liver, is a major regulator of platelet production. Serum TPO levels are generally increased in thrombocytopenia. We report a case of a 12-year-old boy with chronic severe thrombocytopenia, achondroplasia and nephritis. Severe chronic thrombocytopenia was found at 9 months of age. It was resistant to any treatment. Studies on megakaryocytic colonies in vitro revealed that the marrow cells responded well to TPO and no plasma inhibitor was found. Although hepatic function test results were normal, serum TPO levels in the patient (0.94 fmol/ml) were consistent with those in age-matched children (0.49 1.75 fmol/ml). Chronic thrombocytopenia requires individual evaluation before clinical trials with TPO.

    DOI PubMed

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    5
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  • Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21 (WAF1/Cip1) by STAT5

    Itaru Matsumura, Jun Ishikawa, Koichi Nakajima, Kenji Oritani, Yoshiaki Tomiyama, Jun Ichiro Miyagawa, Takashi Kato, Hiroshi Miyazaki, Yuji Matsuzawa, Yuzuru Kanakura

    Molecular and Cellular Biology   17 ( 5 ) 2933 - 2943  1997.05

     View Summary

    Although thrombopoietin (TPO) is known to play a fundamental role in both megakaryopoiesis and thrombopoiesis, the molecular mechanism of TPO- induced megakaryocytic differentiation is not known. In a human megakaryoblastic leukemia cell line, CMK, that showed some degree of megakaryocytic differentiation after culture with TPO, the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/Cip1), but not p27(Kip1), p16(INK4A), p15(INK4B), or p18(INK4C), was found to be upregulated in an immediately early response to TPO. The expression of p21 was found to be sustained over a period of 5 days by treatment with TPO in large polyploid cells that developed in response to TPO, but not in small undifferentiated cells, indicating a close correlation between the ligand-induced differentiation and p21 induction in CMK cells. To examine potential roles of Cdk inhibitors in megakaryocytic differentiation, CMK cells were transfected with the p21, p27, or p16 gene, together with a marker gene, β-galactosidase, and were cultured with medium alone for 5 days. The ectopic expression of p21 or p27 but not of p16 led to induction of megakaryocytic differentiation of CMK cells. Overexpression of the N-terminal domain (amino acids [aa] 1 to 75) of p21 was sufficient to induce megakaryocytic differentiation, whereas that of the C- terminal domain (aa 76 to 164) had little or no effect on morphological features. Furthermore, we found that although TPO induced tyrosine phosphorylation of both STAT3 and STAT5 in CMK cells, only STATS showed binding activities to potential STAT-binding sites that locate in the promoter region of p21 gene (p21-SIE sites), thereby leading to transactivation of p21. These results suggested that p21 induction, possibly mediated through activated STATS, could play an important role in TPO- induced megakaryocytic differentiation.

    DOI PubMed

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  • Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin

    Takashi Kato, Atsushi Oda, Yoshimasa Inagaki, Hideya Ohashi, Atsushi Matsumoto, Katsutoshi Ozaki, Yoshitaka Miyakawa, Hiroshi Watarai, Kazumi Fuju, Atsuko Kokubo, Toshihiko Kadoya, Yasuo Ikeda, Hiroshi Miyazaki

    Proceedings of the National Academy of Sciences of the United States of America   94 ( 9 ) 4669 - 4674  1997.04

     View Summary

    A heterogeneity in the molecular weight (M(r)) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low M(r) peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.

    DOI PubMed

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  • Protein kinase C and c-myc gene activation pathways in thrombopoietin signal transduction

    Masae Kunitama, Ritsuko Shimizu, Minami Yamada, Takashi Kato, Hiroshi Miyazaki, Koji Okada, Yasusada Miura, Norio Komatsu

    Biochemical and Biophysical Research Communications   231 ( 2 ) 290 - 294  1997.02

     View Summary

    Thrombopoietin (TPO) is the major regulator of the proliferation and differentiation of megakaryocyte precursors through interaction with its receptor encoded by the c-mpl protooncogene. We established the human TPO-dependent leukemia cell line, UT-7/TPO. In these cells, TPO activated protein kinase C (PKC) in a time dependent manner. Subsequently, the c-myc gene was transiently induced to a maximal level 60-90 minutes after TPO exposure. In addition, we found that stimulating UT-7/TPO cells with TPO rapidly induces the significant accumulation of inositol 1,4,5-trisphosphate (Ins-P3), leading to the mobilization of calcium from intracellular stores. Taken together, the activation of PKC and subsequent c-myc gene induction are involved in the TPO-induced cellular response(s), presumably through the activation of PLC.

    DOI PubMed

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    21
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  • Erratum: Improvement of thrombocytopenia following bone marrow transplantation by pegylated recombinant human megakaryocyte growth anti development factor in mice (Bone Marrow Transplantation (1996) 18 (1035-1041))

    K. Kabaya, K. Shibuya, Y. Torii, Y. Nitta, M. Ida, H. Akahori, T. Kato, M. Kusaka, H. Miyazaki

    Bone Marrow Transplantation   20 ( 7 ) 619 - 620  1997

    DOI

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  • Increased serum levels of thrombopoietin in patients with thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, or disseminated intravascular coagulation

    K. Hiyoyama, H. Wada, M. Shimura, T. Nakasaki, N. Katayama, M. Nishikawa, H. Shiku, T. Tahara, T. Kato

    Blood Coagulation and Fibrinolysis   8 ( 6 ) 345 - 349  1997

     View Summary

    The serum levels of thrombopoietin (TPO) were measured in 16 patients with thrombotic thrombocytopenic purpura (TTP), 12 with hemolytic uremic syndrome (HUS), 10 with aplastic anemia (AA), 10 with disseminated intravascular coagulation (DIC), and 71 with idiopathic thrombocytopenic purpura (ITP). The serum TPO levels were measured with a sensitive sandwich enzyme-linked immunosorbent assay. The serum TPO level in the ITP group (1.68 ± 0.85 fmol/ml) were not significantly increased compared with those of the normal subjects. The TPO levels in the TTP (2.77 ± 1.38 fmol/ml) and HUS groups (5.77 ± 4.41 fmol/ml) were higher than those of the normal subjects. The patients with AA (12.7 ± 8.0 fmol/ml) and those with DIC (13.3 ± 5.7 mol/ml) had significantly higher serum TPO levels than did the normal subjects and ITP patients. The TPO levels were well correlated with the platelet counts in the TIP patients, and were negatively correlated with the platelet counts in the ITP patients. These results suggest that the serum TPO levels in some thrombocytopenic diseases are regulated not only by the platelet count and the megakaryocyte mass, but also by other factors.

    DOI PubMed

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    35
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  • Role for C-MPL and its Ligand Thrombopoietin in Early Hematopoiesis

    Naoyuki Katayamaa, Ryugo Itoh, Takashi Kato, Takayuki Sugawara, Nadim Mahmud, Kohshi Ohish, Masahiro Masuya, Masatoshi Aoki, Nobuyuki Minami, Hiroshi Miyazaki, Hiroshi Shiku

    Leukemia and Lymphoma   28 ( 42006 ) 51 - 56  1997.01

     View Summary

    Proto-oncogene c-mpl is structurally homologous with the hematopoietic growth factor receptor superfamily. The ligand for c-mpl was purified and its gene cloned. Extensive functional studies revealed that the ligand for c-mpl plays a crucial role in megakaryocytopoiesis and platelet production, hence, this ligand proved to be the long-sought hematopoietin, thrombopoietin (TPO). We briefly review here the role for TPO in early hematopoiesis, based on our in vitro data obtained using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) but not TPO alone supported the growth of murine primitive hematopoietic progenitors. Studies on lineage expression indicated that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of primitive hematopoietic progenitors. TPO also hastened IL-3-dependent growth of progenitors by shortening the time required for cell-cycling. While size of the colonies did not differ between colonies supported by IL-3 alone and those supported by IL-3 plus TPO, the incidence of megakaryocyte-containing colonies in cultures supported by IL-3 plus TPO was higher than that in cultures supported by IL-3 alone. Taken together, TPO as a single factor can support the survival of hematopoietic progenitors and TPO synergizes with SF or IL-3 to be active on early multipotential hematopoietic progenitors. These findings suggest that the function of TPO initially thought to be restricted to the megakaryocytic lineage proved to be greater in hematopoiesis. Reports from other laboratories regarding the involvement of TPO in early hematopoiesis are also discussed. © 1997, Informa UK Ltd All rights reserved: reproduction in whole or part not permitted. All rights reserved.

    DOI PubMed

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    7
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  • Measurement of endogenous plasma thrombopoietin in patients with acquired aplastic anaemia by a sensitive enzyme-linked immunosorbent assay

    Seiji Kojima, Takaharu Matsuyama, Yoshihisa Kodera, Tomoyuki Tahara, Takashi Kato

    British Journal of Haematology   97 ( 3 ) 538 - 543  1997

     View Summary

    We measured and the endogenous plasma concentration of thrombopoietin (TPO) in 76 patients with acquired aplastic anaemia (AA) by a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). The plasma TPO concentrations were significantly higher in AA patients when compared to healthy control subjects (P<0.0001) and there was a significant negative correlation between plasma TPO concentrations and platelet counts in 54 AA patients who had not received any platelet transfusions prior to sampling. On the other hand, there was no statistically significant correlation between the TPO concentrations and platelet counts in 22 AA patients who had previously received platelet transfusions. We studied serial changes of plasma TPO concentration in 24 patients showed an increase in their platelet counts following bone marrow transplantation or immunosuppressive (IS) therapy. Although a decrease in plasma TPO concentration as observed, levels remained above the range of normal healthy controls even in the patients who attained normal platelet counts following therapy. A decrease in TPO concentrations was observed in only half of the responders to IS therapy. Whether exogenous TPO will result in increased platelet counts in AA patients with high TPO levels remain to be determined.

    DOI PubMed

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    30
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  • Megakaryocytes derived from CD34-positive cord blood cells produce interleukin-8

    Tsukasa Higuchi, Kenichi Koike, Nobukuni Sawai, Hadija Hemed Mwamtemi, Kouichi Takeuchi, Masaaki Shiohara, Toshimi Kikuchi, Kozo Yasui, Susumu Ito, Osamu Yamagami, Yumiko Sasaki, Nobuo Okumura, Takashi Kato, Hiroshi Miyazaki, Masayuki Ikeda, Muneo Yamada, Atsushi Komiyama

    British Journal of Haematology   99 ( 3 ) 509 - 516  1997

     View Summary

    In a serum-free liquid culture, thrombopoietin (TPD) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 x 105) secreted > 1000 pg/ml of interleukin (IL)-8, in contrast to small amounts of IL-1β and IL-6. A time-course study showed that the IL-8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. The combination of TPO and IL-1α was required for a significant augmentation of the IL-8 secretion. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse transcription-polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL-8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL-8 release by the megakaryocytic cells with the aid of IL-1.

    DOI PubMed

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    15
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  • Action of thrombopoietin at the megakaryocyte progenitor level is critical for the subsequent proplatelet production

    K. Horie, H. Miyazaki, T. Hagiwara, E. Tahara, A. Matsumoto, T. Kadoya, K. Ogami, T. Kato

    Experimental Hematology   25 ( 2 ) 169 - 176  1997

     View Summary

    Formation of proplatelets from megakaryocytes is believed to be the first step of platelet production in vitro. In this study, we evaluated the effects of recombinant human thrombopoietin (hTPO) on the development of proplatelets from a GpIIb/IIIa+ population of rat bone marrow cells highly enriched for late megakaryocyte progenitors (GpIIb/IIIa+ CFU-MK) that we recently found to be a primary target population of TPO. Quantitative measurement of hTPO-induced proplatelet formation was performed in liquid cultures. Proplatelet formation from megakaryocytes derived from GpIIb/IIIa+ CFU-MK in the presence of hTPO began on day 4 of culture and peaked the following day. On day 5 of culture, lower concentrations of hTPO expanded the number of megakaryocytes, increased the number of proplatelets and the percentage of proplatelet-developing megakaryocytes. Increasing hTPO concentrations resulted in a modest decrease in proplatelet development. We next used hTPO to derive immature or mature megakaryocytes from GpIIb/IIIa+ CFU-MK. These populations of cultured megakaryocytes spontaneously formed proplatelets when recultured in the absence of exogenous hTPO. The addition of hTPO at higher concentrations modestly augmented proplatelet production from immature megakaryocytes derived from 2-day liquid cultures. However, either murine interleukin-6 (IL-6) or human IL-11, but not rat IL-3, was more potent than hTPO in augmenting proplatelet formation from immature megakaryocytes. Each of these four cytokines had an inhibitory effect on proplatelet formation from more differentiated megakaryocytes derived from 3-day liquid cultures. These results indicate that TPO enhances proplatelet production primarily by stimulating CFU-MK to increase the number of proplatelet-forming megakaryocytes and that its action is clearly different from those of other cytokines that also stimulate megakaryocytopoiesis.

    PubMed

  • Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization

    S. Nomura, K. Ogami, K. Kawamura, I. Tsukamoto, Y. Kudo, Y. Kanakura, Y. Kitamura, H. Miyazaki, T. Kato

    Experimental Hematology   25 ( 7 ) 565 - 572  1997

     View Summary

    The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

    PubMed

  • Recombinant human c-Mpl ligand (thrombopoietin) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro

    S. Tanimukai, T. Kimura, H. Sakabe, Y. Ohmizono, T. Kato, H. Miyazaki, H. Yamagishi, Y. Sonoda

    Experimental Hematology   25 ( 10 ) 1025 - 1033  1997

     View Summary

    To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma- containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU- Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst- promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.

    PubMed

  • Purification and biochemical properties of thrombopoietin

    T. Kato

    Seikagaku   69 ( 6 ) 433 - 438  1997

    PubMed

  • Circulating endogenous thrombopoietin, interleukin-3, interleukin-6 and interleukin-11 levels in patients undergoing allogeneic bone marrow transplantation

    Akaru Ishida, Yoshitaka Miyakawa, Ryuji Tanosaki, Masatoshi Wakui, Hironori Ueno, Reiko Watanabe, Norihiro Awaya, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Atsushi Oda, Masahiro Kizaki, Shinichiro Okamoto, Yasuo Ikeda

    International Journal of Hematology   65 ( 1 ) 61 - 69  1996.12

     View Summary

    To elucidate the physiologic role of thrombopoietin (TPO) for hematologic reconstitution following allogeneic bone marrow transplantation (BMT), serum TPO levels as well as interleukin-3 (IL-3), IL-6 and IL-11 were serially measured in 55 samples from 3 patients who underwent allogeneic BMT using an enzyme-linked immunosorbent assay (ELISA). The TPO level was higher in the serum taken during marrow aplasia than in the pretransplant serum. The serum TPO levels and platelet counts showed a strong inverse relationship in all patients examined. We also sequentially measured endogenous serum TPO levels before and within 36 h after platelet transfusions. Endogenous serum TPO levels were inversely correlated with platelet mass following platelet transfusions. Serum levels of IL-3 had no apparent correlation with platelet counts and serum levels of IL-11 remained below the detection levels (31.3 pg/ml) in all samples. Serum levels of IL-6 were high during myeloaplasia and more upregulated in the febrile period. These findings support the view that TPO is the central regulator for megakaryopoiesis in vivo and the rationale for its clinical use after allogeneic BMT.

    DOI PubMed

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  • Soluble thrombopoietin receptor (Mpl) and granulocyte colony-stimulating factor receptor directly stimulate proliferation of primitive hematopoietic progenitors of mice in synergy with steel factor or the ligand for Flt3/Flk2

    Hsun Ku, Fumiya Hirayama, Takashi Kato, Hiroshi Miyazaki, Masaharu Aritomi, Yoshimi Ota, Alan D. D'Andrea, Stewart D. Lyman, Makio Ogawa

    Blood   88 ( 11 ) 4124 - 4131  1996.12

     View Summary

    In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony-stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.

    DOI PubMed

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    34
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  • Improvement of thrombocytopenia following bone marrow transplantation by pegylated recombinant human megakaryocyte growth and development factor in mice

    K. Kabaya, K. Shibuya, Y. Torii, Y. Nitta, M. Ida, H. Akahori, T. Kato, M. Kusaka, H. Miyazaki

    Bone Marrow Transplantation   18 ( 6 ) 1035 - 1041  1996.12

     View Summary

    We examined whether pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is capable of improving thrombocytopenia and promoting thrombopoietic reconstitution following lethal irradiation and bone marrow transplantation (BMT) in mice. Immediately after receiving 10 Gy whole body irradiation (day 0), male C3H/HeN mice were inoculated with 106 bone marrow cells obtained from syngeneic mice. Circulating platelet counts decreased to below 4% of the normal counts with a nadir on day 10, and then returned to the normal level on day 28 in the control mice undergoing BMT. Subcutaneous consecutive treatment with PEG-rHuMGDF at doses from 10 to 300 μg/kg/day from day 1 for 13 days significantly improved the platelet nadir and promoted platelet recovery. The white blood cell counts and hemoglobin concentration following BMT were not influenced by the PEG-rHuMGDF. PEG-rHuMGDF-injection starting from day 5 did not improve the platelet nadir following BMT. Furthermore, administration with PEG-rHuMGDF on alternate days at 55.7 μg/kg/day for 7 days or at an interval of 3 days at 78 μg/kg/day for 4 days (twice a week for 2 weeks) had a significant efficacy, but these administration regimens had less efficacy than consecutive administration at 30 μg/kg/day for 13 days. The numbers of megakaryocytes and megakaryocyte progenitor cells decreased to 5 and 0.2% of normal level, respectively, in the control mice. Consecutive administration of PEG-rHuMGDF enhanced the recovery of the mean number of these cells compared to those in vehicle-treated mice, although such effects were not statistically significant except for the number of megakaryocyte progenitors on day 12. These results suggest that consecutive treatment with PEG-rHuMGDF beginning from the day after BMT may be effective in improving thrombocytopenia following BMT.

    PubMed

  • Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice

    Hiromichi Akahori, Kazunori Shibuya, Masako Ozai, Masumi Ida, Koji Kabaya, Takashi Kato, Hiroshi Miyazaki

    Stem Cells   14 ( 6 ) 678 - 689  1996.11

     View Summary

    Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal rule in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-Mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy- induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by i.v. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia: it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG- CSF had an additive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.

    DOI PubMed

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  • In vivo effects of pegylated recombinant human megakaryocyte growth and development factor on hematopoiesis in normal mice

    Koji Kabaya, Hiromichi Akahori, Kazunori Shibuya, Yuko Nitta, Masumi Ida, Masaru Kusaka, Takashi Kato, Hiroshi Miyazaki

    Stem Cells   14 ( 6 ) 651 - 660  1996.11

     View Summary

    The in vivo effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human thrombopoietin modified with polyethylene glycol, were investigated in normal Balb/c mice. PEG-rHuMGDF was more potent in producing platelets and the dose-response curve was sleeper compared with the case of the nonpegylated form of this molecule. Five consecutive injections with PEG- rHuMGDF caused a dose-dependent increase in peripheral platelet counts with a peak on day 8. There was a dose-dependent rise in platelet counts on day 8 at daily doses from 0.333 to 30 μg/kg. Intermediate doses of PEG-rHuMGDF (1.111 to 10 μg/kg/day) caused a significant decrease in mean platelet volume, and conversely, higher doses of PEG-rHuMGDF (30 to 270 μg/kg/day) induced a dose-dependent increase in mean platelet volume. There was a dose-dependent decrease in hemoglobin concentration with a minimum on day 8 but no significant reduction in reticulocyte counts following PEG-rHuMGDF administration. White blood cell counts were unchanged by PEG-rHuMGDF treatment. Marrow megakaryocyte size enlarged in 1.5-fold and the number of marrow megakaryocytes increased to sixfold by consecutive administration of PEG-rHuMGDF at 30 μg/kg/day. A twofold increase in the number of marrow megakaryocytic progenitor cells (colony-forming units-megakaryocyte) was also observed. Marrow erythroid progenitor (colony-forming units-erythroid) counts decreased but splenic colony-forming units-erythroid, marrow and splenic erythro/myeloid progenitor cell counts, and splenic granulocyte/macrophage progenitor cell counts increased with PEG-rHuMGDF treatment. Marrow and splenic erythroid burst-forming cells were unchanged. These results indicate that PEG-rHuMGDF, a truncated molecule of thrombopoietin, is a potent stimulator for megakaryopoiesis and thrombopoiesis, and also affects the development of other hematopoietic cells in normal mice.

    DOI PubMed

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    36
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  • Serum thrombopoietin (TPO) levels in patients with amegakaryocytic thrombocytopenia are much higher than those with immune thrombocytopenic purpura

    Harumi Y. Mukai, Hiroshi Kojima, Kazuo Todokoro, Tomoyuki Tahara, Takashi Kato, Yuichi Hasegawa, Toshitaka Kobayashi, Haruhiko Ninomiya, Toshiro Nagasawa, Tsukasa Abe

    Thrombosis and Haemostasis   76 ( 5 ) 675 - 678  1996.11

     View Summary

    We assayed serum thrombopoietin (TPO) levels in amegakaryocytic thrombocytopenia (AMT) and immune thrombocytopenic purpura (ITP) patients by using a newly established enzyme-linked immunosorbent assay (ELISA). TPO levels in AMT patients were quite high (mean ± SD = 13.7 ± 11.2 fmoles/ml, n = 4), whereas those in ITP patients were only slightly higher (1.25 ± 0.39, n = 12) than those of the healthy donors (0.55 ± 0.2, n = 20). Furthermore, in ITP patients no correlation was observed between platelet counts and serum TPO levels (correlation coefficient = 0.14). We further assayed serum TPO levels sequentially during steroid treatment in patients with AMT and ITP. In one AMT patient serum TPO levels started to decrease in accordance with the increase of megakaryocyte counts, which preceded the increase in platelet counts. However, in ITP patients serum TPO levels did not change significantly throughout the course of the treatment despite the recovery of platelet counts. Based on these findings, we conclude that serum TPO levels may be regulated at least in part by megakaryocyte counts.

    DOI PubMed

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    65
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  • Functional analysis of the cytoplasmic domain of the human Mpl receptor for tyrosine-phosphorylation of the signaling molecules, proliferation and differentiation

    Haruhiko Morita, Tomoyuki Tahara, Atsushi Matsumoto, Takashi Kato, Hiroshi Miyazaki, Hideya Ohashi

    FEBS Letters   395 ( 2-3 ) 228 - 234  1996.10

     View Summary

    To investigate the functional domains for signal transduction of human Mpl, we constructed a series of human c-mpl cDNAs with various deletions in the cytoplasmic domain, and then introduced each cDNA into murine IL3-dependent myeloid leukemia FDC/P2 cells to establish stable transformants. We examined the growth and differentiation responses and tyrosine phosphorylation of the intracellular signaling proteins including Jak2, Tyk2, Stat3, Stat5, Vav, SHPTP2, Cbl, Shc and Shc-associated p145 when receptor stimulation occurred after thrombopoietin (TPO) binding. TPO stimulated cell proliferation and induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens and tyrosine phosphorylation of the signaling proteins in transformants expressing full length human Mpl. These results suggested that Mpl not only induced proliferation but also transduced megakaryocyte-specific differentiation signals into FDC/P2 cells. Mutational analysis of human Mpl indicated that the N-terminal region of its cytoplasmic domain is necessary and sufficient to transduce proliferation and differentiation signals into cells, while the C-terminal region may also play important roles in transducing the differentiation signals.

    DOI PubMed

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    60
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  • The biologic properties of recombinant human thrombopoietin in the proliferation and megakaryocytic differentiation of acute myeloblastic leukemia cells

    Itaru Matsumura, Yuzuru Kanakura, Takashi Kato, Hirokazu Ikeda, Yoko Horikawa, Jun Ishikawa, Hitoshi Kitayama, Tetsuo Nishiura, Yoshiaki Tomiyama, Hiroshi Miyazaki, Yuji Matsuzawa

    Blood   88 ( 8 ) 3074 - 3082  1996.10

     View Summary

    Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic levels in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.

    DOI PubMed

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  • Serum thrombopoietin (c-Mpl ligand) levels in patients with liver cirrhosis

    Shigetaka Shimodaira, Fumihiro Ishida, Naoaki Ichikawa, Tomoyuki Tahara, Takashi Kato, Hiroshi Kodaira, Toshiro Ito, Eiji Tanaka, Takeshi Sodeyama, Kendo Kiyosawa, Kiyoshi Kitano

    Thrombosis and Haemostasis   76 ( 4 ) 545 - 548  1996.10

     View Summary

    To clarify the role of c-Mpl ligand (thrombopoietin: TPO) in liver cirrhosis (LC), we examined serum TPO levels (sTPO) in patients with LC (N = 44), chronic hepatitis (CH; N = 13) and healthy controls (N = 41) by an enzyme-linked immunosorbent assay. Although platelet counts of all LC patients (89 ± 59 X 109/l; mean ± SD) were lower than those of controls and CH patients, sTPO levels in LC patients (1.23 ± 0.51 fmol/ml) were the same as those in controls (1.22 ± 0.37) and CH patients (1.18 ± 0.36). Platelet counts were significantly higher in splenectomized patients than in unsplenectomized patients, but the sTPO level did not differ between these two groups. In LC patients, the sTPO level was not correlated with the platelet count, but was correlated with prothrombin time, activated partial thromboplastin time, and total bilirubin, indicating that production of TPO in the liver decreases slightly with the development of liver dysfunction. Our findings suggest that production of TPO is maintained in LC patients and their thrombocytopenia is not due to a defect in platelet production.

    DOI PubMed

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    57
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  • Serum thrombopoietin level in various hematological diseases

    Kensuke Usuki, Tomoyuki Tahara, Seiko Iki, Mitsue Endo, Mayumi Osawa, Koichi Kitazume, Takashi Kato, Hiroshi Miyazaki, Akio Urabe

    Stem Cells   14 ( 5 ) 558 - 565  1996.09

     View Summary

    To investigate the pathophysiological role of thrombopoietin (TPO) in thrombopoiesis, we measured its serum levels in 15 healthy individuals, 84 patients with various hematological diseases and 2 patients with liver cirrhosis using an enzyme immunoassay procedure. The TPO level was 0.84 ± 0.40 f mol/ml in normal individuals. TPO levels were considerably elevated in patients with myelosuppression after intensification chemotherapy of acute leukemia in complete remission (postchemotherapy group: n = 18; 18.46 ± 9.70) f mol/ml). When the data of normal individuals and the postchemotherapy group were combined, TPO levels were inversely correlated with the platelet count in this combined group. We compared these data of normal individuals and the postchemotherapy group with various hematological disease states. In aplastic anemia (n = 13; 16.03 ± 9.44 f mol/ml, acute lymphoblastic leukemia (n = 5; 10.36 ± 5.57 f mol/ml), malignant lymphoma (n = 6; 2.79 ± 2.27 f mol/ml), multiple myeloma (n = 3; 3.34 ± 0.20 f mol/ml and chronic lymphocytic leukemia (n = 21 1.71 ± 3.91 f mol/ml), the relationship of serum TPO levels and platelet counts was almost the same as in the combined group with normal individuals and the postchemotherapy group. However, the TPO levels were slightly higher in myeloproliferative disorders (n = 12; 1.99 ± 1.47 f mol/ml) and lower in acute myelogenous leukemia (n = 8; 2.27 ± 1.25 f mol/ml), hypoplastic leukemia (n = 3; 2.76 ± 2.23 f mol/ml, myelodysplastic syndrome (n = 2; 0.42 ± 0.60 f mol/ml), liver cirrhosis (n = 2; 1.50 ± 0.92 f mol/ml) and idiopathic thrombocytopenicpurpura (n = 12; 2.08 ± 1.41 f mol/ml), when compared to the regression line for the combined group with normal individuals and postchemotherapy group. These findings suggest that TPO might play an important role in regulation of the platelet count in normal and pathological conditions.

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  • Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3

    Masao Kobayashi, Joseph H. Laver, Takashi Kato, Hiroshi Miyazaki, Makio Ogawa

    Blood   88 ( 2 ) 429 - 436  1996.07

     View Summary

    We have studied the effects of recombinant human thrombopoietin (TPO; mpl ligand) on the proliferation of human primitive hematopoietic progenitors in vitro. CD34+ cells were enriched for cell-cycle-dormant primitive progenitors by separation on the basis of expression of c-kit and CD38. In the presence of varying combinations of TPO, Steel factor (SF), and interleukin-3 (IL-3), CD34+/c-kit(low)/CD38(neg/low) cells produced fewer colonies than CD34+/c-kit(low)/CD38(high) cells. However, when cultured in suspension for 7 days and replated in methylcellulose culture for measurement of colony-forming cells, the former population generated more colony-forming cells than the latter. In suspension culture of CD34+/c- kit(low)/CD38(neg/low) cells, TPO acted synergistically with SF and/or IL-3 in support of the production of colony-forming cells for granulocyte/macrophage colonies, erythroid colonies, and mixed colonies. Culture studies of individual CD34+/c-kit(low)/CD38(neg/low) cells provided the evidence for the direct nature of the effects of TPO. When combined with SF, TPO showed stronger stimulation of production of progenitors in suspension culture than other early-acting factors, such as IL-6, IL-11, and granulocyte colony-stimulating factor (G-CSF). TPO may be an important cytokine for in vitro manipulation of human hematopoietic stem cells.

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  • Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO

    Norio Komatsu, Masae Kunitama, Minami Yamada, Tetsuya Hagiwara, Takashi Kato, Hiroshi Miyazaki, Mitsuoki Eguchi, Masayuki Yamamoto, Yasusada Miura

    Blood   87 ( 11 ) 4552 - 4560  1996.06

     View Summary

    UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT- 7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT- 7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c- mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NFE2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein, These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.

    DOI PubMed

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  • Thrombopoietin primes human platelet aggregation induced by shear stress and by multiple agonists

    Atsushi Oda, Yoshitaka Miyakawa, Brian J. Druker, Katsutoshi Ozaki, Katsumi Yabusaki, Yoshiaki Shirasawa, Makoto Handa, Takashi Kato, Hiroshi Miyazaki, Akihiro Shimosaka, Yasuo Ikeda

    Blood   87 ( 11 ) 4664 - 4670  1996.06

     View Summary

    Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 μmol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not effected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 μmol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 μmol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 μg/mL), thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin- induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL- 3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

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  • Coexpression of thrombopoietin and c-mpl genes in human acute myeloblastic leukemia cells

    I. Matsumura, Y. Kanakura, H. Ikeda, J. Ishikawa, H. Yoshida, Y. Horikawa, T. Nishiura, T. Tahara, T. Kato, H. Miyazaki, Y. Matsuzawa

    Leukemia   10 ( 1 ) 91 - 94  1996.01

     View Summary

    Thrombopoietin (TPO) is a recently identified hematopoietic growth factor that is essential for the growth and development of megakaryocytes. We have previously shown that TPO induces proliferation of acute myeloblastic leukemia (AML) cells in vitro. In this study, we have examined the expression of TPO and its receptor c-mpl in a series of AML cases and human leukemia cell lines. The mRNA transcripts of TPO were detectable in 18 of 50 AML cases and in some myeloid leukemia cell lines (HEL, M07E and CMK) by means of reverse transcriptase polymerase chain reaction (RT-PCR). In addition, TPO transcripts were coexpressed with c-mpl transcripts in 10 of 50 AML cases and in HEL, M07E and CMK cells. With regard to the French-American-British (FAB) classification, coexpression of TPO and c-mpl was observed with high frequency in AML cases of M7-type. Despite the TPO expression in a substantial fraction of leukemia cells, biological activity of TPO was not found in the conditioned medium that was obtained from cultivation of TPO mRNA-positive leukemia cells. These results suggest that TPO may not commonly participate in the abnormal growth of AML cells as an extracellular autocrine growth factor.

    PubMed

  • Immunocytochemical localization of prohormone convertases PC1/PC3 and PC2 in rat pancreatic islets

    Shigeyasu Tanaka, Shingo Kurabuchi, Hiroshi Mochida, Takashi Kato, Senye Takahashi, Tsuyoshi Watanabe, Kazuhisa Nakayama

    Archives of Histology and Cytology   59 ( 3 ) 261 - 271  1996

     View Summary

    The prohormone convertases PC1/PC3 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. To determine the cellular and subcellular distribution of PC1/PC3 and PC2 in the rat pancreas, we generated their polyclonal antisera in rabbits, using as immunogens two synthetic peptide antigens corresponding to amino acids 442- 459 (ST-28) of PC1/PC3 and 613-629 (ST-29) of PC2 and two bacterially expressed antigens covering amino acids 145-414 (KN-1) of PC1/PC3 and 385- 637 (KN-2) of PC2. Western blot analysis revealed the presence of PC1/PC3 (87 and 68 kDa) and PC2 (75 and 70 kDa) in rat pancreatic islets, indicating that the antisera are specific for the corresponding antigens. Immunocytochemical staining of serial sections demonstrated that the antibody against PC1/PC3 immunostained only insulin-producing cells, whereas the PC2 antibody stained insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells. Double-immunolabeling of the prohormone convertases and pancreatic hormones with gold particles of different sizes revealed that insulin-positive secretory granules were also immunolabeled with PC1/PC3 and PC2 antibodies, whereas glucagon-, somatostatin-, or pancreatic polypeptide-positive granules were labeled only with the PC2 antibody. This differential localization of PC1/PC3 and PC2 provides a further problem on the substrate-specificity of these enzymes in the processing of pancreatic prohormones.

    DOI PubMed

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    48
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  • Regulation of serum thrombopoietin levels by platelets and megakaryocytes in patients with aplastic anaemia and idiopathic thrombocytopenic purpura

    Naoaki Ichikawa, Fumihiro Ishida, Shigetaka Shimodaira, Tomoyuki Tahara, Takashi Kato, Kiyoshi Kitano

    Thrombosis and Haemostasis   76 ( 2 ) 156 - 160  1996

     View Summary

    To clarify the regulatory mechanism of thrombopoietin (TPO, c-Mpl ligand) in chronic thrombocytopenic conditions, we determined TPO levels in the sera of patients with aplastic anaemia (AA; n = 26) and idiopathic thrombocytopenic purpura (ITP; n = 32) by an enzyme-linked immunosorbent assay. Despite a similarity in platelet counts, serum TPO levels in the AA group were markedly higher than those in the ITP group: 20.41 ± 9.71 f mol/ml (mean ± SD) and 1.66 ± 0.55 f mol/ml, respectively, both of which were significantly elevated compared to normal subjects (n = 41; 1.22 ± 0.37). In both groups, serum TPO level showed an inverse correlation with the platelet count. We determined the megakaryocyte volume using bone marrow clot section and found that it was markedly small in the AA group; while in the ITP group it was augmented with a correlation to serum TPO level. Our findings suggest that TPO levels may be regulated not only by platelets but also megakaryocytes in AA and ITP.

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  • Serum thrombopoietin and plasma glycocalicin concentrations as useful diagnostic markers in thrombocytopenic disorders

    S. Kunishima, T. Tahara, T. Kato, S. Kobayashi, H. Saito, T. Naoe

    European Journal of Haematology   57 ( 1 ) 68 - 71  1996

     View Summary

    Using enzyme-linked immunosorbent assays, we measured the concentrations of serum thrombopoietin (TPO) and plasma glycocalicin, a proteolytic fragment of platelet glycoprotein Ibα, in 13 patients with myelodysplastic syndrome (MDS), aplastic anaemia (AA) or idiopathic thrombocytopenic purpura (ITP). In the patients with AA or MDS, the TPO concentrations were remarkably increased, and their glycocalicin concentrations were decreased compared with the normal control individuals. In the patients with ITP; however, the TPO and glycocalicin levels were not changed as much as in the AA/MDS patients in spite of the same degree of thrombocytopenia. During immunosuppressive treatment of ITP patients, there was an inverse relationship between the level of TPO and the platelet count. Thus, measurements of TPO and glycocalicin levels are useful for the diagnosis of thrombocytopenia, and our results from ITP patients did not support the model which suggested the simple feedback regulation of TPO in thrombocytopenia.

    DOI PubMed

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  • Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis

    Ryugo Itoh, Naoyuki Katayama, Takashi Kato, Nadim Mahmud, Masahiro Masuya, Kohshi Ohishi, Nobuyuki Minami, Horoshi Miyazaki, Horoshi Shiku

    British Journal of Haematology   94 ( 2 ) 228 - 235  1996

     View Summary

    We examined the role of the ligand for c-mpl, thrombopoietin (TPO), in murine early haemopoiesis, using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice, whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO, as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.

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  • Cyclic change of cytokines in a patient with cyclic thrombocytopenia

    Fumihiko Kimura, Yukistugu Nakamura, Ken Sato, Naoki Wakimoto, Takashi Kato, Tomoyuki Tahara, Muneo Yamada, Naokazu Nagata, Kazuo Motoyoshi

    British Journal of Haematology   94 ( 1 ) 171 - 174  1996

     View Summary

    The serial change of various cytokines in the serum from a patient with cyclic thrombocytopenia is described, Interleukin 7, stem cell factor, and transforming growth factor β1 synchronized with the platelet count, and there was a significant positive correlation between the three cytokines and the platelet count. Levels of macrophage colony-stimulating factor, thrombopoietin, platelet-associated IgG and erythropoietin changed reciprocally with the platelet count, and there was a significant negative correlation between the platelet count and these cytokines except erythropoietin. No cyclic change was observed in IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor, or leukaemia inhibitory factor. These observations suggest that this disease involves two cyclic changes: megakaryocytopoiesis and platelet destruction, in both of which the cytokines play an important role.

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    29
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  • Erratum: Purification and Characterization of Thrombopoietin (Journal of Biochemistry (1995) Vol 118, No 1 (Page 229-236))

    T. Kato, K. Ogami, Y. Shimada, A. Iwamatsu, Y. Sohma, H. Akahori, K. Horie, A. Kokubo, Y. Kudo, E. Maeda, K. Kobayashi, H. Ohashi, T. Ozawa, H. Inoue, K. Kawamura, H. Miyazaki

    Journal of Biochemistry   119 ( 1 ) 208  1996

  • Protein characteristics of thrombopoietin

    Takashi Kato

    Stem Cells   14 ( SUPPL. 1 ) 139 - 147  1996

     View Summary

    Thrombopoietin (TPO) was purified from irradiated thrombocytopenic rat plasma. In the process of purification, some biochemical and biological characteristics were investigated. Rat plasma TPO was extremely hydrophobic and exhibited multiple peaks of activity on gel filtration. Both the low and high molecular weight fractions were separately subjected to further purification. Consequently, a rat TPO cDNA was cloned based on the amino acid sequences of purified rat plasma TPO. It revealed that each final purified rat plasma TPO was not a full-length form. In addition, rat hepatocytes and three rat hepatoma cell lines were found to produce rat TPO. Each native TPO derived from cultured cells was also partially purified, and hepatocyte-derived TPOs were shown to be heterogeneous in molecular weight. To study the structure of TPO, various recombinant TPO molecules were generated. Two disulfide bonds (Cys7-Cys151 and Cys29-Cys85) located in the N-terminal domain of TPO have an important effect on its biological activity. The human TPO muteins, sequentially deleted from the C-terminal, were expressed in COS-1 cells. TPO (1-151) was active, but TPO (1-150), which lacks Cys151, did not exhibit TPO activity. These findings indicate that the region essential for TPO activity is the N-terminal domain, which contains two disulfide bonds. Although the role(s) of the C-terminal domain is not clear at present, the potential N-glycosylation in the C-terminal domain is not directly required for exhibiting TPO activity. ©AlphaMed Press.

    DOI PubMed

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    7
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  • A sensitive sandwich ELISA for measuring thrombopoietin in human serum: Serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders

    Tomoyuki Tahara, Kensuke Usuki, Hiroaki Sato, Hideya Ohashi, Haruhiko Morita, Haruhiko Tsumura, Atsushi Matsumoto, Hiroshi Miyazaki, Akio Urabe, Takashi Kato

    British Journal of Haematology   93 ( 4 ) 783 - 788  1996

     View Summary

    A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) has been established to estimate serum thrombopoietin (TPO) concentrations in healthy volunteers and patients with haemopoietic disorders. The ELISA uses a mouse monoclonal antibody (Ab) as the capture Ab and a biotinylated rabbit polyclonal Ab as the detector. The ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intra- and interassay variation were from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The quantitative limit of the ELISA was 0.09 fmol/ml in serum. The quantitative limit was lower than the normal level. The dose-response curves of serum samples from healthy volunteers and patients with haemopoietic disorders were parallel to the standard curves. The ELISA did not cross-react with a variety of blood components and cytokines to produce false-positive results. The serum TPO concentrations from 29 normal males and 21 females were 0.79 ± 0.35 and 0.70 ± 0.26 fmol/ml, respectively. Serum TPO levels in patients with aplastic anaemia (AA), acute lymphocytic leukaemia (ALL) and essential thrombocythaemia (ET) were measured using the ELISA. The serum TPO levels in the patients with ET (n = 6, 2.80 ± 1.55 fmol/ml) were higher than the normal level. The patients with AA (n = 7, 18.53 ± 12.37 fmol/ml) and ALL (n = 5, 10.36 ± 5.57 fmol/ml) had significantly higher serum TPO levels than normal individuals. These results indicate that the ELISA specific to TPO should prove useful in measuring the TPO concentration in serum samples.

    DOI PubMed

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    199
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  • Circulating thrombopoietin level in chronic immune thrombocytopenic purpura

    Satoru Kosugi, Yoshiyuki Kurata, Yoshiaki Tomiyama, Tomoyuki Tahara, Takashi Kato, Seiji Tadokoro, Masamichi Shiraga, Shigenori Honda, Yuzuru Kanakura, Yuji Matsuzawa

    British Journal of Haematology   93 ( 3 ) 704 - 706  1996

     View Summary

    The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system, The TPO level (mean ± SD) in ITP patients was mildly elevated (186 ± 1.17 fmol/ml) compared to that in normal subjects (0.76 ± 0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35 ± 642 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.

    DOI PubMed

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    169
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  • Effect of recombinant human thrombopoietin in nonhuman primates with chemotherapy-induced thrombocytopenia

    Hiromichi Akahori, Kazunori Shibuya, Masako Obuchi, Yoshito Nishizawa, Akio Tsuji, Koji Kabaya, Masaru Kusaka, Hideya Ohashi, Haruhiko Tsumura, Takashi Kato, Hiroshi Miyazaki

    British Journal of Haematology   94 ( 4 ) 722 - 728  1996

     View Summary

    We examined the effects of recombinant human thrombopoietin (rhTPO) on myelosuppressive chemotherapy-induced thrombocytopenia in cynomolgus monkeys. After treatment with nimustine (ACNU) on day 0, the monkeys intravenously received rhTPO at a dose of 0.04, 0.2 or 1 μg/kg/d or monkey's serum once each day from day 1 to day 2.8. Administration of rhTPO reduced the severity of thrombocytopenia and accelerated the rate of platelet recovery in a dose-dependent fashion. Treatment with the highest rhTPO dose completely prevented thrombocytopenia and stimulated a marked increase in platelet counts over the normal values. Animals treated with ACNU also became neutropenic and slightly anaemic. Administration of rhTPO following ACNU treatment significantly improved neutropenia with increasing doses of rhTPO, but had no effect on anaemia. Compared to the control animals, rhTP0-treated animals exhibited no significant-changes in several serum parameters, C-reactive protein concentration and some blood coagulation profiles within the study period. These results suggest a therapeutic efficacy of rhTPO in improving chemotherapy-induced thrombocytopenia.

    DOI PubMed

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  • GpIIb/IIIa+ subpopulation of rat megakaryocyte progenitor cells exhibits high responsiveness to human thrombopoietin

    T. Kato, K. Horie, T. Hagiwara, E. Maeda, H. Tsumura, H. Ohashi, H. Miyazaki

    Experimental Hematology   24 ( 10 ) 1209 - 1214  1996

     View Summary

    The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIlb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa+ population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/III- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIla+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

    PubMed

  • Plasma thrombopoietin concentrations in patients with acquired aplastic anemia

    S. Kojima, T. Matsuyama, Y. Kodera, T. Tahara, T. Kato

    Experimental Hematology   24 ( 9 ) 1063  1996

     View Summary

    Thrombopoietin (TPO) stimulates proliferation and maturation of megakaryocytes in vivo and in vitro. In an attempt to better define the regulation and clinical significance of TPO concentrations, we measured endogenous TPO concentrations in 75 patients with acquired aplastic anemia (AA) and 12 normal controls by a sensitive ELJSA. The mean plasma TPO concentration is 1.3 ±0.5 fmoles/ml in normal controls. The plasma TPO concentrations were significantly higher in patients with AA than in normal controls (p<0.05). (VSAA: 26.5 ±10.5, SAA: 21.3 ±9.9, NSAA: 16.9 ±7.6 fmoles/ml, respectively). TPO levels were serially measured in 23 patients undergoing BMT and in 20 patients receiving immunosuppressive (IS) therapy. Although a decrease in TPO concentrations was observed in patients who achieved self-sustaining hematopoiesis following BMT, we did not find decreases of TPO levels in responders to IS therapy. Whether exogenous TPO will result in increased platelet counts in AA patients with high TPO levels remains to be determined.

  • Serum thrombopoietin level in various hematological diseases

    A. Urabe, K. Usuki, S. Iki, M. Endo, T. Tahara, T. Kato, H. Mivazaki

    Experimental Hematology   24 ( 9 ) 1068  1996

     View Summary

    To investigate the pathophysioJogical role of thrombopoietin (TPO) in thrombopoiesis, we measured its serum levels in 15 healthy individuals, 87 patients with various hematological diseases and 2 patients with liver cirrhosis using an enzymeimmunoassay procedure. The TPO level was negatively correlated with the platelet count in all 104 subjects investigated. The TPO levels was 0.84 ±0.04 f mol/mL in the normal individuals. TPO levels were considerably elevated in patients with myelosuppression after intensification chemotherapy of acute leukemia in complete remission. When the data of normal individuals and patients with myelosuppression were combined as a control, TPO levels showed a significant negative correlation with the platelet count in this group (r= -0.768, p<0.0001). In aplastic anemia, acute lymphoblasuc leukemia, malignant lympnoma, multiple myeloma and chronic lymphocytic leukemia, the relationship between serum TPO levels and platelet counts was almost the same as in the control group. However, the TPO levels were slightly higher in myelpproliferative disorders, and lower in acute myelogenous leukemia, hypoplastic leukemia, myelodysplastic syndrome, liver cirrhosis and idiopaUiic thrombocytopenic purpura, when compared with the regression line for the control group. These findings suggest that TPO might play an important role in regulation of the platelet count in normal and pathological conditions.

  • A highly sensitive and specific ellsa for measuring thrombopoietin in human serum

    T. Tahara, K. Usuki, H. Sato, H. Ohashi, H. Monta, H. Taiimura, H. Mivazaki', T. Kato

    Experimental Hematology   24 ( 9 ) 1068  1996

     View Summary

    We developed a sandwich enzyme-linked imrnuiiosorbent assay (ELISA) to quantify serum levels of human thrombopoietin (TPO) by using monoclonal and polyclonal antibodies. The. ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intraand inter-assay variation were from 3.0 10 4.9 % and from 5.9 to 6.1 %. respectively, This quantitative limit of the ELISA was 0.09 f moVml m scrum, which represents 3.0 pg of a fulllength TPO peptide in I ml scrum. The quantitative limit was lower than the normal level. The dose-response curves of serum samples from healthy volunteers and patients with hematopoieüc disorders were parallel to the standard curves. The ELISA did no! cross-react with a variety of Wood components and seven other cytokines including rhIL-3. rhlL-6. rhlL-7. rhG-CSF. rhGMCSF, rhEPO and rhSCF. The addition of extracellular domain of (he human c-MpI that may coexist in the blood samples had only a minimal effect on the ELISA. The estimated TPO levels m normal donors and in patients with hematopoietic disorders using the ELISA were as follows. The serum TPO concentrations from 29 normal males aged 24-38 (mean, 31 years) and 21 females aged 20-35 (mean, 26 years) were 0.79±0.35 <mean±SD; range 0.33 10 1.72) and 0.70±0.26 fmol/ml (range 0.33 to 1.33), respectively. No statistical difference in the serum TPO levels between males and females was noted (P-0.710). m addition, there was no statistically significant correlation between the serum TPO levels and platelet counts in the normal individuals. The serum TPO levels in the patients with idiopathic thromboeylopenic purpura (n-5, î.70±0.51 fmol/ml) were higher than the normal level. The patients with plastic anemia (n-7, 18.53± 12.37 fmol/ml) and acute lymphocytic leukemia (n-5, I0.36±5.57 fmoi/ml) had significantly higher serum TPO levels than the normal. Interestingly, in patients with essential thrombocythemia whose platelet counts were increased, the TPO lvcls(n-6, 2.80+L55 fmol/ml) were not decreased but elevated above normal. The established ELISA specific to TPO should prove useful to measure the TPO concentration in serum samples.

  • Analysis of functional domains of the human tpo receptor encoded by C-MPL

    M. Takatoku, N. Komateu, R. Shimizu, T. Kato, H. Miyazakis, Y. Miuräï

    Experimental Hematology   24 ( 9 ) 1109  1996

     View Summary

    c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin (TPO). To establish a model system for analyzing functional domains of c-Mpl, we searched for a cell line not expressing endogenous c-Mpl. Previously we reported that c-mpl mRNA could not be detected in a human EPO-responsive cell line, UT7/EPO (Blood 82:456,1993; Blood in press). This was confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Based on these observations, we prepared an expression vector containingfulllengthc-mp/ cDNA and transfected it into the UT-7/EPO cells. We isolated 3 clones and examined whether these clones exogenously expressed c-Mpl on the surface of the cells by FACS analysis with an anti-c-Mpl antibody. All these transfectants expressed c-Mpl and grew in response to TPO. Moreover, after incubation with TPO for one week, some of the cells became large and multinucleated, and expressed platelet factor-4 mRNA, suggesting that the TPO signalling pathway mediated through c-Mpl is involved in not only the proliferation but also megakaryocytic differentiation of the transfectants. Therefore, this model system may be useful for analyzing the functional domains of human c-Mpl. We are now preparing several kinds of deletion mutants.

  • Thrombopoietin has a direct effect on erythroid differentiation

    M. Yamada, N. Komatsu, K. Kirito, R. Shimizu, T. Kato, R. Mivazak, P. K. Abe, Y. Miura

    Experimental Hematology   24 ( 9 ) 1108  1996

     View Summary

    The administration of thrombopoietin (TPO) to mice after chemotherapy and total body irradiation not only promotes recovery from chemotherapy-induced thrombocytopenia, but also relieves anemia. In addition, TPO has burst promoting activity (BPA) in vitro. These observations suggest the influence of TPO on erythropoiesis. To elucidate whether TPO has a direct effect on erythroid differentiation, we established a subclone of a human cytokine-dependent cell line UT7, UT-7/GM Hl. UT-7/GM HI is maintained by GM-CSF and can grow in response to erythropoietin (EPO) or TPO. After a 1 week culture in IMDM medium containing 10% FCS in the presence of I U/ml of EPO or 10 ng/ml of TPO, more than 90 % and 45 % of UT-7/GM HI cells, respectively, were dianisidine-stained. Even after treatment of UT7/GM HI cells with up to 100 ng/ml of TPO, the EPO receptor was not tyrosine phosphorylated. These findings suggest that TPO directly induced erythroid differentiation and that the effect was not achieved via an erythropoietin receptor.

  • Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors

    Masao Kobayashi, Joseph H. Laver, Takashi Kato, Hiroshi Miyazaki, Makio Ogawa

    Blood   86 ( 7 ) 2494 - 2499  1995.10

     View Summary

    We have studied the effects of recombinant human thrombopoietin (TPO, Mpl ligand) on human hematopoiesis in vitro. TPO alone did not support erythroid burst formation but, in the presence of erythropoietin, it enhanced erythroid burst formation from CD34+ bone marrow and cord blood cells. The burst- promoting activity (BPA) was stronger under 5% serum than 30% serum conditions. The direct nature of BPA effects was documented by replating studies of early erythroid bursts. The BPA of TPO was less than that of interleukin-3 but was comparable with that of granulocyte/macrophage colony- stimulating factor and steel factor. The soluble form of Mpl receptor inhibited burst enhancing effects of TPO, suggesting that the BPA effects of TPO are mediated through the Mpl receptor. These results further delineate the physiologic roles of TPO and may aid in the determination of the clinical usages of TPO.

    DOI PubMed

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  • Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin

    Itaru Matsumura, Yuzuru Kanakura, Takashi Kato, Hirokazu Ikeda, Jun Ishikawa, Yoko Horikawa, Koji Hashimoto, Yasuhiro Moriyama, Tohru Tsujimura, Tetsuo Nishiura, Hiroshi Miyazaki, Yuji Matsuzawa

    Blood   86 ( 2 ) 703 - 709  1995.07

     View Summary

    Thrombopoietin (TPO) is e newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto- oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c- mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French-American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL-3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors.

    DOI PubMed

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    88
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  • Purification and characterization of thrombopoietin

    Takashi Kato, Kinya Ogami, Yoshihiro Shimada, Akihiro Iwamatsu, Yoshiaki Sohma, Hiromichi Akahori, Kaori Horie, Atsuko Kokubo, Yoko Kudo, Emiko Maeda, Keiko Kobayashi, Hideya Ohashi, Tadashi Ozawa, Hideo Inoue, Kazuo Kawamura, Hiroshi Miyazaki

    Journal of Biochemistry   118 ( 1 ) 229 - 236  1995.07

     View Summary

    A thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1, 100 rats by measuring the production of megakaryo-cytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49%108, respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO cDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO cDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl. © 1995 by the Journal of Biochemistry.

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    249
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  • Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets

    Yoshitaka Miyakawa, Atsushi Oda, Brian J. Druker, Takashi Kato, Hiroshi Miyazaki, Makoto Handa, Yasuo Ikeda

    Blood   86 ( 1 ) 23 - 27  1995.07

     View Summary

    A cDNA for the thrombopoietin has been cloned by several groups. The recombinant thrombopoietin has been reported to stimulate the megakaryocytopoiesis and thrombopoiesis. Little is known regarding the molecular basis of its effects. To elucidate the molecular mechanism involved in signal transduction, we have investigated the effects of thrombopoietin on platelet tyrosine phosphorylation. We report here that thrombopoietin induced time- and dose-dependent tyrosine phosphorylation of several proteins including Janus kinase 2 (Jak2) and a 52-kD protein, Shc, in human blood platelets. Both Jak2 and Shc were tyrosine phosphorylated within 15 seconds after stimulation. The tyrosine phosphorylation of Jak2 was accompanied by increased kinase activity, whereas Shc tyrosine phosphorylation induced its association with a 25-kD protein, Grb2. Thus, our data suggest that Jak2, Shc, and Grb2 may be involved in signal transduction after ligand binding to c-mpl in human platelets.

    DOI PubMed

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    134
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  • The sequence of a rat cDNA encoding thrombopoietin

    Kinya Ogami, Yoshihiro Shimada, Yoshiaki Sohma, Hiromichi Akahori, Takashi Kato, Kazuo Kawamura, Hiroshi Miyazaki

    Gene   158 ( 2 ) 309 - 310  1995.06

     View Summary

    Overlapping cDNA clones encoding rat thrombopoietin (TPO) were isolated from liver-derived cell line cDNA libraries and the nucleotide sequences were determined. The deduced 326-amino-acid rat TPO showed significant homology to the known TPO of other species, especially in the N-terminal sequence. © 1995.

    DOI PubMed

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    36
    Citation
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  • A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK

    H. Miyazaki, K. Horie, Y. Shimada, A. Kokubo, E. Maeda, H. Inoue, T. Kato

    Experimental Hematology   23 ( 11 ) 1224 - 1228  1995

     View Summary

    A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5x104 cells/ml (104 cells/well) with test samples in 96-well tissue culture plates for 4 days at 37°C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples-including a wide variety of cytokines and unknown growth factors, alone or in combinations-and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

    PubMed

  • Recombinant human megakaryocyte growth and development factor (rhumgdf), a ligand for c‐mpl, produces functional human platelets in vitro

    Esther S. Choi, Martha Hokom, Tim Bartley, Yue‐Sheng ‐S Li, Janet L. Nichol, Jim Skrine, Andrew Knudten, Janice Chen, Alex Hornkohl, Gustavo Grampp, Lisa Sleeman, Sean Cole, Geri Trail, Pamela Hunt, Hideya Ohashi, Takashi Kato

    STEM CELLS   13 ( 3 ) 317 - 322  1995

     View Summary

    Platelet formation, occurring from bone marrow or lung megakaryocytes, has been difficult to study mechanistically. Recombinant human megakaryocyte growth and development factor (rHuMGDF), a recently described cytokine, has now been used to establish an in vitro system in which this important and little understood process occurs. CD34+ cells cultured with rHuMGDF develop into megakaryocytes which form long cytoplasmic extensions (proplatelets) that fragment into platelet‐sized particles (in vitro platelets). Morphologically, in vitro and human plasma‐derived platelets (control platelets) are virtually identical with respect to size, dense granule distribution and ultrastructural features. Functionally, in vitro and control platelets have similar aggregation and activation responses, and similarly incorporate mepacrine into dense granules. These findings suggest that rHuMGDF is sufficient to generate platelet‐synthesizing megakaryocytes from CD34+ cells and provide an experimental setting in which the study of human platelet formation can be adequately performed. Copyright © 1995 AlphaMed Press

    DOI PubMed

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    91
    Citation
    (Scopus)
  • Megakaryocyte growth and development factor: Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy- induced thrombocytopenia

    J. L. Nichol, M. M. Hokom, A. Hornkohl, W. P. Sheridan, H. Ohashi, T. Kato, Yue Sheng Li, T. D. Bartley, E. Choi, J. Bogenberger, J. D. Skrine, A. Knudten, J. Chen, G. Trail, L. Sleeman, S. Cole, G. Grampp, P. Hunt

    Journal of Clinical Investigation   95 ( 6 ) 2973 - 2978  1995

     View Summary

    The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r-HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis.

    DOI PubMed

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    193
    Citation
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  • Thrombopoietin induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in a human thrombopoietin-dependent cell line

    Minami Yamada, Norio Komatsu, Koji Okada, Takashi Kato, Hiroshi Miyazaki, Yasusada Miura

    Biochemical and Biophysical Research Communications   217 ( 1 ) 230 - 237  1995

     View Summary

    Thrombopoietin (TPO) is a cytokine which can support the proliferation and differentiation of megakaryocyte progenitor cells, and the maturation of megakaryocytes. We show here that mitogen-activated protein (MAP) kinases, Erk1 and Erk2, are involved in TPO signal transduction in the human TPO-dependent megakaryocytic cell line, UT-7/TPO. TPO induced tyrosine phosphorylation of Erk1 and Erk2 proteins in a dose and time-dependent manner. Moreover, the activation of MAP kinases was actually induced by TPO. These results suggest that MAP kinase activation is involved in the signalling pathway of TPO, as it is for other cytokines, one of which is erythropoietin. © 1995 by Academic Press, Inc.

    DOI PubMed

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    48
    Citation
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  • Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines

    Y. Shimada, T. Kato, K. Ogami, K. Horie, A. Kokubo, Y. Kudo, E. Maeda, Y. Sohma, H. Akahori, K. Kawamura, H. Miyazaki

    Experimental Hematology   23 ( 13 ) 1388 - 1396  1995

     View Summary

    Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.

    PubMed

  • Molecular cloning and chromosomal localization of the human thrombopoietin gene

    Yoshiaki Sohma, Hiromichi Akahori, Naohiko Seki, Tada aki Hori, Kinya Ogami, Takashi Kato, Yoshihiro Shimada, Kazuo Kawamura, Hiroshi Miyazaki

    FEBS Letters   353 ( 1 ) 57 - 61  1994.10

     View Summary

    The complete gene for human thrombopoietin (TPO) has been cloned by screening a human genomic library using human TPO cDNA as a probe. This gene is 6.2 kb in length and contains six exons and five introns. It is shown that the human genome contains a single copy of the human TPO gene according to Southern blotting analysis. The transcription initiation site was determined by S1 nuclease mapping. The human TPO gene expressed TPO activity when transfected into COS-1 cells. The human TPO gene has been mapped to chromosome 3q27 by in situ hybridization using a biotin-labeled probe. © 1994.

    DOI PubMed

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    242
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  • Identification of a protein, SPY75, with repetitive helix-turn-helix motifs and an SH3 domain as a major substrate for protein tyrosine kinase(s) activated by FcεRI cross-linking

    Hiromi Fukamachi, Nobuo Yamada, Toru Miura, Takashi Kato, Masaharu Ishikawa, Erich Gulbins, Amnon Altman, Yuko Kawakami, Toshiaki Kawakami

    Journal of Immunology   152 ( 2 ) 642 - 652  1994.01

     View Summary

    Cross-linking of the high affinity receptor for IgE (FcεRI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the FcεRI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on FcεRI cross- linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast cells, despite their responsiveness to these cytokines. SPY75 was not physically associated with the receptor or other known signaling molecules. This protein, the mouse homologue of the human HS1 gene product, has putative repetitive helix-turn-helix motifs found in many DNA-binding proteins and a putative nuclear transport signal. It also has a Src homology 3 domain, which is found in many signaling molecules and cytoskeletal proteins. These structural features and the rapid tyrosine phosphorylation on FcεRI cross-linking suggest that the signal generated by FcεRI cross-linking is transmitted through tyrosine phosphorylation of SPY75.

    PubMed

  • Growth factor‐induced process formation of megakaryocytes derived from CFU‐MK

    Hideo Inoue, Hiromi Ishii, Miki Tsutsumi, Naomi Takahashi, Masahiro Sato, Yoshihiro Shimada, Takashi Kato, Tadashi Sudo, Hiroshi Miyazaki

    British Journal of Haematology   85 ( 2 ) 260 - 269  1993.10

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  • Structure and Expression of Novel Protein-Tyrosine Kinases, EMB and EMT, in Hematopoietic Cells

    Nobuo Yamada, Yuko Kawakami, Hitoshi Kimura, Hiromi Fukamachi, Gottfried Baier, Amnon Altman, Takashi Kato, Yoshimasa Inagaki, Toshiaki Kawakami

    Biochemical and Biophysical Research Communications   192 ( 1 ) 231 - 240  1993

     View Summary

    Two novel tyrosine kinase cDNAs were obtained from murine mast cells. These kinases, Emb and Emt, constitute a novel tyrosine kinase subfamily which may also include Tec, a kinase preferentially expressed in liver, and Dsrc28, a fruit fly kinase. Both lack hydrophobic stretches characteristic of the transmembrane domains found in growth factor receptor tyrosine kinases and carboxyl-terminal, negative regulatory tyrosine residue found in Src family kinases. In addition to the Src homology region 2 (SH2) and SH3 domains characteristic of the Src family kinases and other signaling molecules, Emb and Emt share a similar amino-terminal domain comprised mainly of two repeat segments. The emb 2.7-kb transcript was expressed in mast cells, myeloid cells and B lymphocytes while the emt 4.6-kb mRNA in mast cells, myeloid cells and T lymphocytes. The evidence for in vitro tyrosine kinase activity of Emb and Emt proteins is also provided. © 1993 Academic Press.

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  • Production of monoclonal antibodies against human erythropoietin and their use in the purification of human urinary erythropoietin

    Hiroshi Miyazaki, Hiroyuki Kozutsumi, Takashi Kato, Sakuo Hoshi, Setsuko Tamura, Mamoru Kubota, Takamoto Suzuki

    Journal of Immunological Methods   113 ( 2 ) 261 - 267  1988.10

     View Summary

    Several murine monoclonal antibodies (MAbs) specific for human erythropoietin (HuEpo) were produced by hybridomas obtained from the fusion of murine myeloma cells, P3X63-Ag.8-653, with the splenocytes of mice immunized with recombinant human Epo (rHuEpo). Based on epitope analysis by a competitive binding assay, these MAbs could be classified into at least three groups: (1) 1E10, (2) 1H7, (3) 2D6, 3D6 and 3D8. In a sandwich enzyme-linked immunosorbent assay (ELISA), using these MAbs as the solid-phase antibodies, MAb-bound HuEpo was detected with rabbit anti-HuEpo sera. Some combinations of two different classes of MAbs, such as 1H7 and 3D8, were found to capture much more HuEpo than each MAb used individually. Urinary HuEpo (U-HuEpo) was highly purified from the urine of patients with severe aplastic anemia with about 50% final recovery using an immunoaffinity column on which a mixture of 1H7 and 3D8 was immobilized. The purified U-HuEpo had a specific activity of 77 340 U/mg in a radioimmunoassay (RIA) and of 76 673 U/mg using an in vivo bioassay. © 1988.

    DOI PubMed

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    11
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  • Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells

    M. Takeuchi, S. Takasaki, H. Miyazaki, T. Kato, S. Hoshi, N. Kochibe, A. Kobata

    Journal of Biological Chemistry   263 ( 8 ) 3657 - 3663  1988

     View Summary

    The asparagine-linked sugar chains of human erythropoietin produced by recombinant Chinese hamster ovary cells and naturally occurring human urinary erythropoietin were liberated by hydrazinolysis and fractionated by paper electrophoresis, lectin affinity chromatography, and Bio-Gel P-4 column chromatography. Both erythropoietins had three asparagine-linked sugar chains in one molecule, all of which were acidic complex type. Structural analysis of them revealed that the sugar chains from both erythropoietins are quite similar except for sialyl linkage. All sugar chains of erythropoietin produced by recombinant Chinese hamster ovary cells contain only the NeuAcα2→3Gal linkage, while those of human urinary erythropoietin contain the NeuAcα2→6Gal linkage together with the NeuAcα2→3Gal linkage. The major sugar chains were of fucosylated tetraantennary complex type with and without N-acetyllactosamine repeating units in their outer chain moieties in common, and small amounts of 2,4- and 2,6-branched triantennary and biantennary sugar chains were detected. This paper proved, for the first time, that recombinant technique can produce glycoprotein hormone whose carbohydrate structures are common to the major sugar chains of the native one.

    PubMed

  • Unique Pattern of Gene Expression in the Erythroid Precursor Cells: gene expression/erythroid/CFU‐E/fetal liver

    YUJI MISHINA, TAKASHI KATO, AKIO URABE, FUMIMARO TAKAKU, SHUNJI NATORI, MASUO OBINATA

    Development, Growth &amp; Differentiation   28 ( 1 ) 1 - 6  1986.02

     View Summary

    Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU‐E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt‐genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt‐genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU‐E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt‐genes may be induced after the stimulation of erythropoietin. Copyright © 1986, Wiley Blackwell. All rights reserved

    DOI

    Scopus

  • Binding of sex steroid to oviduct cytosol of the newt, Cynops pyrrhogaster ensicauda

    Takashi Kato, Sakae Kikuyama, Tetsuo Noumura

    Proceedings of the Japan Academy Series B: Physical and Biological Sciences   62 ( 5 ) 153 - 156  1986

    DOI

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    2
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  • Production of erythropoietin‐like activity by human renal and hepatic carcinomas in cell culture

    Tetsuro Okabe, Akio Urabe, Takashi Kato, Shyozo Chiba, Fumimaro Takaku

    Cancer   55 ( 9 ) 1918 - 1923  1985.05

     View Summary

    Two types of human cancers, a renal cell carcinoma and a hepatocellular carcinoma, were investigated in vitro; both produced a marked erythrocytosis in each patient. These tumors, when transplanted into athymic nude mice, produced a remarkable erythrocytosis in the host mice. To analyze this phenomenon, the primary cultures from these xenotransplanted tumors were performed. To obtain pure tumor cell cultures, cells derived from host nude mice were eliminated by the treatment with the antiserum raised against nude mouse cells. Epithelial cells derived from each tumor attached and grew in the cultures. The conditioned media from both tumor cells revealed high erythropoietic stimulatory activities. We have characterized these erythropoietin‐like activities by size‐exclusion high‐performance liquid chromatography. Three peaks of erythropoietin‐like activities were noted after bovine serum albumin region. The molecular weights were estimated at about 55,000, 40,000, and 33,000, respectively. The results suggested that the human renal cell and hepatocellular carcinomas produced erythropoietin‐like activities in vitro in culture and that erythrocytosis found in patients with cancer and in nude mice transplanted with the tumors was attributable to production of the erythropoietin‐like activities by the tumor cells themselves. Copyright © 1985 American Cancer Society

    DOI PubMed

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  • 早稲田大学が創る教師教育

    早稲田大学教育総合研究所

    学文社  2017.03 ISBN: 9784762027215

  • いちばんやさしい生理学

    加藤, 尚志, 南沢, 享

    成美堂出版  2015.12 ISBN: 9784415320717

  • 震災と教育 : 学び、将来へ伝える

    早稲田大学教育総合研究所, 柴山, 知也, 細川, 光洋, 本田, 恵子, 三上, 貴仁, 鴨川, 光, 中島, 好美, 加藤, 尚志, 堀, 誠

    学文社  2012.03 ISBN: 9784762022838

Misc

  • 腎臓と造血の関係における普遍性と多様性

    加藤 尚志, 小俣 和輝, 小川 斐女

    日本腎臓学会誌   63 ( 4 ) 385 - 385  2021.06

  • 造血因子の創薬から四半世紀を経て カエルやメダカに学ぶ探索研究

    加藤 尚志

    日本臨床分子医学会学術総会プログラム・抄録集   57回   35 - 35  2020.04

  • 比較血液学からみる造血の謎 巨核球・血小板造血を中心に

    加藤 尚志

    臨床血液   60 ( 9 ) 1063 - 1069  2019.09

     View Summary

    現代血液学において,鳥類,爬虫類,両生類,魚類といった脊椎動物全般を俯瞰する造血や血球の研究の取り組みは遅れている。ヒトやマウス以外を研究対象に選ぶと,多くの実験的制約が立ちはだかる現実があった。しかし,フローサイトメトリーによる有核血球の血算の自動化や,全ゲノム情報の利用などが実現した。そして例えば両生類造血の解析から新たな知見が獲得されつつある。末梢に有核栓球が循環し,血小板をもたないアフリカツメガエルにも巨核球が存在する。ヒト分子にみられる長大なC末端領域は,鳥類,両生類,魚類のTPO分子にはない。それはなぜか?変異型CALRとMPLの結合や,EPOとEphB4の結合は動物種に依存せず普遍的なものなのか?未解明の課題は尽きないが,比較血液学的視点はいろいろな謎解きへのアプローチを提供する。(著者抄録)

  • トロンボポエチンの発見から四半世紀を経て

    加藤 尚志

    日本輸血細胞治療学会誌   65 ( 2 ) 233 - 233  2019.04

  • 低温刺激によるツメガエル血球と血管内皮細胞の接着(The adhesion of thrombocytes to vascular endothelium by cold stimulation in a Xenopus model)

    加藤 奈菜, 蜷尾 はるか, 野村 一騎, 佐藤 圭, 村瀬 絢香, 加藤 尚志

    臨床血液   59 ( 9 ) 1539 - 1539  2018.09

  • アフリカツメガエルにおける経口投与鉄と赤血球造血の関連(The effect of oral administration of SFC on erythropoiesis and iron metabolism in a Xenopus model)

    佐藤 圭, 加藤 康太, 野村 一騎, 加藤 尚志

    臨床血液   59 ( 9 ) 1562 - 1562  2018.09

  • アフリカツメガエル肺におけるエリスロポエチンとその受容体の発現及び機能探索(Pulmonary expressions and functions of erythropoietin and its receptor in African clawed frogs)

    加藤 康太, 佐藤 圭, 野村 一騎, 加藤 尚志

    臨床血液   59 ( 9 ) 1563 - 1563  2018.09

  • アフリカツメガエルにおける分化が限定された造血巣に存在する造血幹/前駆細胞の解析(Biological properties of HSPCs in the lineage-biased hematopoietic organ in African clawed frogs)

    野村 一騎, 加藤 康太, 佐藤 圭, 加藤 尚志

    臨床血液   59 ( 9 ) 1693 - 1693  2018.09

  • 非哺乳類モデルにおけるマイクロパーティクルを介する有核栓球の血栓形成(Microparticles-associated thrombus formation of nucleated thrombocytes in a non-mammalian model)

    蜷尾 はるか, 加藤 奈菜, 佐藤 圭, 加藤 尚志

    臨床血液   59 ( 9 ) 1747 - 1747  2018.09

  • アフリカツメガエル抗栓球モノクローナル抗体が認識する細胞表面抗原の解析

    林 優太, 佐藤 圭, 渡会 敦子, 加藤 尚志

    生命科学系学会合同年次大会   2017年度   [1P - 0902]  2017.12

  • Live-cell single-molecule imaging of the cytokine receptor MPL for analysis of dynamic dimerization

    Akihiko Sakamoto, Takashi Tsukamoto, Yuji Furutani, Yuki Sudo, Kazuyuki Shimada, Akihiro Tomita, Hitoshi Kiyoi, Takashi Kato, Takashi Funatsu

    Journal of Molecular Cell Biology   8 ( 6 ) 553 - 555  2016.12

    Rapid communication, short report, research note, etc. (scientific journal)  

    DOI PubMed

  • 中和抗体結合反応におけるヒト・トロンボポエチンの構造学的・熱力学的変化

    新井栄揮, 柴崎千枝, 安達基泰, 玉田太郎, 前田宜丈, 田原知幸, 加藤尚志, 宮崎洋, BLABER Michael, 黒木良太

    日本結晶学会年会講演要旨集   2016   79  2016.11

    J-GLOBAL

  • 貧血誘導およびエリスロポエチン投与によって出現するツメガエル末梢未熟赤血球の諸性質

    佐藤 圭, 平田 昭人, 上原 あずさ, 谷崎 祐太, 加藤 尚志

    日本生化学会大会プログラム・講演要旨集   89回   [2T14 - 262)]  2016.09

  • 造血と環境 両生類モデルから探る造血の環境応答

    加藤 尚志, 佐藤 圭, 相曾 卓樹, 谷崎 祐太

    日本生化学会大会プログラム・講演要旨集   89回   [2S08 - 6]  2016.09

  • 【トロンボポエチン受容体作動薬の臨床応用〜現況と展望〜】トロンボポエチンとトロンボポエチン受容体の構造と作用

    黒木 良太, 宮崎 洋, 加藤 尚志

    血液フロンティア   25 ( 2 ) 171 - 180  2015.01

     View Summary

    トロンボポエチンは巨核球前駆細胞に作用し、巨核球の成熟と血小板への分化を促進する液性因子であり、1994年にキリンビール社をはじめ、複数の研究グループによって発見された。ヒトのトロンボポエチンの全長分子は、332のアミノ酸残基からなる糖蛋白質である。アミノ末端側領域は4ヘリックスバンドル構造を取り、受容体c-Mplと結合する相互作用部位を2ヶ所持つ。トロンボポエチン1分子と受容体2分子が会合して、細胞内シグナル伝達系の活性化が生じると推定される。トロンボポエチン受容体作動薬ロミプロスチムとエルトロンボパグは、内因性トロンボポエチンとは全く異なる構造を持つが、c-Mplと結合して生物活性を発揮する。(著者抄録)

  • Diverse of Erythropoiesis Responding to Hypoxia and Low Environmental Temperature in Vertebrates

    Shun Maekawa, Takashi Kato

    BioMed Research International   2015  2015

    Book review, literature introduction, etc.  

     View Summary

    Erythrocytes are responsible for transporting oxygen to tissue and are essential for the survival of almost all vertebrate animals. Circulating erythrocyte counts are tightly regulated and respond to erythrocyte mass and oxygen tension. Since the discovery of erythropoietin, the erythropoietic responses to environment and tissue oxygen tension have been investigated in mice and human. Moreover, it has recently become increasingly clear that various environmental stresses could induce the erythropoiesis via various modulating systems, while all vertebrates live in various environments and habitually adapt to environmental stress. Therefore, it is considered that investigations of erythropoiesis in vertebrates provide a lead to the various erythropoietic responses to environmental stress. This paper comparatively introduces the present understanding of erythropoiesis in vertebrates. Indeed, there is a wide range of variations in vertebrates' erythropoiesis. This paper also focused on erythropoietic responses to environmental stress, hypoxia, and lowered temperature in vertebrates.

    DOI PubMed

  • 一分子イメージングで明らかになった骨髄増殖性腫瘍関連MPL変異体の動的な二量体化(Dynamic dimerization of the myeloproliferative neoplasm-relevant MPL mutant revealed by single-molecule imaging)

    坂本 明彦, 加藤 尚志, 島田 和之, 冨田 章裕, 清井 仁, 船津 高志

    日本癌学会総会記事   73回   J - 3054  2014.09

  • Iron,Heme and Inflammation 赤血球産生と鉄恒常性における環境応答系の探索

    加藤 尚志

    臨床血液   55 ( 7 ) 735 - 743  2014.07

     View Summary

    ユニークな動物モデルを確立して、mRNA、蛋白質、miRNAなどの非翻訳RNA、代謝化合物など、分子クラスによる調節作用の多様性を正確に捉えた検証の展開は、今後の実験血液学研究で新知見を獲得するプロセスに非常に重要である。造血の環境応答の探索で経験した二つの実験事例、miRNAによる鉄恒常性調節パスウェイと、アフリカツメガエル造血巣の低温応答モデルについて述べた。

  • 【赤血球造血の基礎と臨床】低酸素および低温環境ストレスと赤血球造血

    前川 峻, 加藤 尚志

    血液フロンティア   24 ( 4 ) 549 - 557  2014.03

     View Summary

    酸素を末梢組織に運搬する末梢赤血球数は、個体環境の酸素分圧の変動に応答して精密に調節される。環境に応答する造血生理研究の先駆者の取組みには学ぶことが非常に多い。腎臓で産生される赤血球産生因子エリスロポエチンが純化され、遺伝子組換え医薬が登場して以後、赤血球造血の捉えられ方は大きく変貌を遂げた。赤血球造血の低酸素応答に関しても膨大な知見が蓄積されてきた。一方、酸素環境だけではなく赤血球造血に影響を与えるストレスとして、環境温度についても様々な動物で報告されている。(著者抄録)

  • アフリカツメガエル未熟赤血球を認識するモノクローナル抗体の作出(Generation of the monoclonal antibody to erythroid progenitors of Xenopus laevis)

    入江 達也, 武藤 洋史, 永澤 和道, 奥井 武仁, 加藤 尚志

    日本生化学会大会プログラム・講演要旨集   86回   1P - 256  2013.09

  • アフリカツメガエルの赤血球膜蛋白質群の同定

    竹島 功将, 永澤 和道, 谷崎 祐太, 渡会 敦子, 加藤 尚志

    日本生化学会大会プログラム・講演要旨集   86回   2P - 302  2013.09

  • 【巨核球・血小板の細胞運命制御機構】巨核球・血小板産生におけるTPO-c-Mpl系の分子動態

    加藤 尚志, 坂本 明彦, 船津 高志, 宮崎 洋

    日本血栓止血学会誌   23 ( 6 ) 544 - 551  2012.12

  • 【がんと代謝 何故がん細胞が好んで解糖系を使うのか?メタボローム解析が明かすがん細胞の本質から代謝研究がもたらす創薬・診断まで】 (第3章)低酸素、酸化ストレス がん細胞の代謝異常とmicroRNA制御

    吉岡 祐亮, 小坂 展慶, 加藤 尚志, 落谷 孝広

    実験医学   30 ( 15 ) 2455 - 2461  2012.09

     View Summary

    がん細胞の代謝制御は正常細胞とは異なることが古くから知られている。それはがん細胞の存在する環境が、正常細胞と異なる点も影響しており、その1つが低酸素環境である。低酸素環境下における代謝制御の変化はがん悪性化に繋がることが示され、その詳細な分子機構が徐々に解明されはじめ、microRNA(miRNA)の関与も認められた。miRNA、代謝制御、がん、という3つのキーワードを結びつける報告はまだ少ないが、今後報告が増えることが予想され、本稿ではその先駆けとして、われわれの研究成果も交えて最新の知見を解説する。(著者抄録)

  • 【過渡的複合体が関わる生命現象の統合的理解-生理的準安定状態を捉える新技術と応用-】細胞膜上におけるトロンボポエチン受容体の一分子ダイナミクス

    坂本 明彦, 加藤 尚志, 船津 高志

    生化学   83 ( 10 ) 912 - 919  2011.10

     View Summary

    トロンボポエチン(TPO)は、巨核球系列細胞の分化と血小板の産生を担うサイトカインである。TPOが細胞へシグナルを伝達する上で受容体Mplの二量体化が必要だが、その動的な過程は不明である。本研究では、Mpl二量体化の動的な過程と制御機構を、一分子イメージングで解明することにした。全反射蛍光顕微鏡で細胞膜上のMpl分子を一分子レベルで観察した。Mpl分子に対応する個々の輝点の蛍光強度を解析した結果、Mplが単量体・二量体・多量体の平衡状態で存在することが分かった。また、リガンドTPOの結合や細胞のコレステロールに依存してMpl二量体の安定性が向上し、平衡が二量体・多量体側へ移動することが分かった。TPOの結合やコレステロールによるMpl二量体の安定化は、TPOシグナルに必須のMplリン酸化の制御に重要だと思われる。(著者抄録)

  • ヒトトロンボポエチン受容体を構成する2つのサイトカイン受容体相同性領域の調製とリガンド相互作用

    松本富美子, 安達基泰, 清水瑠美, 目黒瑞枝, 玉田太郎, 加藤尚志, 黒木良太

    日本蛋白質科学会年会プログラム・要旨集   11th   78  2011.05

    J-GLOBAL

  • 染色体・核・遺伝子発現・シグナル伝達 細胞膜上におけるトロンボポエチン受容体の一分子ダイナミクス(Signal transduction/ Chromosome/ Cell nucleus/ Gene expression Single Molecule Dynamics of The Thrombopoietin Receptor on The Plasma Membrane)

    坂本 明彦, 加藤 尚志, 船津 高志

    日本細胞生物学会大会講演要旨集   63回   110 - 110  2011.05

  • 赤血球造血因子エリスロポエチンの結晶構造から見える受容体結合部位の種間保存

    目黒瑞枝, 永澤和道, 小坂(野川)菜美, 安達基泰, 岡崎伸生, 玉田太郎, 黒木良太, 加藤尚志

    日本比較内分泌学会大会及びシンポジウムプログラム・講演要旨   36th   34  2011

    J-GLOBAL

  • Erythropoietin-Inducible MicroRNA-362 Contributes to Erythropoiesis Via the Suppression of Histone Deacetylase 3

    Yusuke Yoshioka, Keiichi Sugiura, Nobuyoshi Kosaka, Yusuke Yamamoto, Norio Komatsu, Hiroshi Miyazaki, Takahiro Ochiya, Takashi Kato

    BLOOD   116 ( 21 ) 1076 - 1076  2010.11

    Research paper, summary (international conference)  

  • アフリカツメガエルエリスロポエチンのX線結晶構造解析

    目黒瑞枝, 安達基泰, 岡崎伸生, 玉田太郎, 黒木良太, 加藤尚志

    日本蛋白質科学会年会プログラム・要旨集   10th   99  2010.05

    J-GLOBAL

  • トロンボポエチンシグナルの上流部は脂質ラフトによって制御されている(Thrombopoietin upstream signaling is regulated by lipid rafts)

    坂本 明彦, 加藤 尚志, 船津 高志

    日本細胞生物学会大会講演要旨集   62回   149 - 149  2010.05

  • 低温曝露で誘導されるアフリカツメガエル末梢赤血球数減少の機序

    家村 仁美, 前川 峻, 倉持 裕子, 小坂 菜美[野川], 會沢 洋一, 加藤 尚志

    臨床血液   50 ( 9 ) 969 - 969  2009.09

  • トロンボポエチンシグナルの初期段階は脂質ラフトによって制御されている(Initial thrombopoietin signaling is regulated by lipid rafts)

    坂本 明彦, 加藤 尚志, 船津 高志

    日本生化学会大会プログラム・講演要旨集   82回   4T4p - 12  2009.09

  • ヒト巨核芽球性白血病細胞株UT‐7における非翻訳RNAの一種miR‐210の発現制御

    吉岡祐亮, 小坂展慶, 山本雄介, 杉浦圭一, 落谷孝広, 小松則夫, 宮崎洋, 加藤尚志

    臨床血液   49 ( 9 ) 1088 - 1088  2008.09

    J-GLOBAL

  • 赤血球産生因子エリスロポエチンにより誘導されるマイクロRNAの同定

    小坂展慶, 杉浦圭一, 山本雄介, 吉岡祐亮, 宮崎洋, 小松則夫, 落谷孝広, 加藤尚志

    臨床血液   49 ( 9 ) 909 - 909  2008.09

    J-GLOBAL

  • アフリカツメガエル肝臓及び脾臓における栓球系細胞の解析

    古川 翔介, 下地 美也子, 石田 貴子, 加藤 尚志

    臨床血液   49 ( 9 ) 987 - 987  2008.09

  • 低温環境におけるアフリカツメガエルの慢性貧血とその生理機序の検討

    前川 峻, 倉持 裕子, 小坂 菜美[野川], 家村 仁美, 西川 裕展, 會沢 洋一, 加藤 尚志

    臨床血液   49 ( 9 ) 1035 - 1035  2008.09

  • N結合型糖鎖を導入したアフリカツメガエルのエリスロポエチンの生物活性

    永澤 和道, 小坂 菜美[野川], 上原 信明, 會沢 洋一, 加藤 尚志

    臨床血液   49 ( 9 ) 1035 - 1035  2008.09

  • Regulation of miR-210 generation in response to hypoxia in erythrocytic cells.

    Nobuyoshi Kosaka, Yusuke Yamamoto, Keiichi Sugiura, Hiroshi Miyazaki, Norio Komatsu, Takahiro Ochiya, Takashi Kato

    BLOOD   110 ( 11 ) 376A - 376A  2007.11

    Research paper, summary (international conference)  

  • Expression of miR-188 and 362 induced by erythropoietin stimulation in a human erythrocytic leukemia cell line.

    Keiichi Sugiura, Nobuyoshi Kosaka, Yusuke Yamamoto, Yasuke Yoshioka, Hiroshi Miyazaki, Norio Komatsu, Takahiro Ochiya, Takashi Kato

    BLOOD   110 ( 11 ) 656A - 656A  2007.11

    Research paper, summary (international conference)  

  • Alternation of cellular morphology and proliferation induced by microRNA in human leukemia cell line, UT-7/EPO.

    Nobuyoshi Kosaka, Yusuke Yamamoto, Nami Nogawa, Keiichi Sugiura, Hiroshi Miyazaki, Norio Komatsu, Takahiro Ochiya, Takashi Kato

    BLOOD   108 ( 11 ) 130B - 130B  2006.11

    Research paper, summary (international conference)  

  • Asn結合型糖鎖をもたないエリトロポエチンによる両生類の赤血球造血

    野川 菜美, 小坂 展慶, 会沢 洋一, 小松 則夫, 宮崎 洋, 加藤 尚志

    臨床血液   47 ( 9 ) 1216 - 1216  2006.09

  • Gene expression of Limb girdle muscular dystrophy type 2A

    Yoko Keira, Rumi Kurokawa, Masako Fujita, Narihiro Minami, Yukiko K. Hayashi, Satoru Noguchi, Takashi Kato, Ichizo Nishino

    NEUROMUSCULAR DISORDERS   16   S72 - S72  2006.07  [Refereed]

    Research paper, summary (international conference)  

  • Preparation of recombinant proteins derived from recombinant organisms

    Haruhiko Tsumura, Takashi Kato

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   49 ( 11 Suppl ) 1506 - 1513  2004.08

    Book review, literature introduction, etc.  

    PubMed

  • 可溶性ヒトトロンボポエチンレセプターの同定 血小板数との相関

    桑木 知朗, 上田 周二, 山城 賢太郎, 松本 篤志, 田原 知幸, 加藤 尚志, 金倉 譲, 宮崎 洋

    臨床血液   44 ( 8 ) 776 - 776  2003.08

  • 母乳中のトロンボポイエチン値

    松原 康策, 加藤 尚志, 宮崎 洋

    臨床血液   44 ( 8 ) 776 - 776  2003.08

  • Therapeutically-induced autoantibodies in patients treated with recombinant hematopoietic growth factors: A brief summary

    Takashi Kato, Hiroshi Miyazaki

    Current Pharmaceutical Design   9 ( 14 ) 1129 - 1132  2003

    Book review, literature introduction, etc.  

     View Summary

    Since recombinant gene technology was established to provide rare important regulatory proteins as recombinant molecules, cytokine therapies have widely developed and enormously contributed to the treatment of various diseases; nevertheless, it has been revealed that recombinant therapeutic molecules are not always perfect because of side-effects related to pharmacological functions of cytokines and/or potential antigenicity observed in some clinical cases. Although studies on the antigenicity of recombinant proteins have initiated, and observations in clinical studies have been accumulated over this decade, mechanisms of the autoantibody production are not clarified yet. Among various hematopoietic growth factors introduced into clinical trials, this report summarizes current issues of autoantibodies to primary regulators for terminal hematopoiesis.

    DOI PubMed

  • 最新創薬 薬の分子メカニズム 血小板産生機構を制御する造血因子トロンボポエチン

    宮崎 洋, 加藤 尚志

    Molecular Medicine   39 ( 10 ) 1190 - 1195  2002.09

  • Thrombopoietin in human milk [3]

    Kousaku Matsubara, Takashi Kato, Hiroshi Miyazaki

    International Journal of Hematology   75 ( 1 ) 109 - 110  2002.01

    Rapid communication, short report, research note, etc. (scientific journal)  

    DOI PubMed

  • 【ヒト肝細胞を利用した研究とその臨床応用】血小板産生と肝臓

    加藤 尚志, 森田 晴彦, 宮崎 洋

    細胞   33 ( 11 ) 414 - 417  2001.10

  • Serum thrombopoietin concentration and peripheral platelet counts in essential thrombocythemia

    H. Kawasaki, T. Nakano, U. Kohdera, T. Tahara, T. Kato, Y. Kobayashi

    Annals of Hematology   80 ( 1 ) 62 - 63  2001

    Rapid communication, short report, research note, etc. (scientific journal)  

    DOI PubMed

  • Autoantibodies neutralizing thrombopoietin in a patient with amegakaryocytic thrombocytopenic purpura [4]

    Hiroko Shiozaki, Shuichi Miyawaki, Tomoaki Kuwaki, Tetsuya Hagiwara, Takashi Kato, Hiroshi Miyazaki

    Blood   95 ( 6 ) 2187 - 2188  2000.03

    Rapid communication, short report, research note, etc. (scientific journal)  

    DOI PubMed

  • ヒト肝細胞の機能発現とその応用 Thrombopoietinの発見と肝臓における発現

    加藤 尚志, 島田 佳宏, 宮崎 洋

    組織培養研究   18 ( 3 ) 253 - 263  1999.09

  • 血小板増多症に先行する血中TPO値の増加は川崎病に加え感染症でも見られる

    石黒 精, 新保 敏和, 松原 康策, 加藤 尚志

    臨床血液   40 ( 9 ) 920 - 920  1999.09

  • 担癌患者における血小板増多とTPO,及び新たなTPO isoform(TPO-4,5,6)

    高橋 隆幸, 佐々木 豊, 中村 紀士子, 宮崎 洋, 松本 篤志, 加藤 尚志

    臨床血液   40 ( 9 ) 981 - 981  1999.09

  • 感染症における血小板増多症と血中トロンボポエチン値の増加

    石黒 精, 加藤 尚志, 松原 康策, 新保 敏和

    日本小児血液学会雑誌   13 ( 4 ) 272 - 272  1999.08

  • 血小板の産生とトロンボポエチン

    加藤 尚志

    日本輸血学会雑誌   45 ( 4 ) 576 - 577  1999.08

  • PEG-rHuMGDFによる長期再構築能を有する幹細胞動員作用-rhG-CSFとの併用効果

    俵 知紀, 赤堀 弘典, 井田 ますみ, 加藤 尚志, 宮崎 洋

    International Journal of Hematology   69 ( Suppl.1 ) 177 - 177  1999.04

  • マウスITPモデル(NZWxBXSB)F1)に対するPEG-rHuMGDFの効果

    渋谷 和憲, 赤堀 弘典, 田原 恵美子, 加藤 尚志, 宮崎 洋

    International Journal of Hematology   69 ( Suppl.1 ) 184 - 184  1999.04

  • 巨核球系細胞に対するTPOのApoptosis抑制効果

    堀江 かおり, 萩原 哲也, 加藤 尚志, 宮崎 洋

    International Journal of Hematology   69 ( Suppl.1 ) 185 - 185  1999.04

  • TPOの多核白血球(PMN)活性化に関する検討

    結城 千鶴, 桑木 知朗, 加藤 尚志, 宮崎 洋

    International Journal of Hematology   69 ( Suppl.1 ) 186 - 186  1999.04

  • CHO細胞により産生されたTPOの投与による抗TPO中和抗体の出現

    桑木 知朗, 赤堀 弘典, 加藤 尚志, 宮崎 洋

    International Journal of Hematology   69 ( Suppl.1 ) 186 - 186  1999.04

  • 【血小板の臨床】c-Mplの発現とシグナル伝達

    加藤 尚志, 宮崎 洋

    臨床医   25 ( 1 ) 70 - 74  1999.01

  • 劇症肝炎の新しい治療 ラジアルフロー型バイオリアクターで培養しヒト由来株化肝細胞のトロンボポイエチン産生

    永森 静志, 蓮村 哲, 松浦 知和, 川田 雅昭, 清水 英佑, 加藤 尚志, 大上 欽也, 池永 裕

    肝臓   39 ( 11 ) 880 - 880  1998.11

  • トロンボポエチンの開発

    宮崎 洋, 加藤 尚志

    BIO Clinica   13 ( 9 ) 776 - 781  1998.08

  • Native thrombopoietin: structure and function.

    T. Kato, A. Matsumoto, K. Ogami, T. Tahara, H. Morita, H. Miyazaki

    Stem cells (Dayton, Ohio)   16 Suppl 2   11 - 19  1998

    Book review, literature introduction, etc.  

     View Summary

    Thrombopoietin (TPO), the c-Mpl ligand, is produced constitutively in liver and other organs, circulates in the bloodstream, and is delivered to bone marrow, where it stimulates the early development of multiple hematopoietic lineages and megakaryocytopoiesis. The concentration of TPO in blood is regulated by c-Mpl mass on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including the possible modulation of TPO mRNA abundance in bone marrow, megakaryocytopoiesis and platelet production may be regulated as a result of modulation of TPO activity by proteolytic processing that generates truncated forms of the molecule. Characterization of TPO partially purified from human plasma, however, revealed that the full-length molecule was the predominant form in the blood of both normal individuals and thrombocytopenic patients, although small amounts of truncated species were detected. Thus, truncation of TPO, at least that in the circulation examined, does not appear to contribute to the direct regulation of platelet production in response to increased demand. Given that native TPO isolated from the plasma of thrombocytopenic animals comprises truncated forms, the truncation of TPO is likely of physiological importance in the life history of this molecule.

    DOI PubMed

  • Identification of the biologically active domains of human thrombopoietin using neutralizing antibodies.

    T Tahara, T Kuwaki, A Matsumoto, Y Inagaki, H Ohashi, H Watarai, H Morita, K Ohgami, H Miyazaki, T Kato

    BLOOD   90 ( 10 ) 3538 - 3538  1997.11

    Research paper, summary (international conference)  

  • トロンボポエチンの性状

    宮崎 洋, 加藤 尚志

    炎症と免疫   5 ( 5 ) 507 - 514  1997.08

  • トロンボポエチンの単離とその機能解析

    加藤 尚志

    生化学   69 ( 6 ) 433 - 438  1997.06

  • 造血因子 トロンボポエチンの構造と遺伝子

    大上 欽也, 加藤 尚志, 宮崎 洋

    現代医療   29 ( 4 ) 775 - 782  1997.04

  • トロンビンによるトロンボポエチン(TPO)の切断

    加藤 尚志

    International Journal of Hematology   65 ( Suppl.1 ) 50 - 50  1997.04

  • トロンボポエチンの産生臓器と薬理作用

    島田 佳宏, 加藤 尚志, 宮崎 洋

    最新医学   52 ( 2 ) 157 - 162  1997.02

  • Development of supersensitive enzyme immunoassay for gene recombination type human Thrombopoietin.

    後藤真澄美, 川田浩美, 渡部好実, 佐藤博章, 美細津正, 新開寛, 田原知幸, 加藤尚志

    日本薬学会年会要旨集   117th ( 4 )  1997

    J-GLOBAL

  • Cloning and expression of feline thrombopoietin and its physiological activities.

    松代はるか, 加藤大智, 加藤尚志, 岩田晃, 亘敏広, 辻本元, 長谷川篤彦

    日本獣医学会学術集会講演要旨集   124th  1997

    J-GLOBAL

  • トロンボポエチンの発見の経緯

    宮崎 洋, 加藤 尚志

    日常診療と血液   7 ( 1 ) 7 - 15  1996.12

  • Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin.

    T Kato, A Oda, Y Inagaki, H Ohashi, A Matsumoto, K Ozaki, Y Miyakawa, H Watarai, K Fuju, A Kokubo, T Kadoya, Y Ikeda, H Miyazaki

    BLOOD   88 ( 10 ) 2162 - 2162  1996.11

    Research paper, summary (international conference)  

  • Thrombopoietinの精製・遺伝子クローニングと生物学的活性

    加藤 尚志

    最新医学   51 ( 10 ) 1807 - 1816  1996.10

  • 造血幹細胞 トロンボポエチン(Thrombopoietin;TPO)

    堀江 かおり, 宮崎 洋, 加藤 尚志

    臨床検査   40 ( 8 ) 948 - 950  1996.08

  • トロンボポエチンの構造と作用

    加藤 尚志, 大上 欽也, 宮崎 洋

    血液・腫瘍科   33 ( 1 ) 1 - 10  1996.07

  • An introduction of an asparagine binding sugar chain to a human TPO ( thrombopoietin ) activity expression indispensable region.

    武藤隆則, 日高祐子, 石田功, 加藤尚志, 宮崎洋, 黒木良太

    日本分子生物学会年会プログラム・講演要旨集   19th  1996

    J-GLOBAL

  • Identification of thrombopoietin productive cells in liver by in situ hybridization.

    大上欽也, 野村慎太郎, 川村和男, 塚本幾代, 工藤容子, 金倉譲, 北村幸彦, 宮崎洋, 加藤尚志

    日本分子生物学会年会プログラム・講演要旨集   19th  1996

    J-GLOBAL

  • Thrombopoietin(c-Mplリガンド)

    加藤 尚志, 宮崎 洋

    Surgery Frontier   2 ( 3 ) 307 - 310  1995.09

  • トロンボポエチンのクローニングと生物活性

    宮崎 洋, 加藤 尚志

    医学のあゆみ   174 ( 9 ) 689 - 693  1995.08

  • ラット肝細胞株で分泌されるスロンボポエチン(TPO)の性質

    加藤 尚志

    生化学   67 ( 7 ) 789 - 789  1995.07

  • トロンボポエチン(TPO)の精製とその性質

    加藤 尚志

    International Journal of Hematology   61 ( Suppl.1 ) 231 - 231  1995.06

  • トロンボポエチンの発見 その構造と機能

    加藤 尚志, 宮崎 洋

    実験医学   13 ( 3 ) 347 - 349  1995.02

  • Promoter analysis and transcriptional regulation of the human thrombopoietin(TPO) gene.

    大上欽也, 川岸真由美, 工藤容子, 加藤尚志, 宮崎洋, 川村和男

    日本分子生物学会年会プログラム・講演要旨集   18th  1995

    J-GLOBAL

  • Molecular cloning and expression of hyman thrombopoietin(TPO) cDNA.

    大上欽也, 加藤尚志, 島田佳宏, 岩松明彦, 相馬良明, 赤堀弘典, 堀江かおり, 小久保敦子, 宮崎洋

    日本分子生物学会年会プログラム・講演要旨集   17th  1994

    J-GLOBAL

  • Molecular cloning and expression of rat thrombopoietin cDNA.

    島田佳宏, 加藤尚志, 大上欽也, 岩松明彦, 相馬良明, 赤堀弘典, 堀江かおり, 小久保敦子, 宮崎洋

    日本分子生物学会年会プログラム・講演要旨集   17th  1994

    J-GLOBAL

▼display all

Research Projects

  • Regulation of amphibian hematopoiesis: the distribution of hematopoietic stem cells in the liver, spleen, and bone marrow.

    Project Year :

    2020.04
    -
    2023.03
     

  • Studies on the response to cold stress in cellular functions and morphology of blood cells and hematopoiesis in amphibia

    Project Year :

    2017.04
    -
    2020.03
     

  • Characterization and physiological relevance of the myeloid cells newly discovered in the amphibian tadpole

    Project Year :

    2016.04
    -
    2019.03
     

     View Summary

    In the present study, we analyzed the origin of the myeloid cell cluster found in the mesonephric mesoderm area by the immuno-histochemical approach and concluded that they derived from the tail mesoderm of the tailbud embryo. Second, we identified the enhancer sequence of mpo gene expressed specifically in the myeloid cells and made a transgenic frog that expresses GFP in the myeloid cells. This frog will enable us to select the live myeloid cells in the fractionation of blood cells. Third, we established a new method for fractionation of leukocytes of adult peripheral blood cells. Finally, we elucidated the transcriptional control mechanism of gene, hb4, which is expressed specifically in the frog oocytes

  • Amphibian hematopoietic regulation under environmental stress

    Project Year :

    2014.04
    -
    2017.03
     

     View Summary

    To develop a better comprehension of diversity and universality in vertebrate physiological regulations, we focus on amphibian hematopoietic responses against environmental stress in African clawed frog (Xenopus laevis) and Western clawed frog (Xenopus (Silurana) tropicalis). We established an in vivo Xenopus hypoxic model that caused a pulmonary defect. This model should be vital to investigate non-hematopoietic functions of erythropoietin. In the latter model, low-temperature exposure induced pancytopenia. We revealed that one of the causes of thrombocytopenia was due to the reversible cellular adhesion between thrombocytes and the vascular wall cells

  • Shell pigments of mollusks: from chemical structures to mechanisms of pattern formation

    Project Year :

    2014.04
    -
    2017.03
     

     View Summary

    Identifications of chemical structures of animal pigments are essential to reveal their evolutionary history. We investigated pigment compounds in mollusk and brachiopod shells using Raman microspectroscopy. The compounds tended to be specific for taxa and/or food habits. Based on the results, we have identified putative genes and proteins involved in the metabolism of pigment compounds from the mantle tissues

  • Molecular basis of maternal behavior~using Usp46 mutant mice~

    Project Year :

    2013.04
    -
    2016.03
     

     View Summary

    Biological investigation (such as using animal models) of generating mechanisms of child abuse has not been well performed although this has been treated in a field of social science (psychology and education) and clinical science (psychiatry). In this study, therefore, we examined the role of Usp46 on cellular and nervous system using Usp46 mutant mice which display poor maternal behavior. As a result, we found that the level of maternal care is transmitted to their pups and proper maternal behaviors can be shaped if adequate postpartum maternal care is given, even in genetically vulnerable mice. In addition, it is suggested that Usp46 affects several behavioral phenotypes through GABAergic system

  • Study of cellular protein aggregates involved in the expression of harmful heavy metal toxicity.

    Project Year :

    2013.04
    -
    2016.03
     

     View Summary

    To elucidate properties and significance of intracellular protein aggregates related to cytotoxicity of harmful heavy metals, in the analysis of aggregated components, we improved the purity of the aggregated protein fraction by reviewing the composition of cell extraction buffers. In addition, identification of aggregated components was carried out by a method based on shotgun analysis using high-resolution HPLC / Q-TOF. To study the formation mechanism of protein aggregation, we introduced the cell culture experiments using stable isotope-containing amino acids, and analyzed it in a time-dependent manner. Additionally, we improved the method of evaluation system of the protein aggregation using the hardly soluble polyubiquitinated proteins as an indication of cellular protein aggregation, and by this efficient method we verified and analyzed the effect of heavy metals on human epithelial cells

  • 両生類における血球産生調節の環境応答に関する研究

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(C))

    Project Year :

    2014
    -
    2016
     

  • Comprehensive approaches to mechanisms underlying the development of blood islands in amphibian embryo

    Project Year :

    2012.04
    -
    2015.03
     

     View Summary

    The present research has attempted to elucidate development of the blood islands and myeloid cells in Xenopus embryo. First, we found a physiological role of Nkx2.5 and GATA4, which are the transcription factors for heart specification, are involved in the myeloid cell differentiation in the anterior blood islands. Second, we showed the distribution of Rdd (repeated D domain-like) protein in the developing embryo. Third, we discovered a novel population of myeloid cells in the mesonephric rudiment at an early tadpole stage

  • 有害重金属の毒性発現に関与する細胞内タンパク凝集体の研究

    科学研究費助成事業(東京慈恵会医科大学)  科学研究費助成事業(基盤研究(C))

    Project Year :

    2013
    -
    2015
     

     View Summary

    研究代表者は平成25年度の当初より東京慈恵会医科大学国領校の生物学研究室に赴任したため、始めに本研究に不可欠な細胞培養室や生化学実験室などの整備を行った。また、新たな研究環境の検証を兼ねて有害重金属による細胞内タンパク凝集体の形成に適する条件を検討した。
    有害重金属としてカドミウムを選択し、ヒト腎由来HK-2細胞に対するその細胞毒性を評価するため、12,24,48時間の各曝露条件での半致死的濃度(EC50)を求めたところ、その値は順に200,85,70 μMと見積もられた。飽和密度を超えたHK-2細胞に対する長期間(1週間以上)曝露においてもEC50値は70 μMに収束した。
    カドミウム曝露で形成される凝集体はポリユビキチン化タンパク質を含有する。そこで曝露時間と凝集体の関係を解明するため、細胞内ポリユビキチン鎖をELISAで定量してその経時的変化を分析した。その結果、HK-2細胞に毒性を示さない40 μM カドミウムでは48時間以上の長時間曝露でもポリユビキチン鎖の量に変動を認めなかった。一方、先の検討で曝露時間依存性の毒性を与えると判明した70,85,200 μMのカドミウムでは、何れの場合も曝露6時間の時点から、細胞死に先行したポリユビキチン鎖量の顕著な増加が観察された。さらにその後の推移を比較したところ、200μMは6時間、85 μMでは24時間の曝露をピークとしてポリユビキチン鎖量が減少に転じたが、70 μMでは曝露24時間以降も高いレベルを維持した。
    以上の検討から、有害重金属の毒性発現に先立って出現するHK-2細胞のタンパク凝集体の調製には、70 μM カドミウムの24~48時間曝露が適切と結論された。

  • Development of super-resolution fluorescence microscopy and single-molecule analysis of signal transduction mechanism of a receptor protein in cell membrane

    Project Year :

    2009.04
    -
    2014.03
     

     View Summary

    The thrombopoietin (TPO) receptor, Mpl, is a cytokine receptor regulating platelet production and early hematopoiesis. Although TPO signaling is initiated by phosphorylation of optimally oriented Mpl dimers, dimerization of Mpl is poorly understood. Here, we describe the regulatory mechanism of dimerization of Mpl by single-molecule fluorescence imaging in live cells. Mpl molecules were associated and dissociated repeatedly on the plasma membrane. As a result, Mpl existed as a mixture of monomers, dimers, and oligomers. TPO increased Mpl dimers or oligomers by stabilizing Mpl dimers. The stabilization of Mpl dimers was dependent on their phosphorylation and an adaptor protein, Shc, suggesting that phosphorylated Mpl dimers were crosslinked by Shc. In contrast, knockdown of Shc, an Mpl transmembrane mutant, influenced their phosphorylation. Thus, phosphorylation of Mpl was regulated by the stability of Mpl dimers in a positive feedback manner

  • 両生類胚における血島の分化制御に関する総合的研究

    科学研究費助成事業(新潟大学)  科学研究費助成事業(基盤研究(C))

    Project Year :

    2012
    -
    2014
     

     View Summary

    本課題では、アフリカツメガエルにおける胚血島および造血の制御機構を理解することを目的として研究に取り組んでおり、平成25年度には以下のような成果を得た。第一に、心臓原基に付随して出現する骨髄球の分化制御のしくみを、前年度に引き続いて解析を進めた。これまでに知られている骨髄球転写因子CEBPαに加え、心臓形成にかかわる転写因子NKx2.5およびGATA4が骨髄球の分化にかかわることを示し、論文をまとめ投稿した。第二に、この延長上の実験として、cebpαの発現制御にかかわるエンハンサーの同定を試み、ネッタイツメガエルcebpα遺伝子 5’フランキング配列中にdkk1の過剰発現による骨髄球分化誘導に特異的に反応するエレメントが存在することを見いだした。第三に、骨髄球特異的に働くエンハンサー配列をネッタイツメガエルmpo遺伝子に同定し、その配列を用いたトランスジェニックガエルの作成をおこなった。現在、成体となったカエルを飼育中であり、F1個体の作成の準備を進めている。第四に、過去に獲得していたモノクローナル抗体の再評価を含め,アフリカツメガエルの白血球を特異的に認識する抗体の調達を進めた。末梢全血球より白血球-栓球画分(除赤血球画分)を分離し,これを免疫したマウスの脾臓とミエローマを細胞融合して得られたハイブリドーマのうち,白血球を認識する抗体(T5)の認識性の評価を進めた。T5抗体は一部の栓球も認識するため白血球特異的とはいえなかったが,分化段階あるいは血球系譜の異なる白血球を分別する可能性がある。また,赤血球系細胞を免疫抗原にして作出したモノクローナル抗体のうち,たまたま白血球を認識する抗体を見出した。これらの発生胚や幼生の細胞の認識性を調べる予定である。

  • Establishment of assessment system for toxic chemicals based onabnormal protein recognition mechanism in eukaryotic cells.

    Project Year :

    2010.04
    -
    2013.03
     

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    We focused accumulation of polyubiquitinated proteins in cells which reflect protein abnormality to evaluate chemical-induced toxicity with a new index. By analyzing changes in hardly-soluble polyubiquitin chainsdue to exposure to each test substance using six established cell lines, toxic chemicals could be divided into three groups in terms of their toxic effects on levels of cellular abnormal proteins, which were(1) increased in all cells(2) increased in a cell type-dependent manner(3) unchanged in all cellsThe present evaluation system is useful for safety management of chemicals based on the understanding of toxic mechanism

  • Establishment of assessment system for toxic chemicals based onabnormal protein recognition mechanism in eukaryotic cells.

    Project Year :

    2010
    -
    2012
     

     View Summary

    We focused accumulation of polyubiquitinated proteins in cells which reflect protein abnormality to evaluate chemical-induced toxicity with a new index. By analyzing changes in hardly-soluble polyubiquitin chainsdue to exposure to each test substance using six established cell lines, toxic chemicals could be divided into three groups in terms of their toxic effects on levels of cellular abnormal proteins, which were
    (1) increased in all cells
    (2) increased in a cell type-dependent manner
    (3) unchanged in all cells
    The present evaluation system is useful for safety management of chemicals based on the understanding of toxic mechanism.

  • Mechanical stimulation-induced vascular elastogenesis

    Project Year :

    2010
    -
    2011
     

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    Vascular elastic fibers play a very important role in maintaining the structure of the arteries. The impairment of elastic fiber formation is closely related with aging and vascular diseases. We hypothesized that mecanical stresses onto the arteries would affect elastic fiber formation. Therefore, we tried to perform the quantitative analysis of the relation between elastic fiber formation and the types and strength of mechanical stresses. We found that elastic fiber formation was promoted in multi-layers of smooth muscle cells or by cycle stretch.

  • Studies on development, structure and function of pituitary gland in amphibians

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    1.Ontogenic study of pituitary glandAs a tool for tracing the cells that migrate with pituitary primordium but remain in the foregut region, transgenic toads (Xenopus laevis) that express GFP were prepared.2.Central action of prolactinAn adenohypophyseal hormone prolactin is involved in eliciting courtship behavior in the newt, genus Cynops, acting centrally. As a step to elucidate the mechanism of the prolactin action, distribution of prolactin receptors in the newt (Cynops pyrrhogaster) brain was studied by immunohistochemistry and in situ hybridization. Immunohistochemically detectable prolactin receptors as well as prolactin receptor mRNAs were observed in several region of hypothalamic nuclei, and most notably in the choroid plexus. Involvement of choroid plexus in transporting prolactin molecules into the brain was suggested.3.Regulation of pituitary function by hypothalamic neuropeptidesNewly developed radioimmunoassay for amphibian TSH made it possible to detect TSH-releasing factors in the amphibian brain. As a result, CRF was revealed to be the most potent substance to stimulate the release of TSH from the bullfrog pituitary. Using CRF antagonists and agonists, CRF receptor type 2 was demonstrated to mediate the action of CRF to release TSH

  • Hematopoietic tissue environment and molecular interactions in erythropoiesis

     View Summary

    In African clawed frog, Xenopus laevis, mammalian homologues of erythropoietin (EPO) and its receptor (EPO) express in the adult liver. Xenopus EPO had only 38% identity with mammalian EPO protein. By establishing a colony assay for the first time in amphibian, we were able not only to demonstrate the presence of erythroid progenitors in the liver capable of forming erythroid colonies but also the specific function of Xenopus EPO and the presence of EPO in anemic serum. Under conditions of hypothermic anemia, new erythrocytes were retained in the liver. This is the first study to describe the direct evidences in a physiological animal model to investigate the erythroid progenitor in amphibian hematopoietic tissues

  • Isolation and analyses of novel regulatory factors in differentiation of blood islands of frog embryo

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    The physiological and biochemical properties of Val and Rdd proteins found in the Xenopus embryo have been investigated. We examined the biological activity and biochemical property of the various deletion mutants of Val. We also examined the association of Rdd proteins in the vascular system in the developing embryo. Furthermore, two distinct origins and differential regulations of primitive myeloid cells appeared in the tailbud embryo have been analyzed

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Specific Research

  • 両生類の造血幹細胞の表現型と臓器分布に関する研究

    2020  

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    本年度はコロナ禍の影響を受けて,当初予定していた研究の中でも実施可能な内容を吟味し,ツメガエル赤血球と栓球の細胞学的特徴と機能を精査することに集中した。抗ツメガエルEPORモノクローナル抗体を用いたフローサイトメトリーでは、ネッタイツメガエルの末梢赤血球の95%がEPORを発現していた。赤血球をEPOとともに室温でインキュベートしたところ,1.8±0.6個のEPO分子が1つの赤血球に結合できることがわかった。ツメガエル末梢栓球に関しては,ヒト血小板発現糖蛋白質のアミノ酸配列をゲノムデータベース上でBLAST検索したところ、GPIIbとGPIbβのオルソログ遺伝子の登録を確認した。ツメガエル末梢栓球におけるこれらの遺伝子発現は栓球・白血球層で発現を認めたが、赤血球では発現を認めなかった。またGPIIbは、アフリカツメガエルの脾臓で発現を認めた。

  • 低温刺激で作動する脂肪髄の造血スイッチの研究

    2017   佐藤圭

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    ツメガエル(ゼノパス)成体の造血は主に肝臓が担い,骨髄は脂肪髄化している。しかし低温刺激により,骨髄は赤色髄化し造血を開始する。このモデルを基礎にして,造血系に作用する組織環境の諸要素と分子機序を明らかにしたい。本研究では,加齢に伴う骨髄の脂肪髄化と環境低温応答の共役系の探索を進めた。本期間内では特に,低温曝露アフリカツメガエル(Xenopus laevis)モデルで,脂肪髄における血管新生と造血能亢進と,間葉系幹細胞からの脂肪細胞分化・骨形成能との間で連鎖する分子機序を探索した。

  • 造血制御における低温応答系の分子機序の探索

    2016   谷崎祐太, 佐藤圭

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    変温あるいは恒温動物とも,in vivo低温曝露によって,急性に末梢血球数が変動するが,造血や血球数調節の低温応答の分子機序は不明である。本研究では,両生類アフリカツメガエルに加え,新たにネッタイツメガエル(ナショナル・バイオリソース・プロジェクトより分与)の末梢血球数変化の低温応答を解析した。これらより,低温ストレスによる栓球の血管内皮への可逆的接着,骨髄造血の変動等の現象の詳細を明らかにした。

  • 質量分析による血球特異的抗体の抗原分子の解析

    2015   谷崎祐太(Yuta Tanizaki)

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    脊椎動物の血液には多種多様の血球が存在し,これらの血球の形態や細胞腫には多様性が見いだされる。例えば哺乳動物は,無核赤血球,白血球,無核血小板をもつ。しかし魚類,両生類,爬虫類,鳥類は,赤血球は有核であり,小型細胞の血小板のかわりに有核栓球をもつ。このような血球あるいは血球産生(造血)の仕組みの違いは,脊椎動物の進化を考察する上で極めて重要な指標になると考えられる。ヒトやマウスの細胞の種類や性質を調べるためには,細胞発現蛋白質を特異的に認識する抗体が作出されて市販され,免疫染色によるフローサイトメトリーや組織染色が一般に普及している。あるいは全自動血算装置が市販されており機会計測による鑑別と計数が可能である。また,ゲノム情報を活用して分取した細胞に特異的に発現する遺伝子も容易に同定可能である。ところがヒトやマウス以外の動物の血球鑑別では利用可能な特異抗体は殆ど存在せず,免疫染色の適用はできず,機会計測法も利用できずにいる。我々はアフリカツメガエル,ネッタイツメガエル,メダカを対象に造血制御解析の新しいモデル動物確立を展開してきた(現在遂行中の科学研究費課題)。本研究では,表現型となる遺伝子が未知の動物血球に対して,その血球そのものを免疫抗原としてマウスに免疫(細胞免疫)してモノクローナル抗体を作出し,その未知抗原分子を同定する手法の開発を展開した。抗体の抗原分子決定法には,ペプチドオーバーラップ法,発現クローニング法など複数の選択肢がある。本研究では,我々が得意とするショットガン質量分析法(LC-MS/MS)を選択した。そして次の1)~5)の手順にて分析を進めた。1)抗体が認識する抗原細胞と抗体とを結合させる,2)両者をビーズ破砕する。3)Protein Gビーズによる免疫沈降法により,抗原抗体複合体を回収する,4)核酸・脂質除去後のトリプシン消化部分ペプチド群を質量分析へ進める,5)in silico解析し,抗原を同定する。このプロトコルでは界面活性剤の使用の有無,ビーズ破砕の条件,抗原抗体複合体の回収方法などについて,かなり厳密な最適化が必要であった。他にも細胞膜分子の糖鎖をビオチン化し,抗体が認識するビオチン化分子を濃縮後,同様の手順で質量分析し抗原分子を同定するなどの検討も必要だとおもわれた。本年度では,モデル実験によって再現性あるモノクローナル抗体の抗原分子同定までは至らずにいる。しかし細胞の前処理や蛋白質分子調製に関して,最適化に至る課題を洗い出すことができたため,どの研究室でも実施可能な標準手法の確立に向けて努力したい。

  • スポンジモデル検証を基礎とする両生類の血球産生調節の研究

    2013  

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    本研究の最終目標は,動物における血球や血球産生(造血)の仕組みの多様性と普遍性を明らかにすることである。血球産生(造血)は,脊椎動物に共通した生命機構であり,地球上の様々な動物の多様性と普遍性の探求のすぐれた題材である。酸素運搬を担う赤血球,生体防御を担う白血球,止血血栓形成を担う栓球(哺乳類の脱核した血小板も栓球に含まれる)のそれぞれは,いずれも造血幹細胞を源とする細胞である。本研究期間では,両生類(アフリカツメガエル;Xenopus laevis,以後ツメガエル)の造血を精査するために,血球関連分子と抗体の作出/赤血球造血因子エリスロポエチン(EPO)定量系/in vitro及び in vivoモデル確立など,各種の予備検討を進めた。ツメガエル赤血球造血器の肝臓には,ヒトやマウスに比較して1細胞あたりのEPO受容体数が圧倒的に多いと考えられ,この結果,過剰のEPO受容体がフリーのEPOを吸着し,赤血球産生量を決定する造血因子量を規定している可能性がある(スポンジモデル)。このことを実験的に検証するためには,様々な生物学的実験資源の調製が必要になる。その中でも,赤血球系細胞を特異的に検出するモノクローナル抗体と,EPO受容体を認識するモノクローナル抗体(xlEPOR MoAb)を作出することができた。特に後者に関しては,抗原免疫マウス脾臓とミエローマ細胞を融合させて得た約3000クローンのハイブリドーマの中から,本研究に有益を考えられる4クローンを選別した。それぞれのクローンが産生するモノクローナル抗体の化学的性質を精査し,さらにツメガエル肝臓,末梢赤血球,ツメガエルEPO受容体強制発現ヒト白血病細胞株などのEPO受容体に関して,免疫染色像,フローダイトグラムを検討することができた。今後,さらに獲得抗体の抗原特異性,細胞認識特異性,結合特異性を調べ,ツメガエル造血系解析に積極的に利用展開する。

  • 造血器の多様性に関する生物種間の比較解析

    2013  

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    動物成体の主な造血器は,魚類は腎臓,両生類は肝臓や脾臓,哺乳類は骨髄が主となる。無尾両生類アフリカツメガエル(Xenopus laevis)は偽4倍体の染色体をもちゲノム量がヒトよりも多い。このため全ゲノム解読は,現在,文部科学省ナショナルバイオリソースプロジェクト(NBRP)の指定動物となっている2倍体染色体のネッタイツメガエル(Xenopus tropicalis)が先となった。両種の外観形態,発生様式などは類似し,ゲノム情報をもち成長速度も速いネッタイツメガエルは,長い歴史をもつアフリカツメガエルモデルに代替される,と期待されてきた。実際,我々もアフリカツメガエルの造血研究を進める過程で,ネッタイツメガエルのゲノム情報を頻用してきた。アフリカツメガエルの肝臓は,赤血球造血をおこなうために赤色を呈する。ところが我々は偶然,ネッタイツメガエルの肝臓が黒色であることに気がついた。そしてネッタイツメガエルの造血器は,魚類のように腎臓なのではないか?という仮説を立てるに至った。本研究ではまず,両種の肝臓,脾臓,腎臓,肺,心臓の各組織を同一条件で顕微観察し,形態的所見を比較した。その結果,肝臓と腎臓に組織像に差異が認められた。造血能の差異について解析するため,造血器候補臓器より細胞を採取し,造血因子添加培養下,実際に血球が出現するかを調べた。また造血関連分子(造血因子,造血因子受容体,血球増殖・分化関連転写因子,血球系譜特異的表現型分子)について,in silico解析で造血関連分子の遺伝子配列を確定し,PCR法によって赤血球造血関連分子群の発現を比較した。以上の一連の実験によって,両種は近縁種ではあるが,肝臓と腎臓のそれぞれの造血能は必ずしも同等ではなく,アフリカツメガエルは造血能をより一層,肝臓にシフトさせており,一方,造血能を腎臓に残している所見が得られた。しかしNBRPからのフォローアップ情報によると,今回の検討の対象となったネッタイツメガエルが完全に健康ではなかった可能性があり,今後も引き続き精査の継続し,モデル生物構築の一環としてネッタイツメガエルの情報蓄積に貢献したい。

  • 両生類造血巣が育む血球前駆細胞の成熟と末梢血球数調節

    2012   前川峻

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    ほとんど全ての脊椎動物は,血球をもつ。したがって血球産生(造血)の科学は,地球上の様々な動物の多様性と普遍性の探求のすぐれた題材である。脊椎動物の血球は,酸素運搬を担う赤血球,生体防御を担う白血球,止血血栓形成を担う栓球(哺乳類の脱核した血小板も栓球に含まれる)に大別される。それぞれ細胞形態も機能も異なるが,いずれも造血幹細胞を源として恒常的に派生する血球前駆細胞が増殖,分化,成熟し,末梢血管へ放出された細胞である。この過程を「造血」と呼ぶ。動物造血は基礎生物学・比較生物学の謎の宝庫の場であると確信する一方,全体を俯瞰する科学蓄積が乏しい。本研究は,進化序列と,造血の環境応答の興味から,特に両生類(アフリカツメガエル;Xenopus laevis,以後ツメガエル)の造血に焦点を当て,解析系立上げに尽力した。環境刺激としては,外温動物の特性を活かして,低温環境に暴露したツメガエルの汎血球減少症の機序の解明に焦点を当てた。特に赤血球減少について詳細な分析を進めたところ,低温環境に暴露されたツメガエルは,その急性期(低温暴露後1日以内)に,肝臓への循環赤血球の集積と破壊が起こることを突き止めた。この結果,赤血球造血の亢進を待たずに循環赤血球数が低下する。一方,恒温動物のマウスで同様の低温環境暴露モデルを解析したところ,一過性の末梢赤血球数の低下は起こるが,外温動物とは異なり,造血器そのものは体温に維持され,腎臓における赤血球産生因子エリスロポエチンのmRNA発現が亢進することが判明した。本研究の取り組みによって,ツメガエルにおける造血解析が徐々に精緻化できるようになり,このような比較実験生物学的な検討が可能となった。本研究の取り組みをはじめて10年を経過したが,研究成果を紹介する機会が徐々に増えてきた。特に最近,新しい題材として両生類造血に感心を寄せる研究者が増えつつあると実感している。

  • 両生類血球の起源と造血組織動態の解明

    2011   前川峻

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    脊椎動物の調節系の多様性と普遍性の研究が各種進められる中で,動物の血球産生調節の仕組みの統合的理解は進んでいない。膨大な種を含む脊椎動物のうち,赤血球とヘモグロビンとを持たない生物は,ウナギの幼生と,酸素を血中に溶存させる南極のコオリウオ科魚類数種だけとされる(シュミット=ニールセン著,動物生理学~環境への適応,1997;沼田・中嶋監訳:東大出版,2007)。造血は脊椎動物共通の基幹系であり,種間で比較すれば,調節系の普遍性や多様性を論ずる良い題材である。本研究では,両生類アフリカツメガエルを対象に,造血発生や成体血球産生の機序の詳細な検討とともに,組織形態解析に立脚した造血解析に取り組んだ。アフリカツメガエル幼生(オタマジャクシ)では,赤血球造血発生に関して組織観察と遺伝子発現を検討した。発生段階NF52の組織をHE染色したところ肝臓の組織内に赤血球を認めた。NF56,60,66および成体の各組織の血球関連遺伝子の発現を解析したところ,NF56では赤血球分化に関わるgata-1・gata-2・エリスロポエチン(EPO)・EPO受容体・β-グロビンが肝臓・腎臓で発現していた。変態期の組織リモデリングの伴うNF60では腎臓でのgata-2・EPO・β-グロビンの発現が消失し,脾臓でこれらの遺伝子が発現するようになることから腎臓から脾臓へ赤血球造血巣の移行が示唆された。一方,これまでの我々の検討の結果,アフリカツメガエル成体の肝臓には,エリスロポエチン受容体発現細胞の約90%が類洞内に局在していることが判明している。そこでさらに成体肝臓における解析を進めた。o-dianisidine染色および透過型電子顕微鏡(TEM)観察により、ヘモグロビン含有赤芽球の局在を調べ、赤血球造血の微小環境を形成しうる接着分子vcam1, integrin alpha4, alpha5, beta1, macrophage erythroblast attacher(maea) 発現細胞との位置関係を明らかにした。また,栓球系の出現(前駆細胞からの増殖と分化)について,詳細な解析をした。ヒト,マウスと類似配列をもつTPO受容体(c-Mpl)を介した増殖分化をおこなう栓球前駆細胞が肝臓に存在したことから,栓球造血におけるTPO/c-Mpl系の生物種間の保存と生物活性の交叉を考察した。白血球系については,アフリカツメガエル顆粒球コロニー刺激因子(xlG-CSF)とその受容体G-CSFR(xlG-CSFR)の相同遺伝子配列を同定し、生物活性を検討した。xlG-CSF遺伝子をヒト胎児腎臓細胞株HEK293Tに導入し、組換え蛋白質を得た。一方、xlG-CSFR遺伝子を恒常発現するマウスFDC-P2細胞を得て、組換えxlG-CSFを本細胞株に添加したところ,明らかな増殖活性を示した。さらに、造血器由来細胞を組換えxlG-CSF存在下で培養し、出現する血球細胞中の好中球数を調べ,作用特異性を確認した。幼生,成体とも,肝臓で赤血球造血の直接証拠を獲得しつつあるが,それらの造血組織環境に,細胞接着因子ESAMが関与する可能性がある,ESAMは、動物44 種で報告されたが、アフリカツメガエルでは報告がない。近縁種ネッタイツメガエルESAM の遺伝子配列情報を利用して、アフリカツメガエルESAM の成熟蛋白質をコードする遺伝子配列を決定した。ヒトやメダカなどの一次構造と比較すると、塩基、アミノ酸それぞれの配列の一致率は低いが、機能領域、Cys 残基、N 結合型糖鎖の位置は保存されていた。造血巣である肝臓、脾臓、貧血成体の末梢血で発現することから、アフリカツメガエルのESAM 分子の発現は造血動態と関連することが示唆された。

  • メダカ造血と連鎖する甲状腺内分泌因子の研究

    2011   前川 峻

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    造血系と内分泌系の研究は,これまでにそれぞれ独立した領域で展開されてきた。その結果,生理調節にいて相互に干渉・交差する分子機序の理解には至っていない。そこで我が国が世界に誇るべきモデル生物である小型魚類メダカを基礎生物学研究所ナショナルバイオリソースプロジェクト(NBRP)(http://www.nbrp.jp/report/reportProject.jsp?project=medaka)より分与を受け,発生工学的手法を用いてin vivoで赤血球系譜特異的に甲状腺ホルモンの作用を阻害する実験系構築を行うこととした。その実験的手法の確立を目指し,本年度は,赤血球系細胞特異的に蛍光標識したトランスジェニック(Tg)メダカ作出を試みた。1.EPOR:DsRed Tgの作出赤血球造血因子エリスロポエチンの受容体(EPOR)遺伝子の5’上流転写調節領域(約4 kbp)を,メダカゲノムDNAから単離した。次いで単離遺伝子下流に,蛍光蛋白質DsRed遺伝子を含むベクターを構築した。本ベクターにはホーミングメガヌクレアーゼI-SceI認識配列を含んでおり,高効率的にゲノム内に組み込まれるように設計した。本遺伝子を,一細胞期のメダカ受精卵に,I-SceI酵素とともに共注入した。結果,蛍光顕微鏡観察により,受精後5日目には,血流を循環するDsRed陽性血球が多数確認された。2.GATA1:EGFP Tgの作出GATA1遺伝子座を含むBACクローン(基礎生物学研究所NBRPより譲渡)を用いて,大腸菌(SW102株, National Cancer Instituteより提供)内での相同組換え法によりGATA1遺伝子5’転写調節領域10 kbpを蛍光蛋白質EGFP遺伝子の上流に組み込んだ。本方法は,従来方と比較し,広領域の遺伝子を短期間でクローニング可能である。本遺伝子を1と同様にメダカ受精卵に微小注入した。結果,蛍光顕微鏡観察により受精後1日目の腹部血統領域にEGFP陽性細胞を確認できた。各Tgメダカは現在,世代交代により系統化を進めている。今後は上記Tgメダカ造血巣に存在す各赤血球系譜細胞をフローサイトメトリー法による同定・単離法の確立を進める。また,EPORやGATA1遺伝子の転写調節領域下流に甲状腺ホルモン受容体のドミナントネガティブ領域を組み込み,赤血球系細胞特異的に甲状腺ホルモンの効果を阻害するTgメダカの作出も合わせて行う予定である。

  • 血球細胞の最終分化の場における分子間作用と組織特性

    2007  

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    本研究は,分化方向が決定した血球細胞の最終分化の組織環境(場の形成)について注目し,以下の課題に取り組んだ。1)新規の動物モデルを構築する2)血球数異常を呈する動物個体の血球産生の動態を多面的に解析する3)細胞の最終分化の場の組織特性と,細胞相互および分子相互の関係を解析する1)について鋭意展開した結果,各種の造血因子とその受容体の構造を明らかにしつつあり,分子的な理解を進めることが可能となる世界的にも貴重な新しい造血解析モデルが誕生しつつある。またゼノパスのEPOは,種を超えてヒトEPO受容体強制発現細胞の増殖を刺激することを確認した。2)については,主要三血球系統の前駆細胞が増殖して最終分化に至るには,造血因子が必要であるので,既に取得したEPO,G-CSF,TPOのゼノパス相同遺伝子を動物細胞あるいは大腸菌で発現させて組換え蛋白質を取得した。一方,糖鎖付加,組織発現,活性などの基本を調べ,特に,哺乳類のin vivo活性に必須のN-結合型糖鎖が付加されないゼノパスEPOについては大腸菌発現系に注力し,結晶構造解析による種間交差性活性構造相関の解析の準備を進めた。同時にこれらの造血因子と受容体の抗体作製も進めた。さらに正常個体や溶血性貧血個体の肝臓,脾臓,骨髄,腎臓などの造血器における血球産生局在の全体像を,特異発現遺伝子を用いたin situ染色組織像と,フローサイトメトリーの両者を併用して明らかにしつつある。3)については,ゼノパスEPO分子には,N型糖鎖の付加がないことを見出し,肝臓においてEPO産生細胞と,血管外にあるEPO受容体を発現する赤血球前駆細胞が肝微小環境で相互に作用しあって赤血球産生の場を形成しているとするという新たな造血モデルを検証した。また,胎生期のヒトやマウスの肝臓でも,細胞接着因子群も加わって同様の環境が機能している可能性があり,種固有性に十分留意しつつ,種を超えた解釈を進める実験展開を進めた。

  • 血小板産生調節におけるトロンボポエチン-Mpl系の分子動態

    2005  

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    巨核球や巨核球前駆細胞に発現する細胞膜受容体c-Mplは、血液循環中にある血小板産生因子トロンボポエチン(Thrombopoietin ; TPO)と会合し、標的細胞の増殖と分化を引起こすシグナル伝達系を作動させる。新たに血液循環に供給される血小板の産生は、このTPO-c-Mpl系が主要な役割を担う。1細胞の中の、TPO-c-Mplの1複合体が実際に局在転移を起こして細胞に内在化し、その後c-Mplのみが再び細胞膜上にリサイクリングされる過程を直接観察するため、まず蛍光で可視化可能なc-Mplを発現する細胞を作出した。具体的にはFDCPマウス白血病細胞株を発現宿主とし、遺伝子クローニングしたマウスc-mplを強制発現導入した細胞株を樹立した。この時、重合体を形成しにくい緑色蛍光蛋白質eGFPの変異遺伝子をタンデムに連結させ、c-MplのN末端側(細胞外領域)あるいはC末端側(細胞内領域)に緑色蛍光をもつc-Mpl誘導体発現キメラ遺伝子を導入した。その結果、共焦点レーザー蛍光顕微鏡によって、双方のキメラ遺伝子ともにそれぞれが発現する細胞株を取得することに成功したことが確認された。一方、特殊な無細胞蛋白質合成系を利用して、c-MplのリガンドとなるTPO部分長分子のC末端側に蛍光色素を共有結合させた分子の取得に成功した。これらの細胞は遺伝子組換えTPOの存在下で細胞増殖能を獲得した。続いて生物物理学分野の共同研究者である東京大学薬学部船津高志教授とともに、レーザーを光源とした精密な顕微分子計測系(生体分子の一分子計測・可視化技術)による、1細胞における蛍光標識TPOあるいは蛍光標識c-Mplの観察に着手した。顕微観察系は構築可能であり、蛍光標識c-Mpl発現量の異なる細胞亜株をサブクローニングして、観察する細胞株のファインチューニングを継続して実施している。以上のことからサイトカインおよびその受容体の双方に異なる蛍光をもつ分子を利用した解析系構築に目処がたち、【標的細胞におけるTPO-c-Mpl複合体の局在変化】の解明へ向けた世界初の実験検証系の確立となった。

  • 造血巣の形成と臓器間移動に関する解析

    2005  

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    骨髄、脾臓、肝臓、腎臓を造血巣として使い分ける両生類成体(ゼノパス)を実験モデルとして、造血巣の個体内移動における血球系細胞とその環境の質的変化を、造血微小環境を視点の中心として明らかにすることが本研究の目的である。ゼノパスの造血幹細胞のマーカーの同定と分離法、諸造血関連分子の同定などの技術基盤が大幅に進展し、造血巣を構成する分化血球の分布について、1)in situハイブリダーゼーション法による発現遺伝子(mRNA)の細胞組織形態解析を進めた、2)非哺乳類で初めて主要造血因子の遺伝子を分離同定し、それらの構造を明らかにした(データベースへ登録)、3)両生類赤血球前駆細胞のin vitroコロニー細胞培養の最適化に成功し、レトロスペクティブな手法によって、個体内の造血幹細胞の局在や、血球産生の動態を正確にとらえる手法を確立した。また個体内のダイナミックな造血動態を解析するために、全く新たな造血解析モデルを作出した。これらは低温飼育による慢性血球減少症モデルや抗ゼノパス血球モノクローナル抗体投与モデルであるが、これら各モデルの末梢血球変動や、血球系細胞の寿命変動に関して、独自に作成した各種のモノクローナル抗体を適用して、レーザー蛍光細胞分析分離装置によるフローサートメトリーや蛍光免疫細胞染色によって解析した。同時に、ゼノパスの赤血球寿命が100日以上であることや、栓球寿命が1週間以上であるなどの基礎生物学における新たな学術成果も得た。また血球数減少期や回復期にある時、骨髄、脾臓、肝臓、腎臓にある血球細胞の分布(位置と種類)の変動、あるいは各造血巣と周辺組織の細胞相互の位置について、組織連続切片や細胞染色標本を用いた精査に着手することができた。以上の取組みは、現在ヒト造血幹細胞移植療法や再生医療の将来へ向けて課題になっている造血巣の性質に関する基礎的解明と連鎖する成果となり、造血巣に出現する造血幹細胞の由来や、生物種に普遍的な造血能を代償する未知の制御系の発見へ繋がるユニークな取組みとして国内外より注目されつつある。

  • ナノ・レベルで血液に存在する生体因子の発掘と解析

    2004   菊山榮

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    内分泌ホルモンや細胞増殖・分化誘導因子等(サイトカイン・インターロイキン等)などを代表例として、生体試料に存在する微量蛋白質は、生体調節機能を有する場合が多い。しかし微量であるがゆえに、それらの蛋白質を単離し分子構造を同定する作業は古くから非常に困難な作業であって、このことは全ゲノム配列が判明した生物種を対象としても変ることがない。むしろ後者の場合は、ポストゲノム情報として今後益々、蛋白質の機能情報の獲得は重要な課題となってきた。本研究は、血液循環中に微量(サブμg・サブng/ml)に存在する生体機能蛋白質、即ち、大量の主要血液蛋白質の存在に隠れ、通常の方法では分離・検出されずにいるか、未発見である微量蛋白質の分離を免疫学的手法を活用することにより、簡便かつ網羅的な高純度濃縮・精製を実現する手法概念の実証を目的とした。さらに進化生物学上、種として重要な位置にありながらゲノム解析が未着手であり、個体からの採取血液量が限られる両生類(アフリカツメガエル、ウシガエル、イモリ等)を実験対象とし、存在の検証に続いて、生物学的研究を進める意義の深い分子群について、蛋白質化学的な分子性状を解明する手段として適用する手法を目指した。実験モデルとして、無尾両生類アフリカツメガエルの血中微量蛋白質の分離を試行した。まず血液中の主要蛋白質を認識するポリクローナルウサギ抗体の作製を行い、精製抗体を固相に固定化し、主要蛋白質群を抗体吸着画分として出発材料から除くクロマトグラフィーを調製した。第一段階の抗体結合性吸着画分には約90%の蛋白質が吸着した。更に、第一段階の抗体カラムの非吸着画分を抗原にして抗体を作製し、第二段階のクロマトグラフィーを組み立て、得られる画分の成分変化を調べた。この結果、血液総蛋白質の90%以上の除去に成功し、2ステップで得られた蛋白質中にプロラクチンなどの生体微量分子が高い回収率で濃縮可能であった。以上のように、精製初期段階で夾雑蛋白質を簡便迅速に除去できれば、精製ステップ数の低減や、活性の維持や精製純度を高める有効な手段となることが示された。今後は他のクロマトグラフィーとの併用によって、高純度の微量蛋白質画分を調製し、質量分析などを適用することによって、分子同定を進める道が拓かれた。

  • 生物種を超える造血制御の環境応答

    2004  

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    血球産生は動物の生存に重要な役割を担う。本研究では個体の置かれる環境ストレスに対する造血系の応答に着目した。例えば、低酸素体外培養後のヒト骨髄幹細胞の移植後再構築能の向上(Danet, et al. J .Clin Invest, 2003)、造血微小環境の変化による移植造血幹細胞の体内生着率変動など、生体内外の造血環境は、血球産生に大きく作用する。また、脊椎動物、特に両生類では、季節・性周期や棲息環境・発生段階の変化によって、主要造血器は、骨髄、腎臓、脾臓、肝臓などの臓器間で移動する。その仕組みを外温生物である無尾両生類アフリカツメガエル(ゼノパス:Xenopus laevis)をモデルにして、包括的理解を進めるために必要とする基礎的手法の開発、ならびに実際に個体への温度環境負荷に依存する末梢血球数の変動について調べた。環境ストレスとして、まず低温環境を選択し、飼育温度の変化に応答するin vivo造血変動を調べた。<造血制御の温度依存性>ゼノパスを25℃(常温)で飼育後、10℃の低温下へ飼育環境を移行して経時採血を行ったところ、各種末梢血球数は徐々に減少し始め、特に赤血球は約3週間で常温時の40%ほどに減少し、その後一定数に保たれた。再び常温飼育下に移行することで、これら血球数は、ほぼ元の値まで可逆的に回復した。血球数回復時の末梢血球のMGG(メイ・グリュンワルド-ギムザ)染色像を観察すると、常温飼育では末梢血中にはほとんど観察できない幼若な赤血球が多く出現しことから、低温応答によって血球産生が亢進していることが示された。<ゼノパス造血における脾臓の機能解析>脾摘ゼノパスを作出し、脾摘後の末梢血球数変動を観察した。脾摘直後、一旦減少した栓球数は術後20日目までに大幅に増加し、約一ヵ月後に正常値に回復した。また、脾内細胞をMGG染色にて観察すると、栓球様の細胞が多数見られる。これらの結果から、脾臓は環境変動負荷における末梢血中の栓球数調節に密接な役割を担っていることが示された。今後は、以上の結果を踏まえて、環境ストレスに暴露された血球系細胞の増殖・分化・機能や細胞局在を観察し、細胞質と核内で発現が変動する分子を発掘していく予定である。注目すべき分子を蛋白質化学・分子生物学的な手法によって探査選別、同定を進め、次いで当該遺伝子の機能発現動物モデルにおいて、造血器の組織形態の変化、末梢血球数の変動を調べ、これらの分子機能の検証へ進めていきたい。

  • 両生類等における造血制御解析法の実験的確立

    2002  

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    本研究では、代表的な外温生物である両生類アフリカツメガエル(Xenopus laevis laevis)を実験動物とし、新たな造血制御機構の探索を展開するために必要となる、血球系細胞の基礎的解析手法を検討した。成体アフリカツメガエル末梢血を心採血し、塗沫標本ををMay-Gr&uuml;nwald-Giemsa染色、Hadji-Azimiら(1987)の分類に従って、各種血球の鑑別分類を行った。一方、臨床で用いられている自動血球計数装置(Sysmex F802)による末梢血球数の自動計数も検討したが、両生類のインタクトな血球の分別測定には粒度分布パラメータの設定や、血球前処理工程などに尚も工夫が必要であった。従って浮遊血球試料についてはcrystal violet染色血球をNeubauer血算盤で顕微観察により測定することとした。Percoll密度勾配法を実施し、赤血球と白血球は明確な分離が可能となった。また脾臓、肝臓、骨髄より細胞を回収し、染色標本で血球系細胞の存在を検討したところ、骨髄には脂肪が多く血球系細胞が少ないが、脾臓、肝臓には血球系細胞が多く見出された。すなわち、アフリカツメガエルの主たる造血巣は骨髄でなく、通常は脾臓や肝臓が造血の場であることを確認した。in vitroにおけるアフリカツメガエル血球系細胞の培養基礎条件を検討したところ、ウシ胎仔血清存在下、哺乳類で用いる細胞培養液を浸透圧調整後(7/9希釈)の培地によって、コロニーアッセイで必要とされる、少なくとも数日程度の培養が可能であることを確認した。外部環境に応答して造血系が変化する仕組みを解析する端緒として、アフリカツメガエルの末梢血球数変動の温度感受性を調べたところ、低温飼育後のアフリカツメガエルの末梢血球は半減することを確認した。以上の一連の研究は、生物における新たな造血制御系を探索を想定して、両生類による造血解析モデルの基礎的検討であり、今後、多様な実験系を確立する必要がある。この過程で解明される両生類造血の仕組みは、基礎生物学にとっても重要な知見を生むものと考えられた。本成果を基盤として、遺伝子組換えヒト造血因子への応答試験を含めた具体的な展開を予定している。

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Overseas Activities

  • 血球の性質と造血動態に関する総合的研究

    2013.10
    -
    2014.03

    アメリカ   スミソニアン博物館

 

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