| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
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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. We previously reported that cells positive for either Kit, tyrosinase, Tyrp1 or Tyrp2 did not appear in neural crest cell (NCC) primary cultures in Mitfmi・ew mice even when Kitl and/or Edn3 were added to the cultures as well as previous reports but that some Tyrp1-positive cells and fewer numbers of Kit-positive cells appeared when they were cultured with TPA and cholera toxin. These results suggest that Tyrp1 may play a role in melanocytes survival. To clarify the mechanisms of Mitf and Tyrp1 in maintaining melanocytes, we knocked-down the expression of Mitf or Tyrp1 in a melanoblast cell line (NCCmelb4M5: Kawa, et. Al., 2005) and a melanocyte cell line (NCCmelan5: Ooka, et. Al., 2001) using the RNA interference method. NCCmelb4M5 is a Kit- and DOPA-negative cell line. NCCmelan5 is a Kit- and DOPA-positive cell line. In transfection of Mitf-M or Tyrp1 siRNA, these two cell lines did not survive and were completely abolished by 96 hrs. Immunostaining showed that caspase 3-positive cells increased in time-dependently. Real-time PCR analysis also revealed that interferon-stimulated transcription factor 3 and Fas mRNA greatly enhanced at 48 hours in the both cells. In contrast, Kit and bcl-2 decreased in NCCmelan5 cells but not in NCCmelb4M5 cells. We did not detect any change in the cells transfected GL3 as negative control. Based on these findings, we suggest that Tyrp1 also survival factor as well as Mtf for melanocyte and their survival mechanisms in both cells could mainly correlate with Fas pathway.
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