Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells

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Abstract

Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E2 (PGE2) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE2-producing enzymes, cyclooxygenase-2 and microsomal PGE2 synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-κB) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-κB pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

Highlights

► OxLDL-induced responses in human gingival epithelial cells were studied. ► OxLDL enhanced the production of IL-8, IL-1β and PGE2 in Ca9-22 cells. ► An NF-κB inhibitor suppressed the expression of COX-2 and mPGES1 induced by oxLDL. ► Unlike the case in macrophages, oxLDL did not increase the CD36 level.

Introduction

Epidemiological studies have demonstrated a critical link between periodontitis and cardiovascular disease. For example, patients with acute myocardial infarction have significantly higher risks for periodontitis, and vise versa [1], [2]. It was reported that the levels of total cholesterol, triglycerides and low-density lipoprotein (LDL)-cholesterol in blood from periodontal patients were higher than from normal subjects [3], and the ratio of total cholesterol to high-density lipoprotein (TC/HDL) was associated with changes in the gingival index in the hyperlipidemic group [4]. Chronic periodontitis is associated with an increased incidence of coronary heart disease (CHD) among young men which is independent of established cardiovascular risk factors [5]. Successful treatment of periodontitis was shown to significantly improve endothelial function and other surrogate markers of CHD [6]. Improved oral hygiene and non-surgical periodontal treatment are reportedly effective in decreasing plasma oxidized LDL (oxLDL), and are positively associated with a significant reduction in circulating reactive oxygen species [7]. However, the mechanistic relationship between the development of periodontitis and abnormalities in lipid metabolism is not well understood.

Periodontal tissue is composed of gingival tissue and underlying jaw bone. The former is constituted of an epithelial layer and connective layer surrounding the dental necks, and the latter is comprised of alveolar bone, which supports the teeth. Periodontitis is a chronic inflammatory disease elicited by the accumulation of dental plaque and infection by periodontopathic bacteria, which together ultimately result in the destruction of periodontal tissues. The elevated production in gingival tissues of inflammatory mediators, such as interleukin-8 (IL-8) and prostaglandin E2 (PGE2), is associated with both the initiation and progression of periodontitis [8].

An increased oxLDL level in plasma is a well established risk factor for cardiovascular disease. In addition to the induction of foam cell formation by macrophages, oxLDL also enhances the production of pro-inflammatory cytokines and chemokines in endothelial cells [9]. Various studies, including our own, have shown that oxLDL exist in atherosclerotic lesions and in circulating plasma [10], [11], [12]. Recently, it was shown that auto-antibodies against oxLDL were significantly higher in periodontitis patient [13], implicating oxLDL in the progression of periodontal disease. We discovered that oxLDL is present in human gingival crevicular fluid (GCF) from healthy sites, and this ratio of oxLDL/LDL appeared to be higher than in peripheral plasma [14]. These observations led us to investigate whether oxLDL, a well-known inflammatory stimulant in vascular cells, played a role in the inflammatory responses which take place in the gingival tissues. In this study, we show that treatment of the human gingival epithelial cell line Ca9-22 with oxLDL induced the production of IL-8 and PGE2. Similar oxLDL-induced inflammatory responses were also demonstrated in primary human oral keratinocytes (HOK). In addition, we show that the nuclear factor- kappa B (NF-κB) pathway is involved in oxLDL-induced IL-8 and PGE2 production in gingival epithelial cells.

Section snippets

Cells and reagents

Ca9-22 cells and primary HOK from gingival mucosa tissue were purchased from RIKEN BioResource Center (Ibaraki, Japan, RCB1976) and ScienCell (Carlsbad, CA, USA, #2610), respectively. BAY11-7082, an inhibitor of NF-κB, was purchased from Calbiochem-Merck (Darmstadt, Germany). The antibodies (Abs) to cyclooxygenase-2 (COX-2), CD36, inhibitor of kappa B-α (IkB-α) and β-actin were purchased from Santa Cruz Biotechnology Inc. (CA, USA), and the Abs for microsomal PGE2 synthase-1 (mPGES1) and mPGES2

Effect of OxLDL on the IL-8 and PGE2 production in cells of the human gingival epithelial cell line Ca9-22

When Ca9-22 cells were incubated with 50 μg/mL of oxLDL for 6 h, IL-8 production was up-regulated 7.5-fold higher than in those treated with native LDL (Fig. 1A). Under the same conditions, oxLDL induced an up-regulation of IL-1β and PGE2 production which was 3-fold higher than the control in Ca9-22 cells (Fig. 1B and C). It was reported that the COX-2 protein is expressed in the periodontal tissues of patients with periodontitis [15], and also that it is induced by the treatment of human

Discussion

It is established that oxLDL induces cellular inflammatory responses in vessel walls, and our previous finding that oxLDL is present in GCF [14] strongly suggests that oxLDL may have a role as inflammatory stimulant in periodontitis. Inflammatory responses in periodontal tissues are characterized by the production of cytokines and inflammatory mediators. However, the effects of oxLDL on epithelial cells have not been reportedly well studied and the mechanism of these inflammatory responses

Acknowledgments

This work was supported in part by grants-in-aid from the Ministry of Education, Technology, Sports, Sciences and Technology of Japan (MEXT) (Nos. 21592634, 21792128, 21890257, 21590073 and 21790092), grants for the Private University High Technology Research Center Projects from the MEXT, Research on Publicly Essential Drugs and Medical Devices from Japan Health Sciences Foundation, and Showa University Research Grant for Young Researchers. Pacific Edit reviewed the manuscript prior to

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