Nicotine stimulates the expression of cyclooxygenase-2 mRNA via NFκB activation in human gingival fibroblasts

doi:10.1016/j.archoralbio.2008.11.006

Archives of Oral Biology

Volume 54, Issue 3, March 2009, Pages 251-257

https://doi.org/10.1016/j.archoralbio.2008.11.006 Get rights and content

Abstract

Nicotine, a major component in tobacco smoke, stimulates the synthesis of prostaglandin E₂. We investigated the involvement of the transcription nuclear factor κ B (NFκB) in the nicotine-induced expression of cyclooxygenase-2 (COX-2), a key enzyme for prostaglandin synthesis, in human gingival fibroblasts. Nicotine-stimulated release of prostaglandin E₂ and expression of COX-2 mRNA in a time- and dose-dependent manner. The nicotine-stimulated release of prostaglandin E₂ and expression of COX-2 mRNA and protein were inhibited by an NFκB inhibitor, pyrrolidine dithiocarbamate (PDTC), by approximately 50%. Nicotine stimulation of IκBα, an inhibitor of NFκB degradation, was also characterized by Western blotting. Mecamylamine, a specific antagonist of the nicotinic acetylcholine receptor, failed to inhibit the effect of nicotine on prostaglandin E₂ release. When human gingival fibroblasts were incubated with [³H]-nicotine, uptake of nicotine was observed. These results suggest that nicotine is taken up by human gingival fibroblasts and that it then stimulates COX-2 expression via the activation of NFκB and the subsequent release of prostaglandin E₂.

Introduction

Tobacco has been recognized as a significant risk factor for the development and progression of periodontal disease.1, 2, 3 Tobacco smokes directly affect periodontal tissues because the oral cavity is an area exposed to the smoke. More than 4000 chemicals are present in tobacco smoke and they significantly contribute to the progression of periodontal disease and to delayed healing after periodontal therapy.⁴

Nicotine is a major component of tobacco smoke and is recognized for its modulation of the pathological effects of tobacco. Nicotine inhibits the attachment and growth of human periodontal ligament fibroblasts.⁵ In human gingival fibroblasts, nicotine inhibits cell growth and the production of fibronectin, collagen and extracellular matrix proteins, and increases collagenase activity.⁶ Nicotine also provokes the expression of multiple cytokines, including inflammatory cytokines such as interleukin-6 (IL-6) and IL-8, in human gingival fibroblasts.7, 8 Therefore, nicotine has been considered to cause the development and progression of periodontal disease via augmented destruction of the gingival extracellular matrix and the expression of cytokines.

One common feature in the pathogenesis of various smoking-associated diseases is inflammation. In inflammation processes, prostaglandin E₂ is considered likely to play crucial roles, since chemical mediators invoke prostaglandin E₂ synthesis. Cyclooxygenase (COX) catalyzes the first step in the synthesis of prostanoids from arachidonic acid. Two forms of COX have been characterized, a constitutively expressed form (COX-1) and an inducible form (COX-2).⁹ COX-1 may be responsible for basal prostanoid biosynthesis and is necessary for the maintenance of physiological functions. In contrast, COX-2, which is rapidly induced by a variety of mitogenic and inflammatory stimuli, may produce the prostanoids involved in immune and/or inflammatory responses. In human gingival fibroblasts, it has been previously demonstrated that the expression of COX-2 mRNA and protein is stimulated by nicotine.10, 11

Nuclear factor κ B (NFκB) is a transcription factor that plays a regulatory role in inflammation. Activation of NFκB is caused by the phosphorylation of specific serine residues of an inhibitory protein, inhibitor of NFκB (IκBα), by IκB kinase (IKK), which then allows the active form of NFκB to translocate into the nucleus to initiate gene transcription.¹² Cytokines such as IL-1β and TNF-α have been demonstrated to activate NFκB.13, 14 In human gingival fibroblasts, the involvement of NFκB activation in COX-2 mRNA expression stimulated by the proinflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α) has been demonstrated.15, 16

In this study, we investigated the involvement of NFκB activation in the nicotine-induced production of prostaglandin E₂ and the expression of COX-2 mRNA and protein in order to elucidate the mechanism of nicotine effects on prostaglandin E₂ synthesis in human gingival fibroblasts.

Section snippets

Materials

Nicotine was purchased from Sigma (St. Louis, MO), IL-1β was from R&D Systems (Minneapolis, MN) and [³H]-nicotine was from GE Healthcare UK Ltd. (Buckinghamshire, England). D-Minimal essential medium (D-MEM) and One-Step Reverse transcription-polymerase chain reaction (RT-PCR) with Platinum Taq were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was from Irvine Scientific (Santa Ana, CA) and RNeasy was from QIAGEN (Tokyo, Japan). The monoclonal anti-COX-2 antibody and the

Nicotine-induction of prostaglandin E₂ release

We first determined the effect of nicotine on prostaglandin E₂ release from human gingival fibroblasts. When human gingival fibroblasts were incubated with 5 mM nicotine, prostaglandin E₂ release was detected after 6 h and increased up to 24 h (2.5 ± 0.26 ng/ml, Fig. 1A). When the cells were incubated with various concentrations of nicotine for 6 h, 1–10 mM nicotine induced prostaglandin E₂ release in a dose-dependent manner (Fig. 1B). The choice of nicotine concentration used in subsequent studies

Discussion

Nicotine has been previously reported to provoke COX-2 expression in human gingival fibroblasts.10, 11 In this study, we showed that nicotine induces the release of prostaglandin E₂ and simultaneously provokes COX-2 expression in human gingival fibroblasts, which suggests that the nicotine-induced release of prostaglandin E₂ is caused by COX-2 expression, as previously demonstrated in the case of IL-1β and TNF-α stimulation.15, 16

In the presence of the NFκB inhibitor PDTC, the nicotine-induced

Acknowledgements

This study was supported in part by a Smoking Research Foundation Grant and by a Grant-in Aid for Scientific Research from JSPS (#17592166).

Funding: Smoking Research Foundation Grant. Grant-in Aid for Scientific Research from JSPS (#17592166).

Conflict of interest: No conflict of interest declared.

Ethical Approval: Ethics Committee of Nihon University School of Dentistry at Matsudo (No. EC 03-041).

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The median (interquartile range [IQR]) quality assessment score of the 42 assessed papers was 8.0 [1.0], out of a maximum possible score of 15. The lowest score was six [38,39] and the highest score was nine [14,16,19,20,22,32–37,40–45]. Regarding the key methodological domains assessed by the modified CONSORT checklist, all of the studies failed to achieve: sample size determination, random sequence generation, allocation concealment, implementation details, blinding and publication of the full study protocol.
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This report followed the PRISMA statement. Two reviewers performed a literature search up to October 2018 in 3 databases: MEDLINE, EMBASE and Web of Science, supplemented by manual searching. Inclusion criteria comprised: in vitro studies evaluating human gingival fibroblasts, human periodontal ligament cells or human gingival/oral epithelial cells; nicotine exposure as a variable; including an appropriate control (no nicotine); published in English. Quality assessment was based on a 15-item checklist.
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According to findings from in vitro studies, nicotine, at levels found in tobacco smokers, nicotine replacement therapy users and e-cigarette users, is unlikely to be cytotoxic to human gingival and periodontal cells, though saliva levels in smokeless tobacco users may be high enough to achieve cytotoxicity. There was limited and contradictory evidence for nicotine effects on cell attachment, proliferation and inflammatory mediator production.
It is well established that whole tobacco smoke is highly damaging to oral tissues. The specific effects of nicotine are not well understood but are of increasing importance given the recent popularity of novel nicotine products. Increased knowledge on this topic will help to better inform dental professionals and patients.
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Nicotine stimulates the expression of cyclooxygenase-2 mRNA via NFκB activation in human gingival fibroblasts

Abstract

Introduction

Section snippets

Materials

Nicotine-induction of prostaglandin E2 release

Discussion

Acknowledgements

J Am Dent Assoc

Biomed Pharmacother

Eur J Pharmacol

Anal Biochem

Eur J Pharmacol

J Pharm Sci

Cigarette smoking in a periodontal practice

J Periodontol

Exposure to tobacco smoking and periodontal health

J Clin Periodontol

The impact of cigarette smoking on periodontal disease and treatment

Periodontol 2000

Effects of the tobacco products on the attachment and growth of periodontal ligament fibroblasts

J Periodontol

Effects of nicotine on proliferation and extracellular matrix production of human gingival fibroblasts in vitro

J Periodontol

Regulation of cytokine production in human gingival fibroblasts following treatment with nicotine and lipopolysaccharide

J Periodontol

Nicotine and lipopolysaccharide affect cytokine expression from gingival fibroblasts

J Periodontol

Cyclooxygenase: structural cellular, and molecular biology

Annu Rev Biochem

Induction of cyclooxygenase-2 mRNA and protein expression in human gingival fibroblasts stimulated with nicotine

J Periodont Res

Regulation of nicotine-induced cyclooxygenase-2 protein expression in human gingival fibroblasts

Acta Pharmacol Sin

The IL-1 receptor signaling pathway

J Leukoc Biol

Nicotine-induction of prostaglandin E₂ release