Wnt3a signaling induces murine dental follicle cells to differentiate into cementoblastic/osteoblastic cells via an osterix-dependent pathway

Background and Objective Dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwig's epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial–mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells. Material and Methods The expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression. Results We identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix–small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels. Conclusion These findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/β-catenin signaling pathway.