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• W2041012906 abstract "Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1β (IL-1β) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1β release and dissected the signaling pathways leading to LPS-induced IL-1β secretion. We demonstrate that both the NF-κB and the C/EBPβ-binding elements of the IL-1β promoter drive LPS-induced IL-1β gene expression. NF-κB activation requires the classical TLR4-initiated signaling cascade leading to IκB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPβ activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPβ, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPβ. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1β. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1β gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1β gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer. Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1β (IL-1β) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1β release and dissected the signaling pathways leading to LPS-induced IL-1β secretion. We demonstrate that both the NF-κB and the C/EBPβ-binding elements of the IL-1β promoter drive LPS-induced IL-1β gene expression. NF-κB activation requires the classical TLR4-initiated signaling cascade leading to IκB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPβ activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPβ, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPβ. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1β. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1β gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1β gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer. Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis and also peptic ulcer, gastric carcinoma, and gastric lymphoma. H. pylori-associated gastritis is characterized by severe infiltration of neutrophils and mononuclear cells in the gastric mucosa (1Suerbaum S. Michetti P. N. Engl. J. Med. 2002; 347: 1175-1186Crossref PubMed Scopus (2204) Google Scholar). Accumulation and activation of these cells is induced by the local production of chemokines and cytokines. Recent studies have demonstrated that mucosal levels of interleukin (IL) 1The abbreviations used are: IL, interleukin; LPS, lipopolysaccharide; TLR, Toll-like receptor; MyD88, myeloid differentiation factor 88; PI-3K, phosphatidylinositol 3-kinase; IRAK, IL-1 receptor-associated kinase; TRAF, tumor necrosis factor receptor-associated factor; IKK, IκB kinase; PAK, p21-activated kinase; MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; z-YVAD-FMK, z-Tyr-Val-Ala-Asp(OMe)-CH2F; z, benzyloxycarbonyl; DTT, dithiothreitol; NTA, nitrilotriacetic acid; C/EBP, CCAAT/enhancer-binding protein; ELISA, enzyme-linked immunosorbent assay; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; RT, reverse transcription; GSK, glycogen synthase kinase; ERK, extracellular signal-regulated kinase; dn, dominant-negative.-1β, IL-6, and IL-8 are significantly higher in H. pylori-positive patients than in H. pylori-negative patients (2Noach L.A. Bosma N.B. Jansen J. Hoek F.J. van Deventer S.J. Tytgat G.N. Scand. J. Gastroenterol. 1994; 29: 425-429Crossref PubMed Scopus (489) Google Scholar, 3Crabtree J.E. Shallcross T.M. Heatly R.V. Wyatt J.I. Gut. 1991; 32: 1473-1477Crossref PubMed Scopus (490) Google Scholar). ELISA and RT-PCR analyses suggest that IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α play important roles in gastric inflammation caused by H. pylori infection. Several potential soluble virulence factors derived from H. pylori are considered to attract or activate neutrophils and mononuclear cells, and there is evidence that H. pylori strain genotype as well as host factors determine the clinical outcome. H. pylori does not invade mononuclear cells. Evidence from our own laboratory (4Bhattacharyya A. Pathak S. Datta S. Chattopadhyay S Basu J. Kundu M. Biochem. J. 2002; 368: 121-129Crossref PubMed Scopus (92) Google Scholar) as well as other laboratories (5Innocenti M. Svennerholm A-M. Quiding-Jarbrink M. Infect. Immun. 2001; 69: 3800-3808Crossref PubMed Scopus (24) Google Scholar) suggests that H. pylori lipopolysaccharide (LPS) mediates release of cytokines and chemokines from human monocytes. The biological effects of these cytokines may result in the recruitment, influx, and activation of neutrophils in gastric mucosa during H. pylori infection. H. pylori LPS differs from that of Enterobacteriaceae (6Moran A.P. Aliment. Pharmacol. Ther. 1996; 10: 39-50Crossref PubMed Scopus (80) Google Scholar) in its ability to activate inflammatory cells possibly because of structural differences between the lipid A molecules. H. pylori LPS activates inflammatory cells to produce IL-1β, IL-8, and TNF-α (7Birkholz S. Knipp U. Nietzki C. Adamek R.J. Opferkuch W. FEMS Immunol. Med. Microbiol. 1993; 6: 317-324Crossref PubMed Scopus (90) Google Scholar). IL-1β, TNF-α, and IL-8 are increased in the antral mucosa of individuals with H. pylori gastritis (8Yamaoka Y. Kodama T. Kita M. Imanishi J. Kashima K. Graham D.Y. Helicobacter. 2001; 6: 116-124Crossref PubMed Scopus (74) Google Scholar, 9Ernst P.B. Gold B.D. Annu. Rev. Microbiol. 2000; 54: 615-640Crossref PubMed Scopus (485) Google Scholar). However, the molecular mechanisms associated with H. pylori LPS-induced cytokine/chemokine production have not been extensively studied. IL-1β is a potent inflammatory cytokine that is released as a component of the host response against bacterial infection. It is primarily expressed by activated monocytes/macrophages. IL-1β is produced as a precursor molecule, pro-IL-1β, in the cytosol of macrophages. Pro-IL-1β is a 31–34-kDa inactive form of the cytokine, which is later cleaved by caspase-1 to active 17-kDa IL-1β (10Cerretti D.P. Kozlosky C.J. Mosley B. Nelson N. Ness VanK. Green-street T.A. March C.J. Kronheim S.R. Druck T. Cannizzaro L.A. Huebner K. Black R.A. Science. 1992; 256: 97-100Crossref PubMed Scopus (1007) Google Scholar). The active IL-1β is released and exhibits its diverse biological functions. Soluble mediators of H. pylori are known to induce IL-1β. Of particular significance is the finding that IL-1β gene cluster polymorphisms suspected of enhancing production of IL-1β are associated with an increased risk of gastric cancer (11El-Omar E.M. Carrington M. Chow W.H. McColl K.E. Bream J.H. Young H.A. Herrera J. Lissowska J. Yuan C.C. Rothman N. Lanyon G. Martin M. Fraumeni Jr., J.F. Rabkin C.S. Nature. 2000; 404: 398-402Crossref PubMed Scopus (2058) Google Scholar). This makes it worthwhile to explore the mechanism of induction of IL-1β by H. pylori, and in particular, the role of LPS. The expression of IL-1β is regulated at the level of transcription (12Cahill C.M. Waterman W.R. Xie Y. Auron P.E. Calderwood S.K. J. Biol. Chem. 1996; 271: 24874-24879Abstract Full Text Full Text PDF PubMed Google Scholar, 13Godambe S.A. Chaplin D.D. Takova T. Bellone C.J. Mol. Cell. Biol. 1995; 15: 112-119Crossref PubMed Google Scholar, 14Hiscott J. Marois J. Garoufalis J. D'Addario M. Roulston A. Kwan I. Pepin N. Lacoste J. Nguyen H. Bensi G. Mol. Cell. Biol. 1993; 13: 6231-6240Crossref PubMed Google Scholar), mRNA stabilization, and post-translational proteolytic processing (15Auron P.E. Webb A.C. Eur. Cytokine Net. 1994; 5: 573-592PubMed Google Scholar). C/EBPβ (13)- and NF-κB (14)-binding sites have been characterized in the human IL-1β promoter. Toll-like receptors (TLRs) play central roles in innate immunity by recognition and discrimination of specific conserved patterns of molecules derived from bacteria, fungi, or viruses (16Medzhitov R. Nat. Rev. Immunol. 2001; 1: 135-145Crossref PubMed Scopus (3284) Google Scholar, 17Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar, 18Akira S. J. Biol. Chem. 2003; 278: 38105-38108Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Activation of TLRs results in stimulation of signaling pathways widely involving recruitment of the adaptor molecule myeloid differentiation factor 88 (MyD88) (19Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (988) Google Scholar, 20Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 21Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1311) Google Scholar). The serine/threonine kinase IL-1 receptor-associated kinase 1 (IRAK1) is subsequently recruited, becomes phosphorylated, dissociates from the complex and associates with tumor necrosis factor receptor-associated factor 6 (TRAF6) (22Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar, 23Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1123) Google Scholar, 24Bradley J.R. Pober J.S. Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (527) Google Scholar) leading to the activation of mitogen-activated protein kinases (MAPKs), transcription factors such as NF-κB and concomitant production of cytokines (25Swantek J.L. Tsen M.F. Cobb M.H. Thomas J.A. J. Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (234) Google Scholar, 26Zhang G Ghosh S. J. Clin. Investig. 2001; 107: 13-19Crossref PubMed Scopus (620) Google Scholar). MAPKs comprise an important group of serine/threonine signaling kinases that transduce a variety of extracellular stimuli through a cascade of protein phosphorylations, which lead to the activation of transcription factors (27Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2897) Google Scholar, 28Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar, 29Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar). There are three principal MAPK family members: (i) p46 and p54 c-Jun N-terminal kinase or stress-activated protein kinase (JNK or SAPK, respectively) with multiple subisoforms, (ii) p38 MAPK with α, β, γ, and δ isoforms, and (iii) p42 and p44 extracellular signal-regulated kinase (ERK). MAPKs are activated by specific upstream MAPK kinases (MKKs) (30Derijard B. Raingeaud J. Barrett T. Wu L.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar). Since TLR- and MAPK signaling lead to the control of gene transcription, we explored the role of these signaling pathways in H. pylori-LPS-mediated IL-1β release, and its regulation at the level of transcription. We also explored the pathways regulating proteolytic processing of pro-IL-1β. The study described here provides evidence that H. pylori LPS signals through TLR4 to regulate IL-1β transcription driven by NF-κB and C/EBPβ elements in the IL-1β promoter. Whereas regulation at the level of NF-κB involves the canonical TLR4-directed phosphorylation of IκB-α leading to NF-κB activation, regulation of C/EBPβ involves a TLR4/phosphatidylinositol 3-kinase (PI-3K)/Akt/p38 MAPK pathway. We observed a direct interaction between phospho-p38MAPK and C/EBPβ, suggesting that p38MAPK is the immediate upstream kinase responsible for activating C/EBPβ. In addition, PI-3K/Rac1/p21-activated kinase 1(PAK1) signaling also regulates NF-κB, as well as the caspase-1-mediated processing of pro-IL-1β to -IL-1β. We present the novel finding that PAK1 interacts with caspase-1, and that this interaction is blocked in dominant-negative (dn)-Rac1-transfected cells. PAK1 immunoprecipitated from H. pylori LPS-challenged cells, phosphorylated caspase-1 at Ser376 and activation of caspase-1 was abrogated in caspase-1 (S376A)-transfected cells. We hypothesize that PAK1 is a key upstream kinase regulating the activation of caspase-1 at least in some cell types. Chemicals—Akt inhibitor (1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate), SB203580, U0126, SP 600125, z-Tyr-Val-Ala-Asp(OMe)-CH2F (z-YVAD-FMK) N-acetyl-Tyr-Val-Ala-Asp-(7-amino 4-trifluoromethylcoumarin) (Ac-YVAD-AFC), phorbol 12-myristate 13-acetate (PMA), and wortmannin, were products of EMD Biosciences, San Diego, CA. Protease inhibitors were from Roche Applied Science. All other reagents were of analytical grade. The human interleukin-1β ELISA kit was from Amersham Biosciences. Escherichia coli LPS was purchased from Sigma. [γ-32P]ATP was from Jonaki, BRIT, Hyderabad, India. Antibodies—Anti-p38 MAPK, -PAK1, -phospho-p38MAPK, -phospho-PAK1, -interleukin-1β, and -p-Tyr were from Cell Signaling Technology, Beverly, MA. Anti-p85 and supershift antibodies against p50, p65, and C/EBPβ were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Anti-Rac1 and anti-caspase-1 were from BD Biosciences, San Jose, CA. Neutralizing TLR-4 antibody (HTA 125) was purchased from Imgenex Biotech, Bhubaneswar, India. LPS Preparations—H. pylori 26695 was cultured for 3 days on horse blood agar plates in a microaerophilic milieu (10% CO2,5%O2, and 85% N2) at 37 °C. The bacterial cells were harvested; LPS was prepared by the hot phenol-water method described by Westphal and Jann (31Westphal O. Jann K. Witler R. Methods in Carbohydrate Chemistry. Academic Press, New York1965: 83-92Google Scholar), dialyzed, and freeze-dried. LPS was estimated using the E-TOXATE amoebocyte lysate assay kit from Sigma. Cell Culture—THP1 cells (derived from a patient with acute monocytic leukemia) are mature cells from the monocyte/macrophage lineage. These were obtained from the National Center for Cell Science (Pune, India). The cell line was maintained in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, and 20 mm sodium bicarbonate. The cells were incubated at 5% CO2 and 95% humidity in a 37 °C chamber. THP-1 cells were treated with PMA to induce maturation of the monocytes to a macrophage-like adherent phenotype (32Tsuchiya S. Kobayashi Y. Goto Y. Okumura H. Nakae S. Konno T. Tada K. Cancer Res. 1982; 42: 1530-1536PubMed Google Scholar). Cells were washed thrice with culture medium and cell viability was determined to be >95% by the trypan blue dye exclusion method. The inhibitors U0126, SB203580, SP600125, Akt inhibitor, and wortmannin, were dissolved in Me2SO. Appropriate vehicle controls were maintained in all experiments in which these inhibitors were used. Plasmids and Transient Transfections—TLR4 was amplified from RNA isolated from THP-1 cells using the sense and antisense primers 5′-ATGGATCCGCATGGAGCTGAATTTCTA-3′ and 5′-ATAAAGCTTCTAAGATGTTGCTTCCTG-3′, respectively and cloned between the BamHI and HindIII sites of pcDNA3.1. TLR4-(1–643) (TLR4-dn) encoding the first 643 amino acids of TLR4, was generated using the same sense primer and the antisense primer 5′-ATAAAGCTTCTACTGGCTTGAGTAGAT-3′. RNA was isolated from THP-1 (using the Qiagen RNeasy Mini kit) and used as template to amplify the cDNA of the coding sequence for caspase-1 by RT-PCR using the sense and antisense primers 5′-TAGAATTCATGGCCGACAAGGTCCTG-3′(a) and 5′-TTGGATCCATGTCCTGGGAAGAGGTA-3′(d), respectively. Mutant caspase-1 (S376A) was generated by overlap extension PCR. The initial rounds of PCR were carried out with primer pairs a and b (5′-GTTCGATTTGCATTTGAGCAG-3′), and c (5′-CTGCTCAAATGCAAATCGAAC-3′) and d. The products of each PCR were purified and used as templates for the second round of PCR using the primers a and d. The caspase-1 gene was cloned between the EcoRI and BamHI of pTrc6His. For transfection experiments, caspase-1 constructs were generated by cloning between the EcoRI and BamHI sites of pFlagCMV6c. Myc-tagged IRAK1-(1–215) (IRAK1-dn) in pcDNA3.1 was obtained from Klaus Ruckdeschel, Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Munich, with the consent of Marta Muzio. MyD88-(152–196) (MyD88-dn) in pcDNA3.1 was obtained from Marta Muzio and Alberto Mantovani, Mario Negri Institute for Pharmacological Research, Milan, Italy. pRK5-FLAG-TRAF6, pRK5-Flag TRAF6- (289–522) (TRAF6-dn), pRK5-FLAG-IKKβ, pRK5-FLAG-IKKβ K44A (IKKβ-dn) were gifts from Tularik Inc. FLAG-tagged p38 and JNK MAPKs and their dominant-negative mutants (p38 (agf) and JNK (afp), respectively), were obtained from Roger Davis, University of Massachusetts Medical School, Worcester, MA. The dominant negative mutant of the phosphatidylinositol 3-kinase p85 subunit deleted in the inter-SH2 region of wild-type p85α (PI-3KΔ85) was a gift from Robert Farase, J. A. Haley Veterans Hospital, Tampa, FL and the kinase-deficient mutant of Akt (Akt-Kd) carrying the mutation K179M was a gift from Kenneth Walsh, Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA. pCMV-PAK1(wt) and pCMV-PAK1 (K299R) (PAK1-KD) were gifts from Jeffrey Frost, University of Texas Southwestern Medical Center, Dallas, TX. GST-C/EBPβ- (22–227) was a gift from Jean-Rene Cardinaux, University of Lausanne, Switzerland. Transient transfections were carried out using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. β-Gal reporter plasmid was used to normalize transfection efficiencies. Treatment with LPS and Preparation of Cell Lysates—THP-1 cells were treated with PMA and cultured in 24-well tissue culture plates at 5 × 105 cells per well followed by treatment with H. pylori LPS (or E. coli LPS where indicated). The wells were washed with ice-cold phosphate-buffered saline and lysed with lysis buffer (20 mm Tris-HCl pH 7.4, 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 137 mm NaCl, 20 mm NaF, 1 mm EDTA, 40 mm Na-β-glycerophosphate, 4 μg/ml each of leupeptin, pepstatin, and aprotinin, 1 mm Na3VO4,1mm pefabloc, 1 mm benzamidine) (buffer A) on ice for 15 min. Cell lysates were boiled for 5 min after the addition of 5× Laemmli sample buffer and subjected to Western blotting. Where necessary, THP-1 cells were first treated with pharmacological inhibitors or vehicle (Me2SO) alone, prior to incubation with LPS. Western Blotting—Proteins were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene difluoride membranes. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature and subsequently incubated overnight at 4 °C with primary antibodies (in TBS-Tween 20 (1%, v/v) (TBST) with 5% (w/v) bovine serum albumin). Following three washes of 5 min each with TBST, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) or appropriate secondary antibodies) in blocking buffer for 1 h at room temperature. After three washes with TBST, the blots were developed by chemiluminescence using the phototope-HRP Western Detection kit (Cell Signaling Technology) and exposed to x-ray film (Kodak XAR5). Western Analysis of Caspase-1 Activation—Transfected or non-transfected THP-1 cells after treatment with H. pylori LPS (20 ng/ml) were pelleted and freeze-thawed three times in 20 μl of cell extraction buffer (50 mm PIPES/NaOH, pH 6.5, 2 mm EDTA, 0.1% (w/v) CHAPS, 5 mm DTT, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mm pefabloc). The lysates were centrifuged at 10,000 × g for 5 min at 4 °C, and the supernatants were boiled for 5 min after addition of 5× Laemmli sample buffer before subjecting to Western blotting using anticaspase-1 antibody. Akt Kinase Assay—The activity of Akt protein kinase was determined by using the Akt kinase assay kit (Cell Signaling Technology) using glycogen synthase kinase (GSK)-3 as substrate, according to the manufacturer's instructions. Briefly, lysates were incubated with immobilized Akt monoclonal antibody. The beads after washing, were incubated with 2 μg of GSK-3 fusion protein as substrate in 20 μl of kinase reaction buffer containing 200 μm ATP. After incubation for 30 min at 30 °C, the reaction was terminated by the addition of 5× Laemmli sample buffer. Phosphorylated proteins were resolved by SDS-PAGE followed by Western blotting. Blots were probed with anti-phospho-GSK-3 antibody and visualized by chemiluminescence. Affinity Precipitation Assay for Rac—This was done as described by Benard et al. (33Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). Cell extracts after treatment were incubated with GST-PBD (the p21-binding domain (PBD) of PAK1 fused to GST) in a maximal final volume of 500 μl for 1 h at 4 °C. The bead pellet was then centrifuged for 2 min at 2,000 rpm at 4 °C and washed three times with washing buffer (25 mm Tris-HCl pH 7.6, 1 mm DTT, 30 mm MgCl2, 40 mm NaCl, 1% Nonidet P-40) and twice with the same buffer without Nonidet P-40. The bead pellet was finally suspended in 20 μl of Laemmli sample buffer. Proteins bound to the beads were separated on SDS-polyacrylamide (12%) gels, transferred onto nitrocellulose membrane and blotted for Rac1. Assays of C/EBPβ-p38 MAP Kinase Interaction—In order to study the in vivo interaction between C/EBPβ and p38 MAP kinase, C/EBPβ was immunoprecipitated from lysates of cells treated without or with H. pylori LPS. The immunoprecipitate was boiled in Laemmli buffer, separated on SDS-PAGE, transferred onto polyvinylidene difluoride membrane and blotted for p38 MAPK. In separate experiments, lysates from cells treated without or with H. pylori LPS were incubated with immobilized phospho-p38 MAP kinase (Cell Signaling Technology) overnight at 4 °C. Bead-bound proteins were separated by SDS-PAGE, electrotransferred, and probed with anti-C/EBPβ antibody. In an alternative approach, GST-C/EBPβ-(22–227) was expressed in E. coli BL21, purified over glutathione-Sepharose beads and GST-C/EBPβ-bound beads were incubated for 2 h at 4 °C with lysates from cells treated without or with H. pylori LPS as described by Kovacs et al. (34Kovacs K.A. Steinmann M. Magistretti P.J. Halfon O. Cardinaux J-R. J. Biol. Chem. 2003; 278: 36959-36965Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The bead pellets were washed, suspended in Laemmli buffer, separated on SDS-polyacrylamide (10%) gels, transferred onto nitrocellulose membrane, and blotted for p38 MAP kinase. Affinity Precipitation Assay to Study the Interaction of Caspase-1 and PAK1—His-tagged caspase-1 was expressed in E. coli DH5α by inducing cells using 200 μm isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 2 h and immobilized on Ni2+-NTA-agarose. The agarose beads were incubated for 2 h at 4 °C with lysates from cells treated without or with H. pylori LPS. The bead pellets were washed, suspended in Laemmli buffer, separated on SDS-polyacrylamide (7.5%) gels, transferred onto nitrocellulose membranes and blotted for PAK1. In order to study the in vivo interaction between PAK1 and caspase-1, lysates from cells treated without or with H. pylori LPS were incubated overnight with PAK1 antibody at 4 °C, followed by incubation with protein A/G-agarose for 3 h. Agarose-bound proteins were separated by SDS-PAGE, electro-transferred, and probed with anti-caspase-1 antibody. Caspase-1 Activity Assays—Transfected or non-transfected THP-1 cells were either left untreated or were treated with LPS (20 ng/ml) and then lysed by incubating with 25 μl of lysis buffer (25 mm HEPES, pH 7.5, 2 mm EDTA, 0.1% (w/v) CHAPS, 5 mm DTT, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 20 μg/ml leupeptin, 1 mm pefabloc) on ice for 15 min. The supernatant was kept frozen at–70 °C until used. Lysates (20 μg of protein) were taken in 100 μl of assay buffer containing 50 mm HEPES, pH 7.5, 100 mm NaCl, 10 mm DTT, 10% (v/v) glycerol, 0.1% CHAPS, and 200 μm caspase-1 substrate Ac-YVAD-AFC. Fluorescence of the released AFC was measured using excitation and emission wave-lengths of 400 nm and 505 nm, respectively. Phosphorylation of Caspase-1 in Vitro—PAK1 was immunoprecipitated from lysates of cells challenged with H. pylori LPS by incubating with PAK1 antibody overnight followed by incubation with protein A/G-agarose for another 3 h. The immunoprecipitate was washed twice in kinase buffer (300 mm Tris, 1 m KCl, 1 mm CaCl2,60mm MgCl2,1mm Na3VO4, 10 mm DTT, pH 7.5) and incubated with 5 μg of purified His-tagged caspase-1 as substrate in 20 μl of kinase buffer containing 1 μCi of [γ32P]ATP and 7.5 μm cold ATP for 15 min. The reaction was terminated by the addition of 5× Laemmli buffer and phosphorylated caspase-1 was detected by autoradiography of proteins or by Western blotting using phosphoserine-specific antibody. RNA Isolation and Reverse Transcription (RT)-PCR—Total RNA was prepared from cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. 100 ng of RNA was reverse-transcribed using the Titan 1-tube RT-PCR kit (Roche Applied Sciences). The sense primer 5′-AAA CAG ATG GCT TAT TAC AGT-3′, and the antisense primer 5′-TGG AGA ACA CCA CTT GTT GCT CCA-3′, were used to amplify 391 bp of IL-1β mRNA. Glyceraldehyde-3-phosphate dehydrogenase was amplified using the following primers: sense, 5′-CCA TCA ATG ACC CCT TCA TTG ACC-3′; and antisense, 5′-GAA GGC CAT GCC AGT GAG CTT CC-3′ to generate a 604-bp product. The PCR conditions for IL-1β mRNA were: denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min, extension at 68 °C for 1 min for 35 cycles. Electrophoretic Mobility Shift Assay—To prepare nuclear extracts, cells after treatment were washed twice with ice-cold phosphate-buffered saline and scraped in ice-cold TNE buffer (40 mm Tris-HCl, pH 7.5, 0.1 m NaCl, 1 mm EDTA), pelleted by centrifugation at 600 × g for 1 min, resuspended in 400 μl of lysis buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 0.5 mm pefabloc, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 0.5 mg/ml benzamidine) and lysed by the addition of 12.5 μl of 10% (v/v) Nonidet P-40. The nuclear pellet was suspended in 25 μl of ice-cold extraction buffer (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm pefabloc, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 0.5 mg/ml benzamidine). The supernatant (nuclear extract) was immediately frozen and stored at–70 °C. Typically, 15 μg of nuclear extract was incubated on ice for 5 min in a volume of 20 μl containing 4 μl of 5× binding buffer (125 mm HEPES, pH 8, 2.5 mm EDTA, 2.5 mm DTT, 5% Nonidet P-40, 20 mm NaCl, 20% (v/v) glycerol) and 0.75 μg of poly (dI-dC) as nonspecific competitor DNA. 32P-end-labeled double-stranded oligonucleotides (80,000 counts per minute per reaction) containing the IL-1β gene-specific C/EBP-binding site (5′-TTTTGAAAG-3′) or the mutated oligonucleotide with the mutated sites underlined (5′-GTTTTAAGG-3); IL-1β-specific Spi1 site (5′-TACTTCTGCTTT-3′) or the mutated oligonucleotide with the mutated sites underlined (5′-TACTTAGGCTTT-3′); IL-1β gene-specific NFκB site (5′-GGGAAAATCC-3′) or the mutated oligonucleotide with mutated sites underlined (5′TCTAGAATCC-3′), was added and incubated for 15 min at 25 °C. The DNA-protein complex that formed was separated from the oligonucleotide on a 4% native polyacrylamide gel, which was dried and analyzed by autoradiography. For supershift assays nuclear extracts were incubated for 1 h at" @default.
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• W2041012906 title "NF-κB- and C/EBPβ-driven Interleukin-1β Gene Expression and PAK1-mediated Caspase-1 Activation Play Essential Roles in Interleukin-1β Release from Helicobacter pylori Lipopolysaccharide-stimulated Macrophages" @default.
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