AmeloD regulates cell proliferation and differentiation process of ameloblasts through modulation of the sonic hedgehog (shh) signaling pathway.

Tooth morphogenesis is controlled by various molecules, and the enamel knot plays a important role as a signaling center. Here, we aimed to analyze the role of AmeloD in the development of the enamel knot. The AmeloD-stably expressing dental epithelial cell line, SF2, was established and RNA sequencing analysis was performed. The cusp morphology of AmeloD-deficient mouse molars was analyzed by micro-computed tomography. AmeloD-binding molecules were screened using the yeast two-hybrid methods. AmeloD was expressed in the enamel knot during tooth development. Proliferation was suppressed in cells overexpressing AmeloD, which was consistent with the cessation of proliferation in the enamel knot. RNA sequencing analysis revealed that the expression of proliferation-related molecules, such as Mki67, Ccnd1, and Ccnd2, decreased in AmeloD-overexpressing cells. Shh was expressed in the enamel knot, and in AmeloD-overexpressing cells, Shh promoted the expression of ameloblastin and Sox21. Furthermore, AmeloD-deficient mice had reduced intercuspal distance, mesiodistal and buccolingual diameters, and enamel hypoplasia. Odam, Selenof, Kct2, and TCF4 were identified as AmeloD-binding molecules using the yeast two-hybrid methods. AmeloD and Odam were expressed in similar regions during the earlier stages of ameloblast differentiation. To clarify how Odam regulates AmeloD, the Odam gene was transfected into AmeloD-overexpressing cells. In Odam-expressing cells, the suppression of proliferation and migration by AmeloD was inhibited. AmeloD promotes cell cycle arrest and activation of Shh signaling in the enamel knot, and it binds to Odam in the early stages of ameloblast differentiation, inhibiting the proliferation activity of Odam through these interactions.