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Periodontal ligament cells play important roles in the homeostasis of periodontal tissue by mechanical stress derived from mastication, such as tension, compression, fluid shear, and hydrostatic force. In the present study, we showed that cyclic tensile force increased the gene expression level of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization, in human periodontal ligament cells using real-time PCR. Signaling inhibitors, PD98059/U0126 (extracellular signal-regulated kinase (ERK) inhibitors) and SB203580/SB202190 (p38 inhibitors), revealed that tensile force-mediated BMP-2 expression was dependent on activation of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways. Cyclic tensile force also induced cyclooxygenase-2 (COX-2) gene expression in a manner dependent on ERK1/2 and p38 MAP kinase pathways, and induced prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced tensile force-mediated BMP-2 expression, indicating that PGE2 synthesized by COX-2 may be involved in the BMP-2 induction. The inhibitory effect of NS-398 was completely restored by the addition of exogenous PGE2. However, stimulation with PGE2 alone in the absence of tensile force had no effect on the BMP-2 induction, indicating that some critical molecule(s) other than COX-2/PGE2 may be required for cyclic tensile force-mediated BMP-2 induction. Collectively, the results indicate that cyclic tensile force activates ERK1/2 and p38 MAP kinase signaling pathways, and induces COX-2 expression, which is responsible for the sequential PGE2 biosynthesis and release, and furthermore, mediates the increase in BMP-2 expression at the transcriptional level.

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells.