Investigating WDR72's intracellular function in enamel mineralization

Objectives: Our overall aim is to determine the function of WDR72 in enamel mineralization. We previously characterized a Wdr72 knockout mouse model and linked a human WDR72 mutation to a hypomaturation type of Amelogenesis Imperfecta, both showing decreases in mineralized enamel. Ours and other published works have shown that WDR72 is needed for protein removal from the enamel matrix in order for enamel crystals to mature (fully mineralize). Wdr72-/- mice have retained matrix proteins, largely amelogenins, which suggests that amelogenins are (1) not being hydrolyzed to make room for the growing enamel crystals, or (2) not being reabsorbed by the ameloblasts. In this study, we therefore tested whether activity of KLK4, an essential protease secreted during enamel maturation, was decreased in the absence of WDR72. In addition, we tested whether the WDR72 deletion affects endocytosis, a major event occurring during this stage of enamel formation.
Methods: Ameloblasts from Wdr72^+/+, Wdr72^+/- and Wdr72^-/- mouse incisors were microdissected from the enamel surface and used to compare relative amounts of KLK4 mRNA. The underlying enamel was assayed for KLK4 protease activity using a quenched peptide assay. To test for an endocytosis-related function, mice were injected with HRP, a tracer molecule for constitutive endocytosis, and relative HRP immunofluorescence was compared in Wdr72^+/+, Wdr72^-/- ameloblasts.
Results: KLK4 mRNA expression and proteinase activity were not significantly different among Wdr72^+/+, Wdr72^+/- and Wdr72^-/- mice. HRP uptake appeared to be reduced at the apical ends of Wdr72^-/- maturation-stage ameloblasts with no obvious effect in secretory ameloblasts.
Conclusions: The retention of enamel matrix proteins in maturation stage Wdr72^-/- mice is not due to changes in KLK4 activity. However, the reduced HRP uptake in Wdr72 ^-/- maturation-stage ameloblasts suggests either a direct function of WDR72 in endocytosis, or an indirect effect secondary to matrix retention.