Citrate regulation in amelogenesis - PROJECT SUMMARY Proper citrate regulation is critical for development of mineralized tissues, notably tooth enamel. Forming the outer layer of tooth crowns, enamel, when compromised, can lead to dental caries, weakened tooth structure, oral infections, and in severe cases, tooth loss. Ablation of Solute Carrier Family 13 Member 5 (SLC13A5), which transports citrate over other carboxylates from extracellular to intracellular spaces, results in severe tooth enamel defects. Despite its association with enamel defects, mechanisms of action for SLC13A5 in amelogenesis (enamel development) are largely unknown. Recently, progressive ankylosis protein (ANK), a regulator of pyrophosphate (PPi) and osteogenesis, has been proposed as a citrate regulator. PPi is an inhibitor of hydroxyapatite formation, and ANK loss results in decreased plasma PPi levels, ectopic joint calcifications, and tooth root hypercementosis. Additionally, Ank-/- mice exhibited reduced plasma and urine citrate levels. We generated an Ank and Slc13a5 dual ablation mouse model, and despite no reports of Ank expression in ameloblasts and no detected enamel defects in Ank-/- mice, we found that enamel defects present in Slc13a5-/- mice were markedly reduced in Ank-/-;Slc13a5-/- mice. Accordingly, ANK and SLC13A5 are hypothesized to modulate amelogenesis through common citrate regulatory pathways. Aims were developed to investigate local genetic and systemic contributions from ANK and SLC13A5 in amelogenesis. Histological studies will be conducted on WT mice, Slc13a5-/- mice, Slc13a5 conditional knockout (cKO) mice targeted to ameloblasts, Ank-/- mice, and Ank-/-,Slc13a5-/- mice. Mandibular first molar ameloblasts will be analyzed secretory, mid-maturation, and late maturation stages. MicroCT analysis and scanning electron microscopy will be employed to assess enamel volume, density, microarchitecture and elemental composition. Enamel matrix proteins expressed in secretory and maturation stages will be assessed with immunohistochemistry. RNA-Seq will be employed on secretory and maturation ameloblasts to identify candidate genes affected by Slc13a5 loss. To determine whether systemic levels of citrate and other TCA metabolites are normalized with dual ablation of Ank and Slc13a5, TCA cycle targeted metabolomics on plasma and ameloblast samples is proposed. Profiles will be compared to identify correlations between cellular and systemic metabolites. This proposal provides a multifaceted approach to elucidate mechanisms by which SLC13A5 and ANK modulate citrate in amelogenesis. Results will help address knowledge gaps in citrate regulation in amelogenesis, aid in development of new approaches to study amelogenesis, and yield information that may be extrapolated to other mineralized tissues.
