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

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。加入TPA和CT后72小时，蛋白质印迹法显示KIT的表达水平达到最大，流式细胞仪分析显示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。FACS分析显示，在添加钙磷蛋白C和H-89后72小时，NCCmelb 4中KIT阳性细胞减少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。

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.