Dentin sialoprotein promotes endothelial differentiation of dental pulp stem cells through DSPaa34–50–endoglin–AKT1 axis

Dentin sialoprotein (DSP), a major dentin extracellular matrix noncollagenous protein, is well recognized as an important regulator for dentinogenesis. DSP as a secreted protein can interact with membrane receptors, activate intracellular signaling, and initiate the odontoblastic differentiation of dental papilla cells. In a recent study, we have demonstrated that DSP can induce the endothelial differentiation of dental pulp stem cells (DPSCs), a type of tooth pulp-derived multipotent stem cells, dependent on membrane receptor endoglin (ENG). However, the intimate mechanisms by which DSP-ENG association facilitates the endothelial differentiation of DPSCs remain enigmatic. Here, we find that the amino acid (aa) residues 34-50 of DSP (DSP_aa34-50) is responsible for its association with ENG using a series of co-immunoprecipitation assays. Immunofluorescent staining and in situ proximity ligation assay demonstrate that overexpressed ENG in human embryonic kidney 293T cells shows codistribution and proximity ligation assay signals to the supplemented DSP_aa34-50 protein but not to DSP without aa34-50 (DSP_Δ34-50) on cell surfaces. Moreover, the zona pellucida domain of ENG mediates its association with DSP_aa34-50. Further experiments indicate that DSP_aa34-50 exhibits equivalent effects to the full-length DSP on the migration and endothelial differentiation of DPSCs dependent on ENG but DSP_Δ34-50 does not. Mechanistically, DSP_aa34-50 activates AKT1 and triggers the expression of blood vessel development-related genes in DPSCs. Multiple experiments demonstrate that AKT1 inhibition suppresses the DSP_aa34-50-induced migration and endothelial differentiation of DPSCs. Thus, AKT1 mediates the cellular and molecular functions of DSP_aa34-50-ENG association. Collectively, these findings identify that DSP promotes the endothelial differentiation of DPSCs through the DSP_aa34-50-ENG-AKT1 signaling axis.