NRP1裂解片段调控DDR2稳定性促进牙髓干细胞成牙本质向分化的研究

Directed differentiation of dental pulp stem cells (DPSCs) is controlled by posttranslational modification of membrane proteins. NRP1 is an important membrane protein involved in odontoblastic differentiation of DPSCs, but its regulation remains to be explored. Our previous study indicated that the expression of NRP1, glycosylated NRP1 and its cleaved fragments increased paralleling with that of DDR2 during early steps of odontoblastic differentiation of DPSCs. N-linked glycosylation inhibitors partially inhibited the expression of glycosylated NRP1, its cleaved fragments and DDR2 and inhibited differentiation. It has been reported that NRP1 promoted osteogenic differentiation of preosteoblasts by stabilizing DDR2. The hypothesis is proposed:membrane protein NRP1 is glycosylated to promote the proteolytic processing of its carboxyl terminal, and the cleaved fragments of NRP1 containing the intracellular region bind to the intracellular domain of DDR2 to stabilize DDR2. Finally, odontoblastic differentiation of DPSCs was enhanced by ERK-OSX signaling pathway. This project intends to clarify the role and regulation of NRP1 glycosylation and its cleavage fragments during odontoblastic differentiation of DPSCs, and to investigate the interaction mode of NRP1 cleavage fragments and DDR2. This research will study odontoblastic differentiation of DPSCs from the novel perspective of glycosylation and hydrolysis cleavage of membrane proteins, providing new ideas for exploring its biological mechanism.

牙髓干细胞（DPSCs）定向分化受膜蛋白翻译后修饰的调控，NRP1是参与DPSCs成牙本质向分化的重要膜蛋白，但其调控方式尚不明确。项目组前期发现，NRP1、其糖基化蛋白和裂解片段及膜蛋白DDR2在DPSCs成牙本质向分化前期表达增加；N-连接糖基化抑制剂部分抑制NRP1糖基化蛋白、裂解片段及DDR2的表达，且抑制分化。据报道NRP1通过稳定DDR2促前成骨细胞成骨向分化。据此本研究提出假说：DPSCs膜蛋白NRP1被糖基化后促其羧基端裂解，包含胞内段的裂解产物与DDR2胞内段结合后稳定DDR2，最终通过ERK-OSX信号通路促进DPSCs成牙本质向分化。本课题拟明确NRP1糖基化和其裂解片段在DPSCs成牙本质向分化中的作用和调控方式，并探讨NRP1裂解片段与DDR2的作用模式。本研究从膜蛋白糖基化和剪切水解机制这个新视点探讨DPSCs成牙本质向分化，为完善其生物学机制提供新思路。

牙髓干细胞（DPSCs）定向分化受膜蛋白翻译后修饰的调控，神经毡蛋白1（NRP1）是参与DPSCs 成牙本质向分化的重要膜蛋白，但其调控方式尚不明确。本研究发现NRP1的糖基化片段、裸蛋白和裂解片段在DPSCs成牙本质向分化前期表达增加，在此现象基础上探究了NRP1糖基化形式、NRP1裂解片段产生所依赖的蛋白酶和可能与NRP1发生相互作用的下游蛋白，主要结论如下：（1）NRP1在DPSCs成牙本质向分化中糖基化片段、裸蛋白和裂解片段表达增加。（2） NRP1的糖基化修饰类型包含N-连接糖基化修饰和硫酸软骨素（CS）修饰的蛋白聚糖形式。（3）NRP1裂解片段的产生由去整合素-金属蛋白酶10（ADAM10）介导。（4）NRP1和盘状结构域受体2（DDR2）可能在DPSC成牙本质向分化中发挥协同作用，但不是以直接结合的方式。（5）NRP1和JNK相互作用蛋白4（JIP4）可能以直接结合的方式在DPSC成牙本质向分化中发挥作用。.为进一步完善NRP1在干细胞多向分化中的作用，本项目进一步探讨了NRP1裸蛋白和糖基化片段在 C3H10T1/2细胞，即一种小鼠胚胎来源的间充质干细胞中成脂向、成骨向和成软骨向分化中的作用，主要结论如下：（1）NRP1在C3H10T1/2中成脂向、成骨向和成软骨向分化中裸蛋白和糖基化片段表达增加。（2）下调NRP1的蛋白表达，抑制C3H10T1/2的成脂向、成骨向和成软骨向分化。（3）C3H10T1/2的成脂向分化中，NRP1蛋白与JIP4蛋白相互作用。（4）NRP1的糖基化修饰抑制NRP1裸蛋白对C3H10T1/2的成脂向分化的促进作用。.本研究从NRP1糖基化和剪切水解这个新视点探讨DPSCs成牙本质向分化和间充质干细胞的多向分化作用机制，为完善膜蛋白在干细胞多向分化中的生物学机制提供新思路。