SLIT3: a Novel Regulator of Odontogenic Differentiation through Akt/Wnt/β-catenin Signaling Pathway

Abstract

Abstract The odontogenic differentiation of Stem cells from apical papilla (SCAP) is regulated by many extracellular matrix proteins, which plays a crucial role in dentin formation and regeneration. Extracellular matrix protein SLIT3, a classical axon guidance molecule, can link bone resorption to formation as clastokine. However, there is little information about SLIT3 in odontogenesis. Therefore, our study is aimed to explore the effects and possible mechanism of SLIT3 on the proliferation and differentiation of SCAP. Through Immunohistochemical staining and re-analysis of single-cell RNA sequencing and microarray datasets, we found that SLIT3 was expressed in the dental papilla and odontoblast layer of the developing molar tooth of mice. Real time polymerase chain reaction (RT-PCR) and Western blot assays also revealed an increased expression of SLIT3 during the odontogenic differentiation of SCAP. Afterwards, SLIT3 siRNA was used to knockdown SLIT3 and recombinant human SLIT3 (rhSLIT3) protein was used to treat SCAP. Cell Counting Kit-8 assays (CCK8) assays showed SLIT3 promoted proliferation of SCAP. Alkaline phosphatase (ALP) staining and Alizarin red staining were decreased/increased accordingly. Odontogenic markers DMP-1 and DSPP were also down-regulated/up-regulated. In addition, p-Akt and p-GSK3β levels were increased in rhSLIT3-treated SCAP and the movement into cell nucleus of β-catenin was promoted. The effect of SLIT3 was canceled after treatment with the inhibitor of Akt/Wnt/β-catenin signaling pathway. Taken together, our data show that SLIT3 could promote the proliferation and odontogenic differentiation of SCAP by activating Akt/Wnt/β-catenin signaling pathway.