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Luckprom P, Wongkhantee S, Yongchaitrakul T, Pavasant P. Adenosine triphosphate stimulates RANKL expression through P2Y 1 receptor–cyclo-oxygenase-dependent pathway in human periodontal ligament cells. J Periodont Res 2010; 45: 404–411. © 2010 John Wiley & Sons A/S 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/P2Y1 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 μm). The expressions of RANKL and cyclo-oxygenase 2 (COX-2) were examined by RT-PCR and western blot analysis. The level of prostaglandin E2 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 κB (NFκB) inhibitor, pyrrolidine dithiocarbamate, inhibited RANKL expression, prostaglandin E2 production and NFκB translocation. In addition, a specific P2Y1 receptor antagonist, MRS2179, and P2Y1 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 P2Y1 receptor, cAMP-dependent protein kinase, NFκB 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 P2Y1 receptor and the NFκB–COX–RANKL axis in periodontal tissue. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase 2 (MMP-2) has been implicated to play a role in pathogenesis of periodontal disease. We recently reported thatPorphyromonas gingivalissupernatant could activate MMP-2 in human periodontal ligament (HPDL) cells. In this study, activation of MMP-2 byActinobacillus actinomycetemcomitanssupernatant and the mechanism was investigated.HPDL cells were treated with eitherA. actinomycetemcomitansorP. gingivalissupernatant for 48 h. To verify the mechanism, pretreated inhibitors were used. Gelatin zymography, RT-PCR and Western blot analysis were used to detect the activation of MMP-2, expression of MT1-MMP and TIMP-2 mRNA and the proteins, respectively.The supernatant fromA. actinomycetemcomitanscould activate MMP-2 in HPDL cells similar to that fromP. gingivalisbut by a different mechanism. Activation byA. actinomycetemcomitanssupernatant was correlated with a reduction of TIMP-2 secretion without any alteration of MT1-MMP, while activation byP. gingivalisincreased MT1-MMP but no change of TIMP-2 was found.The supernatant fromA. actinomycetemcomitansandP. gingivaliscould induce the activation of MMP-2 possibly through the imbalance of MT1-MMP and TIMP-2 in HPDL cells but by different mechanisms. The imbalance of MT1-MMP and TIMP-2 may be another factor that is involved in the severity of periodontal disease. [ABSTRACT FROM AUTHOR]

The study aims to report annual demographic characteristics and to compare the differences of socioeconomic inequality, type of motor vehicles, and seating relating to major bone injuries among hospitals in and outside Bangkok. Six public hospitals in Bangkok and six regional hospitals in the provinces were studied over a one year period (2008-2009). There were 3,650 cases: 3,596 injured patients and 54 deaths. Patients with a lower education level accounted for the largest number of cases, both in the provinces (46.3%) and Bangkok (17.1%). Their incomes were less than THB 10,000/yearly. Total number of motorcycle cases (3,360) was higher (11.6:1) than 290 cases of motor vehicles. Pickup cars were used more commonly. Riding a motorcycle was likely to be fatal. The front seat was the most common involvement. Passengers occupying the middle and rear seat of the motorcycle were involved in 16.0% and 1.0% cases, respectively. Long bone and joints were the most common injuries. The results strongly confirmed the striking contribution of motorcycles and pickups to road traffic accidents. People with a low educational level, in conjunction with low income, and in areas outside of Bangkok were more at risk. Specific education on road safety should be delivered, preferably in primary schools.

Background: Mechanical stress such as orthodontic forces can produce mechanical damage and inflammatory reaction in the periodontium. Osteopontin (OPN) is a multifunctional cytokine that has been correlated with periodontal disease progression. Because the periodontal ligament (PDL) can be affected by stress and PDL cells are involved in periodontal destruction and remodeling, we aimed to study the influence of mechanical stress on the expression and regulation of OPN in human PDL (HPDL) cells., Methods: The mechanical stress was generated by continuous compressive force, and the expression of OPN was examined by reverse transcription-polymerase chain reaction and Western analysis. The application of inhibitors was used to examine the mechanism involved., Results: Both mRNA and protein expression of OPN significantly increased in a force-dependent manner. Increase of receptor activator of nuclear factor-kappa B ligand (RANKL) was also observed. Interestingly, application of indomethacin could abolish the induction of RANKL but not that of OPN, suggesting the cyclooxygenase-independent mechanism for stress-induced OPN expression. In addition, the upregulation of OPN was diminished by Rho kinase inhibitor but not by cytochalasin B., Conclusions: Mechanical stress affects OPN expression in HPDL cells through the Rho kinase pathway. Because OPN participates in bone resorption and remodeling induced by mechanical and biologic signals, these results suggest the significance of stress-induced OPN in HPDL cells in alveolar bone resorption and remodeling.

Background: Human periodontal ligament (HPDL) cells may support osteoclastogenesis by expressing receptor activator of nuclear factor-kappa B ligand (RANKL) in response to periopathogenic factors and inflammatory cytokines. Because osteoclastogenesis requires the presence of macrophage colony-stimulating factor (M-CSF), we examined whether HPDL cells secrete M-CSF in response to tumor necrosis factor-alpha (TNF-alpha)., Methods: Cultured HPDL cells were treated with TNF-alpha in serum-free condition. The expression of M-CSF and RANKL was determined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Inhibitors and anti-TNF receptor (TNFR) neutralizing antibodies were used for the inhibitory experiments. A migration assay was performed., Results: TNF-alpha upregulated M-CSF and RANKL in HPDL cells. The effect on M-CSF expression could be partially blocked by pyrrolidine-dithiocarbamate ammonium salt and LY294002 but not by NS398. Neutralizing antibody to TNFR1 could diminish the effect of TNF-alpha. In addition, TNF-treated culture medium exhibited chemotactic effect for RAW264.7., Conclusions: HPDL cells are capable of secreting M-CSF and expressing RANKL in response to TNF-alpha. The upregulation of M-CSF is possibly one of the mechanisms essential for periodontal tissue destruction in response to inflammatory cytokines. The upregulation is partly through nuclear factor-kappa B (NF-kappaB) and phosphatidylinositol 3'-kinase and possibly involves TNFR1.

Extracellular matrix and growth factors are the crucial factors that regulate healing and regenerating processes in human periodontal ligament cells. The purpose of this study was to examine the effects of type I collagen and insulin-like growth factor-I (IGF-I) on osteopontin (OPN) expression. The data showed that OPN expression was significantly decreased when cells were cultured on collagen-coated plates. Addition of IGF-I obviously induced OPN expression only in a collagen-coated condition, suggesting an attenuating effect of IGF-I on the decrease of OPN expression. Cells treated with a combination of inhibitory antibody to beta1 integrin and IGF-I showed the same level of OPN expression as those treated with either inhibitory antibody to beta1 integrin or IGF-I alone. These results indicate that IGF-I counteracts with the inhibitory signal from type I collagen through beta1 integrin receptor.

Background: The lipopolysaccharide (LPS) of A. actinomycetemcomitans is one of the major pathogenic factors in periodontal disease. It induces secretion of proinflammatory cytokines and is involved in alveolar bone destruction. We hypothesized that the LPS of A. actinomycetemcomitans could affect the activation of matrix metalloproteinase (MMP)-2 and the expression of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin in human periodontal ligament (HPDL) cells leading to the destruction of periodontium., Methods: HPDL cells were cultured in serum-free medium with or without the LPS of A. actinomycetemcomitans for 36 hours. The activation of MMP-2 was analyzed by zymography. Changes of the expression of RANKL and osteoprotegerin (OPG) were examined by reverse transcription-polymerase chain reaction and supported by Western blot analysis., Results: The activation of MMP-2 could be induced by the LPS of A. actinomycetemcomitans in HPDL cells and could be inhibited by a serine protease inhibitor. This result suggested that the LPS might activate MMP-2 through a serine protease-dependent pathway. This activation was also blocked by NF-kappaB inhibitor, which indicated the involvement of NF-kappaB. The upregulation of RANKL but not OPG by the LPS was found in both transcription and translation and could be reduced by indomethacin. In addition, serine protease inhibitor also inhibited the upregulation of RANKL, suggesting the activity of serine protease., Conclusions: The effect of the LPS of A. actinomycetemcomitans on HPDL cells is serum-independent and the induction of the activation of MMP-2 and the expression of RANKL are serine protease-dependent pathways. The results suggest the role of HPDL cells in the pathogenesis of periodontitis.

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.

It has been reported that matrix metalloproteinase (MMP) produced by host cells plays a major role in periodontal tissue destruction. In addition, secreted virulence factors from Porphyromonas gingivalis can alter MMP secretion and cause activation in host cells that lead to the tissue degradation. In this study, we examine the effects of P. gingivalis supernatant on matrix metalloproteinase-2 (MMP-2) activation in human periodontal ligament (HPDL) cells. Cultures of HPDL cells were treated with P. gingivalis supernatant for 48 h and the level of MMP-2 activation was monitored by gelatin zymography. The profound activation of MMP-2 was seen only in the treated group. The activation of MMP-2 was inhibited by MMP inhibitors phenanthroline and EDTA, but not serine protease or cysteine protease inhibitors. To study the correlation between the expression of membrane-type-1 matrix metalloproteinase (MT1-MMP) and the activation of MMP-2, the level of MT1-MMP was analyzed. The results from reverse-transcription polymerase chain reaction (RT-PCR) and Western analysis indicated that P. gingivalis supernatant up-regulated the expression of MT1-MMP in both transcription and translation levels within 48 h. These results suggest that P. gingivalis supernatant can activate MMP-2 in HPDL cells and the mechanism of activation may involve the increased amount of MT1-MMP. It is possible that the activation of MMP-2 by P. gingivalis plays a role in the process of chronic periodontitis.