Critical roles for WDR72 in calcium transport and matrix protein removal during enamel maturation.

Defects in WDR72 (WD repeat-containing protein 72) cause autosomal recessive hypomaturation amelogenesis imperfecta. We generated and characterized Wdr72-knockout/lacZ-knockin mice to investigate the role of WDR72 in enamel formation. In all analyses, enamel formed by Wdr72 heterozygous mice was indistinguishable from wild-type enamel. Without WDR72, enamel mineral density increased early during the maturation stage but soon arrested. The null enamel layer was only a tenth as hard as wild-type enamel and underwent rapid attrition following eruption. Despite the failure to further mineralize enamel deposited during the secretory stage, ectopic mineral formed on the enamel surface and penetrated into the overlying soft tissue. While the proteins in the enamel matrix were successfully degraded, the digestion products remained inside the enamel. Interactome analysis of WDR72 protein revealed potential interactions with clathrin-associated proteins and involvement in ameloblastic endocytosis. The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts. Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane. Fewer blood vessels were observed in the papillary layer supporting ameloblasts. Specific WDR72 expression by maturation stage ameloblasts explained the observation that enamel thickness and rod decussation (established during the secretory stage) are normal in the Wdr72 null mice. We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

FAM20C functions intracellularly within both ameloblasts and odontoblasts in vivo.

FAM20C, also known as Golgi casein kinase (G-CK), is proposed to be the archetype for a family of secreted kinases that phosphorylate target proteins in the Golgi and in extracellular matrices, but FAM20C serving an extracellular function is controversial. FAM20C phosphorylates secretory calcium-binding phosphoproteins (SCPPs), which are associated with the evolution of biomineralization in vertebrates. Current models of biomineralization assume SCPP proteins are secreted as phosphoproteins and their phosphates are essential for protein conformation and function. It would be a radical departure from current theories if proteins in mineralizing matrices were dephosphorylated as part of the mineralization mechanism and rephosphorylated in the extracellular milieu by FAM20C using ATP. To see if such mechanisms are possible in the formation of dental enamel, we tested the hypothesis that FAM20C is secreted by ameloblasts and accumulates in the enamel extracellular matrix during tooth development. FAM20C localization was determined by immunohistochemistry in day 5 mouse incisors and molars and by Western blot analyses of proteins extracted from pig enamel organ epithelia (EOE) and enamel shavings. FAM20C localized intracellularly within ameloblasts and odontoblasts in a pattern consistent with Golgi localization. Western blots detected FAM20C in the EOE extracts but not in the enamel matrix. We conclude that FAM20C is not a constituent of the enamel extracellular matrix and functions intracellularly within ameloblasts.