Xu Ximin, Fu Jing, Yang Guobin, Chen Zhi, Chen Shuo, Yuan Guohua
State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China; Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan, Hubei, China.
State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei, China.
J Biol Chem. 2025 Apr;301(4):108380. doi: 10.1016/j.jbc.2025.108380. Epub 2025 Mar 4.
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) 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 protein but not to DSP without aa34-50 (DSP) on cell surfaces. Moreover, the zona pellucida domain of ENG mediates its association with DSP. Further experiments indicate that DSP exhibits equivalent effects to the full-length DSP on the migration and endothelial differentiation of DPSCs dependent on ENG but DSP does not. Mechanistically, DSP activates AKT1 and triggers the expression of blood vessel development-related genes in DPSCs. Multiple experiments demonstrate that AKT1 inhibition suppresses the DSP-induced migration and endothelial differentiation of DPSCs. Thus, AKT1 mediates the cellular and molecular functions of DSP-ENG association. Collectively, these findings identify that DSP promotes the endothelial differentiation of DPSCs through the DSP-ENG-AKT1 signaling axis.
牙本质涎蛋白（DSP）是牙本质细胞外基质中的一种主要非胶原蛋白，被公认为是牙本质形成的重要调节因子。作为一种分泌蛋白，DSP可与膜受体相互作用，激活细胞内信号传导，并启动牙乳头细胞的成牙本质细胞分化。在最近的一项研究中，我们已经证明，DSP可以诱导牙髓干细胞（DPSCs）的内皮分化，DPSCs是一种牙髓来源的多能干细胞，其依赖于膜受体内皮糖蛋白（ENG）。然而，DSP-ENG相互作用促进DPSCs内皮分化的具体机制仍然不明。在这里，我们通过一系列免疫共沉淀实验发现，DSP的34-50位氨基酸残基负责其与ENG的结合。免疫荧光染色和原位邻近连接分析表明，在人胚肾293T细胞中过表达的ENG与补充的DSP蛋白在细胞表面共分布且有邻近连接信号，但与没有34-50位氨基酸的DSP（DSP）没有。此外，ENG的透明带结构域介导其与DSP的结合。进一步的实验表明，DSP对依赖ENG的DPSCs的迁移和内皮分化表现出与全长DSP等效的作用，但DSP则没有。机制上，DSP激活AKT1并触发DPSCs中血管发育相关基因的表达。多项实验表明，抑制AKT1可抑制DSP诱导的DPSCs迁移和内皮分化。因此,AKT1介导了DSP-ENG相互作用的细胞和分子功能。总的来说，这些发现表明DSP通过DSP-ENG-AKT1信号轴促进DPSCs的内皮分化。
