Research ArticleExperimental Studies

Effect of Melatonin on Cyclooxygenase-2 Expression and Nuclear Factor-Kappa B Activation in RAW264.7 Macrophage-like Cells Stimulated with Fimbriae of Porphyromonas gingivalis

YUKIO MURAKAMI, KENJI YUHARA, NAOKI TAKADA, TOMOE ARAI, SEIKO TSUDA, SAYAKO TAKAMATSU, MAMORU MACHINO and SEIICHIRO FUJISAWA
In Vivo July 2011, 25 (4) 641-647;

Abstract

Background: The possible link between melatonin and anti-inflammatory activity is currently a focus of interest. In the present study, COX-2 expression and NF-κB activation in RAW264.7 macrophage-like cells stimulated with the fimbriae of Porphyromonas gingivalis, an oral anaerobe, in the absence and presence of melatonin were investigated. Materials and Methods: The cytotoxicity of melatonin and indole against RAW264.7 cells was determined using a cell counting kit. The regulatory effect of melatonin, and of indole on the expression of COX-2 mRNA stimulated by exposure to the fimbriae was investigated by Northern blot analysis. NF-κB activation was evaluated by both electrophoretic mobility-shift assay and Western blot analysis. Results: The half maximal (50%) effective concentration (EC50) values for melatonin and indole were 3300 μM and 130 μM, respectively. Melatonin at non-cytotoxic concentrations significantly inhibited the fimbria-induced expression of COX-2. The fimbria-stimulated binding of NF-κB to its consensus sequence and phosphorylation-dependent proteolysis of inhibitor κB-α were markedly inhibited by melatonin. However, indole did not inhibit COX-2 expression and NF-κB activation. Conclusion: Melatonin may be able to prevent diseases induced by oral bacteria.

Melatonin is the major secretory product of the pineal gland and is mostly associated with regulation of the circadian dark/light rhythm of the human body (1). Recently, melatonin has also been recognized as a potent antioxidant and immunomodulator, and is considered to be an important natural oncostatic agent (1, 2). Melatonin enters saliva by passive diffusion from circulating blood. The function of melatonin as an antioxidant has recently received attention with regard to its involvement in oral cavity disorders, oxidative stress-related oral diseases, and periodontal inflammation (3-5). Melatonin may exert beneficial effects in certain types of oral pathology including periodontal disease, herpes viral infection and candidiasis, local inflammatory processes, xerostomia, oral ulcers and oral cancer (6). Such beneficial effects of melatonin may be related to its ability to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) (7).

Cyclooxygenase (COX)-2 is the key enzyme that catalyzes the two sequential steps responsible for biosynthesis of prostaglandins (PGs) from arachidonic acid. The inducible isoform of COX, namely COX-2, plays a critical role in the inflammatory response, and its overexpression has been associated with several types of pathology, including neurodegenerative diseases and various cancers. Using lipopolysaccharide (LPS)-activated RAW264.7 macrophages as a model, it has been shown that melatonin and its metabolites exert a suppressive effect on the activities of COX-2 and inducible nitric oxide synthase (iNOS) (8). In addition, it has been demonstrated that melatonin, but not tryptophan or serotonin, reduces the protein levels and promoter activities of LPS-induced COX-2 and iNOS in RAW264.7 cells in a time- and concentration-dependent manner (9). Furthermore, COX-2-dependent PGE2 down-regulates interleukin-1-alpha-induced matrix metallo-proteinase-13 (MMP-13) production via the E-type prostaglandin receptor EP1 in human periodontal ligament cells (10). Since PGE2 may be involved in regulating the destruction of extracellular matrix components in periodontal lesions, the above findings suggest that melatonin may help to prevent various oral diseases, and even neoplastic diseases such as precancerous leukoplakia, lichen planus, and oral cancer (6). Although melatonin has been studied in relation to oral diseases, its mechanism of action remains unclear.

We have previously reported that the dimer of butylated hydroxyanisole (bis-BHA: 3,3’-di-tert-butyl-5,5’-dimethoxy-1,1’-biphenyl-2,2’-diol), a potent antioxidant, exerts a preventive effect against activator protein-1 (AP-1) activation and COX-2 expression in macrophages stimulated with fimbriae of Porphyromonas gingivalis. This experimental model using macrophages has been shown to be reliable for evaluating the anti-inflammatory activity of phenolic antioxidants (11). A possible etiologic link between Buerger disease and chronic infections such as those due to oral bacteria has been reported previously (12). Therefore, it would be of interest to determine whether COX-2 expression and nuclear factor kappa B (NF-κB) activation could be suppressed by the antioxidant action of melatonin in RAW 264.7 cells stimulated with P. gingivalis fimbriae. If this were the case, then melatonin might contribute not only to the prevention of oral diseases, but also the regeneration of alveolar bone through stimulation of type I collagen fiber production and modulation of osteoblastic and osteoclastic activity derived from cellular mediator proteins such as receptor activator of NF-κB ligand (RANKL) caused by P. gingivalis and related bacteria. Melatonin may also help to prevent chronic infections induced by oral bacteria that are possibly linked to the initiation of atherosclerosis. Recently, we have investigated the radical-scavenging activity of melatonin and related compounds, and found that melatonin is a potent antioxidant (13).

In the present study, we investigated whether indole hormone melatonin and indole are able to inhibit P. gingivalis fimbria-induced COX-2 expression and NF-κB activation.

Materials and Methods

Materials. Melatonin and indole were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan. The chemical structures of melatonin and indole are shown in Figure 1. Test samples were prepared by dissolving melatonin and indole in dimethyl sulfoxide, and then the indicated concentration was reached by dilution with serum-free RPMI-1640 (Sigma-Aldrich Co., Japan). A Megaprime DNA labeling system, 5’-[α-32P]dCTP, and [γ-32P]ATP were purchased from Amersham Biosciences Co. (Piscataway, NJ, USA). A 5’-end labeling system was purchased from Promega Co. (Madison, WI, USA). A mouse COX-2 cDNA probe with a length of approximately 1.2 kbp was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). A 25-mer β-actin oligonucleotide (single-stranded DNA) probe was purchased from GeneDetect.com Ltd. (Bradenton, FL, USA). COX-2 goat polyclonal antibody, β-actin rabbit polyclonal antibody, and horseradish peroxidase (HRP)-conjugated mouse anti-goat IgG were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Phospho-specific anti-alpha of inhibitory kappa B (IkB-α) antibody (recognizing phospho-Ser 32) and anti-IκB-α, both rabbit polyclonal antibodies, as well as HRP-conjugated goat anti-rabbit IgG, and a Phototope-HRP Western blot detection kit were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). RPMI-1640 was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA).

Figure 1.
Figure 1.

The chemical structures of melatonin and indole.

Cell culture. The murine macrophage-like cell line RAW264.7, obtained from Dainippon Sumitomo Pharma Biomedical Co. Ltd. (Osaka, Japan), was used. The cells were cultured to a subconfluent state in RPMI-1640 medium supplemented with 10% FBS at 37°C and 5% CO2 in air, washed, and then incubated overnight in serum-free RPMI-1640. They were then washed again and treated with the test samples.

Cytotoxicity. The relative number of viable cells was determined using a Cell Counting Kit-8 (CCK-8) (Dojindo Co., Kumamoto, Japan) (14). In brief, RAW264.7 cells (3×104 per well) were cultured in NUNC 96-well plates (flat-well-type microculture plates) for 48 hours, after which the cells were incubated with test samples for 24 hours. CCK-8 solution was added to each well and then the absorbance was measured at 450 nm with a microplate reader (Biochromatic, Helsinki, Finland). The 50% cytotoxic concentration (CC50) was determined from the dose–response curves. Data are expressed as means of three independent experiments. Statistical analyses were performed by Student's t-test.

Preparation of P. gingivalis fimbriae. P. gingivalis ATCC33277 were prepared and purified from cell washings by the method of Yoshimura et al. (15). As documented previously, purified fimbria-induced biological activities were not attributable to LPS contaminants in the preparation (16, 17). Viability of the cells after exposure to the fimbriae at the concentrations used was over 90% by CCK-8. The protein content of the fimbriae was measured by the method of Smith et al. (18).

Northern blot analysis. The procedure employed was similar to that reported previously (19). Briefly, 106 cells were placed in Falcon 5-cm-diameter dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and pretreated for 30 min with the melatonin or indole (at 10, 100, 1000 μM). They were then incubated in the presence or absence of fimbriae (4 μg/ml), and their total RNA was prepared 3 h later by the acid guanidine-phenol-chloroform procedure (20). The RNA was electrophoresed in 1% agarose gels with 0.2 M sodium phosphate as a running buffer, and then blotted onto nylon membranes (Micron Separations, Inc., Westboro, MA, USA). The membranes were then hybridized with a COX-2 cDNA probe labeled with 5’-[alpha-32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences Co.) and a β-actin oligonucleotide probe labeled with [γ-32P]ATP using a 5’-end labeling system purchased from Promega Co. After hybridization, the membranes were washed and dried, then exposed overnight to Kodak X-ray film (Eastman Kodak Co., Rochester, NY, USA) at −70°C. β-Actin was used as an internal standard for quantification of total RNA in each lane of the gel. Quantification of COX-2 expression was carried out by densitometry. The data were expressed as the relative signal intensity (percentage of maximum).

Western blot analysis. Cells in Falcon 5-cm-diameter dishes (106 cells per dish) were treated with test samples. The cells were then solubilized with lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% [v/v] Triton X-100, 2.5 mM sodium pyrophosphate, 2 mM Na3VO4, 10 mM NaF, 1 mM β-glycerophosphate, 1 μg/ml aprotinin, 1 mM phenyl-methylsulfonyl fluoride [PMSF]). Protein concentrations were measured by the method of Smith et al. (18). Each sample (10 μg of protein) was subjected to SDS-PAGE in a 12.5% polyacrylamide gel, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Co., Bedford, MA, USA). The blots were then blocked with 5% skim milk, washed and incubated with anti-COX-2 antibody as the primary antibody diluted 1:1000 in working solution (5% bovine serum albmim, 1× TBS [50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl] and 0.1% Tween 20) at 4°C. β-Actin antibody was used at 0.1 μg/ml after dilution with working solution. After incubation, the blots were treated at room temperature with HRP-conjugated secondary antibody diluted 1:4000. Proteins were detected with a Phototope-HRP Western blot detection kit (Cell Signaling Technology, Inc.), and the blots were exposed to Kodak X-ray film for 10 min. β-Actin was used as a loading control in each lane of the gel.

Preparation of nuclear extract and electrophoretic mobility-shift assay (EMSA). Nuclei were extracted and prepared for the gel mobility-shift assay as reported previously (19). In brief, the cells in Falcon 15-cm-diameter dishes (107 cells per dish) were pretreated for 30 min with or without the indicated doses of melatonin or indole and then treated with the fimbriae at 4 μg/ml for 1 h. Thereafter, the cells were scraped into phosphate-buffered saline, pelleted, and suspended in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 10 mM NaCl and 0.5% Nonidet P-40. The nuclei were separated from the cytosol by centrifugation at 3,000 × g for 15 min. The extracted nuclei were then treated with buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT]) and further treated by stirring for 60 min at 47°C in buffer B (20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 0.2 mM EDTA, 0.42 M NaCl, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF). Nuclear extracts were obtained by centrifugation for 60 min at 25,000 ×g and demineralized through a Sephadex G-25 column equilibrated with buffer C (20 mM HEPES [pH 7.9], 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF). Protein concentrations were measured using a method reported previously (19). Binding reactions were performed for 20 min at room temperature with 10 μg of the nuclear proteins in 2 mM Tris (pH 7.5) containing 8 mM NaCl, 0.2 mM EDTA, 0.8% (v/v) glycerol, 0.2 mM DTT, 0.5 mM PMSF, 1 μg of poly (dI-dC) and 20,000 cpm of 32P-labeled NF-κB oligonucleotide in a final volume of 20 μl. Poly (dI-dC) and nuclear extract were incubated at 47°C for 10 min before addition of the labeled oligonucleotide. Double-stranded oligonucleotide containing a tandem repeat of the consensus sequence for the respective binding site, -GGGGACTTTCCC- for NF-κB was end-labeled by the T4 polynucleotide kinase and [γ-32P]ATP method. DNA-protein complexes were electrophoresed in native 6% polyacrylamide gel in 0.25× Tris borate EDTA buffer (22 mM Tris-HCl [pH 8.0], 22 mM boric acid, 0.6 mM EDTA). The gel was then dried, and exposed to Kodak X-ray film at −70°C.

Figure 2.
Figure 2.

The cytotoxicity of melatonin and indole on RAW264.7 cells. The procedures employed are described in the Materials and Methods. The results are presented as the means±standard error (SE). SE <15%. There was a significant difference between melatonin and indole for each concentration of 100 μM and 1000 μM. *p<0.001.

Results

Cytotoxicity. First, using CCK-8, we tested the cytotoxicity of melatonin and indole to RAW264.7 cells. The results are shown in Figure 2. The half maximal (50%) effective concentration (EC50) of melatonin and indole was 3300 μM and 130 μM, respectively. The cytotoxicity of melatonin was 25-fold lower than that of indole.

COX-2 inhibition. We then investigated the inhibitory effects of melatonin and indole on P. gingivalis fimbria-stimulated COX-2 mRNA expression in RAW264.7 cells. Figure 3 shows that melatonin at non-cytotoxic concentrations significantly inhibited the fimbria-induced expression of COX-2. In contrast, the inhibitory effect of indole was not complete within a concentration range of 10-100 μM. At 1,000 μM, melatonin and indole inhibited COX-2 expression completely. The inhibitory effect of indole at this concentration may have been at least partly attributable to cytotoxicity, as the EC50 is 130 μM. Furthermore, using Western blotting, we tested whether melatonin and indole inhibited fimbria-induced COX-2 production. Figure 4 shows that melatonin inhibited the production of COX-2 strongly, whereas indole at 100 μM had only a weak inhibitory effect. These results suggested that in RAW264.7 cells, melatonin exerts a markedly stronger inhibitory effect on COX-2 expression than indole.

Figure 3.
Figure 3.

Regulatory effect of melatonin and indole on fimbria-stimulated expression of the COX-2 gene in RAW 264.7 cells. Left hand panel: The cells were pretreated for 30 min with the indicated dosages of melatonin or indole. They were then incubated for 3 h with or without fimbriae at 4 μg/ml, and their total RNA was prepared. Northern blot analysis was performed with radiolabeled COX-2 cDNA and β-actin antisense oligonucleotide probes. Three independent experiments were performed and similar results were obtained. Right hand panel: Bar graph showing the level of COX-2 mRNA. The results are presented as means±standard error (SE) of three independent experiments. SE <15%. A significant difference between samples at 10 μM and 100 μM for melatonin and indole was observed for inhibition of COX-2 gene expression. *p<0.001.

Inhibition of NF-κB activation. To clarify whether melatonin and indole are inhibitors of fimbria-stimulated NF-κB, we examined their inhibitory effects on the binding of NF-κB to its consensus sequence in fimbria-stimulated RAW cells using EMSA. As shown in the left hand panel of Figure 5, the fimbria-stimulated binding of NF-κB was markedly inhibited by melatonin, whereas indole inhibited the binding only weakly. In addition, the fimbria-stimulated binding of NF-κB was completely inhibited by an unlabeled oligonucleotide (data not shown). We also investigated whether melatonin was able to inhibit fimbria-stimulated phosphorylation-dependent proteolysis of IκB-α in these cells. As shown in the right hand panel of Figure 5, melatonin clearly inhibited both the phosphorylation and degradation of IκB-α stimulated by the fimbriae. These findings suggest that melatonin is a potent inhibitor of fimbria-triggered cellular signaling in RAW264.7 cells.

Figure 4.
Figure 4.

Regulatory effects of melatonin and indole on fimbria-stimulated expression on COX-2 protein in RAW 264.7 cells. The cells were pretreated for 30 min with or without the indicated doses of melatonin or indole, and then incubated with or without the fimbriae at 4 μg/ml. The cells were solubilized with lysis buffer at 6 h after the start of treatment. Equal amounts of cell lysates were analyzed by Western blotting after SDS-PAGE with anti-COX-2 antibody. The blotting analysis was performed to determine the level of COX-2 protein. Three independent experiments were performed, and similar results were obtained.

Figure 5.
Figure 5.

Inhibition of fimbria-stimulated NF-κB activation by melatonin and indole in RAW264.7 cells. Left hand panel: Inhibitory effects of melatonin and indole on the fimbria-stimulated NF-κB binding. The cells were pretreated for 30 min with or without the indicated doses of melatonin or indole, and then incubated with or without the fimbriae at 4 μg/ml for 1 h. A gel mobility shift assay was performed with the nuclear proteins and 32P-labeled oligonucleotide containing the NF-κB consensus sequence. Three independent experiments were performed, and similar results were obtained. Right hand panel: Inhibitory effects of melatonin and indole on phosphorylation-dependent proteolysis of fimbria-stimulated IκB-α. The cells were pretreated for 30 min with or without melatonin or indole at 10 μM and then incubated with or without the fimbriae at 4 μg/ml. Equal amounts of cell lysates were analyzed by Western blotting after SDS-PAGE with phospho-specific anti-IκB-α antibody, anti-IκB-α antibody or anti-β-actin antibody. Three independent experiments were performed, and similar results were obtained.

Discussion

Melatonin at 1 mM was previously reported to exert a moderate cytotoxic effect on CMK, Jurkat and MOLT-4 cells (21). In the present study, the EC50 for melatonin on RAW264.7 cells was approximately 3 mM. The cytotoxic effects of melatonin on RAW264.7 cells appeared to be of a magnitude similar to that on the above leukemia cells, which was associated with ROS (21). In the present study, the cytotoxicity of melatonin on RAW264.7 cells was markedly less than that of indole. The cytotoxicity of chemicals is related to their lipophilicity, i.e. log P, the octanol:water partition coefficient (22). In general, as log P increases, then so does the cytotoxicity. Log P for melatonin and indole is reported to be 1.2 (23) and 2.14 (22), respectively. The difference in cytotoxicity between melatonin and indole observed in the present study may be related to this difference in lipophilicity. On the other hand, the effect of melatonin reducing the generation of superoxide may be involved in its cytotoxicity, and in fact, melatonin has been reported to reduce ischemia/reperfusion-induced superoxide generation in arterial walls (24). By contrast, it has also been reported that indole dissolves in membrane lipids, causing membrane disruption that enables direct interaction with redox-cycling isoprenoid quinones and dioxygen, due to the generation of superoxide (25). Thus it appears there is a considerable difference between melatonin and indole in terms of superoxide generation, the inhibitory effect of melatonin being at least partly attributable to its potent antioxidant activity.

Our results showed that melatonin, but not indole, significantly inhibited expression of the COX-2 gene induced by exposure to P. gingivalis fimbriae. In addition, melatonin, but not indole, inhibited the fimbria-stimulated phosphorylation-dependent proteolysis of IκB-α and the transcriptional activity of NF-κB in the cells. Deng et al. reported that melatonin, but not tryptophan or serotonin, inhibited the level of COX-2 and iNOS proteins in RAW264.7 cells after treatment with LPS or LPS/interferon γ (9). In the same study, they also demonstrated that melatonin, but not tryptophan or serotonin, inhibited iNOS promoter activity. The most well-known derivative of indole is the amino acid tryptophan, the precursor of the neurotransmitter serotonin. Melatonin, serotonin and tryptophan share a common backbone. Among them, however, melatonin alone exhibits anti-inflammatory activity, and a requirement for the side chains of melatonin for inhibition of COX-2 and iNOS expression at both the protein and gene levels has been suggested. Structurally, melatonin is composed of an indole ring with a methoxy group at position 5 and an acylaminoethyl side chain at position 3, whereas indole has no side chains. The two side chains on the indole ring may play an important role in the inhibition of COX-2 and iNOS expression.

The adhesion of P. gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds and invades epithelial cells, and the fimbriae are known to be intrinsically involved in the first step of this process (26). The dynamic systemic proinflammatory cellular response to localized periodontal bacteria can occasionally lead to widespread organ damage or death. We have previously demonstrated that fimbriae bind to cellular receptors (27), induce the production of proinflammatory cytokines (16, 28), and stimulate bone resorption in vitro through signal transduction mechanisms (17, 29). Interestingly, P. gingivalis fimbriae as well as LPS can stimulate COX-2 expression in RAW264.7 cells. We have also reported the anti-inflammatory effect of phenolic antioxidants such as bis-BHA, a dimer of the synthetic antioxidant BHA, which was able to inhibit AP-1 transcriptional activity stimulated by P. gingivalis fimbriae in RAW264.7 cells (11). Furthermore, bis-BHA significantly inhibits fimbria-induced expression of the COX-2 gene followed by phosphorylation-dependent proteolysis of IκB-α and transcriptional activity of NF-κB (30). By contrast, BHA exerts no anti-inflammatory effect, possibly due to its pro-oxidative action (30). The potent anti-inflammatory effect of bis-BHA may be due to its high antioxidant activity. Bis-BHA inhibits NF-κB activation through inactivation of IκB-α kinase, leading to down-regulation of COX-2 synthesis. Similarly, dimeric ferulic acid, but not monomeric ferulic acid, inhibits LPS-induced COX-2 gene expression in RAW 264.7 cells (31). The existing data related to the anti-inflammatory activity of phenolic dimers suggest that the activity may be at least partly attributable to their potent antioxidant action. Melatonin scavenges a variety of reactive oxygen and nitrogen species, including the hydroxyl radical, hydrogen peroxide, singlet oxygen, nitric oxide and peroxynitrite anion (1). Melatonin also scavenges alkyl and peroxy radicals to a markedly higher degree than indole (13). The potent anti-inflammatory activity of melatonin may lead to a high degree of endogenous radical-scavenging and anti-inflammatory activity.

In conclusion, melatonin, but not indole, has been shown to significantly inhibit the expression of the COX-2 gene induced by exposure to the fimbriae of P. gingivalis through suppression of NF-κB activation in RAW264.7 cells. These findings suggest that melatonin may be able to prevent periodontopathic bacteria-induced oral diseases and chronic infection in the body.

  • Received January 25, 2011.
  • Revision received April 4, 2011.
  • Accepted April 5, 2011.

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Effect of Melatonin on Cyclooxygenase-2 Expression and Nuclear Factor-Kappa B Activation in RAW264.7 Macrophage-like Cells Stimulated with Fimbriae of Porphyromonas gingivalis
YUKIO MURAKAMI, KENJI YUHARA, NAOKI TAKADA, TOMOE ARAI, SEIKO TSUDA, SAYAKO TAKAMATSU, MAMORU MACHINO, SEIICHIRO FUJISAWA
In Vivo Jul 2011, 25 (4) 641-647;
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