Wnt/β-catenin signaling has been known to influence bone formation and homeostasis. In this study, we investigated the canonical Wnt signaling regulation of osteogenic differentiation from periodontal ligament (PDL) fibroblasts. Stimulating PDL fibroblasts with lithium chloride (LiCl), a canonical Wnt activator, significantly increased mineralized nodule and alkaline phosphatase (ALP) activity in a time- and dose-dependent manner. LiCl up-regulated protein expression of osteogenic transcription factors, including the runt-related gene 2, Msx2, and Osterix 2, in the PDL fibroblasts. Treatment of these cells with LiCl also increased the mRNA levels of ALP, FosB, and Fra1 in a dose-dependent manner. Blockage of canonical Wnt signaling by treating the cells with DKK1 inhibited Wnt1-stimulated mRNA expression of these osteogenic factors. Furthermore, pretreatment with DKK1 reduced the ALP activity and matrix mineralization stimulated by Wnt1. Collectively, these results suggest that canonical Wnt signaling leads to the differentiation of PDL fibroblasts into osteogenic lineage with the attendant stimulation of osteogenic transcription factors.

Type I collagen (COL I) and matrix metalloproteinase-1 (MMP-1) are the predominant matrix proteins in the extracellular matrix of the human periodontal ligament (PDL). The expression of these proteins in PDL fibroblasts (PLF) is sensitive to physiological and mechanical stress and is critical for PDL remodeling accompanied by alveolar bone remodeling. This study examined how dose tensile force regulates the expression of COL I and MMP-1 and explored the possible roles of mitogen-activated protein kinases (MAPKs) and transcription factors, such as activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). Tensile force stimulated the mRNA expression of COL I and MMP-1 in the cells and also activated MAPKs including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 MAPK. A pharmacological inhibitor of ERK or JNK prevented the expression of matrix genes and the nuclear translocation of c-Jun proteins in the force-applied PLF. The knockdown of c-Jun by transfecting the cells with its antisense oligonucleotides reduced the force-induced increase in matrix gene expression. In particular, the ERK inhibitor but not JNK or p38 MAPK inhibitor attenuated the force-mediated stimulation of NF-κB-DNA binding and MMP-1 expression. Overall, these results highlight the mechanotransduction pathways involved in matrix gene expression in PLF, where the tension-stimulated expression of COL I and MMP-1 is controlled by the ERK/JNK-AP-1 and ERK-NF-κB signaling pathways.

Numerous studies have reported that inflammatory cytokines are important mediators for osteoclastogenesis, thereby causing excessive bone resorption and osteoporosis. Acteoside, the main active compound of Rehmannia glutinosa, which is used widely in traditional Oriental medicine, has anti-inflammatory and antioxidant potentials. In this study, we found that acteoside markedly inhibited osteoclast differentiation and formation from bone marrow macrophages (BMMs) and RAW264.7 macrophages stimulated by the receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL). Acteoside pretreatment also prevented bone resorption by mature osteoclasts in a dose-dependent manner. Acteoside (10 µM) attenuated RANKL-stimulated activation of p38 kinase, extracellular signal-regulated kinases, and c-Jun N-terminal kinase, and also suppressed NF-κB activation by inhibiting phosphorylation of the p65 subunit and the inhibitor κBα. In addition, RANKL-mediated increases in the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and in the production of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were apparently inhibited by acteoside pretreatment. Further, oral acteoside reduced ovariectomy-induced bone loss and inflammatory cytokine production to control levels. Our data suggest that acteoside inhibits osteoclast differentiation and maturation from osteoclastic precursors by suppressing RANKL-induced activation of mitogen-activated protein kinases and transcription factors such as NF-κB, c-Fos, and NFATc1. Collectively, these results suggest that acteoside may act as an anti-resorptive agent to reduce bone loss by blocking osteoclast activation.