BMP-4 regulates melanocyte differentiation in mouse neural crest cells just before they enter the Kit-dependent cycle of melanogenesis

Kit和Kit配体编码基因(Kit1)在黑素母细胞分化过程中起着重要作用。我们以前建立了三个永生但不同的小鼠神经脊(NC)细胞群体。NCCmelb4M5细胞不表达Kit并独立于Kit1生长；它们有分化为NCCmelb4细胞的潜力，NCCmelb4细胞是Kit阳性的黑素细胞前体。NCCmer5细胞具有分化黑素细胞的特征。我们研究了BMP-4对未成熟黑素细胞前体细胞分化的影响。三种细胞系均有BMP受体表达。BMP-4上调大多数未成熟NCCmelb4M5细胞Kit蛋白和mRNA的表达。BMP-4拮抗剂noggin可显著降低BMP-4诱导的Kit表达。Western印迹分析表明，外源性BMP-4导致Smads的磷酸化并激活NCCmelb4M5细胞中的信号转导。结论：在NCCmelb4M5细胞中，BMP-4通过Kit表达促进黑素细胞前体分化。我们进一步研究了BMP-4对体外培养的野生型小鼠NC细胞的影响。与未经处理的对照组相比，在培养液中添加BMP-4可增加Kit阳性细胞的数量。在野生型NC外植体中加入过量的BMP-4会抑制NC细胞的生长。因此，我们已经确定BMP-4是青春期前Kit阴性的黑素母细胞在进入Kit依赖的黑素合成周期之前的一个分化因子。

Genes encoding Kit and Kit ligand (Kit1) play essential roles in the differentiating melanoblasts. We previously established three immortal but distinct cell populations of mouse neural crest (NC) cells. NCCmelb4M5 cells do not express Kit and grow independently of Kit1 ; they have the potential to differentiate into NCCmelb4 cells, which are Kit-positive melanocyte precursors. NCCmelan5 cells show the characteristics of differentiated melanocytes. We investigated the effects of BMP-4 on the differentiation of immature melanocyte precursors. Three cell lines showed BMP-receptor expression. BMP-4 up-regulated Kit protein and mRNA expression in most immature NCCmelb4M5 cells. Noggin, a BMP-4 antagonist, dramatically decreased the Kit expression induced by BMP-4. Western blot analysis revealed that extrinsic BMP-4 leads to the phosphorylation of Smads and activates signaling in NCCmelb4M5 cells. We conclude that in NCCmelb4M5 cells, BMP-4 promotes melanocyte precursor differentiation via Kit expression. We further investigated the influence of BMP-4 in vitro using primary NC cells cultured from wild-type mice. Addition of BMP-4 to the medium increased the number of Kit-positive cells compared to untreated controls. An excess of BMP-4 added to wild-type NC explants abolished the growth of NC cells. Thus, we have identified BMP-4 as a differentiation factor for prepubertal Kit-negative melanoblasts just before they enter the Kit-dependent cycle of melanogenesis.