NRP1 promotes osteo/odontogenic differentiation via shroom3 in dental pulp stem cells

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.