Protein behavior of the ligand of Numb-protein X 2 (LNX2) in mouse ameloblasts at the maturation stage

OBJECTIVES: Enamel formation comprises two main stages: matrix formation (secretory), during which ameloblasts secrete an immature enamel matrix; and maturation, during which the matrix is degraded and remodeled. During maturation, the position of the cellular tight junctions change between the ruffle- and smooth-end phases; however, the regulatory mechanisms underlying this morphological alteration remain unclear. We focused on the Ligand of Numb-protein X (LNX): We would like to delete " family of proteins" and add "PDZ-domain containig" to define the LNXs have ability to bind tight junctional proteins, which have been identified as PDZ-domain containing E3 ubiquitin ligases, and examined the changes in their expression and localization in ameloblasts. METHODS: Enamel organs were isolated from 5- and 13-day-old mouse molars, and rat incisors were sectioned at the secretory and maturation stages to evaluate mRNA expression. Double-immunofluorescence staining was performed using antibodies against LNX2 and other LNX2-related proteins in paraformaldehyde-fixed, decalcified mouse incisors. RESULTS: Among the two LNXs, LNX1 and LNX2, LNX2 had higher mRNA expression levels at the secretory and maturation stages, in the molars and in the incisors. Double immunofluorescence staining of the LNX-associated protein, Numb, an interaction partner of LNX2, was performed. LNX2 staining in ruffle-end ameloblasts during maturation was observed in two forms: (1) aggregated intracellular vesicles co-localized with Numb and (2) diffuse cytoplasmic labeling not co-localized with Numb. The lysosomal marker, lysosome-associated membrane protein-1, and claudin-7, a tight-junction membrane protein that predominantly localizes to the basolateral membrane, were present in the proximal and distal cytoplasmic regions, where LNX2 was also detected. CONCLUSIONS: These results suggest that LNX2 contributes to the degradation of Numb and claudin-7 in ruffle-end ameloblasts during maturation.