Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. MATERIAL AND METHODS: hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. RESULTS: Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. CONCLUSION: These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.

Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells.

The aim of this study was to investigate the response of human periodontal ligament (hPDL) fibroblasts to an intermittent compressive force and its effect on the expression of SOST, POSTN, and TGFB1. A computerized cell compressive force loading apparatus was introduced, and hPDL cells were subjected to intermittent compressive force. The changes in messenger RNA (mRNA) and protein expression were monitored by real-time polymerase chain reaction and Western blot analysis, respectively. An increased expression of SOST, POSTN, and TGFB1 was observed in a time-dependent fashion. Addition of cycloheximide, a transforming growth factor (TGF)-ß inhibitor (SB431542), or a neutralizing antibody against TGF-ß1 attenuated the force-induced expression of SOST and POSTN as well as sclerostin and periostin, indicating a role of TGF-ß1 in the pressure-induced expression of these proteins. Enzyme-linked immunosorbent assay analysis revealed an increased level of TGF-ß1 in the cell extracts but not in the medium, suggesting that intermittent compressive force promoted the accumulation of TGF-ß1 in the cells or their surrounding matrix. In conclusion, an intermittent compressive force regulates SOST/POSTN expression by hPDL cells via the TGF-ß1 signaling pathway. Since these proteins play important roles in the homeostasis of the periodontal tissue, our results indicate the importance of masticatory forces in this process.

Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2.

BACKGROUND AND OBJECTIVE: Prostaglandin E2 (PGE2) has been shown to be able to influence both bone formation and resorption. The purpose of this study was to investigate the effect of PGE2 on the osteogenic differentiation of human periodontal ligament (HPDL) cells. MATERIAL AND METHODS: HPDL cells were cultured with 0.001-1 µm PGE2 in osteogenic medium. In-vitro mineral deposition was determined by Alizarin Red S staining, and gene expression was determined by real-time PCR. RESULTS: PGE2 inhibited in-vitro mineral deposition by HPDL cells in a dose-dependent manner. PCR analyses showed that PGE2 upregulated the expression of Runt-related transcription factor 2 (RUNX2), but had no effect on osteocalcin expression. Upregulation of TWIST-related protein1 (TWIST1), a functional antagonist of RUNX2, was also observed. In addition, increased levels of RUNX2 and TWIST1 proteins, induced by PGE2, were detected by western blot analysis. Using a chemical activator of E prostanoid (EP) receptors as well as small interfering RNA against an EP receptor, it was shown that PGE2 regulated RUNX2 and TWIST1 via the EP2 receptor. The role of protein kinase A in the inductive effect of PGE2 was also demonstrated. CONCLUSION: The results of this study revealed that PGE2 modulates the osteogenic differentiation of HPDL cells via regulating the expression of RUNX2 and TWIST1. The results suggest a possible role for PGE2 in regulating the homeostasis of periodontal ligament tissue.

Transient receptor potential vanilloid-1 regulates osteoprotegerin/RANKL homeostasis in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Increasing evidence has shown the presence of transient receptor potential vanilloid-1 (TRPV1) in a variety of nonneuronal tissues; however, the function of TRPV1 in these cells is not well understood. In this study, we aimed to investigate the expression and function of TRPV1 in human periodontal ligament (HPDL) cells. As HPDL cells are known to play an important role in the bone-remodeling process, we hypothesized that TRPV1 might be implicated in the regulation of osteoprotegerin (OPG) and RANKL expression. MATERIAL AND METHODS: TRPV1 expression was examined by western blot analysis. The function of TRPV1 was studied using capsaicin, a well-known TRPV1 agonist. RT-PCR was performed to study the expression of OPG and RANKL mRNAs. The expression of OPG and RANKL proteins was analyzed by ELISA and western blotting, respectively. The mechanisms of capsaicin-induced OPG expression in HPDL cells were studied using inhibitors. RESULTS: In this study we found that TRPV1 was present in HPDL cells. Treatment with capsaicin induced OPG expression in a dose-dependent manner but did not affect the expression of RANKL. The increase of the OPG/RANKL ratio was also found in human osteoblasts, but not in MC3T3-E1 cells, a mouse osteoblastic cell line, suggesting species specificity. Capsazepine, the competitive TRPV1 antagonist, significantly abolished the effect of capsaicin on OPG expression in HPDL cells. In addition, studies investigating the effects of a calcium chelator and a phospholipase C inhibitor indicated that calcium ions and phospholipase C were required for the induction. Interestingly, capsaicin was able to increase the OPG/RANKL ratio, even in the presence of prostaglandin E2, a potent inducer of RANKL. CONCLUSION: Our study demonstrates that activation of TRPV1 leads to an increase of the OPG/RANKL ratio in HPDL cells. These findings suggest the novel function of TRPV1 in periodontal tissues, at least, as the regulator of the OPG/RANKL axis.

Osteoprotegerin induces osteopontin via syndecan-1 and phosphoinositol 3-kinase/Akt in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Our previous study found that thrombin induced osteoprotegerin synthesis in human periodontal ligament cells. As elevated levels of osteoprotegerin can exert biological effects on various cell types, in the present study we investigated the effect of osteoprotegerin on the expression of osteopontin in human periodontal ligament cells. MATERIAL AND METHODS: Cultured human periodontal ligament cells were treated with recombinant human osteoprotegerin (0-100 ng/mL) for 24-48 h. The expression of osteopontin mRNA and protein was analyzed using reverse transcription-polymerase chain reaction and western blot analyses, respectively. Phosphoinositol 3-kinase inhibitor, Akt inhibitor, heparinase, neutralizing antibody against receptor activator of nuclear factor-kappaB ligand (RANKL) and syndecan-1, and small interfering RNA against syndecan-1, were used to determine the mechanism involved. RESULTS: Osteoprotegerin up-regulated the mRNA and protein expression of osteopontin in human periodontal ligament cells in a dose-dependent manner. Addition of neutralizing antibody against RANKL attenuated the inductive effect of osteoprotegerin on osteopontin expression. In addition, the inductive effect of osteoprotegerin was abolished by phosphoinositol 3-kinase and Akt inhibitors, as well as by syndecan-1 antibody or syndecan-1 small interfering RNA. None of the inhibitors had any effect on the background level of osteopontin expression. CONCLUSION: An increased level of osteoprotegerin can generate signals via a RANKL/syndecan-1/phosphoinositol 3-kinase/Akt pathway. The results also suggest that osteopontin is one of the downstream targets of the pathway mediated by osteoprotegerin in human periodontal ligament cells. Thus, in addition to counteracting RANKL in the RANKL-osteoprotegerin system, osteoprotegerin may play a role in periodontal tissue remodeling through modulation of the extracellular matrix.