Nucleolin Mediates Endocytosis of Copine-7 During Odontoblast Differentiation

Objectives: Our previous studies demonstrated that Copine-7 (Cpne7), a dental epithelium-derived protein, is a signaling molecule that is secreted by preameloblasts and regulates the differentiation of dental or non-dental mesenchymal cells into odontoblasts. However, action mechanisms of Cpne7 during odontoblast differentiation are not well known. Therefore, we aimed to investigate the mechanisms of cellular uptake of Cpne7 during odontoblast differentiation.
Methods: To elucidate the characteristics of the endocytic pathway of Cpne7, MDPC-23 cells were pretreated with the endocytic inhibitors for 60 min before treatment with Flag-Cpne7 recombinant protein. And also to confirm that Nucleolin is a receptor responsible for mediating endocytosis of Cpne7, MDPC-23 cells were treated with Nucleolin siRNA for 48 h and analysed by western blotting and confocal analysis.
Results: Here, we show that cellular uptake of Cpne7 occurs through receptor-mediated endocytosis. This process is initiated by Cpne7 binding to Nucleolin as an important endocytic receptor for Cpne7 in odontoblast cells. We further demonstrate that lipid raft-mediated endocytosis is the major routes, by which Cpne7 enter the cells. It is possible that lipid raft-mediated endocytosis of Cpne7 might initiate signaling cascades necessary for downstream events leading to odontoblast differentiation.
Conclusions: Our data suggests that binding of Cpne7 with their receptor Nucleolin might be an important function by which Cpne7 is internalized and transported to the nucleus during odontoblast differentiation.