Influence of clodronate and compressive force on IL-1ß-stimulated human periodontal ligament fibroblasts

Objectives

The aim of this study was to investigate in vitro the effect of clodronate on interleukin-1ß (IL-1ß)–stimulated human periodontal ligament fibroblasts (HPdLFs) with the focus on inflammatory factors of orthodontic tooth movement with and without compressive force.

Materials and methods

HPdLFs were incubated with 5 μM clodronate and 10 ng/mL IL-1ß. After 48 h, cells were exposed to 3 h of compressive force using a centrifuge. The gene expression of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), matrix metalloproteinase 8 (MMP-8), and the tissue inhibitor of MMP (TIMP-1) was analyzed using RT-PCR. Prostaglandin E2 (PGE-2), IL-6, and TIMP-1 protein syntheses were quantified via ELISA.

Results

Compressive force and IL-1ß induced an overexpression of COX-2 gene expression (61.8-fold; p < 0.05 compared with control), diminished by clodronate (41.1-fold; p < 0.05 compared with control). Clodronate slowed down the compression and IL-1ß induced IL-6 gene expression (161-fold vs. 85.6-fold; p < 0.05 compared with control). TNF-α was only slightly affected without statistical significance. Clodronate reduced IL-1ß-stimulated MMP-8 expression with and without compressive force. TIMP-1 on gene and protein level was downregulated in all groups. Analyzing the MMP-8/TIMP-1 ratio, the highest ratio was detected in IL-1ß-stimulated HPdLFs with compressive force (21.2-fold; p < 0.05 compared with control). Clodronate diminished IL-1ß-induced upregulation of MMP-8/TIMP-1 ratio with (11.5-fold; p < 0.05 compared with control) and without (12.5-fold; p < 0.05 compared with control) compressive force.

Conclusion

Our study demonstrates a slightly anti-inflammatory effect by clodronate under compressive force in vitro. Additionally, the periodontal remodeling presented by the MMP-8/TIMP-1 ratio seems to be diminished by clodronate.

Clinical relevance

Reduction of pro-inflammatory factors and reduction of periodontal remodeling might explain reduced orthodontic tooth movement under clodronate intake.

     

Statins regulate interleukin-1β-induced RANKL and osteoprotegerin production by human gingival fibroblasts

Stein SH, Dean IN, Rawal SY, Tipton DA. Statins regulate interleukin‐1β ‐ induced RANKL and osteoprotegerin production by human gingival fibroblasts. J Periodont Res 2011; 46: 483–490. © 2011 John Wiley & Sons A/S Background and Objective: Three‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase competitive inhibitors, or ‘statins’, are widely used for lowering cholesterol and thereby reducing the risk of a heart attack. Recent data suggest that statins influence metabolic bone activity by their actions on three molecules: RANKL; RANK; and osteoprotegerin (OPG), the soluble decoy receptor for RANKL. The purpose of this study was to evaluate OPG and RANKL production in resting and interleukin‐1β (IL‐1β)‐activated human gingival fibroblasts (HGFs), and to determine the effect of statins on their production. Material and Methods: Fibroblasts were pre‐incubated with atorvastatin or simvastatin for 24 h in serum‐free medium, and then incubated with IL‐1β for 6 d. The concentration of OPG or RANKL in culture supernatants was measured by specific ELISA. Data were analyzed using analysis of variance and Scheffe’s F procedure for post hoc comparison. Results: IL‐1β (1 × 10^?8 m ) stimulated a significant increase in the production of OPG on days 1, 3 and 6. There was a trend towards an increase in RANKL production as a result of stimulation with IL‐1β. Both statins, at multiple concentrations, significantly increased the constitutive RANKL/OPG ratio. Only atorvastatin at the highest concentration (5 × 10^?6 m ) significantly increased the IL‐1β‐stimulated RANKL/OPG ratio. Conclusion: IL‐1β significantly increased OPG production by HGFs. The statins differed minimally in their effects on OPG and RANKL production by resting and IL‐1β‐activated HGFs. Both statins increased constitutive RANKL/OPG ratios, but generally not IL‐1β‐stimulated ratios. Thus, statins may influence the production of RANKL and OPG by HGFs to favor bone catabolism, under noninflammatory conditions.     

Effects of glucosamine-chondroitin combination on synovial fluid IL-1β, IL-6, TNF-α and PGE2 levels in internal derangements of temporomandibular joint

Background

The aim of the present study was to evaluate the effects of glucosamine-chondroitin sulphate combination on internal derangements of temporomandibular joint in clinical and biochemical manners.

Material and Methods

This randomized clinical study included 31 cases reporting joint tenderness, in which disc displacement was detected on MR imaging. In all patients, synovial fluid sampling was performed under local anesthesia. In the study group, the patients were prescribed a combination of 1500 mg glucosamine and 1200 mg chondroitin sulphate, while patients in the control group were only prescribed 50 mg tramadol HCl (twice daily) for pain control. After 8 weeks, synovial fluid sampling was repeated in the same manner. The levels of pain, maximum mouth opening (MMO), synovial fluid IL-1ß, IL-6, TNF-α and PGE2 measured before and after pharmacological intervention were compared.

Results

The reduction in pain levels was significant in both groups. There was no significant difference between two groups in terms of pain reduction. The improvement in MMO was significant in the study group but it was not in the control group. The MMO improvement was significantly higher in the study group compared to the control group. In the study group, significant decrease was observed in PGE2 level, while the decreases in IL-1β, IL-6 and TNF-α levels were not significant. In the control group, no significant decrease was observed in any of the inflammatory cytokines after 8 weeks, moreover IL-1ß and IL-6 levels were increased. Alterations of IL-1ß and IL-6 levels were significant in study group while TNF-α and PGE2 levels were not, compared to control group.

Conclusions

In conclusion, these results might suggest that glucosamine-chondroitin combination significantly increases the MMO and decreases the synovial fluid IL1β and IL6 levels in internal derangements of TMJ compared to tramadol. The modifications of synovial fluid TNF-α and PGE2 levels do not reach statistical significance. This combination also provides efficient pain relief in similar level with tramadol, a narcotic analgesic. Key words: Chondroitin sulphate, glucosamine, internal derangement, TMJ, tramadol.     

Involvement of angiotensin II type 1 receptors in interleukin-1β-induced interleukin-6 production in human gingival fibroblasts

The renin-angiotensin system is thought to be involved in inflammatory processes such as periodontitis. However, its precise role is still unclear. Therefore, in the present study the expression of the angiotensin II type 1 receptor (AT1R) was investigated in inflamed human gingival tissue, and the possible involvement of the AT1R in interleukin-1β (IL-1β)-induced interleukin-6 (IL-6) production by cultured human gingival fibroblasts (HGFs) was also studied. Immunohistochemical staining revealed that inflammatory cells and fibroblast-like cells were positive for the AT1R. However, in healthy gingival tissue, AT1R staining was very weak. The levels of AT1R mRNA and AT1R protein increased in HGFs after stimulation with IL-1β. The levels of IL-1β-induced IL6 mRNA and IL-6 protein were significantly reduced in AT1R gene-silenced HGFs compared with control HGFs. The data suggest that the AT1R may be involved in the regulation of gingival inflammation by modulating IL-1β-induced IL-6 production in HGFs.     

Periodontal ligament and gingival fibroblasts participate in the production of TGF-β, interleukin (IL)-8 and IL-10

The aim of this study was to quantify and compare the production of transforming growth factor beta (TGF-β), interleukin (IL)-8 and IL-10 by human cultured periodontal ligament and gingival fibroblasts both obtained from the same donors challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Fibroblasts were exposed to 0.1-10 μg/mL of LPS from P. gingivalis and after 24 h the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). TGF-β protein production was upregulated in a concentration-dependent manner, mainly in gingival fibroblasts, which was statistically significant when challenged by 10 μg/mL LPS. Additionally, at this concentration, gingival fibroblasts had almost a two-fold increase in the amount of TGF-β when compared to periodontal ligament fibroblasts. Both periodontal ligament and gingival fibroblasts showed an increase in IL-8 production when challenged with 1 μg/mL and 10 μg/mL LPS. IL-10 production remained unaffected when challenged by any of the LPS concentrations tested in either periodontal ligament or gingival fibroblasts. Our results demonstrate that periodontal ligament and gingival fibroblasts when challenged by LPS from P. gingivalis with 24 h may play a critical role in producing TGF-β and IL-8 but not IL-10.     

Pulp capping materials exert an effect on the secretion of IL-1β and IL-8 by migrating human neutrophils

Pulp repair is a complex process whose mechanisms are not yet fully understood. The first immune cells to reach the damaged pulp are neutrophils that play an important role in releasing cytokines and in phagocytosis. The objective of this study was to analyze the effect of different pulp-capping materials on the secretion of interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) by migrating human neutrophils. Neutrophils were obtained from the blood of three healthy donors. The experimental groups were calcium hydroxide [Ca(OH)(2)], an adhesive system (Single Bond), and mineral trioxide aggregate (MTA). Untreated cells were used as control. Transwell chambers were used in performing the assays to mimic an in vivo situation of neutrophil chemotaxis. The pulp-capping materials were placed in the lower chamber and the human neutrophils, in the upper chamber. The cells were counted and the culture medium was assayed using ELISA kits for detecting and quantifying IL-1β and IL8. The data were compared by ANOVA followed by Tukey's test (p < 0.05). The secretion of IL-8 was significantly higher in all groups in comparison to the control group (p < 0.05). The adhesive system group showed higher IL-8 than the MTA group (p < 0.05). The secretion of IL-1β was significantly greater only in the MTA group (p < 0.001). It was concluded that only MTA is able to improve the secretion of IL-1β, and all materials tested increased IL-8 secretion. These results combined with all the other biological advantages of MTA indicate that it could be considered the material of choice for dental pulp capping.     

Salivary levels of IL-1β, IL-6, IL-8, and TNF-α in patients with burning mouth syndrome

Objective

To compare salivary IL-1β, IL-6, IL-8, and TNF-α levels between patients with burning mouth syndrome (BMS) and controls.

Design

Forty female patients with BMS (mean age: 61.6 ± 10.1 years) and 20 female control subjects (mean age: 65.1 ± 9.0 years) were included in the study. Unstimulated (UWS) and stimulated whole saliva samples (SWS) were collected and their flow rates were determined. Salivary IL-1β, IL-6, IL-8, and TNF-α levels and total protein concentration were also determined. Salivary transferrin level was determined to investigate the level of blood contamination in saliva samples. Gingival index of the subjects was also examined. Student's t-test, Pearson's correlation analysis, and analysis of covariance were used.

Results

No significant differences were found in the salivary levels of IL-1β, IL-6, IL-8, and TNF-α in BMS patients compared with controls. Salivary flow rates and their total protein concentrations did not differ significantly between the groups. The levels of salivary cytokines and total protein concentration correlated significantly with the level of blood contamination in both UWS and SWS.

Conclusion

There were no differences in the salivary levels of IL-1β, IL-6, IL-8, and TNF-α in BMS patients compared with controls. Cytokine levels in whole saliva were affected mainly by the amount of blood contamination.     

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.     

Induction of interleukin (IL)-1β, IL-10, IL-8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans

Trindade SC, Olczak T, Gomes‐Filho IS, Moura‐Costa LF, Cerqueira EMM, Galdino‐Neto M, Alves H, Carvalho‐Filho PC, Xavier MT, Meyer R. Induction of interleukin (IL)‐1β, IL‐10, IL‐8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans. J Periodont Res 2012; 47: 27–32. © 2011 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis, an anaerobic gram‐negative bacterium, is associated with chronic periodontitis. This study was undertaken to evaluate the production of interleukin (IL)‐1β, IL‐8 and IL‐10 by human peripheral blood mononuclear cells (PBMC) stimulated with P. gingivalis antigens and to assess the levels of serum immunoglobulin (Ig)G, IgA and IgG subclasses raised against P. gingivalis HmuY protein. Material and Methods: PBMC from patients with chronic periodontitis (CP) and from nonperiodontitis (NP) control subjects were stimulated with P. gingivalis antigens, and the cytokine levels in the culture supernatants were determined by ELISA. The specificity of serum antibodies raised against HmuY was analyzed by Western blotting and by ELISA. Results: Compared with the NP controls, the CP patients produced higher levels of total serum IgG and IgG1 specific for P. gingivalis HmuY. No differences were found between CP and NP groups in the production of IL‐1β and IL‐8 by PBMC stimulated with total P. gingivalis antigens. Only P. gingivalis lipopolysaccharide (LPS) induced higher levels of IL‐10 in the CP group. Higher levels of IL‐1β and IL‐10 were induced by HmuY than by other antigens derived from the wild‐type P. gingivalis strains. In contrast, total antigens derived from the hmuY‐deletion mutant strain induced the production of significantly higher levels of IL‐8 and significantly lower levels of IL‐1β. Conclusion: Our data suggest that P. gingivalis HmuY may be considered an immunogenic protein associated with host–pathogen interactions.     

Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β

Rawal SY, Dabbous MKh, Tipton DA. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β. J Periodont Res 2012; 47: 320–329. © 2011 John Wiley & Sons A/S Background and Objective: Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix‐degrading activities. Material and Methods: Fibroblasts were incubated with CBD in serum‐free medium for 1–6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor β and the extracellular matrix molecule fibronectin were measured by ELISA. Pro‐MMP‐1 and total MMP‐2 were measured by ELISA. Activity of MMP‐2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP‐2. Data were analysed using ANOVA and Scheffe’s F procedure for post hoc comparisons. Results: Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor β production by as much as 40% (p < 0.001), while higher concentrations decreased it by as much as 40% (p < 0.0001). Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP‐2 activity (p < 0.02). Conclusion: The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor β and fibronectin, while decreasing MMP production and activity.     

Effect of titanium surface on secretion of IL1β and TGFβ1 by mononuclear cells

Mononuclear cells play an important role in the modulation of healing. The characteristics of implant surface topography may alter the production of signaling molecules such as cytokines. The aim of this in vitro study was to evaluate the effects of commercially available titanium surface treatments on both cell viability and the secretion of the antagonist cytokines, IL1β and TGFβ1. Human mononuclear cells were cultured on 10 mm diameter commercially pure titanium (cpTi) disks that were prepared using a turning procedure (control = machined surface) and either acid etched or bio-anodized for 1-7 days. Adhered cells were investigated with respect to cell viability using an MTT assay, and cytokine production was verified using an ELISA assay. The results indicate that surface characteristics did not alter the cell viability at days 1 and 4, although the machined surface presented the highest absorbance values at day 7 (p = 0.0084). Cell viability was reduced throughout the time course for all analyzed surfaces (p < 0.05). On day 4, IL1β levels were significantly higher on bio-anodized compared to acid etched surfaces (p = 0.0097). TGFβ1 did not show differences among the surfaces at days 1 and 4. The responses of non-stimulated mononuclear cells to titanium surfaces suggest only modest effects of the surface treatment and roughness on pro-inflammatory cytokine (IL1β) release.     

Hydrogen sulfide synergistically upregulates Porphyromonas gingivalis lipopolysaccharide-induced expression of IL-6 and IL-8 via NF-κB signalling in periodontal fibroblasts

Objectives

The periodontal pathogen Porphyromonas gingivalis produces hydrogen sulfide (H₂S). H₂S in the oral cavity is positively correlated with periodontitis but the mechanism by which H₂S contributes to periodontal diseases is obscure. We investigated the effect of H₂S in combination with P. gingivalis lipopolysaccharide (LPS) on expression of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 in periodontal fibroblasts and the underlying mechanism of action.

Material and methods

Gingival fibroblasts (GFs) and periodontal ligament cells (PDLCs) were treated with different concentrations of the H₂S donor NaHS in the presence/absence of P. gingivalis LPS for different time periods. Expression of IL-6 and IL-8 was detected by real-time PCR and ELISA. The activity of nuclear factor-kappa B (NF-κB) signalling was investigated using western blotting, EMSA and pathway blockade assays.

Results

Real-time PCR and ELISA results showed that H₂S not only upregulated expression of IL-6 and IL-8 at mRNA and protein levels in a dose- and time-dependent manner, but also aggravated P. gingivalis LPS-induced expression of IL-6 and IL-8 in GFs and PDLCs. Western blotting and EMSA showed that NF-κB signalling was activated by NaHS, P. gingivalis LPS, and both, which was in accordance with the expression levels of IL-6 and IL-8 in GFs and PDLCs. These results were confirmed using a NF-κB pathway blockade assay.

Conclusions

H₂S synergistically upregulated P. gingivalis LPS-induced expression of IL-6 and IL-8 in GFs and PDLCs via activation of NF-κB signalling, which could promote the development of periodontitis.     

Effect of scaling and root planing on interleukin-1β, interleukin-8 and MMP-8 levels in gingival crevicular fluid from chronic periodontitis patients

Konopka ?, Pietrzak A, Brzezińska‐B?aszczyk E. Effect of scaling and root planing on interleukin‐1β, interleukin‐8 and MMP‐8 levels in gingival crevicular fluid from chronic periodontitis patients. J Periodont Res 2012; 47: 681–688. © 2012 John Wiley & Sons A/S Background and Objective: There are few data concerning the effect of scaling and root planing on the levels of immune and inflammatory mediators in gingival crevicular fluid from patients with chronic periodontitis. Therefore, in this study the influence of scaling and root planing was determined on amounts of interleukin (IL)‐1β, IL‐8 and MMP‐8 in gingival crevicular fluid from patients with chronic periodontitis, in relation to clinical parameters. Material and Methods: A total of 51 patients were enrolled in this study. The study population consisted of 30 patients with generalized advanced chronic periodontitis, while 21 periodontally healthy subjects were recruited for the control group. The clinical parameters included approximal plaque index, gingival index, pocket depth and clinical attachment loss. The amounts of IL‐1β, IL‐8 and MMP‐8 in gingival crevicular fluid were measured by ELISA. Periodontal parameters as well as gingival crevicular fluid humoral factor amounts were evaluated in the control group and in chronic periodontitis patients at baseline and at 1 and 4 wk after scaling and root planing treatment. Results: At baseline, there were significant differences between control subjects and chronic periodontitis patients in terms of clinical attachment loss, pocket depth, gingival index (p < 0.001) and approximal plaque index (p < 0.01). The amounts of IL‐1β, MMP‐8 (p < 0.001) and IL‐8 (p < 0.01) in gingival crevicular fluid were significantly lower in healthy subjects than in chronic periodontitis patients. Scaling and root planing led to improvement in all examined clinical parameters, apart from clinical attachment loss. Periodontal treatment also resulted in a significant decrease in the amounts of IL‐1β, IL‐8 and MMP‐8 in comparison to baseline, especially 4 wk after scaling and root planing (p < 0.001); however, the amounts of these humoral factors were still higher than those in control group. Conclusion: Our observations indicated that short‐term nonsurgical therapy resulted in a significant improvement in periodontal indices and in a marked decrease of IL‐1β, IL‐8 and MMP‐8 gingival crevicular fluid levels. Nevertheless, no significant correlations were found between clinical parameters and amounts of humoral factors after therapy.     

Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E(2) by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells

Kim Y‐S, Shin S‐I, Kang K‐L, Herr Y, Bae W‐J, Kim E‐C. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E₂ by hypoxia‐inducible factor‐1α up‐regulation in human periodontal ligament cells. J Periodont Res 2012; 47: 719–728. © 2012 John Wiley & Sons A/S Background and Objective: Although hypoxia‐inducible factor 1α (HIF‐1α) is up‐regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF‐1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF‐1α and on the production of its target genes, including cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂), MMP‐2 and MMP‐9 in PDLCs. Material and Methods: The expression of COX‐2 and HIF‐1α proteins was evaluated using western blotting. The production of PGE₂ and MMPs was evaluated using enzyme immunoassays and zymography, respectively. Results: LPS and nicotine synergistically induced the production of PGE₂, MMP‐2 and MMP‐9, and increased the expression of MMP‐2, MMP‐9, COX‐2 and HIF‐1α proteins. Inhibition of HIF‐1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS‐ and nicotine‐stimulated PGE₂ and MMPs, as well as the expression of COX‐2 and HIF‐1α. Furthermore, pretreatment with inhibitors of COX‐2, p38, extracellular signal‐regulated kinase, Jun N‐terminal kinase, protein kinase C, phosphatidylinositol 3‐kinase and nuclear factor‐kappaB decreased the expression of nicotine‐ and LPS‐induced HIF‐1α and COX‐2, as well as the activity of PGE₂ and MMPs. Conclusion: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF‐1α is a potential target in periodontal disease associated with smoking and dental plaque.