Cell biological analysis of extracellular-signal regulated kinase (ERK) in the differentiation of ameloblasts

我们观察了MEK-1的抑制剂PD98059诱导的成釉细胞的形态变化，以阐明ERK在成釉细胞分化过程中的作用。同时检测细胞外调节激酶(ERK)和MAP/ERK激酶(MEK)在釉质器官中的免疫定位。用釉器裂解液进行免疫印迹显示，釉器细胞表达ERK和MEK。PD98059作用后1h和3h，成釉细胞各期形态均无明显变化。6h时，过渡期成釉细胞形态明显异常。成釉细胞层外观不规则。这一层可见大量核固缩的凋亡细胞。然而，其他时期的成釉细胞没有表现出严重的异常。过渡期成釉细胞远端有强的ERK免疫反应。免疫电子显微镜显示ERK定位于靠近细胞黏附结构的胞浆中。ERK被认为在细胞的增殖、分化和凋亡中发挥重要作用。我们的结果提示ERK参与了过渡期成釉细胞的形态变化和细胞凋亡。我们还评估了其他转录调控分子，如Sp家族和核因子-B。这些分子的定位导致他们被认为与釉质发生过程密切相关。这些结果表明，几种信号转导途径可能调节成釉细胞的分化。

We investigated the morphological changes of ameloblasts induced by PD98059, an inhibitor of MEK-1, to clarify a role of ERK in the process of ameloblast differentiation. Immunolocalization of extracellular regulated kinase (ERK) and MAP/ERK kinase (MEK) in enamel organ was also examined. Western blotting using the lysate of enamel organ revealed that cells in enamel organ expressed ERK and MEK. At 1 and 3 h after PD98059 administration, there was no morphological change in ameloblasts at any stages. At 6 h, abnormal morphology of ameloblasts was evident at the transitional stage. The layer of ameloblasts showed irregular appearance. Numerous apoptotic cells with nuclear condensation were seen in this layer. However, ameloblasts at other stages did not exhibit severe abnormality. Intense immunoreactivity for ERK was detected in the distal region of ameloblasts at the transitional stage. Immunoelectron microscopy revealed that ERK localized in cytoplasm close to cell adhesion structures. ERK is thought to play an important role in cell proliferation, differentiation and apoptosis. Our results suggest that ERK is involved in morphological changes and apoptosis of ameloblasts at the transitional stage. We also assessed other transcription regulating molecules, such as Sp family and NF-□B. The localization of these molecules leads to the consideration that they are closely related with the process of amelogenesis. These results indicate that several signal transduction may regulate the differentiation of ameloblasts.