JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M311279200 on March 8, 2004

J. Biol. Chem., Vol. 279, Issue 20, 20889-20897, May 14, 2004
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Peptidoglycan Induces Nuclear Factor-{kappa}B Activation and Cyclooxygenase-2 Expression via Ras, Raf-1, and ERK in RAW 264.7 Macrophages*

Bing-Chang Chen{ddagger}, Ya-Sheng Chang§, Ju-Chiun Kang¶, Ming-Jen Hsu§, Joen-Rong Sheu¶, Ta-Liang Chen||, Che-Ming Teng**, and Chien-Huang Lin{ddagger}§{ddagger}{ddagger}

From the {ddagger}School of Respiratory Therapy, §Graduate Institute of Biomedical Technology, the Department of Pharmacology, School of Medicine, Taipei Medical University, the ||Department of Anesthesiology, Taipei Medical University-Wan Fang Hospital, and **Pharmacological Institute, College of Medicine, National Taiwan University, Taipei 110, Taiwan

Received for publication, October 14, 2003 , and in revised form, March 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated I{kappa}B kinase {alpha}/{beta} (IKK{alpha}/{beta}), I{kappa}B{alpha} phosphorylation, I{kappa}B{alpha} degradation, and {kappa}B-luciferase activity. Treatment of macrophages with an NF-{kappa}B inhibitor (pyrrolidine dithiocarbamate), an I{kappa}B{alpha} phosphorylation inhibitor (Bay 117082), and I{kappa}B protease inhibitors (L-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKK{alpha}/{beta} and {kappa}B-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85{alpha} and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKK{alpha}/{beta} and NF-{kappa}B activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptidoglycan (PGN),1 a cell wall component of Gram-positive bacteria, is an alternating {beta}-linked N-acetylmuramyl and N-acetylglucosaminyl glycan whose residues are cross-linked by short peptides (1, 2). Like lipopolysaccharide (LPS) as a cell wall component of Gram-negative bacteria, PGN induces most of the clinical manifestations of bacterial infections, including inflammation, fever, septic shock, etc. (3). Most of these effects are due to the activation of macrophages and generation of proinflammatory cytokines, such as tumor necrosis factor {alpha}, interleukin (IL)-6, and IL-8 (4–8). PGN binds CD14 and Toll-like receptor 2 (TLR2), which activates transcription factors and induces gene expression (4, 9). Even though it is well known that bacterial products have multiple and various effects on the regulation of host defense and immune responses by macrophages, little is known about how PGN regulates induction of the cyclooxygenase-2 (cox-2) gene.

COX is the key enzyme that synthesizes prostaglandins and thromboxane from arachidonic acid (10, 11). Two COX isozymes, COX-1 and COX-2, have been cloned and identified to have 60% homology in humans (12, 13). COX-1, which is constitutively expressed in most tissues, mediates physiological responses and regulates renal and vascular homeostasis. COX-2, an inducible gene for cell synthesis, can be up-regulated by inflammatory stimuli including IL-1{beta}, lipoteichoic acid (LTA), and LPS to produce proinflammatory prostaglandins in inflammation (14–19). Several consensus sequences, including those for nuclear factor-{kappa}B (NF-{kappa}B), NF-IL6, and the activating transcription factor/cyclic AMP-responsive element in the 5' region of the cox-2 gene, have been identified as regulatory sequences that can induce COX-2 in response to various stimuli (20–22). NF-{kappa}B, the most important transcription factor for regulating COX-2 expression, is a dimer of the transcription factors p65 or p50 (23). In a resting state, this dimer is associated with I{kappa}Bs to retain NF-{kappa}B in the cytosol (24). I{kappa}B kinase (IKK), which is activated through stimulation by cytokines and bacterial products, phosphorylates I{kappa}B{alpha} at Ser-32 and Ser-36 and I{kappa}B{beta} at Ser-19 and Ser-23 (25–27), to produce ubiquitination of I{kappa}B{alpha}/{beta} at lysine residues and degradation by the 26 S proteasome (28). This process releases active NF-{kappa}B, which is then translocated from the cytosol to the nucleus, to bind specific DNA enhancer sequences and to induce gene transcription (23). A previous report showed that PGN-induced NF-{kappa}B activation is mediated through TLR2-dependent multiple signaling molecules including myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), tumor necrosis factor receptor-associated factor 6 (TRAF6), NF-{kappa}B-inducing kinase, and the IKK signaling pathway (4, 5), but little information is available about the role of Ras in regulating NF-{kappa}B signaling following PGN stimulation.

Ras has been found to couple with multiple effector systems to activate distinct physiological and pathological responses such as cell proliferation and release of proinflammatory mediators (22, 29–31). An important class of Ras effectors is the mitogen-activated protein kinase (MAPK) family. The classic Ras-mediated pathway involves the binding of Raf-1 and subsequent phosphorylation of Raf-1 at Ser-338 by many kinases (32, 33), which in turn activates extracellular signal-regulated kinases (ERKs) (34), and consequently phosphorylates many target proteins including transcription factors and protein kinases (35). Although a role for Ras in COX-2 induction has been implied by many cell types (22, 30, 31), however, PGN-induced COX-2 expression has not been investigated in macrophages. This study was intended to identify the signaling pathway of PGN-induced Ras activation and its roles in PGN-mediated NF-{kappa}B activation and COX-2 expression in RAW 264.7 macrophages. Our hypothesis is that PGN might induce TLR2, p85{alpha}, and Ras complex formation and subsequently activate the Ras/Raf-1/ERK pathway, which in turn increases IKK{alpha}/{beta} activity and NF-{kappa}B activation and finally causes COX-2 expression in RAW 264.7 macrophages.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Peptidoglycan (PGN derived from Staphylococcus aureus) was purchased from Fluka (Buchs, Switzerland). Manumycin A, PD 098059, and SB 203580 were obtained from Calbiochem. GW 5074 was obtained from Tocris (Bristol, UK). Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/Ham's F-12), fetal calf serum, and penicillin/streptomycin were purchased from Invitrogen. Antibodies specific for {alpha}-tubulin and COX-2 were purchased from Transduction Laboratories (Lexington, KY). Protein A/G beads, I{kappa}B{alpha} protein (amino acids 1–317), antibodies specific for ERK, ERK phosphorylated at Tyr-204, I{kappa}B{alpha}, I{kappa}B{alpha} phosphorylated at Ser-32, IKK{alpha}, IKK{beta}, Raf-1, TLR2, Ras, p85{alpha}, and anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific for Raf-1 phosphorylated at Ser-338 was purchased from Cell Signaling and Neuroscience (St. Louis, MO). Anti-mouse and anti-rabbit IgG-conjugated alkaline phosphatases were purchased from Jackson ImmunoResearch (West Grove, PA). pGL2-ELAM-Luc (which is under the control of a single NF-{kappa}B-binding site) and pBK-CMV-LacZ were kindly provided by Dr. W.-W. Lin (National Taiwan University, Taipei, Taiwan). A Ras dominant negative mutant (RasN17) and a Ras activity assay kit were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). [{gamma}-32P]ATP (6,000 Ci/mmol) was purchased from Amersham Biosciences. Bay 117082 was obtained from Alexis (Lausen, Switzerland). GenePORTERTM 2 was purchased from Gene Therapy System (San Diego, CA). All materials for SDS-PAGE were purchased from Bio-Rad. All other chemicals were obtained from Sigma.

Cell Culture—The mouse macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection (Livingstone, MT), and cells were maintained in DMEM/Ham's F-12 nutrient mixture containing 10% fetal calf serum, 100 units/ml of penicillin G, and 100 µg/ml streptomycin in a humidified 37 °C incubator. After reaching confluence, cells were seeded onto either 6-cm dishes for immunoblotting, co-immunoprecipitation, or kinase assays, or 12-well plates for transfection and {kappa}B luciferase assays.

Transfection and {kappa}B Luciferase Assays—For these assays, 2 x 105 RAW 264.7 cells were seeded onto 12-well plates, and cells were transfected the following day using GenePORTERTM 2 with 0.5 µg of pGL2-ELAM-Luc and 0.5 µg of pBK-CMV-LacZ. After 24 h, the medium was aspirated, replaced with fresh DMEM/Ham's F12 containing 10% FBS, and then stimulated with PGN (30 µg/ml) for another 24 h before harvest. To assess the effects of Ras, Raf-1, and ERK inhibitors, drugs were added to cells 20 min before PGN addition. To assay the effect of RasN17, cells were co-transfected with RasN17, pGL2-ELAM-Luc, and pBK-CMV-LacZ for 24 h and then treated with PGN. Luciferase activity was determined with a luciferase assay system (Promega) and was normalized on the basis of LacZ expression. The level of induction of luciferase activity was compared as a ratio to cells with and without stimulation.

Immunoblot Analysis—To determine the expressions of COX-2, {alpha}-tubulin, ERK phosphorylated at Tyr-204, ERK2, IKK{alpha}/{beta}, I{kappa}B{alpha} phosphorylated at Ser-32, I{kappa}B{alpha}, Ras, Raf-1 phosphorylated at Ser-338, and Raf-1 in RAW 264.7 macrophages, proteins were extracted, and Western blotting analyses were performed as described previously (36). Briefly, RAW 264.7 macrophages were cultured in 6-cm dishes. After reaching confluence, cells were treated with vehicle and PGN or pretreated with specific inhibitors as indicated followed by PGN. After incubation, cells were washed twice in ice-cold phosphate-buffered saline (PBS) and solubilized in extraction buffer containing 10 mM Tris (pH 7.0), 140 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, 0.5% Nonidet P-40, 0.05 mM pepstatin A, and 0.2 mM leupeptin. Samples of equal amounts of protein (60 µg) were subjected to SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane that was then incubated in TBST buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.02% Tween 20 (pH 7.4)) containing 5% non-fat milk. Proteins were visualized by specific primary antibodies and then incubated with horseradish peroxidase- or alkaline phosphatase-conjugated second antibodies. Immunoreactivity was detected using enhanced chemiluminescence or nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate following the manufacturer's instructions. Quantitative data were obtained using a computing densitometer with scientific imaging systems (Eastman Kodak Co.).

Immunoprecipitation and Protein Kinase Assays—RAW 264.7 cells were grown in 6-cm dishes. After reaching confluence, cells were treated with 30 µg/ml PGN for the indicated time intervals or pretreated with specific inhibitors as indicated followed by PGN. After incubation, cells were washed twice with ice-cold PBS, lysed in 1 ml of lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 125 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 mM {beta}-glycerophosphate, 50 mM NaF, and 100 µM sodium orthovanadate, and centrifuged. The supernatant was then immunoprecipitated with respective polyclonal antibodies against IKK{alpha}, IKK{beta}, or ERK2 in the presence of A/G-agarose beads overnight. The beads were washed three times with lysis buffer and two times with kinase buffer containing 20 mM HEPES (pH 7.4), 20 mM MgCl2, and 2 mM dithiothreitol. The kinase reactions were performed by incubating immunoprecipitated beads with 20 µl of kinase buffer supplemented with 20 µM ATP and 3 µCi of [{gamma}-32P]ATP at 30 °C for 30 min. To assess IKK{alpha}/{beta} and ERK activities, 0.5 µg of GST-I{kappa}B{alpha} protein (amino acids 1–317) and 50 µg/ml of myelin basic protein (MBP) were respectively added as the substrates. The reaction mixtures were analyzed by 12 (IKK{alpha}/{beta}) or 15% (ERK) SDS-PAGE followed by autoradiography.

Ras Activity Assay—Ras activity was measured using a Ras activity assay kit. The assay was performed according to the manufacturer's instructions. Briefly, cells were washed twice with ice-cold PBS, lysed in magnesium lysis buffer (MLB) (25 mM HEPES (pH 7.5), 150 mM NaCl, 5% Igepal CA-630, 10 mM MgCl2, 5 mM EDTA, 10% glycerol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), and centrifuged. An equal volume of lysate was incubated with 5 µg of Ras-binding domain for Raf-1 (Raf-1 RBD) at 4 °C overnight, and the beads were washed three times with MLB lysis buffer. Bound Ras proteins were then solubilized in 2x Laemmli sample buffer and quantitatively detected by Western blotting (10% SDS-PAGE) using mouse monoclonal Ras with the ECL system and by densitometry of corresponding bands using scientific imaging systems.

Co-immunoprecipitation of TLR2 Complex Formation—RAW 264.7 cells were grown in 6-cm dishes. After reaching confluence, cells were treated with 30 µg/ml PGN for the indicated time intervals. The cells were harvested, lysed in 1 ml of PD buffer (40 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1% Nonidet P-40, 6 mM EGTA, 10 mM {beta}-glycerophosphate, 10 mM NaF, 300 µM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM dithiothreitol), and centrifuged. The supernatant was then immunoprecipitated with specific antibodies against TLR2, p85{alpha}, or Ras in the presence of protein A/G beads at 4 °C overnight. The immunoprecipitated beads were washed three times with PD buffer. The samples were fractionated on 15 (for Ras) or 8% (for TLR2 or p85{alpha}) SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and subjected to immunoblot analysis with antibodies specific for Ras, TLR2, or p85{alpha}.

Statistical Analysis—Results are presented as the mean ± S.E. from at least three independent experiments. One-way analysis of variance followed by, when appropriate, Bonferroni's multiple range test was used to determine the statistical significance of the difference between means. A p value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PGN Induces Macrophage COX-2 Expression—Murine RAW 264.7 macrophages were chosen to investigate the signal pathways of PGN in COX-2 expression, an inflammatory response gene. Treatment with PGN (1–100 µg/ml) for 24 h induced COX-2 protein expression in a concentration-dependent manner (Fig. 1A); this induction also occurred in a time-dependent manner (Fig. 1B). After 24 h of treatment with 30 µg/ml PGN, the COX-2 protein had increased by ~640 ± 14% (n = 3) (Fig. 1B). To further confirm this stimulation-specific mediation by PGN without LPS contamination, polymycin B, an LPS inhibitor, was tested. We found that polymycin B (0.3 µg/ml) did not change PGN-induced COX-2 protein levels. In contrast, polymycin B completely inhibited LPS (1 µg/ml)-induced COX-2 protein expression (data not shown).



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  		FIG. 1.
Concentration- and time-dependent increases in COX-2 expression by PGN in RAW 264.7 macrophages. A, macrophages were incubated with various concentrations of PGN for 24 h, and then COX-2 or {alpha}-tubulin protein levels were determined. B, cells were incubated with 30 µg/ml PGN for the indicated time intervals, and then COX-2 or {alpha}-tubulin protein levels were determined. Equal loading in each lane is shown by the similar intensities of {alpha}-tubulin. Traces represent results from three independent experiments, which are presented as the mean ± S.E. *, p < 0.05 as compared with the control group.

 
Augmentation of COX-2 Expression Occurred at the Level of Transcription—RAW 264.7 macrophages were pretreated with either actinomycin D (a transcriptional inhibitor) or cycloheximide (a translational inhibitor) and then treated with 30 µg/ml PGN. As a result, the PGN-induced elevation of COX-2 expression was inhibited by actinomycin D (30 nM) and cycloheximide (100 nM) by about 55 ± 4 and 72 ± 6%, respectively (n = 3) (Fig. 2A). The results suggest that the increase in COX-2 protein in RAW 264.7 macrophages responsive to PGN may have been due to COX-2 transcriptional expression.



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  		FIG. 2.
NF-{kappa}B is involved in PGN-mediated COX-2 induction in RAW 264.7 macrophages. A, transcriptional activity is involved in PGN-induced COX-2 expression. Cells were pretreated for 30 min with vehicle, 10–30 nM actinomycin D (Act. D), and 30–100 nM cycloheximide (CHX) and then stimulated with 30 µg/ml PGN for 24 h. Cells were lysed and then immunoblotted for COX-2 or {alpha}-tubulin. B–D, the NF-{kappa}B signal pathway was necessary for PGN-induced COX-2 expression. Cells were pretreated for 30 min with 5–50 µM PDTC (B), 1–10 µM Bay 117082 (C), or 3 µM TPCK and 10 µM calpain inhibitor I (Calp) (D) and then stimulated with 30 µg/ml PGN for 24 h. Cell were lysed and then immunoblotted for COX-2 or {alpha}-tubulin. Equal loading in each lane is demonstrated by the similar intensities of {alpha}-tubulin. Results are representative of three independent experiments. Traces represent results from three independent experiments, which are presented as the mean ± S.E. *, p < 0.05 as compared with PGN treatment.

 
Involvement of NF-{kappa}B in PGN-induced COX-2 Expression—As mentioned previously, NF-{kappa}B activation is necessary for COX-2 induction. Pyrrolidine dithiocarbamate (PDTC), an antioxidant, has been shown to inactivate NF-{kappa}B in macrophages (37). To examine whether NF-{kappa}B activation is involved in the signal transduction pathway leading to COX-2 expression caused by PGN, the NF-{kappa}B inhibitor PDTC was used. Fig. 2B shows that PGN-induced COX-2 protein levels were inhibited by PDTC (5–50 µM) in a concentration-dependent manner. When cells were treated with 50 µM PDTC, PGN-induced COX-2 expression was inhibited by 64 ± 4% (n = 3). Furthermore, macrophage pretreatment with an I{kappa}B{alpha} phosphorylation inhibitor (Bay 117082) (38) inhibited PGN-induced COX-2 protein expression in a concentration-dependent manner. Pretreatment of cells with 10 µM Bay 117082 inhibited the PGN response by 53 ± 3% (n = 3) (Fig. 2C). In parallel with the inhibition by PDTC and Bay 117082, I{kappa}B protease inhibitors (L-1-tosylamido-2-phenylenylethyl chloromethyl ketone (TPCK, 3 µM) and calpain inhibitor I (10 µM)) (39, 40) also inhibited PGN-induced COX-2 protein expression by 35 ± 3 and 65 ± 5%, respectively (n = 3) (Fig. 2D). These results suggest that NF-{kappa}B activation is necessary for PGN-induced COX-2 expression in RAW 264.7 macrophages.

PGN Causes Increases in IKK{alpha}/{beta} Activity, I{kappa}B{alpha} Phosphorylation, I{kappa}B{alpha} Degradation, and {kappa}B-Luciferase Activity—We further determined the upstream molecules of NF-{kappa}B in PGN-induced NF-{kappa}B activation. Stimulation of cells with 30 µg/ml PGN induced increases in IKK{alpha}/{beta} activity and I{kappa}B{alpha} phosphorylation in a time-dependent manner, reaching maximums after 10 and 10–30 min of treatment, respectively (Fig. 3, A and B). In parallel with IKK{alpha}/{beta} activity and I{kappa}B{alpha} phosphorylation, I{kappa}B{alpha} degradation was apparent after 10 min of treatment with 30 µg/ml PGN, and the I{kappa}B{alpha} protein was resynthesized after 60 min of treatment (Fig. 3C). To directly determine NF-{kappa}B activation after PGN treatment, RAW 264.7 macrophages were transiently transfected with pGL2-ELAM-{kappa}B-luciferase as an indicator of NF-{kappa}B activation. As shown in Fig. 3D, macrophage treatment with PGN (1–100 µg/ml) for 24 h caused a concentration-dependent increase in {kappa}B luciferase activity.



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  		FIG. 3.
PGN induced IKK{alpha}/{beta} activation, I{kappa}B{alpha} phosphorylation, I{kappa}B{alpha} degradation, and {kappa}B-luciferase activity in RAW 264.7 macrophages. A, RAW 264.7 cells were incubated with 30 µg/ml PGN for 0–60 min, and cell lysates were then immunoprecipitated with antibodies specific for IKK{alpha} and IKK{beta}. One set of immunoprecipitates was subjected to the kinase assay (KA) described under "Experimental Procedures" using the GST-I{kappa}B{alpha} fusion protein as a substrate (top panel). The other set of immunoprecipitates was subjected to 10% SDS-PAGE and analyzed by immunoblotting (IB) with the anti-IKK{alpha}/{beta} antibody (bottom panel). Equal amounts of the immunoprecipitated kinase complex present in each kinase assay were confirmed by immunoblotting for IKK{alpha}/{beta}. B and C, following incubation for 0–120 min with 30 µg/ml PGN, I{kappa}B{alpha} phosphorylation (B) and I{kappa}B{alpha} degradation (C) were determined by immunoblotting using phospho-I{kappa}B{alpha}- and I{kappa}B{alpha}-specific antibodies, respectively. Typical traces are representative of three experiments with similar results. D, RAW 264.7 macrophages were transiently transfected with 0.5 µg of pGL2-ELAM-Luc and 0.5 µg of pBK-CMV-LacZ for 24 h, and then cells were incubated with 1–100 µg/ml PGN for another 24 h. Luciferase activities were determined as described under "Experimental Procedures." The level of induction of luciferase activity was compared with that of cells without PGN treatment. Data represent the mean ± S.E. of three to four experiments performed in duplicate. *, p < 0.05 as compared with the control without PGN treatment.

 
Ras Is Involved in PGN-induced COX-2 Expression—To explore whether Ras might mediate PGN-induced COX-2 expression, manumycin A, a Ras inhibitor (41), was used. As shown in Fig. 4A, pretreatment of RAW 264.7 macrophages with manumycin A inhibited PGN-induced COX-2 expression in a concentration-dependent manner. When cells were treated with 3 µM manumycin A, PGN-induced COX-2 expression was inhibited by 58 ± 2% (n = 3). However, manumycin A had no effect on the basal level of COX-2 expression (Fig. 4A). Moreover, we found that 3 µM manumycin A also inhibited 1 µg/ml LPS-stimulated COX-2 expression by 72 ± 8% (n = 3) (Fig. 4A). Next, we directly measured the Ras activity in response to PGN and LPS. Fig. 4, B and C, shows that treatment of RAW 264.7 cells with 30 µg/ml PGN or 1 µg/ml LPS induced an increase in Ras activity in a time-dependent manner, as assessed by immunoblotting samples for Ras immunoprecipitated from lysates using Raf-1-RBD. Maximal activation was detected after 5 min of stimulation, and the response continued until 30 min after PGN stimulation (Fig. 4B). Similarly, the LPS-induced increase in Ras activity began at 3 min, peaked at 5 min, and then declined at 10 min after LPS treatment (Fig. 4C). Furthermore, the PGN-induced increase in Ras activity was markedly inhibited by pretreatment of cells for 30 min with manumycin A (1–3 µM) in a concentration-dependent manner (Fig. 4D). Taken together, these results imply that Ras activation is involved in PGN- or LPS-induced COX-2 expression.



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  		FIG. 4.
Effects of manumycin A on PGN-induced COX-2 expression and Ras activation in RAW 264.7 macrophages. A, RAW 264.7 macrophages were pretreated with vehicle and 0.3–3 µM manumycin A (Manu) for 30 min followed by stimulation with 30 µg/ml PGN or 1 µg/ml LPS for another 24 h, and COX-2 expression was determined by immunoblotting with an antibody specific for COX-2. Equal loading in each lane is demonstrated by the similar intensities of {alpha}-tubulin. Typical traces are representative of three experiments with similar results, which are presented as the mean ± S.E. *, p < 0.05 as compared with PGN or LPS treatment. B and C, RAW 264.7 macrophages were incubated with 30 µg/ml PGN or 1 µg/ml LPS for indicated time intervals, and cell lysates were then immunoprecipitated with an antibody specific for Raf-1 RBD. The Ras activity assay is described under "Experimental Procedures." Typical traces represent three experiments with similar results. D, cells were pretreated with 1–3 µM manumycin A (Manu) for 30 min and then treated with 30 µg/ml PGN for another 10 min. Cells were then lysed for Ras activity assay as described above. Typical traces represent two experiments with similar results.

 
Raf-1 Is Involved in PGN-induced COX-2 Expression—To examine whether Raf-1, a target protein for Ras, might play a crucial role in PGN-induced COX-2 expression, the Raf-1 inhibitor GW 5074 (42) was used. As shown in Fig. 5A, 1 µM of GW 5074 alone did not affect the basal COX-2 level, but it significantly inhibited PGN-induced COX-2 expression by 43 ± 3% (n = 3). Furthermore, we found that GW 5074 (1 µM) inhibited LPS-induced COX-2 expression by 76 ± 3% (n = 3) (Fig. 5A). Raf-1 is associated with Ras-GTP and then by additional modifications such as phosphorylation at Ser-338 to become the active form (33). The activated Raf-1 then triggers sequential activation of downstream molecules. Thus, phosphorylation of Raf-1 at Ser-338 is a critical step in Raf-1 activation. Next, we further examined Raf-1 Ser-338 phosphorylation by PGN stimulation in RAW 264.7 macrophages using the anti-phospho-Raf-1 antibody at Ser-338. When cells were treated with 30 µg/ml PGN for various time intervals, Raf-1 Ser-338 phosphorylation increased at 5 min and peaked at 10 min. After 20 min of treatment, the PGN-induced increase in Raf-1 Ser-338 phosphorylation declined (Fig. 5B). In addition, PGN-induced Raf-1 Ser-338 phosphorylation was also inhibited by treatment with 3 µM manumycin A and 1 µM GW 5074 (Fig. 5C). Results indicate that Raf-1 is a downstream molecule of Ras and is involved in Ras-mediated COX-2 protein expression.



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  		FIG. 5.
Effects of GW 5074 on PGN-induced COX-2 expression and Raf-1 phosphorylation in RAW 264.7 macrophages. A, RAW 264.7 macrophages were pretreated with vehicle and GW 5074 (0.3–1 µM) for 30 min followed by stimulation with 30 µg/ml PGN or 1 µg/ml LPS for another 24 h, and COX-2 expression was determined by immunoblotting with an antibody specific for COX-2. Equal loading in each lane is demonstrated by the similar intensities of {alpha}-tubulin. Typical traces represent three experiments with similar results, which are presented as the mean ± S.E. *, p < 0.05 as compared with PGN or LPS treatment. B, RAW 264.7 macrophages were incubated with 30 µg/ml PGN for 0–30 min, and then Raf-1 phosphorylation (upper panel) and Raf-1 (bottom panel) protein levels were determined. The presence of equal loading in each lane is shown by the similar intensities of Raf-1. Traces represent results from three independent experiments. C, cells were pretreated with 3 µM manumycin A (Manu) or 0.3 µM GW 5074 for 30 min, and then treated with 30 µg/ml PGN for another 10 min. Cells were then lysed for Raf-1 phosphorylation (upper panel) and Raf-1 (bottom panel) protein levels as described above. Typical traces represent two experiments with similar results.

 
PGN Mediates Phosphorylation of ERK—We next wished to determine whether PGN is able to activate ERK, a critical downstream target of Raf-1 (43), which has been shown to induce gene expression (44). We tested the role of ERK in mediating PGN-induced COX-2 expression by using the specific MEK inhibitor PD 098059. As shown in Fig. 6A, PGN-induced COX-2 expression was markedly attenuated by pretreatment of cells with PD 098059 (3–30 µM) in a concentration-dependent manner. Pretreatment of cells with 30 µM PD 098059 inhibited PGN-induced COX-2 expression by 85 ± 7% (n = 3) (Fig. 6A). Moreover, we also found that 30 µM PD 098059 inhibited LPS-induced COX-2 expression by 76 ± 5% (n = 3) (Fig. 6A). To directly confirm the crucial role of ERK in COX-2 expression, we determined ERK phosphorylation and kinase activity in response to PGN. As shown in Fig. 6B, treatment of RAW 264.7 macrophages with 30 µg/ml PGN resulted in a time-dependent phosphorylation of ERK. The ERK phosphorylation began at 10 min, peaked at 30–60 min, and then declined to 120 min after PGN treatment (Fig. 6B, upper panel). The protein level of ERK2 was not affected by PGN treatment (Fig. 6B, bottom panel). In parallel, using MBP as an ERK substrate, an increase in ERK activity was observed within 10 min and reached a peak 30 min after PGN stimulation, thereafter declining to 60 min after stimulation (Fig. 6C). To determine the relationships among Ras, Raf-1, and ERK in the PGN-mediated signaling pathway, we found that pretreatment of RAW 264.7 cells for 30 min with 3 µM manumycin A, 1 µM GW 5074, and 30 µM PD 098059 markedly inhibited the PGN-induced increase in ERK activity (Fig. 6D). Based on these results, we suggest that activation of Ras and Raf-1 occurs upstream of ERK in the PGN-induced signaling pathway.



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  		FIG. 6.
Effects of PD 098059 on PGN-induced COX-2 expression, ERK phosphorylation, and ERK activity in RAW 264.7 macrophages. A, RAW 264.7 macrophages were pretreated with vehicle and PD 098059 (3–30 µM) for 30 min before treatment with 30 µg/ml PGN or 1 µg/ml LPS for another 24 h, and COX-2 expression was determined by immunoblotting with an antibody specific for COX-2. Equal loading in each lane is shown by the similar intensities of {alpha}-tubulin. Typical traces represent three experiments with similar results, which are presented as the mean ± S.E. *, p < 0.05 as compared with PGN or LPS treatment. B, after cells were treated with 30 µg/ml PGN for different intervals, ERK phosphorylation was shown by immunoblotting with an antibody specific for phosphorylated ERK (upper panel). Equal loading in each lane is shown by the similar intensities of ERK2 (bottom panel). Typical traces represent three experiments with similar results. C, for ERK kinase activity, cell lysates were immunoprecipitated with ERK2 antibody. One set of immunoprecipitates was subjected to the kinase assay (KA) using MBP as a substrate. The other set of immunoprecipitates was subjected to 12% SDS-PAGE and analyzed by immunoblotting (IB) with an anti-ERK2 antibody. Equal amounts of the immunoprecipitated kinase complexes present in each kinase assay were confirmed by immunoblotting for ERK2. Shown are representative results from three independent experiments. D, cells were pretreated with 3 µM manumycin A (Manu), 0.3 µM GW 5074, and 30 µM PD 098059 for 20 min and then incubated with 30 µg/ml PGN for another 10 min. Cell lysates were immunoprecipitated with an antibody specific for ERK2. The kinase assay (KA) and immunoblotting (IB) were as described above. Equal amounts of the immunoprecipitated kinase complexes present in each kinase assay were confirmed by immunoblotting for ERK2. The data shown represent three experiments.

 
Ras, Raf-1, and ERK Mediated PGN-induced IKK{alpha}/{beta} and NF-{kappa}B Activation—We further examined whether the activation of IKK{alpha}/{beta} and NF-{kappa}B occurs through the Ras/Raf-1/ERK signaling pathway. As shown in Fig. 7A, pretreatment of cells for 30 min with manumycin A (3 µM), GW 5074 (1 µM), and PD 098059 (30 µM) markedly attenuated the PGN-induced increase in IKK{alpha}/{beta} activity. None of these inhibitors affected the basal IKK{alpha}/{beta} activity (Fig. 7A). Similarly, the PGN-induced increase in {kappa}B-luciferase activity was also inhibited by 3 µM manumycin A, 1 µM GW 5074, and 30 µM PD 098059 by 74 ± 11, 71 ± 6, and 58 ± 13%, respectively (n = 3) (Fig. 7B). To confirm further the role of Ras in PGN-mediated NF-{kappa}B activation, RasN17 was tested. Transfection of RAW 264.7 cells with RasN17 inhibited the PGN-induced increase in {kappa}B-luciferase activity in a dose-dependent manner, with 1 µg of RasN17 reducing the basal {kappa}B-luciferase activity and PGN response by about 64 ± 15 and 82 ± 7%, respectively (n = 3) (Fig. 7B). Taken together, these data suggest that activation of Ras/Raf-1/ERK pathway is also required for PGN-induced IKK{alpha}/{beta} and NF-{kappa}B activation in RAW 264.7 macrophages.



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  		FIG. 7.
The Ras/Raf-1/ERK signal pathway is involved in PGN-mediated IKK{alpha}/{beta} and NF-{kappa}B activation in RAW 264.7 macrophages. A, RAW 264.7 macrophages were pretreated with vehicle, 3 µM manumycin A (Manu), 0.3 µM GW 5074, and 30 µM PD 098059 for 30 min followed by stimulation with 30 µg/ml PGN for another 20 min; cell lysates were then immunoprecipitated with antibodies specific to IKK{alpha} and IKK{beta}. One set of immunoprecipitates was subjected to the kinase assay (KA) by using the GST-I{kappa}B{alpha} fusion protein as a substrate (top panel). The other set of immunoprecipitates was subjected to 10% SDS-PAGE and analyzed by immunoblotting (IB) with anti-IKK{alpha}/{beta} antibody (bottom panel). Equal amounts of the immunoprecipitated kinase complex present in each kinase assay were confirmed by immunoblotting for IKK{alpha}/{beta}. B, RAW 264.7 macrophages were either transiently transfected with 0.5 µg of pGL2-ELAM-Luc and 1 µg of pBK-CMV-LacZ for 24 h and were co-transfected with 0.5 and 0.5 µg of RasN17, or were pretreated with 3 µM manumycin A, 0.3 µM GW 5074, and 30 µM PD 098059 for 20 min and then stimulated with 30 µg/ml PGN for another 24 h. Cells were harvested for luciferase assay as described in Fig. 4C. Data represent the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05 as compared with the corresponding control response of PGN treatment.

 
Ras Is Associated with TLR2 by p85{alpha} upon PGN Stimulation—The rapid activation of Ras by PGN stimulation suggested that Ras activation might occur close to TLR2 in the PGN signal pathway. Therefore, we investigated whether PGN can induce the interaction between Ras and TLR2. As shown in Fig. 8A, treatment of RAW 264.7 macrophages with 30 µg/ml PGN led to the rapid association of Ras and TLR2, as detected by immunoblotting using the antibody to Ras after immunoprecipitation of TLR2. The association of TLR2 and Ras occurred at 1 min and peaked at 3–5 min (Fig. 8A). Control experiments using an unrelated polyclonal antibody for immunoprecipitation showed no Ras binding (Fig. 8A). The interaction between TLR2 and Ras was further confirmed by converse experiments in which the TLR2 and Ras complex was immunoprecipitated with the Ras antibody and immunoblotted with the TLR2 antibody (Fig. 8B). Previous reports (45) showed that p85 can interact with TLR2 and then induces Rac activation. Therefore, we further examined the role of p85{alpha} involvement in TLR2 and Ras complex formation by PGN stimulation. Fig. 8C shows that PGN (30 µg/ml) treatment rapidly increased the formation of the TLR2 and p85{alpha} complex at as early as 30 s and then it declined at 1–5 min. We also found that treatment of macrophages with PGN induced the association of p85{alpha} and Ras within 1 min, and this was sustained for 5 min (Fig. 8D). These results suggested that PGN induces Ras activation by interacting with TLR2 and p85{alpha} in macrophages.



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  		FIG. 8.
Ras is associated with p85{alpha} and TLR2 upon PGN stimulation in RAW 264.7 macrophages. A, RAW 264.7 macrophages were stimulated with 30 µg/ml PGN for 0–5 min and were then lysed. Cells extracts were immunoprecipitated (IP) with anti-TLR2 or anti-isotype IgG antibody and then immunoblotted (IB) with anti-Ras anti-body. IgG LC (IgG light chain). B, RAW 264.7 macrophages were treated with PGN for the indicated time intervals. Cells extracts were immunoprecipitated with the anti-Ras antibody and then immunoblotted with the anti-TLR2 antibody. C, cells were treated with PGN for 0–5 min, then immunoprecipitated with the anti-TLR2 antibody, and immunoblotted with the anti-p85{alpha} antibody. D, cells were stimulated with PGN for 0–5 min, then immunoprecipitated with the anti-p85 {alpha} antibody, and immunoblotted with the anti-Ras antibody. Typical traces are representative of three experiments with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The findings of this study showed that PGN induces COX-2 expression through activation of the Ras/Raf-1/ERK/IKK{alpha}/{beta}/NF-{kappa}B signal pathway in RAW 264.7 macrophages. In mice and humans, the COX-2 promoter has many transcription factors including NF-{kappa}B in the 5' region of the cox-2 gene (20–22). Transcription factor NF-{kappa}B has been reported to induce macrophage COX-2 in response to various inflammatory stimuli (46). Previous studies have demonstrated that NF-{kappa}B activation is necessary for LPS-induced COX-2 induction in RAW 264.7 macrophages (47–50). Hwang et al. (47, 48) reported that in LPS-treated RAW 264.7 macrophages, NF-{kappa}B plays a crucial role in TLR4-induced cox-2 gene expression. The results of this study showed that NF-{kappa}B activation also contributed to PGN-induced COX-2 induction in RAW 264.7 macrophages (Fig. 2, and that all inhibitors of the NF-{kappa}B-dependent signaling pathway, including PDTC, Bay 117082, TPCK, and calpain inhibitor I, inhibited PGN-induced macrophage COX-2 expression. Furthermore, PGN induced IKK{alpha}/{beta} activation, I{kappa}B{alpha} phosphorylation, and I{kappa}B{alpha} degradation, as well as an increase in {kappa}B-luciferase activity. These findings are in agreement with those from studies on LTA (16, 51), another cell wall component of Gram-positive bacteria. NF-{kappa}B activation is required to induce COX-2 transcription in epithelial cells. The results of this study also showed that Ras, Raf-1, and ERK are involved in NF-{kappa}B activation through an increase in IKK{alpha}/{beta} activity. A previous report showed that in transformed liver epithelial cells, Ras and Raf-1 lead to constitutive activation of NF-{kappa}B through the IKK{alpha}/{beta} complex (52). These pathways may mediate PGN effect. As shown in Fig. 7, manumycin A, GW 5074, and PD 098059 blocked PGN-induced IKK{alpha}/{beta} activity. In addition, RasN17 and these inhibitors attenuated induction of NF-{kappa}B reporter activity or cox-2 gene expression, implying a role in the pathway of transactivation by PGN stimulation.

The TLR family now consists of 10 different TLRs (TLR1-TLR10), but only TLR2 and TLR4 are known to have biological and pathological functions (53, 54). The cytoplasmic portion of TLRs shows high similarity to that of the IL-1 receptor family and is now called the Toll/IL-1 receptor (TIR) domain (53). Upon recognizing respective ligands, the TIR domain recruits MyD88/IRAK/TRAF6 and activates downstream signaling molecules such as MAPK and NF-{kappa}B (53). In addition to the common MyD88/IRAK/TRAF6 pathway, the TIR domain family can activate downstream signal components through small G proteins such as Rac and Ras to mediate NF-{kappa}B activation and COX-2 expression (55, 56). To date, how the TIR domain family activates Ras, especially TLR2, is still not clear. Recently, Ras activation has been shown to be involved in the signaling pathway of IL-1-induced p38 MAPK activation and occurred through associating with IRAK, IRAK2, TRAF6, and TAK1 multiprotein complex in EL4.NOB-1 cells and HeLa cells (57, 58). Furthermore, Ras was found to participate in CpG oligonucleotide-induced COX-2 expression through association with TLR9 and to activate ERK and NF-{kappa}B in RAW 264.7 macrophages (56). In addition, a previous study also showed that the TIR domain of TLR2 contains a p85-binding motif (YXXM), which contributes to Rac mediation of TLR2-induced NF-{kappa}B activation via the recruitment of p85 and Rac to the TLR2 (45). The findings of our experiments showed that PGN can rapidly induce TLR2 association with p85{alpha} as early as 30 s, and subsequently Ras interacted with TLR2 at 1 min following PGN treatment. Simultaneously, we also found that PGN induced the association of Ras and p85{alpha} during the interaction of Ras and TLR2. Based on these findings, we suggest that PGN-induced Ras activation may occur through the recruitment of p85{alpha} and Ras to TLR2 in RAW 264.7 macrophages.

Ras, an oncogenic protein, plays a critical role in the induction of COX-2 protein (59, 60). Ras might activate a number of signal pathways, including the Raf-1/MEK/ERK pathway and the phosphatidylinositol 3-kinase/Akt/NF-{kappa}B pathway (34, 59, 60). In RAW 264.7 macrophages, LPS induces tumor necrosis factor gene expression through the Ras/Raf-1/MEK/ERK pathway (61). In murine fibroblasts, oncogene- and growth factor-induced COX-2 transcription requires Ras-dependent Raf-2/MAPKK/ERK activation (60). In this study, we found that treatment of RAW 264.7 macrophages with PGN caused sequential activations of Ras, Raf-1, and ERK, and that manumycin A, GW 5074, and PD 098059 all inhibited PGN-induced ERK activation and COX-2 expression. These results suggested that the Ras/Raf-1/ERK signal pathway is important for PGN-induced COX-2 expression. This suggestion is further supported by our previous report that LTA induces COX-2 expression through the ERK pathway to induce NF-{kappa}B activation in human lung epithelial cells (62). However, the contribution of Ras or ERK to LPS-mediated RAW 264.7 macrophages COX-2 expression is controversial. Most reports (47, 50, 63–65) have shown that LPS-induced RAW 264.7 macrophages cox-2 gene expression is dependent on the ERK signaling pathway. However, Wadleigh et al. (66) showed that overexpression of a dominant negative mutant of Ras or ERK did not inhibit LPS-induced COX-2 luciferase reporter activity. Nevertheless, they did not directly measure Ras or ERK activity. In the present study, we found that LPS induced an increase in Ras activity and that manumycin A, GW 5074, and PD 098059 all attenuated LPS-mediated COX-2 expression. Taken together, our results indicate that the Ras/Raf-1/ERK signaling pathway is also necessary for LPS-induced COX-2 expression in RAW 264.7 macrophages.

In conclusion, the findings of our study for the first time showed that PGN induced COX-2 transcription through the Ras/Raf-1/ERK signaling pathway to increase in IKK{alpha}/{beta} activity, NF-{kappa}B activation, and COX-2 expression in RAW 264.7 macrophages. This is the first study showing PGN-induced Ras activation may occur through the recruitment of p85{alpha} and Ras to TLR2 in RAW 264.7 macrophages. Fig. 9 is a schematic representation of the signaling pathway of PGN-induced COX-2 expression in RAW 264.7 macrophages. With an understanding of these signal transduction pathways, we can design therapeutic strategies to reduce inflammation caused by Gram-positive organisms.



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  		FIG. 9.
Schematic summary of signal transduction by PGN induces COX-2 expression in RAW 264.7 macrophages. PGN-induced Ras activation may occur through the recruitment of p85{alpha} and Ras to TLR2 in RAW 264.7 macrophages. The PGN-induced activation of the Ras/Raf-1/ERK cascade results in an increase in IKK{alpha}/{beta} activity, I{kappa}B{alpha} degradation, and NF-{kappa}B activation, which leads to expression of the macrophage COX-2.

 

   FOOTNOTES
 
* This work was supported by Grant NSC 92-2314-B-038-059 from the National Science Council of Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed: School of Respiratory Therapy, Taipei Medical University, 250 Wu-Hsing St., Taipei 110, Taiwan. Tel.: 886-2-27361661 (ext. 3510); Fax: 886-2-27397313; E-mail: chlin{at}tmu.edu.tw.

1 The abbreviations used are: PGN, peptidoglycan; COX-2, cyclooxygenase-2; DMEM/Ham's F-12, Dulbecco's modified Eagle's medium/Ham's F-12; ERK, extracellular signal-regulated kinase; IKK, I{kappa}B kinase; IL, interleukin; IRAK, IL-1 receptor-associated kinase; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MEK, MAPK/ERK kinase; MyD88, myeloid differentiation protein; NF-{kappa}B, nuclear factor-{kappa}B; PBS, phosphate-buffered saline; PDTC, pyrrolidine dithiocarbamate; Raf-1 RBD, Ras-binding domain for Raf-1; TIR, toll/IL-1 receptor; TLR2, Toll-like receptor 2; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone, TRAF6, tumor necrosis factor receptor-associated factor 6. Back



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