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

KIT对黑素细胞的发育是必不可少的，但诱导黑素母细胞表达KIT的因素尚未得到很好的鉴定。为了阐明KIT表达的机制，我们分别使用了KIT阳性和KIT阴性的黑素母细胞系NCCmelb4和NCCmelb4M5。这些细胞是从我们实验室的神经脊细胞中建立的。RT-PCR检测到NCCmelb4M5细胞中有KIT基因的表达，但未检测到KIT蛋白。免疫组织化学染色仅检测到少数KIT阳性细胞(约0.1%)。加入12-o-十四酰-13-乙酸酯(TPA)和霍乱毒素(CT)后，NCCmelb4M5对KIT呈弱阳性反应。TPA的最佳浓度为50 ng/ml，CT的最佳浓度为1 nM。在加入TPA和CT后72小时，Western blotting检测KIT的表达水平最高，FACS检测21%的NCCmelb4M5细胞KIT呈阳性。免疫染色显示α-MSH(10 0 NM)和DBcAMP(0.5 mM)可微弱诱导NCCmelb4M5的KIT表达，而免疫印迹法未检测到该表达。免疫组织化学染色显示，干细胞因子(50 ng/ml)、内皮素3(100 NM)、转化生长因子-β(10 ng/ml)对KIT表达无明显影响。免疫组织化学染色显示，当PKC抑制剂Calphostin C(20 NM)和PKA抑制剂H-89(2μM)处理NCCmelb4细胞时，其KIT表达下降。流式细胞仪分析显示，在加入Calphostin C和H-89后72小时，NCCmelb4中KIT阳性细胞减少了26%。这些结果表明，PKC或PKA部分参与了黑素细胞前体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.