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

Abstract

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.

Store-operated Ca2+ entry controls ameloblast cell function and enamel development

“…Therefore, the cellular infoldings of the ruffled ameloblasts are critical for NCKX4 to carry out its main function. These data are consistent with the phenotype reported in a mouse model with a mutation in WD repeat-containing protein 72 (Wdr72), in which NCKX4 also adopts an intracellular localization similar to what we have described here and the cyclic modulation from ruffled to smooth ameloblasts was abnormal (31).…”

Maturation stage enamel malformations in Amtn and Klk4 null mice

“…This pattern suggests that the endocytosis mechanism, in the absence of KLK4, is still able to reabsorb much of the protein near the enamel surface, but the protein away from the surface is unable to work its way back to the cell for reabsorption. Interestingly, a pathological accumulation of enamel proteins also occurs in Wdr72 −/− mice, whose enamel shows more severe reductions in mineral density and hardness than those observed in Klk4 −/− mice [45,46]. WDR72 is an intracellular protein and defects in WDR72 contribute to the etiology of autosomal recessive amelogenesis imperfecta [47,48].…”

“…WDR72 is an intracellular protein and defects in WDR72 contribute to the etiology of autosomal recessive amelogenesis imperfecta [47,48]. Identification of proteins that co-immunoprecipitated with WDR72 suggested that WDR72 is involved in endocytosis or vesicle trafficking [45,46]. These findings, and the discovery that Klk4 is a recent (evolutionary) addition to the process of enamel maturation, suggest that endocytosis is the primary mechanism for the removal of enamel matrix proteins and that secretion of the KLK4 enzyme is a later refinement that facilitates that process by degrading extracellular proteins and thereby allowing proteins in the deeper layers to more rapidly diffuse to the enamel surface and be reabsorbed by ameloblasts.…”

Fam83h null mice support a neomorphic mechanism for human ADHCAI

“…2015). Briefly, D14 first molars were incubated in 1 mL of 0.17 N HCl/0.95% formic acid for 2 h at 4°C.…”

“…2015). In brief, hemimandibles from 7‐week‐old mice were cleaned free of soft tissue and embedded in Epon resin following graded acetone dehydration.…”