Objective: Amelogenins are known as a major constituent of the enamel matrix secreted by ameloblasts and play an important role in enamel formation. Amelogenin knockout mice exhibit enhanced osteoblast formation and resorption of tooth cementum. Recent studies have revealed that amelogenins also have cell signaling properties. However, the biological functions of amelogenin in osteoblasts remain unclear. In this study, we examined the effects of recombinant human full-length amelogenin (rh174) on the proliferation of cultured normal human osteoblasts (NHOst).
Methods: NHOst cells were cultured and treated with 100 ng/ml rh174. Cell proliferation was evaluated using MTS assay in a time-dependent manner. Expression of Lysosomal-associated membrane protein 1 (LAMP 1), a possible amelogenin receptor, in NHOst cells was analyzed. NHOst cells were cultured and treated with 100 ng/ml rh174 in the presence or absence of LAMP 1-blocking antibody. Cell proliferative activity was analyzed by BrdU assay. Phosphorylation of extracellular signal regulated kinases (ERK) 1/2 was measured by ELISA and western blotting analysis.
Results: Proliferation of NHOst cells was enhanced significantly (p<0.01) by treatment with rh174, and was inhibited significantly (p<0.01) by addition of anti-LAMP 1-blocking antibody. In addition, the ratio of phosphorylated ERK1/2 to total ERK1/2 was significantly larger (p<0.01) with rh174 treatment, and was reduced significantly by the addition of anti-LAMP 1-blocking antibody in NHOst cells.
Conclusion: These results confirmed that rh174 interacts with LAMP 1, and rh174/LAMP 1 interaction activates the ERK1/2 signaling pathway, enhancing the proliferation activity of NHOst cells.Author(s): Ryo Kunimatsu, Yuki Yoshimi, Tetsuya Awada, Kotaro Tanimoto