Function of Amelogenins in Periodontal Regeneration Induced by Enamel Matrix Derivative

Abstract

Amelogenins are a complex mixture of hydrophobic proteins that are the major organic components of developing enamel. The principal functions of the amelogenins and their degradation products are structural roles in creating the space and milieu for promoting enamel mineralization. Enamel matrix derivative (EMD) has been used clinically for periodontal regeneration, and its therapeutic effectiveness has been attributed to amelogenin, non-amelogenin enamel matrix proteins and growth factors. While EMD is believed to induce periodontal regeneration, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast and cementoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. EMD, recombinant porcine amelogenin (rAmelogenin) and purified porcine amelogenins (25-, 20-, 13- and 6-kDa amelogenins) increased BSP mRNA levels in ROS 17/2.8 osteoblast-like cells. In transient transfection analyses, EMD increased BSP promoter activities (luciferase activities) of pLUC4 (nts - 425 to + 60) and pLUC5 (nts - 801 to + 60) constructs. On the other hand, rAmelogenin and purified amelogenins increased pLUC3 (nts -116 to + 60), pLUC4 and pLUC5 activities, transfected into ROS 17/2.8 cells. Within the pLUC3, 4 and 5, a fibroblast growth factor 2 response element (FRE), a homeodomain protein binding site (HOX) and a TGF-β activation element (TAE) are present. Gel mobility shift analyses showed that purified amelogenins increased the binding of FRE, HOX and TAE. These results demonstrate that EMD, rAmelogenin and purified amelogenins stimulate BSP transcription in osteoblast-like cells by targeting FRE, HOX and TAE in the rat BSP gene promoter, and that EMD and amelogenins are able to regulate BSP transcription via different signaling pathways.