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The role of Mitf and Tyrp1 as survival factor on melanocytes

Mitf 和 Tyrp1 作为黑色素细胞存活因子的作用

基本信息

批准号：
17591187
负责人：
KAWA Yoko
金额：
$ 2.24万
依托单位：
St.Marianna University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591187/
关键词：
melanocyte Mitf Tyrpl neural crest cell RNA iterference melanocyte development NCCmelb4M5 NCCmelb4 NCCmelan5 Tyrp 1 development Mitf^<mi-ew> mouse

项目摘要

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.

研究表明，Mitf不仅是酪氨酸酶、Tyrp1和Tyrp2的转录因子，也是黑素细胞的存活因子。我们以前报道过，在Mitfmi·new小鼠的神经嵴细胞（NCC）原代培养中，即使在培养中添加Kitl和/或Edn3，也没有出现Kit、酪氨酸酶、Tyrp1或Tyrp2阳性的细胞，以及以前的报道，但是当用TPA和霍乱毒素培养时，出现了一些Tyrp1阳性细胞和较少数量的Kit阳性细胞。这些结果提示Tyrp1可能在黑素细胞存活中发挥作用。为了阐明Mitf和Tyrp1维持黑素细胞的机制，我们使用RNA干扰方法在一个黑色素母细胞细胞系（NCCmelb4M5: Kawa, et. Al., 2005）和一个黑素细胞细胞系（NCCmelan5: Ooka, et. Al., 2001）中敲低Mitf或Tyrp1的表达。NCCmelb4M5是Kit和dopa阴性细胞系。NCCmelan5是Kit和dopa阳性细胞系。转染Mitf-M或Tyrp1 siRNA后，这两种细胞系无法存活，96小时后完全消失。免疫染色显示caspase 3阳性细胞呈时间依赖性增加。Real-time PCR分析还显示，干扰素刺激的转录因子3和Fas mRNA在48小时后显著增强。相比之下，Kit和bcl-2在NCCmelan5细胞中下降，而在NCCmelb4M5细胞中没有下降。作为阴性对照，我们未检测到转染GL3的细胞有任何变化。基于这些发现，我们认为Tyrp1和黑色素细胞的存活因子以及Mtf及其在两种细胞中的存活机制可能主要与Fas通路相关。