Main

Helicobacter pylori (H. pylori) infection is an important risk factor for chronic gastritis, peptic ulcer, and gastric carcinoma.1, 2 Since H. pylori exhibits chemotactic activity for neutrophils, one of the potential toxic factors involving H. pylori-induced gastric injury is oxygen radicals released from activated neutrophils.3 However, our previous study showed that H. pylori-induced oxygen radicals in gastric epithelial cells in the absence of inflammatory cells.4, 5 These results demonstrate the possibility that H. pylori directly turns on transcription levels of inflammatory genes in gastric epithelial cells prior to the recruitment of inflammatory cells.

Chemoattractant cytokines (chemokines) form a superfamily of closely related secreted proteins that specialize in mobilizing leukocytes to areas of immune challenge.6 These inducible proinflammatory peptides potently stimulate leukocyte migration along a chemotactic gradient. They also modulate leukocyte adhesion, activate signal transduction cascades leading to novel gene expression programs, and mediate other leukocyte function necessary for leukocytes to leave the circulation and infiltrate tissues. Chemokines are divided into groups that are defined by characteristic cysteine motifs. Four families of chemokines, such as CXC, CC, C and CX3C (C is a conserved cysteine residue and X is any other amino acid), have been described.7 Among these chemokines, interleukin-8 (IL-8), a prototype CXC chemokine, seems to play an important role in recruiting and activating neutrophils in the gastric mucosa.8 Several reports suggest that gastric epithelial cells represent an important source of IL-8.8, 9 In addition to chemotactic potential, IL-8 is capable of activating polymorphonuclear leukocyte degranulation, the respiratory burst, and the 5-lipoxygenase pathway.6 Thus, IL-8 may be a component of the inflammatory cascade. Indeed, gastric mucosal levels of IL-8 correlates with histological severity in patients with H. pylori-induced gastritis.8 Prolonged IL-8 production by gastric epithelial cells during H. pylori infection could result in the recruitment of leukocytes to infected tissues and therefore may be important in the regulation of inflammatory and immune processes in response to H. pylori.10 Since chronic H. pylori-associated gastritis is accompanied by monocyte and lymphocyte infiltration, in addition to neutrophil infiltration, we were interested in determining whether members of the CC subfamily of chemokines, which recruit monocytes ad lymphocytes, participated in H. pylori-associated pathogenesis. Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that stimulates mononuclear leukocytes. Like IL-8 induction, the in vivo expression of MCP-1 is elevated in the gastric mucosa following H. pylori infection.11 H. pylori infection of gastric epithelial cell lines also stimulates MCP-1 expression.11 Expression of the inflammatory gene such as IL-8 and MCP-1 is primarily controlled at the transcriptional level. Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are inducible transcription factors and their binding sites are found in the promoter region of both IL-8 and MCP-1 genes.12 Although the importance of these transcription factors in chemokine production is apparent, it remains unclear as to how H. pylori could activate these transcription factors and induce chemokine expression. Since these transcription factors are thought to be activated by oxygen radicals,13 oxygen radicals generated by H. pylori may trigger the activation of these transcription factors in gastric epithelial cells.

NF-κB is a member of the Rel family including p50 (NF-κB1), p52(NF-κB2), Rel A (p65), c-Rel, Rel B, and Drosophila morphogen dorsal gene product.14 Its activity is controlled by its cytoplasmic inhibitory protein IκB-α.15 Previously, we found that H. pylori increased lipid peroxidation, an indication of oxidative damage, and induced the activation of two species of NF-κB dimers (a p50/p65 heterodimer and a p50 homodimer) in gastric epithelial cells.4, 5 Pyrrolidine dithiocarbamate (PDTC), a proven free radical scavenger and NF-κB inhibitor, potentially inhibits NF-κB interaction with its upstream regulatory binding site thereby preventing NF-κB-mediated transcriptional activation in H. pylori-infected gastric epithelial cells.16 AP-1 is composed of homodimers or heterodimers of members of Fos and Jun families, which binds to the TRE (TPA responsive element) motif to mediate gene transcription. In relation to H. pylori infection, transcription of IL-8 gene was reported to require the activation of NF-κB and, to a lesser extent, AP-1 in human gastric epithelial MKN 45 cells,17 Kato III cells,18 and AGS cells.19 H. pylori-induced MCP-1 expression is mediated by NF-κB in gastric epithelial cells.20, 21 H. pylori strongly induced AP-1 DNA binding and selectively activated the mitogen-activated protein kinase (MAPK) cascade in gastric epithelial cells.22 Therefore, chemokine gene transcription may require the activation of the combination of NF-κB and AP-1, or that of NF-κB and CCAAT/enhancer binding protein (C/EBP). This may depend on the types of cell or stimuli since there are binding sites for NF-κB, AP-1, and C/EBP in the promoter regions of IL-8 and MCP-1 genes.

Three major subfamilies of MAPKs have been identified in mammalian cells: the extracellular signal-regulated kinases (ERKs), the c-Jun NH2-terminal protein kinases (JNKs) or stress-activated protein kinases (SAPKs), and the p38 MAPKs.23 AP-1 activity is regulated by all three MAPK pathways.24 Transcriptions of c-fos25 and c-jun26 are upregulated by activated MAPK. Post-transcriptionally, c-Jun activity is potentiated through phosphorylation of the transcriptional activator domain by SAPK/JNK.27 H. pylori activated AP-1 through ERK signaling22, 28 and JNK signaling29 in gastric epithelial cells. A variety of extracellular stimuli induce ras activation, resulting in activation of the ras/raf/ERK kinase/MAPK cascade.30 The Ras–Raf pathway increases the transcriptional activity of transcription factor such as c-Jun, an important component of AP-1.31 Ras dominant-negative gene expression almost blocked p38 phosphorylation in smooth muscle cells.32 These studies suggest that Ras is the upstream activator for MAPK, and thus may be related to AP-1 activation.

Molecular genetic analysis of H. pylori has shown that approximately 50–60% of strains have a 40 kb DNA segment called the cytotoxin-associated gene (cagA) pathogenecity island (PAI).33 Some of the proteins encoded by cagA PAI genes are responsible for oxidant-sensitive transcription factor NF-κB and MAPK activation in gastric epithelial cells.34 Infection by cagA strain is more likely to result in peptic ulceration, atrophic gastritis, and gastric carcinoma.35, 36 H. pylori isolates show a high degree of genetic variability.1, 37 These genetic differences may play a role in the clinical outcome of the infection, particularly H. pylori-virulence-associated genes such as vacA, cagA, and iceA genes.38, 39 The predominant genotype of H. pylori in Korea has been reported as cagA-positive and vacA-positive genotype.40, 41

We conducted the present study to evaluate whether cagA+,vacA+H. pylori strain from a Korean isolate induces the expression of chemokines (IL-8, MCP-1), which is mediated by MAPK activation (ERK, JNK, p38) and oxidant-sensitive transcription factors, NF-κB and AP-1. We also assessed whether H. pylori-induced chemokine expression is inhbited by transfection with mutant genes for Ras, c-Jun, and IκBα genes to inhibit Ras, AP-1, and NF-κB activation, respectively, and treatment of specific inhibitors for ERK (U0126) and p38 (SB203580) in gastric epithelial AGS cells. Prior to the experiment, virulence factors (cagA, vacA, iceA) of H. pylori from a Korean isolate were characterized by polymerase chain reaction (PCR) analysis.

Materials and methods

Bacterial Strain and Culture Condition

An H. pylori strain (HP99) was isolated from gastric antral mucosa obtained from a Korean patient with duodenal ulcer at Seoul National University. HP99 was identified as cagA+, vacA+ positive strain.42, 43 HP99 was kindly provided by Dr HC Jung (Seoul National University College of Medicine, Seoul, Korea). These bacteria were inoculated onto chocolate agar plates (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) at 37°C under microaerophilic conditions using an anaerobic chamber (BBL Campy Pouch® System, Becton Dickinson Microbiology Systems).

Determination of Virulence Factors of H. pylori (HP99)

H. pylori genomic DNA was extracted with the QIAGEN Genomic DNA purification kit (QIAGEN, Inc., Valencia, CA, USA) according to the manufacturer's instruction. PCR was performed using specific primers for cagA, vacA, and iceA genes because these genes have been reported to be virulence factors of H. pylori.38, 39 Bacterial genomic DNA was amplified in a 50 μl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 2 μl of genomic DNA, 2.5 U of Taq DNA polymerase, and 25 pmol of specific primer sets described below. PCR amplification was performed for 50 cycles, with one cycle consisting of 45 s at 95°C, 1 min at 55°C, and 1 min 30 s at 72°C. The final cycle included a 5 min extension step to ensure full extension of the PCR products. For detection of the cagA gene, primers CAGAF and CAGAR which yield a fragment of 349 bp from the middle conservative region of the cagA gene, were used).41 For analysis of the vacA s region, primers VA1-F, VA1-R were used as described by Atherton et al44, 45 Primers VA1-F and VA1-R yielded a fragment of 259 bp for s1 variants. Each isolate was typed s1a or s1b or s1c by performing PCR using primers S1A-F—VA1-R, SS3-F—VA1-R, and S1C-F—VA1-R, repectively. For analysis of the vacA m region, primers VA3-F—VA3-R, and VA4-F—VA4-R yielded a fragment of 290 bp for m1 variants and a fragment of 352 bp for m2 variants.44 For analysis of the iceA genotype, primers iceA1F, iceA1R, iceA2F, iceA2R were used as previously described.46, 47 Primers iceA1F and iceA1R yielded a fragment of 247 bp for the iceA1 allele, and primers iceA2F and iceA2R yielded a fragment of 229 or 334 bp depending on the presence of repeated sequences of 105 nucleotides.

Cell Line and H. pylori Infection

Human gastric cancer AGS cells (gastric adenocarcinoma, ATCC CRL 1739) were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium (pH 7.4; Sigma, St Louis, MO, USA), supplemented with 10% fetal bovine serum, 4 mM glutamine (GIBCO-BRL, Grand Island, NY, USA), antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). The cells were seeded in 12-well cell culture plates at 105 cells per well in a volume of 1 ml and cultured to reach 80% confluency. Prior to stimulation, each well was washed twice with 1 ml of fresh cell culture medium containing no antibiotics. Bacterial cells were harvested, washed with phosphate-buffered saline (PBS), and then resuspended in antibiotic-free cell culture medium. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500:1 in a 1 ml volume. A ratio of bacterium/cell was adapted from previous studies.4, 5, 48 Supernatants were collected for the determination of IL-8 and MCP-1 at the indicated time points. The cells were homogenized and RNA was extracted for the determination of IL-8 and MCP-1 mRNA expression. Cell lysates were analyzed for control and phospho-specific ERK1/2, JNK1/2, and p38 level by Western blot analysis and nuclear extracts were used for the levels of NF-κB, AP-1, and C/EBP by electrophoretic mobility shift assay (EMSA).

Experimental Protocol

To determine whether HP99 induces activation of transcription factors (NF-κB, AP-1, C/EBP) and MAPK (ERK1/2, JNK2/1, p38), and the expression of chemokines (IL-8, MCP-1) in AGS cells, AGS cells were cultured in the presence of HP99 for 12 h. To determine the involvement of Ras, AP-1, and NF-κB on chemokine expression, AGS cells were treated with MAPK inhibitors and transfected with mutant genes for Ras, c-Jun, and IκBα and cultured in the absence and presence of HP99 for 12 h. mRNA and protein expression of chemokines were determined by RT-PCR analysis and enzyme-linked immunosorbent assay (ELISA) (at 12 hour). The activation of transcription factors was assessed by electrophoretic mobility shift assay (EMSA) using nuclear extracts of the cells (at 1 h). MAPK activation was evaluated by Western blot analysis at the indicated time points (up to 180 min). The MAP kinase inhibitors, U0126 (an ERK inhibitor, Catalog # 9903, Cell Signaling Technology, Inc., Beverly, MA, USA) and SB203580 (a p38 inhibitor, Catalog # 559389, Calbiochem Biochemicals, San Diego, CA, USA), were purchased and dissolved in dimethylsulfoxide at 50 mM of stock solution. The inhibitors, at 20 μM final concentration, were pretreated to the culture medium 1 h before the treatment of H. pylori.

Transfection with Mutant Genes for Ras, c-Jun, and IκBκ

A mutated IκBα gene, MAD-3 double point mutant (substitution of two serine residues at positions 32 and 36 by alanine residues) construct was prepared as described previously.49 By transfection of MAD-3 into the cells, IκBα could not be phosphorylated, which inhibits NF-κB activation. A dominant-negative mutant of C-Jun, called TAM-67, is a potent inhibitor of AP-1-mediated transactivation.50 A dominant-negative mutant lacking the transactivation domain of c-Jun can dimerize with c-Jun or c-Fos family members, and can bind DNA. Thus, such a mutant could inhibit the function of wild-type c-Jun and c-Fos through either a blocking or quenching mechanism.51 TAM-67 was a kind gift from Dr Andreas von Knethen (University of Erlangen, Erlangen, Germany). Transfection of a dominant-negative mutant of ras, called ras N-17, into the cells interferes with ras function by the expression of a dominant inhibitory mutation in c-Ha-ras. This mutation changes serine-17 to arginine-17 in the gene product and thus inhibits ras activity.52 Ras N-17 was kindly provided by Dr SY Song (Yonsei University College of Medicine, Seoul, Korea). The control vector pcDNA3 (Invitrogen Corp., Carlsbad, CA, USA) was transfected to the cells instead of mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3). These cells were considered as a relative control and named pcN-3.

Subconfluent AGS cells, plated in a 10 cm culture plate (6 × 105 cells per plate), were transfected with each 10 μg of expression construct using DOTAP (N-[1-(2,3-dioleoyloxy) propyl]-N,N,N trimethyl ammonium methylsulfate) (Boehringer-Mannheim, Pentzberg, Germany) for 16 h. After transfection, the cells were trypsinized and plated at 1 × 105cells per 12-well plate. The cells were cultured in the presence or absence of H. pylori. Electrophoretic mobility shift assays for NF-κB and AP-1 were performed at 1 h-culture (Figure 6) while the levels of mRNA and protein for chemokines (Figure 5) were determined at 12 h culture (Figure 5) in the cells transfected with control vector (pcN-3) or mutant genes (ras N-17, TAM-67, MAD-3). AGS cells without transfection were cultured in the presence of H. pylori (control) or absence of H. pylori (none).

Figure 6
figure 6

Activation of NF-κB and AP-1 in AGS cells transfected with or without mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3). AGS cells transfected with or without mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3) were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1 for 1 h. The cells were harvested and nuclear extracts were subjected to electrophoretic mobility shift assay (EMSA) for the activation of NF-κB, AP-1, and C/EBP. The control vector pcDNA3 was transfected to the cells (pcN-3) instead of mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3). These cells were considered as a relative control. pcN-3, the cells transfected control vector (pcDNA) and cultured in the presence of H. pylori; none, AGS cells without transfection and cultured in the absence of H. pylori; control, AGS cells without transfection and cultured in the presence of H. pylori.

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Figure 5
figure 5

Chemokine mRNA and protein expression in AGS cells transfected with or without mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3). AGS cells transfected with or without mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3) were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1 for 12 h. The control vector pcDNA3 was transfected to the cells (pcN-3) instead of mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3). These cells were considered as a relative control. pcN-3, the cells transfected control vector (pcDNA) and cultured in the presence of H. pylori; none, AGS cells without transfection and cultured in the absence of H. pylori; Control, AGS cells without transfection and cultured in the presence of H. pylori. Each bar represents means±s.e. of four separate experiments. *P<0.05 vs none; +P<0.05 vs pcN-3.

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Western Blot Analysis for ERK, JNK, and p38

The AGS cells were cultured in the presence of H. pylori for 180 min (Figure 4). At an indicated time point, the cells were trypsinized, washed, and homogenized in Tris-HCl (pH 7.4) buffer containing 0.5% Triton X-100 and protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN, USA) for determination of control and phospho-specific ERK1/2 (p44/p42), JNK 2/1 (p54/p46), and p38. The protein concentration of each sample was determined by Bradford assay (Bio-Rad laboratories, Hercules, CA, USA). Cellular protein (100 μg) was loaded per lane, separated by 10% SDS-polyacrylamide gel electrophoresis under reducing conditions, and transferred onto nitrocellulose membranes (Amersham Inc., Arlington Heights, IL, USA) by electroblotting. The transfer of protein and equality of loading in all lanes was verified using reversible staining with Ponceau S. The membranes were blocked using 5% nonfat dry milk in TBS-T (Tris-buffered saline and 0.15% Tween 20) for 3 h at room temperature. The proteins were detected with polyclonal antibodies for pan-ERK1/2 (p44/p42) (Catalog # 9102), phospho- ERK1/2 (Catalog # 9101), pan-JNK 2/1 (p54/p46) (Catalog # 9252), phospho- JNK 2/1 (p54/p46) (Catalog # 9251), pan-p38 (Catalog # 9212), and phospho-p38 (Catalog # 9211) at 1:2000 dilation (all from Cell Signaling Technology, Inc., Bevery, MA, USA) diluted in TBS-T containing 5% dry milk, and incubated at 4oC overnight. After washing in TBS-T, the immunoreactive proteins were visualized using goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase, which was followed by enhanced chemiluminescence (Amersham). Exactly equal amount of protein, determined by Bradford assay (Bio-Rad laboratories), was loaded in each lane. The Western blot result presented in each figure is representative of five separate experiments.

Figure 4
figure 4

Time-dependent activation of MAPK by H. pylori in AGS cells. The cells were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1. Cell lysates were analyzed for control (b) and phospho-specific (a) ERK1/2, JNK2/1, and p38 level by Western blot analysis at the indicated time points. For the detection of control MAPKs, antibodies to pan-ERK1/2, pan-JNK2/1, and pan-p38 were used. Antibodies to p-ERK1/2, p-JNK2/1, and p-p38 were used for the determination of phospho-specific levels of ERK1/2, JNK2/1, and p38. MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal protein kinases.

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Extraction of Nuclei

The cells treated with inhibitors for ERK (U0126) and p38 (SB203580) or transfected with mutant genes (ras N-17, TAM-67, MAD-3) or control vector (pcN-3) were cultured in the presence or absence of H. pylori for 1 h. The cells were rinsed with ice-cold PBS, harvested by scraping into PBS, and pelleted by centrifugation at 1500 g for 5 min. The cells were lysed in buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM MgCl2, 0.2% Nonidet P-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonylfluoride (PMSF). The nuclear pellet was resuspended on ice in nuclear extraction buffer containing 20 mM HEPES, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF53 and the nuclear protein concentration was determined by Bradford assay (Bio-Rad laboratories). AGS cells without transfection were cultured in the presence of H. pylori (control) or absence of H. pylori (none) in Figures 6 and 8. For time-course experiment, AGS cells were cultured in the presence of H. pylori for 4 hours (Figure 3).

Figure 8
figure 8

Activation of NF-κB and AP-1 in AGS cells treated with MAPK inhibitors. AGS cells were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The MAP kinase inhibitors, U0126 (an ERK inhibitor) and SB203580 (a p38 inhibitor) (at 20 μM final concentration), were pretreated to the culture medium 1 h before the treatment of H. pylori. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1 for 12 h. None, AGS cells without transfection and cultured in the absence of H. pylori; control, AGS cells without transfection and cultured in the presence of H. pylori.

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Figure 3
figure 3

Time course of H. pylori-dependent activation of transcription factors in AGS cells. The cells were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1. The cells were harvested and nuclear extracts were subjected to electrophoretic mobility shift assay (EMSA) for the activation of NF-κB, AP-1, and C/EBP at the indicated time points. C/EBP, CCAAT/enhancer binding protein.

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Electrophoretic Mobility Shift Assay

NF-κB gel shift oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′), AP-1 gel-shift oligonucleotide (5′-CGCTTGAT AGTCAGCCGGAA-3′), or C/EBP (CCAAT/enhancer binding protein) gel-shift oligonucleotide (5′-GTACACATTGCACAATCTTA-3′) (all from Promega Corp, Madison, WI, USA) was labeled with [32P]dATP (Amersham) using T4 polynucleotide kinase (GIBCO-BRL). End-labeled probe was purified from unincorporated [32P]dATP using a Bio-Rad purification column (Bio-Rad Laboratories, Hercules, CA, USA) and recovered in Tris-EDTA buffer (TE). Nuclear extracts (5 μg) were preincubated in buffer containing 12% glycerol; 12 mM Hepes, pH 7.9; 4 mM Tris-HCl, pH 7.9; 1 mM EDTA; 1 mM DTT; 25 mM KCl; 5 mM MgCl2; 0.04 μg/ml poly[d(I-C)](Boehringer Mannheim, Indianapolis, IN, USA); 0.4 mM PMSF; and TE. The labeled probe was added and samples were incubated on ice for 10 min. The samples were subjected to electrophoretic separation at room temperature on a nondenaturing 5% acrylamide gel at 30 mA using 0.5 × Tris borate EDTA buffer. The gels were dried at 80°C for 1 h and exposed to a radiography film for 6–18 h at −70°C with intensifying screens.53

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis for Chemokines

Gene expressions of IL-8 and MCP-1 mRNA were assessed using RT-PCR standardized by coamplifying this gene with the housekeeping gene β-actin, which served as an internal control. The cells, treated with inhibitors for ERK (U0126) and p38 (SB203580) or transfected with mutant genes (ras N-17, TAM-67, MAD-3) or control vector (pcN-3), were cultured in the presence or absence of H. pylori for 12 h. Total RNA was isolated from the cells by guanidine thiocyanate extraction method. Total RNA was reverse transcribed into cDNA and used for PCR with human specific primers for IL-8, MCP-1, and β-actin. Sequences of IL-8 primers were 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ (forward primer) and 5′-TCTCAGCCCTCTTCAAAAACTTCT-3′ (reverse primer), giving a 297 bp PCR product. Sequences of MCP-1 primers were 5′-AAGCTGTGATCTTCAAGACCATTG-3′ (forward primer), 5′-TTAAGGCATAATGTTTCACATCAACAA-3′ (reverse primer) giving a 269 bp PCR product. For β-actin, the forward primer was 5′-ACCAACTGGGACGACATGGAG-3′ and the reverse primer was 5′-GTGAGGATCTTCATGAGGTAGTC-3′, giving a 349 bp PCR product. Briefly, the PCR was amplified by 25–30 repeat denaturation cycles at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. During the first cycle, the 95°C step extended to 2 min, and on the final cycle the 72°C step extended to 5 min. PCR products were separated on 1.5% agarose gels containing 0.5 μg/ml ethidium bromide and visualized by UV transillumination. AGS cells without transfection were cultured in the presence of H. pylori (control) or absence of H. pylori (none) in Figures 5 and 7. For time-course experiment, AGS cells were cultured in the presence of H. pylori for 12 h (Figure 2).

Figure 7
figure 7

Chemokine mRNA and protein expression in AGS cells treated with MAPK inhibitors. AGS cells were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The MAP kinase inhibitors, U0126 (an ERK inhibitor) and SB203580 (a p38 inhibitor) (at 20 μM final concentration), were pretreated to the culture medium 1 h before the treatment of H. pylori. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1 for 12 h. None, AGS cells without transfection and cultured in the absence of H. pylori; control, AGS cells without transfection and cultured in the presence of H. pylori. Each bar represents means±s.e. of four separate experiments. *P<0.05 vs none; +P<0.05 control.

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Figure 2
figure 2

Time-dependent induction of chemokine mRNA (a) and protein (b) by H. pylori in AGS cells. The cells were seeded in 12-well culture plates at 105 cells per well and cultured to reach 80% confluency. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500 : 1. The cells were homogenized and RNA was extracted for the determination of IL-8 and MCP-1 mRNA expression by RT-PCR. Supernatants were collected for the determination of IL-8 and MCP-1 at the indicated time points. Protein level in the medium was determined by ELISA.The concentration and loading of RNA in each lane was standardized by hybridization with cDNA probe for the constitutively expressed β-actin (a). Protein level in the medium was expressed as means±s.e. of four separate experiments (b). *P<0.05 vs value at corresponding 0 h.

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Determination of IL-8 and MCP-1

The cells treated with inhibitors for ERK (U0126) and p38 (SB203580) or transfected with mutant genes (ras N-17, TAM-67, MAD-3) or control vector (pcN-3) were plated at 105 cells/well in a 12 well culture plate and cultured in the presence or absence of H. pylori for 12 h. The levels of IL-8 and MCP-1 were determined in the medium by enzyme-linked immunosorbent assay (ELISA) kits (R&D System, Minneapolis, MN, USA). AGS cells without transfection were cultured in the presence of H. pylori (control) or absence of H. pylori (none) in Figures 5 and 7. For time-course experiment, AGS cells were cultured in the presence of H. pylori for 12 h (Figure 2).

Statistical Analysis

The results are expressed as means±s.d. of four separate experiments. Analysis of variance (ANOVA) followed by Newman–Keul's test was used for statistical analysis. P<0.05 was considered to be statistically significant.

Results

Virulence factors in H. pylori (HP99)

An H. pylori strain (HP99) was isolated from gastric antral mucosa obtained from a Korean patient with duodenal ulcer. HP99 was previously identified as cagA+, vacA+ positive strain.4, 5 VacA, encoding the vacuolating toxin, is present in all strains,45, 54 and comprises two regions of allelic variation.44 The s-region, located at the 5′ end of the gene, and the m-region, located in the middle of vacA.46, 47 The production of the cytotoxin is related to the mosaic combination of s- and m-region alleles in vacA. The cytotoxin-associated gene (cagA) is expressed by the majority of the cytotoxin (vacA)-positive isolates.55 Another virulence factor iceA (induced by contact with epithelium) locus comprises two main variants, iceA1 and iceA2.56 Even though the function of these variants is not yet clear, the combination for the virulence factors and their allelic variants may affect clinical outcome associated with H. pylori infection. H. pylori in a Korean isolate (HP99) used in the present study is identified as cagA+, vacA s1b, m2, iceA1 H. pylori strain, determined by PCR analysis for genomic DNA isolated from HP99 (Figure 1).

Figure 1
figure 1

Determination of virulence factors in H. pylori (HP99). H. pylori genomic DNA was extracted and PCR was performed using specific primers for cagA, vacA, and iceA genes. Bacterial genomic DNA was amplified in a 50 μl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 2 μl of genomic DNA, 2.5 U of Taq DNA polymerase, and 25 pmol of specific primer sets for cagA, vacA (s1a, s1b, s1c, m1, m2), and iceA1 and iceA2 genes. H. pylori in Korean isolates (HP99) is identified as cagA+, vacA s1b, m2, iceA1 H. pylori strain.

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Time-dependent Expression of IL-8 and MCP-1 by H. pylori

mRNA expression (Figure 2a) and protein level (Figure 2b) of IL-8 and MCP-1 were determined in H. pylori-treated AGS cells. RT-PCR analysis shows that IL-8 and MCP-1 mRNA expression were observed at 4 h and 8, respectively, and increased at 12 h incubation (Figure 2a). As shown in Figure 2b, IL-8 and MCP-1 protein increased in a time-dependent manner up to 24 h. IL-8 levels in medium (pg/ml) released from the cells at the start of experiment was 5.8±0.4, which increased by H. pylori to 15.1±1.1, 24.6±4.7, and 51.2±6.3 at culture time of 4, 8, and 12 h, respectively. MCP-1 levels in medium (pg/ml) released from the cells at the start of experiment was 0.6±0.2, which increased by H. pylori to 9.5±0.3, 14.1±0.7, and 44.5±1.9 at culture times of 4, 8 and 12 h, respectively. Thus, for the following experiments using transfection with mutant genes and treatment with MAPK inhibitors, the time point of 12 h, at which chemokine mRNA and protein expression were evident, was used. β-Actin was constitutively expressed in AGS cells and not changed with incubation time.

H. pylori-induced Activation of NF-κB and AP-1

The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500:1. The cells were harvested and nuclear extracts were subjected to EMSA for the activation of NF-κB, AP-1, and C/EBP at the indicated time points. Figure 3 shows autoradiographs from EMSA for NF-κB, AP-1, and C/EBP in AGS cells cultured in the presence of H. pylori for 4 h. H. pylori resulted in the activation of NF-κB and AP-1 while C/EBP was not affected. NF-κB activation was observed at 1 h culture with maximum activation, which declined at 4 h. An increased amount of activated AP-1 was detected at 1 h and even higher level of activated AP-1 was observed at 4 h. Since C/EBP was not changed by H. pylori infection, NF-κB and AP-1 may contribute to the expression of IL-8 and MCP-1 by H. pylori in AGS cells. For the following experiment on transfection with mutant genes and treatment with MAPK inhibitors, 1 h culture was used for the activation of NF-κB and AP-1 by H. pylori.

Time-dependent Activation of ERK, JNK and p38 by H. pylori

After addition of bacterial cells to the cultured cells, cell lysates were analyzed for control (Figure 4b) and phospho-specific (Figure 4a) ERK1/2, JNK2/1, and p38 levels by Western blot analysis at the indicated time points. H. pylori significantly increased in MAPK phosphorylation. Kinetic analysis indicates that an increase in phospho-specific ERK1/2 was observed at 15 min, with maximum induction achieved 30 min after H. pylori infection and declining thereafter. JNK2/1 phosphorylation was first detected at 15 min with the maximum level at 30 min and continuously detected until 180 min after H. pylori infection. p38 phosphorylation was also observed at 15 min after H. pylori infection and continued up to 180 min. As shown in Figure 4b, ccntrol MAPKs, determined using antibodies to pan-ERK1/2, pan-JNK2/1, and pan-p38, were not changed by H. pylori in AGS cells.

Chemokine mRNA and Protein Expression in AGS Cells Transfected with or without Mutant Genes

AGS cells transfected with or without mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3) were cultured in the presence of H. pylori for 12 h. The control vector pcDNA3 was transfected to the cells (pcN-3) instead of mutant genes, which was considered as a relative control. RT-PCR showed that H. pylori-induced mRNA expression of IL-8 and MCP-1 in control cells without transfection and pcN-3 cells. No cells represent AGS cells without transfection and cultured in the absence of H. pylori. H. pylori- induced IL-8 mRNA expression was not observed in the cells transfected with mutant genes (ras N-17, TAM-67, MAD-3). MCP-1 mRNA expression by H. pylori was not detected in the cells transfected with ras N-17 and TAM-67, but slightly detected in the cells transfected with MAD-3, even though the level was low (Figure 5a). Similar phenomena were shown in chemokine protein expression in the cells transfected with or without mutant genes (ras N-17, TAM-67, MAD-3), determined by ELISA (Figure 5b). Even though MCP-1 protein expression was relatively slightly affected by NF-κB, transcription of IL-8 and MCP-1 may be regulated by ras, AP-1, and NF-κB in H. pylori-infected AGS cells.

Activation of NF-κB and AP-1 in AGS Cells Transfected with or without Mutant Genes

AGS cells treated with or without mutant genes for ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3) were cultured in the presence of H. pylori for 1 h (Figure 6). Nuclear exctracts were subjected to EMSA for the activation of NF-κB and AP-1. The control vector pcDNA3 was transfected to the cells (pcN-3) instead of mutant genes (Figure 6). H. pylori-induced activation of NF-κB and AP-1, shown in control cells without transfection and pcN-3 cells. H. pylori- induced activation of NF-κB and AP-1 was not observed in the cells transfected with mutant genes (ras N-17, TAM-67, MAD-3). The results show that inhibition of AP-1, by transfection with mutant genes for ras (ras N-17) and c-Jun (TAM-67), may inhibit NF-κB as transfection with mutant gene for IκBα (MAD-3) in H. pylori-infected AGS cells. Similarly, inhibition of NF-κB, by transfection with mutant genes for IκBα (MAD-3), may inhibit AP-1 as transfection with mutant genes for ras (ras N-17) and c-Jun (TAM-67), by H. pylori in AGS cells. The possible suggestion is that NF-κB and AP-1 are cross-coupled for the transcription of chemokines, IL-8 and MCP-1, in H. pylori-infected AGS cells.

Chemokine mRNA and Protein Expression in AGS Cells Treated with MAPK Inhibitors

The MAP kinase inhibitors, U0126 (an ERK inhibitor) and SB203580 (a p38 inhibitor) (at 20 μM final concentration), were pretreated to the culture medium 1 h before the treatment of H. pylori. The bacterial cells were added to the cultured cells at a bacterium/cell ratio of 500:1 for 12 h. RT-PCR showed that H. pylori-induced mRNA expression of IL-8 and MCP-1 in control cells. H. pylori-induced mRNA expression of IL-8 and MCP-1 was inhibited by treatment of an ERK inhibitor and a p38 inhibitor (Figure 7a). Treatment of an ERK inhibitor and a p38 inhibitor also inhibited chemokine protein expression, determined by ELISA (Figure 7b). The results suggest the involvement of ERK and p38 in the expression of IL-8 and MCP-1 in H. pylori-infected AGS cells.

Activation of NF-κB and AP-1 in AGS Cells Treated with MAPK Inhibitors

AGS cells were pretreated with the MAP kinase inhibitors, U0126 (an ERK inhibitor) and SB203580 (a p38 inhibitor) (at 20 μM final concentration), to the culture medium 1 h before the treatment of H. pylori at a bacterium/cell ratio of 500:1. After 1 h, nuclear extracts were extracted and subjected to EMSA for the activation of NF-κB and AP-1. H. pylori-induced activation of NF-κB and AP-1, which was shown in control cells cultured in the presence of H. pylori as compared to no cells cultured in the absence of H. pylori. H. pylori-induced activation of NF-κB and AP-1 was inhibited by treatment of an ERK inhibitor and a p38 inhibitor (Figure 8). The results suggest the involvement of MAPK such as ERK and p38 in the activation of NF-κB and AP-1 in H. pylori-infected AGS cells.

Discussion

The main finding of this study is that Ras and MAPK cascade may act as the upstream signaling for the activation of AP-1 and NF-κB, which induce chemokine expression in H. pylori-infected AGS cells. We used an H. pylori strain HP99 that was isolated from gastric antral mucosa obtained from a Korean patient with duodenal ulcer and identified as cagA+, vacA s1b, m2, iceA1. Expressions of IL-8 and MCP-1 were markedly upregulated at the levels of mRNA and protein, in parallel with the activation of NF-κB and AP-1 and increase in phospho-specific ERK1/2, JNK2/1 and p38 by HP99 in AGS cells. Expression levels of IL-8 and MCP-1 in HP99-infected AGS cells, as well as activation of NF-κB and AP-1, were inhibited by transfection with mutant genes for Ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3) or treatment with MAPK inhibitors (U0126 as an ERK inhibitor or SB203580 as a p38 inhibitor). These results suggest that induction of IL-8 and MCP-1 by H. pylori in a Korean isolate may depend on the activation of Ras, MAPK cascade, AP-1, and NF-κB in gastric epithelial cells. In addition, inhibition of NF-κB by transfection with mutant genes for IκBα (MAD-3) was closely related to inhibition on AP-1 activation by transfection with mutant genes for ras (ras N-17) and c-Jun (TAM-67) in H. pylori-infected AGS cells. Both inhibition of NF-κB and AP-1 by transfection with mutant genes (ras N-17, TAM-67, MAD-3) similarly inhibited the expression of IL-8 and MCP-1 in H. pylori-infected AGS cells. The results suggest that NF-κB and AP-1 may be cross-coupled for the transcription of chemokines IL-8 and MCP-1 in H. pylori-infected AGS cells.

The crossactivity between NF-κB and AP-1 activation was reported in human keratinocytes,53 JB6 cells,53 and mammary carcinoma cells.57 The expression of dominant-negative Jun inhibited elevated AP-1 and NF-κB transactivation and reduced anchorage-independent growth in human keratinocytes.53 These results suggest that activation of AP-1 and NF-κB contributes to neoplastic progression of immortalized human keratinocytes and that specific targeting of the elevated levels seen in benign or malignant tumors might be effective for prevention of treatment of human cancer.53 Even though NF-κB and AP-1 represent distinct mammalian transcription factors that target unique DNA enhancer element, the heterodimer of NF-κB (p65/p50) shares structural homology with the c-rel proto-oncogene product. Similarly, the AP-1 transcription factor complex is composed of dimmers of c-fos and c-jun proto-oncogenes products.57 c-Fos and c-Jun are capable of physically interacting with NF-κB p65 through the Rel homology domain. This complex of NF-κB p65 and Jun or Fos exhibits enhanced DNA binding and biological function via both the κB and AP-1 response elements.57 Besides, oxidative events regulate AP-1 and NF-κB transactivation, these oxidative events can be important molecular targets for cancer prevention.23 The induction of IL-8 and MCP-1 occurs at the transcriptional level. The region contains three cis elements important for the induction of IL-8 and MCP-1 gene expression: NF-κB, AP-1, and C/EBP binding sites. IL-8 and MCP-1 gene transcriptions require the activation of the combination of transcription factor NF-κB and AP-1 or that of NF-κB and C/EBP, depending on the types of cell or stimuli).58, 59 The present study showed that C/EBP was not involved in H. pylori-induced IL-8 and MCP-1 expression in AGS cells, which was determined by EMSA.

Keates et al48 reported that direct contact of H. pylori with gastric epithelial cells activates NF-κB in vitro. Both the phosphorylation and proteolytic degradation of IκBα allows the release and nuclear transmigration of NF-κB, which may be induced by oxygen radicals.14, 15 Some evidence shows that H. pylori-induced MCP-1 expression is mediated by NF-κB in gastric epithelial cells.20, 21 Oxygen radicals were produced by the association of H. pylori with gastric epithelial cells. We previously demonstrated that lipid peroxidation, an index of oxidative membrane damage, increased by H. pylori in gastric epithelial AGS and Kato III cells, which was in parallel with a time-course stimulation of IL-8 production.4, 5 More recently, Shimoyama et al60 reported that oxygen radicals are important mediators for chemokine expression in human neutrophils stimulated by H. pylori. Since oxidative stress is an important regulator of chemokine gene expression and an inducer of the NF-κB and AP-1,14, 15, 23 antioxidants might be beneficial for the treatment of H. pylori-induced gastric mucosal injury and inflammation caused by oxidant-mediated chemokine production.

H. pylori-induced expressions of IL-8 and MCP-1 were shown in gastric epithelial cells11, 61 and gastric mucosa tissues of the patients.62 AP-1 has been known as an oxidant-sensitive or antioxidant-responsive factor depending on cell types.63 In H. pylori-induced IL-8 gene transcription in MKN 45 cells17 and KATO III cells,18 the activation of both NF-κB and, to a lesser extent, AP-1 was induced for IL-8 gene transcription. Our previous study19 demonstrated the importance of AP-1 as much as NF-κB for IL-8 expression by H. pylori in AGS cells. We characterized the AP-1 component activated by H. pylori as a heterodimer of c-Fos/c-Jun, which might be induced by oxygen radicals.19 This hypothesis was proven by the inhibitory effect of N-acetylcysteine on the activation of these transcription factors, NF-κB and AP-1, and IL-8 expression in AGS cells. N-acetylcysteine increases the intracellular stores of glutathione in the cells, thereby enhancing endogenous antioxidative defense mechanism.64 IL-8 gene transcription requires the activation of the combination of transcription factor NF-κB and AP-1 or that of NF-κB and C/EBP, depending on the types of cells or stimuli.58, 59 In this study, we show that C/EBP is not involved in H. pylori-induced IL-8 and MCP-1 expression in AGS cells. This is supported by the observation that there is no decrease in the induction of luciferase activity from AGS cells transfected with luciferase expression vector linked to IL-8 gene, but containing mutant C/EBP-binding site as described in our previous study.19

AP-1 activity is regulated by all three MAPK pathways.24 Regulation occurs both at the transcriptional level and at the post-transcriptional level. The expression of c-fos, which is nearly absent in quiescent cells, is controlled at the level of transcription. All three MAPKs, ERK1/2, SAPK/JNK, and p38, can phosphorylate the transcription factor Elk-1, a member of the complex family.65, 66 Elk-1 binds the serum response element motif in the c-fos promoter, thereby inducing c-fos transcription.25 In contrast, c-jun is regulated both transcriptionally and post-transcriptionally. A few c-Jun homodimers pre-exist in resting cells. In addition, c-jun transcription is upregulated by activated MAPK.26 Subsequently, c-Jun can increase its own transcription by binding to the TRE motif in its promoter.67 Novel cFos synthesis leads to the formation of Jun/Fos heterodimers, which have a 10-fold higher DNA binding affinity than Jun/Jun homodimers, resulting in increased AP-1 activity.68 Post-transcriptionally, c-Jun activity is potentiated through phosphorylation of the transcriptional activator domain by SAPK/JNK.27 A variety of extracellular stimuli induce ras activation, resulting in activation of the ras/raf/ERK kinase (MEK)/MAPK cascade.30 The Ras–Raf pathway increases the transcriptional activity of transcription factors, such as c-Jun, and important components of AP-1.31 Ras dominant-negative gene expression almost blocked p38 phosphorylation in smooth muscle cells.32 H. pylori activated AP-1 through ERK signaling22, 28 and JNK signaling29 in gastric epithelial cells. H. pylori- induced IL-8 expression was reported to be mediated by AP-1 through ERK in gastric cancer cells.28, 69 H. pylori cagA induced ras activation and Elk-phosphorylation in gasrtic epithelial cells.70 The present activated AP-1 by H. pylori was inhibited by treatment of the MAP kinase inhibitors, U0126 (an ERK inhibitor) and SB203580 (a p38 inhibitor), and transfection with ras dominant-negative mutant gene (ras N-17) and c-Jun dominant-negative mutant gene (TAM-67). The results clearly show that H. pylori-induced activation of AP-1 involves Ras, MAPK, and c-Jun in AGS cells.

NF-κB activation is tightly regulated by its endogenous inhibitor, IκB, which complexes NF-κB in the cytoplasm. To date, the most extensively studied IκB protein is IκBα (36 kDa) encoded by the human MAD-3 gene or its homologues in different species.71 The mechanisms that lead to the degradation of IκBα proteins are poorly understood, but involve changes in the phosphorylation state of IκBα.15 Two serines in the N-terminal domain of IκBα, serine residues 32 and 36, were shown to be critical for IκBα stability.72 Substitution of these two serine residues by alanine residues rendered IκBα undegradable by cellular activators.72 Among the many proteins exhibiting IκB function, IκBα is the only inhibitor that, in response to cell stimulation, dissociates from the NF-κB complex, with kinetics matching NF-κB translocation to the nucleus.73 It was therefore suggested that the activation of NF-κB is mainly regulated by NF-κB/IκBα dissociation. Such a mutant IκBα has been shown to act as an NF-κB super-repressor.74 A dominant-negative mutant of c-jun, called TAM-67, is a potent inhibitor of AP-1-mediated transactivation.50 A dominant-negative mutant lacking the transactivation domain of c-Jun can dimerize with c-jun or c-fos family members, and can bind DNA. Thus, such a mutant could inhibit the function of wild-type c-Jun and c-Fos through either a blocking or quenching mechanism.51 Transfection of a dominant-negative mutant of ras, called ras N-17, into the cells interferes with ras function by the expression of a dominant inhibitory mutation in c-Ha-ras. This mutation changes serine-17 to arginine-17 in the gene product and thus inhibits ras activity.52

For the virulence factors known in H. pylori strains, vacA, encoding the vacuolating toxin, is present in all strains45, 54 and comprises two regions of allelic variation.44 The s-region, located at the 5′ end of the gene, and the m-region, located in the middle of vacA, has three allelic forms.46, 47 The production of the cytotoxin is related to the mosaic combination of s- and m-region alleles in vacA. The vacA s1/m1 strains have higher cytotoxic activity than s1/m2 strains,44 vacA m1-type strains are associated with greater gastric epithelial damage than m2 strains.45 Even though we did not compare the cytotoxicity between present HP99 (vacA s1b/m2 strain) and other vacA s1/m1 strains, HP99 significantly activated NF-κB and AP-1 and thus induced chemokine expression in AGS cells. The cytotoxin-associated gene (cagA) is expressed by the majority of the cytotoxin (vacA)-positive isolates.55 This gene is a marker for the presence of a multigenic 40 kb region of the genome, called the cag pathogenicity island.55 Infection by cagA+ strains is more likely to result in peptic ulceration,55 atrophic gastritis,35 and gastric carcinoma.36 Meer-ter-Vehn et al22 determined that H. pylori induces AP-1 activity in gastric epithelial cells. H. pylori strongly induced AP-1 DNA binding and selectively activated the ERK/MAPK cascade. The stimulation of ERK led to phosphorylation of the transcription factor Elk-1 and markedly increased c-fos transcription. At the protein level, the expression of c-Fos and phosphorylation of c-Jun was strongly induced. H. pylori strains that did not express cagA did not induce AP-1, MAPK activity, c-fos, or c-jun expression. Another putative virulence gene has recently been described and is designated iceA.56 The iceA locus comprises two main variants, iceA1 and iceA2. The function of these variants is not yet clear. The expression of iceA1 is upregulated by contact of the bacteria with human gastric epithelial cells and in some populations is associated with peptic ulcer disease.47, 56 After examining 94 gastric biopsy specimens from the patients in the Netherlands, van Doorn et al47 reported that the iceA allelic type was independent of the cagA and vacA status, and there was a significant association between the presence of the iceA1 allele and peptic ulcer disease. Figueiredo et al75 demonstrated that vacA s1 and cagA+ H. pylori strains are associated with duodenal ulcer, gastric ulcer, or gastric carcinoma in Portugal. VacA m1 is associated with gastric ulcer or carcinoma, but not with duodenal ulcer. Since HP99 is cagA+, vacA s1b, m2, iceA1 H. pylori strain and induced significant activation of NF-κB and AP-1 and thus, induction of chemokines, IL-8 and MCP-1, in AGS cells, this strain may be cytotoxic to gastric epithelial cells.

The present results demonstrate that the expressions of IL-8 and MCP-1 by H. pylori in a Korean isolate (HP99; cagA+, vacA s1b, m2, iceA1 H. pylori strain) are regulated by the transcription factors NF-κB and AP-1, mediated by Ras and MAPK, in gastric epithelial cells. Further studies on the signaling pathways and mediators induced by H. pylori infection, depending on the virulence factors and H. pylori strains, are necessary to provide better understanding and rational approaches for the control of this inflammation process.