Amelogenin Stimulates Bone Sialoprotein (BSP) Expression Through Fibroblast Growth Factor 2 Response Element and Transforming Growth Factor‐β1 Activation Element in the Promoter of the BSP Gene

Abstract

Amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. The principal function of the amelogenins and their degradation products has been assigned to 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. In this study, we examined the ability of amelogenin to regulate BSP gene transcription in osteoblast like cells. We conducted Northern hybridization, transient transfection analyses, and gel mobility shift assays using full-length recombinant amelogenin to determine the molecular basis of the transcriptional regulation of BSP gene by amelogenin. Recombinant amelogenin (1 microg/ml, 12 hours) increased BSP mRNA levels approximately 2.4-fold. In transient transfection analyses, amelogenin (1 microg/ml, 12 hours) increased luciferase activity approximately 1.5-fold in pLUC3 (nucleotides -116 to +60) and further increased pLUC5 (nucleotides -801 to +60) activity approximately 2.3-fold transfected into ROS 17/2.8 cells. Amelogenin also increased luciferase activities in rat stromal bone marrow cells. The effect of amelogenin was inhibited by the tyrosine kinase inhibitor herbimycin A. Transcriptional stimulation by amelogenin was almost completely abrogated in cells expressing a BSP promoter construct with a mutation in the fibroblast growth factor 2 (FGF2) response element (FRE). Gel mobility shift assays with radiolabeled FRE and transforming growth factor-beta1 (TGF-beta1) activation element (TAE) ds-oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells. Amelogenin stimulation alters BSP gene transcription by inducing nuclear proteins that bind to the FRE and TAE in the rat BSP gene promoter.