Abstract：
Neuropilin-1 (NRP1) is a single transmembrane glycoprotein involved in a variety of physiological events. However, the exact mechanisms by which NRP1 regulates dental pulp stem cells (DPSCs) to differentiate toward an osteo/odontogenic phenotype are poorly understood. Here, we determined the significantly increased expression of full-length NRP1 and glycosaminoglycan (GAG)-modified NRP1 during osteo/odontogenesis in DPSCs. NRP1 was confirmed to promote alkaline phosphatase (ALP) activity, mineralized nodule deposition, protein and mRNA expression of Runx2, DSPP and DMP1 in DPSCs via the loss-of-function and gain-of-function approaches. Further, a non-GAG-modified NRP1 mutant (NRP1 S612A) was generated and the suppression of osteo/odontogenic differentiation was observed in the NRP1 S612A overexpression cells. Knockdown of the adaptor protein shroom3 resulted in the inhibition of osteo/odontogenesis. The protein-protein interaction network, the protein-protein docking and confocal analyses indicated the interactions between NRP1 and shroom3. Furthermore, immunoprecipitation followed by western analysis confirmed the binding of NRP1 to shroom3, but overexpression of NRP1 S612A greatly influenced the recruitment of shroom3 by NRP1. These results provide strong evidence that NRP1 is a critical regulator for osteo/odontogenesis through interacting with shroom3. Moreover, our results indicate that NRP1 S612A attenuates osteo/odontogenesis, suggesting that GAG modification is essential for NRP1 in DPSCs.
摘要：
Neuropilin-1(NRP1)是参与多种生理事件的单一跨膜糖蛋白。然而,NRP1调节牙髓干细胞(DPSC)向骨/牙源性表型分化的确切机制尚不清楚.这里,我们确定了全长NRP1和糖胺聚糖（GAG）修饰的NRP1在DPSC中骨/牙本质形成过程中的表达显着增加。NRP1被证实促进碱性磷酸酶（ALP）活性，矿化结节沉积，通过功能丧失和功能获得方法在DPSC中Runx2，DSPP和DMP1的蛋白质和mRNA表达。Further,产生非GAG修饰的NRP1突变体(NRP1S612A),并且在NRP1S612A过表达细胞中观察到骨/牙源性分化的抑制。接头蛋白shroom3的敲除导致骨/牙形成的抑制。蛋白质-蛋白质相互作用网络，蛋白质-蛋白质对接和共聚焦分析表明NRP1和shroom3之间的相互作用。此外，免疫沉淀，然后进行Western分析，证实了NRP1与shroom3的结合，但是NRP1S612A的过表达极大地影响了NRP1对shroom3的募集。这些结果提供了强有力的证据，表明NRP1通过与shroom3相互作用是骨/牙本质形成的关键调节因子。此外，我们的结果表明，NRP1S612A减弱骨/牙形成，提示GAG修饰对DPSC中的NRP1至关重要。
