Phosphate and Enamel Formation

PROJECT SUMMARY/ABSTRACT Dental enamel is the most highly mineralized tissue in mammals and has no self-repair mechanism if damaged. The major components to enamel are calcium (Ca2+), inorganic phosphate (PO43-; Pi) and hydroxide (OH-). While several mammalian Pi cellular import channels are known, little is understood of Pi transcellular and intracellular movements, and even less is known as to Pi cellular export as it relates to bone and teeth mineralization. In enamel development two distinct formative stages are recognized; an extracellular protein matrix-rich “secretory-stage”, immediately followed by the rapidly mineralizing “maturation-stage”. Preliminary data indicate PiT1 is expressed in secretory-stage amelogenesis. Published data show that the Pi importer NaPi2b, and the Pi exporter Xpr1 are both expressed during maturation-stage, suggesting that these proteins contribute significantly to enamel mineralization. This leads to the hypothesis of this grant application that “active transcellular and intracellular Pi movements dictate enamel mineralization processes”. If this can be shown, a major paradigm shift in our basic understanding of enamel formation would transpire, including the genetic controls involved in enamel biomineralization. To study the process of Pi movements in amelogenesis we have access to five unique mouse models: K14-Cre, Odam-IRESCre, floxed Slc20a1/PiT1 (f PiT1), floxed Slc34a2/NaPi2b (f NaPi2b) and floxed Xpr1 (f Xpr1) mice. In five Specific Aims we will: SA1) perform in situ hybridization (ISH), RT-PCR, and immuno-fluorescence (IF) to study Cre-recombinase expression in Odam- IRESCre heterozygous and homozygous mice; SA2) examine the contributions of PiT1 on secretory-stage enamel mineralization; 3) examine the contributions of NaPi2b (Slc34a2) and Xpr1 (Slc53a1/Xpr1) in maturation-stage amelogenesis by breeding Odam-IRESCre mice with respective floxed mutant mice strains; SA4) use transmitted polarized light microscopy, reflected metallography, SEM, EDS, fracture toughness and microhardness testing, performed on PN8 week fully mature mandibular incisor enamel, to study the architecture/organization and mechanical properties of mutant teeth and in these same samples define the atomic composition of the mineral composition; and SA5) ameloblast-like cell (ALC) culture will be examined to study the dynamics of mineralization using siRNAs silencing of Slc20a1, Slc34a2 and Slc53a1/Xpr1 coupled with Alizarin Red and von Kossa staining, and alkaline phosphatase activity, and also coupled with a thorough total in silico RNA-seq analysis. In this study the clear focus is on the roles of PiT1, NaPi2b and Xpr1 in enamel formation. With a recently developed enamel-specific Odam Cre-recombinase mouse line (Odam-IRESCre) we are now in a unique position to study Pi-related activities in amelogenesis. The roles of Pi transporters in teeth have had little prior attention, but for all dental researchers and practicing dentists, controls of Pi movements in enamel Hap formation must be understood to better define amelogenesis.