Nephronectin promotes ameloblast differentiation through its RGD motif

Nephronectin (Npnt) is a multifunctional basement membrane protein containing EGF-like repeats and an RGD motif. While we previously reported that Npnt regulates dental epithelial stem cell proliferation via its EGF-like repeats, the specific role of its RGD motif in ameloblast differentiation remained to be elucidated. In this study, we investigated the domain-specific functions of Npnt and identified its functional receptors during tooth development. Using the dental epithelial cell line M3H1, we demonstrated that overexpression of full-length Npnt and an EGF-deleted mutant significantly induced the expression of the ameloblast differentiation marker ameloblastin (Ambn), whereas an RGD-deleted mutant failed to induce differentiation. Competitive inhibition assays using RGD peptides further confirmed that Npnt-mediated differentiation and cell adhesion are RGD-dependent. Analysis of single-cell RNA sequencing data from mouse incisors revealed that Integrin αV and β6 are highly enriched in differentiating ameloblasts. siRNA-mediated knockdown experiments confirmed that Integrin β6 is essential for Npnt-induced Ambn expression. Furthermore, we found that EGF signaling negatively regulates Npnt-mediated differentiation, suggesting a mechanism to prevent premature maturation. These findings suggest that Npnt orchestrates a functional switch in tooth development: it promotes ameloblast differentiation via the integrin αVß6 pathway through its RGD motif, a mechanism distinct from its EGF-mediated regulation of stemness.