PROJECT SUMMARY/ABSTRACT Mineralized enamel is the hardest tissue in the human body, and its proper formation is critical for the protection and maintenance of healthy teeth for a lifetime. Enamel biomineralization is directed by the epithelial-derived ameloblasts. Ameloblasts secrete enamel matrix proteins first, which are then hydrolyzed and replaced by hydroxyapatite crystals in the maturation process. During the maturation process, the pH of the enamel matrix modulates periodically to be acidic in approximately 80% of the maturation stage, with the remaining approximately 20% of the time remaining neutral. Maturation stage ameloblasts also modulate periodically along with the matrix pH cycling, changing cell morphology and functions. When pH cycling is disrupted by gene mutants or environmental factors that dysregulate ameloblast modulation, enamel maturation is dysregulated, resulting in hypomineralized, poorly formed enamel. However, the mechanisms that drive both matrix pH cycling and ameloblast modulation interactions are poorly understood. In preliminary studies, we found amelogenins can differentially affect cell morphology and adhesion in a pH-dependent manner; and ameloblast modulation is associated with changes in focal adhesion-P13k-AKT signaling pathways. Furthermore, we identified possible contributions of matrix pH to the function of the pH-dependent multifunctional enzyme, transglutaminase 2, in regulating amelogenin hydrolysis in the maturation of enamel matrix. In these proposed studies we will address the central hypothesis that enamel matrix pH regulates ameloblast function, protein structure, and matrix mineralization. We will use both in vitro and in vivo models with alterations in endocytosis, calcium transport, matrix hydrolysis and pH, to test our central hypothesis with the following specific aims. 1) To determine the effect of extracellular pH on adhesion and modulation of maturation stage ameloblasts; 2) To determine the effect of pH and calcium on integrin-mediated focal adhesion - PI3K-AKT-mTOR signaling pathways of ameloblasts; 3) To determine the significance of matrix pH on Transglutaminase 2 activity in the maturation stage enamel matrix. These studies will allow us to better understand the etiology of enamel hypomineralization, and they will allow us to apply this knowledge to reduce the risk of enamel defects. Furthermore, the findings from these studies will contribute to our long-term goal to identify strategies for enamel tissue bioengineering, repair, and the use of enamel proteins therapeutic purposes.