Augmentation of enamel mineralization by APin

Abstract

APin was previously suggested to have a functional role in the mineralization and maturation of enamel, which is mediated by MMP-20 and tuftelin expression. In the present study, the biological function of APin was examined during ameloblast differentiation and enamel mineralization in vitro. An APin recombinant protein was produced and stable APin transgenic cell lines were also established using ameloblast-lineage cells (ALCs). The cells were cultured for 2 weeks with or without the APin recombinant protein in a differentiation and mineralization medium. The alkaline phosphatase (ALP) activity was measured and the level of mineralization was determined by staining the calcified nodules with Alizarin red S stain. A high-density protein microarray was also performed to identify the other proteins interacting with APin. A subset of genes that reacted strongly with APin was selected. The expression of these genes was evaluated by RT-PCR after the inactivation or overexpression of APin. Mineralization was augmented by the ALCs treated with the APin recombinant protein and the sense APin overexpressing cells. The ALP activity was also increased markedly in the sense APin overexpressing cells and the ALCs treated with APin recombinant protein. The inactivation of APin in the ALCs down-regulated the expression of BMP receptor 1B (BMPR-1B) and the EF hand calcium binding protein 2 (CBP2), whereas its expression was up-regulated markedly when APin was overexpressed. However, APin did not affect the mineralization of osteoblasts and periodontal ligament cells (PDL cells). These results provide deeper insights into the process of ameloblast maturation and in enamel mineralization. It also suggested that APin augmented enamel mineralization.