Screening for New Regulatory Factors in Ameloblast Differentiation

Objectives: Differentiated ameloblasts secret enamel matrix proteins like amelogenin, ameloblastin and enamelin and the expression of these proteins are regulated by various factors. In this study, we aimed to find a new regulatory protein that relates to ameloblast differentiation and enamel biomineralization.
Methods: Mouse ameloblast lineage cells (mALC) were cultured with or without mineralizing medium, including 50 μg/mL ascorbic acid and 5 mM β-glycerophosphate, for 8 days and their protein lysates were prepared. 2D-PAGE and peptide mass fingerprinting analyses were performed to search candidate proteins. Real-time PCR, histology and gain- or loss-of function experiments were performed to examine functions of the protein.
Results: Ten candidate proteins were identified by 2D-PAGE. Of them, family with sequence similarity 50, member A (Fam50a) was significantly increased during differentiation of mALC and also Fam50a protein was highly expressed in secretory ameloblasts of mouse tooth germs. In mALC cultures, forced expression of Fam50a up-regulated the expression of the enamel matrix protein genes with alkaline phosphatase activity and mineralized nodule formation. In contrast, knockdown of Fam50a decreased the expression of enamel matrix protein genes, ALP activity and mineralized nodule formation.
Conclusions: These results suggest that Fam50a is a new positive regulator of ameloblast differentiation.