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Engineering of Artificial Biomatrix through Extracellular Matrix Targeting of Functional Proteins

Research Project

Project/Area Number 11558081
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Structural biochemistry
Research InstitutionOsaka University

Principal Investigator

SEKIGUCHI Kiyotoshi  Institute for Protein Research, Osaka University Professor, 蛋白質研究所, 教授 (50187845)

Co-Investigator(Kenkyū-buntansha) NOZAKI Chikahide  Research Division, KAKETSUKEN Chief, 室長
NISHIDA Teruo  Faculty of Medicine, Yamaguchi University Professor, 医学部, 教授 (80036475)
LI Shaoliang  Institute for Protein Research, Osaka University Instructor, 蛋白質研究所, 助手 (40252720)
GU Jianguo  Institute for Protein Research, Osaka University Instructor, 蛋白質研究所, 助手 (40260369)
顧 建国  大阪大学, 蛋白質研究所, 助手 (30314420)
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥9,900,000 (Direct Cost: ¥9,900,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1999: ¥4,400,000 (Direct Cost: ¥4,400,000)
KeywordsExtracellular matrix / basement membrane / matrix engineering / growth factor / wound healing / laminin / cell adhesion / HGF / インテグリン / フィブロネクチン
Research Abstract

Novel protocols for engineering of artificial biomatrix by targeting of proteins to the extracellular matrices have been developed. First, we established a protocol to target transforming growth factor-alpha (TGF-a) to the fibronectin matrix via chimerization with the marix assembly domain of fibronectin. The resulting chimeric protein retains the mitogenic activity of TGF-a without-compromising its matrix assembly activity. The chimeric protein was more potent than authentic, soluble TGF-a in promoting would healing of rabbit cornea, demonstrating its potency as a new protein drug for accelerating, wound healing. Second, we developed another protocol to target functional proteins to the basement membrane, the scaffold for growth and differentiation of stem cells. We utilized the N-terminal laminin-binding domain of agrin to target hepatocyte growth factor (HGF) or green fluorescent protein to the basement membrane. The resulting chimeric protein retained the motogenic activity of HGF … More as well as the laminin-binding activity of agrin. When cells expressing the chimeric protein were grown on the laminin-containing basement membrane-like gel (I.e., Matrigel), the chimeric protein was found to be deposited around the cells. This protocol will be applicable for targeting of various growth factors and/or cell differentiation-inducing factors to the basement membranes of vasculature and other organs. Third, we purified new laminin isoforms, I.e., laminin-8 and -10, and characterized their physiologic activities with special reference to integrin-mediated signaling events. We also established the expression system for laminin-6, -8, and -10 by cDNA transfection into human 293 cells. We also succeeded in expression of the functional domains of laminin chains with putative activities for integrin/alpha-dystroglycan binding as well as laminin selfassembly. These domains of laminin chains will be useful for construction of tailor-made, artificial basement membranes optimized for proliferation and differentiation of particular cell types, e.g., embryonic and tissue stem cells. Less

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (37 results)

All Other

All Publications (37 results)

  • [Publications] Kikkawa, Y. et al.: "Integrin binding specificity Ci : laminin-10/11 : laminin-10/11 are recognized by α3β1,α6β1,and α6β4 integrins"J. Cell Sci.. 113. 869-876 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Naito, M. et al.: "The domains of human fibronectin mediating the binding of α antigen, the most immuno-potent antigen of mycobacteria that induces protective immunity against mycobacteria"Biochem. J.. 347. 725-731 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fleischmajor, R. et al.: "Differential expression of laminin a chains during proliferative and differentiation stages of skin morphogenesis"Matrix Biol.. 19. 637-647 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fujiwara, H. et al.: "Purification and characterization of human laminin-8:laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins"J. Biol. Chem.. 276. 17550-17558 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Gu, J. et al.: "Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130Cas-Crkll-DOCK180 pathway"J. Biol. Chem.. 276. 27090-27097 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Gu, J. et al.: "Lmainin-10/11 aqnd fibronectin differentially prevent apoptosis induced by serum removal via Pl 3-kinase/Akt-and MEK^1/ERK-dependent pathways"J. Biol. Chem.. (in press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 関口 清俊: "多細胞体の構築と細胞接着システム"共立出版. 208 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Manabe, R., Oh-e, N., and Sekiguchi, K.: "The alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction"J. Biol. Chem.. 274. 5919-5924 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tanaka, M., Tamaki, T., Kawamura, A., Yokota, N., Hayashi, T., Konoeda, Y., Yonekasa, M., Sanzen, N., and Sekiguchi, K.: "A novel monitoring of kidney allograft rejection with plasma EDA(+) fibronectin"Transplantation Proc.. 31. 310-311 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Satoi, S., Hiramatsu, Y., Kitade, H., Kwon, A.-H., Matsui, K., Miyashita, K., Sakashita, E., Sekiguchi, K., Takahashi, H., and Kamiyama, Y.: "Different responses to surgical stress between extra domain A+ and plasma fibronectin"Clin. Exp. Pharmcol. Physiol.. 26. 225-229 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kikkawa, Y., Sanzen, N., Fujiwara, H., and Sekiguchi, K.: "Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by α3β1, α6β1, and α6β4 integrins"J. Cell Sci.. 113. 869-876 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Naito, M., Fukuda, T., Sekiguchi, K., and Yamada, T.: "The domains of human fibronectin mediating the binding of α antigen, the most immuno-potent antigen of mycobacteria that induces protective immunity against mycobacterial infection"Biochem. J.. 347. 725-731 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fleischmajor, R., Kuroda, K., Utani, A., MacDonald, E. D., Perlish, J. S., Arikawa-Hirasawa, E., Sekiguchi, K., Sanzen, N., Timpl, R., and Yamada, Y.: "Differential expression of laminin α chains during proliferative and differentiation stages of skin morphogenesis"Matrix Biol.. 19. 637-647 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Satoi, S., Kitabe, H., Hiramatsu, Y., Kwon, A-H., Yakahashi, H., Sekiguchi, K., Uehara, M., Oda, M., Yanagimoto, Y., Miyashita, K., Sakashita, E., and Kamiyama, Y.: "Increased extra domain-A containing fibronectin and hepatic dysfunction during septic response: an in vivo and invitro study"SHOCK. 13. 492-496 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fujiwara, H., Kikkawa, Y., Sanzen, N., and Sekiguchi, K.: "Purification and characterization of human laminin-8: laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins"J. Biol. Chem.. 276. 17550-17558 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Matsui, Y., Nakata, K., Araki, N., Ozono, K., Fujita, Y., Tsumaki, N., Kawabata, H., Yasui, N., Sekiguchi, K., and Yoshikawa, H.: "Altemative splicing of fibronectin transcripts in osteochondrogenic tumors"Anticancer Res.. 21. 1103-1106 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Gu, J., Sumida, Y., Sanzen, N., and Sekiguchi, K.: "Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130Cas-CrkII-DOCK180 pathway"J. Biol. Chem.. 276. 27090-27097 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Gu, J., Fujibayashi, A., Yamada, K.M., and Sekiguchi, K.: "Lmainin-10/11 aqnd fibronectin differentially prevent apoptosis induced by serum removal via PI 3-kinase/Akt- and MEK1/ERK-dependent pathways"J. Biol. Chem.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fujiwara, H., et al.: "Purification and characterization of human laminin-8:laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins"J.Biol.Chem.. 276. 19550-19558 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Matsui, Y., et al.: "Altemative splicing of fibronectin transcripts in osteochondrogenic tumors"Anticancer Res.. 21. 1103-1106 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Gu, J., et al.: "Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130Cas-CrkII-DOCK180 pathway"J.Biol.Chem.. 276. 27090-27097 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Nakamura M, et al.: "Role of the Small GTP-Binding Protein Rho in Epithelial Cell Migration in the Rabbit Cornea"Invest Ophthalmol Vis Sci.. 73. 1207-1214 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Gu, J., et al.: "Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via PI 3-kinase/Akt-and MEK1/ERK-dependent pathways"J.Biol.Chem.. (in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Fukuda K, et al.: "Characterization of the interleukin-4 receptor complex in human corneal fibroblasts"Invest Ophthalmol. Vis. Sci.. (in press).

    • Related Report
      2001 Annual Research Report
  • [Publications] 関口 清俊: "多細胞体の構築と細胞接着システム"共立出版. 208 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kikkawa,Y et al.: "Integrin binding specificity of laminin-10/11 : laminin-10/11 are recognized by a3b1, a6b1, and a6b4 integrins."J.Cell Sci.. 113. 869-876 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Naito,M. et al.: "The domains of human fibronectin mediating the binding of alpha antigen the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterial infection."Biochem.J.. 348. 725-731 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Satoi,S. et al.: "Increased extra domain A-containing fibronectin and hepatic dysfunction during septic response."Shock. 13. 492-496 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Fleischmajor,R. et al.: "Differential expression of laminin a chains during proliferative and differentiation stages of skin morphogenesis."Matrix Biol.. 19. 637-647 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Fujiwara,H. et al.: "Purification and characterization of human laminin-8: laminin-8 stimulates cell adhesion and migration through a3b1 and a6b1 integrins."J.Biol.Chem.. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Matsui,Y. et al.: "Alternative splicing of fibronectin transcripts in osteochondrogenic tumors."Anticancer Res.. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Manabe, R. et al.: "Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction"J. Biol. Chem.. 274. 5919-5924 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Satoi, S. et al.: "Different responses to surgical stress between extra domain A+ and plasma fibronectin"Clin. Exp. Pharmacol. Physiol.. 26. 225-229 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Gu, J. et al.: "Shc and FAX differentially regulate cell mobility and directionality modulated by PTEN"J. Cell Biol.. 146. 389-403 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tamura, M. et al.: "PTEN interactions with focal adhesion kinase and suppression of the extra-cellular matrix-dependent PI 3-kinase/Akt cell survival pathway"L. Biol. Chem.. 274. 20693-20703 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kikkawa, Y. et al.: "Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by α3β1,α6β1,and ,α6β4 integrins"J. Cell Sci.. 113. 869-876 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Naito, M. et al.: "The domains of human fibronectin mediating the binding of α antigen of mycobacteria that induces protective immunity against mycobacterial infection"Biochem. J.. (in press). (2000)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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