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
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ABSTRACT |
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IB kinase; NF-
B-inducing kinase; nitric oxide; superrepressor
Many of the genes that are activated in gastrointestinal epithelial cells after bacterial infection are target genes of NF-B (8, 23, 30). NF-
B is a dimeric transcription factor, which is normally held in the cytoplasm in an inactive state by inhibitory proteins, the I
B kinases (I
Bs) (37). The stimulation of epithelial cells with several pathogens or inflammatory cytokines can induce I
B degradation and subsequently cause the NF-
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-B requires the phosphorylation of I
B
(Ser32 and Ser36) and I
B
(Ser19 and Ser23) (40). This phosphorylation leads to ubiquitination and the 26S proteosome-mediated degradation of I
B
, thereby releasing NF-
B from the complex to enter the nucleus and activate genes (37). Two cytokine-inducible kinases, IKK
and IKK
, have been cloned and characterized (2, 27, 44). IKK
and IKK
phosphorylate I
B
at Ser32 and Ser36 in response to proinflammatory cytokines. NF-
B-inducing kinase (NIK) is a novel member of the MEKK family and was found to activate both IKK
and IKK
by interacting with adapter proteins associated with receptors for TNF-
and IL-1 (26). Recently, it was demonstrated that H. pylori could activate NF-
B via a signaling pathway involving IKK and NIK (25). Given that many cell types become more sensitive to TNF-
or H. pylori-induced apoptosis after the suppression of NF-
B activity (36, 39), NF-
B may function antiapoptotically. In contrast, other studies showed that the suppression of NF-
B activation inhibited H. pylori-induced apoptosis in gastric epithelial cells (22). These conflicting observations indicate that the role of NF-
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-B signals were decreased and the population of apoptosis was increased. Because NF-
B activates within 1 h after infection in gastric epithelial cells, there is a possibility that NF-
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-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-B signaling pathway. To substantiate this hypothesis, we investigated whether the inhibition of NF-
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.
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METHODS |
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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 (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 [32P]-labeled oligonucleotide probe corresponding to a consensus NF-
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, in which lysine at position 44 was replaced by alanine (IKK
-AA), was used to block NF-
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-
B-luciferase, p
-actin-luciferase, and pRSV-
-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
-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 IB
(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
B
-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 -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
-actin primers were used to amplify a 384-bp fragment from human cDNA. Reverse transcription and PCR amplification of the internal RNA standards using
-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.
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RESULTS |
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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-IB
-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-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-
B influenced the kinetics of apoptosis in gastric epithelial cells. As shown in Fig. 3, the apoptosis of NF-
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-
B activity, and this continued to increase over the next 24 h. However, no significant differences were evident 48 h postinfection in the NF-
B-suppressed groups and NF-
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
B
-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-
B activation were not noted after infection with other bacteria such as C. jejuni, C. fetus subsp. fetus, and nonpathogenic E. coli DH5
(Fig. 5).
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NF-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-
B activation involves the phosphorylation of I
B
following IKK and NIK activation, which is in turn followed by I
B degradation and the subsequent migration of NF-
B dimers from the cytoplasm to the nucleus (25, 37). We assayed the apoptosis of gastric epithelial cells with suppressed NF-
B activity, which had been transfected with the retrovirus-I
B
-AA, IKK
-AA, or NIK-AA. To confirm that the transfection with several superrepressors or retrovirus-I
B
-AA was related to a decrease of NF-
B signal, luciferase assays were performed. Activation of NF-
B transcriptional reporters was inhibited in MNK-45 cell lines cotransfected with retrovirus-I
B
-AA, IKK
-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-
B-suppressed MKN-45 cell lines (Fig. 6B). However, H. pylori infection significantly increased gastric epithelial cell apoptosis when the NF-
B activity was suppressed (Fig. 6C). In addition, the other gastric epithelial Hs746T cell lines transfected with IKK
-AA, NIK-AA, or retrovirus-I
B
-AA also showed increased apoptosis and caspase-3 activity compared with H. pylori-infected cells expressing intact NF-
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-
B signaling pathway via I
B
degradation and IKK/NIK activation may play a role in protecting gastric epithelial cells from the apoptosis induced by H. pylori infection.
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Effects of NF-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-
B activation in human gastric epithelial cells infected with H. pylori. As shown in Table 2, the suppression of the NF-
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-
B signaling pathway was suppressed by transfection with IKK
-AA, NIK-AA, or retrovirus-I
B
-AA (Fig. 7). These results indicate that iNOS expression is regulated by an NF-
B signal in H. pylori-infected gastric epithelial cells.
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In contrast to iNOS expression, caspase-3 activity was significantly increased in Hs746T and MKN-45 cell lines when the NF-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-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-
B activity using transfection with retrovirus-I
B
-AA. We asked whether exogenous NO might influence caspase-3 activity and apoptosis in NF-
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-
B signals were blocked by transfection with IKK
-AA, NIK-AA, or retrovirus-I
B
-AA (Fig. 8A). Moreover, the addition of SNAP significantly inhibited the increased apoptosis induced by suppressing the NF-
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
B
-AA, 4.9 ± 0.6; H. pylori + retrovirus-I
B
-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
B
-AA also showed that the addition of SNAP (100 µM) inhibited the increased apoptosis induced by suppressing NF-
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
B
-AA, 5.6 ± 0.4; H. pylori + retrovirus-I
B
-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.
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DISCUSSION |
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One of the NF-B signaling pathways consists of NIK, IKK, and I
B degradation (34, 37). Moreover, H. pylori infection was found to induce this NF-
B signaling pathway in gastric epithelial cells (25). Therefore, we used NIK-AA, IKK
-AA, or retrovirus-I
B
-AA to block the NF-
B signaling pathway in gastric epithelial cells. The present study shows that an NF-
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-B activation inhibited H. pylori-induced apoptosis (22), whereas another study demonstrated antiapoptotic effects mediated by NF-
B activation in H. pylori-infected gastric epithelial cells (24). Therefore, it is far from clear as to whether NF-
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-
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-
B activation induced by H. pylori. In the present study, the apoptosis of gastric epithelial cells with intact NF-
B activity was first apparent 18 h after H. pylori infection. In contrast, the apoptosis of the cells without NF-
B activity was first apparent 6 h after H. pylori infection. These results indicated that H. pylori-induced NF-
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
B
-AA vs. nontransfected cells at 48 h after H. pylori infection. The activation of NF-
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-
B activity in nontransfected MKN-45 cell lines. Therefore, we hypothesized that the molecules regulated by NF-
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-
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-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-
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-
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-B activity, we hypothesized that the antiapoptotic effects of NF-
B might be mediated by the expression of iNOS/NO in response to H. pylori infection. In gastric epithelial cells transfected with retrovirus-I
B
-AA, the addition of exogenous NO donor SNAP (100 µM) partially downregulated the increased apoptosis induced by NF-
B suppression. These results provide strong evidence to support the functional role of iNOS/NO via the NF-
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-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-
B also regulates COX-2 expression (7).
In conclusion, our results suggest that NF-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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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