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Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells
Biochemical and Biophysical Research Communications 360 (2007) 346–351 www.elsevier.com/locate/ybbrc
Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells Shamima Islam, Ferdaus Hassan, Gantsetseg Tumurkhuu, Jargalsaikhan Dagvadorj, Naoki Koide, Yoshikazu Naiki, Isamu Mori, Tomoaki Yoshida, Takashi Yokochi * Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan Received 5 June 2007 Available online 12 June 2007
Abstract Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calciumphosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-a antibody, but not antibodies to macrophagecolony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-jB ligand (RANKL). TNF-a might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-jB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed. 2007 Elsevier Inc. All rights reserved. Keywords: Osteoclast; Lipopolysaccharide; TNF-a; Receptor activator of nuclear factor-jB ligand (RANKL); Jun-N-terminal kinase 1/2 (JNK1/2); Macrophage-colony stimulating factor; Tartrate-resistant acid phosphatase (TRAP)
Osteoclasts are exclusive bone resorbing multinucleated cells that originate from hemopoietic progenitors of the monocyte/macrophage lineage [1–4]. Osteoclasts have a unique morphology and function to resorb calcified bone by making resorption pits (Howship’s lacunae) [5]. Severe bone loss due to excessive bone resorption is observed in bacterial infection-related inflammatory diseases, such as periodontitis, osteomyelitis, and some types of arthritis [6]. Osteoclasts are formed through multiple steps including cell to cell contact, fusion, and differentiation. Receptor activator of nuclear factor (NF)-jB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) are considered as essential and sufficient for osteoclast formation [4,7,8]. Lipopolysaccharide (LPS) is a component of Gram-negative bacterial cell wall and is a well-known pathogen of
*
Corresponding author. Fax: +81 0561 63 9187. E-mail address: [email protected] (T. Yokochi).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.023
inflammatory bone loss. LPS induces the production of various cytokines and mediators, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, and prostaglandin E2 (PGE2) in macrophages and they play an important role in maturation of osteoclast progenitors and bone loss [4,7,9,10]. Thus, LPS seems to affect osteoclast formation in a complicated fashion. However, the action of LPS in the maturation of osteoclast progenitors to osteoclasts is not entirely elucidated. In the present study, we investigated if and how LPS-induced osteoclast formation in murine RAW 264.7 macrophage cells. Here, we demonstrate that LPS may induce the formation of osteoclasts with bone resorbing activity in RAW 264.7 cells. Materials and methods Materials. LPS from Escherichia coli O55:B5 and parthenolide, an inhibitor of NF-jB were purchased from Sigma Chemicals (St. Louis, MO, USA). Recombinant murine RANKL was purchased from Pepro-
S. Islam et al. / Biochemical and Biophysical Research Communications 360 (2007) 346–351 tech EC (London, UK). Tartrate-resistant acid phosphatase (TRAP) staining kit was obtained from Wako, Osaka, Japan. Recombinant mouse M-CSF, anti-mouse M-CSF antibody, monoclonal anti-RANKL antibody, and M-CSF enzyme-linked immunosorbent assay (ELISA) kit were purchased from R and D systems (Minneapolis, MN, USA). PD98059, an extracellular signal regulated kinase (ERK) 1/2 inhibitor; SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor; SP600125, a stress activated protein kinase (SAPK/JNK) inhibitor and NS398 an inhibitor of cyclooxygenase-2 were purchased from Calbiochem (San Diego, CA, USA). Cell culture. The murine macrophage cell line, RAW 264.7, was obtained from Riken Cell Bank (Tsukuba, Japan) and maintained in RPMI 1640 medium containing 5% heat inactivated fetal calf serum (Gibco-BRL, Gaithersburg, MD, USA) and antibiotics at 37 C under 5% CO2. RAW 264.7 cells were cultured in 96-well plates at a density of 1 · 106 cells and treated with LPS at100 ng/ml unless otherwise stated. TRAP staining. TRAP staining was carried out according to the manufacturer’s instruction. Briefly, cells were washed with phosphatebuffered saline (PBS) and treated with a fixation solution at room temperature for 5 min. The cells were washed with distilled water and treated with TRAP-reagent at 37 C for 20–60 min. After washing with distilled water, the cells were observed under microscope. The images were taken with a digital camera attached to the microscope. For the frequency of TRAP-positive cells, at least more than 150 cells/well were counted under microscope. Bone resorption assay. The osteoclastic bone resorption assay was performed by using a commercially available OAAS kit (Osteotec, Choongnam, Korea). Bottoms of the plates were coated with thin films of calcium carbonate as substrates for osteoclasts. RAW 264.7 cells were cultured on the plate and stimulated with various concentrations of LPS for 3 days. TRAP staining was carried out in situ to identify osteoclasts. After staining, the plate was washed with PBS, and 5% sodium hypochlorite (100 ll/well) was added for 5 min to detach the cells. The plate was washed again with PBS and dried. The images were taken with a digital camera attached to the microscope. Alkaline phosphatase assay. RAW 264.7 cells were cultured in 96-well plates and stimulated with various concentrations of LPS for 3 days. The alkaline phosphatase activity was determined as described elsewhere [11]. In brief, an enzyme assay solution containing 8 mM p-nitrophenyl phosphate, 12 mM MgCl2, and 0.1 mM ZnCl2 in 0.1 M glycine–NaOH buffer at pH 10.5 (200 ll) were added to each wells 3 days after the cultivation and the plate was incubated at 37 C for 10 min. The enzyme reaction was terminated by 0.5 M NaOH and the amount of p-nitrophenol released by the enzyme reaction was determined with the absorbance at 405 nm. Data represent mean value of triplicates ± SD. Immunoblotting. RAW 264.7 cells were cultured in a 35 mm plastic dish (4 · 105 cells/dish) and stimulated with LPS at 100 ng/ml, RANKL at 10 ng/ml or M-CSF at 5 ng/ml for 24 h. The immunoblotting method was performed as described previously [12]. Briefly, the cell lysates were extracted by a lysis buffer and boiled at 80 C for 5 min. The protein concentration of the samples was determined by the BCA protein assay reagent (Pierce, Rockford, IL, USA). An equal amount of protein (20 lg) was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to a membrane filter. The membranes were treated with an appropriately diluted antibody for overnight. The immune complexes were detected with a 1:5000 dilution of horseradish peroxidase-conjugated protein G for 1 h and the bands were visualized with a chemiluminescent reagent (Pierce, Rockford IL, USA). Determination of M-CSF. RAW 264.7 cells were cultured in 96-well plates at a density of 1 · 106 cells and treated with LPS at 10, 100, and 1000 ng/ml for 6 h or 24 h. After removal of the supernatants, the M-CSF concentration in the supernatants was determined by a M-CSF ELISA kit according to manufacturer’s instruction. Statistical analysis. Statistical analysis was performed by the Student t test, with p < 0.05 considered significant. All experiments were carried out at least three independent times. Data represent mean value of triplicates ± SD.
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Results Formation of TRAP-positive multinucleated giant cells (MGC) in LPS-stimulated RAW 264.7 cells The effect of various concentrations of LPS on MGC formation in RAW 264.7 cells was examined (Table 1). RAW 264.7 cells were cultured with LPS at 10, 100, or 1000 ng/ml for various days and the TRAP activity, which is a specific histochemical marker of osteoclasts [13,14], was detected by TRAP staining. LPS-induced various numbers of TRAP-positive mononuclear or multinucleated cells. LPS at 100 ng/ml led to more TRAP-positive cells than LPS at 10 ng/ml, but there was no significant difference in the number between LPS at 100 and 1000 ng/ml. More than 20% of cultured cells were TRAP-positive in stimulation with LPS at 100 and 1000 ng/ml for 3 days. LPS at 100 ng/ml was used for further characterization of osteoclast formation unless otherwise stated. Subsequently, the kinetics in the appearance of TRAP-positive MGC was examined in cultures with the addition of LPS (Fig. 1a). The morphological changes of LPS-induced osteoclasts were also followed with TRAP staining (Fig. 1b). No MGC formation was seen at day 1 after LPS stimulation, although approximately 23% of RAW 264.7 cells were TRAP-positive. Those TRAP-positive cells were still mononuclear. The frequency of TRAP-positive cells gradually declined after day 1 and subsequently MGC appeared in LPS-stimulated RAW 264.7 cells. At day 4 most of TRAP-positive cells were MGC but there were still a small number of TRAP-positive mononuclear cells. The number of TRAP-positive MGC decreased significantly at day 5, probably due to the cell death (Fig. 1a).
Bone resorption activity and alkaline phosphatase activity in LPS-induced MGC The bone resorption activity of LPS-induced MGC was examined by utilizing a commercially available OAAS plate, the bottom of which is coated with calcium carbonate thin film to mimic bone. RAW 264.7 cells were cultured with LPS for 3 days on OAAS plate. LPS-induced MGC clearly formed pits on the bottom of the plate, suggesting Table 1 Induction of TRAP-positive cells by various concentrations of LPS LPS (ng/ml)
Frequency of TRAP-positive cellsa (%)
0 10 100 1000
0 13.08 ± 3 21.3 ± 5 24.0 ± 2
a The frequency of TRAP-positive cells was determined by counting more than 150 cells/well in triplicate. The value is expressed as mean with SD.
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Participation of TNF-a in LPS-induced osteoclast formation The possibility was raised that some cytokine or mediator induced by LPS might cause osteoclast formation in RAW 264.7 cells. Therefore, the effect of several anti-cytokine antibodies on LPS-induced osteoclast formation was examined (Fig. 3A). RAW 264.7 cells were cultured with LPS in the presence or absence of various anti-cytokine antibodies. Among anti-cytokine antibodies tested, antiTNF-a antibody (1 lg/ml) remarkably inhibited LPSinduced osteoclast formation. Therefore, we studied the effect of recombinant TNF-a in place of LPS on osteoclast formation in RAW 264.7 cells (Fig. 3B). RAW 264.7 cells were cultured with recombinant TNF-a at 1 ng/ml for 3 days. Recombinant TNF-a induced TRAP-positive mononuclear cells, but not MGC. The combination of LPS and recombinant TNF-a definitely induced the formation of TRAP-positive MGC, suggesting participation of LPS in the MGC formation. No requirement of M-CSF and RANKL in LPS-induced osteoclast formation
Fig. 1. LPS-induced osteoclastogenesis in RAW 264.7 cells. RAW 264.7 cells were incubated with LPS at 100 ng/ml for various days and stained by TRAP staining. The frequency of TRAP-positive cells at day 1, 2, 3, 4, and 5 after LPS treatment is expressed as mean of triplicate determinations ± SD (a). The morphological changes of LPS-induced TRAPpositive cells are shown (b). (A) none; (B) 1 day; (C) 2 days; (D) 3 days after LPS stimulation.
that the bone resorption activity of LPS-induced MGC were functionally active as osteoclasts (Fig. 2A). Next, we examined the alkaline phosphatase activity in LPS-simulated RAW 264.7 cells. Alkaline phosphatase hydrolyzes the ester bond of organic phosphates compounds under alkaline condition and plays an important role in the calcification of bone [15]. RAW 264.7 cells were cultured with LPS at 10, 100, and 1000 ng/ml for 3 days. The alkaline phosphatase activity in LPS-stimulated RAW 264.7 cells was lower than that in untreated control (Fig. 2B). The decrease in the bone remodeling might be reasonable because LPS-induced osteoclast formation in RAW 264.7 cells, which causes bone destruction.
Osteoclast formation is reported to require M-CSF and RANKL [4,7,8]. Therefore, we examined whether M-CSF and RANKL were involved in LPS-induced osteoclast formation or not. The effect of anti-RANKL antibody and anti-M-CSF antibody on LPS-induced osteoclast formation was examined (Fig. 3A). RAW 264.7 cells were treated with anti-RANKL antibody (1 lg/ml) or anti-M-CSF antibody (5 lg/ml) in the presence of LPS (100 ng/ml) for 3 days. TRAP-positive MGC were counted as osteoclasts. Neither anti-M-CSF antibody nor anti-RANKL antibody inhibited LPSinduced osteoclast formation, suggesting no involvement of M-CSF and RANKL. Further, we tried to detect M-CSF in the culture supernatant at 6 and 24 h after stimulation of LPS. No M-CSF was detected in the supernatants (data not shown). Furthermore, we examined the expression of M-CSF and RANKL in LPS-stimulated RAW 264.7 cells (data not shown). RAW 264.7 cells were stimulated with LPS, M-CSF (5 ng/ml) or recombinant RANKL (10 ng/ml) for 24 h and the expression of M-CSF and RANKL was examined by immunoblotting. LPS failed to induce the expression of M-CSF and RANKL at any time tested. On the other hand, RANKL induced the expression of M-CSF and M-CSF also induced the RANKL expression. LPSinduced osteoclast formation did not require M-CSF and RANKL. Furthermore, we investigated the effect of NS398, a cyclooxygenase-2 inhibitor, on LPS-induced osteoclast formation since LPS stimulates osteoclast formation and PGE2 production in co-cultures of mouse osteoblasts and bone marrow cells, and the stimulation is completely inhibited by NS398 [10]. However, NS398 did not inhibit LPS-induced osteoclast formation in our experimental system (data not shown).
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Fig. 2. Osteoclastic bone resorption activity and alkaline phosphatase activity. RAW 264.7 cells were cultured on OAAS plate and stimulated with LPS (100 ng/ml) for 3 days. After removal of cells, osteoclastic pits were visualized under microscope (A). Effect of LPS on alkaline phosphatase activity was examined (B). RAW 264.7 cells were cultured with 10, 100, and 1000 ng/ml LPS for 72 h. The alkaline phosphatase activity is expressed as mean of triplicate determinations ± SD.
The signaling pathway triggering LPS-induced osteoclast formation It has been shown that a series of MAPKs are essential for osteoclast differentiation and activation [16–18]. The effect of various signal inhibitors on LPS-induced osteoclast formation was examined (Fig. 4). First, we tried to examine whether a series of MAPKs, such as ERK1/2, p38 or SAPK/JNK pathway might be involved in LPSinduced osteoclast formation or not. RAW 264.7 cells were pretreated with PD98059 (10 lM) an ERK1/2 inhibitor, SB203580 (10 lM) a p38 inhibitor and SP600125 (2.5 lM) a SAPK/JNK inhibitor for 30 min and then stimulated with LPS at 100 ng/ml for 3 days. SP600125, a SAPK/JNK inhibitor definitely prevented LPS-induced osteoclast formation, although PD98059 and SB203580 did not affect it. Further, we examined the effect of an inhibitor of NF-jB on LPS-induced osteoclast formation. RAW 264.7 cells were pretreated with parthenolide (5 lM) for 30 min and then stimulated with 100 ng/ml LPS for 3 days. Parthenolide, a NF-jB inhibitor, definitely abrogated LPS-induced osteoclast formation (Fig. 4). It was consistent with the fact that NF-jB is an essential factor for osteoclastogenesis and that mice lacking p50 and p52 NF-jB subunits fail to generate osteoclasts and are osteopetrotic [19].
Discussion In the present study, we demonstrate that LPS causes the formation of TRAP-positive MGC in RAW 264.7 cells and that they exhibit a pit-forming activity on calcium carbonate-coated plates. Therefore, LPS is strongly suggested to induce osteoclast formation in RAW 264.7 cells. RAW 264.7 cells have been utilized for analysis of RANKLinduced osteoclast formation [19,20]. We demonstrate that RAW 264.7 cells also act as osteoclast progenitors and differentiate to osteoclasts in response to LPS. LPS is reported to stimulate the survival and fusion of osteoclast progenitors, independent of RANKL and induces pit-forming activity in the presence of M-CSF [21]. Further, LPS promotes osteoclast formation in co-cultures of mouse osteoblasts and bone marrow cells. LPS enhances osteoclast formation in cultures of whole bone marrow cells with dexamethasone and markedly enhances it in the presence of 1,25-dihydroxyvitamin D3 and dexamethasone [22]. On the other hand, LPS inhibits osteoclast formation in whole bone marrow cells with 1,25dihydroxyvitamin D3 via GM-CSF production [22]. Thus, LPS regulates osteoclast formation in a complicated fashion. The present study demonstrate that LPS itself induces osteoclastogenesis in RAW 264.7 cells. The LPS-induced osteoclastogenesis might be closely associated with the bone resorbing activity of LPS.
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Fig. 4. Effect of various signal inhibitors on LPS-induced osteoclastogenesis. RAW 264.7 cells were pretreated with PD98059 (10 lM), SB203580 (10 lM), SP600125 (2.5 lM) and parthenolide (5 lM) for 30 min, and stimulated with LPS (100 ng/ml) for 3 days. TRAP staining was carried out to determine the number of osteoclasts. Experimental data represent mean of triplicate determinations ± SD.
Fig. 3. Participation of TNF-a in LPS-induced osteoclastogenesis in RAW 264.7 cells. Effect of anti-TNF-a antibody on LPS-induced osteoclastogenesis was examined (A). RAW cells were cultured with LPS (100 ng/ml) with or without anti-TNF-a antibody (1 lg/ml), anti-MCSF antibody(5 lg/ml), and anti-RANKL antibody(1 lg/ml) for 3 days. TRAP staining was carried out to determine the number of osteoclasts. Experimental data represent mean of triplicate determinations ± SD. Effect of recombinant TNF-a on LPS-induced osteoclastogenesis was examined (B). RAW 264.7 cells were cultured with LPS (100 ng/ml) and/ or recombinant TNF-a (1 ng/ml) for 3 days. TRAP staining was carried out to determine the number of osteoclasts. Experimental data represent mean of triplicate determinations ± SD.
During preparation of the manuscript, Hotokezaka et al. have just reported RANKL-independent cell fusion of osteoclast-like cells induced by TNF-a, LPS, and peptidoglycan [23]. TNF-a as well as LPS is reported to induce cell fusion for osteoclastogenesis [23]. In the present study, we demonstrate that recombinant TNF-a leads to TRAPpositive mononuclear cells but not their cell fusion although LPS has the ability to induce the cell fusion. Hotokezaka et al. [23] stimulated RAW 264.7 cells with LPS in the presence of U0126 as an ERK1/2 inhibitor and RANKL whereas we stimulated the cells with LPS alone in the absence of such reagents. The difference in the experimental systems might cause the different action of TNF-a on the osteoclast formation in RAW 264.7 cells. RANKL and M-CSF are considered essential and sufficient for osteoclastogenesis [4,7,8]. In our experimental system, however, M-CSF did not participate in LPS-induced
osteoclast formation because anti-M-CSF antibody did not inhibit LPS-induced osteoclast formation and M-CSF was not detected in the supernatant from cultures of LPS-stimulated RAW 264.7 cells. Further, RANKL and PGE2 do not seem to be involved in LPS-induced osteoclast formation. LPS itself is suggested to induce osteoclast formation in RAW 264.7 cells, independent of RANKL, M-CSF, and PGE2. Signaling by RANKL is essential for terminal differentiation of osteoclast progenitors into osteoclasts. The TRAF6 signaling pathway plays an important role downstream of RANKL. Moreover, RANKL induces the activation of NF-jB and SAPK/JNK through TRAF6 signaling and subsequently the activation of the transcription factor NFAT2 [24–28]. LPS as well as RANKL induces the activation NF-jB and SAPK/JNK in RAW 264.7 cells [29]. LPS may mimic RANKL-induced osteoclast formation via activation of NF-jB and SAPK/JNK. There is one report concerning a role of SAPK/JNK pathway in the secretion of inflammatory cytokine TNF-a [30] and selective inhibition of SAPK/JNK blocked LPSinduced TNF expression [31]. Inhibition of SAPK/JNK by the selective inhibitor (SP600125) inhibited LPS-induced osteoclastogenesis. It is still unclear whether NF-jB and SAPK/JNK signaling is required for LPS-induced osteoclast formation or the production of TNF-a as the effector molecule. LPS induces osteoclast formation in RAW 264.7 cells, but not in mouse peritoneal cells (data not shown). Therefore, RAW 264.7 cells may possess the profile of monocytic progenitors. Otherwise, they may produce the factor(s) lacking in mature peritoneal cells. LPS-induced osteoclast formation might be a useful experimental system for characterization of osteoclastogenesis. Acknowledgments This work was supported by in part by a Grant-in-Aid for Scientific Research from the Ministry of Education,
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Science, Sports and Culture of Japan. We are grateful to K. Takahashi and A. Morikawa for the technical assistance.
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Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells Shamima Islam, Ferdaus Hassan, Gantsetseg Tumurkhuu, Jargalsaikhan Dagvadorj, Naoki Koide, Yoshikazu Naiki, Isamu Mori, Tomoaki Yoshida, Takashi Yokochi * Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan Received 5 June 2007 Available online 12 June 2007
Abstract Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calciumphosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-a antibody, but not antibodies to macrophagecolony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-jB ligand (RANKL). TNF-a might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-jB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed. 2007 Elsevier Inc. All rights reserved. Keywords: Osteoclast; Lipopolysaccharide; TNF-a; Receptor activator of nuclear factor-jB ligand (RANKL); Jun-N-terminal kinase 1/2 (JNK1/2); Macrophage-colony stimulating factor; Tartrate-resistant acid phosphatase (TRAP)
Osteoclasts are exclusive bone resorbing multinucleated cells that originate from hemopoietic progenitors of the monocyte/macrophage lineage [1–4]. Osteoclasts have a unique morphology and function to resorb calcified bone by making resorption pits (Howship’s lacunae) [5]. Severe bone loss due to excessive bone resorption is observed in bacterial infection-related inflammatory diseases, such as periodontitis, osteomyelitis, and some types of arthritis [6]. Osteoclasts are formed through multiple steps including cell to cell contact, fusion, and differentiation. Receptor activator of nuclear factor (NF)-jB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) are considered as essential and sufficient for osteoclast formation [4,7,8]. Lipopolysaccharide (LPS) is a component of Gram-negative bacterial cell wall and is a well-known pathogen of
*
Corresponding author. Fax: +81 0561 63 9187. E-mail address: [email protected] (T. Yokochi).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.023
inflammatory bone loss. LPS induces the production of various cytokines and mediators, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, and prostaglandin E2 (PGE2) in macrophages and they play an important role in maturation of osteoclast progenitors and bone loss [4,7,9,10]. Thus, LPS seems to affect osteoclast formation in a complicated fashion. However, the action of LPS in the maturation of osteoclast progenitors to osteoclasts is not entirely elucidated. In the present study, we investigated if and how LPS-induced osteoclast formation in murine RAW 264.7 macrophage cells. Here, we demonstrate that LPS may induce the formation of osteoclasts with bone resorbing activity in RAW 264.7 cells. Materials and methods Materials. LPS from Escherichia coli O55:B5 and parthenolide, an inhibitor of NF-jB were purchased from Sigma Chemicals (St. Louis, MO, USA). Recombinant murine RANKL was purchased from Pepro-
S. Islam et al. / Biochemical and Biophysical Research Communications 360 (2007) 346–351 tech EC (London, UK). Tartrate-resistant acid phosphatase (TRAP) staining kit was obtained from Wako, Osaka, Japan. Recombinant mouse M-CSF, anti-mouse M-CSF antibody, monoclonal anti-RANKL antibody, and M-CSF enzyme-linked immunosorbent assay (ELISA) kit were purchased from R and D systems (Minneapolis, MN, USA). PD98059, an extracellular signal regulated kinase (ERK) 1/2 inhibitor; SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor; SP600125, a stress activated protein kinase (SAPK/JNK) inhibitor and NS398 an inhibitor of cyclooxygenase-2 were purchased from Calbiochem (San Diego, CA, USA). Cell culture. The murine macrophage cell line, RAW 264.7, was obtained from Riken Cell Bank (Tsukuba, Japan) and maintained in RPMI 1640 medium containing 5% heat inactivated fetal calf serum (Gibco-BRL, Gaithersburg, MD, USA) and antibiotics at 37 C under 5% CO2. RAW 264.7 cells were cultured in 96-well plates at a density of 1 · 106 cells and treated with LPS at100 ng/ml unless otherwise stated. TRAP staining. TRAP staining was carried out according to the manufacturer’s instruction. Briefly, cells were washed with phosphatebuffered saline (PBS) and treated with a fixation solution at room temperature for 5 min. The cells were washed with distilled water and treated with TRAP-reagent at 37 C for 20–60 min. After washing with distilled water, the cells were observed under microscope. The images were taken with a digital camera attached to the microscope. For the frequency of TRAP-positive cells, at least more than 150 cells/well were counted under microscope. Bone resorption assay. The osteoclastic bone resorption assay was performed by using a commercially available OAAS kit (Osteotec, Choongnam, Korea). Bottoms of the plates were coated with thin films of calcium carbonate as substrates for osteoclasts. RAW 264.7 cells were cultured on the plate and stimulated with various concentrations of LPS for 3 days. TRAP staining was carried out in situ to identify osteoclasts. After staining, the plate was washed with PBS, and 5% sodium hypochlorite (100 ll/well) was added for 5 min to detach the cells. The plate was washed again with PBS and dried. The images were taken with a digital camera attached to the microscope. Alkaline phosphatase assay. RAW 264.7 cells were cultured in 96-well plates and stimulated with various concentrations of LPS for 3 days. The alkaline phosphatase activity was determined as described elsewhere [11]. In brief, an enzyme assay solution containing 8 mM p-nitrophenyl phosphate, 12 mM MgCl2, and 0.1 mM ZnCl2 in 0.1 M glycine–NaOH buffer at pH 10.5 (200 ll) were added to each wells 3 days after the cultivation and the plate was incubated at 37 C for 10 min. The enzyme reaction was terminated by 0.5 M NaOH and the amount of p-nitrophenol released by the enzyme reaction was determined with the absorbance at 405 nm. Data represent mean value of triplicates ± SD. Immunoblotting. RAW 264.7 cells were cultured in a 35 mm plastic dish (4 · 105 cells/dish) and stimulated with LPS at 100 ng/ml, RANKL at 10 ng/ml or M-CSF at 5 ng/ml for 24 h. The immunoblotting method was performed as described previously [12]. Briefly, the cell lysates were extracted by a lysis buffer and boiled at 80 C for 5 min. The protein concentration of the samples was determined by the BCA protein assay reagent (Pierce, Rockford, IL, USA). An equal amount of protein (20 lg) was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to a membrane filter. The membranes were treated with an appropriately diluted antibody for overnight. The immune complexes were detected with a 1:5000 dilution of horseradish peroxidase-conjugated protein G for 1 h and the bands were visualized with a chemiluminescent reagent (Pierce, Rockford IL, USA). Determination of M-CSF. RAW 264.7 cells were cultured in 96-well plates at a density of 1 · 106 cells and treated with LPS at 10, 100, and 1000 ng/ml for 6 h or 24 h. After removal of the supernatants, the M-CSF concentration in the supernatants was determined by a M-CSF ELISA kit according to manufacturer’s instruction. Statistical analysis. Statistical analysis was performed by the Student t test, with p < 0.05 considered significant. All experiments were carried out at least three independent times. Data represent mean value of triplicates ± SD.
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Results Formation of TRAP-positive multinucleated giant cells (MGC) in LPS-stimulated RAW 264.7 cells The effect of various concentrations of LPS on MGC formation in RAW 264.7 cells was examined (Table 1). RAW 264.7 cells were cultured with LPS at 10, 100, or 1000 ng/ml for various days and the TRAP activity, which is a specific histochemical marker of osteoclasts [13,14], was detected by TRAP staining. LPS-induced various numbers of TRAP-positive mononuclear or multinucleated cells. LPS at 100 ng/ml led to more TRAP-positive cells than LPS at 10 ng/ml, but there was no significant difference in the number between LPS at 100 and 1000 ng/ml. More than 20% of cultured cells were TRAP-positive in stimulation with LPS at 100 and 1000 ng/ml for 3 days. LPS at 100 ng/ml was used for further characterization of osteoclast formation unless otherwise stated. Subsequently, the kinetics in the appearance of TRAP-positive MGC was examined in cultures with the addition of LPS (Fig. 1a). The morphological changes of LPS-induced osteoclasts were also followed with TRAP staining (Fig. 1b). No MGC formation was seen at day 1 after LPS stimulation, although approximately 23% of RAW 264.7 cells were TRAP-positive. Those TRAP-positive cells were still mononuclear. The frequency of TRAP-positive cells gradually declined after day 1 and subsequently MGC appeared in LPS-stimulated RAW 264.7 cells. At day 4 most of TRAP-positive cells were MGC but there were still a small number of TRAP-positive mononuclear cells. The number of TRAP-positive MGC decreased significantly at day 5, probably due to the cell death (Fig. 1a).
Bone resorption activity and alkaline phosphatase activity in LPS-induced MGC The bone resorption activity of LPS-induced MGC was examined by utilizing a commercially available OAAS plate, the bottom of which is coated with calcium carbonate thin film to mimic bone. RAW 264.7 cells were cultured with LPS for 3 days on OAAS plate. LPS-induced MGC clearly formed pits on the bottom of the plate, suggesting Table 1 Induction of TRAP-positive cells by various concentrations of LPS LPS (ng/ml)
Frequency of TRAP-positive cellsa (%)
0 10 100 1000
0 13.08 ± 3 21.3 ± 5 24.0 ± 2
a The frequency of TRAP-positive cells was determined by counting more than 150 cells/well in triplicate. The value is expressed as mean with SD.
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Participation of TNF-a in LPS-induced osteoclast formation The possibility was raised that some cytokine or mediator induced by LPS might cause osteoclast formation in RAW 264.7 cells. Therefore, the effect of several anti-cytokine antibodies on LPS-induced osteoclast formation was examined (Fig. 3A). RAW 264.7 cells were cultured with LPS in the presence or absence of various anti-cytokine antibodies. Among anti-cytokine antibodies tested, antiTNF-a antibody (1 lg/ml) remarkably inhibited LPSinduced osteoclast formation. Therefore, we studied the effect of recombinant TNF-a in place of LPS on osteoclast formation in RAW 264.7 cells (Fig. 3B). RAW 264.7 cells were cultured with recombinant TNF-a at 1 ng/ml for 3 days. Recombinant TNF-a induced TRAP-positive mononuclear cells, but not MGC. The combination of LPS and recombinant TNF-a definitely induced the formation of TRAP-positive MGC, suggesting participation of LPS in the MGC formation. No requirement of M-CSF and RANKL in LPS-induced osteoclast formation
Fig. 1. LPS-induced osteoclastogenesis in RAW 264.7 cells. RAW 264.7 cells were incubated with LPS at 100 ng/ml for various days and stained by TRAP staining. The frequency of TRAP-positive cells at day 1, 2, 3, 4, and 5 after LPS treatment is expressed as mean of triplicate determinations ± SD (a). The morphological changes of LPS-induced TRAPpositive cells are shown (b). (A) none; (B) 1 day; (C) 2 days; (D) 3 days after LPS stimulation.
that the bone resorption activity of LPS-induced MGC were functionally active as osteoclasts (Fig. 2A). Next, we examined the alkaline phosphatase activity in LPS-simulated RAW 264.7 cells. Alkaline phosphatase hydrolyzes the ester bond of organic phosphates compounds under alkaline condition and plays an important role in the calcification of bone [15]. RAW 264.7 cells were cultured with LPS at 10, 100, and 1000 ng/ml for 3 days. The alkaline phosphatase activity in LPS-stimulated RAW 264.7 cells was lower than that in untreated control (Fig. 2B). The decrease in the bone remodeling might be reasonable because LPS-induced osteoclast formation in RAW 264.7 cells, which causes bone destruction.
Osteoclast formation is reported to require M-CSF and RANKL [4,7,8]. Therefore, we examined whether M-CSF and RANKL were involved in LPS-induced osteoclast formation or not. The effect of anti-RANKL antibody and anti-M-CSF antibody on LPS-induced osteoclast formation was examined (Fig. 3A). RAW 264.7 cells were treated with anti-RANKL antibody (1 lg/ml) or anti-M-CSF antibody (5 lg/ml) in the presence of LPS (100 ng/ml) for 3 days. TRAP-positive MGC were counted as osteoclasts. Neither anti-M-CSF antibody nor anti-RANKL antibody inhibited LPSinduced osteoclast formation, suggesting no involvement of M-CSF and RANKL. Further, we tried to detect M-CSF in the culture supernatant at 6 and 24 h after stimulation of LPS. No M-CSF was detected in the supernatants (data not shown). Furthermore, we examined the expression of M-CSF and RANKL in LPS-stimulated RAW 264.7 cells (data not shown). RAW 264.7 cells were stimulated with LPS, M-CSF (5 ng/ml) or recombinant RANKL (10 ng/ml) for 24 h and the expression of M-CSF and RANKL was examined by immunoblotting. LPS failed to induce the expression of M-CSF and RANKL at any time tested. On the other hand, RANKL induced the expression of M-CSF and M-CSF also induced the RANKL expression. LPSinduced osteoclast formation did not require M-CSF and RANKL. Furthermore, we investigated the effect of NS398, a cyclooxygenase-2 inhibitor, on LPS-induced osteoclast formation since LPS stimulates osteoclast formation and PGE2 production in co-cultures of mouse osteoblasts and bone marrow cells, and the stimulation is completely inhibited by NS398 [10]. However, NS398 did not inhibit LPS-induced osteoclast formation in our experimental system (data not shown).
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Fig. 2. Osteoclastic bone resorption activity and alkaline phosphatase activity. RAW 264.7 cells were cultured on OAAS plate and stimulated with LPS (100 ng/ml) for 3 days. After removal of cells, osteoclastic pits were visualized under microscope (A). Effect of LPS on alkaline phosphatase activity was examined (B). RAW 264.7 cells were cultured with 10, 100, and 1000 ng/ml LPS for 72 h. The alkaline phosphatase activity is expressed as mean of triplicate determinations ± SD.
The signaling pathway triggering LPS-induced osteoclast formation It has been shown that a series of MAPKs are essential for osteoclast differentiation and activation [16–18]. The effect of various signal inhibitors on LPS-induced osteoclast formation was examined (Fig. 4). First, we tried to examine whether a series of MAPKs, such as ERK1/2, p38 or SAPK/JNK pathway might be involved in LPSinduced osteoclast formation or not. RAW 264.7 cells were pretreated with PD98059 (10 lM) an ERK1/2 inhibitor, SB203580 (10 lM) a p38 inhibitor and SP600125 (2.5 lM) a SAPK/JNK inhibitor for 30 min and then stimulated with LPS at 100 ng/ml for 3 days. SP600125, a SAPK/JNK inhibitor definitely prevented LPS-induced osteoclast formation, although PD98059 and SB203580 did not affect it. Further, we examined the effect of an inhibitor of NF-jB on LPS-induced osteoclast formation. RAW 264.7 cells were pretreated with parthenolide (5 lM) for 30 min and then stimulated with 100 ng/ml LPS for 3 days. Parthenolide, a NF-jB inhibitor, definitely abrogated LPS-induced osteoclast formation (Fig. 4). It was consistent with the fact that NF-jB is an essential factor for osteoclastogenesis and that mice lacking p50 and p52 NF-jB subunits fail to generate osteoclasts and are osteopetrotic [19].
Discussion In the present study, we demonstrate that LPS causes the formation of TRAP-positive MGC in RAW 264.7 cells and that they exhibit a pit-forming activity on calcium carbonate-coated plates. Therefore, LPS is strongly suggested to induce osteoclast formation in RAW 264.7 cells. RAW 264.7 cells have been utilized for analysis of RANKLinduced osteoclast formation [19,20]. We demonstrate that RAW 264.7 cells also act as osteoclast progenitors and differentiate to osteoclasts in response to LPS. LPS is reported to stimulate the survival and fusion of osteoclast progenitors, independent of RANKL and induces pit-forming activity in the presence of M-CSF [21]. Further, LPS promotes osteoclast formation in co-cultures of mouse osteoblasts and bone marrow cells. LPS enhances osteoclast formation in cultures of whole bone marrow cells with dexamethasone and markedly enhances it in the presence of 1,25-dihydroxyvitamin D3 and dexamethasone [22]. On the other hand, LPS inhibits osteoclast formation in whole bone marrow cells with 1,25dihydroxyvitamin D3 via GM-CSF production [22]. Thus, LPS regulates osteoclast formation in a complicated fashion. The present study demonstrate that LPS itself induces osteoclastogenesis in RAW 264.7 cells. The LPS-induced osteoclastogenesis might be closely associated with the bone resorbing activity of LPS.
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Fig. 4. Effect of various signal inhibitors on LPS-induced osteoclastogenesis. RAW 264.7 cells were pretreated with PD98059 (10 lM), SB203580 (10 lM), SP600125 (2.5 lM) and parthenolide (5 lM) for 30 min, and stimulated with LPS (100 ng/ml) for 3 days. TRAP staining was carried out to determine the number of osteoclasts. Experimental data represent mean of triplicate determinations ± SD.
Fig. 3. Participation of TNF-a in LPS-induced osteoclastogenesis in RAW 264.7 cells. Effect of anti-TNF-a antibody on LPS-induced osteoclastogenesis was examined (A). RAW cells were cultured with LPS (100 ng/ml) with or without anti-TNF-a antibody (1 lg/ml), anti-MCSF antibody(5 lg/ml), and anti-RANKL antibody(1 lg/ml) for 3 days. TRAP staining was carried out to determine the number of osteoclasts. Experimental data represent mean of triplicate determinations ± SD. Effect of recombinant TNF-a on LPS-induced osteoclastogenesis was examined (B). RAW 264.7 cells were cultured with LPS (100 ng/ml) and/ or recombinant TNF-a (1 ng/ml) for 3 days. TRAP staining was carried out to determine the number of osteoclasts. Experimental data represent mean of triplicate determinations ± SD.
During preparation of the manuscript, Hotokezaka et al. have just reported RANKL-independent cell fusion of osteoclast-like cells induced by TNF-a, LPS, and peptidoglycan [23]. TNF-a as well as LPS is reported to induce cell fusion for osteoclastogenesis [23]. In the present study, we demonstrate that recombinant TNF-a leads to TRAPpositive mononuclear cells but not their cell fusion although LPS has the ability to induce the cell fusion. Hotokezaka et al. [23] stimulated RAW 264.7 cells with LPS in the presence of U0126 as an ERK1/2 inhibitor and RANKL whereas we stimulated the cells with LPS alone in the absence of such reagents. The difference in the experimental systems might cause the different action of TNF-a on the osteoclast formation in RAW 264.7 cells. RANKL and M-CSF are considered essential and sufficient for osteoclastogenesis [4,7,8]. In our experimental system, however, M-CSF did not participate in LPS-induced
osteoclast formation because anti-M-CSF antibody did not inhibit LPS-induced osteoclast formation and M-CSF was not detected in the supernatant from cultures of LPS-stimulated RAW 264.7 cells. Further, RANKL and PGE2 do not seem to be involved in LPS-induced osteoclast formation. LPS itself is suggested to induce osteoclast formation in RAW 264.7 cells, independent of RANKL, M-CSF, and PGE2. Signaling by RANKL is essential for terminal differentiation of osteoclast progenitors into osteoclasts. The TRAF6 signaling pathway plays an important role downstream of RANKL. Moreover, RANKL induces the activation of NF-jB and SAPK/JNK through TRAF6 signaling and subsequently the activation of the transcription factor NFAT2 [24–28]. LPS as well as RANKL induces the activation NF-jB and SAPK/JNK in RAW 264.7 cells [29]. LPS may mimic RANKL-induced osteoclast formation via activation of NF-jB and SAPK/JNK. There is one report concerning a role of SAPK/JNK pathway in the secretion of inflammatory cytokine TNF-a [30] and selective inhibition of SAPK/JNK blocked LPSinduced TNF expression [31]. Inhibition of SAPK/JNK by the selective inhibitor (SP600125) inhibited LPS-induced osteoclastogenesis. It is still unclear whether NF-jB and SAPK/JNK signaling is required for LPS-induced osteoclast formation or the production of TNF-a as the effector molecule. LPS induces osteoclast formation in RAW 264.7 cells, but not in mouse peritoneal cells (data not shown). Therefore, RAW 264.7 cells may possess the profile of monocytic progenitors. Otherwise, they may produce the factor(s) lacking in mature peritoneal cells. LPS-induced osteoclast formation might be a useful experimental system for characterization of osteoclastogenesis. Acknowledgments This work was supported by in part by a Grant-in-Aid for Scientific Research from the Ministry of Education,
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Science, Sports and Culture of Japan. We are grateful to K. Takahashi and A. Morikawa for the technical assistance.
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