Rankl Upregulation Associated With Periodontitis And Porphyromonas Gingivalis

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RANKL Upregulation Associated With Periodontitis and Porphyromonas gingivalis Nawarat Wara-aswapati,* Rudee Surarit,† Anek Chayasadom,*† Jason A. Boch,‡ and Waranuch Pitiphat§

Background: Receptor activator of nuclear factor-kappa B (NFkB) ligand (RANKL) and osteoprotegerin (OPG) are critical for homeostatic control of osteoclast activity, suggesting their vital roles in the progression of bone loss in periodontitis. In this study, the expression of RANKL and OPG mRNA and the relationship between these factors and periodontopathic bacteria in periodontal tissue were studied. Methods: Gingival tissue and subgingival plaque samples were collected from 15 patients with chronic periodontitis and 15 periodontally healthy subjects. RNA was extracted from the tissue and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for RANKL or OPG. b-actin was amplified as a control to ensure equal loading. The intensity of RT-PCR products was analyzed by a densitometer in proportion to the intensity of b-actin. The numbers of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were determined by quantitative real-time PCR. Results: Our results showed increased levels of RANKL mRNA in chronic periodontitis tissues. The RANKL/OPG expression ratio was significantly higher in the periodontitis group compared to the healthy control group (P = 0.001). Interestingly, the expression of RANKL (r = 0.64; P <0.001), but not OPG (r = -0.24; P = 0.20), was significantly correlated with increased numbers of P. gingivalis. A. actinomycetemcomitans was detected in only 6.7% of all sites. Conclusions: Chronic periodontitis was associated with RANKL mRNA upregulation and increased RANKL/OPG mRNA expression ratio. In addition, our data showed for the first time to our knowledge an association between upregulated RANKL levels and the number of P. gingivalis in clinically obtained periodontal tissues. J Periodontol 2007;78:1062-1069. KEY WORDS Bacteria; osteoprotegerin; periodontal disease; Porphyromonas gingivalis; receptor activator of nuclear factor kappa B ligand.

* Department of Periodontology, Faculty of Dentistry, Khon Kaen University, Khon Kaen, Thailand. † Department of Physiology and Biochemistry, Faculty of Dentistry, Mahidol University, Bangkok, Thailand. ‡ Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA. § Department of Community Dentistry, Faculty of Dentistry, Khon Kaen University.

C

hronic periodontitis is an inflammatory disease caused by Gram-negative periodontopathic bacteria. Porphyromonas gingivalis, Tannerella forsythensis, and Actinobacillus actinomycetemcomitans have been shown to be among the major etiologic agents.1 These organisms express a number of potential virulence factors and induce host inflammatory mediators, eventually leading to connective tissue breakdown and alveolar bone resorption. Osteoclasts are the main cell type responsible for bone resorption and require receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL) for their formation.2,3 RANKL is a transmembrane molecule of the tumor necrosis factor (TNF) ligand superfamily that is expressed in osteoblasts, stromal cells, and T cells.2-5 RANKL-/- mice exhibit a phenotype with defective tooth eruption and severe osteopetrosis associated with the absence of osteoclasts, supporting the essential role of RANKL in osteoclast differentiation.3 Osteoprotegerin (OPG) is a natural inhibitor of RANKL that plays an important role in the homeostatic control of osteoclast activity.6,7 OPG is able to bind to RANKL and neutralize its activity by inhibiting the cell-to-cell signaling between osteoblast/bone stromal doi: 10.1902/jop.2007.060398

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cells and osteoclast precursor cells, resulting in the inhibition of osteoclast formation. OPG-/- mice show severe osteoporosis, resulting from unopposed RANKL activity, which leads to excessive osteoclast differentiation and activity.8,9 Recent studies10-13 have shown the effects of several components of P. gingivalis and A. actinomycetemcomitans on the mRNA expression of RANKL and OPG. However, the association between periodontopathic bacteria and these factors has not been examined using clinically obtained tissues. In this study, we compared the levels of RANKL and OPG mRNA in chronic periodontitis tissues to those in healthy control tissues. In addition, we studied the relationship between these molecules and the levels of specific periodontopathic bacteria. MATERIALS AND METHODS Study Population and Clinical Examination The Khon Kaen University and Mahidol University review committee approved the protocol for human subjects. Thirty subjects were enrolled during a 2-month period (September to October 2005) at the Faculty of Dentistry, Mahidol University. After having signed informed consent forms, samples of gingival tissues and subgingival plaque were obtained from 15 periodontally healthy individuals (control group) and 15 patients with chronic periodontitis. Patients with chronic periodontitis were diagnosed according to the 1999 International Workshop for a Classification of Periodontal Diseases and Conditions14 and had ‡14 natural teeth. The patients exhibited at least four sites in different teeth with probing depth (PD) ‡6 mm and clinical attachment loss (CAL) ‡3 mm. In addition, at least one tooth was planned for extraction because of severe periodontal destruction. All subjects were systemically healthy. The PD, CAL, and bleeding on probing (BOP) of the sampling sites were evaluated using a periodontal probe.i Samples were collected from sites with the deepest PD from each subject. Sampling of Gingival Tissue and Subgingival Plaque For both groups, teeth were gently dried with sterile cotton swabs. After removing supragingival plaque by cotton pellets and air-drying, subgingival plaque was collected with two sterile paper points inserted into the bottom of the periodontal pocket or gingival crevice for 20 seconds. The paper points were stored at -80C until DNA extraction. Gingival tissue samples from patients with chronic periodontitis were collected during extraction of teeth because of severe periodontal destruction and inflammation. Periodontally healthy gingival tissue samples were obtained during extraction of teeth

for orthodontic treatment. Gingival tissue was immediately submerged in RNA stabilization reagent¶ and stored at -80C until RNA extraction. Evaluation of mRNA Expression of RANKL and OPG Total RNA from gingival tissue was extracted using an RNA isolation kit# according to the manufacturer’s instructions and treated with DNase.** RNA purity was confirmed with the spectrophotometric absorbance ratio at 260/280, and RNA quantity was estimated by the absorbance at 260 nm.†† Reverse transcription-polymerase chain reaction (RT-PCR) was performed with an RT-PCR kit‡‡ using a thermal cycler.§§ Specific oligonucleotides were synthesizedii based on the published sequences of RANKL (sense 59-CTATTTCAGAGCGCAGATGGAT-39, antisense 59-TATGAGAACTTGGGATTTTGATGC-39); OPG (sense 59-TGACAAATGTCCTCCTGGTA-39, antisense 59TGTGTTGCATTTCCTTTCTG-39); and b-actin (sense 59-TGACGGGGTCACCCACACTGTGCCCATCTA-39, antisense 59-CTAGAAGCATTTGCGGTGGACGATGGAGGG-39). A dynamic test and cycle determination of RT-PCR protocols were carried out to determine the linear range of products and ensure an accurate semiquantitative analysis. The RNA was reverse transcribed by a reaction at 50C for 30 minutes followed by 95C for 20 minutes. There were 35 cycles of denaturation at 94C for 1 minute, annealing at 60C for 45 seconds (65C, 1 minute for b-actin), and primer extension at 68C for 45 seconds (72C, 1.5 minutes for b-actin). The reactions were finally extended at 72C for 10 minutes. The blank control (RT-PCR without RNA template) and RT(-) reactions (PCR without reverse transcription) were executed along with all RT-PCRs. The expected PCR products for RANKL, OPG, and b-actin were 557, 420, and 661 base pairs (bp), respectively. The PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. For b-actin, the PCR products were diluted 10-fold before loading. Two series of experiments were carried out for each tissue sample to ensure reproducibility. Quantitative analysis was performed using photographs and image analysis software.¶¶ The relative amount of RANKL and OPG mRNA expression was calculated as its ratio to b-actin from the same template.

i ¶ # ** †† ‡‡ §§ ii ¶¶

PCPUNC 15 periodontal probe, Hu-Friedy, Leimen, Germany. RNAlater, Qiagen, Valencia, CA. Rneasy Mini Kit, Qiagen. Rnase-free DNase set, Qiagen. Spectronic 601, Milton Roy, Rochester, NY. One-step RT-PCR kit, Qiagen. GeneAmp PCR system 2400, Perkin-Elmer/Roche, Branchburg, NJ. Qiagen. Quality One software, Bio-Rad Laboratories, Hercules, CA.

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Evaluation of Number of A. actinomycetemcomitans and P. gingivalis Plaque samples were suspended in 1 ml sterile double-distilled water, pelleted, and resuspended in 200 ml DNA isolation reagent.## Total DNA was extracted according to the manufacturer’s instructions. The suspension was centrifuged, and 5 ml of resulting supernatant was used for real-time PCR. The real-time PCR (Syber green) was performed using a detection system.*** The reactions were carried out as previously described15 using oligonucleotide primers specific for P. gingivalis (sense 59-CTTGACTTCAGTGGCGGCAG-39, antisense 59-AGGGAAGACGGTTTTCACCA-39) and A. actinomycetemcomitans (sense 59-CTTACCTACTCTTGACATCCGAA-39, antisense 59-ATGCAGCACCTGTCTCAAAGC-39). The PCR included 40 cycles of 95C for 15 seconds and 60C for 1 minute, with an initial cycle of 95C for 10 minutes. To determine the quantitative range of real-time PCR, DNA was prepared from 10 to 107 cells of cultured P. gingivalis and A. actinomycetemcomitans. Statistical Analysis Characteristics of the patients with periodontitis and controls were compared using the x2 test for categorical variables and the t or Mann-Whitney U tests for continuous variables. The Mann-Whitney U test was used to identify whether differences in the levels of RANKL and OPG and RANKL/OPG ratio existed between subject groups. Linear regression analysis adjusted for age and gender was used to determine the association of periodontitis with the levels of RANKL, OPG, and RANKL/OPG ratio. The gene expression data were log-transformed to achieve normal distribution in the regression analyses. Spearman correlation coefficient was calculated to evaluate the correlation between RANKL, OPG and RANKL/OPG expression with number of bacteria. Two-tailed P <0.05 was considered statistically significant. RESULTS Fifteen periodontally healthy individuals (control group; two men and 13 women; mean age, 34.8 – 16.0 years) and 15 patients with chronic periodontitis (nine men and six women; mean age, 51.9 – 13.1 years) were included in the study. Patients with periodontitis were significantly older (P <0.01) and more likely to be men (P <0.01) than the control group. Table 1 summarizes the clinical characteristics of the sampling sites. The mean PD, mean CAL, and proportion of BOP-positive sites were significantly higher in the periodontitis group than in the control group. Expression of RANKL mRNA and OPG mRNA was detected in all tissue samples (Fig. 1A). The median level of RANKL mRNA in the periodontitis group (1.63; interquartile range, 0.91 to 2.07) was signifi1064

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Table 1.

Characteristics of the Study Subjects Comparison Group Characteristic

Control Group Periodontitis (n = 15) Group (n = 15)

P Value

Age in years (mean – SD)

34.8 – 16.0

51.9 – 13.1

<0.01

Female [n (%)]

13 (86.7)

6 (40.0)

<0.01

PD* (mm; mean – SD)

2.7 – 0.6

7.4 – 1.8

<0.001

CAL* (mm; mean – SD)

1.9 – 0.7

10.4 – 2.0

<0.001

BOP* [n (%)]

5 (33.3)

15 (100.0)

<0.001

* Measured at the sampling sites.

cantly higher than in the control group (0.60; interquartile range, 0.44 to 1.30; P = 0.001; Fig. 1B). In contrast, the median level of OPG mRNA expression was lower in the periodontitis group (0.87; interquartile range, 0.51 to 1.14) than in the control group (1.21; interquartile range, 0.75 to 1.56), although the difference was not statistically significant (P = 0.25; Fig. 1C). When the ratio of RANKL to OPG mRNA was calculated, it was found to be significantly higher in the periodontitis group (1.53; interquartile range, 1.01 to 2.44) compared to the control group (0.69; interquartile range, 0.48 to 1.00; P = 0.001; Fig. 1D). Additional regression analyses to control the effects of age and gender confirmed these results. Periodontitis was associated with increased RANKL (P = 0.004) and RANKL/OPG ratio (P <0.001) but not with OPG (P = 0.07). The number of A. actinomycetemcomitans and P. gingivalis in the study sites was evaluated by realtime PCR. A. actinomycetemcomitans was detected in only 6.7% of all sites. Therefore, we cannot examine the relationship between this microorganism and the mRNA expression levels of RANKL and OPG for statistical significance. We further studied the association of RANKL and OPG mRNA levels with the number of P. gingivalis. It was shown that the upregulation of RANKL mRNA was positively correlated with the number of P. gingivalis (r = 0.64; P <0.001; Fig. 2A). However, no significant correlation was observed between the expression level of OPG mRNA and the number of P. gingivalis (r = -0.24; P = 0.20; Fig. 2B). The RANKL/OPG expression ratio showed a ## InstaGene Matrix, Bio-Rad Laboratories. *** GeneAmp 7000 Sequence Detection System, PE Applied Biosystems, Foster City, CA.

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Figure 1. Significant differences are demonstrated in the mRNA levels of RANKL and RANKL/OPG ratio in gingival tissues between the control and periodontitis groups. A) Amplified bands of RANKL, OPG, and b-actin of representative samples from control (lanes 1 through 3) and periodontitis (lanes 4 through 6) groups. B through D) The relative mRNA levels of RANKL, OPG, and RANKL/OPG ratio, respectively. The boxes represent the interquartile range, with the lower edge and upper edge corresponding to the 25th and 75th percentiles, respectively. The horizontal line within each box represents the median expression level in each group. Circles indicate outliers.

significantly positive correlation with the number of P. gingivalis (r = 0.67; P <0.001; Fig. 2C). DISCUSSION Periodontitis is a chronic inflammatory disease caused by periodontopathic bacteria including P. gingivalis, T. forsythensis, and A. actinomycetemcomitans and eventually leads to connective tissue destruction and alveolar bone loss.1,16 Recent studies17-19 have suggested the roles of RANKL and OPG in the progression of bone loss in periodontal disease. RANKL and OPG are key molecules that act as positive and negative regulators, respectively, in osteoclastogenesis and bone resorption.2,3,6,20 However, the association between these two factors and periodontopathic bacteria in periodontal tissue has not been studied. In this study, we showed that RANKL mRNA was upregulated in periodontitis tissues. OPG mRNA showed a trend for being decreased but was not statistically significant. Marked differences in age and gender between the control and periodontitis groups were observed. To our knowledge, there are no prior studies investigating whether age or gender influences the expression of RANKL or OPG mRNA specifically in gingival tissue. However, other tissues have been studied, and results suggested that age could affect

expression of these molecules. There is no evidence that gender could affect expression of RANKL and OPG mRNA. It was shown that expression of RANKL mRNA increased with advancing age in whole bone, cultured marrow cells, and stromal/osteoblastic cells from both humans and animals, whereas expression of OPG mRNA either decreased or remained unchanged.21-23 In human cancellous bone, there was no difference for RANKL and OPG mRNA expression between women and men.21 To address the significant differences in age and gender between the study groups in the present study, these two factors were taken into consideration when conducting the statistical analysis. Therefore, linear regression analysis adjusted for age and gender was used to determine the association of periodontitis with the levels of RANKL, OPG, and RANKL/OPG ratio. The periodontitis group had a significantly increased RANKL/OPG ratio compared to the healthy group, suggesting that the balance of these two factors is critical for regulating bone destruction in periodontitis. Our results are consistent with a previous report.18 In that study, the authors compared the mean rank values of RANKL and OPG mRNA expression among comparison groups without taking into account the internal control (GAPDH) expression level. In our study, we determined the 1065

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Figure 2. Scatter plot showing the association between the number of P. gingivalis and the mRNA expression levels of RANKL, OPG, and RANKL/OPG ratio in gingival tissues. A) The relative mRNA expression level of RANKL was significantly correlated with the number of P. gingivalis (r = 0.64; P <0.001). B) There was no relationship between the relative mRNA expression level of OPG and the number of P. gingivalis (r = -0.24; P = 0.20). C) Elevated RANKL/OPG expression ratio was significantly related to an increased number of P. gingivalis (r = 0.67; P <0.001). Each mark represents each sample from both control and periodontitis groups.

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levels of RANKL and OPG mRNA relative to the internal control (b-actin) from the same template. This method allows for a more accurate expression value and more precise comparison. Our finding of elevated RANKL expression, but reduced OPG expression, in periodontitis tissues is not surprising because these alterations support osteoclast formation and activity. Although the levels of mRNA may correlate with expressed protein, several other steps can modulate protein levels, including translational control and posttranslational modification. Recent studies have shown increased levels of RANKL mRNA and protein in diseased gingival tissues24,25 and in gingival crevicular fluid from diseased tissues.19,26 RANKL is found in both membrane-bound and soluble forms. Several studies19,24-26 have examined the involvement of RANKL in periodontal disease. However, the specific form involved in periodontal disease pathogenesis remains unclear. Further study is warranted to resolve this issue. Studies2,3,5,17,27,28 have reported that RANKL mRNA is expressed in periodontal ligament (PDL) cells, osteoblasts, osteoclasts, stromal cells, T lymphocytes, endothelial cells, and epithelial cells. OPG mRNA is expressed in gingival fibroblasts, PDL cells, dental pulp cells, and endothelial cells.27-29 Moreover, a recent report used in situ hybridization to localize RANKL mRNA in gingival tissues and observed expression in epithelial cells and inflammatory cells including lymphocytes and macrophages.18 Therefore, we speculate that these cell types may account for the RANKL mRNA upregulation found in our present study. However, additional work is needed to resolve which cells in the periodontal microenvironment upregulate RANKL mRNA. Two recent studies24,25 determined the cellular source of RANKL protein production in the gingival tissues. It was shown that activated T and B lymphocytes were the major sources of RANKL protein in periodontally diseased gingival tissues. We evaluated the association of mRNA levels of RANKL and OPG with the numbers of P. gingivalis and A. actinomycetemcomitans. Real-time PCR was used to quantify the number of bacteria following a standard protocol.15 However, this method does not differentiate between live and dead bacteria, making it possible that some dead bacteria could have been measured in our findings. The absolute numbers of P. gingivalis and A. actinomycetemcomitans were used rather than ratios of these to the total bacterial numbers. Using absolute numbers allowed for a more precise determination of the association between the specific bacteria and the mRNA expression levels of RANKL and OPG. To our knowledge, our study is the first to show that upregulation of RANKL mRNA was significantly correlated with increased numbers of

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P. gingivalis in clinically obtained periodontal tissues. In contrast, the OPG mRNA expression level was not significantly affected by the presence of this pathogen. A recent study12 showed that viable P. gingivalis is a potent stimulant for the induction of RANKL mRNA through the activator protein 1 (AP-1) pathway. Several studies have shown that inflammatory cytokines including interleukin (IL)-1, TNF-a, and prostaglandin E2 (PGE2) induce RANKL expression and that some of these cytokines downregulate OPG expression.30-34 During inflammatory processes in periodontal disease, IL-1, TNF-a, and PGE2 are major cytokines produced at diseased sites and are involved in the propagation of connective tissue destruction and alveolar bone resorption. A variety of periodontopathic bacteria and their products, especially lipopolysaccharide (LPS), trigger synthesis of these cytokines. Therefore, it is reasonable to postulate that the upregulation of RANKL and downregulation of OPG observed in periodontitis tissues in the present study may be a consequence of both the direct effect of P. gingivalis and also the indirect effect of inflammatory cytokines stimulated by P. gingivalis. This suggests a critical role of RANKL and OPG in P. gingivalis–mediated alveolar bone resorption. The relationship between A. actinomycetemcomitans and mRNA expression of RANKL and OPG could not be evaluated in this study because of the low prevalence of this microorganism in our study population. However, recent studies11,13 have shown that the expression of RANKL mRNA was upregulated by A. actinomycetemcomitans LPS. In addition, it was shown that A. actinomycetemcomitans directly induced production of RANKL in T cells and modulated A. actinomycetemcomitans–induced alveolar bone destruction.10 Examination of additional putative periodontal pathogens such as T. forsythensis and Treponema denticola is needed to determine whether the effect on RANKL/OPG is unique to specific pathogens or a more generalized phenomenon. CONCLUSIONS Our data showed that the upregulation of RANKL mRNA and increased RANKL/OPG mRNA ratio were associated with chronic periodontitis. In addition, to the best of our knowledge, this study is the first to show a positive correlation between P. gingivalis and upregulation of RANKL in clinically obtained human gingival tissues. P. gingivalis may modulate the RANKL/ OPG expression in periodontal tissues by direct or indirect mechanisms and stimulate osteoclastogenesis, leading to alveolar bone resorption. The interactions between P. gingivalis, RANKL, and OPG are important in the pathogenesis of periodontitis, and these findings may help to provide insight essential for development of novel therapeutic approaches aimed 1067

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at controlling P. gingivalis–induced alveolar bone loss. ACKNOWLEDGMENTS The authors thank Prof. Isao Ishikawa and Dr. Toshiyuki Nagasawa, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan, for technical advice and helpful discussion and Prof. Sutas Rakprasitkul, Faculty of Dentistry, Mahidol University, for technical assistance. This study was supported by grants from the Faculty of Dentistry, Khon Kaen University, and Mahidol University. REFERENCES 1. Zambon JJ. Periodontal diseases: Microbial factors. Ann Periodontol 1996;1:879-925. 2. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998; 95:3597-3602. 3. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999; 397:315-323. 4. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999;20:345-357. 5. Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 1999; 402:304-309. 6. Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 1997;89:309-319. 7. Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998;139:1329-1337. 8. Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerindeficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998;12:1260-1268. 9. Mizuno A, Amizuka N, Irie K, et al. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/ osteoprotegerin. Biochem Biophys Res Commun 1998; 247:610-615. 10. Teng YT, Nguyen H, Gao X, et al. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 2000;106:R59-R67. 11. Kikuchi T, Matsuguchi T, Tsuboi N, et al. Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Tolllike receptors. J Immunol 2001;166:3574-3579. 12. Okahashi N, Inaba H, Nakagawa I, et al. Porphyromonas gingivalis induces receptor activator of NFkappaB ligand expression in osteoblasts through the activator protein 1 pathway. Infect Immun 2004;72: 1706-1714. 13. Tiranathanagul S, Yongchaitrakul T, Pattamapun K, Pavasant P. Actinobacillus actinomycetemcomitans 1068

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29. Nagasawa T, Kobayashi H, Kiji M, et al. LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin. Clin Exp Immunol 2002;130:338344. 30. Hofbauer LC, Lacey DL, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S. Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 1999;25:255-259. 31. Nukaga J, Kobayashi M, Shinki T, et al. Regulatory effects of interleukin-1beta and prostaglandin E2 on expression of receptor activator of nuclear factorkappaB ligand in human periodontal ligament cells. J Periodontol 2004;75:249-259. 32. Wei S, Kitaura H, Zhou P, Ross FP, Teitelbaum SL. IL-1 mediates TNF-induced osteoclastogenesis. J Clin Invest 2005;115:282-290.

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33. Fukushima H, Jimi E, Okamoto F, Motokawa W, Okabe K. IL-1-induced receptor activator of NFkappaB ligand in human periodontal ligament cells involves ERK-dependent PGE2 production. Bone 2005;36:267-275. 34. Choi BK, Moon SY, Cha JH, Kim KW, Yoo YJ. Prostaglandin E(2) is a main mediator in receptor activator of nuclear factor-kappaB ligand-dependent osteoclastogenesis induced by Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii. J Periodontol 2005;76:813-820. Correspondence: Dr. Nawarat Wara-aswapati, Department of Periodontology, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand. Fax: 66-43-348-152; e-mail: [email protected]. Accepted for publication January 6, 2007.

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