Method: CPNE7 expression was evaluated by RT-PCR, western blot and immunocytochemistry in ameloblasts and odontoblasts. To evaluate the functional roles of CPNE7 in odontoblasts, dentin sialoprotein (DSP) expression, mineralized nodule formation and dentin sialophosphoprotein (DSPP) promoter assay were performed with or without CPNE7 stimulation/inactivation. Co-immunoassay and ChIP assay were performed to assess the capability of nucleolin on CPNE7. Dentin formation was evaluated by transplantation of human dental pulp cells (hDPCs) with or without CPNE7 treatment/inactivation into subcutaneous spaces of nude mice.
Result: In this study, CPNE7 protein was expressed in preameloblast and secreted to the extracellular matrix during ameloblast differentiation. We detected translocation of CPNE7 protein from preameloblast to odontoblast by co-culture system and immunohistochemistry. In addition, CPNE7 expression in odontoblasts was gradually increased during odontoblast differentiation and located in nucleus of odontoblastic MDPC-23 cells. In odontoblasts, expression of DSP was significantly increased by overexpression of CPNE7, treatment of recombinant CPNE7 and PA-CM, but decreased by CPNE7 siRNA treatment and inactivation CPNE7 in PA-CM. Meanwhile, we found that the CPNE7 showed preferential binding to nucleolin protein in nucleus using co-immunoassay, and the CPNE7 and nucleolin proteins bound on DSPP promoter using ChIP assay. Furthermore, CPNE7 regulated DSPP transcriptional responses with nucleolin. According to in vivo results, hDPCs generated more mineralized tissue upon CPNE7 stimulation, while inactivation of CPNE7 in PA-CM group showed less mineralized tissue, and produced reparative dentin-like structures and showed bone sialoprotein expression in root canal spaces.
Conclusion: These studies suggest that CPNE7 participates in epithelial-mesenchymal interaction during odontogenesis and confirm that CPNE7 plays important functional roles in odontoblast differentiation and mineralization. Furthermore, these results elucidate regulatory mechanisms underlying odontogenesis through secreted CPNE7 protein from epithelial cells, which is able to induce odontoblast differentiation-related gene expression.