Amelogenins are enamel matrix proteins that play a crucial role in enamel formation. Recent studies have revealed that amelogenins also have cell signaling properties. Although amelogenins had been described as specific products of ameloblasts, recent research has demonstrated their expression in bone marrow stromal cells. In this study, we examined the effect of recombinant human full-length amelogenin (rh174) on the proliferation of human mesenchymal stem cells (MSCs) derived from bone marrow and characterized the associated changes in intracellular signaling pathways. MSCs were treated with rh174 ranging in dose from 0 to 1,000 ng/ml. Cell proliferative activity was analyzed by bromodeoxyuridine (BrdU) immunoassay. The expression of lysosomal-associated membrane protein 1 (LAMP1), a possible amelogenin receptor, in MSCs was analyzed. Anti-LAMP1 antibody was used to block the binding of rh174 to LAMP1. The MAPK-ERK pathway was examined by Cellular Activation of Signaling ELISA (CASE) kit and western blot analysis. A specific MAPK inhibitor, U0126, was used to block ERK activity. It was shown that rh174 increased the proliferation of MSCs and MAPK-ERK activity. The MSC proliferation and MAPK-ERK activity enhanced by rh174 were reduced by the addition of anti-LAMP1 antibody. Additionally, the increased proliferation of MSCs induced by rh174 was inhibited in the presence of U0126. In conclusion, it is demonstrated that rh174 increases the proliferation of MSCs by interaction with LAMP1 through the MAPK-ERK signaling pathway, indicating the possibility of MSC application to tissue regeneration in the orofacial region.

The present study shows that rh174 enhances mineralization accompanied by upregulation of mineralization markers in HCEMs, whereas it has no effect on that in HPDLs, suggesting different effects of amelogenin on PDL and cementum.

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Amelogenins are the major constituent of developing extracellular enamel matrix proteins and are understood to have an exclusively epithelial origin. Recent studies have demonstrated that amelogenins can be detected in other tissues, including bone marrow mesenchymal stem cells (MSCs), but the role of amelogenins in MSCs remains unclear. The purpose of this study was to examine the effect of recombinant human full-length amelogenin (rh174) on the osteogenic differentiation of cultured human MSCs. MSCs isolated from human bone marrow were cultured in osteoblastic differentiation medium with 0, 10 or 100 ng/ml rh174. The mRNA levels of bone markers were examined by real-time PCR analysis. Alkaline phosphatase (ALP) activity and calcium concentration were determined. Mineralization was evaluated by alizarin red staining. The mRNA levels of ALP, type I collagen, osteopontin and bone sialoprotein in the MSCs treated with rh174 became significantly higher than those in non-treated controls. Treatment of MSCs with rh174 also enhanced ALP activity and calcium concentration, resulting in enhanced mineralization, as denoted by high intensity of alizarin red staining. In conclusion, the present study showed that rh174 enhances the mineralization accompanied by the upregulation of bone markers in human bone marrow MSCs during osteogenic differentiation, suggesting a certain role of amelogenin in the modulation of osteogenic differentiation of MSCs.

The results show that rh174 interacts with CD63, and rh174/CD63 interaction activates the ERK1/2 signaling pathway, enhancing the proliferation activities of HCEM and HPDL cells.