Prediabetes Enhances Periodontal Inflammation Consistent With Activation of Toll‐Like Receptor–Mediated Nuclear Factor‐κB Pathway in Rats

Background: Clinical studies have showed that prediabetes (preDM) is a predisposing factor for periodontitis. However, the pathogenic mechanism involved is unclear. Because it is known that the activation of Toll‐like receptor (TLR)‐mediated nuclear factor‐kappa B (NF‐κB) signaling pathway plays a crucial role in periodontitis, it is hypothesized that preDM enhances periodontal inflammation by activation of the TLR‐mediated NF‐κB pathway. Methods: In this study, a preDM rat model is established by feeding a high‐fat diet (HFD). HFD‐induced rats with preDM (n = 7) and normal chow–fed rats (n = 7) were studied. The animal model was characterized in terms of body weight and the glycemic and insulinemic profiles. The following parameters were assessed to evaluate possible early periodontal alterations and underlying mechanisms: 1) histology analysis of periodontal tissue; and 2) serum and mRNA levels and/or the tissue protein expression of TLRs, myeloid differentiation factor 88 (MyD88), tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6), NF‐κB, cytokines, advanced glucose ends (AGEs), and free fatty acids (FFAs). Results: Rats with preDM presented higher expression of TLR2 and TLR4 in periodontal tissue in the HFD group compared with the control group. The TLR2 and TLR4 was mostly expressed in gingiva, and TLR4 was expressed in periodontal ligament in rats. Furthermore, the MyD88 and TRAF6 protein levels were significantly increased in gingiva in rats with preDM compared with normal rats. The activity of NF‐κB signals was higher in rats with preDM than in normal rats. Regarding cytokines expression, the TNF‐α protein levels and interleukin‐1β mRNA levels were significantly increased in the HFD group compared with the control group. In the serum, AGEs levels were significantly increased in the rats with preDM. Mean FFAs concentrations were increased in rats with preDM compared with normal rats, but it did not reach statistical significance. Conclusion: In rats with preDM, TLR2 and TLR4 gene and protein levels were higher in periodontal tissue, and the activation of NF‐κB may, through TLRs/MyD88, cause more cytokine secretion, which is associated with the onset or development of periodontal disease.     

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.     

Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells

Lee S‐I, Kang K‐L, Shin S‐I, Herr Y, Lee Y‐M, Kim E‐C. Endoplasmic reticulum stress modulates nicotine‐induced extracellular matrix degradation in human periodontal ligament cells. J Periodont Res 2012; 47: 299–308. © 2012 John Wiley & Sons A/S Background and Objective: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine‐induced cytotoxicity and ECM degradation. Material and Methods: Cytotoxicity and cell death were measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT‐PCR and western blot analysis. Results: Treatment with nicotine reduced cell viability and increased the proportion of annexin V‐negative, propidium iodide‐positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase‐like ER‐resident kinase, phosphorylated eukaryotic initiation factor‐2α and glucose‐regulated protein‐78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP‐1, MMP‐2, MMP‐8 and MMP‐9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine‐induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor‐κB. Conclusion: These results indicate that nicotine‐induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.     

Streptococcus cristatus modulates the Fusobacterium nucleatum-induced epithelial interleukin-8 response through the nuclear factor-kappa B pathway

Zhang G, Chen R, Rudney JD.Streptococcus cristatusmodulates theFusobacterium nucleatum‐induced epithelial interleukin‐8 response through the nuclear factor‐kappa B pathway. J Periodont Res 2011; 46: 558–567.©2011 John Wiley & Sons A/S Background and Objective: We previously reported that the interleukin‐8 (IL‐8) response to Fusobacterum nucleatum was attenuated in the presence of Streptococcus cristatus. Here, we further examined the underlying mechanism(s) involved in the modulating effect of S. cristatus by looking specifically at its impact on the nuclear factor‐kappa B (NF‐κB) pathway under the toll‐like receptor (TLR) signaling background. Material and Methods: OKF6/TERT‐2 and KB cells were co‐cultured with F. nucleatum and S. cristatus, either alone or in combination. Secretion of IL‐8 protein was measured by ELISA. The nuclear translocation of NF‐κB was evaluated by confocal microscopy, while DNA‐binding activity was quantified using TransAM? ELISA kits. Western blot analysis was performed to determine whether the anti‐inflammatory effect of S. cristatus is related to the modulation of the NF‐κB inhibitory protein IκB‐α. Results: Incubation with F. nucleatum significantly enhanced the nuclear translocation of NF‐κB. Exposure to S. cristatus alone did not cause detectable NF‐κB translocation and was able to inhibit the F. nucleatum‐induced NF‐κB nuclear translocation. The TransAM assay further confirmed that S. cristatus blocked the nuclear translocation of NF‐κB in response to F. nucleatum stimulation. In contrast to the nearly complete degradation of IκB‐α induced by F. nucleatum alone, the presence of S. cristatus stabilized IκB‐α. Pre‐incubation with TLR2 and TLR4 antibodies, however, did not affect the epithelial response to either species alone or in combination. Conclusion: The mechanism by which S. cristatus attenuates F. nucleatum‐induced proinflammatory responses in oral epithelial cells appears to involve blockade of NF‐κB nuclear translocation at the level of IκB‐α degradation.     

High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts

Background: Diabetes mellitus (DM) is a major risk factor for periodontal disease and affects various cellular functions. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration; however, the effect of hyperglycemia on PDLSCs is unclear. The aim of this study is to investigate whether hyperglycemia affects periodontal tissue regeneration, using human PDLSCs and high‐glucose medium as a model of DM. Methods: PDLSCs were obtained from healthy adult human mandibular third molars. Cell proliferation, osteoblastic differentiation, and proinflammatory cytokine expression were investigated by culturing PDLSCs in media supplemented with four different glucose concentrations representative of control patients (5.5 mM), patients with postprandial or controlled DM (8.0 mM), and patients with uncontrolled DM (12.0 and 24.0 mM). The molecular effects of hyperglycemia on PDLSC physiology were examined with a focus on the nuclear factor (NF)‐(κB signaling pathway. The involvement of NF‐κB was investigated with a specific NF‐κB inhibitor in PDLSCs under hyperglycemic conditions. Results: High glucose levels inhibited PDLSC proliferation and differentiation into osteoblasts but induced NF‐κB activation and subsequent interleukin (IL)‐6 and IL‐8 expression. Treatment with an NF‐κB inhibitor rescued the defects in cell proliferation and osteoblastic differentiation and inhibited the IL‐6 expression caused by the high‐glucose environment. Conclusion: The results of this study demonstrate that hyperglycemia inhibits human PDLSC proliferation and osteoblastic differentiation.     

Moesin‐induced signaling in response to lipopolysaccharide in macrophages

Zawawi KH, Kantarci A, Schulze‐Späte U, Fujita T, Batista EL Jr, Amar S, Van Dyke TE. Moesin‐induced signaling in response to lipopolysaccharide in macrophages. J Periodont Res 2010; 45: 589–601.©2010 John Wiley & Sons A/S Background and Objective: Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane‐organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)‐mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS‐induced moesin signaling pathways in macrophages. Material and Methods: Macrophages were stimulated with 500 ng/mL LPS in macrophage serum‐free medium. For blocking experiments, cells were pre‐incubated with anti‐moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real‐time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD‐2 and Toll‐like receptor 4 (TLR4) and co‐immunoprecipitation of MyD88–interleukin‐1 receptor‐associated kinase (IRAK) and IRAK–tumor necrosis factor receptor‐activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor κB (NF‐κB) and IκBα were studied. Tumor necrosis factor α, interleukin‐1β and interferon β were measured by ELISA. Results: Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl‐terminus. Moesin is temporally associated with TLR4 and MD‐2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide‐induced signaling is transferred downstream to p38, p44/42 MAPK and NF‐κB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF‐κB activation and translocation to the nucleus. Conclusion: These results suggest an important role for moesin in the innate immune response and TLR4‐mediated pattern recognition in periodontal disease.     

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

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.     

Calcium Phosphate Particles Induce Interleukin‐8 Expression in a Human Gingival Epithelial Cell Line via the Nuclear Factor‐κB Signaling Pathway

Background: Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)‐8 in human gingival epithelial cells. Methods: Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9‐22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real‐time polymerase chain reaction analysis and enzyme‐linked immunosorbent assay, respectively. The activity of nuclear factor (NF)‐κB signaling was measured by an immunofluorescence assay to evaluate NF‐κB p65 nuclear translocation. Results: The results show that nano/microsized particles stimulate IL‐8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL‐8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40‐nm and 5‐μm diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF‐κB activity. Pretreatment with BMS‐345541, an NF‐κB signaling inhibitor, inhibited the particle‐mediated IL‐8 gene induction, suggesting a requirement for the NF‐κB signaling pathway. Conclusion: These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.     

Carbon monoxide releasing molecule‐3 inhibits concurrent tumor necrosis factor‐α‐ and interleukin‐1β‐induced expression of adhesion molecules on human gingival fibroblasts

Song H, Zhao H, Qu Y, Sun Q, Zhang F, Du Z, Liang W, Qi Y, Yang P. Carbon monoxide releasing molecule‐3 inhibits concurrent tumor necrosis factor‐α‐ and interleukin‐1β‐induced expression of adhesion molecules on human gingival fibroblasts. J Periodont Res 2011; 46: 48–57. © 2010 John Wiley & Sons A/S Background and Objective: Carbon monoxide releasing molecule‐3 (CORM‐3) is a newly reported compound that has shown anti‐inflammatory effects in a number of cells. In this study, we aimed to investigate the influence of CORM‐3 on concurrent tumor necrosis factor‐α (TNF‐α)‐ and interleukin (IL)‐1β‐induced expression of adhesion molecules on human gingival fibroblasts (HGF). Material and Methods: HGF were cultured from the explants of normal gingival tissues. Cells were costimulated with TNF‐α and IL‐1β in the presence or absence of CORM‐3 for different periods of time. The expression of adhesion molecules, nuclear factor‐kappaB (NF‐κB) and phosphorylated p38 was studied using western blotting. RT‐PCR was applied to check the expression of the adhesion molecules at the mRNA level. The activity of NF‐κB was analysed using a reporter gene assay. Results: CORM‐3 inhibited the up‐regulation of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule in HGF after costimulation with TNF‐α and IL‐1β, which resulted in the decreased adhesion of peripheral blood mononuclear cells to these cells. Sustained activation of the NF‐κB pathway by costimulation with TNF‐α and IL‐1β was suppressed by CORM‐3, which was reflected by a reduced NF‐κB response element‐dependent luciferase activity and decreased nuclear NF‐κB‐p65 expression. CORM‐3 inhibited MAPK p38 phosphorylation in response to stimulation with proinflammatory cytokines. Conclusion: The results of this study bode well for the application of CORM‐3 as an anti‐inflammatory agent to inhibit NF‐κB activity and to suppress the expression of adhesion molecules on HGF, which suggests a promising potential for CORM‐3 in the treatment of inflammatory periodontal disease.     

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats

Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti‐inflammatory effect. However, there are few reports about its anti‐inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell‐based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)‐alpha, interleukin (IL)‐1β, IL‐6, and IL‐8 were determined. The anti‐inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS‐stimulated hPDLCs via nuclear factor (NF)‐kappa B, p38/extracellular signal‐regulated kinase, and mitogen‐activated protein kinase/activator protein‐1 pathways. Moreover, in the LPS and ligature‐induced periodontitis rat model, BTZ suppressed expression of TNF‐α, IL‐1β, IL‐6, and IL‐8, reduced the ratio of receptor activator of NF‐κB ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti‐inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.