|
Sign In to gain access to subscriptions and/or personal tools.
|
Cementoblast Gene Expression is Regulated by Porphyromonas gingivalis Lipopolysaccharide Partially via Toll-like Receptor-4/MD-2
F.H. Nociti, Jr.1,2,
B.L. Foster2,
S.P. Barros3,
R.P. Darveau2 and
M.J. Somerman2,*
1 Department of Prosthodontics/Periodontics, Division of Periodontics, School of Dentistry at Piracicaba, University of Campinas, Brazil;
2 Department of Periodontics, School of Dentistry, 1959 NE Pacific, D322-Health Science Center, University of Washington, Seattle, WA 98195-7444, USA; and
3 Department of Morphology, Division of Histology, School of Dentistry at Piracicaba, University of Campinas, Brazil;
Correspondence: * corresponding author, somerman{at}u.washington.edu
 |
ABSTRACT
|
|---|
Lipopolysaccharides are potent inflammatory mediators considered to contribute to destruction of periodontal tissues. Here, we hypothesized that Porphyromonas gingivalis lipopolysaccharide (P-LPS) treatment would regulate gene expression in murine cementoblasts through Toll-like receptor 4. Real-time (RT)-PCR and Northern blot analysis indicated that P-LPS decreased expression of transcripts for osteocalcin (OCN) and receptor activator of nuclear factor  B ligand (RANKL). In contrast, a dose-dependent up-regulation in mRNA levels for osteopontin (OPN) and osteoprotegerin (OPG) was observed. Similarly, ELISA demonstrated decreased RANKL and increased OPG levels. A monoclonal antibody specific for mouse TLR-4/MD-2 partially neutralized the P-LPS effect on cementoblasts. These results indicate that exposure of cementoblasts to P-LPS can alter cell function by regulating markers of osteoclastic activity (e.g., RANKL/OPG), thereby potentially affecting the inflammation-associated resorption of mineralized tissues.
Key Words: lipopolysaccharides • cementoblasts • cytokines • Porphyromonas gingivalis
|
INTRODUCTION
|
|---|
Periodontitis is a chronic inflammatory disease characterized by gingival inflammation and alveolar bone resorption. Lipopolysaccharides released from Gram-negative bacteria, including Porphyromonas gingivalis, are reported to elicit inflammatory responses that may contribute to the destruction of periodontal tissues. The ability of several varieties of lipopolysaccharides to regulate synthesis and release of cytokines has been associated with their marked osteolytic properties in different cell types (Nair et al., 1996; Chiang et al., 1999; Jiang et al., 2002; Nagasawa et al., 2002; Choi et al., 2003; Tsukahara et al., 2003); however, no information on lipopolysaccharide effect on cementoblasts is available.
The availability of cementoblast cell lines has allowed for the investigation of cellular and molecular mechanisms by which root-surface cells control the homeostasis of periodontal tissues (Grzesik et al., 1998). Our laboratory developed a cementoblast cell line (OC-CM) that reflects genes and proteins (such as bone sialoprotein [BSP], osteocalcin [OCN], and type 1 collagen) expressed by these cells in situ (D’Errico et al., 2000), providing an excellent model for determining the role of cementoblasts in health and disease. Additional studies have demonstrated that these cells respond to factors associated with the formation and/or regeneration of the periodontium (Zhao et al., 2003), and also factors associated with bone metabolism (Ouyang et al., 2000).
Here we show that cementoblasts express mRNA for Toll-like receptors (TLR)-2 and -4, and for CD-14 and MD-2, molecules shown to enhance Porphyromonas gingivalis lipopolysaccharide (P-LPS) responsiveness. In addition, we demonstrate that P-LPS regulates genes associated with cementum formation. Partial neutralization of this P-LPS effect was achieved with the use of monoclonal antibodies against the TLR-4/MD-2 complex.
|
MATERIALS & METHODS
|
|---|
Cell Line
An immortalized murine cementoblast cell line, OC-CM.30, was used. These cells were established by the isolation of tooth root-surface cells from transgenic mice containing a SV40 large T-antigen (TAg) under the control of the osteocalcin (OCN) promoter (D’Errico et al., 2000). All procedures were approved by the University Committee on Use and Care of Animals and were in compliance with state and Federal laws.
Preparation of Lipopolysaccharides
Porphyromonas gingivalis lipopolysaccharide (P-LPS) was purified from P. gingivalis ATCC 33277 by the cold MgCl2/ethanol procedure (Coats et al., 2003). P-LPS preparations were suspended in lipopolysaccharide-free water (Sigma Chemical Co., St. Louis, MO, USA). P-LPS preparations were determined to be free of contaminating nucleic acid and protein, and were analyzed by gas chromatographic analysis for sugar and fatty acid composition. MALDI-TOF analysis of isolated P-LPS revealed two lipid species at M/Z1435 and 1450.
Proliferation Assay
Cells were seeded in 35-mm tissue-culture dishes at a density of 15,000 cells/cm2 and cultured for 24 hrs in Dulbecco’s Modified Eagle Medium (DMEM) with 5% FBS and antibiotics. Cells were counted and treated with P-LPS (100 ng/mL) plus ascorbic acid (AA) (50 µg/mL). Cells were harvested at 2, 4, and 6 days, with medium changed on day 4. Cell number was obtained by hemacytometry, and viability evaluated by trypan blue staining. Experiments were carried out two times with comparable results.
Northern Blot Analysis
Dose-response assay
Cells were seeded at 30,000 cells/cm2 in 60-mm dishes in triplicate and were maintained in DMEM plus 10% FBS, antibiotics, and L-glutamine (Gibco BRL, Gaithersburg, MD, USA) in a humidified atmosphere of 5% CO2 at 37°C. Upon reaching confluence, cells were switched to DMEM with 5% FBS and AA (50 µg/mL, control), or plus P-LPS (1–1000 ng/mL) for 24 hrs. Total RNA was isolated with the use of Trizol® reagent (Gibco, BRL, Gaithersburg, MD, USA), and expression of transcripts for OCN, osteopontin (OPN), bone sialoprotein (BSP), and collagen I (Col I) was examined by Northern blot as previously described (Zhao et al., 2002). Experiments were carried out two times with comparable results.
Time-course assay
Cells were seeded and maintained as described above, with P-LPS at a concentration of 100 ng/mL. After 1, 6, 12, and 24 hrs, total RNA was isolated and gene expression determined by Northern blot analysis.
Real-time PCR
Reverse transcription
Total RNA was DNase-treated (DNA-freeTM, Ambion Inc., Austin, TX, USA), and 1 µg was used for cDNA synthesis. The reaction was carried out with the use of the first-strand cDNA synthesis kit (Roche Diagnostic Co., Indianapolis, IN, USA) according to the manufacturer’s recommendations.
Primer design
Alkaline phosphatase (AP) and GAPDH primers were prepared as suggested by Locklin et al.(2003). Primers for TLR-2, TLR-4, MD-2, CD-14, OPG, RANKL, Cbfa-1, IL-1, and IL-6 were designed with the use of LightCycler probe design software (Roche Diagnostics GmbH, Mannheim, Germany). Experiments were run twice with comparable results. Amplification profile was 95/0; 55/7; 72/20 [temperature (°C)/time (sec)] and 35 cycles. Primer sequences were as follows (F/R):
- RANKL (CATGACGTTAAGCAACGG/AGGGAAGGGTTGGACA);
- OPG (TGAATGCCGAGAGTGTAG/CTGCTCGCTCGATTTG);
- TLR-4 (AGCCGTTGGTGTATCT/GGCTCTCGGTCCATAG);
- TLR-2 (AGTCTAAAGTCGATCCGC/CTCGCTCACTACGTCT);
- MD-2 (TCGAGTCCGATGGTCT/CCTTACGCTTCGGCAA);
- CD-14 (TGCGAGCTAGACGAGG/AGATGTTGAGATCGGGT);
- IL-1 (GTATGCCTACTCGTCGG/GTCATAGAGGGCAGTCC);
- IL-6 (ATGATGGATGCTACCAAAC/AGGCATAACGCACTAGG); and
- Cbfa-1 (CTTCATTCGCCTCACAAAC/GTCACTGCGCTGAAGA).
Optimization of PCR conditions
Reaction efficiency was optimized, and final concentrations of 3 mM MgCl2 and 0.5 µM primer were chosen.
RT-PCR reactions
RT-PCR was performed in the LightCycler system (Roche Diagnostics GmbH) with the FastStart DNA Master SYBR Green I kit (Roche Diagnostic Co.). For each run, water was the negative control. Reaction product was quantified (Relative Quantification Software, Roche Diagnostics GmbH), with GAPDH as the reference (housekeeping) gene.
Antibody against TLR-4/MD-2
Cells were seeded and maintained as described above (dose-response assay). Cells were switched to DMEM with 5% FBS and AA (50 µg/mL, control) or plus P-LPS (100 ng/mL). Before the addition of P-LPS, cells were incubated for 1 hr with MTS510 antibody (eBioscience, San Diego, CA, USA). The negative control was omission of P-LPS, and positive control was omission of antibodies. After 24 hrs, total RNA was isolated, and BSP, OCN, Col I, and OPN transcripts were examined by Northern blot, with RANKL/OPG levels assessed by RT-PCR.
Enzyme-linked Immunoabsorbent Assay (ELISA) for the Detection of OPG and RANKL
ELISA assays were performed as recommended (R&D Systems, Inc., Minneapolis, MN, USA). Cells were seeded and maintained as described above (dose-response assay). At 24 hrs after treatment, medium was collected and used for OPG detection at a 1:10 dilution. Cells were harvested in 1 mL lysis buffer (150 mM NaCl, 10 mM phosphate buffer, pH 7, 1% NP40, 0.1% SDS, 1% sodium deoxycholate), vortexed and centrifuged, and supernatant was used for RANKL detection.
Statistical Analysis
Data were analyzed by one-way ANOVA ( = 0.05). If statistical difference was detected, we used the Bonferroni t test to identify the groups that differed from the control. The Student t test ( = 0.05) compared P-LPS-treated groups with respective controls in the time-course assay.
|
RESULTS
|
|---|
TLR-2/-4 and CD-14/MD-2 in Mouse Cementoblasts
P-LPS has the potential to interact with TLR-2 and -4 and promote cell activation in vitro (Bainbridge et al., 2002). Both TLR-2 and TLR-4 mRNAs were expressed constitutively in cementoblasts (Fig. 1A ). Furthermore, these cells expressed mRNA for CD-14 and MD-2, both known to be part of the TLR-4 signaling complex (Figs. 1A, 1B).
View larger version (13K):
[in this window]
[in a new window]
|
Figure 1. Characterization of cementoblasts: mRNA expression of (A) Toll-like receptors 2 and 4 (TLR-2/TLR-4), and (B) MD-2 and CD-14. Bars represent mean ± SD from two separate experiments, with similar results (n = 2). Cementoblasts (OCCM-30) were seeded, and upon reaching confluence (4 days), mRNA was extracted and analyzed by real-time RT-PCR; the results were normalized to GAPDH and to calibrator (OCCM-30) values. (C) Proliferation assay: Cementoblasts (OCCM-30) were seeded and, after 24 hrs, were treated with P. gingivalis lipopolysaccharide at 100 ng/mL. Cells were counted before treatment (D0), and after 2, 4, and 6 days. Graph illustrates the proportion of viable (open bar) vs. non-viable cells (dark bar) (Trypan blue staining) for a representative experiment with similar results seen in two separate experiments (n = 2). *P < 0.001 and **P < 0.05 vs. control within the same period of time. CN = control group and P-LPS = Porphyromonas gingivalis-treated group.
|
|
The Effect of P-LPS on Cementoblast Proliferation
Having established that cementoblasts express TLRs, our next step was to determine whether P-LPS exposure would influence cell proliferation and viability. On D2 and D4, no negative effect of P-LPS was observed on either cell proliferation or viability. On D6, while cells exposed to P-LPS still proliferated, there was a small but statistically significant decline in total cell number and an increase in cell death (Fig. 1C ).
Effect of P-LPS on Gene Expression: Dose-response
Next, effects of P-LPS on genes associated with cell differentiation, maturation, and mineral formation (e.g., BSP, OCN, OPN, Col I, Cbfa-1, and AP), and genes associated with osteoclast activation (e.g., OPG, RANKL, IL-1, and IL-6) were determined. P-LPS reduced OCN/RANKL and increased OPN/OPG mRNA levels in a dose-dependent manner. Col I was increased slightly, while BSP was minimally affected by P-LPS (Figs. 2A, 2B). Transcripts for AP, IL-1, IL-6, and Cbfa-1 were not altered (data not shown).
View larger version (30K):
[in this window]
[in a new window]
|
Figure 2. Effect of P. gingivalis lipopolysaccharide (P-LPS) on gene expression: (A) Cementoblasts (OCCM-30) were cultured in DMEM (5% FBS) with ascorbic acid (AA) (50 µg/mL) (control - CN) or plus P-LPS (1–1000 ng/mL). mRNA was extracted after 24 hrs, and expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col I), and osteopontin (OPN) was analyzed by Northern blot. Results from two separate experiments (n = 2) showed that P-LPS regulates expression of mineral-associated genes in a dose-dependent manner, with a marked decrease in OCN and an increase in OPN and Col I transcripts. Effect of P-LPS on genes/proteins associated with bone resorption: (B) Cementoblasts (OCCM-30) were cultured as described above (n = 2). RNA was extracted after 24 hrs and analyzed by real-time PCR, and values were normalized to GAPDH and calibrator values. P-LPS significantly decreased RANKL and increased OPG expression in a dose-dependent manner. (C) Protein levels evaluated by ELISA. Cell lysate and medium were used to obtain RANKL and OPG levels, respectively. Note that the impact of P-LPS on protein levels parallels findings on gene expression. Bars represent mean ± SD from two separate experiments, with similar results (n = 2). *P < 0.001 and **P < 0.05 vs. control.
|
|
ELISA: OPG and RANKL
To determine whether changes in RANKL/OPG protein levels paralleled changes in gene expression, we collected conditioned medium and cell lysate from cells treated for 24 hrs with P-LPS (1–1000 ng/mL) and examined them by ELISA. In agreement with the observed effects on RANKL and OPG mRNA levels, P-LPS reduced RANKL protein levels in cell lysates and increased OPG protein levels in media. A significant reduction in RANKL levels was observed at all P-LPS concentrations, while up-regulation of OPG protein levels was significant at 10 and 100 ng/mL (Fig. 2C).
Effect of P-LPS on Gene Expression: Time Course
BSP
Untreated cells exhibited a gradual increase in BSP expression at 1 hr and 6 hrs, with down-regulation by 12 hrs, and P-LPS did not alter this pattern (Fig. 3).
View larger version (36K):
[in this window]
[in a new window]
|
Figure 3. Effect of P. gingivalis lipopolysaccharide (P-LPS) on gene expression: Time-course assay: Cementoblasts (OCCM-30) were cultured in DMEM (5% FBS) with ascorbic acid (AA) (50 µg/mL) (control-CN) or plus P-LPS (100 ng/mL). RNA was extracted after days 2, 4, and 6, and expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col I), and osteopontin (OPN) was analyzed by Northern blot. Values were normalized to 18S, and bars represent mean ± SD from two separate experiments, with similar results (n = 2). Results indicated that the effect of P-LPS on gene expression was observed as early as 6 hrs. OCN mRNA levels were markedly decreased at 12 and 24 hrs, whereas OPN levels were significantly noted by 6 hrs. Col I mRNA levels were slightly and consistently up-regulated by P-LPS at 24 hrs, whereas minimal changes were observed in BSP expression when the control group was compared with the P-LPS group. *P < 0.001 and **P < 0.05 vs. control within the same period of time.
|
|
OCN
Untreated cells showed a time-dependent up-regulation of OCN mRNA, resulting in the highest levels at 24 hrs. In contrast, with exposure to P-LPS, a significant time-dependent down-regulation in OCN expression was observed (Fig. 3).
OPN
Under control conditions, OPN levels increased over time up to 12 hrs, but by 24 hrs, its level was lower than at 12 hrs. Upon exposure to P-LPS, OPN expression was increased beyond the control, with the most dramatic effect noted at 6 hrs (Fig. 3).
Col I
For both untreated and P-LPS-treated cells, Col I transcripts showed a slight decline from 1 hr to 12 hrs, with an increase at 24 hrs. P-LPS increased Col I mRNA levels at 24 hrs, and although this was a slight effect, it was reproduced in 3 separate experiments (Fig. 3).
RANKL/OPG
Time-course RT-PCR analysis indicated that P-LPS down-regulated RANKL and up-regulated OPG by 6 hrs vs. untreated cells (data not shown).
TLR-4/MD-2 Antibodies Partially Neutralized the Effect of P-LPS on Gene Expression.
TLR-4 knockout mice have been reported to be hypo-responsive to lipopolysaccharides (Poltorak et al., 1998), suggesting that TLR-4 is required for cells to respond to lipopolysaccharides. The effect of P-LPS on gene expression can be partly attributed to the TLR-4/MD-2 complex (Fig 4). Incubating cells with antibodies against TLR-4/MD-2 complex, prior to LPS treatment, partially blocked P-LPS-mediated effects on transcripts for Col I, OCN, OPN, RANKL, and OPG (Fig. 4).
View larger version (28K):
[in this window]
[in a new window]
|
Figure 4. Lipopolysaccharide-receptor interactions: Cementoblasts (OCCM-30) were cultured in DMEM (5% FBS) with ascorbic acid (AA) (50 µg/mL) (control-CN), plus P-LPS (100 ng/mL), or P-LPS plus Antibody (against TLR-4/MD-2 complex-MTS510) (20 µg/mL). (A) RNA was extracted after 24 hrs, and expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col I), and osteopontin (OPN) was assessed. Values were normalized to 18S, and bars represent mean ± SD from two separate experiments, with similar results (n = 2). Note that antibody treatment partially blocked the effect of P-LPS on OCN/OPN/Col I mRNA levels. (B) Real-time PCR for RANKL/OPG mRNA expressions. Results illustrate that antibody also partially blocked the effect of P-LPS on RANKL/OPG. Antibody treatment resulted in a significant reduction of the effect of P-LPS on OPG, whereas its effect on RANKL expression was not statistically significant. Bars represent mean ± SD from two separate experiments (n = 2). *P < 0.001 and **P < 0.05 vs. control.
|
|
|
DISCUSSION
|
|---|
Our results suggest that P-LPS has the ability to influence cementoblasts by regulating the genes and proteins associated with mineralization and bone resorption. OPN, Col I, and OPG mRNA levels were increased, while OCN and RANKL mRNA levels were decreased. No significant effect was observed for BSP, Cbfa-1, AP, IL-1, and IL-6 mRNA levels. Importantly, no toxic effects of P-LPS on cell behavior were observed (Fig. 1C).
Several cell types have been reported to be responsive to lipopolysaccharides, including neutrophils (Tsukahara et al., 2003), monocytes (Darveau et al., 2002), lymphocytes (Kong et al., 1999), and bone cells (Chiang et al., 1999). Bone cells, e.g., osteoblasts and osteoclasts, are the most extensively investigated with respect to lipopolysaccharide-mediated inflammatory responses and subsequent bone loss. Bacterial lipopolysaccharide can induce bone resorption in vivo, and the effects have been partly attributed to IL-1 and tumor necrosis factor (TNF) activities (Chiang et al., 1999). However, other studies support direct effects of lipopolysaccharides on the induction of osteoclastogenesis (Suda et al., 2002). Since the discovery of the RANKL-RANK-OPG signaling system (Simonet et al., 1997; Yasuda et al., 1998), the ability of lipopolysaccharides to modify these genes/proteins has been examined, with conflicting findings reported (Kikuchi et al., 2001; Jiang et al., 2002; Suda et al., 2002; Zou et al., 2002; Choi et al., 2003).
We now report that P-LPS affects cementoblasts and may also regulate the genes associated with matrix formation. The significance of our findings cannot be immediately determined, but it is clear that cementoblasts play a role in regulating the health/disease states of the periodontium. In fact, while we can only speculate at this time, analysis of the data suggests that cementoblasts elicit a protective response, i.e., OPG mRNA/protein levels were significantly increased, while RANKL mRNA/protein, known as a factor involved with osteoclast function (Katagiri and Takahashi, 2002), was decreased. In support of these findings, untreated periodontitis is associated with marked bone loss, but rarely with root resorption. Increased levels of OPG have also been reported when human gingival fibroblasts were treated with lipopolysaccharides, indicating that these cells may protect against lipopolysaccharide-mediated bone resorption (Nagasawa et al., 2002).
Besides stimulating bone resorption, the role of lipopolysaccharides in regulating bone formation has been investigated. P-LPS was reported to inhibit osteogenesis (Loomer et al., 1994) and to decrease AP activity and OCN and OPN expression (Kadono et al., 1999). In contrast, analysis of data from other groups indicates that lipopolysaccharides have no effect on AP activity (Murata et al., 1997) and increase OPN expression in MC3T3-E1 osteoblastic cells (Jin et al., 1990). Nair et al.(1996) reported down-regulation of Col I after lipopolysaccharide exposure. Here, a dramatic influence of P-LPS on cementoblasts was noted, resulting in a dose-dependent down-regulation of OCN and up-regulation of OPN expression and a slight up-regulation of Col I. Different experimental designs, cell types, stages of cell maturation, and lipopolysaccharide preparation methods may explain the diversity of these published results.
Among the many roles proposed for OPN, one is as a protector from cell death (Denhardt et al., 2001). Therefore, it is possible that increased OPN transcripts are an attempt to protect the cell against P-LPS-induced apoptosis. In contrast, down-regulation of OCN may be related to the ability of P-LPS to retard normal cell function, since OCN is a marker of cell maturation and is normally expressed by cementoblasts. Future studies will address the significance of these findings in more detail.
The molecular basis of lipopolysaccharide-host interactions and the identity of functional lipopolysaccharide receptors have been under investigation for several decades. Evidence from TLR-4-/MD-2-deficient mice (Poltorak et al., 1998; Nagai et al., 2002) supports TLR-4/MD-2 as essential for lipopolysaccharide responsiveness. Here, we demonstrated that TLR-2, TLR-4, CD-14, and MD-2 mRNAs were expressed in cementoblasts. P-LPS up-regulated all these genes in a dose-dependent manner (data not shown), as reported for other cell types (Kadono et al., 1999; Akashi et al., 2000; Kikuchi et al., 2001). As previously reported (Akashi et al., 2000; Kikuchi et al., 2001), we demonstrated that antibodies against TLR-4/MD-2 complex partially blocked the effects of P-LPS. Because TLR-2 mRNA is expressed by cementoblasts (Fig. 1A), these receptors may also be involved in cell recognition of P-LPS, and thus the extent to which TLR-2 may be involved needs to be explored. Defining the role of cementoblasts in development/regeneration and health/disease will assist in the design of new therapeutic approaches for the treatment of periodontal disease.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by NIH grants DE09532 (MJS) and DE012768-05 (RPD). Dr. Nociti was supported by the National Board of Research (CNPq-Brazil).
Received for publication September 8, 2003.
Revision received May 25, 2004.
Accepted for publication May 25, 2004.
|
REFERENCES
|
|---|
- Akashi S, Shimazu R, Ogata H, Nagai Y, Takeda K, Kimoto M, et al. (2000). Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. J Immunol 164:3471–3475.[Abstract/Free Full Text]
- Bainbridge BW, Coats SR, Darveau RP (2002). Porphyromonas gingivalis lipopolysaccharide displays functionally diverse interactions with the innate host defense system. Ann Periodontol 7:29–37.[CrossRef][Medline]
[Order article via Infotrieve]
- Chiang CY, Kyritsis G, Graves DT, Amar S (1999). Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide. Infect Immun 67:4231–4236.[Abstract/Free Full Text]
- Choi BK, Lee HJ, Kang JH, Jeong GJ, Min CK, Yoo YJ (2003). Induction of osteoclastogenesis and matrix metalloproteinase expression by the lipooligosaccharide of Treponema denticola. Infect Immun 71:226–233.[Abstract/Free Full Text]
- Coats SR, Reife RA, Bainbridge BW, Pham TT, Darveau RP (2003). Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells. Infect Immun 71:6799–6807.[Abstract/Free Full Text]
- D’Errico JA, Berry JE, Ouyang H, Strayhorn CL, Windle JJ, Somerman MJ (2000). Employing a transgenic animal model to obtain cementoblasts in vitro. J Periodontol 71:63–72.[CrossRef][Medline]
[Order article via Infotrieve]
- Darveau RP, Arbabi S, Garcia I, Bainbridge B, Maier RV (2002). Porphyromonas gingivalis lipopolysaccharide is both agonist and antagonist for p38 mitogen-activated protein kinase activation. Infect Immun 70:1867–1873.[Abstract/Free Full Text]
- Denhardt DT, Giachelli CM, Rittling SR (2001). Role of osteopontin in cellular signaling and toxicant injury. Annu Rev Pharmacol Toxicol 41:723–749.[CrossRef][Medline]
[Order article via Infotrieve]
- Grzesik WJ, Kuzentsov SA, Uzawa K, Mankani M, Robey PG, Yamauchi M (1998). Normal human cementum-derived cells: isolation, clonal expansion, and in vitro and in vivo characterization. J Bone Miner Res 13:1547–1554.[CrossRef][Medline]
[Order article via Infotrieve]
- Jiang Y, Mehta CK, Hsu TY, Alsulaimani FF (2002). Bacteria induce osteoclastogenesis via an osteoblast-independent pathway. Infect Immun 70:3143–3148.[Abstract/Free Full Text]
- Jin CH, Miyaura C, Ishimi Y, Hong MH, Sato T, Abe E, et al. (1990). Interleukin 1 regulates the expression of osteopontin mRNA by osteoblasts. Mol Cell Endocrinol 74:221–228.[CrossRef][Medline]
[Order article via Infotrieve]
- Kadono H, Kido J-I, Kataoka M, Yamauchi N, Nagata T (1999). Inhibition of osteoblastic cell differentiation by lipopolysaccharide extract from Porphyromonas gingivalis. Infect Immun 67:2841–2846.[Abstract/Free Full Text]
- Katagiri T, Takahashi N (2002). Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis 8:147–159.[CrossRef][Medline]
[Order article via Infotrieve]
- Kikuchi T, Matsuguchi T, Tsuboi N, Mitani A, Tanaka S, Matsuoka M, et al. (2001). Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via toll-like receptors. J Immunol 166:3574–3579.[Abstract/Free Full Text]
- Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. (1999). OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397:315–323.[CrossRef][Medline]
[Order article via Infotrieve]
- Locklin RM, Khosla S, Turner RT, Riggs BL (2003). Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone. J Cell Biochem 89:180–190.[CrossRef][Medline]
[Order article via Infotrieve]
- Loomer PM, Sigush B, Sukhu B, Ellen RP, Tenenbaum HC (1994). Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro. Infect Immun 62:1289–1297.[Abstract/Free Full Text]
- Murata T, Ansai T, Takehara T, Kobayashi S, Haneji T (1997). Extracts of Prevotella intermedia and Actinobacillus actinomycetemcomitans inhibit alkaline phosphatase activity in osteoblastic cells in vitro. Oral Dis 3:106–112.[Medline]
[Order article via Infotrieve]
- Nagai Y, Akashi S, Nagafuku M, Ogata M, Iwakura Y, Akira S, et al. (2002). Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 3:667–672.[Medline]
[Order article via Infotrieve]
- Nagasawa T, Kobayashi H, Kiji M, Aramaki M, Mahanonda R, Kojima T, et al. (2002). LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin. Clin Exp Immunol 130:338–344.[CrossRef][Medline]
[Order article via Infotrieve]
- Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B (1996). Bacterially induced bone destruction: mechanisms and misconceptions. Infect Immun 64:2371–2380.[Abstract]
- Ouyang H, Franceschi RT, McCauley LK, Wang D, Somerman MJ (2000). Parathyroid hormone-related protein down-regulates bone sialoprotein gene expression in cementoblasts: role of the protein kinase A pathway. Endocrinology 141:4671–4680.[Abstract/Free Full Text]
- Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, et al. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088.[Abstract/Free Full Text]
- Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, et al. (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319.[CrossRef][Medline]
[Order article via Infotrieve]
- Suda K, Woo JT, Takami M, Sexton PM, Nagai K (2002). Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-alpha, IL-1, and RANKL. J Cell Physiol 190:101–108.[CrossRef][Medline]
[Order article via Infotrieve]
- Tsukahara Y, Lian Z, Zhang X, Whitney C, Kluger Y, Tuck D, et al. (2003). Gene expression in human neutrophils during activation and priming by bacterial lipopolysaccharide. J Cell Biochem 89:848–861.[CrossRef][Medline]
[Order article via Infotrieve]
- Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et al. (1998). Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602.[Abstract/Free Full Text]
- Zhao M, Xiao G, Berry JE, Franceschi RT, Reddi A, Somerman MJ (2002). Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype. J Bone Miner Res 17:1441–1451.[CrossRef][Medline]
[Order article via Infotrieve]
- Zhao M, Berry JE, Somerman MJ (2003). Bone morphogenetic protein-2 inhibits differentiation and mineralization of cementoblasts in vitro. J Dent Res 82:23–27.
- Zou W, Bar-Shavit Z (2002). Dual modulation of osteoclast differentiation by lipopolysaccharide. J Bone Miner Res 17:1211–1218.[CrossRef][Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 83, No. 8,
602-607 (2004)
DOI: 10.1177/154405910408300804
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter What's this?
This article has been cited by other articles:
|
|
|
|
 
J. Yang, Y. H. Ryu, C.-H. Yun, and S. H. Han
Impaired osteoclastogenesis by staphylococcal lipoteichoic acid through Toll-like receptor 2 with partial involvement of MyD88
J. Leukoc. Biol.,
October 1, 2009;
86(4):
823 - 831.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION