Helicobacter pylori infection activates NF-{kappa}B signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Jung Mogg Kim,1 Joo Sung Kim,2 Hyun Chae Jung,2 Yu-Kyoung Oh,3 Hee-Young Chung,1 Chul-Hoon Lee,4 and In Sung Song2

1Department of Microbiology and Institute of Biomedical Science, Hanyang University College of Medicine, Seoul 133-791; 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul 110-744; 3Department of Microbiology, Pochon CHA University College of Medicine, Kyunggi-do 487-800; and 4Department of Medical Genetics, Hanyang University College of Medicine, Seoul 133-791, Korea

Submitted 25 November 2002 ; accepted in final form 24 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-{kappa}B activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-{kappa}B significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-{kappa}B signaling pathway via NF-{kappa}B-inducing kinase and I{kappa}B kinase-{beta} activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing I{kappa}B{alpha} superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-{kappa}B. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 µM), decreased caspase-3 activity and apoptosis in NF-{kappa}B-suppressed cells infected with H. pylori. These results suggest that NF-{kappa}B activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.

I{kappa}B kinase; NF-{kappa}B-inducing kinase; nitric oxide; superrepressor


THE SINGLE LAYER of epithelial cells that lines the gastric mucosa is the initial site of interaction between the host and Helicobacter pylori. Gastric epithelial cells respond to H. pylori infection by activating NF-{kappa}B (11, 25, 30), upregulating the expression of a proinflammatory gene program, which includes the chemokine IL-8, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) (10, 14, 15), and by finally undergoing apoptosis (13, 41). These relationships suggest that the signals produced by gastric epithelial cells can influence the inflammatory responses and apoptosis of gastric epithelial cells following H. pylori infection.

Many of the genes that are activated in gastrointestinal epithelial cells after bacterial infection are target genes of NF-{kappa}B (8, 23, 30). NF-{kappa}B is a dimeric transcription factor, which is normally held in the cytoplasm in an inactive state by inhibitory proteins, the I{kappa}B kinases (I{kappa}Bs) (37). The stimulation of epithelial cells with several pathogens or inflammatory cytokines can induce I{kappa}B degradation and subsequently cause the NF-{kappa}B complex to migrate to the nucleus and to bind to DNA recognition sites in the regulatory regions of target genes (4, 37).

The activation of NF-{kappa}B requires the phosphorylation of I{kappa}B{alpha} (Ser32 and Ser36) and I{kappa}B{beta} (Ser19 and Ser23) (40). This phosphorylation leads to ubiquitination and the 26S proteosome-mediated degradation of I{kappa}B{alpha}, thereby releasing NF-{kappa}B from the complex to enter the nucleus and activate genes (37). Two cytokine-inducible kinases, IKK{alpha} and IKK{beta}, have been cloned and characterized (2, 27, 44). IKK{alpha} and IKK{beta} phosphorylate I{kappa}B{alpha} at Ser32 and Ser36 in response to proinflammatory cytokines. NF-{kappa}B-inducing kinase (NIK) is a novel member of the MEKK family and was found to activate both IKK{alpha} and IKK{beta} by interacting with adapter proteins associated with receptors for TNF-{alpha} and IL-1 (26). Recently, it was demonstrated that H. pylori could activate NF-{kappa}B via a signaling pathway involving IKK and NIK (25). Given that many cell types become more sensitive to TNF-{alpha} or H. pylori-induced apoptosis after the suppression of NF-{kappa}B activity (36, 39), NF-{kappa}B may function antiapoptotically. In contrast, other studies showed that the suppression of NF-{kappa}B activation inhibited H. pylori-induced apoptosis in gastric epithelial cells (22). These conflicting observations indicate that the role of NF-{kappa}B in H. pylori-induced apoptosis in gastric epithelial cells has not been clarified.

It is not well known why the total amount of the cells undergoing apoptosis is so low in H. pylori-infected gastric epithelial cells. Our previous results indicated that the morphologic and biochemical changes characteristic of apoptosis after H. pylori infection were delayed compared with proinflammatory cytokine expression (18), suggesting that epithelial cells activate antiapoptotic mechanisms in early infection period. In addition, as the infection period was prolonged, NF-{kappa}B signals were decreased and the population of apoptosis was increased. Because NF-{kappa}B activates within 1 h after infection in gastric epithelial cells, there is a possibility that NF-{kappa}B activation in the early infection period may be associated with a low population of apoptosis in H. pylori-infected gastric epithelial cells.

NO is generated by the conversion of L-arginine to L-citrulline by NOS, which exists in three isoforms, namely, iNOS, endothelial (e)NOS, and neuronal (n)NOS, each encoded by a separate gene (28, 29). We previously demonstrated that H. pylori infection regulates iNOS expression in gastric epithelial cells in vitro (14). iNOS/NO affects the apoptosis of gastrointestinal epithelial cells in response to several bacterial infections (12, 22). However, the role of iNOS/NO in apoptosis is both diverse and complex, because NO can be considered as either a proapoptotic or an antiapoptotic molecule (3, 20). In this regard, it is not clear how the NF-{kappa}B signaling pathway and the iNOS expression of gastric epithelial cell apoptosis are related in response to H. pylori infection.

On the basis of the above findings, we hypothesized that the iNOS expression induced by H. pylori infection may be linked to the apoptotic response of human gastric epithelial cells via NF-{kappa}B signaling pathway. To substantiate this hypothesis, we investigated whether the inhibition of NF-{kappa}B signaling pathway may influence apoptosis in H. pylori-infected gastric epithelial cells and whether NO could change the apoptosis of gastric epithelial cells in response to H. pylori infection in an in vitro model.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Bacterial strains. The H. pylori strains used in this study were isolated from human gastric biopsy samples obtained from patients with gastric or duodenal ulcers at Seoul National University Hospital, Seoul, Korea. H. pylori was cultured under microaerophilic conditions (5% O2-10% CO2-85% N2) as previously described (16). The virulence factors of the H. pylori strains, such as the cagA gene and the production of vacuolating cytotoxin, were determined as previously described (16).

Bacterial strains closely related to H. pylori were used for the control experiment, including Campylobacter jejuni, Campylobacter fetus subspecies fetus (12), and nonpathogenic Escherichia coli DH5{alpha} (Promega, Madison, WI). These strains were obtained from a clinical laboratory at Seoul National University Hospital and were identified by standard microbiological techniques.

Cultivation of epithelial cells and infection protocol. The gastric epithelial cell lines were cultured in DMEM (pH 7.4, Sigma, St. Louis, MO) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT) and antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin), as described previously (14). In these experiments, we used the gastric epithelial cell lines MKN-45 (JCRB) and Hs746T (ATCC HTB 135). After the antibiotics were removed, the cells were cultured in six-well tissue culture plates for 24 h. Confluent monolayers of the cells in the six-well plates were incubated with H. pylori at a ratio of 1:150. Uninfected controls for each cell type were included in every experiment performed.

Human gastric mucous cells were isolated and cultured, as previously described (35). Briefly, gastric tissues were obtained from patients undergoing surgical gastrectomy. The surgical specimens were washed twice in serum-free media, pinned down on polymerized Sylgard, and the epithelium was removed by scraping the surface with a glass slide. The scraped tissue pieces were minced using razor blades and then washed three times at 100 g for 3 min in serum-free media. The pellets were then transferred to siliconized 125-ml screw-cap Erlenmeyer flasks containing 20 ml of serum-free culture media with 20 mg/ml of Type I collagenase and 0.1% bovine albumin. The flasks were then gassed with 95% O2-5% CO2, put into a 37°C shaking water bath, and gyrated at 120 oscillations/min for 45 min. At the end of the incubation period, the collagenase-digested mixture was put into a 50-ml syringe with an attached 15-gauge luer-stub adapter and the contents were pushed through a 200-µm nylon mesh screen. The mesh-filtered suspension was washed twice in serum-free media, centrifuged at 100 g for 3 min, then the pellet was resuspended in 15 ml of serum-free culture media, and a 200-µl aliquot was taken for cell counts in a Coulter Counter. The 15-ml suspension was divided into three 5-ml aliquots in 16 x 125-cm Falcon round-bottom tubes and then 5 ml of isosmotic Percoll were added to each tube. The tubes were centrifuged for 15 min at 100 g at 24°C, and the bottom pellet was removed containing the gastric mucous epithelial cells. The pellet was washed three times, centrifuged at 20 g for 3 min in serum-free cell culture media, and then the cells were plated on 24-multiwell plastic dishes (35). Epithelial cell preparations had less than 5% of contaminating B cells and monocytes/macrophages as assessed by flow cytometry using CD19/20 and CD14 as markers.

EMSAs. Cells were harvested, and nuclear extracts were prepared as described previously (17). The concentrations of proteins in the extracts were determined by Bradford assay (Bio-Rad, Hercules, CA). EMSAs were performed according to the manufacturer's instructions (Promega). In brief, 5 µg of nuclear extracts were incubated for 30 min at room temperature with {gamma}[32P]-labeled oligonucleotide probe corresponding to a consensus NF-{kappa}B binding site. After incubation, bound and free DNAs were resolved on 5% native polyacrylamide gels, as described previously (4, 17).

Transfection of plasmids and luciferase assay. An expression vector encoding FLAG-tagged IKK{beta}, in which lysine at position 44 was replaced by alanine (IKK{beta}-AA), was used to block NF-{kappa}B activation, as described before (4, 17). The mutant protein acts as a superrepressor of IKK. The expression vector for NIK catalytic mutant, which has a double replacement of alanine residues to lysine residues at positions 429 and 430 (NIK-AA), was a gift from Dr. Karin of the University of California, San Diego (4). pIL8-luciferase, p2x NF-{kappa}B-luciferase, p{beta}-actin-luciferase, and pRSV-{beta}-galactosidase transcriptional reporters were kindly donated by Dr. Kagnoff of the University of California, San Diego (17). Cells in six-well dishes were transfected with 1.5 µg of plasmid DNA, using Lipofectamine Plus (Gibco-BRL, Life Technologies, Palo Alto, CA), according to the manufacturer's instructions (17). The transfected cells were incubated for 48 h at 37°C in a 5% CO2 incubator. Cells were incubated with H. pylori for the indicated periods. The transfection efficiency was 10-20% into the gastric epithelial cell lines. Luciferase activity was determined and normalized relative to {beta}-galactosidase expression, in accordance with the manufacturer's instructions (Tropix, Bedford, MA). Light release was quantitated for 10 s using a luminometer (MicroLumat Plus, Berthold, Bad Wildbad, Germany), as described before (17).

Recombinant retrovirus and retrovirus infection. Dominant-negative I{kappa}B{alpha} (S32A, S36A) (1) was amplified with sense (5'-aacc ATGGCATACCCATACGACGTCCCAGACTACGCTttccaggcggccgagcgcccccaggag-3') and antisense (5'-aaaaGGATCCtcataacgtcagacgctggcct-3') primers using high-fidelity Taq polymerase (Gibco BRL). The capital letters represent nucleotides encoding an HA tag. The PCR products were digested with Nco-1 and BamH1 restriction enzymes and cloned into the corresponding sites in MFG retroviral vector by replacing the GFP sequence of MFG.GFP.IRES.puro (32). The retroviral plasmids obtained were introduced into 293-gpg retrovirus packaging cell line (33) by transient transfection with Lipofectamine (Gibco BRL). After 72 h, the supernatants were harvested and used for retroviral infection. The virus titers, measured in NIH3T3 cell line by puromycin-resistant colony formation, were between 105 and 5 x 105/ml (retrovirus-I{kappa}B{alpha}-AA). The infection and selection of target cells with puromycin were performed as described previously (32). The transfection efficiency was ~99% into the gastric epithelial cell lines.

Assay for apoptosis in gastric epithelial cell lines. To assess the number of cells undergoing apoptosis, FITC-conjugated annexin V, which binds to phosphatidylserine, and propidium iodide were added to 105 cells. The cells were then incubated for 15 min at room temperate in the dark, according to the manufacturer's instructions (Apoptosis Detection Kit; R&D Systems, Minneapolis, MN). They were then analyzed by flow cytometry (13). To assess DNA fragmentation, nucleosomes in the cytoplasm were quantified using sandwich ELISA (Cell Death Detection ELISAplus kit; Boehringer Mannheim). In brief, adherent cells were detached and 1 x 105 cells were lysed, after which cytosolic oligonucleosome was quantified using biotin-coupled mouse monoclonal anti-histone antibody as the capturing antibody, peroxidase-conjugated mouse monoclonal anti-DNA antibody as the detecting antibody, and 2,2'-azino-di[3-thylbenzthiazolin-sulfonate] as the developing reagent. The relative increase of nucleosomes in the cytoplasm was expressed as an enrichment factor, which was calculated as the ratio of specific absorbency of lysates from bacteria-infected cells to that of uninfected cells, as described by Boehringer Mannheim.

For TUNEL assay, a complete flow cytometry kit for apoptosis (APO-DIRECT, PharMingen, San Diego, CA) was used in accordance with the manufacturer's instruction.

For transmission electron microscopy (TEM), cells were fixed with 2% cacodylate-buffered glutaraldehyde (pH 7.4) for 2 h and postfixed with 1% osmium tetraoxide for 2 h. The cells were spun down and stained en bloc with 1% uranyl acetate in malate buffer (pH 5.2). Samples were dehydrated in ethanol/propylene oxide and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate, followed by analysis using JEOL JEM-100 CX.

Determination of caspase-3 activity in H. pylori-infected gastric epithelial cell lines. Activation of caspase-3 (CPP32) was determined by detecting the chromophore p-nitroanilide (pNA) liberated from the labeled substrate Asp-Glu-Val-Asp-pNA after cleavage (DEVD-pNA; ApoAlert CPP32 colorimetric assay, Clontech, Palo Alto, CA). In brief, adherent gastric epithelial cell lines were detached from the tissue culture plates, 3 x 106 of the cells were lysed, and incubated with DEVD-pNA for 1 h at 37°C. Optical density was measured at 405 nm. The human T cell lymphoma cell line, Jurkat, incubated with anti-Fas monoclonal antibody (clone CH-11, IgM, Upstate Biotechnology, Lake Placid, NY) (50 ng/ml) for 8 h was used as a positive control for caspase-3 activation. Control reactions in each experiment involved the caspase-3 inhibitor DEVD-FMK (Asp-Glu-Val-Asp-CH2F), which was added before the addition of DEVD-pNA (13). This inhibitor completely blocked caspase-3 activation.

RNA extraction and RT-PCR analysis. The total cellular RNA was extracted using the acid guanidinium-phenol-chloroform method (TRIzol; Gibco BRL). Quantitative RT-PCR using an internal standard RNA was used to quantify the mRNA levels of iNOS, as described previously (14). The primer sequences used for the iNOS were as follows: 5'-CGG TGC TGT ATT TCC TTA CGA GGC GAA GAA GG-3' (sense) and 5'-GGT GCT GCT TGT TAG GAG GTC AAG TAA AGG GC-3' (antisense) (14). The sizes of the iNOS PCR products for standard and target RNAs were 356 and 259 bp, respectively. PCR amplification was performed in a thermal cycler (GenAmp PCR system 9600; Perkin Elmer Cetus, Norwalk, CT). The PCR amplification of iNOS consisted of 35 cycles of: -45 s of denaturation at 94°C, 45 s of annealing at 60°C, and 2 min of extension at 72°C, followed by a final 10-min extension step. RNA isolated from human peripheral blood mononuclear cells (PBMCs) was used as the positive control for iNOS. The negative control was obtained by omitting the RNA from the reverse transcription mixture. After amplification, the PCR products were separated on a 1% agarose gel (Ultrapure; Gibco BRL) and visualized by ethidium bromide staining. Photographs of the gels were taken, and the density of the bands was used to calculate the number of cellular RNA transcripts, as described previously (10, 14). This method can detect 103 mRNA transcripts per microgram of cellular RNA. The PCR amplification of {beta}-actin consisted of 32 cycles of: -45 s of denaturation at 95°C, and 2 min 30 s of annealing and extension at 60°C. The {beta}-actin primers were used to amplify a 384-bp fragment from human cDNA. Reverse transcription and PCR amplification of the internal RNA standards using {beta}-actin primers yielded a 520-bp fragment (9, 10).

Western blot analysis. Confluent epithelial monolayers in six-well plates were washed with ice-cold PBS and lysed in a 0.5 ml/well lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 0.1% Triton X-100, 1 mM PMSF, and 10 µg/ml aprotonin). The lysates were sonicated for 5 s on ice and centrifuged at 12,000 g for 20 min. Protein concentrations in the lysates were determined by Bradford method (Bio-Rad) using bovine serum albumin (Sigma) as a standard. Fifteen micrograms of protein/lane were size fractionated on a denaturing, nonreducing 6% polyacrylamide minigel (Mini-PROTEIN II; Bio-Rad) and electrophoretically transferred to a nitrocellulose membrane (0.1 µm pore size). Specific proteins were detected using mouse antihuman caspase-3 (PharMingen), iNOS, or actin (Santa Cruz Biotechnology) as a primary antibody and peroxidase-conjugated antimouse IgG (Transduction Laboratories, Lexington, KY) as a secondary antibody. Specifically bound peroxidase was detected by enhanced chemiluminescence (ECL system; Amersham Life Science, Buckinghamshire, UK) and exposure to X-ray film (XAR5; Eastman Kodak, Rochester, NY) for 10 to 30 s.

Statistical analysis. Data are presented as the means ± SE of separate experiments. Wilcoxon's rank sum test was used for statistical analysis. A P value of <0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Blockage of NF-{kappa}B signal increases apoptosis in gastric epithelial cells infected with H. pylori. H. pylori infection induced apoptosis in gastric epithelial cell lines such as Hs746T and MKN-45 cells using TEM analysis (Fig. 1, A and B). Because the transcription factor NF-{kappa}B is activated by human gastric epithelial cells on infection with H. pylori (25, 30), we evaluated whether NF-{kappa}B activation by H. pylori infection influences the apoptosis of human gastric epithelial cells. MKN-45 or Hs746T cell lines were transfected with retrovirus-I{kappa}B{alpha}-AA. The transfected cells were then infected with H. pylori for 3 h and NF-{kappa}B DNA binding activity was assessed by EMSA. As shown in Fig. 2A, transfection with retrovirus-I{kappa}B{alpha}-AA blocked NF-{kappa}B activity in H. pylori-infected MNK-45 cell lines; however, the control retrovirus containing GFP plasmid did not change NF-{kappa}B activation. In the MKN-45 cell lines transfected with retrovirus-I{kappa}B{alpha}-AA, cagA+cytotoxin+ H. pylori were infected for 24 h and the level of apoptosis was then determined by flow cytometry using annexin V and propidium iodide staining. As shown in Fig. 2B, the numbers of apoptotic cells induced by H. pylori infection were increased when NF-{kappa}B signal was blocked (control, 0.8 ± 1.2%; H. pylori, 2.8 ± 0.5%; retrovirus-I{kappa}B{alpha}-AA + H. pylori, 4.2 ± 0.8%; retrovirus-GFP + H. pylori, 2.5 ± 0.6%; n = 5, means ± SE). Similar results, as assayed by flow cytometry using annexin V and propidium iodide staining, were shown in Hs746T cells infected with H. pylori (control, 1.1 ± 0.5%; H. pylori, 3.4 ± 0.6%; retrovirus-I{kappa}B{alpha}-AA + H. pylori, 5.6 ± 0.8%; retrovirus-GFP + H. pylori, 3.6 ± 0.5%; n = 5, means ± SE).



View larger version (135K):
[in this window]
[in a new window]
 
Fig. 1. Apoptosis in Helicobacter pylori-infected Hs746T and MKN-45 monolayers. Monolayers of Hs746T or MKN-45 cell lines were infected with cagA+cytotoxin+ H. pylori for 48 h. The cells were analyzed using transmission electron microscopy. B and D: many apoptotic bodies. A: noninfected Hs746T cells. B: H. pylori-infected Hs746T cells. C: non-infected MKN-45 cells. D: H. pylori-infected MKN-45 cells (x4,000).

 


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 2. NF-{kappa}B activation and apoptosis in H. pylori-infected gastric epithelial cell lines transfected with retrovirus containing the I{kappa}B{alpha} superrepressor. A: MKN-45 cells were transfected with either retrovirus containing I{kappa}B{alpha} superrepressor (retrovirus-I{kappa}B{alpha}-AA) or control virus (retrovirus-GFP). At 48 h after transfection, the cells were infected with H. pylori for 3 h. NF-{kappa}B binding activity was assayed by EMSA. The results are representative of 5 repeated experiments. + Represents a positive control whereby gastric epithelial cells were treated with TNF-{alpha} (20 ng/ml) for 1 h. B: transfected MKN-45 cells were then infected with cagA+cytotoxin+ H. pylori for 24 h. Adherent and nonadherent cells were pooled, incubated with FITC-conjugated annexin V and propidium iodide, and analyzed by flow cytometry. Data are representative of 5 separate experiments. Early apoptotic cells showed increased annexin but no propidium iodide staining, as indicated by the area marked R1. Necrotic and late apoptotic cells were stained with both annexin V and propidium iodide, as indicated by the area marked R2. 1: Noninfected control. 2: H. pylori infected. 3: Retrovirus-I{kappa}B{alpha}-AA + H. pylori. 4: Retrovirus-GFP + H. pylori.

 

To confirm the inhibition of apoptosis obtained by a cell death detection ELISA, TUNEL assay was performed. Similar findings obtained by a cell death detection ELISA were also observed with TUNEL assay, when MKN-45 cell lines were infected with H. pylori for 24 h (noninfected control, 1.8 ± 0.3; H. pylori infected, 7.3 ± 1.5; H. pylori + retrovirus-I{kappa}B{alpha}-AA, 12.8 ± 2.2; means of % ± SE, n = 3).

Apoptosis was a relatively late response of epithelial cells to H. pylori infection compared with proinflammatory cytokine gene expression (13). Apoptosis was first apparent 18 h after H. pylori infection and continued to increase over the next 48 h, as assessed by cell death detection ELISA (Fig. 3). Because NF-{kappa}B was found to prevent the apoptosis of H. pylori-infected gastric epithelial cells (Fig. 2), we speculated as to whether the activation of NF-{kappa}B influenced the kinetics of apoptosis in gastric epithelial cells. As shown in Fig. 3, the apoptosis of NF-{kappa}B-suppressed MKN-45 cell lines was first apparent 6 h after H. pylori infection compared with the 6 h of the cells with intact NF-{kappa}B activity, and this continued to increase over the next 24 h. However, no significant differences were evident 48 h postinfection in the NF-{kappa}B-suppressed groups and NF-{kappa}B-intact groups in response to H. pylori infection. Similar to the gastric epithelial cell lines, primary human gastric epithelial cells transfected with retrovirus-I{kappa}B{alpha}-AA also showed increase of apoptosis 24 h after H. pylori infection (Fig. 4). Primary colon epithelial cells were obtained from five individuals. Interestingly, increases of gastric epithelial cell apoptosis upon blocking NF-{kappa}B activation were not noted after infection with other bacteria such as C. jejuni, C. fetus subsp. fetus, and nonpathogenic E. coli DH5{alpha} (Fig. 5).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Time course of apoptosis after H. pylori infection of MKN-45 cells. MKN-45 cells were transfected with retrovirus-I{kappa}B{alpha}-AA. These transfected cells were then infected with cagA+cytotoxin+ H. pylori for the indicated times. Cells were assessed for apoptosis using cell death detection ELISA. Numbers refer to DNA fragmentation as determined by the enrichment factor. Values are means ± SE of 5 separate experiments. *Value significantly different from H. pylori-infected cells without retrovirus-I{kappa}B{alpha}-AA. {blacktriangledown}, H. pylori-infected cells transfected with retrovirus-I{kappa}B{alpha}-AA. {bullet}, H. pylori-infected cells without retrovirus-I{kappa}B{alpha}-AA. {blacksquare}, Noninfected cells transfected with retrovirus-I{kappa}B{alpha}-AA.

 


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 4. NF-{kappa}B activation and apoptosis in H. pylori-infected primary human gastric epithelial cells transfected with retrovirus containing the I{kappa}B{alpha} superrepressor. A: primary human gastric epithelial cells were transfected with either retrovirus containing I{kappa}B{alpha} superrepressor (retrovirus-I{kappa}B{alpha}-AA) or control virus (retrovirus-GFP). At 48 h after transfection, the cells were infected with H. pylori for 3 h. NF-{kappa}B binding activity was assayed by EMSA. The results are representative of 3 repeated experiments. B: transfected primary human gastric epithelial cells were then infected with cagA+cytotoxin+ H. pylori for 24 h. Cells were assessed for apoptosis using cell death detection ELISA. Numbers refer to DNA fragmentation as determined by the enrichment factor. Values are means ± SE of 5 separate experiments. *Value significantly different from H. pylori-infected cells without retrovirus-I{kappa}B{alpha}-AA. 1: Noninfected control. 2: H. pylori infected. 3: Retrovirus-I{kappa}B{alpha}-AA + H. pylori. 4: Retrovirus-GFP + H. pylori.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Apoptosis of gastric epithelial cells infected with H. pylori, Campylobacter jejuni, Campylobacter fetus subspecies fetus, and nonpathogenic Escherichia coli DH5{alpha}. Culture and transfection conditions of the MKN-45 cells were the same as those shown in Fig 1. Forty-eight-hours later, the transfected cells were infected with cagA+ cytotoxin+ H. pylori for 24 h. Cells were assessed for apoptosis using cell death detection ELISA. Numbers refer to DNA fragmentation as determined by enrichment factor. Values are means ± SE of 5 separate experiments.

 

NF-{kappa}B signaling pathway via IKK and NIK activation is involved in inhibition of apoptosis of gastric epithelial cell lines infected with H. pylori. One of the major pathways of NF-{kappa}B activation involves the phosphorylation of I{kappa}B{alpha} following IKK and NIK activation, which is in turn followed by I{kappa}B degradation and the subsequent migration of NF-{kappa}B dimers from the cytoplasm to the nucleus (25, 37). We assayed the apoptosis of gastric epithelial cells with suppressed NF-{kappa}B activity, which had been transfected with the retrovirus-I{kappa}B{alpha}-AA, IKK{beta}-AA, or NIK-AA. To confirm that the transfection with several superrepressors or retrovirus-I{kappa}B{alpha}-AA was related to a decrease of NF-{kappa}B signal, luciferase assays were performed. Activation of NF-{kappa}B transcriptional reporters was inhibited in MNK-45 cell lines cotransfected with retrovirus-I{kappa}B{alpha}-AA, IKK{beta}-AA, or NIK-AA (Fig. 6A). Consistent with this, the activation of IL-8 reporters in response to H. pylori infection was also decreased in NF-{kappa}B-suppressed MKN-45 cell lines (Fig. 6B). However, H. pylori infection significantly increased gastric epithelial cell apoptosis when the NF-{kappa}B activity was suppressed (Fig. 6C). In addition, the other gastric epithelial Hs746T cell lines transfected with IKK{beta}-AA, NIK-AA, or retrovirus-I{kappa}B{alpha}-AA also showed increased apoptosis and caspase-3 activity compared with H. pylori-infected cells expressing intact NF-{kappa}B activity (Table 1). However, transfection with the control virus retrovirus-GFP did not influence caspase-3 activity or apoptosis in gastric epithelial cells. These results suggest that an NF-{kappa}B signaling pathway via I{kappa}B{alpha} degradation and IKK/NIK activation may play a role in protecting gastric epithelial cells from the apoptosis induced by H. pylori infection.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. Apoptosis in H. pylori-infected human gastric epithelial cells when suppressing NF-{kappa}B signals. MKN-45 cells were transfected with retrovirus-I{kappa}B{alpha} superrepressor, I{kappa}B kinase (IKK){beta} superrepressor, or NF-{kappa}B-inducing kinase (NIK) superrepressor. A and B: MKN-45 cells were transfected with 2x NF-{kappa}B-luciferase or pIL8-luciferase transcriptional reporters together with retrovirus-I{kappa}B{alpha}-AA, IKK{beta}-AA, or NIK-AA, as indicated. Forty-eight hours later, the transfected cells were infected with cagA+cytotoxin+ H. pylori for3h (A) or 18 h (B). Data are expressed as mean fold induction ± SE in luciferase activity relative to noninfected controls (n = 5). C: transfected cells were infected with cagA+cytotoxin+ H. pylori for 24 h. Cells were assessed for apoptosis using cell death detection ELISA. Numbers refer to DNA fragmentation as determined by enrichment factor. Values are means ± SE of 5 separate experiments.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Caspase-3 activity and DNA fragmentation in Hs746T gastric epithelial cells infected with H. pylori when NF-{kappa}B was suppressed

 

Effects of NF-{kappa}B suppression on iNOS and caspase-3 activation in human gastric epithelial cells infected with H. pylori. Because H. pylori infection induced iNOS expression and NO production in gastric epithelial cells (14), we tested whether iNOS expression could be linked with NF-{kappa}B activation in human gastric epithelial cells infected with H. pylori. As shown in Table 2, the suppression of the NF-{kappa}B signaling pathway resulted in the downregulation of iNOS mRNA expression in H. pylori-infected MKN-45 or Hs746T gastric epithelial cell lines. Moreover, the intracellular iNOS proteins were also decreased in response to H. pylori infection in MKN-45 cell lines when the NF-{kappa}B signaling pathway was suppressed by transfection with IKK{beta}-AA, NIK-AA, or retrovirus-I{kappa}B{alpha}-AA (Fig. 7). These results indicate that iNOS expression is regulated by an NF-{kappa}B signal in H. pylori-infected gastric epithelial cells.


View this table:
[in this window]
[in a new window]
 
Table 2. Quantification of iNOS mRNA expression in gastric epithelial cells infected with H. pylori

 


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7. Western blot of caspase-3, inducible nitric oxide synthase (iNOS), and actin proteins in H. pylori-infected human gastric epithelial cells. Culture and transfection conditions of the MKN-45 cells were the same as those shown in Fig 4. At 48 h later, the transfected cells were infected with cagA+cytotoxin+ H. pylori. MKN-45 cell lysates were prepared 18 h postinfection for caspase-3 and 24 h postinfection for iNOS and actin. Caspase-3, iNOS, and actin were detected with specific antibodies and by enhanced chemiluminescence. Representative examples of X-ray films are shown. Major bands representing caspase-3, iNOS, and actin were present at 32, 130, and 40 kDa, respectively.

 

In contrast to iNOS expression, caspase-3 activity was significantly increased in Hs746T and MKN-45 cell lines when the NF-{kappa}B-suppressed gastric epithelial cells were infected with H. pylori; however, transfection with the control virus retrovirus-GFP did not change caspase-3 activation (Table 1 and Fig. 7). Considering that caspase-3 activation is a step of apoptosis (5), caspase-3 activation and apoptosis may be associated with the iNOS expression of gastric epithelial cells in response to H. pylori infection.

NO protects gastric epithelial cells from apoptosis induced by H. pylori infection. NO promotes apoptosis in intestinal epithelial cells infected with bacteria (12) or protects several cell types from apoptosis (43). In this study, we demonstrated that iNOS expression is linked with NF-{kappa}B signaling pathways in human gastric epithelial cells infected with H. pylori (Fig. 6). In addition, NO production, as determined by assaying its stable end products nitrite and nitrate by Griess reaction (14), was ~35 µM 48 h postinfection. NO production was <5 µM in H. pylori-infected MKN-45 cell lines, which were without NF-{kappa}B activity using transfection with retrovirus-I{kappa}B{alpha}-AA. We asked whether exogenous NO might influence caspase-3 activity and apoptosis in NF-{kappa}B-suppressed gastric epithelial cells infected with H. pylori. The addition of S-nitroso-N-acetyl-penicillamine (SNAP; 100 µM) in H. pylori-infected MKN-45 cells downregulated the caspase-3 activity when the NF-{kappa}B signals were blocked by transfection with IKK{beta}-AA, NIK-AA, or retrovirus-I{kappa}B{alpha}-AA (Fig. 8A). Moreover, the addition of SNAP significantly inhibited the increased apoptosis induced by suppressing the NF-{kappa}B signaling pathway in H. pylori-infected MKN-45 cells (Fig. 8B). However, a higher concentration of SNAP (500 µM) did not protect H. pylori-infected MKN-45 cells from apoptosis (H. pylori, 2.2 ± 0.4; H. pylori + 500 µM of SNAP, 3.2 ± 0.8; H. pylori + retrovirus-I{kappa}B{alpha}-AA, 4.9 ± 0.6; H. pylori + retrovirus-I{kappa}B{alpha}-AA + 500 µM of SNAP, 5.3 ± 0.6; mean of enrichment factor ± SE, n = 5). In contrast, the addition of NG-nitro-L-arginine methyl ester (L-NAME; 1.0 mM), an NO inhibitor, significantly increased apoptosis of MKN-45 cell lines infected with H. pylori for 24 h (H. pylori infected, 2.3 ± 0.2; H. pylori + L-NAME, 4.1 ± 0.6; mean of enrichment factor ± SE, n = 5). Similar to the gastric epithelial cell lines, primary human gastric epithelial cells transfected with retrovirus-I{kappa}B{alpha}-AA also showed that the addition of SNAP (100 µM) inhibited the increased apoptosis induced by suppressing NF-{kappa}B but the addition of L-NAME (1.0 mM) increased apoptosis of primary gastric epithelial cells infected with H. pylori for 24 h (H. pylori, 3.1 ± 0.5; H. pylori + retrovirus-I{kappa}B{alpha}-AA, 5.6 ± 0.4; H. pylori + retrovirus-I{kappa}B{alpha}-AA + SNAP, 4.2 ± 0.6; H. pylori + L-NAME, 6.2 ± 0.8; mean of enrichment factor ± SE, n = 3). These results suggest that endogenous NO may play a role in the protection of gastric epithelial cells from H. pylori-induced apoptosis.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 8. Effects of an NO donor S-nitroso-N-acetylpenicillamine (SNAP) on caspase-3 and apoptosis in gastric epithelial cells infected with H. pylori. A: culture and transfection conditions of MKN-45 cells were the same as those shown in Fig 4. MKN-45 cells were infected with cagA+cytotoxin+ H. pylori for 18 h in the presence or absence of SNAP (100 µM). Cells were then assessed for caspase-3 activity. Values are means ± SE of 5 separate experiments. In comparison, caspase-3 activation was increased ~3.5-fold in Jurkat cells treated with anti-Fas monoclonal antibody (clone CH-11, 50 ng/ml) for 18 h. B: parallel cultures were assessed for apoptosis using cell death detection ELISA 24 h after infection. Numbers refer to DNA fragmentation as determined by enrichment factor. Values are means ± SE of 5 separate experiments. *Values significantly different from H. pylori-infected cells transfected with retrovirus-I{kappa}B{alpha}-AA (P < 0.05).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In this study, we showed that NF-{kappa}B activation plays a role in protecting H. pylori-infected gastric epithelial cells from apoptosis and suggest that the NF-{kappa}B signaling pathway can regulate the expression of iNOS in H. pylori-infected gastric epithelial cells. Moreover, exogenous NO could downregulate increased apoptosis by NF-{kappa}B suppression, indicating that an antiapoptotic effect by NF-{kappa}B activation may depend on iNOS expression in gastric epithelial cells.

One of the NF-{kappa}B signaling pathways consists of NIK, IKK, and I{kappa}B degradation (34, 37). Moreover, H. pylori infection was found to induce this NF-{kappa}B signaling pathway in gastric epithelial cells (25). Therefore, we used NIK-AA, IKK{beta}-AA, or retrovirus-I{kappa}B{alpha}-AA to block the NF-{kappa}B signaling pathway in gastric epithelial cells. The present study shows that an NF-{kappa}B signaling pathway via IKK and NIK activation is involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells.

One study showed that the suppression of NF-{kappa}B activation inhibited H. pylori-induced apoptosis (22), whereas another study demonstrated antiapoptotic effects mediated by NF-{kappa}B activation in H. pylori-infected gastric epithelial cells (24). Therefore, it is far from clear as to whether NF-{kappa}B activation by H. pylori infection mediates the apoptosis of gastric epithelial cells. In this regard, the above observations suggest that H. pylori-mediated NF-{kappa}B activation may play either a proapoptotic or antiapoptotic role. In addition, apoptosis was found to be a relatively late response of the epithelial cells to H. pylori infection, compared with, for example, proinflammatory cytokine gene expression. Moreover, a relatively low proportion of infected gastric epithelial cells was found to undergo apoptosis in response to H. pylori infection (13). Therefore, we further tested whether the lack of apoptosis in the early infection period might reflect a protective effect associated with NF-{kappa}B activation induced by H. pylori. In the present study, the apoptosis of gastric epithelial cells with intact NF-{kappa}B activity was first apparent 18 h after H. pylori infection. In contrast, the apoptosis of the cells without NF-{kappa}B activity was first apparent 6 h after H. pylori infection. These results indicated that H. pylori-induced NF-{kappa}B activation exerted antiapoptotic effects in gastric epithelial cells during the early infection period. It is interesting to note that no significant increase of apoptosis was evident in gastric epithelial cells transfected with retrovirus-I{kappa}B{alpha}-AA vs. nontransfected cells at 48 h after H. pylori infection. The activation of NF-{kappa}B in MKN-45 cell lines began within 1 h of infection, increased over the next 6 h, and then decreased, as assessed by luciferase assay (1 h postinfection, 1.8 ± 0.2; 6 h postinfection, 2.6 ± 0.4; 24 h postinfection, 1.2 ± 0.2; 48 h postinfection, 0.9 ± 0.2; means ± SE of fold increase compared with control, n = 5). These results imply that no difference at 48 h postinfection may be, in part, due to the loss of NF-{kappa}B activity in nontransfected MKN-45 cell lines. Therefore, we hypothesized that the molecules regulated by NF-{kappa}B might be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells during the early infection period. However, several other signal transduction pathways, including Fas, Trail, and so on, may be involved in the apoptosis of H. pylori-infected gastric epithelial cells. Therefore, these signal transduction pathways except an NF-{kappa}B pathway cannot be excluded in the apoptosis of gastric epithelial cells infected with H. pylori.

iNOS/NO is an important molecule that affects the apoptosis of intestinal epithelial cells in response to bacterial infection (12, 22, 31). We previously demonstrated that H. pylori infection results in the expression of endogenous iNOS in gastric epithelial cells (14). In this study, the suppression of an NF-{kappa}B signaling pathway resulted in the downregulation of iNOS mRNA and protein expression in H. pylori-infected gastric epithelial cells, indicating that a possible downstream target of NF-{kappa}B is the gene expression of iNOS. Therefore, we next asked whether iNOS/NO could be involved in the inhibition of epithelial cell apoptosis by NF-{kappa}B activation induced by H. pylori infection.

Although iNOS/NO might be involved in the apoptosis of gastric epithelial cells infected with H. pylori (22, 31), iNOS/NO is also known to suppress the expression of proapoptotic genes and apoptosis in several cell lines (37, 42, 43). In addition, considering that iNOS expression was downregulated in gastric epithelial cells without NF-{kappa}B activity, we hypothesized that the antiapoptotic effects of NF-{kappa}B might be mediated by the expression of iNOS/NO in response to H. pylori infection. In gastric epithelial cells transfected with retrovirus-I{kappa}B{alpha}-AA, the addition of exogenous NO donor SNAP (100 µM) partially downregulated the increased apoptosis induced by NF-{kappa}B suppression. These results provide strong evidence to support the functional role of iNOS/NO via the NF-{kappa}B pathway in the inhibition of gastric epithelial cell apoptosis in response to H. pylori infection. However, the mechanistic explanations of this iNOS/NO-induced suppression of apoptosis remain hypothetical in gastric epithelial cells infected with H. pylori. Several mechanisms might contribute to the antiapoptotic effect of NO, including the suppression of the activities of multiple caspases by nitrosylation (19, 21), the inhibition of Bcl-2 cleavage and cytochrome c release (18), a decrease in the formation of reactive oxygen intermediates in mitochondria (6), and the suppression of proapoptotic genes (43). We have not yet evaluated these possibilities in gastric epithelial cells infected with H. pylori. Further investigations are needed to elucidate the apoptotic pathogenesis of gastric mucosal inflammation.

It is interesting to note that the observed increased apoptosis was not completely suppressed by the addition of exogenous NO donor. These findings suggest that other molecules mediated by NF-{kappa}B may be involved in the protection of gastric epithelial cells infected with H. pylori. COX-2 is a possible candidate, because upregulated COX-2 inhibits the apoptosis of human gastric epithelial cells infected with H. pylori (15) and NF-{kappa}B also regulates COX-2 expression (7).

In conclusion, our results suggest that NF-{kappa}B activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis during the early infection period. Moreover, endogenous iNOS expression partially mediated this antiapoptotic effect in gastric epithelial cells.


    DISCLOSURES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by the research fund of Hanyang University (HY-2002-S).


    ACKNOWLEDGMENTS
 
We thank Dr. M. F. Kagnoff and Dr. J. A. DiDonato for gifts of standard RNAs and several plasmids, and Dr. H. K. Yang for gastric tissues to isolate primary gastric epithelial cells.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. C. Jung, Dept. of Internal Medicine, Seoul National Univ. College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea (E-mail: hyunchae{at}plaza.snu.ac.kr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H, Ghosh S, and Karin M. Mapping of the inducible I{kappa}B phosphorylation sites that signal its ubiquitination and degradation. Mol Cell Biol 16: 1295-1304, 1996.[Abstract]
  2. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, and Karin M. A cytokine-responsive I{kappa}B kinase that activates the transcription factor NF-{kappa}B. Nature 388: 548-554, 1997.[ISI][Medline]
  3. Dimmeler S and Zeiher AM. Nitric oxide and apoptosis: another paradigm for the double-edged role of nitric oxide. Nitric Oxide 1: 275-281, 1997.[ISI][Medline]
  4. Elewaut D, DiDonato JA, Kim JM, Troung F, Eckmann L, and Kagnoff MF. Nuclear factor-{kappa}B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J Immunol 163: 1457-1466, 1999.[Abstract/Free Full Text]
  5. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, and Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391: 43-50, 1998.[ISI][Medline]
  6. Hakoda S, Ishikura H, Takeyama N, and Tanaka T. Tumor necrosis factor-{alpha} plus actinomycin D-induced apoptosis of L929 cells is prevented by nitric oxide. Surg Today 29: 1059-1067, 1999.[Medline]
  7. Jobin C, Morteau O, Han DS, and Sartor RB. Specific NF-{kappa}B blockade selectively inhibits tumour necrosis factor-{alpha}-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells. Immunology 95: 537-543, 1998.[ISI][Medline]
  8. Jobin C and Sartor RB. The I{kappa}B/NF-{kappa}B system: a key determinant of mucosal inflammation and protection. Am J Physiol Cell Physiol 278: C451-C462, 2000.[Abstract/Free Full Text]
  9. Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E, and Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 95: 55-66, 1995.[ISI][Medline]
  10. Jung HC, Kim JM, Song IS, and Kim CY. Helicobacter pylori induces an array of proinflammatory cytokines in human gastric epithelial cells: quantification of mRNA for interleukin-8, -1{alpha}/{beta}, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein-1 and tumor necrosis factor-{alpha}. J Gastroenterol Hepatol 12: 473-480, 1997.[ISI][Medline]
  11. Keates S, Hitti YS, Upton M, and Kelly CP. Helicobacter pylori infection activates NF-{kappa}B in gastric epithelial cells. Gastroenterology 113: 1099-1109, 1997.[ISI][Medline]
  12. Kim JM, Eckmann L, Savidge TC, Lowe DC, Witthoft T, and Kagnoff MF. Apoptosis of human intestinal epithelial cells after bacterial invasion. J Clin Invest 102: 1815-1823, 1998.[Abstract/Free Full Text]
  13. Kim JM, Kim JS, Jung HC, Song IS, and Kim CY. Apoptosis of human gastric epithelial cells via caspase-3 activation in response to Helicobacter pylori infection: possible involvement of neutrophils through tumor necrosis factor-{alpha} and soluble Fas ligands. Scand J Gastroenterol 35: 40-48, 2000.[ISI][Medline]
  14. Kim JM, Kim JS, Jung HC, Song IS, and Kim CY. Upregulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-{gamma} in polarized nitric oxide secretion. Helicobacter 7: 116-128, 2002.[ISI][Medline]
  15. Kim JM, Kim JS, Jung HC, Song IS, and Kim CY. Upregulated cyclooxygenase-2 inhibits apoptosis of human gastric epithelial cells infected with Helicobacter pylori. Dig Dis Sci 45: 2436-2443, 2000.[ISI][Medline]
  16. Kim JM, Kim JS, Jung HC, Song IS, and Kim CY. Virulence factors of Helicobacter pylori in Korean strains do not influence proinflammatory cytokine gene expression and apoptosis in human gastric epithelial cells, nor do these factors influence clinical outcome. J Gastroenterol 35: 898-906, 2000.[ISI][Medline]
  17. Kim JM, Oh YK, Kim YJ, Oh HB, and Cho YJ. Polarized secretion of CXC chemokines by human intestinal epithelial cells in response to Bacteroides fragilis enterotoxin: NF-{kappa}B plays a major role in the regulation of IL-8 expression. Clin Exp Immunol 123: 421-427, 2001.[ISI][Medline]
  18. Kim YM, Kim TH, Seol DW, Talanian RV, and Billiar TR. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J Biol Chem 273: 31437-31441, 1998.[Abstract/Free Full Text]
  19. Kim YM, Talanian RV, and Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem 272: 31138-31148, 1997.[Abstract/Free Full Text]
  20. Li J and Billiar TR. The role of nitric oxide in apoptosis. Semin Perinatol 24: 46-50, 2000.[ISI][Medline]
  21. Li J, Billiar TR, Talanian RV, and Kim YM. Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem Biophys Res Commun 240: 419-424, 1997.[ISI][Medline]
  22. Lim JW, Kim H, and Kim KH. NF-{kappa}B, inducible nitric oxide synthase and apoptosis by Helicobacter pylori infection. Free Radic Biol Med 31: 355-366, 2001.[ISI][Medline]
  23. MacDonald TT and Pettersson S. Bacterial regulation of intestinal immune responses. Inflamm Bowel Dis 6: 116-122, 2000.[ISI][Medline]
  24. Maeda S, Yoshida H, Mitsuno Y, Hirata Y, Ogura K, Shiratori Y, and Omata M. Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori. Gut 50: 771-778, 2002.[Abstract/Free Full Text]
  25. Maeda S, Yoshida H, Ogura K, Mitsuno Y, Hirata Y, Yamaji Y, Akanuma M, Shiratori Y, and Omata M. H. pylori activates NF-{kappa}B through a signaling pathway involving I{kappa}B kinases, NF-{kappa}B-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells. Gastroenterology 119: 97-108, 2000.[ISI][Medline]
  26. Malinin NL, Boldin MP, Kovalenko AV, and Wallach D. MAP3K-related kinase involved in NF-{kappa}B induction by TNF, CD95 and IL-1. Nature 385: 540-544, 1997.[ISI][Medline]
  27. Mercurio F, Zhu H, Murry BW, Shevchenko A, Bennett BL, Li J, Young DB, Barbosa M, Mann M, Manning A, and Rao A. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF-{kappa}B activation. Science 278: 860-866, 1997.[Abstract/Free Full Text]
  28. Nathan C and Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915-918, 1994.[ISI][Medline]
  29. Nathan C and Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 269: 13725-13728, 1994.[Free Full Text]
  30. Naumann M. Nuclear factor-{kappa}B activation and innate immune response in microbial pathogen infection. Biochem Pharmacol 60: 1109-1114, 2000.[ISI][Medline]
  31. Neu B, Randlkofer P, Neuhofer M, Voland P, Mayerhofer A, Gerhard M, Schepp W, and Prinz C. Helicobacter pylori induces apoptosis of rat gastric parietal cells. Am J Physiol Gastrointest Liver Physiol 283: G309-G318, 2002.[Abstract/Free Full Text]
  32. Oh SC, Nam SY, Oh SC, Kim CM, Seo JS, Seong RH, Jang YJ, Chung YH, and Chung HY. Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron. Mol Cell 11: 192-197, 2001.
  33. Ory DS, Neugeboren BA, and Mulligan RC. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci USA 93: 11400-11406, 1996.[Abstract/Free Full Text]
  34. Russo MP, Bennett BL, Manning AM, Brenner DA, and Jobin C. Differential requirement for NF-{kappa}B-inducing kinase in the induction of NF-{kappa}B by IL-1{beta}, TNF-{alpha}, and Fas. Am J Physiol Cell Physiol 283: C347-C357, 2002.[Abstract/Free Full Text]
  35. Rutten MJ, Bacon KD, Marlink KL, Stoney M, Meichsner CL, Lee FP, Hobson SA, Rodland KD, Sheppard BC, Trunkey DD, Deveney KE, and Deveney CW. Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells. Am J Physiol Gastrointest Liver Physiol 277: G662-G670, 1999.[Abstract/Free Full Text]
  36. Schmid RM and Adler G. NF-{kappa}B/Rel/I{kappa}B: implications in gastrointestinal diseases. Gastroenterology 118: 1208-1228, 2000.[ISI][Medline]
  37. Thanos D and Maniatis T. NF-{kappa}B: a lesson in family values. Cell 80: 529-532, 1995.[ISI][Medline]
  38. Thippeswamy T, McKay JS, and Morris R. Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro. Eur J Neurosci 14: 1229-1236, 2001.[ISI][Medline]
  39. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, and Verma IM. Suppression of TNF-{alpha}-induced apoptosis by NF-{kappa}B. Science 274: 787-799, 1996.[Abstract/Free Full Text]
  40. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, and Miyamoto S. Rel/NF-{kappa}B/I{kappa}B family: intimate tales of association and dissociation. Genes Dev 9: 2723-2735, 1995.[ISI][Medline]
  41. Wagner S, Beil W, Westermann J, Logan RP, Bock CT, Trautwein C, Bleck JS, and Manns MP. Regulation of gastric epithelial cell growth by Helicobacter pylori: offdence for a major role of apoptosis. Gastroenterology 113: 1836-1847, 1997.[ISI][Medline]
  42. Yoon SJ, Choi KH, and Lee KA. Nitric oxide-mediated inhibition of follicular apoptosis is associated with HSP70 induction and Bax suppression. Mol Reprod Dev 61: 504-510, 2002.[ISI][Medline]
  43. Zamora R, Alarcon L, Vodovotz Y, Betten B, Kim PK, Gibson KF, and Billiar TR. Nitric oxide suppresses the expression of Bcl-2 binding protein BNIP3 in hepatocytes. J Biol Chem 276: 46887-46895, 2001.[Abstract/Free Full Text]
  44. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, and Karin M. The I{kappa}B kinase complex (IKK) contains two kinase subunits, IKK{alpha} and IKK{beta}, necessary for I{kappa}B phosphorylation and NF-{kappa}B activation. Cell 91: 243-252, 1997.[ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
285/6/G1171    most recent
00502.2002v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (4)
Google Scholar
Articles by Kim, J. M.
Articles by Song, I. S.
Articles citing this Article
PubMed
PubMed Citation
Articles by Kim, J. M.
Articles by Song, I. S.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.