Abstract
We examined the responses of human osteoblastic cell line SaOS-2 to bacterial lipid A, a bioactive center of lipopolysaccharide, during osteoclast differentiation of human peripheral blood mononuclear cells (PBMC). SaOS-2 cells expressed mRNA for Toll-like receptor (TLR) 4, MD-2, CD14, and myeloid differentiation factor 88, whereas they failed to express mRNA for TLR2. Escherichia coli-type synthetic lipid A (compound 506) induced cytokine mRNA expression and nuclear factor (NF)-κB activation in SaOS-2 cells. Compound 506 also increased the expression of receptor activator of NF-κB ligand. Further, cells primed with compound 506 augmented the differentiation of PBMC into osteoclastic cells, and the effect was inhibited by anti-TLR4 monoclonal antibody. These findings suggest that the TLR signaling cascade in osteoblastic cells is involved in regulating the function of osteoclastogenesis.
1 Introduction
Osteoclastogenesis has long been a principal subject in the field of bone cell biology, though the molecular determinants were only recently identified. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL), a membrane-bound protein, is induced by osteoblasts and bone marrow stromal cells [1–4]. Mature osteoclasts can be formed in vitro from bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL [5–9]. RANKL and M-CSF mutant mice exhibit severe osteopetrosis associated with a complete absence of osteoclasts [5,10]. Further, the receptor activator of NF-κB (RANK), a type I transmembrane protein [11], was identified as the receptor for RANKL on osteoclasts [12]. In contrast, osteoprotegerin (OPG), a soluble tumor necrosis factor receptor homolog, was shown to be an inhibitor of osteoclastogenesis competing with RANKL for RANK [13].
Bacteria and their metabolic products are known to induce pathological bone remodeling such as periodontitis, osteomyelitis, bacterial arthritis, and bone resorption by infected metal implants [14]. Among various bacterial components, lipopolysaccharide (LPS) from Gram-negative bacteria has been demonstrated to stimulate bone resorption in both in vitro and in vivo studies [15–17]. Differentiation into osteoclasts is influenced by various systemic and local factors, such as hormones, cytokines, growth factors, and eicosanoids, and LPS is thought to stimulate osteoblasts to secrete these factors, as well as indirectly induce osteoclastogenesis [14]. However, the molecular mechanisms of inflammation and subsequent bone resorption by LPS have not been conclusively established.
Toll-like receptors (TLRs) are a family of homologs of Drosophila Toll [18]. Mammalian TLRs comprise a large group with extracellular leucine-rich repeats and a cytoplasmic Toll/interleukin (IL)-1 receptor homology domain, and have been implicated in the recognition of pathogen-associated microbial products [19,20]. Among them, TLR4 has been recently shown to recognize LPS and its active center lipid A [21–23]. In addition, a small secreted protein, MD-2, which is associated with TLR4 on the cell surface of innate immune cells, was shown to be required for TLR4 recognition of LPS [24]. Further, a TLR-related protein, RP105, was identified in B cells, and plays a role as both an LPS sensor and a regulator of B cell proliferation [25]. RP105 like TLR4 requires an MD-2-related protein, MD-1, for its surface expression [26]. TLR2 is also required for the signaling of various bacterial components such as Staphylococcus aureus peptidoglycan [27,28], muramyldipeptide [29], bacterial lipoprotein [30], and Porphyromonas gingivalis fimbriae [29,31].
In the present study, we examined the expression profiles of TLRs in the osteoblastic cell line SaOS-2, and the effect of SaOS-2 cells primed with bacterial lipid A on the differentiation of human peripheral blood mononuclear cells (PBMC) into osteoclastic cells.
2 Materials and methods
2.1 Bacterial and synthetic components
Escherichia coli-type lipid A (compound 506) was synthesized as described previously [32] and the lipid A specimen was dissolved at a concentration of 2 mg ml−1 in a 0.1% triethylamine aqueous solution. A cell wall peptidoglycan specimen of S. aureus was also prepared as described previously [33]. Further, P. gingivalis strain 381 was grown anaerobically in GAM broth (Nissui, Tokyo, Japan) supplemented with hemin and menadione at 37°C for 26 h. Fimbriae were isolated and purified as described previously [34]. These specimens were then dissolved in a pyrogen-free cell culture medium before the assay.
2.2 Reagents
Mouse anti-human TLR4 monoclonal antibody (mAb) HTA125 came from Medical and Biological Laboratories (Nagoya, Japan). Mouse IgG2a (Dako, Glostrup, Denmark) was used as an isotype control for HTA125. Goat anti-human RANKL polyclonal antibody (pAb) (sc-7627) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat IgG (sc-2028; Santa Cruz Biotechnology) was used as an isotype control for anti-human RANKL pAb.
2.3 Cells
SaOS-2, a human osteoblastic cell line derived from an osteosarcoma, was obtained from Dainippon Pharmaceutical (Osaka, Japan). This cell line has been widely used in studies of bone cell differentiation, proliferation, and metabolism and the cells are known to be capable of bone production [35,36]. SaOS-2 cells were cultured in α minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS; Sigma), 50 µg ml−1 of gentamicin (Gibco, Rockville, MD, USA), and 50 ng ml−1 of amphotericin B (Sigma) in a humidified atmosphere of 95% air–5% CO2 at 37°C. In all experiments, SaOS-2 cells were rendered quiescent by 24 h of incubation in serum-free α-MEM before the addition of test specimens. HeLa, an epithelial cell line established from a human cervix cancer, was provided by Dainippon Pharmaceutical, and maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS, and antibiotics (50 µg ml−1 gentamicin, and 50 ng ml−1 amphotericin B). Heparinized venous blood was drawn from healthy donors after receiving informed consent. Human PBMC were collected by gradient centrifugation (500×g, 20 min) using a Histopaque-1077 (Sigma). After washing the PBMC, monocytes were isolated by a magnetic cell sorting system using a monocyte isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany).
2.4 Reverse transcription-polymerase chain reaction (RT-PCR)
Total cellular RNAs from the human cells were extracted with RNAzol™B (Tel-Test, Friendswood, TX, USA) according to the manufacturer's instructions and treated with RNase-free DNase (TaKaRa, Shiga, Japan) based on a method described previously [37]. Reverse transcription and PCR were conducted using AMV reverse transcriptase (TaKaRa) and Ex Taq DNA polymerase (TaKaRa), respectively. For detection of IL-6, IL-8, monocyte chemotactic protein (MCP)-1, M-CSF, RANKL, and OPG mRNA in SaOS-2 cells, total cellular RNA was extracted after stimulation with the indicated doses of the test specimens in α-MEM with 10% FBS. PCR assays were conducted for 30 cycles in a Takara Thermal Cycler MP (TaKaRa) using the primer pairs and conditions described in Table 1. For a negative control, a non-RT sample was amplified by PCR reaction. Following PCR, 10 µl of the total amplified product was electrophoresed on ethidium bromide-stained 1% agarose gels, and visualized under ultraviolet fluorescence.
PCR primer pairs used for amplification of human mRNA
PCR primer pairs used for amplification of human mRNA
2.5 Flow cytometric analysis
Cells were incubated with HTA125 or mouse IgG2b at 25°C for 15 min. After washing with PBS containing 0.1% NaN3 (PBS-azide), the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Dako) at 25°C for 15 min. For RANKL detection, cells were stained with goat pAb to human RANKL or goat IgG, followed by FITC-conjugated rabbit anti-goat IgG (Dako) [38]. The cells were then washed with PBS-azide, and fixed with 1% paraformaldehyde. The stained cells were analyzed using a FACSCalibur with Cell Quest software (BD Biosciences, San Jose, CA, USA).
2.6 Luciferase assay
A single cell suspension of SaOS-2 cells (5×104 cells per well) was seeded in a 24-well flat-bottom microtiter plate (Falcon 3047; BD Biosciences). After incubation at 37°C for 16 h, the monolayers were washed three times with PBS, and then transfected with 0.8 µg of pNF-κB-Luc plasmid (Stratagene, La Jolla, CA, USA) using TransFast Transfection Reagent (Promega, Madison, WI, USA). The pFC-MEKK plasmid was used as a positive control in the assay. After an initial incubation of 24 h, the cells were incubated with 100 ng ml−1 of the test specimens in 500 µl of α-MEM with 10% FBS at 37°C for 4 h. After incubation, the culture supernatants were removed and 100 µl of PBS was added as well as 100 µl of Bright-Glo™ Luciferase Assay Reagent (Promega). Luminescence was quantified with a luminometer (Turner Designs Luminometer Model TD-20/20; Promega).
2.7 Cytokine production
SaOS-2 cells were trypsinized and suspended at a cell density of 2×105 cells ml−1 in α-MEM with 10% FBS. A single cell suspension (2×104 cells per well) was seeded in a 96-well flat-bottom microtiter plate (Falcon 3072; BD Biosciences). After incubation at 37°C for 16 h in humidified air containing 5% (v/v) CO2, the monolayers were washed three times with PBS, and incubated with α-MEM without FBS at 37°C for 24 h. These cells were then stimulated with the indicated doses of test specimens in 200 µl of α-MEM with 10% FBS at 37°C for 24 h. After incubation, the supernatants were collected, and stored at −80°C until the assay for cytokine production. The production of IL-8 was determined by enzyme-linked immunosorbent assay (ELISA), which was performed according to the manufacturer's instructions (ELISA kit system; GT, Minneapolis, MN, USA). The results were estimated using a standard curve prepared for each assay.
2.8 Osteoclast differentiation of PBMC cocultured with SaOS-2 cells
A single cell suspension was seeded subconfluently onto plastic tissue culture slides (no. 177437; Nunc, Roskilde, Denmark) with α-MEM supplemented with 10% FBS at 37°C for 16 h, and then stimulated with 1 ng ml−1 of compound 506 in α-MEM with 10% FBS for 24 h. In some experiments, SaOS-2 cells were incubated with or without 1 µg ml−1 of HTA125 or mouse IgG2a as an isotype control for 30 min before the addition of compound 506. After washing with PBS three times, these cells were cocultured with PBMC (5×105 cells ml−1) in α-MEM supplemented with 10% FBS for 21 days. The medium was changed every 5 days. After incubation, osteoclastic cells in the culture were detected using a TRACP and ALP double-stain kit (TaKaRa) according to the manufacturer's instructions. Three squares (0.25 mm2) were randomly sampled from each coverslip and the nuclei of tartrate-resistant acid phosphatase (TRAP)-positive cells, stained red, were counted in each square under a microscope. An index of fusion was obtained from the ratio of nuclei in multinucleated cells (with three or more nuclei) divided by the total number of counted nuclei in TRAP-positive cells.
2.9 Statistical analysis
Data were analyzed by one-way analysis of variance using the Bonferroni or Dunn method and the results are presented as the mean±S.E.M. When an individual experiment is demonstrated, it is representative of at least three independent experiments.
3 Results
3.1 Expression profiles for TLRs and related molecules of SaOS-2 cells
mRNA expression for the known TLRs, RP105, and several other proteins such as MD-1, MD-2, CD14, and myeloid differentiation factor 88 (MyD88) in SaOS-2 cells was evaluated by RT-PCR. SaOS-2 cells were found to express mRNA for TLR1, TLR4, TLR5, TLR6, TLR9, MD-2, CD14, and MyD88, however, not for TLR2, TLR3, TLR7, TLR8, TLR10, RP105, or MD-1 (Fig. 1A). Human PBMC expressed all of the TLRs and mRNA-related molecules tested. RT-PCR analysis of β-actin expression confirmed the quality of all RNA preparations used for RT-PCR and no band was detected in the non-RT sample by PCR. Further, we examined the cell surface expression of TLR2 and TLR4 in SaOS-2 cells by flow cytometry analysis (Fig. 1B). SaOS-2 cells expressed TLR4, but not TLR2, and human monocytes, as a positive control, demonstrated specific cell surface TLR2 and TLR4 staining (data not shown).
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TLR expression in SaOS-2 cells. A: mRNA expression for human TLRs and their related molecules in SaOS-2 cells was analyzed by RT-PCR as described in Section 2. Human PBMC were used as a positive source of TLR mRNA expression to confirm the specificity of the primers and PCR. The β-actin gene was assayed as a positive control and PCR products of non-RT samples were examined as a negative control. B: TLR2 and TLR4 on SaOS-2 cells were stained with specific antibodies (bold line) or their isotype as a control (thin line) as detailed in Section 2. Experiments were done at least three times and representative results are presented.
TLR expression in SaOS-2 cells. A: mRNA expression for human TLRs and their related molecules in SaOS-2 cells was analyzed by RT-PCR as described in Section 2. Human PBMC were used as a positive source of TLR mRNA expression to confirm the specificity of the primers and PCR. The β-actin gene was assayed as a positive control and PCR products of non-RT samples were examined as a negative control. B: TLR2 and TLR4 on SaOS-2 cells were stained with specific antibodies (bold line) or their isotype as a control (thin line) as detailed in Section 2. Experiments were done at least three times and representative results are presented.
3.2 Induction of cytokine mRNA expression and NF-κB activation
To determine the response of SaOS-2 cells to various bacterial components, we examined cytokine mRNA expression by SaOS-2 cells stimulated with compound 506, S. aureus peptidoglycan, or P. gingivalis fimbriae. Compound 506 clearly induced mRNA expression for IL-6, IL-8, MCP-1, and M-CSF in SaOS-2 cells, whereas S. aureus peptidoglycan and P. gingivalis fimbriae did not induce any cytokine mRNA expression (Fig. 2).
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Induction of cytokine mRNA expression in SaOS-2 cells stimulated with compound 506 (A), S. aureus peptidoglycan (B), or P. gingivalis fimbriae (C). Cells were stimulated with the indicated doses of each test specimen in α-MEM supplemented with 10% FBS at 37°C. After 4 h of incubation, mRNA expression for IL-6, IL-8, MCP-1, and M-CSF was analyzed by RT-PCR as described in Section 2. The β-actin gene was assayed as a positive control and PCR products of non-RT samples were examined as a negative control. Experiments were done at least three times and representative results are presented.
Induction of cytokine mRNA expression in SaOS-2 cells stimulated with compound 506 (A), S. aureus peptidoglycan (B), or P. gingivalis fimbriae (C). Cells were stimulated with the indicated doses of each test specimen in α-MEM supplemented with 10% FBS at 37°C. After 4 h of incubation, mRNA expression for IL-6, IL-8, MCP-1, and M-CSF was analyzed by RT-PCR as described in Section 2. The β-actin gene was assayed as a positive control and PCR products of non-RT samples were examined as a negative control. Experiments were done at least three times and representative results are presented.
Transcription factor NF-κB is involved in the TLR signaling cascade [39]. We examined NF-κB activation in SaOS-2 cells after stimulation with compound 506, S. aureus peptidoglycan, or P. gingivalis fimbriae using a luciferase assay. Compound 506 exhibited significant NF-κB activation in SaOS-2 cells, whereas S. aureus peptidoglycan and P. gingivalis fimbriae showed none (data not shown).
3.3 Induction of IL-8 production
IL-8-producing activities in SaOS-2 cells after stimulation with compound 506, S. aureus peptidoglycan, or P. gingivalis fimbriae were examined (Fig. 3). Compound 506 induced significant IL-8 production in SaOS-2 cells, whereas stimulation with S. aureus peptidoglycan and P. gingivalis fimbriae resulted in none. These results corresponded with our findings of cytokine mRNA expression and NF-κB activation in SaOS-2 cells.
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Induction of IL-8 production in SaOS-2 cells. Cells were stimulated with the indicated doses of compound 506 (closed circle), S. aureus peptidoglycan (open circle), or P. gingivalis fimbriae (open square) in α-MEM supplemented with 10% FBS at 37°C. After 24 h of incubation, culture supernatants were collected and IL-8 production was determined by ELISA. Experiments were done at least three times and representative results are presented. Each assay was done in triplicate wells and the data are expressed as the mean±S.E.M. Significant differences were seen between groups with and without the test specimens (*P<0.01).
Induction of IL-8 production in SaOS-2 cells. Cells were stimulated with the indicated doses of compound 506 (closed circle), S. aureus peptidoglycan (open circle), or P. gingivalis fimbriae (open square) in α-MEM supplemented with 10% FBS at 37°C. After 24 h of incubation, culture supernatants were collected and IL-8 production was determined by ELISA. Experiments were done at least three times and representative results are presented. Each assay was done in triplicate wells and the data are expressed as the mean±S.E.M. Significant differences were seen between groups with and without the test specimens (*P<0.01).
3.4 Induction of RANKL and OPG expression
RANKL is known to be constitutively present as a membrane-bound form in SaOS-2 cells [40]. In the present study, the cells responded to compound 506, but not S. aureus peptidoglycan or P. gingivalis (Figs. 2 and 3). We examined RANKL and OPG mRNA expression and their cell surface products in SaOS-2 cells stimulated with compound 506 (Fig. 4). SaOS-2 cells constitutively expressed mRNA for RANKL and OPG, and an increased expression of RANKL mRNA was observed at 2 and 4 h after stimulation with compound 506, whereas OPG mRNA expression did not change up to 48 h after stimulation (Fig. 4A). Furthermore, stimulation with compound 506 resulted in augmentation of RANKL expression on the cell surface, while anti-human TLR4 mAb HTA125 clearly inhibited RANKL expression stimulated with compound 506 (Fig. 4B). HeLa cells did not express either RANKL or OPG mRNA, or RANKL on the cell surface.
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Induction of RANKL and OPG expression in SaOS-2 cells primed with compound 506. A: Cells were stimulated with 1 ng ml−1 of compound 506 in α-MEM supplemented with 10% FBS at 37°C for 1 and 48 h. After incubation, the mRNA expression for RANKL and OPG was analyzed by RT-PCR as described in Section 2. The β-actin gene was assessed as a positive control and PCR products of non-RT samples were examined as a negative control. HeLa cells were used as RANKL and OPG mRNA-negative cells. B: SaOS-2 cells were incubated with medium alone (a) or 1 ng ml−1 of compound 506 (b) in α-MEM supplemented with 10% FBS at 37°C for 24 h. In some experiments, SaOS-2 cells were preincubated with HTA125 (c) or mouse IgG2a (d) for 30 min, and then stimulated with 1 ng ml−1 of compound 506 for 24 h. The cell surface expression of RANKL on SaOS-2 cells was determined with a specific antibody (bold line) or its isotype control (thin line) as described in Section 2. HeLa cells (e) were used as RANKL-negative cells. Experiments were done at least three times and representative results are presented.
Induction of RANKL and OPG expression in SaOS-2 cells primed with compound 506. A: Cells were stimulated with 1 ng ml−1 of compound 506 in α-MEM supplemented with 10% FBS at 37°C for 1 and 48 h. After incubation, the mRNA expression for RANKL and OPG was analyzed by RT-PCR as described in Section 2. The β-actin gene was assessed as a positive control and PCR products of non-RT samples were examined as a negative control. HeLa cells were used as RANKL and OPG mRNA-negative cells. B: SaOS-2 cells were incubated with medium alone (a) or 1 ng ml−1 of compound 506 (b) in α-MEM supplemented with 10% FBS at 37°C for 24 h. In some experiments, SaOS-2 cells were preincubated with HTA125 (c) or mouse IgG2a (d) for 30 min, and then stimulated with 1 ng ml−1 of compound 506 for 24 h. The cell surface expression of RANKL on SaOS-2 cells was determined with a specific antibody (bold line) or its isotype control (thin line) as described in Section 2. HeLa cells (e) were used as RANKL-negative cells. Experiments were done at least three times and representative results are presented.
3.5 Osteoclast differentiation of PBMC cocultured with SaOS-2 cells primed with compound 506
SaOS-2 cells have been shown to be able to induce the differentiation of PBMC into osteoclastic cells [41]. In the present study, we examined TRAP-positive multinucleated cells from PBMC cocultured with SaOS-2 cells that were primed with and without compound 506 for 24 h. In the absence of compound 506, SaOS-2 cells induced the differentiation of PBMC into TRAP-positive cells after a 21-day cultivation (Fig. 5A). In addition, SaOS-2 cells primed with compound 506 clearly induced TRAP-positive multinucleated cells from PBMC. Compound 506 elicited a 30% augmentation in the level of fusion, whereas the addition of HTA125 clearly inhibited a shift in the fusion rate induced by compound 506 (Fig. 5B).
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Osteoclast differentiation of PBMC cocultured with SaOS-2 cells primed with compound 506. A: A single cell suspension was seeded subconfluently onto plastic tissue culture slides that included α-MEM supplemented with 10% FBS for 16 h, followed by incubation with medium alone or 1 ng ml−1 of compound 506 in α-MEM with 10% FBS for 24 h. After washing with PBS three times, these cells were cocultured with PBMC in α-MEM supplemented with 10% FBS for 21 days, with the medium changed every 5 days. After incubation, osteoclastic cells in the culture were determined using TRAP staining. B: In some experiments, SaOS-2 cells were incubated with 1 µg ml−1 of HTA125 or isotype control mouse IgG2a for 30 min before the addition of 1 ng ml−1 of compound 506. Three squares (0.25 mm2) were randomly sampled from each coverslip, and the nuclei in TRAP-positive cells were counted in each square under a microscope. An index of fusion was obtained from the ratio of nuclei in multinucleated cells (with three or more nuclei) divided by the total number of counted nuclei. Experiments were done at least three times and representative results are presented. Each assay was done in triplicate wells and the data are expressed as the mean±S.E.M. Significant differences were seen between groups with and without HTA125 (*P<0.01).
Osteoclast differentiation of PBMC cocultured with SaOS-2 cells primed with compound 506. A: A single cell suspension was seeded subconfluently onto plastic tissue culture slides that included α-MEM supplemented with 10% FBS for 16 h, followed by incubation with medium alone or 1 ng ml−1 of compound 506 in α-MEM with 10% FBS for 24 h. After washing with PBS three times, these cells were cocultured with PBMC in α-MEM supplemented with 10% FBS for 21 days, with the medium changed every 5 days. After incubation, osteoclastic cells in the culture were determined using TRAP staining. B: In some experiments, SaOS-2 cells were incubated with 1 µg ml−1 of HTA125 or isotype control mouse IgG2a for 30 min before the addition of 1 ng ml−1 of compound 506. Three squares (0.25 mm2) were randomly sampled from each coverslip, and the nuclei in TRAP-positive cells were counted in each square under a microscope. An index of fusion was obtained from the ratio of nuclei in multinucleated cells (with three or more nuclei) divided by the total number of counted nuclei. Experiments were done at least three times and representative results are presented. Each assay was done in triplicate wells and the data are expressed as the mean±S.E.M. Significant differences were seen between groups with and without HTA125 (*P<0.01).
4 Discussion
Bone remodeling is regulated by the balance of bone-forming osteoblasts and bone-resorbing osteoclasts, and these cells are controlled by various hormones and local factors [42]. In most cases of bacterial infection, LPS has been implicated as a causative factor. Lipid A, a lipophilic portion of LPS with a structure that corresponds to those proposed for enterobacterial-type lipid A, has been chemically determined and synthesized [43]. The chemically well-defined synthetic lipid A compounds have been investigated to understand the interaction between the structure and function of the lipid A molecule. Although TLR2 has been shown to mediate LPS-induced cell signaling [44], the responses to enterobacterial LPS in TLR2 knockout mice were found to be comparable to those of wild-type mice [27]. Furthermore, it was suggested that overexpressed TLR2 was extremely sensitive to minor contamination in commercial LPS preparations, and that neither human nor murine TLR2 played a role in LPS signaling in the absence of contaminants [22,45]. In the present study, we used an E. coli-type synthetic lipid A, compound 506, as a pure TLR4 ligand.
Thus far, the expression patterns of TLRs and their related molecules have been examined in various types of tissues and their cells. Human spleen cells and PBMC were shown to express mRNA for all TLRs and their related molecules [46], and epithelial cells derived from normal human vagina, ectocervix, and endocervix tissues also expressed mRNA for TLR1, TLR2, TLR3, TLR5, and TLR6, though they lacked TLR4 and MD-2 [47]. Our present results indicate that SaOS-2 cells express TLR4, MD-2, and MyD88, but not TLR2 (Fig. 1). In contrast, Kikuchi et al. showed that a murine osteoblastic cell line, MC3T3-E1, expressed both TLR2 and TLR4 [48]. In addition, SaOS-2 cells were shown to constitutively express mRNA for TLR5 and TLR9. Further, it was shown that bacterial flagellin and CpG DNA were recognized by TLR5 and TLR9, respectively [49,50].
Osteoblasts induce the production of osteolytic factors such as IL-1, IL-6, M-CSF, MCP-1, prostaglandin E2 (PGE2), and nitric oxide by LPS [14]. IL-1 was also shown to induce IL-8 production in human osteoblasts [51]. In the present study, the responses of SaOS-2 cells to S. aureus peptidoglycan and P. gingivalis fimbriae as a TLR2 ligand, and compound 506 as a TLR4 ligand were examined [23,27,29]. Compound 506 but not S. aureus peptidoglycan or P. gingivalis fimbriae induced cytokine mRNA expression and IL-8 production in SaOS-2 cells (Figs. 2 and 3). These results suggest that SaOS-2 cells recognize compound 506 through TLR4, while TLR2-deficient SaOS-2 cells are unresponsive to S. aureus peptidoglycan and P. gingivalis fimbriae.
The present results also demonstrated that compound 506 clearly induced M-CSF mRNA expression, and increased the expression of RANKL mRNA and its gene product in SaOS-2 cells (Figs. 2 and 4). Human PBMC cocultured with SaOS-2 cells also differentiated into TRAP-positive cells, while SaOS-2 cells primed with compound 506 definitely increased TRAP-positive multinucleated cells (Fig. 5A). Human monocytes in peripheral blood have been shown to be able to differentiate into osteoclastic cells [52–54], however, SaOS-2 cells showed no mRNA expression for cyclo-oxygenase 2, an inducible PGE2 synthase, even after stimulation with compound 506 in the present experiments (data not shown). It was previously shown that PGE2 induced RANKL expression by autocline mechanisms in periodontal ligament cells [55]. These results suggest that the increase of RANKL and M-CSF expression in SaOS-2 cells primed with compound 506 is due to a PGE2-independent mechanism, after which they participate in osteoclastogenesis. Mouse anti-human TLR4 mAb HTA125 definitely reduced the expression of RANKL and the forming rate of TRAP-positive multinucleated osteoclastic cells in human PBMC cocultured with SaOS-2 cells primed with compound 506 (Figs. 4B and 5B). Taken together, the results of our study demonstrate that bacterial synthetic lipid A induced osteoclastogenesis through TLR4 on human osteoblastic cell line SaOS-2 cells, with an increasing expression of RANKL. The recognition of pathogenic factors and their cell responsiveness via TLR4 may play an important role in bone pathology and the effect of bacterial infections.
Acknowledgements
This work was supported in part by grants-in-aid for scientific research (B) from the Japan Society for the Promotion of Science (No. 13470390), and for Encouragement of Young Scientists (No. 13771291). We thank Mr. M. Benton for his critical reading of the manuscript.
