MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Heme oxygenase‐1 mediates nicotine‐ and lipopolysaccharide‐induced expression of cyclooxygenase‐2 and inducible nitric oxide synthase in human periodontal ligament cells

Pi S‐H, Jeong G‐S, Oh H‐W, Kim Y‐S, Pae H‐O, Chung H‐T, Lee S‐K, Kim E‐C. Heme oxygenase‐1 mediates nicotine‐ and lipopolysaccharide‐induced expression of cyclooxygenase‐2 and inducible nitric oxide synthase in human periodontal ligament cells. J Periodont Res 2010; 45: 177–183. © 2010 John Wiley & Sons A/S Background and Objective: Although heme oxygenase‐1 (HO‐1) plays a key role in inflammation, its anti‐inflammatory effects and mechanism of action in periodontitis are still unknown. This study aimed to identify the effects of HO‐1 on the proinflammatory mediators activated by nicotine and lipopolysaccharide (LPS) stimulation in human periodontal ligament (PDL) cells. Material and Methods: The production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2) and HO‐1 proteins was evaluated by Western blot analysis. Results: Lipopolysaccharide and nicotine synergistically induced the production of NO and PGE₂ and increased the protein expression of iNOS, COX‐2 and HO‐1. Treatment with an HO‐1 inhibitor and HO‐1 small interfering RNAs blocked the LPS‐ and nicotine‐stimulated NO and PGE₂ release as well as the expression of iNOS and COX‐2. Conclusion: Our data suggest that the nicotine‐ and LPS‐induced inflammatory effects on PDL cells may act through a novel mechanism involving the action of HO‐1. Thus, HO‐1 may provide a potential therapeutic target for the treatment of periodontal disease associated with smoking and dental plaque.     

Correlation of molecular biomarker concentrations between synovial fluid and saliva of the patients with temporomandibular disorders

Objectives

The synovial membrane and fluid are involved in the pathogenesis of temporomandibular joint (TMJ) disorders. This study aims to assess the relationship between matrix metalloproteinase-2 (MMP-2), chemerin and prostaglandin (PGE₂) levels in the synovial fluid (SF) and saliva of patients with TMJ disorder regarding their role in inflammation and the value of being a candidate for predictive biomarkers in the disease. Also, it is aimed to find out whether chemerin’s main function triggers the formation inflammatory cytokine markers in the associated area.

Materials and methods

Thirty-two samples of SF and saliva were obtained from patients with disc displacement without reduction with limited opening (DDWORwLO). Mann-Whitney-U test was used for the comparisons of the biomarker levels in SF and saliva. The correlation between chemerin and BMI (Body Mass Index) is analyzed by non-parametric Spearman’s rho correlation coefficient.

Results

For all of the three biomarkers, statistically significant differences were found between SF and saliva. An unexpectedly high level expression of chemerin was observed in SF. A statistically significant, positive correlation was observed between PGE₂ -MMP-2, and chemerin-PGE₂ in saliva, chemerin and MMP-2 in SF, respectively (p = 0.031, r = 0.382 / p = 0.039, r = 0.366 / p = 0.032, r = 0.379). A positive correlation was determined between saliva and SF levels of PGE₂ (p = 0.016, r = 0.421).

Conclusions

Chemerin, MMP-2, and PGE₂ can play a role as an inflammatory factor for the development of TMJ disorder.

Clinical relevance

The search for molecular markers in TMJ and the inhibition of the associated molecular signaling mechanism is important to reduce joint inflammation and cartilage degradation.

     

Sodium butyrate induces the production of cyclooxygenases and prostaglandin E₂ in ROS 17/2.8 osteoblastic cells

ObjectiveSodium butyrate (butyric acid; BA) is a major metabolic by-product of the anaerobic periodontopathic bacteria present in subgingival plaque. We examined the effects of BA and/or indomethacin on cell proliferation, the expression of cyclooxygenases (COXs), prostaglandin (PG) receptors (EP1-4), extracellular matrix proteins, such as type I collagen and osteopontin, and PGE₂ production, using ROS17/2.8 cells as osteoblasts.MethodsThe rat clonal cell line ROS 17/2.8 was cultured with 0, 10^?5, 10^?4, and 10^?3 M BA in the presence or absence of 0.5 μM indomethacin, for up to 7 days. The expression of COX-1, COX-2, EP1, EP2, EP3, EP4, type I collagen, and osteopontin was examined at the mRNA and protein levels using real-time PCR and Western blotting, respectively. The amount of PGE₂ in the culture medium was measured by ELISA.ResultsProliferation of ROS 17/2.8 cells was not affected by the addition of BA. However, PGE₂ production and the expression of COX-1 and COX-2 increased with the addition of BA. In contrast, indomethacin, an inhibitor of COX, blocked the stimulatory effect of BA. Furthermore, EP2 expression increased with BA treatment, whereas EP1 expression was not affected and the expression of EP3 and EP4 was not detected. The addition of BA also increased the expression of type I collagen and osteopontin. Indomethacin blocked about 50% of the stimulatory effect of BA on type I collagen, whereas it did not block the effect on osteopontin.ConclusionsThese results suggest that BA induces PGE₂ production by increasing the expression of COX-1 and COX-2 in osteoblasts, and that an autocrine action of the produced PGE₂, via EP1 or BA-induced EP2, is related to an increase in type I collagen expression by BA.     

n-Butanol extracts of Panax notoginseng suppress LPS-induced MMP-2 expression in periodontal ligament fibroblasts and inhibit osteoclastogenesis by suppressing MAPK in LPS-activated RAW264.7 cells

Objective

Periodontitis is a group of inflammatory diseases that affect connective tissue attachments and the supporting bone that surround the teeth. Osteoclasts are responsible for skeletal modeling and remodeling but may also destroy bone in several bone diseases, including osteoporosis and periodontitis. This study examined the anti-inflammatory effects of Panax notoginseng (PN) on periodontal ligament fibroblasts (PDLFs) and RAW264.7 cells under lipopolysaccharide (LPS) induced inflammatory conditions.

Design

The effects of PN on PDLFs were determined by measuring the cell viability and mRNA expression of tissue-destructive proteins. The effects of PN on osteoclasts were examined by measuring the following: (1) the cell viability, (2) the formation of Tartrate-resistant acid phosphatase (TRAP)(+) multinucleated cells, (3) MAPK signaling pathways, (4) mRNA expression of inflammatory-related proteins and (5) nitric oxide (NO) production.

Results

The n-butanol extracts of PN (bPN) increased the cell proliferation of the PDLFs and decreased the mRNA expression of matrix metalloproteinase (MMP)-2 in the PDLFs. bPN inhibited the formation of LPS-stimulated TRAP(+) multinucleated cells. bPN also inhibited the LPS-stimulated activation of JNK and ERK signaling, and inhibited the LPS-stimulated degradation of I_KB in the RAW264.7 cells. In addition, bPN decreased the mRNA expression of MMP-9 and iNOS, which are involved in the range of pathophysiological processes, such as inflammation in the RAW264.7 cells. NO production was also decreased via the inhibition of iNOS.

Conclusions

These findings suggest that bPN has therapeutic effects on bone-destructive processes, such as those that occur in periodontal diseases.     

Intracanal Metformin Promotes Healing of Apical Periodontitis via Suppressing Inducible Nitric Oxide Synthase Expression and Monocyte Recruitment

IntroductionWe have previously shown that intracanal metformin ameliorates apical periodontitis, partially by modulation of osteoblast apoptosis. The action of metformin on other cell types pertinent to the development of apical periodontitis needs to be examined. In the present study, we aimed to analyze whether its effects on the expression of inducible nitric oxide synthase (iNOS) and monocyte recruitment contribute to the therapeutic effect on apical periodontitis.MethodsLipopolysaccharide (LPS)-induced expression of iNOS in a human monocytic cell line, Mono-Mac-6, was assessed by Western blot. The amount of nitrite in culture medium was assessed to quantify nitric oxide (NO) production. C-C motif chemokine ligand-2 (CCL-2) synthesis was measured by enzyme-linked immunosorbent assay. Experimental apical periodontitis in rats was treated with root canal debridement with or without intracanal metformin medication. Lesion progression was assessed by conventional radiography and micro–computed tomographic imaging. Cellular expression of iNOS and the number of monocytes/macrophages were assessed by immunohistochemistry.ResultsMetformin suppressed LPS-induced iNOS and NO production by monocytes. More importantly, metformin inhibited LPS-enhanced CCL-2 synthesis through modulation of the iNOS/NO pathway. Intracanal metformin reduced bone resorption associated with apical periodontitis and suppressed iNOS expression and monocyte recruitment.ConclusionsOur results confirmed the therapeutic efficacy of intracanal metformin for apical periodontitis. Suppression of monocyte recruitment through modulation of iNOS expression and NO production is an important mechanism underlying the beneficial effect of metformin.     

Anti-inflammatory mechanism of PPARγ on LPS-induced pulp cells: role of the ROS removal activity

ObjectivesPPARγ has an anti-inflammatory effect on LPS-induced pulpal inflammation by decreasing the expression of MMPs, ICAM-1 and VCAM-1. However, the anti-inflammatory mechanism of PPARγ on the cell adhesion molecules and their upper signal pathways has not been clarified in pulp cells. The aim of this study is to investigate the anti-inflammatory mechanism of PPARγ in pulpal inflammation.MethodsHuman dental pulp cells (HDPCs) were isolated from freshly extracted third molar and cultured. The over-expression of PPARγ was used by adenoviral PPARγ (Ad/PPARγ). The formation of ROS was analysed using DCFH-DA with FACS, and NO was analysed using colorimetric bioassay. The expression of inflammatory molecules and inflammatory mechanism of PPARγ involved signal pathway were determined by immunoblotting.ResultsLPS-induced HDPC decreased PPARγ expression gradually and strongly activated the ERK1/2 signals amongst the MAPK, and induced NF-κB translocation from the cytosol to the nucleus. On the other hand, the cells to restore PPARγ with Ad/PPARγ were inhibited ERK1/2 despite being stimulated with LPS. In addition, the cells treated with rosiglitazone (PPARγ agonist) also were inhibited ERK1/2 activation, and the expression of ICAM-1, VCAM-1 and NF-κB translocation under LPS stimulation. The GW9667 (PPARγ antagonist)-treated HDPC did not affect the adhesion molecules and signal activation. LPS-induced HDPC produced significant NO and ROS levels, but their production was attenuated in the PPARγ over-expressed cells. Overall, the PPARγ effect under LPS stimulation is due to the removal activity of cellular NO and ROS formation.ConclusionThese results suggest that anti-inflammatory mechanism of PPARγ is due to the removal activity of NO and ROS, and its removal effect suppressed ERK1/2 signal activation and NF-κB translocation. Therefore, the NO and ROS removal activity of PPARγ suggests major anti-inflammatory mechanism in HDPC, and it might offer us a possible molecule for various types of inflammatory inhibition.     

Correlation between nitric oxide concentration (NO2 + NO3) in saliva and CD4+ count in HIV infected adult individuals of Mangalore,Karnataka, India

AimsTo investigate the correlation between nitric oxide concentration (NO₂ + NO₃) in saliva and CD4+ count in HIV infected individuals.Study designA cross-sectional study was carried out among 60 proven HIV-positive patients selected randomly from the ART centres.MethodologyTwo groups – Control (n = 30) and study group (n = 60). The study group was divided into Group I (CD4+ count >200/cu.mm) and Group II (CD4+ count<200/cu.mm). Unstimulated saliva was collected and nitric oxide (NO₃+ NO₃) was estimated by Griess method. Statistical analysis was performed using One-way ANOVA and Tukey's Multiple Comparison Test.ResultsThe mean level of nitric oxide (NO₂ + NO₃) in control group was 43.95 ± 3.64 and in Group I with CD4 >200/cu.mm and Group II with CD4 <200/cu.mm was 51.98 ± 4.08 and 59.78 ± 4.18 respectively. ‘p’ value was statistically significant (P < 0.0001). The mean difference between Control and Group I was −7.937 (P < 0.001), Control and Group II was −15.80 (P < 0.001) and Group I and Group II was −7.867 (P < 0.001) from Tukey's Multiple Comparison Test.ConclusionsFrom our study it is now evident that salivary nitric oxide (NO₂ + NO₃) can serve as an indicator of HIV replication and saliva shows a strong potential to perform tests that are done currently in blood.     

Mangiferin inhibits lipopolysaccharide-induced production of interleukin-6 in human oral epithelial cells by suppressing toll-like receptor signaling

ObjectiveOral epithelial cells have currently been found to play an important role in inflammatory modulation in periodontitis. Mangiferin is a natural glucosylxanthone with anti-inflammatory activity. The aim of this study was to investigate the regulatory effect of mangiferin on lipopolysaccharide (LPS)-induced production of proinflammatory cytokine interleukin-6 (IL-6) in oral epithelial cells and the underlying mechanisms.DesignThe levels of LPS-induced IL-6 production in OKF6/TERT-2 oral keratinocytes were detected using enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor (TLR) 2 and TLR4 was determined using western blot analysis. And the phosphorylation of TLR downstream nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) was examined using cell-based protein phosphorylation ELISA kits.ResultsWe found that mangiferin reduced LPS-upregulated IL-6 production in OKF6/TERT-2 cells. Additionally, mangiferin inhibited LPS-induced TLR2 and TLR4 overexpression, and suppressed the phosphorylation of NF-κB, p38 MAPK and JNK. Moreover, mangiferin repressed IL-6 production and TLR signaling activation in a dose-dependent manner after 24 h treatment.ConclusionsMangiferin decreases LPS-induced production of IL-6 in human oral epithelial cells by suppressing TLR signaling, and this glucosylxanthone may have potential for the treatment of periodontitis.     

Genistein suppresses Prevotella intermedia lipopolysaccharide-induced inflammatory response in macrophages and attenuates alveolar bone loss in ligature-induced periodontitis

ObjectiveGenistein is a major isoflavone subclass of flavonoids found in soybean and a potent tyrosine kinase inhibitor. The present study aimed to assess the effect of genistein on the production of proinflammatory mediators in murine macrophages stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen associated with different forms of periodontal disease, and to evaluate its possible influence on alveolar bone loss in ligature-induced periodontitis using micro-computed tomography (micro-CT) analysis as well.DesignLPS was isolated from P. intermedia ATCC 25611 by using the standard hot phenol–water method. Culture supernatants were analyzed for nitric oxide (NO) and interleukin-6 (IL-6). Inducible NO synthase (iNOS) protein expression was evaluated by immunoblot analysis. Real-time PCR was carried out to measure iNOS and IL-6 mRNA expression. In addition, effect of genistein on alveolar bone loss was evaluated in a rat model of experimental periodontitis using micro-CT analysis.ResultsGenistein significantly attenuated P. intermedia LPS-induced production of iNOS-derived NO and IL-6 with attendant decrease in their mRNA expression in RAW264.7 cells. In addition, when genistein was administered to rats, decreases in alveolar bone height and bone volume fraction induced by ligature placement were significantly inhibited. Genistein administration also prevented ligature-induced alterations in the microstructural parameters of trabecular bone, including trabecular thickness, trabecular separation, bone mineral density and structure model index.ConclusionsWhile additional studies are required, we suggest that genistein could be utilized for the therapy of human periodontitis in the future.     

Effects of prostaglandin E2 on clonogenicity,proliferation and expression of pluripotent markers in human periodontal ligament cells

Background and objectiveBased on our earlier work on the response of periodontal ligament (PDL) cells to mechanical stress by induction of cyclooxygenase expression and production of prostaglandin PGE₂ that could regulate mineralization of PDL cells, it was hypothesized that PGE₂ had potential effects on PDL stemness. In this study, we aimed to investigate clonogenicity, proliferation and expression of certain pluripotent markers, considered to be characteristics of PDL stemness, in response to treatment with exogenously-added PGE₂.Material and methodsHuman PDL cells were cultured and treated with various doses of PGE₂, and the aforementioned characteristics of PDL stemness were analyzed.ResultsThe clonogenicity and proliferation were significantly enhanced by PGE₂ at low concentrations (0.01, 0.1 and 1 ng/ml; P < 0.05), but only the proliferation was significantly diminished by PGE₂ at a high concentration (100 ng/ml; P < 0.05). Expression of NANOG and OCT4 mRNA and protein was increased by PGE₂ treatment at 0.1 and 1 ng/ml. Consistently, expression of stage-specific embryonic antigen 4, a putative stem cell marker, was significantly augmented by PGE₂ treatment at 1 ng/ml (P < 0.05).ConclusionOur findings suggest that although a high dose of PGE₂ (100 ng/ml) inhibits proliferation of PDL cells, PGE₂ at low doses appears to play a role in the maintenance of PDL stemness.     

Prostaglandin E2-Transporting Pathway and Its Roles via EP2/EP4 in Cultured Human Dental Pulp

IntroductionProstaglandin E₂ (PGE₂) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE₂-transporting pathway in human dental pulp and explore the relevance of PGE₂ receptors (EP2/EP4) to angiogenesis and dentinogenesis.MethodsProtein localization of microsomal PGE₂ (mPGES)synthase, PGE₂ transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers.ResultsMRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1).ConclusionsPGE₂-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE₂-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE₂ binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.