Molecular Controls of Ameloblast Differentiation

Stage-specific expression of ameloblast-specific genes is controlled by differential expression of transcription factors, adhesion molecules and growth factors. In addition to stage-specific controls, ameloblasts are subject to daily controls of their main activities i.e. enamel protein secretion and later on enamel mineralization. We hypothesize that these time related controls are orchestrated by daily oscillations of clock proteins involved in circadian rhythms regulation. Objective: To identify the potential links between daily rhythms and molecular controls of ameloblast differentiation. Methods: The effects of key transcription factors (Dlx3 and Runx2) and clock genes (Rev-erbα) on ameloblasts (using differentiation markers amelogenin, enamelin and klk4) were evaluated in HAT-7 ameloblast cell line using computer based promoter analysis, transient transfection, real time quantitative RT-PCR, western-blots, promoter reporter assays and chromatin immunoprecipitation. Results: Enamelin and amelogenin steady-state RNA expression levels were down-regulated in Runx2 over-expressing cells and up-regulated in Dlx3 over-expressing cells. In contrast, Klk4 was up-regulated by Runx2 and Dlx3. Over-expression of Rev-erbα resulted in up-regulation of ameloblast markers (up-regulation of amelogenin and down regulation of Klk4 and Mmp20) but also influenced the expression levels of Runx2. Conclusion: Our study provides novel insights into the regulatory mechanisms controlling ameloblast differentiation and dental enamel formation.