BMP-4 upregulates Kit expression in mouse melanoblasts prior to the Kit-dependent cycle of melanogenesis

Genes encoding Kit and the Kit ligand (KL) play essential roles in the differentiation of 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 KL; they have the potential to differentiate into NCCmelb4 cells, which are Kit-positive melanocyte precursors. NCCmelan5 cells show the characteristics of differentiated melanocytes. All three cell lines demonstrated bone morphogenetic protein (BMP) receptor expression. BMP-4 upregulated 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 in NCCmelb4M5 cells. Using transfected Kit-promoter reporter, we showed BMP-4 could activate Kit promoter in transfected NCCmelb4M5 cells. We conclude that BMP-4 is active and is involved in the regulation of Kit expression on most immature melanocyte precursors. 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 diluent-treated controls. We have identified BMP-4 as an important factor for prenatal Kit-negative melanoblasts just prior to entering the Kit-dependent cycle of melanogenesis.