Tussanee Yongchaitrakul

Mechanical Stress Induces Osteopontin via ATP/P2Y1 in Periodontal Cells

Our previous study showed that mechanical stress induced the expression of osteopontin (OPN) in human periodontal ligament (HPDL) cells through the Rho kinase pathway. The increase of OPN expression via Rho kinase has been demonstrated to be triggered by nucleotide. Therefore, we hypothesized that nucleotides, particularly adenosine triphosphate (ATP), participated in the stress-induced OPN expression in HPDL cells. In the present study, the roles of ATP and P2Y1 purinoceptor were examined. Reverse-transcription polymerase chain-reaction and Western blot analysis revealed that the stress-induced ATP exerted its stimulatory effect on OPN expression. The inductive effect was attenuated by apyrase and completely inhibited by the Rho kinase inhibitor, as well as by the P2Y1 antagonist. We here propose that stress induces release of ATP, which in turn mediates Rho kinase activation through the P2Y1 receptor, resulting in the up-regulation of OPN. Stress-induced ATP could play a significant role in alveolar bone resorption.

The synergistic effect of TGF-β and 1,25-dihydroxyvitamin D3 on SPARC synthesis and alkaline phosphatase activity in human pulp fibroblasts

1,25(OH)2D3 and TGF-beta can influence the function and differentiation of dental pulp fibroblasts. In this study, we examined the effect of 1,25(OH)2D3 and TGF-beta on the synthesis of SPARC and ALP activity in human pulp fibroblasts. Two isoforms of SPARC, the 43 and 38 kDa, were detected in this cell type. TGF-beta increased the synthesis of SPARC about 2.5-fold after 3 days of treatment but had no effect on the ALP activity. On the contrary, 1,25(OH)2D3 increased ALP activity 2-fold but had no effect on SPARC. The combination of TGF-beta and 1,25(OH)2D3 significantly induced SPARC synthesis and ALP activity by 5 and 9 folds, respectively (P<0.05). This finding suggested the synergistic effect between TGF-beta and 1,25(OH)2D3 in dental pulp fibroblasts on the synthesis of SPARC and ALP activity. This interaction could influence the function and differentiation of dental pulp fibroblasts.

Adenosine triphosphate stimulates RANKL expression through P2Y1 receptor-cyclo-oxygenase-dependent pathway in human periodontal ligament cells

BACKGROUND AND OBJECTIVE Our previous study showed that human periodontal ligament cells responded to mechanical stress by increasing adenosine triphosphate (ATP) release, accompanied by the increased expression of RANKL and osteopontin. We found that the signaling pathway of mechanical stress-induced osteopontin was mediated through ATP/P2Y(1) receptor and Rho kinase activation but that of mechanical stress-induced RANKL was different. In this study, we further investigated the effect of extracellular ATP on the expression of RANKL and the mechanism involved. MATERIAL AND METHODS Human periodontal ligament cells were treated with ATP (10-40 microm). The expressions of RANKL and cyclo-oxygenase 2 (COX-2) were examined by RT-PCR and western blot analysis. The level of prostaglandin E(2) was determined using ELISA. Signaling pathways were investigated by using inhibitors and antagonist. RESULTS Adenosine triphosphate induced the expression of RANKL. Indomethacin, an inhibitor of COX, could abolish the induction of RANKL expression, suggesting a COX-dependent mechanism. A cAMP-dependent protein kinase inhibitor, H89, and a nuclear factor kappaB (NF kappaB) inhibitor, pyrrolidine dithiocarbamate, inhibited RANKL expression, prostaglandin E(2) production and NF kappaB translocation. In addition, a specific P2Y(1) receptor antagonist, MRS2179, and P2Y(1) small interfering RNA diminished the effect of ATP. CONCLUSION Extracellular ATP stimulates RANKL expression in human periodontal ligament cells through a pathway dependent on the P2Y(1) receptor, cAMP-dependent protein kinase, NF kappaB and COX. Our results suggest that, among the molecules responsible for the effect of mechanical stress, ATP participates in bone resorption or bone homeostasis by mediating its signal through the P2Y(1) receptor and the NF kappaB-COX-RANKL axis in periodontal tissue.

Role of mechanical stress on the function of periodontal ligament cells.

The periodontium is an important structure that functions to support teeth and mastication. It consists of gingiva, alveolar bone, cementum and periodontal ligament. The periodontal ligament develops from dental follicular cells and becomes a fibrous connective tissue located between cementum and alveolar bone. Periodontal ligament cells reside within the periodontal ligament and secrete extracellular matrix, mainly collagen fibers, that interact with the adjacent hard tissues, alveolar bone and cementum. In the mature jaw bone, the periodontal ligament helps anchor the tooth to alveolar bone via collagen fibers inserted into the hard tissue (known as Sharpey s fibers). The periodontal ligament contains several types of cells, including periodontal ligament fibroblasts, cementoblasts and osteoblasts. In addition, epithelial rests of Malassez, vascular endothelial cells and smooth muscle cells are also found within this tissue. In review, only periodontal ligament fibroblasts are referred to as periodontal ligament cells. The function of periodontal ligament cells is to build and maintain the periodontal ligament. In addition to their role in matrix synthesis, some periodontal ligament cells possess characteristics of progenitor cells and are able to differentiate into cementoblasts or osteoblasts, suggesting a role in repair and regeneration of the periodontium. Interestingly, periodontal ligament cells play an essential role in the response to mechanical loading of the tooth, by remodeling and repairing the damaged matrix components of periodontium. Given these roles, periodontal ligament cells may be described as both builders and caretakers of the periodontium (62). Periodontal ligament cells and mechanical loading

Secreted protein acidic, rich in cysteine induces pulp cell migration via αvβ3 integrin and extracellular signal-regulated kinase

AIM The aim of this study was to investigate the influence of secreted protein acidic, rich in cysteine (SPARC) on the migration of human dental pulp (HDP) cells. METHODS Secreted protein acidic, rich in cysteine was applied in the lower chamber of the chemotaxis apparatus and migration was determined by counting the cells that migrated through the membrane. To determine the signaling pathway involved, cells were incubated with inhibitors for 30 min prior to the migration assay. RESULTS The results indicated that SPARC induced HDP cell migration in a dose-dependent manner via extracellular signal-regulated kinase (ERK). The migration could be inhibited both by the anti-alphavbeta3 integrin antibody and by suramin, a non-selective growth factor receptor and G-protein coupled receptor antagonists. The anti-alphavbeta3 integrin antibody could also inhibit ERK activation, suggesting the possible role of alphavbeta3 integrin on the regulation of ERK and cell migration. Interestingly, both suramin and SB225002, another G-protein coupled receptor antagonist, suppressed ERK activation. CONCLUSIONS Secreted protein acidic, rich in cysteine could act as a chemotactic factor and facilitate migration, possibly through the G-protein coupled receptor, alphavbeta3 integrin and ERK. The data support that SPARC could play a crucial role in dental pulp tissue repair by inducing dental pulp cell migration.

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.