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

本研究通过观察MEK-1抑制剂PD 98059诱导成釉细胞分化的形态学变化，探讨ERK在成釉细胞分化过程中的作用。细胞外调节激酶（ERK）和MAP/ERK激酶（MEK）在成釉器中的免疫定位也进行了研究。Westernblotting检测发现，成釉器细胞表达ERK和MEK。在PD 98059给药后1和3 h，成釉细胞在任何阶段均无形态学变化。6 h时，成釉细胞形态异常，处于过渡期。成釉细胞层外观不规则。该层可见大量凋亡细胞核固缩。而其他阶段的成釉细胞未出现严重异常。ERK在过渡期成釉细胞的远端区域呈强阳性反应。免疫电镜显示ERK定位于细胞浆内，靠近细胞粘附结构。ERK被认为在细胞增殖、分化和凋亡中起重要作用。我们的结果提示ERK参与了成釉细胞在过渡期的形态学改变和凋亡。我们还评估了其他转录调节分子，如Sp家族和NF-□B。这些分子的定位使人们认为它们与釉质形成过程密切相关。这些结果表明，成釉细胞的分化可能受到多种信号转导的调控。

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.