| 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)
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| 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
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