Our research focuses on discovering and characterizing cellular processes that affect key components of craniofacial structures and their functions. Our efforts are concentrated on studying processes that regulate mineralization of teeth, trigger inflammation in teeth and salivary glands, promote tumor growth in the head-and-neck region, and cause painful conditions. We have used a variety of molecular approaches, such as conventional and conditional gene targeting coupled with genomic and proteomic analysis, to specifically investigate molecular roles of amelogenins and dentin sialophosphoprotein in tooth mineralization, TGF- signaling in inflammation of teeth and salivary glands. To pursue our research inquiries, which include some high-risk projects, we have extensively collaborated with many leading researchers in the intramural and extramural communities. Our present studies will not only contribute to a greater understanding of the molecular roles of the candidate genes in development and disease, but should also aid in future efforts to find more effective treatments for many disorders. Tooth Development and Disease Enamel Studies: Functional and enduring dentition is critical to the survival of many species and, not surprisingly, many of the genes involved in tooth formation are highly conserved throughout the animal kingdom. Amelogenin is one such gene that codes for multiple spliced variants of amelogenin in ameloblasts and is important for normal enamel development. Human amelogenin gene mutations result in amelogenesis imperfecta (AI), which is the most common hereditary condition affecting enamel and is characterized by thin and/or poorly mineralized enamel. Our earlier studies demonstrated that amelogenin null (Amel-/-) mice have an enamel phenotype that appears similar to that observed in human AI, confirming the important role of amelogenins in normal development of enamel. Subsequently, we demonstrated that 2 amelogenin isoforms, a full-length M180 amelogenin and a leucine-rich amelogenin peptide (LRAP), are expressed in cementum, and that their absence in aging Amel-/- mice is associated with cementum defects. We have also reported the functions of the amelogenin isoforms in osteoclastogenesis, and in the proliferation and migration of cementoblast/periodontal ligament (CM/PDL) cells. Our results provide strong evidence that LRAP inhibits osteoclastogenesis. In collaboration with Dr. Yoshi Yamada, we developed ameloblastin knockout mice that have severe enamel hypoplasia, detached ameloblasts, and odontogenic tumors of epithelial origin. In order to search for synergistic roles of amelogenin and ameloblastin in enamel development, we generated amelogenin and ameloblastin double-null (Amel-/-/Ambn-/-) mice. These mice showed more severe enamel defects than Amel-/- mice or Ambn-/- mice. SEM analysis showed that enamel structure was completely lost, and that the ameloblast layer was irregular and detached from the basement membrane in Amel-/-/Ambn-/- mice. Proteomic analysis revealed the presence of an increased level of Rho-GDI only in Amel-/-/Ambn-/- tooth buds, suggesting the synergistic effects of amelogenin and ameloblastin. We also initiated studies to analyze amelogenin functions in bone metabolism, by generating transgenic mice that express M180 and LRAP in bones. Our initial analysis revealed that amelogenins may play a role in the maintenance of bone metabolism. We have phased out this project and given all the mouse lines to Dr. Naoto Haruyama, who generated these mice in my laboratory and recently returned to Tokyo University School of Dentistry in Japan. In collaboration with extramural researchers, we also continued to study amelogenin mutations, further characterize the Amel-/- phenotype, and extend our focus to interactions between amelogenin and MMP20. As mentioned above, we have phased out all the amelogenin projects, but we will continue to collaboratively (A) delineate functions of amelogenin spliced variants found in humans and rodents, particularly for their roles in osteoclastogenesis and signaling, and (B) analyze LRAP function in osteoclastogenesis. Dentin Studies: Dentin sialophosphoprotein (DSPP) forms a major constituent of dentin extracellular matrix proteins, and is believed to play an important role in the mineralization process that forms mature dentin. Several mutations have been identified in the DSPP gene of patients with dentinogenesis imperfecta. DSPP is predominantly expressed in dentin-producing odontoblasts, and transiently expressed in enamel-producing ameloblasts. Low levels have also been detected in several other tissues like bone, inner ear, salivary glands, and kidneys. To gain insight into the molecular role of DSPP in dentinogenesis, we previously generated DSPP-/- mice. The structural tooth defects observed in these mice were enlarged pulp chambers, increased width of predentin zone, hypomineralization, pulp exposure, irregular mineralization front, and a lack of uniform coalescence of calcospherites in the dentin. The levels of the proteoglycans biglycan and decorin were increased in the widened predentin zone and in the void spaces among the calcospherites in the null dentin. These enhanced levels correlated well with the regions defective in mineralization, and further indicated that these molecules may adversely affect the dentin mineralization process by interfering with the coalescence of calcospherites. However, type I collagen levels remained unaffected in the null teeth. In order to understand the molecular mechanism underlying this phenotype, we first examined whether the elevated levels of biglycan and decorin were causative factors, or were elevated as a consequence of DSPP deficiency. Towards this goal, we have generated 2 mouse models: DSPP-/-;biglycan-/- and DSPP-/-;decorin-/-. Detailed analysis of these 2 mouse models indicates that the deficiency of decorin, but not biglycan, rescues the enlarged predentin phenotype of DSPP-/- mice. Increased levels of decorin in DSPP-/- predentin contribute to abnormal enlargement of predentin. The DSPP mRNA is translated into a single protein, DSPP, which is cleaved into 3 peptides: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We constructed transgenic vectors that express DSP/DGP under the control of the DSPP promoter and generated independent transgenic mouse lines that express DSP/DGP in odontoblasts and preameloblasts. Two of these lines were bred with DSPP-/- mice to establish 2 mouse lines that express DSP/DGP at low and high levels in the DSPP null background (DPPcKO). DPPcKO teeth show a partial rescue of the DSPP null phenotype with more normal predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars confirmed this partial rescue with significant recovery of the dentin volume, but with no improvement in the dentin mineral density. In addition, a dramatic decrease of chondroitin sulfate proteoglycan in dentin was observed in DSPP null mice that was largely restored in DPPcKO mice, which suggests that DSP is a major chondroitin sulfate chain proteoglycan in dentin. These results indicate the distinct roles of DSP and DPP in dentin mineralization, with DSP possibly regulating initiation of dentin mineralization, and DPP perhaps playing a role in the maturation of mineralized dentin.