Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway,but not through the p38 MAPK pathway

Zhang D, Zheng H, Zhao J, Lin L, Li C, Liu J, Pan Y. Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway, but not through the p38 MAPK pathway. J Periodont Res 2011; 46: 31–38. © 2010 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule‐1 (ICAM‐1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. Material and Methods: We examined the expression of ICAM‐1, IκBα, phospho‐p38 MAPK and nuclear factor‐kappaB (NF‐κB) p65 in an umbilical vein endothelial cell line (ECV‐304) treated with ATCC 33277 and W83, with or without the NF‐κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real‐time PCR, western blotting and immunofluorescence. Results: Both strains could induce ICAM‐1 expression; additionally W83 was able to increase ICAM‐1 expression more significantly than ATCC 33277. In P. gingivalis‐infected endothelial cells, both p38 MAPK and NF‐κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF‐κB. It was found that ICAM‐1 production in endothelial cells was abrogated by inhibition of the NF‐κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. Conclusion: The induction of ICAM‐1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF‐κB pathway, but not by the p38 MAPK pathway.     

PI3K/Akt mediates expression of TNF‐α mRNA and activation of NF‐κB in calyculin A‐treated primary osteoblasts

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor‐α (TNF‐α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF‐α and GAPDH mRNA were determined by RT‐PCR. Cell extracts were subjected to SDS‐PAGE and the activation of Akt and NF‐κB were analyzed by western blotting. Results: Calyculin A‐treatment markedly increased the expression of TNF‐α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF‐α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF‐κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF‐α mRNA and activation of NF‐κB. This NF‐κB activation involves both phosphorylation and nuclear translocation of NF‐κB.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

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.     

Evidence Linking the Role of Placental Expressions of Peroxisome Proliferator‐Activated Receptor‐γ and Nuclear Factor‐Kappa B in the Pathogenesis of Preeclampsia Associated With Periodontitis

Background: Peroxisome proliferator‐activated receptor (PPAR)‐γ activation leads to suppression of production of a broad range of proinflammatory molecules. It plays a role in differentiation of trophoblasts and helps in normal placentation and formation of vascular exchange interface. Activation of nuclear factor‐kappa (NF‐κ) B triggers proinflammatory molecules inducing abnormal placentation and premature labor. This study aims to explore expression of PPAR‐γ and NF‐κB in placentas of women with periodontitis‐associated preeclampsia compared with that in normotensive pregnant women. Methods: Fifty pregnant women were included. Twenty‐five were controls (normotensive pregnant women) and 25 were pregnant women with preeclampsia, including those with gestational hypertension. Demographic data, pregnancy characteristics, and periodontal parameters were recorded, including: 1) plaque index; 2) gingival index; 3) bleeding on probing (BOP); 4) probing depth; and 5) attachment loss (AL). Placental tissue samples were collected from both groups and analyzed to quantify expression of PPAR‐γ and NF‐κB using real‐time polymerase chain reaction. Results: BOP and AL were significantly higher in pregnant women with preeclampsia compared with normotensive pregnant women (P <0.05). Expression of PPAR‐γ was downregulated in patients with preeclampsia compared with that of healthy normotensive patients, which was statistically significant (P <0.05), whereas NF‐κB was significantly activated (P <0.05) in pregnant women with preeclampsia compared with normotensive pregnant women. Conclusions: Higher periodontal disease prevalence is found among pregnant women with preeclampsia, with increased percentage of sites with BOP and greater AL. This study provides novel information on host response to systemic inflammation induced by periodontal pathogens through mechanisms involving downregulation of PPAR‐γ and increased activation of NF‐κB.     

18β‐Glycyrrhetinic acid inhibits periodontitis via glucocorticoid‐independent nuclear factor‐κB inactivation in interleukin‐10‐deficient mice

Sasaki H, Suzuki N, AlShwaimi E, Xu Y, Battaglino R, Morse L, Stashenko P. 18β‐Glycyrrhetinic acid inhibits periodontitis via glucocorticoid‐independent nuclear factor‐κB inactivation in interleukin‐10‐deficient mice. J Periodont Res 2010; 45: 757–763. © 2010 John Wiley & Sons A/S Background and Objective: 18β‐Glycyrrhetinic acid (GA) is a natural anti‐inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study. Material and Methods: Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in interleukin‐10‐deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on lipopolysaccharide (LPS)‐stimulated macrophages, T cell proliferation and osteoclastogenesis was also examined in vitro. Results: 18β‐Glycyrrhetinic acid administered either prophylactically or therapeutically resulted in a dramatic reduction of infection‐induced bone loss in interleukin‐10‐deficient mice, which are highly disease susceptible. Although GA has been reported to exert its anti‐inflammatory activity via downregulation of 11β‐hydroxysteroid dehydrogenase‐2 (HSD2), which converts active glucocorticoids to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, in glucocorticoid‐free conditions, GA potently inhibited LPS‐stimulated proinflammatory cytokine production and RANKL‐stimulated osteoclastogenesis, both of which are dependent on nuclear factor‐κB. Furthermore, GA suppressed LPS‐ and RANKL‐stimulated phosphorylation of nuclear factor‐κB p105 in vitro. Conclusion: These findings indicate that GA inhibits periodontitis by inactivation of nuclear factor‐κB in an interleukin‐10‐ and glucocorticoid‐independent fashion.     

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.     

Porphyromonas endodontalis lipopolysaccharides induce RANKL by mouse osteoblast in a way different from that of Escherichia coli lipopolysaccharide

Introduction

Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts.

Methods

LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control.

Results

Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the up-regulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05).

Conclusions

Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS).     

Resveratrol attenuates the pathogenic and inflammatory properties of Porphyromonas gingivalis

Porphyromonas gingivalis has been strongly associated with chronic periodontitis, which affects tooth‐supporting tissues. This Gram‐negative anaerobic bacterium produces a repertoire of virulence factors that modulate tissue destruction directly or indirectly by the induction of inflammatory processes. The aim of this study was to investigate the effects of resveratrol, a major polyphenol found in grapes and wine, on the growth and virulence properties of P. gingivalis as well as on gingival keratinocyte tight junction integrity and the host inflammatory response. Resveratrol exhibited antibacterial activity that may result from damage to the bacterial cell membrane. Resveratrol also killed a pre‐formed P. gingivalis biofilm and reduced bacterial adherence to matrix proteins. In addition, resveratrol had a protective effect on the integrity of the keratinocyte tight junctions by inhibiting its breakdown by P. gingivalis. This may be related to the ability of resveratrol to inhibit the protease activities of P. gingivalis. Lastly, resveratrol reduced P. gingivalis‐mediated activation of the NF‐κB signaling pathway and attenuated TREM‐1 gene expression as well as soluble TREM‐1 secretion in monocytes. The effect on NF‐κB activation likely results from the ability of resveratrol to act as a PPAR‐γ agonist. In summary, the antibacterial, anti‐adherence, and antiprotease properties of resveratrol, as well as its ability to protect the gingival keratinocyte barrier and attenuate the inflammatory response in monocytes suggest that it may be a promising novel therapeutic agent for treating periodontal disease.     

Lack of Toll‐like receptor 4 decreases lipopolysaccharide‐induced bone resorption in C3H/HeJ mice in vivo

Introduction: Few in vivo studies have demonstrated whether Toll‐like receptor 4 (TLR4) is indispensable for lipopolysaccharide (LPS)‐induced bone resorption and little is known about the receptor activator of nuclear factor‐κB ligand (RANKL) and osteoprotegerin (OPG) expression induced by LPS under conditions of lack of TLR4. Methods: We compared bone resorption histomorphometrically in C3H/HeN and C3H/HeJ mice that were repeatedly injected with Actinobacillus actionmycetemcomitans LPS into their gingiva every 48 h. RANKL‐, interleukin‐1β‐ and OPG‐positive cells in the connective tissue were also compared immunohistochemically. Results: Bone resorption in C3H/HeJ mice in the fourth, seventh, and tenth injection groups was significantly less than that C3H/HeN mice (P < 0.05). The number of RANKL‐positive cells in C3H/HeJ mice in the 10th injection group was significantly smaller than that in C3H/HeN mice (P < 0.05). The numbers of interleukin‐1β‐positive cells in C3H/HeJ mice in the seventh and tenth injection groups were significantly decreased compared with those in C3H/HeN mice (P < 0.05). The numbers of OPG‐positive cells in C3H/HeN and C3H/HeJ mice gradually increased, but there was no significant difference between the two strains of mice. Conclusion: TLR4 is indispensable for LPS‐induced bone resorption in vivo.