During tooth development, odontoblasts modulate dentin formation by producing a predentin matrix into which additional NCPs are subsequently secreted to initiate the mineralization process so necessary for healthy tooth formation. Abnormalities in dentin mineralization due to genetic diseases such as dentinogenesis imperfecta II (DGI-II; affecting 1 in 7,000 newborns) leave these children few choices other than placement of crowns on the teeth or a complete denture. A nonsense mutation, located in the coding sequence for dentin sialoprotein (DSP) is likely responsible for blocking both DSP and phosphophoryn (PP) expression in these individuals because these two NCPs are located on a single DSP-PP transcript. Importantly, some DGI patients also experience tinnitus as well as balancing problems making it necessary to consider problems in DSP-PP transcript and protein expression as an important public health concern. This application proposes a series of studies to better define DSP-PP transcript expression and DSP/PP protein function in dentin mineralization. The specific aims of this proposal are 1) to test the hypothesis that odontoblast-specific factors acting on the DSP-PP gene promoter, regulate DSP-PP expression, 2) to test the hypothesis that multiple DSP-PP transcripts generate functionally distinct PP isoforms with distinct calcium binding capacity and hydroxyapatite forming ability which are required for orderly dentin mineralization, 3) to correlate DSP-PP transcript and DSP-only transcript expression patterns with dentin mineralization, and 4) to validate Aims 2 and 3 by examining mineral formation as a function of DSP and PP isoform expression in cell culture. These ongoing studies, which combine gene regulation and protein function approaches, will reveal underlying mechanisms of dentin mineralization and will later be applicable to reparative dentistry.