Apoptosis signal-regulating kinase 1 in peptidoglycan-induced COX-2 expression in macrophages

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1 Article Apoptosis signal-regulating kinase 1 in peptidoglycan-induced COX-2 expression in macrophages Ming-Jen Hsu,*, Chia-Kai Chang,* Mei-Chieh Chen, Bing-Chang Chen, Hon-Ping Ma, Chuang-Ye Hong, and Chien-Huang Lin*,,,1 *Graduate Institute of Medical Sciences, Departments of Pharmacology and Microbiology and Immunology, and School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei Medical University-Shuang Ho Hospital, Taipei County, and Taipei Medical University-Wang Fang Hospital, Taipei, Taiwan RECEIVED OCTOBER 9, 2009; REVISED FEBRUARY 3, 2010; ACCEPTED FEBRUARY 5, DOI: /jlb ABSTRACT In this study, we investigated the role of ASK1 in PGNinduced C/EBP activation and COX-2 expression in RAW macrophages. The PGN-induced COX-2 expression was attenuated by the DNs of ASK1, JNK1, JNK2, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). PGN caused ASK1 dephosphorylation timedependently at Ser967, dissociation from the ASK complex, and subsequent ASK1 activation. In addition, PGN activated PP2A and suppression of PP2A by okadaic acid markedly inhibited PGN-induced ASK1 Ser967 dephosphorylation and COX-2 expression. PGN induced the activation of the JNK-AP-1 signaling cascade downstream of ASK1. PGN-increased C/EBP expression and DNA-binding activity were inhibited by the ASK1-JNK-AP-1 signaling blockade. COX-2 promoter luciferase activity induced by PGN was attenuated in cells transfected with the COX-2 reporter construct possessing the C/EBP-binding site mutation. In addition, the ASK1-JNK-AP-1-C/EBP cascade was activated in human peripheral mononuclear cells exposure to PGN. The TLR2 agonist Pam 3 CSK 4 was also shown to induce ASK1 Ser967 dephosphorylation, JNK and c-jun phosphorylation, C/EBP activation, and COX-2 expression in RAW macrophages. PGN-induced COX-2 promoter luciferase activity was prevented by selective inhibition of TLR2 and c-jun in RAW macrophages. Our data demonstrate that PGN might activate the TLR2-mediated PP2A-ASK1-JNK-AP-1-C/EBP cascade and subsequent COX-2 expression in RAW macrophages. J. Leukoc. Biol. 87: ; Abbreviations: ASK1 apoptosis signal-regulating kinase 1, ChIP chromatin immunoprecipitation, COX-2 cyclooxygenase-2, DN dominant-negative, IKK I B kinase, LAP 38-kDa C/EBP isoform, LIP 19-kDa C/EBP isoform, m murine, MBP myelin basic protein, NP-40 Nonidet P-40, nsmase neutral sphingomyelinase, p phosphorylated, Pam 3 CSK 4 palmitoyl-3-cysteine-serine-lysine-4, PGN peptidoglycan, PP2A protein phosphatase 2A, SA Staphylococcus aureus, sirna small interfering RNA Introduction Bacteria activate the innate immune system of hosts and induce the release of inflammatory molecules such as chemokines and cytokines [1]. PGN, the main cell-wall component of gram-positive bacteria, is composed of alternating N-acetyl glycosamine and N-acetyl muramic acid long sugar chains that are interlinked by peptide bridges, resulting in a large, complex macromolecular structure [2, 3]. PGN contributes to most of the clinical manifestations of bacterial infections including inflammation, fever, and septic shock [4]. PGN activates the innate immune system of a host through the pattern recognition receptors, TLR2 and CD14, and induces the generation of proinflammatory cytokines, such as TNF-, IL-6, and IL-8 [5 9]. PGN was also shown to induce COX-2 expression in human polymorphonuclear leukocytes [10, 11]. Although 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 cox-2 gene. PGs, lipid mediators, play important roles in various homeostatic and inflammatory processes throughout the body. COX catalyzes the conversion of arachidonic acid to PGH 2, which is then metabolized further to various PGs, prostacyclin, and thromboxane A 2 [12]. Two COX isozymes, COX-1 and COX-2, have been cloned and identified to have 60% homology in humans [13, 14]. COX-1, which is constitutively expressed in most tissues, mediates physiological responses such as regulation of renal and vascular homeostasis and cytoprotection of the stomach. On the other hand, COX-2 is considered to be an inducible, immediate-early gene product whose synthesis in cells can be up-regulated by cytokines, growth factors, or bacterial endotoxin stimulation [15 20]. COX-2 plays a major role in inflammatory processes, and its expression has been linked with several diseases associated with inflammation and colon cancer [21]. 1. Correspondence: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. chlin@tmu.edu.tw /10/ Society for Leukocyte Biology Volume 87, June 2010 Journal of Leukocyte Biology 1069

2 The promoter region of the murine cox-2 gene contains many transcription factor-binding sites. These transcription factors include the C/EBP, CREB, and NF- B [22 24]. The C/EBP element is generally believed to play an important regulatory role in COX-2 expression in macrophages as well as in other cell types [23 25]. In particular, activation of C/EBP leads to the induction of COX-2 [26 28]. We demonstrated previously that NF- B activation is necessary for PGN-induced COX-2 induction and subsequent IL-6 release in RAW macrophages [15, 29]. However, the role of C/EBP in regulating COX-2 expression following PGN stimulation is still unknown. ASK1 is a multifunctional serine/threonine protein kinase involved in regulating diverse physiological processes, including cell differentiation and apoptosis [30, 31]. ASK1 has been reported to be activated by many stress signals or proinflammatory cytokines [32 34]. Into and Shibata [35] also demonstrated that ASK1 may contribute to the TLR2 signaling cascade. However, the molecular mechanism involved in the induction of PGN of ASK1 activation is still unknown. Furthermore, little information is available about the role of ASK1 in regulating C/EBP expression and subsequent COX-2 expression following PGN stimulation. Therefore, we attempted to elucidate the mechanism of PGN-induced ASK1 activation. The role of ASK1 in PGN-induced C/EBP activation and subsequent COX-2 expression was also established. In this study, we demonstrated that PGNinduced ASK1 activation may occur through the induction of ASK1 Ser967 dephosphorylation via PP2A activation, which in turn, causes the dissociation of the ASK complex. Furthermore, PGN activates ASK1, resulting in the activation of the JNK-AP-1 cascade, leading to C/EBP expression and subsequent activation, and ultimately, induces COX-2 expression in RAW macrophages. MATERIALS AND METHODS Reagents PGN purified from SA was purchased from Fluka (Buchs, Switzerland) and another PGN preparation, PGN-SA, was purchased from Invivogen (San Diego, CA, USA). Curcumin, SP600125, okadaic acid, and selective TLR2 ligand, Pam 3 CSK 4, were obtained from Calbiochem (San Diego, CA, USA). DMEM, FCS, and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies specific for -tubulin and COX-2 were purchased from Transduction Laboratories (Lexington, KY, USA). Protein A/G beads, antibodies specific for ASK1, , c-jun, c-jun phosphorylated at Ser73, C/EBP, C/EBP, CREB, and normal rabbit IgG (control IgG) and anti-mouse and anti-rabbit IgG-conjugated HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for JNK1/2, JNK phosphorylated at Thr183/Tyr185, and ASK1 phosphorylated at Ser967 or Thr845 were purchased from Cell Signaling (Beverly, MA, USA). Anti-mouse and anti-rabbit IgG-conjugated alkaline phosphatases were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Taq DNA polymerase was purchased from Takara (Otsu, Japan). ASK1DN, JNK1DN, and JNK2DN were kindly provided by Dr. M-C. Chen (Taipei Medical University, Taipei, Taiwan). AP-1-luc (which contains seven AP-1-binding sites) was purchased from Stratagene (La Jolla, CA, USA). The wild-type murine COX-2 reporter construct ( 966/ 23), pgl3-cox-2-luc, the mutant C/EBP construct, pgl3-mc/ebp-cox-2-luc, in which the C/EBP site ( 138/ 130) sequence TTGCGCAAC was changed to CCGCTCAAC, and pbk-cmv-lac Z were kindly provided by Dr. Wan- Wan Lin (National Taiwan University, Taipei, Taiwan). C/EBP reporter construct, p/t81 C/EBP -luc, was kindly provided by Dr. Kjetil Tasken (University of Oslo, Oslo, Norway). NF- B-Luc, Renilla-luc (coreporter vector), and Dual-Glo luciferase assay system were purchased from Promega Corp. (Madison, WI, USA). The GST-c-jun recombinant protein, an immunoprecipitation phosphatase assay kit, a ChIP assay kit, and Turbofect in vitro transfection reagent were purchased from Upstate Biotechnology (Lake Placid, NY, USA). [ - 32 P]ATP (6000 Ci/mmol) and GFX TM DNA purification spin columns were purchased from GE Healthcare (Little Chalfont, UK). GenePORTER TM 2 was purchased from Gene Therapy System (San Diego, CA, USA). All materials for SDS-PAGE were purchased from Bio-Rad (Hercules, CA, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cell culture The mouse macrophage cell line, RAW 264.7, was obtained from American Type Culture Collection (Livingstone, MT, USA), and cells were maintained in DMEM containing 10% FCS, 100 U/ml penicillin G, and 100 g/ml streptomycin in a humidified 37 C incubator. PBMCs were isolated by standard density gradient centrifugation with Ficoll-Paque (GE Healthcare) from the heparinized whole blood of normal individuals. Subsequently, CD14 cells are purified by high-gradient magnetic sorting using the VARIOMACS technique (Miltenyi Biotec, Bergisch Gladbach, Germany) with anti-cd14 microbeads. Immunoblot analysis Immunoblot analyses were performed as described previously [30]. Briefly, cells were lysed in extraction buffer containing 10 mm Tris (ph 7.0), 140 mm NaCl, 2 mm PMSF, 5 mm DTT, 0.5%, NP-40, 0.05 mm pepstatin A, and 0.2 mm leupeptin. Samples of equal amounts of protein were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, which was then incubated in TBST buffer (150 mm NaCl, 20 mm Tris-HCl, and 0.02% Tween 20, ph 7.4) containing 5% nonfat milk. Proteins were visualized by specific primary antibodies and then incubated with HRP- or alkaline phosphatase-conjugated secondary antibodies. Immunoreactivity was detected using ECL or NBT/5-bromo-4-chloro-3-indolyl phosphate following the manufacturer s instructions. Quantitative data were obtained using a computing densitometer with a scientific imaging system (Kodak, Rochester, NY, USA). Coimmunoprecipitation Cells were lysed in lysis buffer that contained 40 mm Tris-HCl (ph 8.0), 500 mm NaCl, 0.1% NP-40, 6 mm EGTA, 10 mm -glycerophosphate, 10 mm NaF, 300 M sodium orthovanadate, 2 mm PMSF, 10 g/ml aprotinin, 1 g/ml leupeptin, and 1 mm DTT. The cell lysates were subjected to immunoprecipitation using the appropriate antibodies. The resulting immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. PP2A activity assay An immunoprecipitation phosphatase assay kit (Upstate Biotechnology) was used to measure phosphate release as an index of phosphatase activity according to the manufacturer s instructions. Briefly, g cellular proteins were immunoprecipitated with a mouse anti-pp2a catalytic subunit antibody (0.8 g/ l; Upstate Biotechnology) and incubated with substrate, phosphoprotein (amino acid sequence: KRpTIRR, 175 M), in protein phosphatase assay buffer [20 mm 4-morpholinepropanesulfonic acid (ph 7.5), 60 mm 2-ME, 0.1 M NaCl, and 0.1 mg/ml serum albumin]. Reactions were initiated by the addition of the phosphoprotein substrate and carried out for 10 min at room temperature. Reactions were terminated by the addition of 100 l malachite green solution. The absorbance at 650 nm was measured on a microplate reader Journal of Leukocyte Biology Volume 87, June

3 Hsu et al. ASK1-mediated COX-2 expression Immunoprecipitation and protein kinase assays Cell lysates were subjected to immunoprecipitation using the proper antibody, and the immunoprecipitated complexes were washed three times with lysis buffer and two times with kinase buffer containing 20 mm HEPES (ph 7.4), 20 mm MgCl 2, and 2 mm DTT. The kinase reactions were performed by incubating the immunoprecipitated complex with 20 l kinase buffer supplemented with 20 M ATP and 3 Ci [ - 32 P]ATP at 30 C for 30 min. To assess ASK1 and JNK activities, MBP and GST-c-jun were, respectively, added as the substrates. The reaction mixtures were analyzed by 15% (MBP) or 12% (GST-c-jun) SDS-PAGE followed by autoradiography. The ChIP assay was performed following the instructions of Upstate Biotechnology. Briefly, cells were cross-linked with 1% formaldehyde at 37 C for 10 min and then rinsed with ice-cold PBS twice. Cells were then harvested in 0.2 ml SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-Cl, ph 8.1, 1 mm PMSF, 0.05 mm pepstatin A, and 0.2 mm leupeptin) and sonicated five times for 15 s each, followed by centrifugation for 10 min. Supernatants were collected and diluted in ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 1.2 mm EDTA, 167 mm NaCl, 16.7 mm Tris-HCl, ph 8.1), followed by immunoclearing with 80 l protein A-agarose slurry for1hat4 Cwith gentle rotation, and an aliquot of each sample was used as input in the PCR analysis. The remainder of the soluble chromatin was incubated at 4 C overnight with C/EBP, C/EBP, and CREB antibodies (Santa Cruz Biotechnology) or control IgG (Santa Cruz Biotechnology). Immune complexes were collected by incubation with 60 l protein A-agar- Preparation of nuclear extracts and the EMSA The cytosolic and nuclear protein fractions were separated as described previously [15]. Briefly, cells were lysed in hypotonic buffer [10 mm HEPES (ph 7.9), 10 mm KCl, 0.5 mm DTT, 10 mm aprotinin, 10 mm leupeptin, and 20 mm PMSF] for 15 min on ice and vortexed for 10 s. Nuclei were pelleted by centrifugation at 15,000 g for 1 min. A pellet containing nuclei was resuspended in hypertonic buffer [20 mm HEPES (ph 7.6), 25% glycerol, 1.5 mm MgCl 2, 4 mm EDTA, 0.05 mm DTT, 10 mm aprotinin, 10 mm leupeptin, and 20 mm PMSF] for 30 min on ice. Supernatants containing nuclear proteins were collected by centrifugation at 15,000 g for 2 min and then stored at 70 C. A double-stranded oligonucleotide probe containing AP-1 sequences (5 -TGACTCA -3 ; AP-1, Promega Corp.) was end-labeled with [ - 32 P]ATP using T4 polynucleotide kinase. The nuclear extract (5 g) was pretreated with vehicle, control IgG, anti-c-jun, or anti-cfos antibody for 1 h and incubated with 1 ng 32 P-labeled AP-1 probe (50,000 75,000 cpm) in 10 l binding buffer containing 1 g poly(deoxyinosinis-deoxycytidylic), 15 mm HEPES (ph 7.6), 80 mm NaCl, 1 mm EDTA, 1 mm DTT, and 10% glycerol at 30 C for another 25 min. DNA/ nuclear protein complexes were separated from the DNA probe by electrophoresis on 4% polyacrylamide gels, which were vacuum-dried and subjected to autoradiography with an intensifying screen at 80 C. Transfection and AP-1-, COX-2-, or C/EBP-luciferase assays Cells ( /well) were transfected with AP-1-luc, COX-2-luc, or mc/ebp COX-2-luc (with the C/EBP site mutation), plus pbk-cmv-lac Z using GenePORTER TM 2 (Gene Therapy System). Cells were harvested, and the luciferase activity was determined using a luciferase assay system kit (Promega Corp.) and was normalized on the basis of Lac Z expression. In the C/EBP-luciferase assay, cells were transfected with C/EBP-luc plus Renillaluc using Turbofect reagent (Upstate Biotechnology). Cells were harvested, and the luciferase activity was determined using a Dual-Glo luciferase assay system kit (Promega Corp.) and was normalized on the basis of Renilla luciferase activity. The level of induction of luciferase activity was compared as the ratio of cells with and without PGN stimulation. Oligonucleotide pull-down assay The nuclear extract was incubated at 30 C for 60 min with 0.5 nmol 5 biotinylated double-stranded wild-type (5 -GATCCTGCCGCTGCGGTTCTT- GCGCAACTCACT-3 ) or mutant (GATCCTGCCGCTGCGGTTCCCGCT- CAACTCACT) oligonucleotides, coupled previously to streptavidin agarose beads (Sigma, Poole, UK). The wild-type oligonucleotide corresponded to the region of 157 to 125 of the murine COX-2 promoter. After incubation, the biotinylated oligonucleotide-coupled streptavidin beads were washed three times with hypotonic buffer and denatured in SDS-sample buffer. The samples were subjected to immunoblotting analysis for C/EBP using specific antibodies. ChIP assay Figure 1. Involvement of ASK1 in PGN-mediated COX-2 induction in RAW macrophages. (A) Cells were transiently transfected with 1 g pcdna3 (mock transfection) or the ASK1DN mutant for 48 h. Following transfection, cells were treated with vehicle or 30 g/ml PGN for another 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the pcdna3 group in the presence of PGN. (B) Cells were treated with 30 g/ml PGN for various time intervals as indicated. ASK1 activity was then assessed by a kinase assay using MBP as the substrate. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the control group. KA, Kinase assay; IB, immunoblotting. Volume 87, June 2010 Journal of Leukocyte Biology 1071

4 ose slurry for 2 h at 4 C with gentle rotation. The complexes were washed sequentially in the following three washing buffers for 5 min each: a lowsalt immune complex washing buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, ph 8.1, 150 mm NaCl), a high-salt immune complex washing buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, ph 8.1, 500 mm NaCl), and a LiCl immune complex washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, ph 8.1). Precipitates were washed two times with Tris-EDTA buffer. The complexes were eluted twice with two 100 l aliquots of elution buffer (1% SDS, 0.1 M NaHCO 3 ). The cross-linked chromatin complex was reversed in the presence of 0.2 M NaCl and heating at 65 C for 4 h. DNA was purified using GFX TM DNA purification spin columns (GE Healthcare). PCR was carried out using Taq DNA polymerase for hot start (Takara), according to the manufacturer s protocol. Ten percent of the total purified DNA was used for the PCR in a 50- l reaction mixture. The 339-bp COX-2 promoter fragment between 398 and 60 was amplified by using a primer pair, sense: 5 -att ccc tta gtt agg acc tta gat ccc-3 and antisense: 5 -acg tag tgg tga ctc tgt ctt tcc gc-3, in 30 cycles of PCR under the following condition: at 95 C for 30 s, at 56 C for 30 s, and at 72 C for 60 s. The PCR products were analyzed by 1.5% agarose gel electrophoresis Suppression of TLR2 and c-jun expression Protocol for target gene suppression was described previously [30]. For TLR2 suppression, predesigned sirnas targeting the mouse TLR2 gene were purchased from Ambion (Austin, TX, USA). The sirna oligonucleotides targeting the coding regions of mouse TLR2 mrna were as follows: TLR2 sirna, 5 -gggcucacagaagcuguaatt-3 ; for c-jun suppression, predesigned sirnas targeting the mouse c-jun gene were purchased from Sigma Chemical Co. The sirna oligonucleotides targeting the coding regions of mouse c-jun mrna were as follows: c-jun sirna, 5 -guaacccuaagauccuaaa- 3. The negative control sirna comprising a 19-bp scrambled sequence with 3 deoxythymidylic overhangs was also purchased from Ambion. Statistical analysis Results are presented as the mean se from at least three independent experiments. One-way ANOVA 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 0.05 was considered statistically significant. Figure 2. Involvement of PP2A in PGN-induced ASK1 activation and COX-2 expression in RAW macrophages. (A) Cells were treated with 30 g/ml PGN for various time intervals as indicated. Cell lysates were then prepared and subjected to immunoblotting with the anti-pser967-ask1 (p- ASK1) antibody. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the control group. (B) Cells were treated with 30 g/ml PGN for various time intervals as indicated. The interaction of ASK1 and was then determined by coimmunoprecipitation as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the control group. IP, Immunoprecipitation. (C) Cells were pretreated with vehicle or okadaic acid (OA; nm) for 30 min before treatment with 30 g/ml PGN for another 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the group treated with PGN alone. (D) Cells were pretreated with vehicle or 1 nm okadaic acid for 30 min before treatment with 30 g/ml PGN for another 30 min. Cell lysates were then prepared and subjected to immunoblotting with the anti-pser967-ask1 antibody. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the group treated with PGN alone. (E) Cells were pretreated with vehicle or 1 nm okadaic acid for 30 min before treatment with 30 g/ml PGN for another 10 min. Following treatment, the PP2A activity assay was then performed as described in Materials and Methods. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the group treated with PGN alone Journal of Leukocyte Biology Volume 87, June

5 Hsu et al. ASK1-mediated COX-2 expression RESULTS Involvement of ASK1 in PGN-induced COX-2 expression To examine whether ASK1 is involved in the signal transduction pathway leading to COX-2 expression caused by PGN, the ASK1DN mutant was used. As shown in Figure 1A, transfection of RAW macrophages with 1 g ASK1DN markedly inhibited PGN-induced COX-2 expression by 55 16% (n 3). ASK1 kinase activity was also measured after PGN exposure. Treatment of cells with 30 g/ml PGN resulted in a time-dependent increase in ASK1 kinase activity. The response began at 5 min and peaked at 30 min after PGN treatment (Fig. 1B). These results suggest that ASK1 activation is involved in PGNinduced COX-2 expression in RAW macrophages. PP2A is involved in PGN-induced ASK1 activation We next explored the mechanism by which PGN induces ASK1 activation. Dissociation of ASK1 from the inhibitory protein, , may lead to ASK1 activation. Phosphorylation of the ASK1 Ser967 residue was proposed as being a major mode for regulating ASK1 binding to [30, 36]. We examined whether the extent of ASK1 Ser967 phosphorylation is altered after PGN exposure. PGN caused a marked decrease in ASK1 Ser967 phosphorylation at as early as 5 min, and this was sustained to 60 min after PGN exposure (Fig. 2A, upper panel). The protein level of ASK1 was not affected in the presence of PGN (Fig. 2A, lower panel). Coimmunoprecipitation was then used to confirm the hypothesis that PGN-induced ASK1 dephosphorylation was accompanied by dissociation of the ASK complex. As shown in Figure 2B, the PGN-induced dissociation between ASK1 and began at 5 min and was sustained to 60 min after PGN exposure. However, the mechanism that regulates ASK1 Ser967 dephosphorylation remains to be identified. Regulation of the phosphorylated protein is balanced by the activities of kinases and phosphatases [37]. It is conceivable that PGN may activate a protein phosphatase that dephosphorylates Ser967, leading to ASK1 activation and COX-2 expression. PP2A is a serine/threonine-specific protein phosphatase that regulates the activities of several major protein kinase families, such as Akt, ERK, and JNK [38, 39]. Thus, we attempted to determine whether PP2A is involved in ASK1 Ser967 dephosphorylation and COX-2 expression in response to PGN. Okadaic acid is a selective inhibitor of PP2A at low concentrations ( nm) but inhibits PP1 at much higher concentrations [40, 41]. As shown in Figure 2C, okadaic acid at a concentration of 1 nm inhibited COX-2 expression significantly in RAW cells in the presence of PGN. In addition, ASK1 Ser967 dephosphorylation induced by PGN was also inhibited by the presence of okadaic acid at 1 nm (Fig. 2D). Cell viability was not altered significantly by the presence of or okadaic acid (1 nm) in RAW after 24 h treatment (data not shown). Stimulation of cells with 30 g/ml PGN for 10 min induced an increase in PP2A activity (Fig. 2E). Confirming the effects of the PP2A inhibitor, a marked reduction in PP2A enzyme activity was observed in macrophages treated with 1 nm okadaic acid (Fig. 2E). These results suggest that PP2A may be responsible for PGN-induced ASK1 Ser967 dephosphorylation and COX-2 expression in RAW macrophages. JNK is involved in PGN-induced COX-2 expression We next determined whether JNK, a critical downstream molecule of ASK1 [42, 43], participates in PGN-induced COX-2 ex- Figure 3. Involvement of JNK in PGN-mediated COX-2 induction in RAW macrophages. Cells were pretreated for 30 min with various concentrations (1 10 M) of SP (A) or transfected with pcdna3, JNK1DN mutant, JNK2DN, or JNK1DN and JNK2DN for 24 h (B) as indicated. Cells were then stimulated with 30 g/ml PGN for another 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the group treated with PGN alone (A) or the pcdna3 group in the presence of PGN (B). Volume 87, June 2010 Journal of Leukocyte Biology 1073

6 pression. As shown in Figure 3A, the specific JNK inhibitor, SP (1 10 M), markedly attenuated PGN-induced COX-2 expression in a concentration-dependent manner. Pretreatment of cells with 10 M SP inhibited PGN-induced COX-2 expression by 63 12% (n 3). Cell viability was not altered significantly by the presence of SP (10 M) in RAW after 24 h treatment (data not shown). JNK1DN and JNK2DN were then used to block JNK1/2 signaling to confirm further whether JNK1/2 mediates PGN-induced COX-2 expression. As shown in Figure 3B, transfection of RAW cells with JNK1DN, JNK2DN, or cotransfection of JNK1DN and JNK2DN suppressed PGN-induced COX-2 expression significantly by 38 9%, 41 10%, and 47 9%, respectively (n 3). As cotransfection with JNK1DN and JNK2DN did not induce more inhibition of COX-2 expression as compared with the transfection of JNK1DN or JNK2DN, these results suggest that JNK1/2 activation may be partially required for PGN-induced COX-2 expression, and there may be an alternative pathway involved in the PGN response in RAW macrophages. We then examined whether PGN is able to activate JNK1/2. Results from Figure 4A illustrate that 30 g/ml PGN increased JNK1/2 phosphorylation time-dependently with a maximum effect at 30 min after PGN exposure (Fig. 4A, upper panel). The protein level of JNK1/2 was not affected by the presence of PGN (Fig. 4A, lower panel). In parallel, using c-jun as the JNK1/2 substrate, a time-dependent increase in JNK1/2 activity was also observed in PGN-treated RAW cells (Fig. 4B). The increase in JNK1/2 activity was observed beginning at 10 min, peaked at 30 min, and then had declined by 60 min after treatment (Fig. 4B). To ascertain the linkage between ASK1 and the JNK1/2 signaling cascade downstream of PGN, the effect of ASK1DN on PGN-induced JNK1/2 activation was determined. As shown in Figure 4C, transfection of cells with 1 g ASK1DN reduced PGN-induced JNK1/2 activation by 37 18% (JNK1) and 48 14% (JNK2). Neither of these treatments had any effect on the protein level of JNK1/2 (Fig. 4C, lower panel). Together, these findings suggest that the ASK1-JNK1/2 cascade contributed to PGN-induced COX-2 expression in RAW cells. AP-1 is involved in PGN-induced COX-2 expression AP-1, a heterodimer composed of fos and jun, is a transcription factor that plays a key role in inflammatory responses downstream of JNK1/2 [44, 45]. To explore the role of AP-1 in PGN-induced COX-2 expression, an AP-1 inhibitor, curcumin [46, 47], was used. As shown in Figure 5A, pretreatment of RAW macrophages with curcumin (0.1 1 M) attenuated PGN-induced COX-2 expression in a concentrationdependent manner. In addition, curcumin had no effect on the basal level of COX-2 expression (Fig. 5A). Cell viability was not altered significantly by the presence of curcumin (1 M) in RAW after 24 h treatment (data not shown). These results suggest that AP-1 activation might be necessary for PGN-induced COX-2 expression in RAW macrophages. Moreover, treatment of cells with 30 g/ml PGN caused a time-dependent increase in c-jun phosphorylation. This response reached a maximum within min and had declined to the basal level within 60 min after PGN stimulation Figure 4. Involvement of ASK1 in PGN-mediated JNK activation in RAW macrophages. (A) Cells were treated with 30 g/ml PGN for different time intervals as indicated. JNK1/2 phosphorylation status was shown by immunoblotting as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the control group. (B) Cells were treated with 30 g/ml PGN for various time intervals as indicated. JNK activity was then assessed by a kinase assay using GST-c-jun as the substrate as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the control group. (C) Cells were transfected with pcdna3 or ASK1DN for 24 h before incubation with 30 g/ml PGN for another 30 min. JNK1/2 phosphorylation was determined as described in A. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the pcdna3 group in the presence of PGN Journal of Leukocyte Biology Volume 87, June

7 Hsu et al. ASK1-mediated COX-2 expression Figure 5. Involvement of AP-1 in PGNmediated COX-2 induction in RAW macrophages. (A) Cells were pretreated for 30 min with vehicle or various concentrations of curcumin followed by the stimulation with 30 g/ml PGN for another 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the group treated with PGN alone. (B) Cells were treated with 30 g/ml PGN for different time intervals as indicated. Phosphorylation status of c-jun was shown by immunoblotting. Typical traces representative of three independent experiments with similar results are shown. (C) Cells were incubated with 30 g/ml PGN for various time intervals as indicated. EMSA was then performed as described in Materials and Methods. Data shown are representative of three independent experiments with similar results. (D) Cells were treated with PGN for 10 min. EMSA was then performed in the presence of IgG, anti-cjun, or anti-c-fos antibody as described in Materials and Methods. Competition studies were carried out by adding 50- fold excess of an unlabeled AP-1 oligonucleotide (cold) to the nuclear protein fraction from cells treated with PGN. Typical traces representative of three independent experiments with similar results are shown. (Fig. 5B). The nuclear extracts of RAW cells, with or without PGN treatment, were subjected to EMSA using AP-1- specific oligonucleotides as the probes. As shown in Figure 5C, AP-1-specific DNA protein complex formation increased with a maximum effect at 10 min after PGN treatment (Fig. 5C). Formation of the DNA protein complex was reduced completely by the addition of 50 cold AP-1 consensus DNA oligonucleotides (Fig. 5D), indicating that the DNA protein interactions were sequence-specific. To identify the specific subunits involved in the formation of the AP-1 complex after PGN stimulation, EMSA was also performed in the presence of specific antibodies against c-jun or c-fos. As shown in Figure 5D, pretreatment of nuclear extracts with a specific antibody against c-jun or c-fos reduced AP-1-specific DNA protein binding activity, and control antibody (IgG) was without effect, demonstrating that the components of the AP-1 heterodimer are c-jun and c-fos. A reporter assay was then used to determine PGN activation of AP-1 directly. Cells were transiently transfected with an AP-1-luc reporter construct. As shown in Figure 6A, cells treated with PGN ( g/ml) for 24 h caused a concentration-dependent increase in AP-1-luciferase activity. The increase of AP-1-luciferase activity was fold (n 3) after 30 g/ml PGN treatment. Furthermore, the PGN-induced increase in AP-1-luciferase activity was markedly attenuated in cells pretreated for 30 min with 10 M SP and 1 M curcumin by 42 10% and 80 7%, respectively (n 3; Fig. 6B). Based on these results, we suggest that PGN activation of JNK1/2 occurs upstream of AP-1. ASK1-, JNK-, and AP-1-mediated, PGN-induced C/EBP activation and subsequent COX-2 expression We next examined the crucial role of C/EBP in PGN-induced COX-2 expression in RAW macrophages. As shown in Figure 7A, treatment of cells with 30 g/ml PGN markedly increased the protein levels of LIP and LAP in timedependent manners, slightly increasing that of the 35-kDa isoform (LAP*). The increases of LIP and LAP began at 2hand reached a peak at 4 6 h after PGN treatment. To demonstrate physical interaction of C/EBP with the C/EBP-binding sequence of the murine COX-2 promoter, we used biotinylated, double-stranded oligonucleotides coupled to streptavidin agarose beads (wild-type or mutant COX-2 promoter, 157/ 125) to pull-down proteins interacting with the C/EBP-binding sequence in RAW cells in the absence or presence of PGN. The bound protein complex was then analyzed by Western blotting. In the following experiments, the extent of C/EBP binding to the oligonucleotides was reflected by the level of LIP. As shown in Figure 7B, PGN treatment increased the binding of C/EBP to the wild-type oligonucleotide containing binding sequences for C/EBP in a time-dependent manner, which reached a maximum after 6 h of treatment. However, C/EBP did not bind to a similar oligonucleotide Volume 87, June 2010 Journal of Leukocyte Biology 1075

8 Figure 6. Involvement of JNK in the PGN-induced increase in AP-1- luciferase activity in RAW macrophages. (A) Cells were transiently transfected with AP-1-luc and pbk-cmv-lac Z for 24 h and treated with PGN for another 24 h. AP-1-luciferase assay was then determined as described in Materials and Methods. Data represent the mean sem of three independent experiments performed in duplicate. *, P 0.05, as compared with the control group. (B) After transfection as described in A, cells were pretreated with 10 M SP or 1 M curcumin for 30 min before incubation with 30 g/ml PGN for another 24 h. AP-1-luciferase assay was then determined. Data represent the mean sem of three independent experiments performed in duplicate. *, P 0.05, as compared with the group treated with PGN alone. with the mutation of the C/EBP-binding sequence after PGN treatment (Fig. 7B). Transfection of RAW cells with 1 g ASK1DN inhibited PGN-induced C/EBP activation by 60 13% (n 3; Fig. 7C). Furthermore, PGN-induced C/EBP activation was attenuated by 53 12% and 71 20% (n 3) by pretreatment of cells for 30 min with 10 M SP and 1 M curcumin, respectively (Fig. 7D). These results suggest that activations of ASK1, JNK, and AP-1 contributed to PGN activation of C/EBP. To determine whether C/EBP was recruited to the endogenous cox-2 promoter region in response to PGN signaling, we performed ChIP experiments in RAW cells stimulated with PGN. We used primers encompassing the cox-2 promoter region between 398 and 60, which contains the C/EBP-binding site. As shown in Figure 7E, C/EBP binding to the cox-2 promoter region ( 398/ 60) was increased readily after PGN exposure. On the other hand, C/EBP binding to the cox-2 promoter region ( 398/ 60) was also detectable in the absence of PGN, and the interaction of C/EBP and DNA was not altered after PGN exposure. No recruitment of CREB to the cox-2 promoter region ( 398/ 60) was detected. In addition, the cox-2 promoter region ( 398/ 60) was detected in the cross-linked chromatin sample before immunoprecipitation (Fig. 7E, Input, positive control). These results therefore establish that PGN induces the recruitment of C/EBP to the promoter region of the endogenous cox-2 gene. To further confirm the role of C/EBP in the regulation of COX-2 transcription, the wild-type murine COX-2 reporter construct ( 966/ 23, WT COX-2-luc) or a mutant reporter construct with a C/EBP site ( 138/ 130) deletion (mc/ebp COX-2-luc) was transfected into RAW macrophages. As shown in Figure 7F, 30 g/ml PGN induced an increase in COX-2 promoter luciferase activity by fold (n 3) in cells transfected with the murine COX-2 construct. PGN-induced COX-2 promoter luciferase activity was reduced by % (n 4) by transfection with the C/EBP deletion mutant construct. These results suggest that C/EBP activation is necessary for PGN-induced COX-2 expression in RAW macrophages. In addition to PGN, we used PGN-SA, another commercial PGN preparation (Invivogen), to determine whether PGN-SA exerted similar effects on macrophages with those of PGN. As shown in Figure 8A, PGN-SA ( g/ml) increased COX-2 expression significantly in cells after exposure to PGN-SA for 24 h. ASK1 phosphorylation at Thr845 is correlated with ASK1 activity [48]. We thus determined whether the extent of ASK1 Thr845 phosphorylation is altered by PGN-SA. Treatment of cells with PGN-SA increased ASK1 Thr845 phosphorylation significantly at as early as 5 min, and this was sustained to 30 min after PGN-SA exposure (Fig. 8B). Results from Figure 8C illustrated further that 10 g/ml PGN-SA increased JNK1/2 phosphorylation time-dependently. The c-jun phosphorylation downstream of JNK signaling was also observed in PGN-SAtreated RAW cells (Fig. 8C). Similar to PGN, PGN-SA also markedly increased the protein levels of the C/EBP isoforms (LAP and LIP) in time-dependent manners (Fig. 8D). PGN-induced ASK1-JNK-AP-1-C/EBP activation and COX-2 expression in human PBMCs We next determined the role of the ASK1-JNK-AP1-C/EBP cascade in PGN induction of COX-2 in primary human PBMCs. As shown in Figure 9A, PGN ( g/ml) increased COX-2 expression significantly in PBMCs after exposure to PGN for 24 h. Treatment of cells with PGN increased ASK1 Thr845 phosphorylation significantly (Fig. 9B). Results from Figure 9B also illustrated that 30 g/ml PGN increased JNK1/2 phosphorylation and c-jun phosphorylation time-dependently in PGN-treated PBMCs. Similar to the effects of PGN in RAW cells, PGN was shown to increase the protein levels of the C/EBP isoforms (LAP and LIP) in PBMC exposure to PGN ( g/ml) for 6 h (Fig. 9C). TLR2 agonist Pam 3 CSK 4 induced ASK1-JNK-AP-1-C/ EBP activation and COX-2 expression in RAW264.7 macrophages Numerous studies have demonstrated the ability of PGN to induce a proinflammatory response, and PGN has been re Journal of Leukocyte Biology Volume 87, June

9 Hsu et al. ASK1-mediated COX-2 expression Figure 7. C/EBP -mediated PGN-induced COX-2 transcription in RAW macrophages. (A) Cells were treated with 30 g/ml PGN for the indicated time intervals. The protein levels of C/EBP isoforms (38 and 35 kda LAP and 19 kda LIP) and -tubulin were then determined by immunoblotting. (B) After PGN treatment as described in A, the oligonucleotide pull-down assay was performed as described in Materials and Methods. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with the control group. WT, Wild-type. Cells were transfected with pcdna3 or ASK1DN for 48 h (C) or pretreated for 30 min with 10 M SP or 1 M curcumin (D) before incubation with 30 g/ml PGN for another 6 h. The pull-down assay was then performed as described above. Compiled results are shown in the lower panel. Each column represents the mean sem of at least three independent experiments. *, P 0.05, as compared with pcdna3 (C) or the control (D) group in the presence of PGN. (E) Cells were incubated with 30 g/ml PGN for 6 h, and the ChIP assay was performed as described in Materials and Methods. Typical traces representative of three independent experiments with similar results are shown. (F) Cells were transiently transfected with pgl3-cox-2-luc or pgl3-mc/ebp-cox-2-luc (C/EBP site mutant) and pbk-cmv-lac-z for 24 h. Luciferase activity was then determined after treatment of PGN for another 24 h as described in Materials and Methods. Data represent the mean sem of three independent experiments performed in duplicate. *, P 0.05, as compared with the PGN-treated cells transfected with pgl3-cox-2-luc. ported to act as a TLR2 agonist [49 51], although recent studies have suggested that the reported TLR2-activating activity of PGN is a result of contamination of lipoprotein or superantigen-like activity [52, 53]. However, Dziarski and Gupta [54] re-evaluated and concluded that SA PGN is a TLR2 activator. To determine whether TLR2 contributes to PGN or PGN-SA actions on RAW macrophages as described above, we used a selective TLR2 agonist Pam 3 CSK 4 to activate TLR2 signaling cascade. Treatment of cells with Pam 3 CSK 4 (1 10 g/ml) for 24 h markedly increased COX-2 expression (Fig. 10A). We next examined whether the extent of ASK1 Ser967 phosphorylation is altered after Pam 3 CSK 4 exposure. As shown in Figure 10B, Pam 3 CSK 4 caused ASK1 Ser967 dephosphorylation at as early as 5 min, and this was sustained to 30 min after Pam 3 CSK 4 exposure. Results from Figure 10C demonstrated further that 1 g/ml Pam 3 CSK 4 increased JNK1/2 phosphorylation time-dependently, with a maximum effect at 30 min after Pam 3 CSK 4 exposure. In parallel, a time-dependent increase in c-jun phosphorylation was also observed in Pam 3 CSK 4 -treated RAW cells (Fig. 10C). Similar to PGN or PGN-SA, Pam 3 CSK 4 also markedly increased the protein levels of the C/EBP isoforms (LAP and LIP) in time-dependent manners (Fig. 10D). A reporter assay was then used to determine C/EBP activation in cell exposure to Pam 3 CSK 4, PGN, or PGN-SA. As shown in Figure 10E, cells treated with Pam 3 CSK 4 (1 or 10 g/ml), PGN (30 g/ml), or PGN-SA (10 g/ml) for 24 h caused a significant increase in C/EBP -luciferase activity by , , , or fold. These results suggested that Pam 3 CSK 4, like PGN or PGN-SA, may activate the ASK1- JNK-AP1 signaling cascade, leading to C/EBP activation and subsequent COX-2 expression in RAW macrophages. Volume 87, June 2010 Journal of Leukocyte Biology 1077

10 (Fig. 11D). Taken together, these results suggested that the ASK1-JNK1 cascade may be responsible for PGN activation of AP-1, but not NF- B, leading to COX-2 expression in RAW macrophages. DISCUSSION Our previous study has shown that PGN induces TLR2, p85, and Ras complex formation and subsequently activates the Ras-Raf-1-ERK pathway, which in turn, initiates IKK / and NF- B activation and ultimately induces COX-2 expression in RAW macrophages [15]. We also demonstrated that PGN-induced IL-6 production involves COX-2-generated PGE 2, activation of the EP2 and EP4 receptors, camp formation, and activation of protein kinase A, IKK /, p65 phosphorylation, and NF- B [29]. However, the precise molecular mechanism responsible for PGN-induced COX-2 expression remains to be fully characterized. The findings in the present study demonstrated that both commercial PGN preparations (PGN and PGN-SA) might act like Pam 3 CSK 4 to trigger TLR2-mediated activation of the ASK1-JNK-AP-1 cascade and subsequent C/EBP expression and activation, which ultimately induces COX-2 expression in RAW macrophages. Several lines of evidence have shown that PGN is able to activate MAPKs, including ERK1/2, p38 MAPK, and JNK [8]. Figure 8. PGN-SA activates the ASK1-JNK-AP-1-C/EBP signaling cascade and COX-2 expression in RAW macrophages. (A) Cells were treated with vehicle or PGN-SA ( g/ml) for 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting. Cells were treated with 10 g/ml PGN-SA for the indicated time intervals. ASK1 Thr845 phosphorylation (B), JNK1/2 and c-jun phosphorylation (C), or C/EBP isoforms (38 and 35 kda LAP and 19 kda LIP; D) and -tubulin were then determined by immunoblotting. Typical traces representative of three independent experiments with similar results are shown. TLR2 is involved in PGN-induced COX-2 expression To further confirm more specifically that PGN-induced COX-2 expression was mediated by TLR2, a TLR2 sirna oligonucleotide was used. In addition, curcumin (an AP-1 inhibitor) has been shown to be an inhibitor of NF- B [55, 56]. c-jun sirna was thus used to establish the role of AP-1 in PGN induction of COX-2 in RAW macrophages. As shown in Figure 11, sirna experiments revealed that TLR2 sirna or c-jun sirna suppressed the basal level of TLR2 (Fig. 11A) and c-jun (Fig. 11B). Transfection of cells with TLR2 sirna or c-jun sirna reduced PGN-increased COX-2 promoter luciferase activity significantly (Fig. 11C). To determine whether the effects of ASK1-JNK signaling blockade on PGN are a result of an interference with NF- B activation, we also analyzed the effects of ASK1DN or JNK1DN on NF- B-luciferase activity in cell exposure to PGN. As shown in Figure 11, PGN-increased NF- Bluciferase activity was not affected by ASK1DN or JNK1DN Figure 9. PGN activates the ASK1-JNK-AP-1-C/EBP signaling cascade and COX-2 expression in human PBMCs. (A) Cells were treated with vehicle or PGN ( g/ml) for 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting. (B) Cells were treated with 30 g/ml PGN for the indicated time intervals. ASK1 Thr845 phosphorylation, JNK1/2, and c-jun phosphorylation were then determined by immunoblotting. (C) Cells were treated with vehicle or PGN ( g/ml) for 6 h. C/EBP isoforms (38 and 35 kda LAP and 19 kda LIP) and -tubulin were then determined by immunoblotting. Typical traces representative of three independent experiments with similar results are shown Journal of Leukocyte Biology Volume 87, June

11 Hsu et al. ASK1-mediated COX-2 expression Figure 10. Pam 3 CSK 4 activates the ASK1-JNK-AP-1-C/EBP signaling cascade and COX-2 expression in RAW macrophages. (A) Cells were treated with vehicle or Pam 3 CSK 4 (1 10 g/ml) for 24 h. COX-2 and -tubulin protein levels were then determined by immunoblotting. Cells were treated with 1 g/ml Pam 3 CSK 4 for the indicated time intervals. ASK1 Ser967 phosphorylation (B), JNK1/2 and c-jun phosphorylation (C), or C/EBP isoforms (38 and 35 kda LAP and 19 kda LIP; D) and -tubulin were then determined by immunoblotting. Typical traces representative of three independent experiments with similar results are shown. (E) Cells were transiently transfected with C/EBP-Luc and Renilla-luc for 24 h. Luciferase activity was then determined after treatment of Pam 3 CSK 4, PGN, or PGN-SA for another 24 h as described in Materials and Methods. Data represent the mean sem of three independent experiments performed in duplicate. *, P 0.05, as compared with the control group. We demonstrated recently that the Ras-Raf-ERK1/2 signaling pathway is involved in PGN-induced COX-2 expression in RAW macrophages [15]. Activated ASK1 plays a critical role in regulating diverse physiological processes, including cell differentiation and apoptosis, by stimulating downstream signaling events, which include activation of MAPK kinase 4/7 and JNK in sequence [42, 43]. JNK has been proposed as playing a role in AP-1-mediated transcriptional activation of the target genes [44, 45]. In addition, PGN has been shown previously to activate AP-1 in macrophages [57]. However, whether the ASK1- JNK-AP-1 signaling pathway participates in PGN-induced COX-2 expression has not been demonstrated previously. In this study, we found that treatment of RAW macrophages with PGN or PGN-SA caused sequential activations of ASK1, JNK, and AP-1 and that the DN mutants of ASK1, JNK1, and JNK2 or inhibitors of the JNK-AP-1 cascade attenuated PGN-induced COX-2 expression. The PGN-induced increase in AP-1-luciferase activity was inhibited by the JNK inhibitor and the AP-1 inhibitor. Moreover, the DN mutant of ASK1 suppressed PGN-induced JNK1/2 activation. These results suggest that PGN might activate ASK1 to cause JNK activation, which in turn, induces AP-1 activation and ultimately leads to COX-2 expression in RAW macrophages. ASK1 activity is regulated by interactions with various proteins. Phosphorylation of the ASK1 Ser967 residue was shown to be essential for binding to the inhibitory protein, , and for suppressing the ASK1 function [30, 36]. In this study, we found that PGN-induced ASK1 Ser967 dephosphorylation was accompanied by dissociation of the ASK complex. However, the signaling pathways that control the ASK1 function through Ser967 remain unresolved. Activation of an unknown protein phosphatase is required for TNF- -induced ASK1 activation by dephosphorylating ASK1 Ser967 [58]. Goldman et al. [48] demonstrated that an okadaic acid-sensitive phosphatase is required for H 2 O 2 -induced ASK1 Ser967 dephosphorylation in COS7 cells. We demonstrated recently that PP2A might contribute to ASK1 activation in cerebral endothelial cells [30]. In this study, we found that PGN caused an increase in PP2A activity and that okadaic acid, a specific inhibitor of PP2A, inhibited PGN-induced ASK1 Ser967 dephosphorylation and COX-2 expression. These findings suggest that PP2A may play a pivotal role in ASK1 dephosphorylation and subsequent signaling events in macrophages. However, the precise mechanism involved in PGN-induced PP2A activation in RAW macrophages needs to be elucidated further. PP2A, a member of ceramide-activated protein phosphatases family, is known to be activated by ceramide. Cellular ceramide synthesis increases in response to stress signals [59]. One pathway of ceramide formation involves sphingomyelin hydrolysis by nsmase or acidic sphingomyelinase [60]. Another pathway involves ceramide synthase-catalyzed denovo ceramide synthesis [61]. We found recently that nsmase blockade significantly inhibited PGN-induced ceramide accumulation and COX-2 expression. We also found that PGN activates nsmase in RAW cells [62]. These findings raise the possibility that the nsmase-ceramide cascade may mediate PGNinduced PP2A activation in RAW cells. Several consensus sequences, including those for C/EBP, CREB, and NF- B in the 5 flanking region of the cox-2 gene, have been identified in up-regulating COX-2 expression in response to various stimuli [22 24]. A recent study [63] showed that these transcription factors participate in a sequential, coordinated regulation of COX-2 expression in RAW264.7 cell exposure to LPS. C/EBP activation is required for LPS-induced COX-2 induction in RAW macrophages [27, 28]. The results from DNA pull-down and ChIP assay indicated that C/EBP binds to the endogenous cox-2 promoter region ( 398/ 60) and that this binding is augmented by PGN signaling. In contrast, the recruitment of C/EBP to the cox-2 promoter region ( 398/ 60) was readily detected in unstimulated RAW cells. The addition of PGN did not alter the binding of C/EBP to the cox-2 promoter region ( 398/ 60). A Volume 87, June 2010 Journal of Leukocyte Biology 1079