In project 1: My laboratory has uncovered the dentin ECM sialoprotein (DSP) plays an important role in tooth development and formation. DSP mutations in humans and mice cause dentin deficiencies; dentinogenesis imperfecta (DGI) and dentin dysplasia type II (DD-II), which are the most common dentin genetic disorders. Furthermore, we found that DSP acts as a ligand and binds to cellular membrane proteins (receptors), integrin beta 6, occludin (Ocln), CD105 (endologin), activating intracellular signaling transduction for dental cell differentiation and dentin formation. In this proposal, we will study how the DSP domain controls dental stem cell/progenitor cell differentiation and dentin formation in vitro and in vivo. Based on previous data and publications, it will allow us to conduct the proposed project.
In project 2: How does bone morphogenetic protein 2 (Bmp2) control tooth development and formation? We have generated Bmp2 conditional knock out (cKO) mice using Bmp2 floxed/floxed mice bred with OSX-Cre mice. Compared to the wild type mice, Bmp2 cKO mice showed abnormal morphology in tooth development and formation. To understand how Bmp2 effect of tooth formation, in particular, odontoblast cell differentiation and dentin formation, we generated immortalized wild type and Bmp2 KO dental mesenchymal papilla (DPC) cell lines. Using RNA-sequencing, we identified a thousand genes and non-coding RNAs that up- and down-regulated by Bmp2 in the Bmp2 DPC cells. It included Dspp, Dmp1, Dlx3, Osx, and others. Immunohistochemistry showed that expression of Dspp, Dmp1, Dlx3, and Osx was down-regulated in the Bmp2 cKO mice. The Bmp2 phosphorylates Dlx3, Osx, and GCN5 (acetylase) through protein kinases Erk and Akt and induced the three protein translocations into nuclei. Protein-protein interaction demonstrated that GCN5 binds to Osx and Osx interacts with Dlx3, respectively. Osx and Dlx3 bind to Dspp and Dmp1 promoters and up-regulate Dspp and Dmp1 gene transcriptions in mouse DPC cells. Also, GCN5 acetylates histone 3 at different domains and Osx as well as modifies chromatin structure, activating Dspp and Dmp1 gene expression. Furthermore, we are focusing on which domain (s) of Osx is acetylated by GCN5 and phosphorylated by Erk and Akt kinases induced by Bmp2.
In project 3: We study how matrix metalloproteinase 9 (MMP9) controls craniofacial bone metabolisms with aging. We found that MMP-9 is involved in tooth and bone development. MMP-9 is capable of cleaving Dsp and amelogenin into the given fragments. X-ray, Micro-CT, electro-microscope, and histo-chemistry showed that MMP-9 knock out (MMP KO) mice are associated with dentinogenesis imperfecta (DGI) and amelogenesis imperfecta (AI) as well as develop periodontitis with aging. In the next step, we investigate which mechanisms of MMP-9 in tooth/bone formation and bone absorption with aging.