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Originally published In Press as doi:10.1074/jbc.M308898200 on November 20, 2003

J. Biol. Chem., Vol. 279, Issue 8, 7024-7028, February 20, 2004
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Helicobacter pylori VacA Activates the p38/Activating Transcription Factor 2-mediated Signal Pathway in AZ-521 Cells*

Masaaki Nakayama{ddagger}, Miyuki Kimura{ddagger}§, Akihiro Wada{ddagger}, Kinnosuke Yahiro{ddagger}, Ken-ichi Ogushi{ddagger}, Takuro Niidome||, Akihiro Fujikawa**, Daisuke Shirasaka{ddagger}{ddagger}, Nobuo Aoyama{ddagger}{ddagger}, Hisao Kurazono§, Masaharu Noda**, Joel Moss§§, and Toshiya Hirayama{ddagger}¶¶

From the {ddagger}Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523, Japan, the §Department of Medical Technology, School of Health Sciences, Okayama University, Okayama 7008558, Japan, PRESTO, Japan Science and Technology Agency, Saitama 3320012, Japan, the ||Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 8528521, Japan, the **Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki 4448585, Japan, the {ddagger}{ddagger}Department of Endoscopy, Kobe University School of Medicine, Kobe 6578501, Japan, and the §§Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 12, 2003 , and in revised form, November 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Persistent Helicobacter pylori colonization in the stomach induces gastritis and peptic ulcer and interferes with ulcer healing. Most strains of H. pylori produce a cytotoxin, VacA, that induces cytoplasmic vacuolation in epithelial cells with structural and functional changes, leading to gastric injury. VacA is known to cause cell death by mitochondrial damage. We hypothesized that VacA might disrupt other signaling pathways; to that end, we examined the effects of VacA on MAPKs to elucidate their role in the abnormalities seen in VacA-treated cells. VacA stimulated phosphorylation of p38 and Erk1/2, but not JNK, in AZ-521 cells. Both phosphorylation and kinase activation of p38 were maximal 10-30 min after addition of VacA and declined thereafter. Treatment with anti-VacA antibody or the p38 inhibitor SB203580 blocked p38 phosphorylation caused by VacA and inhibited VacA-induced phosphorylation of activating transcription factor 2 (ATF-2), which is implicated in transcriptional control of stress-responsive genes. These data indicate that VacA stimulates a p38/ATF-2-mediated signal pathway. However, 10 µM SB203580, which is sufficient to decrease p38 phosphorylation, did not inhibit VacA-induced cellular vacuolation, decrease in mitochondrial membrane potential, or cytochrome c release from mitochondria. These results suggest that VacA-induced activation of p38/ATF-2-mediated signal pathway is independent of cellular vacuolation, decrease in mitochondrial membrane potential, or cytochrome c release from mitochondria caused by VacA. The cytotoxin may thus act independently on several cellular targets, leading to disruption of signaling, regulatory, and metabolic pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Persistent Helicobacter pylori infection results in the development of chronic gastritis, peptic ulcer, and gastric cancer. Pathogenic strains of H. pylori produce and secrete a potent vacuolating cytotoxin, VacA, that is capable of directly inducing progressive vacuolation (1), mitochondrial damage (2), cytochrome c release (3), and apoptotic death of epithelial cells (4). Both epidemiological study (5) and animal experiments (6-10) have demonstrated that VacA is a major virulence factor associated with the damage to gastric mucosa. VacA is produced as a 140-kDa precursor and cleaved at the bacterial outer membranes, generating the 87-95-kDa mature toxin (11). The mature toxin contains two major domains, the N-terminal region of 37 kDa that exhibits vacuolating activity and the C-terminal region of 58 kDa responsible for toxin binding to target cells (12-15).

In gastric tissues infected with H. pylori, many apoptotic cells were observed due to inflammation resulting from overproduction of cytokines induced by H. pylori (9, 16-19). Recent work on the gastric mucosal inflammatory response to persistent H. pylori infection suggested that the p38 mitogen-activated protein kinase (MAPK)1 signal pathway plays a significant role (20, 21). Activation of p38 MAPK in response to environmental stress and inflammatory cytokines can result in apoptosis (22). In addition, Takahashi et al. (23) demonstrated a role for p38 in H. pylori-induced gastritis in Mongolian gerbils using the p38 inhibitor FR167653, which was discovered as an inhibitor of cytokine production, suggesting a crucial role of p38 in gastritis of both H. pylori-infected individuals and experimentally infected animals. However, it is not clear which virulence factor is responsible for activation of p38 as well as other MAPKs. Nishioka et al. (24) recently reported that neutrophil-activating protein of H. pylori activated MAPK in human neutrophils. Neutrophil-activating protein is a 15-kDa chemotactic protein for neutrophils, monocytes, and mast cells (25). VacA administrated to the mice resulted in epithelial vacuolization and marked infiltrations of mast cells and monocytes into the mucosal epithelium (9). In addition, VacA showed a chemotactic activity for mast cells and caused mast cells to produce proinflammatory cytokines such as tumor necrosis factor-{alpha}, suggesting the contribution of inflammation to H. pylori-induced gastritis.

To elucidate the role of VacA in H. pylori infection, we examined the effect of VacA on phosphorylation and activation of MAPK. Here we report that VacA stimulates phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (Erk1/2) in the gastric cell line AZ-521. p38 phosphorylation was followed by stimulation of activating transcription factor 2 (ATF-2) in vitro and in vivo, suggesting that VacA affects the transcriptional pathway through the p38/ATF-2-mediated signal transduction. VacA-induced activation of the p38/ATF-2-mediated signal pathway is, however, independent of cellular vacuolation, decrease in mitochondrial membrane potential, or cytochrome c release from mitochondria caused by VacA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Mouse anti-p38 monoclonal antibody (clone 24B) was obtained from Transduction Laboratories (catalog no. P98120); anti-phospho-p38 MAPK (Thr-180/Tyr-182) antibody (catalog number 9211), anti-phospho-p44/42 (Erk1/2) MAPK (Thr-202/Tyr-204) antibody (catalog number 9101), anti-phospho-stress-activated protein kinase/c-Jun N-terminal kinase (JNK) (Thr-183/Tyr-185) antibody (catalog number 9251), and p38 MAP kinase assay kit was from Cell Signaling Technology (catalog nos. 9211 and 9820); and mouse anti-cytochrome c monoclonal antibody (clone 7H8.2C12) was from BD Pharmingen (catalog no. 556433). SB203580 was purchased from Calbiochem (catalog no. 559389).

Cell Culture—The human gastric adenocarcinoma cell line AZ-521 (Culture Collection of Health Science Resource Bank, Japan Health Science Fundamental) was grown in Eagle's minimal essential medium containing 10% fetal calf serum under 5% CO2 at 37 °C. To evaluate the effect of VacA on MAPK phosphorylation, AZ-521 cells were grown overnight in medium without fetal calf serum.

VacA Preparation—The toxin-producing H. pylori strain ATCC49503 was used as the source of VacA for purification according to a modification of our published procedure (26, 27). In brief, after growth of H. pylori in Brucella broth containing 0.1% {beta}-cyclodextrin at 37 °C for 4 days with vigorous shaking in a controlled microaerophilic atmosphere of 10% O2 and 10% CO2, VacA was precipitated from culture supernatant with 50% saturated ammonium sulfate and purified by VacA affinity column, which was coupled with anti-VacA-specific IgG antibody and equilibrated with RX buffer (10 mM KCl, 0.3 mM NaCl, 0.35 mM MgCl2, and 0.125 mM EGTA in 1 mM HEPES, pH 7.3). After washing the column with RX buffer, VacA was eluted with 50 mM glycine-HCl, pH 1, and then neutralized with 1 M Tris. After gel filtration by Superose 6HR 10/30 column with TBS buffer (10 mM Tris-HCl buffer, pH 7.5, containing 50 mM NaCl), purified VacA was concentrated and stored (200 µg/ml). VacA concentration was determined by a bead enzyme-linked immunosorbent assay method (28). Protein content was measured by the Bradford method using bovine serum albumin as a standard (29). To quantify vacuolating activity of VacA, uptake of neutral red into the vacuoles in VacA-treated cells was determined by subtracting the absorbance of cells incubated without toxin from that of toxin-treated cells (26, 27).

Detection of MAPK Phosphorylation in AZ-521 Cells Exposed to VacA—AZ-521 cells were treated with 0, 1, 5, 10, or 50 µM SB203580 for 30 min followed by incubation with 120 nM VacA for 0, 30, or 60 min. Cells were solubilized by incubation for 10 min on ice in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. After centrifugation (15 min at 15,000 x g), samples (20 µg of protein) of supernatants were subjected to SDS-PAGE and Western blotting using anti-phospho-MAP kinase antibodies.

Assay for p38 Kinase Activity—p38 kinase activity was measured by using the p38 MAP kinase assay kit (Cell Signaling) according to the manufacturer's instructions. In brief, AZ-521 cells treated with or without VacA were harvested in lysis buffer. The cell lysates were immunoprecipitated with anti-phospho-p38 MAP antibody (Thr-180/Tyr-182). The immunoprecipitate was then incubated with ATF-2 in the presence of ATP and kinase buffer, and phospho-ATF-2 was quantified by Western blotting using a phospho-ATF-2 (Thr-71) antibody.

VacA-induced ATF-2 Phosphorylation in AZ-521 Cells—AZ-521 cells were incubated with 120 nM VacA for 0, 30, 60, or 120 min. Cells were solubilized by incubation for 10 min on ice in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. After centrifugation (15 min, at 15,000 x g), samples (20 µg of protein) of supernatants were subjected to SDS-PAGE and Western blotting using anti-phospho-ATF-2 antibody. For immunofluorescent staining, AZ-521 cells (0.6 x 105 cells/well) were fixed with 2% paraformaldehyde, treated with Triton X-100 in PBS for 10 min, and incubated with Block Ace (Snow Brand Milk Products, Tokyo, Japan), anti-phospho-ATF-2 antibody (primary antibody), and Cy-3-conjugated anti-rabbit IgG antibody (secondary antibody). Samples were inspected using a Nikon Eclipse TE-300 fluorescence microscope (Nikon, Tokyo, Japan). Images were captured and digitized using a Spot charged coupled device camera (Zeiss, AxioCam HRc) and edited using Adobe Photoshop Elements Version 2.0 (Adobe Systems Inc.).

Effect of SB203580 on VacA Vacuolating Activity—AZ-521 cells, grown as monolayers, were incubated with 10 µM SB203580 for 30 min before further incubation with 120 nM VacA for 0, 2, 4, or 8 h. The neutral red assay was used to evaluate the effect of SB203580 on vacuolating activity (26, 27).

Mitochondrial Membrane Potential Induced by VacA—Mitochondrial membrane potential was measured as reported by Vayssiere et al. (30). AZ-521 cells were incubated without or with 120 nM VacA for 6 h, washed twice with PBS, and harvested with 0.25% trypsin. After centrifugation (200 x g for 3 min), the cell pellet was washed with PBS and suspended in 10 ml of Dulbecco's modified Eagle's medium, 0.1 µM 3,3'-dihexyloxacarbocyanine iodide followed by incubation for an additional 30 min at 37 °C, washing three times with PBS, and dispersion in Dulbecco's modified Eagle's medium without fetal calf serum. Cells treated with an uncoupling agent, 5 µM carbonyl cyanide m-chlorophenyl hydrazone, served as a control. Samples were analyzed using a FACScan flow cytometer with excitation at 488 nm and emission at 530 nm.

Cytochrome c Release—AZ-521 cells were incubated with 10 µM SB203580 for 30 min followed by incubation with 120 nM VacA for 24 h. Cells were harvested and washed twice with ice-cold PBS before subsequent procedures at 4 °C. Cells were dispersed in lysis buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin) and broken by 40 passages through a 25-gauge x 5/8-inch needle followed by centrifugation (750 x g for 10 min) at 4 °C. The resulting supernatants were centrifuged (22,000 x g for 15 min) to obtain the mitochondrial fraction (pellet) and then further centrifuged (100,000 x g for 1 h) to prepare the cytosolic fraction (supernatant). Samples (30 µg) of mitochondrial and cytosolic proteins were subjected to SDS-PAGE in 15% gels and immunoblotting analysis using anti-cytochrome c antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VacA Activates MAP Kinases in AZ-521 Gastric Epithelial Cells—Three main groups of MAP kinases have been characterized to date: the p38 MAP kinases, the JNKs, and the ERKs. To determine whether VacA activates MAP kinases in AZ-521 gastric epithelial cells, the AZ-521 cells were incubated with 120 nM VacA. Control cells showed low or undetectable levels of activated MAP kinases (Fig. 1). Phosphorylation of p38 and Erk1/2 was clearly evident within a 30-min incubation of VacA; phospho-p38 declined thereafter, whereas Erk1/2 was maximal at 30 min and declined markedly only after 60 min. JNK was not activated within 120 min.



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  		FIG. 1.
H. pylori VacA activates p38 and Erk1/2 but not JNK in AZ-521 cells. AZ-521 gastric epithelial cells were incubated with 120 nM VacA for the indicated times. Cell lysates were prepared after varying incubation times and subjected to Western blot analyses using phospho-specific MAP kinase antibodies. Cont. refers to the control experiment, i.e. cells treated without VacA for 0 min. There is no change in phosphorylation of p38, Erk1/2, and JNK during incubation. Data are representative of three experiments.

 
VacA Induces p38 Phosphorylation and Activation of Its Kinase Activity—Quantification of effects of VacA indicated a concentration-dependent increase in p38 phosphorylation after 30-min incubation (Fig. 2A). The difference between VacA-treated and untreated cells in p38 phosphorylation was not due to a difference in amounts of p38 protein, which were not significantly different in these two populations of cells, as assessed by Western blot analysis. p38 MAPK phosphorylation in AZ-521 cells treated with 120 nM VacA for 60 min was completely inhibited by addition of 10 µg of anti-VacA IgG (Fig. 2B). p38 kinase activity, assayed in vitro with ATF-2 as substrate after immunoprecipitation from cells incubated with 240 nM VacA for the same times (Fig. 3), paralleled the amounts of phosphorylated p38 (Fig. 3) (31).



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  		FIG. 2.
p38 phosphorylation induced by VacA in AZ-521 cells. A, concentration dependence. Cells were incubated with 0, 12, 120, or 240 nM VacA for 30 min and then in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 µM sodium pyrophosphate, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin for 10 min on ice. After centrifugation (15 min at 15,000 x g), samples of supernatants (20 µg of protein) were subjected to SDS-PAGE and Western blotting using anti-phospho-p38 or anti-p38 antibody. B, effect of anti-VacA antibody. Cells were incubated with 120 nM VacA without (top two panels) or with anti-VacA antibody (bottom two panels) for the indicated time before preparation of cell lysates for Western blot analyses using anti-phospho-p38 antibody and anti-p38 antibody. Data are representative of three experiments.

 



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  		FIG. 3.
VacA activates the kinase activity of p38 MAPK in AZ-521 cells. AZ-521 cells were grown overnight in medium without fetal calf serum. Cells were incubated without (bottom) or with 240 nM VacA (top) for 0, 10, 30, or 60 min. VacA-stimulated cells were solubilized and immunoprecipitated with anti-phospho-p38 antibody. Immunoprecipitates were incubated with 200 µM ATP and 2 µg of ATF-2 fusion protein for 30 min at 30 °C and then analyzed by Western blotting using anti-phospho-ATF-2 antibody. Data are representative of three experiments.

 
Phosphorylation of p38 induced by VacA was blocked by a specific inhibitor of p38 kinase activity, SB203580, in a concentration-dependent manner (10 and 50 µM) (Fig. 4). Among the p38 subfamily of MAPKs, p38{alpha} and p38{beta} can be specifically inhibited by SB203580 through its binding in the ATP pocket (32), whereas p38{gamma} and p38{delta} are not sensitive to this compound (33). Therefore, it appears that VacA induced the phosphorylation of p38{alpha} and/or p38{beta} in AZ-521 cells. Since it is known that 10 µM SB203580 inhibits activity of p38 but not that of JNK or ERK (34), we used 10 µM SB203580 to inhibit p38 phosphorylation induced by VacA.



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  		FIG. 4.
Effect of SB203580 on p38 phosphorylation induced by VacA. Cells were incubated with 0, 10, or 50 µM SB203580 for 30 min and then with 120 nM VacA for 0, 5, or 10 min. Cells were solubilized for 10 min on ice in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. After centrifugation (15 min at 15,000 x g), samples of supernatants (20 µg of protein) were subjected to SDS-PAGE and Western blotting using anti-phospho-p38 or anti-p38 antibody. Data are representative of three experiments.

 
Vacuolization, Mitochondrial Membrane Potential Change, and Cytochrome c Release Caused by VacA Are Not Associated with p38 Phosphorylation—It has been reported that VacA causes vacuolization of target cells through a mechanisms involving the vacuolar ATPase proton pump (35). Our previous work showed that the change in mitochondrial membrane potential by VacA is independent of vacuolization using bafilomycin A1, a specific inhibitor of the vacuolar ATPase proton pump (2). First, to determine whether the stimulation of p38 phosphorylation by VacA led to vacuolization, AZ-521 cells were treated with 10 µM SB203580 for 30 min before incubation with 120 nM VacA for 0-8 h (Fig. 5). Incubation of AZ-521 cells with VacA without prior exposure to 10 µM SB203580 resulted in vacuolization in a time-dependent manner. The increase in vacuolization was similar in cells treated with 10 µM SB203580 before incubation with VacA.



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  		FIG. 5.
Effect of SB203580 on vacuolization induced by VacA. AZ-521 cells were incubated with 10 µM SB203580 for 30 min and then with 120 nM VacA for 0, 2, 4, or 8 h before quantification of vacuolation by neutral red assay. Data are means ± S.D. of values from three separate experiments with assays in duplicate.

 
As VacA induced a decrease in mitochondrial membrane potential (2), we incubated AZ-521 cells with VacA for 1 h after treatment with 10 µM SB203580 for 30 min (Table I). Either 120 nM VacA or an uncoupling agent (5 µM carbonyl cyanide m-chlorophenylhydrazone) diminished the mitochondrial potential to a similar extent as previously reported (2). SB203580 did not significantly inhibit the decrease in mitochondrial potential caused by VacA. A recent report showed that VacA or its 34-kDa fragment induced cytochrome c release from mitochondria and cleavage of nuclear poly(ADP-ribose) polymerase (3). We examined, therefore, whether cytochrome c release from mitochondria in gastric epithelial cells exposed to VacA is related to p38 MAPK phosphorylation (Fig. 6). As reported by Galmiche et al. (3), treatment with VacA induced cytochrome c release from mitochondria of AZ-521 cells. This effect was achieved in cells pretreated with 10 µM SB203580 followed by treatment with 120 nM VacA for 24 h. Thus, other signal mechanisms that did not involve p38 might contribute to the cytochrome c release by VacA.


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  		TABLE I
Effect of SB203580 on the decrease in mitochondrial membrane potential induced by VacA

A2-521 cells were grown overnight in medium without FCS. Cells were preincubated with 10 µM SB203580 for 30 min and further incubated with 120 nM VacA for 1 h. A fluorescence probe of mitochondrial membrane potential, 0.1 µM 3,3'-dihexyloxacarbocyanine iodide, was added to the cells, and cells were incubated for 30 min. As a control, cells were treated with 5 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupling agent. Cells were washed with PBS, suspended in PBS, and then used for FACScan analysis with excitation at 488 nm and emission at 530 nm. Data are representative of three experiments.

 



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  		FIG. 6.
Effect of SB203580 on cytochrome c release from mitochondria induced by VacA. AZ-521 cells were incubated with 10 µM SB203580 for 30 min and then with 120 nM VacA for 24 h before lysis and preparation of mitochondrial (A) and cytosol (B) fractions for determination of cytochrome c as described under "Materials and Methods." Data are representative of three experiments.

 
VacA Activates ATF-2 through Activation of the p38 Pathway—Having shown an increase in ATF-2 phosphorylation in vitro by immunoprecipitated, activated p38 from VacA-treated cells (Fig. 3), we examined the effect of VacA on ATF-2 in intact cells. Phosphorylation of p38 and ATF-2 were clearly evident within 10-30 min of VacA induction with a similar time course by Western blotting (Fig. 7A). Immunostaining analysis of phospho-ATF-2 in intact cells showed that the level of phosphorylated ATF-2 increased after exposure of AZ-521 cells to VacA for 10-30 min and declined thereafter (Fig. 7B). VacA-induced ATF-2 phosphorylation was significantly blocked by prior incubation of cells with SB203580 (data not shown). Taken together, findings from Figs. 3 and 7 suggest that VacA activates ATF-2 through activation of the p38 pathway.



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  		FIG. 7.
ATF-2 phosphorylation in AZ-521 cells treated with VacA. A, cells were incubated with 120 nM VacA for 0, 10, 30, 60, or 120 min before cell lysates were prepared. Cell lysates (20 µg of protein) were subjected to Western blot analyses using anti-phospho-p38 and anti-phospho-ATF-2 antibodies. Cont. refers to the control experiment, i.e. cells treated without VacA for 0 min. There is no change in phosphorylation of p38 and ATF-2 during incubation. B, AZ-521 cells were incubated with 120 nM VacA (upper panels, a-d) or without VacA (lower panels, e-h) for 0, 10, 30, or 60 min before fixation with 2% paraformaldehyde in PBS, staining with anti-phospho-ATF-2 antibody, and reaction with Cy-3-conjugated anti-rabbit IgG antibody. Data are representative of three experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
VacA is known to cause a number of metabolic, regulatory, and morphologic changes in susceptible cells. Although the mechanism by which VacA exerts its cytotoxic effects is not known, it is believed to involve disruption of normal membrane trafficking at or near the level of late endosomes (36-40) as well as pore formation in cellular membranes (41). VacA also induced mitochondrial damage and inhibition of energy metabolism in AZ-521 cells (2). Overexpression of a 34-kDa fragment of VacA in HeLa cells induced cytochrome c release and the cleavage of poly(ADP-ribose) polymerase, known to be a specific substrate for caspase 3, leading to apoptotic cell death (3). VacA applied externally to cells induced release of cytochrome c into the cytosol without induction of a significant cleavage of poly(ADP-ribose) polymerase (3). Studies of a VacA-null mutant (Tox-) strain suggested that VacA as well as the Cag pathogenicity island of H. pylori contributed to apoptosis of AGS cells (42). Furthermore a recombinant VacA lacking vacuolating activity induced the apoptotic response of AGS cells (4), and acid-activated VacA also induced AGS cell apoptosis in the presence of supplemental ammonium chloride (43). These data suggest that VacA-induced vacuolation and apoptosis may proceed by different pathways. Cell loss caused by H. pylori-induced apoptosis could potentially accelerate the development of gastric atrophy or intestinal metaplasia (44)

The signaling pathways involved in apoptotic events have been intensively studied, including the role of key regulatory proteins such as caspases, Bcl-2 family of proteins, and MAP kinases including JNK and p38 in stress conditions-induced apoptotic signaling. (45-47). As shown in Fig. 1, VacA induced increased phosphorylation of p38 and Erk1/2 but not JNK. Since p38 inhibition did not prevent cytochrome c release and depression of mitochondrial membrane potential by VacA, those aspects of VacA toxicity appear to be independent of p38 activation in AZ-521 cells.

By and large, Erk1/2 activation has been linked to cell survival, whereas activation of p38 and JNK is linked to induction of apoptosis (48). ATF-2 is activated both by JNK/stress-activated protein kinase and p38 kinases and is involved in the transcriptional regulation of a number of oncogenes and cell cycle genes (49, 50). As shown in Fig. 7, VacA induced p38 activation and ATF-2 phosphorylation in AZ-521 cells without any change in JNK (Fig. 1). Ronai and coworkers (51, 52) reported that ATF-2 contributed to the resistance of human melanoma cells to apoptosis induced by UV irradiation or chemical treatment by altering the balance between tumor necrosis factor and Fas death signaling cascades and cooperated with v-Jun in promoting proliferation and tumor formation (53). p38 has been implicated in the up-regulation of several genes involved in promoting cell proliferation and tumor progression, including COX-2 and cyclin D1, through the activation of transcriptional factors such as AP-1, cAMP-response element-binding protein, and ATF-2 (54-56).

The roles of the MAP kinase cascade in cell death or survival are highly dependent on cell type and state (57). Another potential signaling pathway for VacA-induced apoptosis is from Erk1/2 through the Bcl-2 family of proteins (Bax, Bak, Bcl-XS, and Bok), which can promote cell death (58). Caputo et al. (59) recently reported that a Tox+ H. pylori strain, but not a Tox- strain, stimulated Erk2 phosphorylation in MKN 28 cells.

H. pylori-stimulated host inflammatory immune responses lead to release of large amounts of cytokines such as tumor necrosis factor-{alpha} and interferon-{gamma}, markedly potentiating apoptosis (60). Numerous molecules produced by H. pylori including VacA, lipopolysaccharide, monochloramine, and nitric oxide may directly induce apoptosis (60). A crucial role for p38 in the inflammation associated with H. pylori-induced gastritis was proven in experimentally infected animals (23). One of the elements involved in the pathogenesis of H. pylori, neutrophil-activating protein, activated neutrophils through activation of p38 and Erk but not JNK (25). Thus, p38 activation by VacA may be involved in the inflammatory response during H. pylori infection. It appears, however, that p38 MAPK phosphorylation by VacA is independent of vacuolization, mitochondrial membrane potential change, and cytochrome c release. Thus, VacA may be responsible for the activation of several independent signaling pathways.


   FOOTNOTES
 
* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Sports, Science, and Technology of Japan and from the Uehara Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

¶¶ To whom correspondence should be addressed. Tel.: 81-95-849-7831; Fax: 81-95-849-7805; E-mail: hirayama{at}net.nagasaki-u.ac.jp.

1 The abbreviations used are: MAPK, mitogen-activated protein kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; ATF-2, activating transcription factor 2; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank I. Kato (Medical School of Chiba University) for helpful discussions and Martha Vaughan (National Institutes of Health, Bethesda, MD) for discussion and critical review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Leunk, R. D., Johnson, P. T., David, B. C., Kraft, W. G., and Morgan, D. R. (1988) J. Med. Microbiol. 26, 93-99[Abstract/Free Full Text]
  2. Kimura, M., Goto, S., Wada, A., Yahiro, K., Niidome, T., Hatakeyama, T., Aoyagi, H., Hirayama, T., and Kondo, T. (1999) Microb. Pathog. 26, 45-52[CrossRef][Medline] [Order article via Infotrieve]
  3. Galmiche, A., Rassow, J., Doye, A., Cagnol, S., Chambard, J. C., Contamin, S., de Thillot, V., Just, I., Ricci, V., Solcia, E., Van Obberghen, E., and Boquet, P. (2000) EMBO J. 19, 6361-6370[CrossRef][Medline] [Order article via Infotrieve]
  4. Kuck, D., Kolmerer, B., Iking-Konert, C., Krammer, P. H., Stremmel, W., and Rudi, J. (2001) Infect. Immun. 69, 5080-5087[Abstract/Free Full Text]
  5. Phadnis, S. H., Ilver, D., Janzon, L., Normark, S., and Westblom, T. U. (1994) Infect. Immun. 62, 1557-1565[Abstract/Free Full Text]
  6. Telford, J. L., Ghiara, P., Dell'Orco, M., Comanducci, M., Burroni, D., Bugnoli, M., Tecce, M. F., Censini, S., Covacci, A., Xiang, Z., Papini, E., Montecucco, C., Parente, L., and Rappuoli, R. (1994) J. Exp. Med. 179, 1653-1658[Abstract/Free Full Text]
  7. Marchetti, M., Arico, B., Burroni, D., Figura, N., Rappuoli, R., and Ghiara, P. (1995) Science 267, 1655-1658[Abstract/Free Full Text]
  8. Eaton, K. A., Cover, T. L., Tummuru, M. K., Blaser, M. J., and Krakowka, S. (1997) Infect. Immun. 65, 3462-3464[Abstract]
  9. Supajatura, V., Ushio, H., Wada, A., Yahiro, K., Okumura, K., Ogawa, H., Hirayama, T., and Ra, C. (2002) J. Immunol. 22, 2603-2606
  10. Fujikawa, A., Shirasaka, D., Yamamoto, S., Ota, H., Yahiro, K., Fukada, M., Shintani, T., Wada, A., Aoyama, N., Hirayama, T., Fukamachi, H., and Noda, M. (2003) Nat. Genet. 33, 375-381[CrossRef][Medline] [Order article via Infotrieve]
  11. Manetti, R., Massari, P., Burroni, D., de Bernard, M., Marchini, A., Olivieri, R., Papini, E., Montecucco, C., Rappuoli, R., and Telford, J. L. (1995) Infect. Immun. 63, 4476-4480[Abstract]
  12. de Bernard, M., Burroni, D., Papini, E., Rappuoli, R., Telford, J., and Montecucco, C. (1998) Infect. Immun. 66, 6014-6016[Abstract/Free Full Text]
  13. Ye, D., Willhite, D. C., and Blanke, S. R. (1999) J. Biol. Chem. 274, 9277-9282[Abstract/Free Full Text]
  14. Reyrat, J. M., Lanzavecchia, S., Lupetti, P., de Bernard, M., Pagliaccia, C., Pelicic, V., Charrel, M., Ulivieri, C., Norais, N., Ji, X., Cabiaux, V., Papini, E., Rappuoli, R., and Telford, J. L. (1999) J. Mol. Biol. 290, 459-470[CrossRef][Medline] [Order article via Infotrieve]
  15. Ye, D., and Blanke, S. R. (2000) Infect. Immun. 68, 4354-4357[Abstract/Free Full Text]
  16. Wagner, S., Beil, W., Westermann, J., Logan, R. P., Bock, C. T., Trautwein, C., Bleck, J. S., and Manns, M. P. (1997) Gastroenterology 113, 1836-1847[CrossRef][Medline] [Order article via Infotrieve]
  17. Jones, N. L., Shannon, P. T., Cutz, E., Yeger, H., and Sherman, P. M. (1997) Am. J. Pathol. 151, 1695-1703[Abstract]
  18. Houghton, J., Korah, R. M., Condon, M. R., and Kim, K. H. (1999) Dig. Dis. Sci. 44, 465-478[CrossRef][Medline] [Order article via Infotrieve]
  19. Houghton, J., Macera-Bloch, L. S., Harrison, L., Kim, K. H., and Korah, R. M. (2000) Infect. Immun. 68, 1189-1195[Abstract/Free Full Text]
  20. Keates, S., Keates, A. C., Warny, M., Peek, R. M., Jr., Murray, P. G., and Kelly, C. P. (1999) J. Immunol. 163, 5552-5559[Abstract/Free Full Text]
  21. Pomorski, T., Meyer, T. F., and Naumann, M. (2001) J. Biol. Chem. 276, 804-810[Abstract/Free Full Text]
  22. Kim, J. S., Kim, J. M., Jung, H. C., and Song, I. S. (2001) Dig. Dis. Sci. 46, 2277-2284[CrossRef][Medline] [Order article via Infotrieve]
  23. Takahashi, S., Keto, Y., Fujita, T., Uchiyama, T., and Yamamoto, A. (2001) J. Pharmacol. Exp. Ther. 296, 48-56[Abstract/Free Full Text]
  24. Nishioka, H., Baesso, I., Semenzato, G., Trentin, L., Rappuoli, R., Del Giudice, G., and Montecucco, C. (2003) Eur. J. Immunol. 33, 840-849[CrossRef][Medline] [Order article via Infotrieve]
  25. Del Giudice, G., Covacci, A., Telford, J. L., Montecucco, C., and Rappuoli, R. (2001) Annu. Rev. Immunol. 19, 523-563[CrossRef][Medline] [Order article via Infotrieve]
  26. Yahiro, K., Niidome, T., Hatakeyama, T., Aoyagi, H., Kurazono, H., Padilla, P. I., Wada, A., and Hirayama, T. (1997) Biochem. Biophys. Res. Commun. 238, 629-632[CrossRef][Medline] [Order article via Infotrieve]
  27. Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699[Abstract/Free Full Text]
  28. Nagata, H., Wada, A., Kurazono, H., Yahiro, K., Shirasaka, D., Ikemura, T., Aoyama, N., Ping, W. A., Makiyama, K., Kohno, S., and Hirayama, T. (1999) Microb. Pathog. 26, 103-110[CrossRef][Medline] [Order article via Infotrieve]
  29. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  30. Vayssiere, J. L., Petit, P. X., Risler, Y., and Mignotte, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11752-11756[Abstract/Free Full Text]
  31. Livingstone, C., Patel, G., and Jones, N. (1995) EMBO J. 14, 1785-1797[Medline] [Order article via Infotrieve]
  32. Gum, R. J., McLaughlin, M. M., Kumar, S., Wang, Z., Bower, M. J., Lee, J. C., Adams, J. L., Livi, G. P., Goldsmith, E. J., and Young, P. R. (1998) J. Biol. Chem. 273, 15605-15610[Abstract/Free Full Text]
  33. Goedert, M., Cuenda, A., Craxton, M., Jakes, R., and Cohen, P. (1997) EMBO J. 16, 3563-3571[CrossRef][Medline] [Order article via Infotrieve]
  34. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233[CrossRef][Medline] [Order article via Infotrieve]
  35. Cover, T. L., Reddy, L. Y., and Blaser, M. J. (1993) Infect. Immun. 61, 1427-1431[Abstract/Free Full Text]
  36. Papini, E., de Bernard, M., Milia, E., Bugnoli, M., Zerial, M., Rappuoli, R., and Montecucco, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9720-9724[Abstract/Free Full Text]
  37. Molinari, M., Galli, C., Norais, N., Telford, J. L., Rappuoli, R., Luzio, J. P., and Montecucco, C. (1997) J. Biol. Chem. 272, 25339-25344[Abstract/Free Full Text]
  38. Papini, E., Satin, B., Bucci, C., de Bernard, M., Telford, J. L., Manetti, R., Rappuoli, R., Zerial, M., and Montecucco, C. (1997) EMBO J. 16, 15-24[CrossRef][Medline] [Order article via Infotrieve]
  39. Suzuki, J., Ohnishi, H., Shibata, H., Wada, A., Hirayama, T., Irii, T., Ueda, N., Kanamura, C., Tsuchida, T., Mashima, H., Yasuda, H., and Fujita, T. (2001) J. Clin. Investig. 107, 15200-15206
  40. Suzuki, J., Ohnishi, H., Wada, A., Hirayama, T., Ohno, H., Ueda, N., Yasuda, H., Iiri, T., Wada, Y., Futai, M., and Mashima, H. (2003) J. Biol. Chem. 278, 25585-25590[Abstract/Free Full Text]
  41. Czajkowsky, D. M., Iwamoto, H., Cover, T. L., and Shao, Z. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2001-2006[Abstract/Free Full Text]
  42. Peek, R. M., Jr., Blaser, M. J., Mays, D. J., Forsyth, M. H., Cover, T. L., Song, S. Y., Krishna, U., and Pietenpol, J. A. (1999) Cancer Res. 59, 6124-6131[Abstract/Free Full Text]
  43. Cover, T. L., Krishna, U. S., Israel, D. A., and Peek, R. M., Jr. (2003) Cancer Res. 63, 951-957[Abstract/Free Full Text]
  44. Correa, P., and Miller, M. J. (1989) Br. Med. Bull. 54, 151-162
  45. Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305-1308[Abstract/Free Full Text]
  46. Evan, G., and Littlewood, T. (1998) Science 281, 1317-1321[Abstract/Free Full Text]
  47. Green, D. R., and Reed, J. C. (1998) Science 281, 1309-1316[Abstract/Free Full Text]
  48. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract/Free Full Text]
  49. van Dam, H., Duyndam, M., Rottier, R., Bosch, A., de Vries-Smits, L., Herrlich, P., Zantema, A., Angel, P., and van der Eb, A. J. (1993) EMBO J. 12, 479-487[Medline] [Order article via Infotrieve]
  50. Tsai, E. Y., Yie, J., Thanos, D., and Goldfeld, A. E. (1996) Mol. Cell. Biol. 16, 5232-5244[Abstract]
  51. Ivanov, V. N., and Ronai, Z. (1999) J. Biol. Chem. 274, 14079-14089[Abstract/Free Full Text]
  52. Ronai, Z., Yang, Y. M., Fuchs, S. Y., Adler, V., Sardana, M., and Herlyn, M. (1998) Oncogene 16, 523-531[CrossRef][Medline] [Order article via Infotrieve]
  53. Huguier, S., Baguet, J., Perez, S., van Dam, H., and Castellazzi, M. (1998) Mol. Cell. Biol. 18, 7020-7029[Abstract/Free Full Text]
  54. Lee, R. J., Albanese, C., Stenger, R. J., Watanabe, G., Inghirami, G., Haines, G. K., III, Webster, M., Muller, W. J., Brugge, J. S., Davis, R. J., and Pestell, R. G. (1999) J. Biol. Chem. 274, 7341-7350[Abstract/Free Full Text]
  55. Ding, M., Shi, X., Dong, Z., Chen, F., Lu, Y., Castranova, V., and Vallyathan, V. (1999) J. Biol. Chem. 274, 30611-30616[Abstract/Free Full Text]
  56. Recio, J. A., and Merlino, G. (2002) Oncogene 21, 1000-1008[CrossRef][Medline] [Order article via Infotrieve]
  57. Chang, L., and Karin, M. (2001) Nature 410, 37-40[CrossRef][Medline] [Order article via Infotrieve]
  58. Adams, J. M., and Cory, S. (1998) Science 281, 1322-1325[Abstract/Free Full Text]
  59. Caputo, R., Tuccillo, C., Manzo, B. A., Zarrilli, R., Tortora, G., Blanco Cdel, V., Ricci, V., Ciardiello, F., and Romano, M. (2003) Clin. Cancer Res. 9, 2015-2021[Abstract/Free Full Text]
  60. Xia, H. H., and Talley, N. J. (2001) Am. J. Gastroenterol. 96, 16-26[Medline] [Order article via Infotrieve]

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