Amelogenin inhibits hard tissue resorption by suppressing RANKL expression

Objectives: Amelogenin, a major enamel protein, plays an important role in enamel formation and mutation in this gene has been reported in the genetic disorder, amelogenesis imperecta. Interestingly, the remarkable resorption of cementum and root dentin was reported in the target disruption of amelogenin gene in mice, suggesting this molecule as an inhibiting factor of hard tissue resorption. To clarify this mechanism, differentiation of cultured osteoclasts and the expression of receptor activator of nuclear factor-κB ligand (RANKL) were examined in this study. Materials and Methods: Two culture systems were performed to generate osteoclastic cell (TRAP-positive multinucleated cell; TRAP(+)MNC). In the first system, the mouse bone marrow cells were cultured in the presence of human M-CSF with prostaglandin (PGE₂) or PTH or 1α25(OH)₂Vit.D₃ . In the second system, mouse bone marrow macrophages were cultured in the presence of human M-CSF and soluble RANKL. In both systems, porcine amelogenin was exogenously added to the culture and the effect on osteoclast formation was examined. Western hybridization of RANKL was performed using the cell lysate of cultured bone marrow cells in the presence of human M-CSF and PGE₂ for 72 hrs. Results: The addition of amelogenin significantly inhibited TRAP(+)MNC formation in the first culture system in which osteoclastogenesis is mediated by endogenous RANKL expression of bone marrow stromal cells. However, amelogenin did not inhibit TRAP(+)MNC formation in the second system in which osteoclastogenesis is induced by exogenous RANKL treatment. Amelogenin inhibited RANKL expression of bone marrow stromal cells in a dose dependent manner. Conclusion: All these results suggest that amelogenin inhibits hard tissue resorption and osteoclastogenesis via suppressing RANKL expression. Supported by a grant from the Minestry of Education, Culture, Sports, Science and Technology of Japan (16390604 and 16659570).