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The role of TPA and/or cholera toxin on the melanocyte development in Mitf^<mi-ew> mouse

Research Project

Project/Area Number 15591199
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Dermatology
Research InstitutionSt.Marianna University School of Medicine

Principal Investigator

KAWA Yoko  St.Marianna University School of Medicine, Faculty of Medicine, Assistant, 医学部, 助手 (10082273)

Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
Keywordsneural crest cell / Kit / Kit ligand / Mitf / melanocyte cell line / mi mutant mouse / TPA / cholera toxin / Kit ligannd / melanocyte cell li / melanocyte / development / SCF / EDN3 / MITF / KIT / choleratoxin
Research Abstract

It has been demonstrated that Mitf is not only a transcription factor for tyrosinase, Tyrp1 and Tyrp2 but also a survival factor for melanocytes. To investigate the role of Mitf in melanocyte survival, we performed neural crest cell(NCC) primary cultures in which explants of neural tubes from Mitf^<mi-ew> mice were cultured in 15% FBS/Eagle MEM. In these cultures, cells positive for either Kit, tyrosinase, Tyrp1 or Tyrp2 did not appear, even when Kitl and/or Edn3 were added to the cultures. When they were cultured with TPA and cholera toxin, some Tyrp1-positive cells and fewer numbers of Kit-positive cells appeared, but tyrosinase and Tyrp2 were still negative in all cells. To clarify the mechanisms of Mitf in maintaining melanocytes, we knocked down the expression of Mitf in NCCmelb4M5,which is a Kit-and DOPA-negative immature melanocyte cell line derived from mouse NCC, by the RNA interference technique. Addition of siRNA of Mitf-M inhibited cell proliferation, and then caused detachment of the cells from the culture flasks. Immunostaining showed that caspase 3-positive cells increased time-dependently, suggesting the induction of apoptosis. Real-time PCR analysis revealed that the addition of siRNA of Mitf-M inhibited Tyrp1 mRNA expression and enhanced bax mRNA expression. Since the expression of bcl-2 was not altered, it is likely that the imbalance of bax/bcl-2 ratio led to apoptosis. When TPA and cholera toxin were added shortly before the addition of siRNA of Mitf-M, the cells were rescued from cell death. In these cells, the mRNA expression of Tyrp1 and bcl-2 were up-regulated, while bax did not change. The present findings suggest that Mitf play a role in melanocyte survival in early developmental stages through the induction of Tyr

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • Research Products

    (4 results)

All 2005 2004 Other

All Journal Article (4 results)

  • [Journal Article] Establishment of a Kit-negative cell line of melanocyte precursors from mouse neural crest cells2005

    • Author(s)
      Yoko Kawa
    • Journal Title

      Pigment Cell Research 18

      Pages: 188-195

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Establishment of a Kit-negative cell line of melanocyte precursors from mouse neural crest cells.2005

    • Author(s)
      Yoko Kawa
    • Journal Title

      Pigment Cell Research 18

      Pages: 188-195

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Establishment by Original Single-cell Cloning Method and Characterization of Immortal Mouse Melanoblast Cell line (NCCmelb4)2004

    • Author(s)
      Msaru Ito, et al.
    • Journal Title

      Pigment Cell Research 17

      Pages: 643-650

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Establishment of a kit-negative cell line of melanocyte precursors from mouse neural crest cells

    • Author(s)
      Yoko Kawa, et al.
    • Journal Title

      Pigment Cell Research (In press)

    • Related Report
      2004 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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