Correspondence: Noboru Yamaguchi, Department of Preventive Dentistry,Kyushu University Faculty of Dental Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: +81 92 642 6353; fax: +81 92 642 6354; e-mail: email@example.com
Previous epidemiologic studies have suggested that periodontal disease is closely related to obesity and glucose tolerance. As the level of adiponectin, an adipocyte-derived cytokine, in plasma had been reported to decrease in obese and type 2 diabetes patients, we explored the role of adiponectin in the etiology of periodontitis using the D clone of RAW264, a clone that exhibits highly efficient osteoclast formation, to determine whether adiponectin acts as a regulatory molecule in osteoclast formation stimulated by lipopolysaccharide of periodontopathic bacteria. We observed that adiponectin acted as a potent inhibitor of osteoclast formation stimulated by Toll-like receptor 4 (TLR4) ligand and receptor activator of NF-κB ligand (RANKL). Because NF-κB is an important transcription factor in osteoclast formation, we examined the effect of adiponectin on its transcriptional activity. A luciferase assay showed that adiponectin was able to inhibit the TLR4-mediated NF-κB activity in RAW264 cells. In addition, we observed that the cytokine was actually able to inhibit TLR4-mediated expression of the gene for inducible nitric oxide synthase and production of nitric oxide in the cells. These observations strongly suggest that adiponectin may function as a negative regulator of lipopolysaccharide/RANKL-mediated osteoclast formation in periodontal disease.
Obesity is a risk factor of systemic diseases, and has been related to these diseases and to adipocytokines [adiponectin, leptin, tumor necrosis factor alpha (TNF-α), etc.], which are physiologically active products derived from adipocytes. Adiponectin is abundantly present in the plasma of healthy humans (Arita et al., 1999). By contrast, it has been shown that the level of adiponectin mRNA and that of its protein in plasma are decreased in obese and type 2 diabetes patients (Hotta et al., 2000; Halleux et al., 2001).
Many studies have recognized that periodontitis is more prevalent in diabetic patients and worsens with the progression of the disease (Page et al., 1997; Rees, 2000). Based on the results of an epidemiologic survey, we previously found a relationship between obesity and periodontitis (Saito et al., 1998). Dalla Vecchia et al. (2005) also showed that obesity was significantly associated with periodontitis in adult women who were nonsmokers. Moreover, our recent epidemiologic study (Saito et al., 2004) showed that periodontal disease could be a risk factor for type 2 diabetes, given that deep periodontal pockets were closely related to the past development of glucose intolerance in nondiabetics. Iwamoto et al. (2003) investigated the effect of antimicrobial periodontal treatment on adiponectin levels in patients with chronic periodontitis. Therefore, it was of interest to explore the etiology of periodontitis with respect to adiponectin.
Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are periodontopathic bacteria that have largely been discussed with regard to the etiology of aggressive periodontitis (Zambon, 1985; Holt et al., 1988). Both species elaborate numerous components that may mediate adherence to mucosal surfaces, inhibit host defense mechanisms, and elicit gingival tissue destruction and alveolar bone resorption (Fives-Taylor et al., 1999; Holt et al., 1999). Lipopolysaccharide is considered to be a potent stimulator of bone loss in inflammatory diseases such as periodontitis and osteomyelitis and some types of arthritis (Ito et al., 1996; Nair et al., 1996; Abu-Amer et al., 1997; Ueda et al., 1998). In the present study, we chose A. actinomycetemcomitans lipopolysaccharide (Aa-LPS) as a potent inducer of osteoclast formation.
Oshima et al. (2005) recently reported that adiponectin inhibited the macrophage colony-stimulating factor (M-CSF)- and receptor activator of NF-κB ligand (RANKL)-induced differentiation of mouse bone marrow macrophages into osteoclasts. By contrast, we show here the inhibitory action of adiponectin for Aa-LPS/RANKL-induced osteoclast formation using the RAW264 D clone (D clone). In addition to showing that it was inhibitory in this regard, we also found that adiponectin was able to suppress Aa-LPS-stimulated NF-κB activity, gene expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in murine macrophage-like cells (RAW264).
Materials and methods
Cells and reagents
Murine macrophage-like cell line RAW264 (RCB0535; RIKEN Cell Bank) was maintained in RPMI 1640 medium (Sigma-Aldrich Corp.) supplemented with 10% fetal bovine serum (FBS; Thermo Trace Ltd), 2 mM l-glutamine and 50 μg mL−1 gentamicin. In order to investigate whether adiponectin would be able to inhibit lipopolysaccharide/RANKL-induced ostoclastogenesis, we used RAW264 cell clone D (D clone), a clone having a high ability to form osteoclasts (Watanabe et al., 2004). Cultures were maintained at 37°C under 5% CO2.
Aa-LPS was extracted from lyophilized cells of A. actinomycetemcomitans strain Y4 by the hot phenol/water procedure, treated with nuclease and washed extensively with pyrogen-free water by ultracentrifugation. The lipopolysaccharide preparation was purified by chromatography on Sephadex G-200 (GE Healthcare Bio-Sciences Corp.) equilibrated with 10 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl, 0.25% (w/v) deoxycholate, 1 mM EDTA and 0.02% (w/v) sodium azide (Yamaguchi et al., 1996). Lipopolysaccharide from Escherichia coli O111 : B4 (E-LPS) and tartrate-resistant acid phosphatase (TRAP) staining kit were purchased from Sigma-Aldrich Corp. Griess-Romijn nitrite reagent and sodium nitrite were purchased from Wako Pure Chemical Industries, Ltd. Soluble RANKL was obtained from PeproTech EC.
Culture conditions for forming osteoclast-like multinucleated cells from D clone
D clone cells were cultured in α-modified Eagle's medium containing 10 % FBS (6.8 × 103 cells in 150 μL per well for 96-well plates) for 3 days in the presence of 5−20 μg mL−1 adiponectin, 20 ng mL−1 soluble RANKL, and 1 μg mL−1 E-LPS or 1 μg mL−1 Aa-LPS.
Purification of recombinant protein
Glutathione S-transferase (GST) fusion vector [pGEX-6P-1 (GE Healthcare Bio-Sciences)] containing the globular domain of mouse 30-kDa adipocyte complement-related protein (gACRP30) was provided by Dr I. Shimomura (Osaka University, Osaka, Japan). Recombinant globular adiponectin (gAd) was prepared as described previously (Maeda et al., 2002). Briefly, GST-gACRP30 protein was produced in E. coli strain BL21 and purified using glutathione Sepharose 4B (GE Healthcare Bio-Sciences). GST was cleaved from GST-gACRP30 protein by PreScission Protease (GE Healthcare Bio-Sciences). The isolated protein was applied to an Affi-Prep polymyxin column (Bio-Rad Laboratories) to remove endotoxin contamination, as described previously (Yamaguchi et al., 1998). According to the cytotoxic assay using a propidium iodide staining and a flow cytometer, gAd did not exhibit the nonspecific toxic effects on RAW264 cells (data not shown).
NF-κB luciferase assay
RAW264 cells (2 × 106 per plate) were incubated with a mixture of pTKκB2Luc (reporter gene, 8 ng), pRL-TK (internal control, 2 ng) and PolyFect transfection reagent (80 μL; QIAGEN K.K.) for 24 h in 10-cm plastic plates with RPMI 1640 medium containing 10% FBS. These cells were harvested, placed in 24-well plastic plates, preincubated with various amounts of gAd, and then incubated for an additional 6 h with or without an lipopolysaccharide stimulant. Thereafter, the treated cells were lysed with Passive Lysis Buffer (Promega). The Dual-Luciferase Reporter Assay System (Promega) was used to quantify the expression of firefly luciferase and Renilla luciferase. Firefly luciferase activity was normalized to that for Renilla and presented as values relative to the control.
Real-time quantitative PCR
Total RNA (5 μg) from RAW264 cells was isolated using an RNeasy Plus Mini Kit (QIAGEN K.K.). The RNA samples were reverse transcribed to cDNA by use of Ready-To-Go You-Prime First-Strand Beads (GE Healthcare Bio-Sciences). β-Actin (internal control) and iNOS mRNA levels were determined by conducting quantitative real-time PCR assays. The probe consisted of an oligonucleotide coupled with a reporter dye (6-carboxyfluorescein; 6FAM) at the 5′ end of the probe and a quencher dye (6-carboxy-tetramethylrhodamine; TAMRA) at an internal thymidine. Following cleavage of the probe, reporter and quencher dyes become separated, resulting in an increased fluorescence of the reporter. Amplification and detection were performed with an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Japan Co., Ltd., Applied Biosystems Division) operated according to the following cycle profile: 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. PCR primers were designed from recently published sequences (Hinz et al., 2001) and were as follows: iNOS forward primer, 5′-TGCCCCTTCAATGGTTGGTA-3′; iNOS reverse primer, 5′-ACTGGAGGGACCAGCCAAAT-3′; iNOS TaqMan probe, 5′-(6FAM)CGCTACAACA(TAMRA)TCCTGGAGGAAGTGG-3′; β-actin forward primer, 5′-TCACCCA CACTGTGCCCATCTACGA-3′; β-actin reverse primer, 5′-GGATGCCACAGGATTCCATACCCA-3′; β-actin TaqMan probe 5′-(6FAM)TATGCTC(TAMRA)TCCCTCACGCCAT CCTGCGT-3′. Quantification of mRNA was performed by determining the threshold cycle as described previously (Hinz et al., 2001). iNOS mRNA levels were normalized to those of the housekeeping gene β-actin.
RAW264 cells (5 × 105) were preincubated with the desired amounts of gAd for 6 h in RPMI 1640 containing 5% FBS. The cells were then washed with phosphate-buffered saline (pH 7.3) and exposed to lipopolysaccharide for 24 h. The levels of NO in the culture supernatants were assessed by performing the Griess reaction (Sosroseno et al., 2004). Each culture supernatant (100 μL) was mixed with an equal volume of the Griess–Romijn nitrite reagent and read in a microplate reader (Benchmark, Bio-Rad Laboratories) at 540 nm. The nitrite concentration was calculated from a standard curve prepared with sodium nitrite.
Student's t-test was used to determine the statistical significance of differences between results obtained for the untreated control group vs. those from gAd-pretreated groups. For the real-time quantitative PCR assay, comparisons between groups were performed with a nonparametric statistical test (Wilcoxon signed-ranks test) using the absolute values from six independent experiments.
gAd inhibition of Aa-LPS/RANKL-induced osteoclast formation by RAW264 D clone
We previously demonstrated that adiponectin inhibits Toll-like receptor (TLR) family-induced signaling (Yamaguchi et al., 2005). In the present study, to study further the involvement of adiponectin in the initiation and progression of periodontitis, we directly investigated whether adiponectin could act as a potent inhibitor of osteoclast formation stimulated by TLR4 signaling. We previously isolated this as a clone that showed a high efficiency in forming osteoclasts from RAW264 cells (Watanabe et al., 2004). Lipopolysaccharide stimulated TRAP-positive osteoclast formation in D clone cells in a dose-dependent manner. The maximal number of osteoclasts was observed at 1 μg mL−1 of either Aa-LPS or E-LPS (data not shown). We then examined the effect of gAd on the Aa-LPS/RANKL-induced formation of TRAP-positive multinucleated cells (MNCs). As shown in Fig. 1, gAd (2.5−20 μg mL−1) strongly inhibited Aa-LPS- and RANKL-induced formation of osteoclast-like MNCs from the D clone cells. Lower concentrations (<2.5 μg mL−1) of gAd did not significantly inhibit Aa-LPS/RANKL-induced osteoclast formation (data not shown). By contrast, the significant inhibitory effect of heat-inactivated (100°C for 10 min) gAd (ΔI gAd) on Aa-LPS/RANKL-induced osteoclastogenesis was not observed (Fig. 1c).
gAd inhibition of Aa-LPS-induced NF-κB activation
Given that, as described above, adiponectin was found to be a potent negative regulator of lipopolysaccharide/RANKL-mediated osteoclast formation, we next measured Aa-LPS-induced NF-κB activity in RAW264 macrophages using a luciferase assay. We included E-LPS as a positive control for this system. The cells were pretreated with gAd (2.5−20 μg mL−1) for 6 h before the addition of Aa-LPS (0.3 μg mL−1) or E-LPS (0.3 μg mL−1) and were then incubated for 6 h. We observed that gAd markedly suppressed Aa-LPS-induced NF-κB activity as well as E-LPS-induced NF-κB activity (Fig. 2). Pretreatment with ΔI gAd did not affect LPS/RANKL-induced NF-κB activation (Fig. 2). The gAd treatment alone had little effect on NF-κB activation (data not shown).
gAd inhibition of Aa-LPS-induced iNOS mRNA expression and NO production
Finally, we examined whether gAd could inhibit lipopolysaccharide-induced iNOS mRNA expression and NO production in RAW264 cells. Our real-time PCR assay using TaqMan probes showed that gAd significantly attenuated LPS-induced iNOS mRNA expression (Fig. 3). Furthermore, gAd markedly inhibited LPS-induced NO production in RAW264 cells (Fig. 4). When the cells were pretreated with ΔI gAd, no changes in LPS/RANKL-induced iNOS mRNA expression or NO production were observed (Figs 3 and 4). These results suggest that adiponectin would also be able to exert an anti-inflammatory effect against A. actinomycetemcomitans infection in periodontal sites.
In this report, we provide the first evidence that adiponectin specifically inhibited lipopolysaccharide/RANKL-mediated osteoclastogenesis by D clone cells, suggesting that adiponectin manipulation could be therapeutically useful for patients with periodontal disease. We previously reported a positive relationship between obesity and periodontitis, based on the results of an epidemiologic study (Saito et al., 1998). The relative risk of periodontitis in a group with a body-mass index (BMI) of over 20 was much higher than that in a group with a BMI of <20. Among the many adipocyte-derived endocrine factors, we noted that the level of the adipocyte-derived plasma protein adiponectin was lower in obese subjects than in nonobese subjects (Arita et al., 1999). If adiponectin could reduce inflammation, this natural plasma component might act as an anti-inflammatory factor to prevent periodontitis. However, the actual role of adiponectin in periodontitis has not yet been demonstrated.
We previously showed that adiponectin functions as a negative regulator of lipopolysaccharide-induced NF-κB activation in murine macrophage-like cells (Yamaguchi et al., 2005). NO has been shown to contribute to immunologic responses given that NO produced by activated macrophages is involved in their tumoricidal and bactericidal actions (Stuehr & Nathan, 1989; Nathan & Hibbs, 1991). There seem to be direct and indirect pathways for macrophage activation by lipopolysaccharide: one is the MyD88-IRAK-TRAF6-TAK1-IKKB-mediated phosphorylation of IκB and TRAF6-mediated C-Jun N-terminal kinase (JNK), p38 activation; the other is an indirect activation of the Janus Kinase (JAK)/STAT pathway (Crespo et al., 2000; Toshchakov et al., 2002). The promoter of iNOS has been shown to possess STAT1 and NF-κB binding sites, and both seem to be required for full activation of this promoter in macrophages (Ganster et al., 2001). Furthermore, NO production from STAT1−/− and MyD88−/− macrophages was decreased compared with that from wild-type macrophages (Kawai et al., 1999; Ohmori & Hamilton, 2001). We have shown here that adiponectin inhibited lipopolysaccharide-mediated iNOS mRNA expression and NO production in macrophage-like cells (Figs 3 and 4). However, we have not yet identified the target molecules of adiponectin in TLR signaling pathways.
NF-κB plays an essential role in osteoclast differentiation (Franzoso et al., 1997; Iotsova et al., 1997). Several researchers have shown that NO can stimulate NF-κB-DNA binding in T cells (Lander et al., 1995), endothelial cells (Umansky et al., 1998) and macrophages (von Knethen et al., 1999). However, NO has also been suggested to be involved in the regulation of osteoclast activity. NO potentiates cytokine-induced bone resorption (Ralston et al., 1995), and high levels of NO can also inhibit bone formation (Damoulis & Hauschka, 1994; Ralston et al., 1994, 1995; Mancini et al., 2000). Gyurko et al. (2005) recently demonstrated that iNOS promotes bone resorption during bone development as well as after a bacterial infection. In our present study, adiponectin strongly blocked Aa-LPS/RANKL-induced osteoclast formation (Fig. 1) and significantly attenuated Aa-LPS-induced iNOS mRNA expression and NO production (Figs 3 and 4). Aa-LPS is known to induce murine calvarial bone resorption (Ishihara et al., 1991). Our preliminary data showed that Aa-LPS itself induced osteoclast formation by the D clone cells, although the extent of osteoclastogenesis was weaker than that induced by Aa-LPS/RANKL (data not shown). These data suggest that Aa-LPS and RANKL may synergistically work on osteoclastogenesis. Moreover, it is possible that Aa-LPS induces TNF-α production and that this cytokine may work together with RANKL to induce osteoclast formation. RANKL itself was also shown to induce the formation of MNCs, but not that of TRAP-positive cells by the D clone cells. Adiponectin was able to inhibit RANKL-induced MNCs as well (data not shown).
In conclusion, the results presented here indicate that adiponectin suppressed lipopolysaccharide/RANKL-mediated osteoclastogenesis. Our data suggest a novel function of adiponectin as a potent negative regulator of the initiation and progression of periodontitis. However, it still remains to elucidate the precise inhibitory mechanism of adiponectin in RANKL-induced osteoclast formation.
We thank Drs I. Shimomura and N. Maeda for generously providing mouse globular adiponectin cDNA. This study was supported by a grant (No. 17592184 to NY) from the program Grants-in-Aid for Scientific Research (C) of the Ministry of Education, Science, Sports, and Culture of Japan.