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Porphyromonas endodontalis Lipopolysaccharides Induce RANKL by Mouse Osteoblast in a Way Different from That of Escherichia coli Lipopolysaccharide
Basic Research—Biology
Porphyromonas endodontalis Lipopolysaccharides Induce RANKL by Mouse Osteoblast in a Way Different from That of Escherichia coli Lipopolysaccharide Yin Tang, PhD,* Feifei Sun, PhD,* Xiaoting Li, PhD,* Yuan Zhou, PhD,* Shihai Yin, PhD, DDS,† and Xuedong Zhou, PhD, DMD* Abstract Introduction: Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-kB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. Methods: LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Tolllike receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Results: Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the upregulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05). Conclusions: Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4–JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS). (J Endod 2011;37:1653–1658)
Key Words Lipopolysaccharide, MC3T3-E1 cells, Porphyromonas endodontalis, RANKL, signaling pathway
A
pical periodontitis is an inflammatory disorder of periradicular tissues caused by bacterial infection of endodontic origin and is characterized by periapical bone resorption (1). Porphyromonas endodontalis is a gram-negative, black-pigmented, anaerobic, rod-shaped bacterium, which is strongly associated with endodontic infection. This species is almost exclusively found in infections of the dental pulp and has been implicated in the etiology of infected root canals and apical periodontitis (2, 3). As a major constituent of outer membrane and a main virulence factor of gramnegative bacteria, lipopolysaccharide (LPS) has been proved to play a critical role in initiating and developing periapical periodontitis and the odontogenic abscess by mediating inflammation and inducing cells to secrete proinflammatory cytokines (4). Diverse inflammatory mediators such as interleukin (IL)-1, IL-2, IL-6, IL-8, IL-12, tumor necrosis factor-a (TNF-a), granulocyte-macrophage colony stimulating factor, nitric oxide, interferon-g, prostaglandins, and metalloproteinases have been associated with periradicular lesions (5–7). A recently identified receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL)/RANK/osteoprotegerin (OPG) has been shown to be the key regulator of bone remodeling and is directly involved in the differentiation, activation, and survival of osteoclasts and osteoclast precursors (8). Membrane-bound or soluble RANKL is primarily produced in osteoblastic lineages and activated T cells and stimulates osteoclast differentiation. The importance of RANKL in osteoclast differentiation underlines the central role played by stromal cells and osteoblasts in the process (9). Apical periodontitis is a multifactorial disease that is resultant of the interplay of many host and bacterial factors, among which LPS is undoubtedly the most studied and quoted virulence factor (10). Studies have revealed that the content of endotoxin or LPS in infected root canals is higher in teeth with symptomatic apical periodontitis, teeth with periradicular bone destruction, or teeth with persistent exudation than in those without them (11). P. endodontalis LPS has been detected in samples from infected root canals or in the pus samples of acute abscesses of about 90% of the patients tested; it can play an integral role as a potent stimulator of inflammatory cytokines that are involved in the formation of acute abscesses (4). Surprisingly, there have been relatively few studies addressing the mechanisms of P. endodontalis LPS in the tissue destruction of apical periodontitis. Although there are
From the *State Key Laboratory of Oral Disease and †Department of Operative Dentistry and Endodontics, West China School and Hospital of Stomatology, Sichuan University, Chengdu, China. Supported by grants from the National Natural Science Foundation of China (grant no. 30973324) and the Sichuan Provincial Science and Technology Department (grant no. 2008sz0202). Address requests for reprints to Dr Xuedong Zhou, State Key Laboratory of Oral Disease, West China School and Hospital of Stomatology, Sichuan University, No. 14, Third Section, Renminnan Road, Chengdu, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2011.08.015
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Basic Research—Biology plenty of studies about Porphyromonas gingivalis or Escherichia coli LPS signaling in monocytes, lymphocytes, endothelial cells, and certain types of fibroblasts, they are rarely associated with endodontic infection. It has also been reported that bacteria preferentially stimulate specific cell types, which may be related to their capacity to induce inflammation in given tissues (12). However, little information is available about the response of osteoblast to LPSs. Osteoblasts are considered as cells primarily concerned with providing physical barriers and structural components in periapical tissues. These cells are important in bone remodeling through expressing RANKL, and they contribute to the bone lesion (13). However, the exact pathway in oesteoblasts induced by P. endodontalis LPS remains unknown. To elucidate the signal transduction pathway of P. endodotalis LPS in the mouse osteoblasts, the present study investigated the expression of RANKL in osteoblasts stimulated by LPS by the real-time polymerase chain reaction and Western blot. The LPS receptor TLR and the intracellular signaling pathways were also evaluated to determine their possible roles in the inflammatory process. E. coli LPS was used as the control.
Materials and Methods Culture of P. endodontalis and Preparation of LPS P. endodontalis ATCC35406 was anerobically cultured (80% N2, 10% H2, and 10% CO2) on brain-heart infusion agar plates containing 5% defibrinated sheep blood enriched with vitamin K3 (1 mg/mL) and hemin (5 mg/mL) for 72 hours at 37 C. Then, P. endodontalis LPS was prepared by the phenol-water method (14), and crude LPS was digested with DNase and RNase at 100 C for 15 minutes and ultracentrifuged at 100,000 g for 2 hours. The purified P. endodontalis LPS bioactivity was measured by the limulus amebocyte lysate test (Sigma-Aldrich, St Louis, MO) (14). The absence of contaminating nucleic acid and protein was verified by measuring the optical densities at 280 nm and 260 nm. The LPS preparations were also examined by infrared spectroscopy using a Perkin-Elmer 467 spectrophotometer (PerkinElmer, Waltham, MA). Cell Culture Mouse osteoblast–derived MC3T3-E1 cells were cultured at 2 105 cells/well in 96-well plates containing DMEM medium (GibcoBRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/ mL penicillin G, and 100 mg/mL streptomycin at 37 C in a humidified atmosphere of 5% CO2. Treat of Osteoblast Cell by P. endodontalis LPS and E. coli LPS The osteoblast cells, stimulated with 1 mg/mL P. endodotalis LPS, 1 mg/mL E. coli LPS (Sigma-Aldrich), and without LPS, were incubated at 37 C in a humidified atmosphere of 5% CO2 for 0, 1, 2, 6, 12, 24, and 48 hours, respectively. Culture cells were collected after various times of incubation and stored at 80 C until use. All time points were performed in triplicate. Blocking Tests Using Anti-TLRs Antibodies To assess the functional role of TLR2 or TLR4 in cytokine production, the osteoblasts were incubated with 2 mg/mL of either anti-TLR2 or anti-TLR4 polyclonal antibody (Santa Cruz Biotechnology Inc, Heidelberg, Germany) for 1 hour before stimulation with 1 mg/mL P. endodontalis LPS or E. coli LPS (15, 16). Cells were harvested at designated time points after stimulation with P. endodontalis LPS or E. coli LPS for 2, 6, and 12 hours, respectively. The positive controls were the cells incubated with LPS but without anti-TLRs, whereas the 1654
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negative controls were without both LPS and anti-TLRs. All time points were performed in triplicate.
Analysis of the Main Intracellular Signaling Pathways To investigate intracellular signaling pathways involved in the expression of RANKL, we used inhibitors of these signaling pathways. The osteoblasts (2 105 cells/well) were incubated with wortmannin (an inhibitor of phosphatidylinositol 3-kinase, 1 mmol/L); SB203580 (an inhibitor of p38 MAPK, 5 mmol/L); and PD98059 (an inhibitor of MEK1/2, 5 mmol/L), SP600125 (the JNK inhibitor, 5 mmol/L), or SN50 (the NF-kB inhibitor, 5 mmol/L) (Calbiochem Bioscences, Inc, La Jolla, CA), respectively, for 1 hour before stimulation with P. endodontalis LPS or E. coli LPS (17). Cells stimulated with P. endodontalis LPS or E. coli LPS for 2, 6, and 12 hours were collected for use. The positive controls were cells incubated with LPS but without signaling pathway inhibitors, whereas the negative controls were without both LPS and signaling pathway inhibitors. All time points were performed in triplicate. Real-time Polymerase Chain Reaction Analysis Total RNA was prepared from the cells by using Trizol reagent (Gibco-BRL), and complementary DNA was synthesized using a RT Reagent Kit (TaKaRa Shuzo Co Ltd, Shiga, Japan) according to the manufacturer’s instructions. To quantify RANKL messenger RNA, real-time polymerase chain reaction was performed by using ABI PRISM 7300 real-time PCR system (Applied Biosystems, Foster, CA) with SYBR Premix EXTaq (TaKaRa Shuzo Co Ltd). The standard polymerase chain reaction conditions were 10 seconds at 95 C followed by 40 cycles at 95 C for 5 seconds, 60 C for 31 seconds, and 72 C for 30 seconds. Each assay was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The normalized data were expressed by the comparative threshold cycle (CT) method to present data as the fold change against the messenger RNA level of unstimulated cells. The primers used are listed in Table 1. Western Blot Analysis The total proteins were extracted from the cells using RIPA lysis buffer (Amresco, Solon, OH), and the protein concentration was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL). The cell extract (20 mg protein) was denatured in sodium dodecyl sulfate sample buffer, resolved by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis, and electrotransferred to a polyvinylidine fluoride membrane. The membranes were blocked in 5% nonfat dry milk and blotted with monoclonal rabbit antimouse RANKL (1:500), monoclonal rabbit antimouse OPG (1:400), and rabbit antimouse GAPDH (1:400) antibodies (Boster, Hubei, China), respectively, followed by horseradish peroxidase–conjugated affinipure goat antirabbit immunoglobulin G (1:5,000) (ZSGB-BIO, Beijing, China). Membrane-bound immune complexes were visualized by the ECL Plus Kit (Amersham Biosciences, Piscataway, NJ). TABLE 1. Sequence and Expected Fragment Sizes of Synthetic Oligonucleotides Used for Polymerase Chain Reaction Target messenger RNA RANKL OPG GAPDH
Primer sequence
Size (bp)
5-TACTTTCGAGCGCAGATGGAT-3 5-ACCTGCGTTTTCATGGAGTCT-3 5-AAACAGCACTGCACAGTGAG-3 5-ACACTGGGCTGCAATACACA-3 5-AACTACATGGTCTACATGTTCCA-3 5-CCATTCTCGGCCTTGACTGT-3
89 108 63
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Basic Research—Biology Statistical Analysis All data are presented as the mean standard deviation and evaluated by analysis of variance (SPSS v.10.0; SPSS Inc, Chicago, IL). Differences between groups were considered significant at the level of P < .05.
Results Assessment of P. endodotalis LPS The limulus amebocyte lysate test for purified P. endodotalis LPS was positive ($15 ng/mL), indicating that this level of LPS has endotoxin bioactivity and could be used in the following study. The infrared spectra revealed that the extraction was endotoxin because the spectrum for P. endodontalis LPS was similar to that of E. coli LPS (Fig. 1), a typical bacterial endotoxin.
Effect of LPS on Expression of RANKL and OPG Our first experiment was to explore the possibility that P. endodontalis LPS induces RANKL and OPG expression in osteoblasts; then, we compared the abilities of P. endodontalis LPS and E. coli LPS to induce the RANKL expression. As shown in Figure 2, RANKL messenger RNA was significantly up-regulated by LPS derived from E. coli or P. endodontalis. When osteoblasts were stimulated for 1 hour with 1 mg/mL P. endodontalis LPS, the level of RANKL was significantly (P < .05) enhanced (Fig. 2). When stimulated with 1 mg/mL E. coli LPS, the expression was noticeably (P < .05) increased after 1 hour (Fig. 2). At some time points, P. endodontalis LPS showed a higher induction activity than E. coli LPS (P < .05), but the RANKL expression at 24 hours and 48 hours induced by P. endodontalis LPS was similar to those induced by E. coli LPS (P > .05) (Fig. 2B). In contrast, the
Figure 1. The infrared spectra of P. endodontalis (Pe) and E. coli (Ec) LPS.
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Functional Role of TLR2 and TLR4 Previous studies have shown that TLR2 or TLR4 serve as the main mediator of responses to LPS in vitro and in vivo (18, 19). Therefore, we next sought to find the functional involvement of TLR2 and TLR4 in our observed LPS-induced RANKL expression by osteoblasts. Cells were incubated with anti-TLR2 or anti-TLR4 polyclonal antibody (2 mg/mL) before stimulation with LPS. The anti-TLR2 antibody had a significant (P < .05) inhibitory effect on RANKL expression (Fig. 3B and D) by osteoblasts stimulated with E. coli LPS, but anti-TLR4 antibody had no noticeable (P > .05) effect. However, both of the anti-TLR2 and antiTLR4 antibodies significantly (P < .05) inhibited the expression of RANKL (Fig. 3A and C) from osteoblasts stimulated with P. endodontalis LPS, and the effect of the anti-TLR2 antibody inhibition was more obvious. The Main Intracellular Signaling Pathways for Expression of RANKL We finally examined the main intracellular signaling pathways for RANKL expression in osteoblasts stimulated with LPS. In the blocking test, pretreatment with wortmannin (an inhibitor of PI3K), SB20350 (an inhibitor of p38 MAPK), and PD98059 (an inhibitor of MEK1/2), or SN50 (the NF-kB inhibitor) failed to reduce the RANKL responses in infected cells stimulated by P. endodontalis LPS (P > .05). On the other hand, SP600125, a potent inhibitor of c-Jun N-terminal kinase
Figure 2. The expression of RANKL and OPG in osteoblasts collected 0, 1, 2, 6, 12, 24, and 48 hours, respectively, after 1 mg/mL P. endodontalis (Pe) or E. coli (Ec) LPS stimulation. (A) Western blot analysis of RANKL and OPG protein. (B) Quantification of RANKL messenger RNA by real-time polymerase chain reaction. Relative messenger RNA levels are expressed as the fold increases compared with the RANKL messenger RNA level in unstimulated cells. The values shown are means standard deviations for triplicate assays. An asterisk indicates that the P value was <.05 in comparison with the RANKL messenger RNA level in cells stimulated with Pe or Ec LPS.
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(JNK), prevented the up-regulation of RANKL expression in infected osteoblasts (P < 0.05) (Fig. 3A and C). In contrast, the inhibitory effect of wortmannin (an inhibitor of phosphatidylinositol 3-kinase) and PD98059 (an inhibitor of MEK1/2) (P < .05) was observed in E. coli LPS-treated mouse osteoblasts (Fig. 3B and D).
Discussion It has been reported that bacteria preferentially stimulate specific cell types, which may be related to their capacity to induce inflammation in given tissues. When it comes to P. endodontalis, it is less potent than Streptococcus mutans in stimulating mononuclear cells but more effective in directly inducing IL-6 and IL-1expression by osteoblastic cells (4). This might be significant because these characteristics may enable P. endodontalis to escape the immune response mounted by leukocytes but possess the ability to cause bone resorption at the sites of endodontic infection. The association of different bacteria with specific pathologic process may be partially explained by their capacities in activating different cell types. In view of this, it was of interest to us to detect the P. endodontalis LPS signaling in osteoblasts, which is closely related to the pathogenic mechanism(s) of the periapical lesion. It is recognized that E. coli LPS is a typical bacterial endotoxin and P. endodontalis is the main pathogenic bacterium of apical periodontitis, so it is of great importance to compare the capacity of P. endodontalis LPS and E. coli LPS to induce the response in osteoblasts. There is no previous study on P. endodontalis LPS in bone lesion but plenty of studies about P. gingivalis LPS, and it has been reported in studies that P. gingivalis LPS is less potent than E. coli LPS in inducing the release of inflammatory cytokines in various cells (20, 21). In the present study, we observed that, at some time points, P. endodontalis LPS showed a higher activity of RANKL induction in osteoblasts than E. coli LPS. P. endodontalis and P. gingivalis belong to the same species; they are both black-pigmented, anaerobic, rod-shaped bacteria, and they have close biological activity and characteristics and also similar pathogenicity. In view of this, we discuss our data with P. gingivalis, and our results are different from those of most other studies to a certain degree. This is possibly related to the P. endodontalis strain used, the LPS extraction method, and the selection of the times for detecting cytokine levels and target cells. It is known that different strains of the same bacterium or different extraction methods have considerable effect on the purity and especially the activity of LPS (22). Another important factor is the LPS concentration in cell culture medium and LPS stimulation time. The LPS concentration we use to treat osteoblasts is 1 mg/mL, which has been confirmed to be a sufficient concentration to induce cell response in this study. According to previous studies (23, 24), higher concentrations have cytotoxicity, which will reduce RANKL expression. The time settlements in the blocking test were less than 8 hours because long-time incubation with LPS will promote the apoptosis of cells. Reports have shown that RANKL messenger RNA in osteoblasts is up-regulated by the bone-resorbing factors, such as vitamin D3, IL-1, IL-11, TNF-a, and parathyroid hormone (25). Because LPS has been shown to stimulate osteoblasts to secrete the osteolytic factors IL-1, IL-6, TNF-a, and prostacyclin (PGI2), it is reasonable to presume that RANKL gene expression by LPS may be indirectly mediated by one of these factors. However, it has been shown that LPS can directly mediate RANKL messenger RNA up-regulation in osteoblasts, and RANKL messenger RNA increase is not secondary to prostacyclin (PGI2) or TNF-a synthesis (24). This has well proved the feasibility of the present study detecting the RANKL expression in osteoblasts induced by P. endodontalis LPS. Our current data have indicated JOE — Volume 37, Number 12, December 2011
Basic Research—Biology
Figure 3. The effects of anti-TLR antibodies, PI3K inhibitor (wortmannin), NF-kB inhibitor (SN50), p38MAPK inhibitor (SB203580), MEK1/2 inhibitor (PD98059), and JNK inhibitor (SP600125) on the expression of RANKL in osteoblasts stimulated with LPS. Before the addition of P. endodontalis (Pe) or E. coli (Ec) LPS (1 mg/mL), osteoblasts were pretreated for 1 hour with anti-TLR2 and anti-TLR4 antibodies (2 mg/mL), wortmannin (1 mmol/L), SN50 (5 mmol/L), SB203580 (5 mmol/L), PD98059 (5 mmol/L), and SP600125 (5 mmol/L), respectively. (A) Western blot analysis of RANKL in osteoblasts stimulated with Pe LPS for 12 hours. (B) Western blot analysis of RANKL in osteoblasts stimulated with Ec LPS for 12 hours. (C) Quantification of RANKL messenger RNA in osteoblasts stimulated with Pe LPS for 2, 6, and 12 hours, respectively. (D) The quantification of RANKL messenger RNA in osteoblasts stimulated with Ec LPS for 2, 6, and 12 hours, respectively. Relative messenger RNA levels are expressed as the fold increases compared with the RANKL messenger RNA level in unstimulated cells. The values shown are means standard deviations for triplicate assays. An asterisk (*) indicates that the P value was <.05 in comparison with the RANKL messenger RNA level in cells stimulated with LPS only.
that LPS rapidly increased RANKL expression in osteoblasts. In contrast, OPG expression remained constant in these cells. These results suggest that LPS potently induce osteoclastogenic activity in mouse osteoblasts. In the signal transduction of LPS, TLR2 and TLR4 are transmembrane receptors that transmit the LPS signal to intracellular components and play important roles in the immune system (26, 27). Initially, it was recognized that TLR4 was the receptor for gram-negative bacterial LPS, and TLR2 was the receptor for gram-positive peptidoglycan and lipopeptides (19). In contrast, we found that P. endodontalis LPS could use both TLR2 and TLR4 (mainly TLR2) in osteoblasts, whereas E. coli LPS stimulated RANKL expression via TLR4 in osteoblasts. It has been reported whether the TLR recognized by LPS (TLR2 or TLR4) is dependent on the structure of the LPS (28). P. gingivalis LPS has been shown to differ from many gram-negative bacterial LPS in structure and various functional activities, and P. gingivalis LPS acts not only via TLR4 but also via TLR2 or other signal transducers in monocytes/macrophages (29). In addition, it has been reported that human periodontitis gingiva expressed TLR2 and TLR4, and the ratio of TLR2-positive cells was higher than that of TLR4 (30). Our findings suggested that there is resemblance between P. endodontalis LPS and P. gingivalis LPS when binding to TLR. After binding to the TLR, LPS can trigger proinflammatory cytokine gene expression by the activation of a number of intracellular JOE — Volume 37, Number 12, December 2011
inflammatory signaling pathways including the PI3K, NF-kB, and various mitogen-activated protein kinase (MAPK) pathways (31, 32). Previous studies have shown that NF-kB is the main signaling pathway of most bacterial LPS (33). We discovered in the present study that the JNK inhibitor was the most effective in blocking RANKL expression in osteoblasts upon stimulation with P. endodontalis LPS. This was in agreement with the report of Darveau et al (34) that P. gingivalis LPS could not activate p38MAPK and ERK pathways in human endothelial cells. Another study has proven that the inhibitor of JNK and activator protein-1 (AP-1) activation can prevent the upregulation of RANKL expression in infected osteoblasts, and P. gingivalis infection resulted in the phosphorylation of c-Junand activation of AP-1 (35). All these findings indicate that JNK is involved in RANKL expression induced by black-pigmented pseudomonas. With E. coli LPS, the story was different; the activation of ERK and PI3K pathways seems to play an important role in the activation of RANKL expression. NF-kB signals did not seem to be involved in RANKL expression, which is consistent with a report showing no obvious NF-kB binding motifs in the promoter regions of the mouse RANKL gene (36). In conclusion, our results show that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4–JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin
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Basic Research—Biology (E. coli LPS). JNK may be an alternative target for screening molecular inhibitors. These findings provide an insight into a therapeutic approach to controlling LPS-induced apical periodontitis.
Acknowledgments The authors deny any conflicts of interest related to this study.
18. 19. 20.
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(TNF-a) induction in lipopolysaccharide (LPS)-stimulated microglia. Brain Res 2006;1073:48–59. Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 2002;14:103–10. Smith MF, Mitchell A, Ding S, et al. Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pyloriinduced NF-kB activation and chemokine expression by epithelial cells. J Biol Chem 2003;278:32552–60. Lu J, Yang Z, Jiang J, et al. Regulative effect of p38MAPK on release of TNF alpha and NO from alveolar macrophages under endotoxin stimulation. Chin J Traumatol 2001;4:75–7. Martin M, Katz J, Vogel SN, et al. Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli. J Immunol 2001;167:5278–85. Mayer H, Tharanathan RN, Weckesser J. Analysis of lipopolysaccharides of gramnegative bacteria. Methods Microbiol 1985;18:157–207. Diya Z, Lili C, Shenglai L, et al. Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS. Innate Immun 2008;14: 99–107. Kikuchi T, Matsuguchi T, Tsuboi N, et al. Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll-like receptors. J Immunol 2001;166:3574–9. Kobayashi K, Takahashi N, Jimi E, et al. Tumor necrosis factor a stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 2000;191:275. Sadeghi K, Berger A, Langgartner M, et al. Immaturity of infection control in preterm and term newborns is associated with impaired Toll-like receptor signaling. J Infect Dis 2007;195:296–302. Hadley JS, Wang JE, Michael LC, et al. Alteration in inflammatory capacity and TLR expression on monocytes and neutrophils after cardiopulmonary bypass. Shock 2007;27:466–73. Coats SR, Reif RA, Bainbridge BW, et al. Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at Toll-like receptor 4 in human endothelial cells. Infect Immun 2003;71:6799–807. Darvean RP, Pham TT, Lemley K, et al. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both Toll-like receptors 2 and 4. Infect Immun 2004;72:5041–51. Mori Y, Yoshimura A, Ukai T, et al. Immunohistochemical localization of Toll-like receptor 2 and 4 in gingival tissue from patients with periodontitis. Oral Microbiol Immunol 2003;18:54–8. Zhou X, Gao XP, Fan J, et al. LPS activation of Toll-like receptor 4 signals CD11b/ CD18 expression in neutrophils. Am J Physiol 2005;288:L655–62. Martin M, Schifferle RE, Cuesta N, et al. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by porphyromonas gingivalis lipopolysaccharide. J Immunol 2003;171:717–25. Hirschfeld M, Weis JJ, Toshchakov V, et al. Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun 2001;69:1477–82. Darveau RP, Arbai S, Garcia I, Bainbridge B, Maier RV. Porphyromonas gingivalis lipopolysaccharide is both agonist and antagonist for p38 mitogen-activated protein kinase activation. Infect Immun 2002;70:1867–73. Okahashi N, Inaba H, Nakagawa I, et al. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway. Infect Immun 2004;72:1706–14. Kitazawa R, Kitazawa S, Maeda S. Promoter structure of mouse RANKL/TRANCE/ OPGL/ODF gene. Biochem Biophys Acta 1999;1445:134–41.
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Porphyromonas endodontalis Lipopolysaccharides Induce RANKL by Mouse Osteoblast in a Way Different from That of Escherichia coli Lipopolysaccharide Yin Tang, PhD,* Feifei Sun, PhD,* Xiaoting Li, PhD,* Yuan Zhou, PhD,* Shihai Yin, PhD, DDS,† and Xuedong Zhou, PhD, DMD* Abstract Introduction: Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-kB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. Methods: LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Tolllike receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. Results: Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the upregulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05). Conclusions: Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4–JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS). (J Endod 2011;37:1653–1658)
Key Words Lipopolysaccharide, MC3T3-E1 cells, Porphyromonas endodontalis, RANKL, signaling pathway
A
pical periodontitis is an inflammatory disorder of periradicular tissues caused by bacterial infection of endodontic origin and is characterized by periapical bone resorption (1). Porphyromonas endodontalis is a gram-negative, black-pigmented, anaerobic, rod-shaped bacterium, which is strongly associated with endodontic infection. This species is almost exclusively found in infections of the dental pulp and has been implicated in the etiology of infected root canals and apical periodontitis (2, 3). As a major constituent of outer membrane and a main virulence factor of gramnegative bacteria, lipopolysaccharide (LPS) has been proved to play a critical role in initiating and developing periapical periodontitis and the odontogenic abscess by mediating inflammation and inducing cells to secrete proinflammatory cytokines (4). Diverse inflammatory mediators such as interleukin (IL)-1, IL-2, IL-6, IL-8, IL-12, tumor necrosis factor-a (TNF-a), granulocyte-macrophage colony stimulating factor, nitric oxide, interferon-g, prostaglandins, and metalloproteinases have been associated with periradicular lesions (5–7). A recently identified receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL)/RANK/osteoprotegerin (OPG) has been shown to be the key regulator of bone remodeling and is directly involved in the differentiation, activation, and survival of osteoclasts and osteoclast precursors (8). Membrane-bound or soluble RANKL is primarily produced in osteoblastic lineages and activated T cells and stimulates osteoclast differentiation. The importance of RANKL in osteoclast differentiation underlines the central role played by stromal cells and osteoblasts in the process (9). Apical periodontitis is a multifactorial disease that is resultant of the interplay of many host and bacterial factors, among which LPS is undoubtedly the most studied and quoted virulence factor (10). Studies have revealed that the content of endotoxin or LPS in infected root canals is higher in teeth with symptomatic apical periodontitis, teeth with periradicular bone destruction, or teeth with persistent exudation than in those without them (11). P. endodontalis LPS has been detected in samples from infected root canals or in the pus samples of acute abscesses of about 90% of the patients tested; it can play an integral role as a potent stimulator of inflammatory cytokines that are involved in the formation of acute abscesses (4). Surprisingly, there have been relatively few studies addressing the mechanisms of P. endodontalis LPS in the tissue destruction of apical periodontitis. Although there are
From the *State Key Laboratory of Oral Disease and †Department of Operative Dentistry and Endodontics, West China School and Hospital of Stomatology, Sichuan University, Chengdu, China. Supported by grants from the National Natural Science Foundation of China (grant no. 30973324) and the Sichuan Provincial Science and Technology Department (grant no. 2008sz0202). Address requests for reprints to Dr Xuedong Zhou, State Key Laboratory of Oral Disease, West China School and Hospital of Stomatology, Sichuan University, No. 14, Third Section, Renminnan Road, Chengdu, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2011.08.015
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Basic Research—Biology plenty of studies about Porphyromonas gingivalis or Escherichia coli LPS signaling in monocytes, lymphocytes, endothelial cells, and certain types of fibroblasts, they are rarely associated with endodontic infection. It has also been reported that bacteria preferentially stimulate specific cell types, which may be related to their capacity to induce inflammation in given tissues (12). However, little information is available about the response of osteoblast to LPSs. Osteoblasts are considered as cells primarily concerned with providing physical barriers and structural components in periapical tissues. These cells are important in bone remodeling through expressing RANKL, and they contribute to the bone lesion (13). However, the exact pathway in oesteoblasts induced by P. endodontalis LPS remains unknown. To elucidate the signal transduction pathway of P. endodotalis LPS in the mouse osteoblasts, the present study investigated the expression of RANKL in osteoblasts stimulated by LPS by the real-time polymerase chain reaction and Western blot. The LPS receptor TLR and the intracellular signaling pathways were also evaluated to determine their possible roles in the inflammatory process. E. coli LPS was used as the control.
Materials and Methods Culture of P. endodontalis and Preparation of LPS P. endodontalis ATCC35406 was anerobically cultured (80% N2, 10% H2, and 10% CO2) on brain-heart infusion agar plates containing 5% defibrinated sheep blood enriched with vitamin K3 (1 mg/mL) and hemin (5 mg/mL) for 72 hours at 37 C. Then, P. endodontalis LPS was prepared by the phenol-water method (14), and crude LPS was digested with DNase and RNase at 100 C for 15 minutes and ultracentrifuged at 100,000 g for 2 hours. The purified P. endodontalis LPS bioactivity was measured by the limulus amebocyte lysate test (Sigma-Aldrich, St Louis, MO) (14). The absence of contaminating nucleic acid and protein was verified by measuring the optical densities at 280 nm and 260 nm. The LPS preparations were also examined by infrared spectroscopy using a Perkin-Elmer 467 spectrophotometer (PerkinElmer, Waltham, MA). Cell Culture Mouse osteoblast–derived MC3T3-E1 cells were cultured at 2 105 cells/well in 96-well plates containing DMEM medium (GibcoBRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/ mL penicillin G, and 100 mg/mL streptomycin at 37 C in a humidified atmosphere of 5% CO2. Treat of Osteoblast Cell by P. endodontalis LPS and E. coli LPS The osteoblast cells, stimulated with 1 mg/mL P. endodotalis LPS, 1 mg/mL E. coli LPS (Sigma-Aldrich), and without LPS, were incubated at 37 C in a humidified atmosphere of 5% CO2 for 0, 1, 2, 6, 12, 24, and 48 hours, respectively. Culture cells were collected after various times of incubation and stored at 80 C until use. All time points were performed in triplicate. Blocking Tests Using Anti-TLRs Antibodies To assess the functional role of TLR2 or TLR4 in cytokine production, the osteoblasts were incubated with 2 mg/mL of either anti-TLR2 or anti-TLR4 polyclonal antibody (Santa Cruz Biotechnology Inc, Heidelberg, Germany) for 1 hour before stimulation with 1 mg/mL P. endodontalis LPS or E. coli LPS (15, 16). Cells were harvested at designated time points after stimulation with P. endodontalis LPS or E. coli LPS for 2, 6, and 12 hours, respectively. The positive controls were the cells incubated with LPS but without anti-TLRs, whereas the 1654
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negative controls were without both LPS and anti-TLRs. All time points were performed in triplicate.
Analysis of the Main Intracellular Signaling Pathways To investigate intracellular signaling pathways involved in the expression of RANKL, we used inhibitors of these signaling pathways. The osteoblasts (2 105 cells/well) were incubated with wortmannin (an inhibitor of phosphatidylinositol 3-kinase, 1 mmol/L); SB203580 (an inhibitor of p38 MAPK, 5 mmol/L); and PD98059 (an inhibitor of MEK1/2, 5 mmol/L), SP600125 (the JNK inhibitor, 5 mmol/L), or SN50 (the NF-kB inhibitor, 5 mmol/L) (Calbiochem Bioscences, Inc, La Jolla, CA), respectively, for 1 hour before stimulation with P. endodontalis LPS or E. coli LPS (17). Cells stimulated with P. endodontalis LPS or E. coli LPS for 2, 6, and 12 hours were collected for use. The positive controls were cells incubated with LPS but without signaling pathway inhibitors, whereas the negative controls were without both LPS and signaling pathway inhibitors. All time points were performed in triplicate. Real-time Polymerase Chain Reaction Analysis Total RNA was prepared from the cells by using Trizol reagent (Gibco-BRL), and complementary DNA was synthesized using a RT Reagent Kit (TaKaRa Shuzo Co Ltd, Shiga, Japan) according to the manufacturer’s instructions. To quantify RANKL messenger RNA, real-time polymerase chain reaction was performed by using ABI PRISM 7300 real-time PCR system (Applied Biosystems, Foster, CA) with SYBR Premix EXTaq (TaKaRa Shuzo Co Ltd). The standard polymerase chain reaction conditions were 10 seconds at 95 C followed by 40 cycles at 95 C for 5 seconds, 60 C for 31 seconds, and 72 C for 30 seconds. Each assay was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The normalized data were expressed by the comparative threshold cycle (CT) method to present data as the fold change against the messenger RNA level of unstimulated cells. The primers used are listed in Table 1. Western Blot Analysis The total proteins were extracted from the cells using RIPA lysis buffer (Amresco, Solon, OH), and the protein concentration was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL). The cell extract (20 mg protein) was denatured in sodium dodecyl sulfate sample buffer, resolved by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis, and electrotransferred to a polyvinylidine fluoride membrane. The membranes were blocked in 5% nonfat dry milk and blotted with monoclonal rabbit antimouse RANKL (1:500), monoclonal rabbit antimouse OPG (1:400), and rabbit antimouse GAPDH (1:400) antibodies (Boster, Hubei, China), respectively, followed by horseradish peroxidase–conjugated affinipure goat antirabbit immunoglobulin G (1:5,000) (ZSGB-BIO, Beijing, China). Membrane-bound immune complexes were visualized by the ECL Plus Kit (Amersham Biosciences, Piscataway, NJ). TABLE 1. Sequence and Expected Fragment Sizes of Synthetic Oligonucleotides Used for Polymerase Chain Reaction Target messenger RNA RANKL OPG GAPDH
Primer sequence
Size (bp)
5-TACTTTCGAGCGCAGATGGAT-3 5-ACCTGCGTTTTCATGGAGTCT-3 5-AAACAGCACTGCACAGTGAG-3 5-ACACTGGGCTGCAATACACA-3 5-AACTACATGGTCTACATGTTCCA-3 5-CCATTCTCGGCCTTGACTGT-3
89 108 63
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Basic Research—Biology Statistical Analysis All data are presented as the mean standard deviation and evaluated by analysis of variance (SPSS v.10.0; SPSS Inc, Chicago, IL). Differences between groups were considered significant at the level of P < .05.
Results Assessment of P. endodotalis LPS The limulus amebocyte lysate test for purified P. endodotalis LPS was positive ($15 ng/mL), indicating that this level of LPS has endotoxin bioactivity and could be used in the following study. The infrared spectra revealed that the extraction was endotoxin because the spectrum for P. endodontalis LPS was similar to that of E. coli LPS (Fig. 1), a typical bacterial endotoxin.
Effect of LPS on Expression of RANKL and OPG Our first experiment was to explore the possibility that P. endodontalis LPS induces RANKL and OPG expression in osteoblasts; then, we compared the abilities of P. endodontalis LPS and E. coli LPS to induce the RANKL expression. As shown in Figure 2, RANKL messenger RNA was significantly up-regulated by LPS derived from E. coli or P. endodontalis. When osteoblasts were stimulated for 1 hour with 1 mg/mL P. endodontalis LPS, the level of RANKL was significantly (P < .05) enhanced (Fig. 2). When stimulated with 1 mg/mL E. coli LPS, the expression was noticeably (P < .05) increased after 1 hour (Fig. 2). At some time points, P. endodontalis LPS showed a higher induction activity than E. coli LPS (P < .05), but the RANKL expression at 24 hours and 48 hours induced by P. endodontalis LPS was similar to those induced by E. coli LPS (P > .05) (Fig. 2B). In contrast, the
Figure 1. The infrared spectra of P. endodontalis (Pe) and E. coli (Ec) LPS.
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Functional Role of TLR2 and TLR4 Previous studies have shown that TLR2 or TLR4 serve as the main mediator of responses to LPS in vitro and in vivo (18, 19). Therefore, we next sought to find the functional involvement of TLR2 and TLR4 in our observed LPS-induced RANKL expression by osteoblasts. Cells were incubated with anti-TLR2 or anti-TLR4 polyclonal antibody (2 mg/mL) before stimulation with LPS. The anti-TLR2 antibody had a significant (P < .05) inhibitory effect on RANKL expression (Fig. 3B and D) by osteoblasts stimulated with E. coli LPS, but anti-TLR4 antibody had no noticeable (P > .05) effect. However, both of the anti-TLR2 and antiTLR4 antibodies significantly (P < .05) inhibited the expression of RANKL (Fig. 3A and C) from osteoblasts stimulated with P. endodontalis LPS, and the effect of the anti-TLR2 antibody inhibition was more obvious. The Main Intracellular Signaling Pathways for Expression of RANKL We finally examined the main intracellular signaling pathways for RANKL expression in osteoblasts stimulated with LPS. In the blocking test, pretreatment with wortmannin (an inhibitor of PI3K), SB20350 (an inhibitor of p38 MAPK), and PD98059 (an inhibitor of MEK1/2), or SN50 (the NF-kB inhibitor) failed to reduce the RANKL responses in infected cells stimulated by P. endodontalis LPS (P > .05). On the other hand, SP600125, a potent inhibitor of c-Jun N-terminal kinase
Figure 2. The expression of RANKL and OPG in osteoblasts collected 0, 1, 2, 6, 12, 24, and 48 hours, respectively, after 1 mg/mL P. endodontalis (Pe) or E. coli (Ec) LPS stimulation. (A) Western blot analysis of RANKL and OPG protein. (B) Quantification of RANKL messenger RNA by real-time polymerase chain reaction. Relative messenger RNA levels are expressed as the fold increases compared with the RANKL messenger RNA level in unstimulated cells. The values shown are means standard deviations for triplicate assays. An asterisk indicates that the P value was <.05 in comparison with the RANKL messenger RNA level in cells stimulated with Pe or Ec LPS.
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(JNK), prevented the up-regulation of RANKL expression in infected osteoblasts (P < 0.05) (Fig. 3A and C). In contrast, the inhibitory effect of wortmannin (an inhibitor of phosphatidylinositol 3-kinase) and PD98059 (an inhibitor of MEK1/2) (P < .05) was observed in E. coli LPS-treated mouse osteoblasts (Fig. 3B and D).
Discussion It has been reported that bacteria preferentially stimulate specific cell types, which may be related to their capacity to induce inflammation in given tissues. When it comes to P. endodontalis, it is less potent than Streptococcus mutans in stimulating mononuclear cells but more effective in directly inducing IL-6 and IL-1expression by osteoblastic cells (4). This might be significant because these characteristics may enable P. endodontalis to escape the immune response mounted by leukocytes but possess the ability to cause bone resorption at the sites of endodontic infection. The association of different bacteria with specific pathologic process may be partially explained by their capacities in activating different cell types. In view of this, it was of interest to us to detect the P. endodontalis LPS signaling in osteoblasts, which is closely related to the pathogenic mechanism(s) of the periapical lesion. It is recognized that E. coli LPS is a typical bacterial endotoxin and P. endodontalis is the main pathogenic bacterium of apical periodontitis, so it is of great importance to compare the capacity of P. endodontalis LPS and E. coli LPS to induce the response in osteoblasts. There is no previous study on P. endodontalis LPS in bone lesion but plenty of studies about P. gingivalis LPS, and it has been reported in studies that P. gingivalis LPS is less potent than E. coli LPS in inducing the release of inflammatory cytokines in various cells (20, 21). In the present study, we observed that, at some time points, P. endodontalis LPS showed a higher activity of RANKL induction in osteoblasts than E. coli LPS. P. endodontalis and P. gingivalis belong to the same species; they are both black-pigmented, anaerobic, rod-shaped bacteria, and they have close biological activity and characteristics and also similar pathogenicity. In view of this, we discuss our data with P. gingivalis, and our results are different from those of most other studies to a certain degree. This is possibly related to the P. endodontalis strain used, the LPS extraction method, and the selection of the times for detecting cytokine levels and target cells. It is known that different strains of the same bacterium or different extraction methods have considerable effect on the purity and especially the activity of LPS (22). Another important factor is the LPS concentration in cell culture medium and LPS stimulation time. The LPS concentration we use to treat osteoblasts is 1 mg/mL, which has been confirmed to be a sufficient concentration to induce cell response in this study. According to previous studies (23, 24), higher concentrations have cytotoxicity, which will reduce RANKL expression. The time settlements in the blocking test were less than 8 hours because long-time incubation with LPS will promote the apoptosis of cells. Reports have shown that RANKL messenger RNA in osteoblasts is up-regulated by the bone-resorbing factors, such as vitamin D3, IL-1, IL-11, TNF-a, and parathyroid hormone (25). Because LPS has been shown to stimulate osteoblasts to secrete the osteolytic factors IL-1, IL-6, TNF-a, and prostacyclin (PGI2), it is reasonable to presume that RANKL gene expression by LPS may be indirectly mediated by one of these factors. However, it has been shown that LPS can directly mediate RANKL messenger RNA up-regulation in osteoblasts, and RANKL messenger RNA increase is not secondary to prostacyclin (PGI2) or TNF-a synthesis (24). This has well proved the feasibility of the present study detecting the RANKL expression in osteoblasts induced by P. endodontalis LPS. Our current data have indicated JOE — Volume 37, Number 12, December 2011
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Figure 3. The effects of anti-TLR antibodies, PI3K inhibitor (wortmannin), NF-kB inhibitor (SN50), p38MAPK inhibitor (SB203580), MEK1/2 inhibitor (PD98059), and JNK inhibitor (SP600125) on the expression of RANKL in osteoblasts stimulated with LPS. Before the addition of P. endodontalis (Pe) or E. coli (Ec) LPS (1 mg/mL), osteoblasts were pretreated for 1 hour with anti-TLR2 and anti-TLR4 antibodies (2 mg/mL), wortmannin (1 mmol/L), SN50 (5 mmol/L), SB203580 (5 mmol/L), PD98059 (5 mmol/L), and SP600125 (5 mmol/L), respectively. (A) Western blot analysis of RANKL in osteoblasts stimulated with Pe LPS for 12 hours. (B) Western blot analysis of RANKL in osteoblasts stimulated with Ec LPS for 12 hours. (C) Quantification of RANKL messenger RNA in osteoblasts stimulated with Pe LPS for 2, 6, and 12 hours, respectively. (D) The quantification of RANKL messenger RNA in osteoblasts stimulated with Ec LPS for 2, 6, and 12 hours, respectively. Relative messenger RNA levels are expressed as the fold increases compared with the RANKL messenger RNA level in unstimulated cells. The values shown are means standard deviations for triplicate assays. An asterisk (*) indicates that the P value was <.05 in comparison with the RANKL messenger RNA level in cells stimulated with LPS only.
that LPS rapidly increased RANKL expression in osteoblasts. In contrast, OPG expression remained constant in these cells. These results suggest that LPS potently induce osteoclastogenic activity in mouse osteoblasts. In the signal transduction of LPS, TLR2 and TLR4 are transmembrane receptors that transmit the LPS signal to intracellular components and play important roles in the immune system (26, 27). Initially, it was recognized that TLR4 was the receptor for gram-negative bacterial LPS, and TLR2 was the receptor for gram-positive peptidoglycan and lipopeptides (19). In contrast, we found that P. endodontalis LPS could use both TLR2 and TLR4 (mainly TLR2) in osteoblasts, whereas E. coli LPS stimulated RANKL expression via TLR4 in osteoblasts. It has been reported whether the TLR recognized by LPS (TLR2 or TLR4) is dependent on the structure of the LPS (28). P. gingivalis LPS has been shown to differ from many gram-negative bacterial LPS in structure and various functional activities, and P. gingivalis LPS acts not only via TLR4 but also via TLR2 or other signal transducers in monocytes/macrophages (29). In addition, it has been reported that human periodontitis gingiva expressed TLR2 and TLR4, and the ratio of TLR2-positive cells was higher than that of TLR4 (30). Our findings suggested that there is resemblance between P. endodontalis LPS and P. gingivalis LPS when binding to TLR. After binding to the TLR, LPS can trigger proinflammatory cytokine gene expression by the activation of a number of intracellular JOE — Volume 37, Number 12, December 2011
inflammatory signaling pathways including the PI3K, NF-kB, and various mitogen-activated protein kinase (MAPK) pathways (31, 32). Previous studies have shown that NF-kB is the main signaling pathway of most bacterial LPS (33). We discovered in the present study that the JNK inhibitor was the most effective in blocking RANKL expression in osteoblasts upon stimulation with P. endodontalis LPS. This was in agreement with the report of Darveau et al (34) that P. gingivalis LPS could not activate p38MAPK and ERK pathways in human endothelial cells. Another study has proven that the inhibitor of JNK and activator protein-1 (AP-1) activation can prevent the upregulation of RANKL expression in infected osteoblasts, and P. gingivalis infection resulted in the phosphorylation of c-Junand activation of AP-1 (35). All these findings indicate that JNK is involved in RANKL expression induced by black-pigmented pseudomonas. With E. coli LPS, the story was different; the activation of ERK and PI3K pathways seems to play an important role in the activation of RANKL expression. NF-kB signals did not seem to be involved in RANKL expression, which is consistent with a report showing no obvious NF-kB binding motifs in the promoter regions of the mouse RANKL gene (36). In conclusion, our results show that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4–JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin
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Acknowledgments The authors deny any conflicts of interest related to this study.
18. 19. 20.
References 1. Nair PNR. On the causes of persistent apical periodontitis: a review. Int Endod J 2006;39:249–81. 2. Gomes BP, Jacinto RC, Pinheiro ET, et al. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR. Oral Microbiol Immunol 2005;20:211–5. 3. Tomazinho LF, Avila-Campos MJ. Detection of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens in chronic endodontic infection. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103: 285–8. 4. Murakami Y, Hanazawa S, Tanaka S, et al. A possible mechanism of maxillofacial abscess formation: involvement of Porphyromonas endodontalis lipopolysaccharide via the expression of inflammatory cytokines. Oral Microbiol Immunol 2001;16: 321–5. 5. Ataoglu T, Ungor M, Ari H, et al. Interleukin-1b and tumor necrosis factor-a levels in periapical exudates. Int Endod J 2002;35:181–5. 6. Shin SJ, Lee JI, Baek SH, et al. Tissue levels of matrix metalloproteinases in pulps and periapical lesions. J Endod 2002;28:313–5. 7. Radics T, Kiss C, Tar I, Marton IJ. Interleukin-6 and granulocyte-macrophage colony-stimulating factor in apical periodontitis: correlation with clinical and histologic findings of the involved teeth. Oral Microbiol Immunol 2003;18:9–13. 8. Mitchell BD, Yerges-Armstrong LM. The genetics of bone loss: challenges and prospects. J Clin Endocrinol Metab 2011;96:1258–68. 9. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397: 315–23. 10. Siqueira JF Jr, R^oc¸as IN. Bacterial pathogenesis and mediators in apical periodontitis. Braz Dent J 2007;18:267–80. 11. Jacinto RC, Gomes BP, Souza-Filho FJ, et al. Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth. J Med Microbiol 2005;54: 777–83. 12. Jiang Y, Schilder H. An optimal host response to a bacterium may require the interaction of leukocytes and resident host cells. J Endod 2002;28:279–82. 13. Yang LC, Huang FM, Lin CS, et al. Induction of interleukin-8 gene expression by black-pigmented Bacteroides in human pulp fibroblasts and osteoblasts. Int Endod J 2003;36:774–9. 14. Nowotny A. Basic Exercises in Immunochemistry. 2nd ed. New York, NY: Springer; 1979. 69–72. 15. Kido J, Kido R, Nagata T, et al. Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD14–Toll-like receptor–nuclear factor kB pathway. J Periodont Res 2003;38:557–63. 16. Tabeta K, Yamzaki K, Akashi S, et al. Toll-like receptors confer responsiveness to lipopolysaccharide from Porphyromonas gingivalis in human gingival fibroblasts. Infect Immun 2000;68:3731–5. 17. Uesugi M, Nakajima K, Tohyama Y, et al. Nonparticipation of nuclear factor kappa B (NF-kB) in the signaling cascade of c-jun N-terminal kinase(JNK) and p38 itogenactivated protein kinase (p38MAPK)-dependent tumor necrosis factor alpha
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JOE — Volume 37, Number 12, December 2011