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The role of SCF, endotheline 3 or MITF on the melanocyte development

SCF、内皮素 3 或 MITF 对黑素细胞发育的作用

基本信息

批准号：
13670902
负责人：
KAWA Yoko
金额：
$ 2.62万
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：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670902/
关键词：
SCF KIT MITF EDNRB neural crest cell mi mutant mouse TPA cholera toxin melanocyte cholera toxin

项目摘要

KIT is essential for melanocyte development, but the factors which induce KIT expression in melanoblasts have not been well identified. To clarify the mechanisms of the KIT expression, we used a KIT-positive, and a KIT-negative melanoblast cell line, NCCmelb4 and NCCmelb4M5, respectively. Those were established from neural crest cells in our laboratory. KIT mRNA by RT-PCR was detected in NCCmelb4M5 cells but KIT protein was not detected. However, only a few KIT positive cells (about 0.1%) were detected by immunostaining. When 12-o-tetradecanoyl-13-acetate (TPA) and cholera toxin (CT) were added to the culture medium, NCCmelb4M5 became weakly positive to KIT by immunostaining. The optimum concentration of TPA was 50 ng/ml and that of CT was 1 nM. At 72 hours after the addition of TPA and CT, the expression level of KIT was maximum by western blotting and 21% of NCCmelb4M5 cells became positive to KIT by FACS analysis. α-MSH (100 nM) and DBcAMP (0.5 mM) weakly induced the KIT expression of NCCmelb4M5 as shown by immunostaining, which was not detected by western blotting. SCF (50 ng/ml), endothelin 3 (100 nM), TGF-β (10 ng/ml) had no effect on the KIT expression as shown by immunostaining. Immunostaining revealed that when NCCmelb4 cells were treated with calphostin C (20 nM), a PKC inhibitor, and H-89 (2 μM), a PKA inhibitor, their KIT expression was decreased. FACS analysis showed a 26% decrease of KIT positive cells in NCCmelb4 at 72 hours after the addition of calphostin C and H-89. These results indicate that PKC or PKA is partially involved in the regulation of KIT expression of melanocyte precursors.

KIT是黑素细胞发育所必需的，但是诱导KIT在成黑素细胞中表达的因子还没有被很好地鉴定。为了阐明KIT表达的机制，我们分别使用了KIT阳性和KIT阴性的成黑素细胞系NCCmelb 4和NCCmelb 4 M5。这些都是在我们实验室里从神经嵴细胞中建立起来的。RT-PCR检测到NCCmelb 4 M5细胞中KIT mRNA的表达，但未检测到KIT蛋白的表达。然而，只有少数KIT阳性细胞（约0.1%）被检测到的免疫染色。当12-o-十四酰基-13-乙酸酯（TPA）和霍乱毒素（CT）加入到培养基中时，通过免疫染色，NCCmelb 4 M5变成对KIT弱阳性。TPA的最适浓度为50 ng/ml，CT的最适浓度为1 nM。在加入TPA和CT后72小时，通过蛋白质印迹法检测到KIT的表达水平最高，并且通过FACS分析，21%的NCCmelb 4 M5细胞对KIT呈阳性。α-MSH（100 nM）和DBcAMP（0.5 mM）弱诱导NCCmelb 4 M5的KIT表达，如免疫染色所示，其未通过蛋白质印迹检测到。免疫组化显示SCF（50 ng/ml）、ET-3（100 nM）、TGF-β（10 ng/ml）对KIT表达无影响。免疫染色显示，当用PKC抑制剂calphostin C（20 nM）和PKA抑制剂H-89（2 μM）处理NCCmelb 4细胞时，其KIT表达降低。流式细胞仪分析显示，添加钙磷蛋白C和H-89后72小时，NCCmelb 4中KIT阳性细胞减少了26%。这些结果表明，PKC或PKA是部分参与黑素细胞前体的KIT表达的调节。