SLIT3: a novel regulator of odontogenic differentiation through Akt/GSK3β/β-catenin signaling pathway

The odontogenic differentiation of Stem Cells from Apical Papilla (SCAP) are governed by various extracellular matrix proteins, playing a crucial role in dentin formation and regeneration. Extracellular matrix protein SLIT3, a classical axon guidance molecule, has been identified as a clastokine linking bone resorption to formation. However, its role in odontogenesis is not well-documented. Thus, our study aimed to explore the effects and mechanisms of SLIT3 on SCAP proliferation and differentiation. Analysis of developing mouse molars showed that while Slit3 mRNA was restricted to the dental mesenchyme, the SLIT3 protein was prominently detected on both odontoblasts and adjacent epithelial ameloblasts. Real time polymerase chain reaction (RT-PCR) and Western blot assays confirmed increased SLIT3 expression during SCAP odontogenic differentiation. SLIT3 siRNA knockdown and recombinant human SLIT3 (rhSLIT3) protein treatments were administered to SCAP. Cell Counting Kit-8 (CCK8) assays indicated that SLIT3 promotes SCAP proliferation, while alkaline phosphatase (ALP) and Alizarin red staining showed increased mineralization. Odontogenic markers DMP-1 and DSPP were also modulated accordingly. Additionally, rhSLIT3 treatment enhanced p-Akt and p-GSK3β levels in SCAP, promoting β-catenin nuclear translocation. The effects of SLIT3 were negated with an Akt/GSK3β/β-catenin signaling pathway inhibitor. Collectively, our data suggest that SLIT3 promotes SCAP proliferation and odontogenic differentiation via the Akt/GSK3β/β-catenin signaling pathway activation.