Effect Of BMP2in Odontoblast Differentiation Through Dlx3-Osx-GCN5Signaling Pathways

Odotoblasts are original from dental papilla mesenchymal cells that have undergone acomplex process of cell lineages. During dentinogenesis,differentiation of dental papillamesenchymal cells into odontoblasts is regulated by many several growth factors,transcription factors,and others. Bone Morphogenetic protein2(BMP2) is necessary fordentin development. Dentin Sialophosphoprotein (DSPP) is an important odontoblastdifferentiation marker. Previous study show BMP2play vital role for odontoblastdifferentiation in vitro. However, the role of BMP2in dentin development in vivo, inparticular during postnatal dentin formation, has not been described. The specificmolecular mechanism of BMP2via its signaling transduction pathways regulating DSPPexpression remain elusive. In this study, we used BMP2conditional knockout mice asanimal model to assess the effect of BMP2in postnatal dentin formation. In vitro assay,an immortalized odontoblast like cell line was established and used for the sdudy of howBMP2modulate DSPP gene transcription in molecular level. The main results we got listbelow:1. BMP2conditional knockout causes dentinogenesis imperfecta in postnatal miceIn this study, the role of Bmp2in the regulation of postnatal dentin formation wasinvestigated via the conditional ablation of Bmp2in dentin using the (Osx-Cre) mouse.Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric andopen forked incisors. Microradiographs revealed broken incisor tips and enlarged dentalpulp chamber with a failure to form proper roots.The dentin layer of incisors and molarswas thin with hypomineralization. Scanning electron microscopy analysis showed thatthe dentin surface was rough and the number of open dentinal tubules was decreased dramaticlly. Most of the dentinal tubules display a disordered arrangement. These resultsdemonstrate that Bmp2is essential for dentin formation.2. Immortalized mouse dental papilla mesenchymal cells preserve odontoblasticphenotype and respond to bone morphogenetic protein2.In this study, immortalized dental papilla mesenchymal cell lines were generated fromthe first mouse mandibular molars at postnatal day3using pSV40. These transformedcells were characterized by RT-PCR, immunohistochemistry, and analyzed for alkalinephosphatase activity and mineralization nodule formation. One of these immortalized celllines, iMDP-3, displayed a high proliferation rate, but retained the genotypic andphenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralizednodules. Furthermore, iMDP-3cells had high transfection efficiency as well as wereinducible and responded to BMP2stimulation. We conclude that the establishment of thestable murine dental papilla mesenchyma cell line might be used for studying themechanisms of dental cell differentiation and dentin formation.3. BMP2mediates DSPP expression through Dlx3-Osx-GCN5Signaling pathwaysIn this study,we report that BMP2induce Dlx3,Osx and GCN5phosphorylation thennuclear translocation via up-regulate Erk and Akt kinase activity. By using antibody andoligonucleotide competition assays in EMSA and ChIP experiments, we show that bothDlx3and Osx physically interacts with the binding sites within the DSPP promoter.Furthermore, forced overexpression of Dlx3and Osx enhances promoter activity iniMDP-3cells. Co-immunoprecipitation verify Osx interact with Dlx3and GCN5respectively. Re-ChIP assay indicates GCN5interaction with RNA Polymerase II bindingto DSPP promoter. Western blot and ChIP experiments reveal GCN5acetylates Histone3for remodeling chromatin. Taken together, this study indicates BMP2regulate Dspp geneexpression and odotoblast differentiation through Dlx3-Osx-GCN5signaling pathways.