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*
Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
Division of Gastroenterology, Vanderbilt University Medical Center, Nashville, TN 37232; and
School of Health Sciences, University of Wolverhampton, Wolverhampton, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
degradation or NF-B activation.
Thus, H. pylori rapidly activates ERK, p38, and JNK MAP
kinases in gastric epithelial cells; cag+
isolates are more potent than cag- strains
in inducing MAP kinase phosphorylation and gene products of the
cag pathogenicity island are required for maximal MAP
kinase activation. p38 and MEK-1 activity are required for H.
pylori-induced IL-8 production, but do not appear to be
essential for H. pylori-induced NF-B activation.
Since MAP kinases regulate cell proliferation, differentiation,
programmed death, stress, and inflammatory responses, activation of
gastric epithelial cell MAP kinases by H. pylori
cag+ strains may be instrumental in inducing
gastroduodenal inflammation, ulceration, and
neoplasia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAP kinases are a family of ubiquitous, highly conserved, cell signaling molecules (7, 8, 9). Upon activation by upstream kinases, MAP kinases phosphorylate downstream kinases and/or mediators, including transcription factors. MAP kinases can be activated by a wide variety of extracellular stimuli and transmit signals from the cell surface to the nucleus to regulate gene expression. Key cellular functions that are regulated at least in part by MAP kinase signaling include cell proliferation, cell survival, and cytokine production. Three main groups of MAP kinases have been characterized to date: the extracellular signal-regulated kinases (ERK), the c-Jun N-terminal kinases (JNK), and the p38 MAP kinases (7, 8, 9). These MAP kinase subfamilies form three parallel cascades that can be activated simultaneously or independently. ERK MAP kinases are strongly activated by growth factors and phorbol ester, but weakly by inflammatory stimuli. In contrast, JNK and p38 MAP kinases are stimulated by inflammatory cytokines and stress stimuli, but minimally by growth factors.
We and others have shown previously that H. pylori infection
activates IL-8 gene expression in gastric epithelial cells in vitro and
in vivo (10, 11, 12, 13, 14, 15, 16). IL-8 mRNA and protein levels are
increased in the gastric mucosa of patients with H. pylori
gastritis, and immunohistochemical studies demonstrate increased IL-8
protein in gastric epithelial cells from infected individuals
(11, 13, 16, 17). IL-8 is a potent neutrophil-activating
chemotactic cytokine or chemokine. Thus, IL-8 release by infected
gastric epithelial cells may be instrumental in regulating neutrophil
infiltration of the gastric mucosa in H. pylori gastritis.
H. pylori also increases IL-8 mRNA levels and protein
production in cultured monolayers of AGS and other gastric epithelial
cell lines (10, 12, 13, 15, 18). Contact with the
bacterium initiates epithelial cell signaling events that result in
activation of the transcription factor NF-B (10, 14, 15, 19, 20, 21, 22). Activated NF-B then translocates to the nucleus
where it up-regulates IL-8 gene transcription. NF-B activation
occurs within minutes of H. pylori infection of AGS gastric
epithelial cells in vitro, and activated NF-B is also evident in
gastric epithelial cells from patients with H. pylori
gastritis (10, 14, 15, 19, 22).
The cag pathogenicity island of H. pylori is a
40-kb region immediately upstream to the cagA gene that
encodes over 40 putative bacterial proteins (12). Based on
sequence homology, cag region gene products appear to
constitute a bacterial secretion system that may be involved in the
export or surface expression of bacterial virulence factors. In the
developed world, approximately 70% of clinical H. pylori
isolates carry the cag pathogenicity island. Carriage of
cag+ strains has been associated in most
published studies with more severe gastritis and a greater risk for
peptic ulceration and gastric cancer than H. pylori
cag- infection (6, 12, 23, 24, 25, 26).
Gene products of the cag pathogenicity island are also known
to participate in epithelial cell activation by H. pylori. H.
pylori cag+ strains are more potent in activating
epithelial cell IL-8 production than cag-
bacteria (16, 27). Furthermore, disruption of specific
cag region genes markedly reduces H.
pylori-mediated tyrosine phosphorylation of gastric epithelial
cell proteins, NF-B activation, and IL-8 gene transcription
(12, 14, 19, 21, 22, 28).
A number of recent studies implicate MAP kinases as upstream mediators
of NF-B activation and cytokine gene expression (9, 29, 30, 31, 32, 33, 34). This led us to examine whether MAP kinases participate
in regulating H. pylori-induced IL-8 production by gastric
epithelial cells. The aims of this study were to determine whether
H. pylori activates MAP kinases in AGS gastric epithelial
cells, whether MAP kinase activation is required for H.
pylori-mediated NF-B activation and IL-8 production, and
whether cag+ and
cag- strains of H. pylori
differ in their ability to activate epithelial cell MAP kinases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AGS gastric epithelial cells (American Type Culture Collection, Manassas, VA) were grown in Ham’s F-12 medium (pH 7.4; Sigma, St. Louis, MO) supplemented with 10% FBS, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (15). MKN-28 cells (provided by R. Peek, Jr., Nashville TN) were grown in DMEM with 10% FBS, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate. All cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cell culture experiments were conducted in 6-, 12-, or 96-well polypropylene tissue culture plates (Corning Costar, Cambridge, MA).
H. pylori strains, clinical isolates, and isogenic mutants
H. pylori were plated onto Brucella agar supplemented with 5% horse blood (BBL, Becton Dickinson Microbiology, Cockeysville, MD) and incubated at 37°C in a microaerophilic environment (15, 35). After 3–7 days the bacteria were harvested into pyrogen-free Dulbecco’s PBS (Cellgro, Mediatech, Herndon,VA). The bacteria were pelleted by centrifugation at 4000 x g for 10 min, and bacterial numbers were determined by resuspension in PBS to an OD600 nm of 1.5, corresponding to 3.6 x 108 CFU/ml as described previously (15). Defined numbers of bacteria were then resuspended in antibiotic-free Ham’s F-12 medium. Unless otherwise stated experiments were performed using the cagA+ and vacA+ H. pylori strain 43504 (American Type Culture Collection) (15).
H. pylori clinical strains were isolated from gastric mucosal biopsies obtained during upper gastrointestinal endoscopy as previously described (16). The presence of cagA was determined by PCR of bacterial genomic DNA and vacuolating cytotoxin activity was determined by neutral red assay (16, 36). All of the cag+ clinical isolates used in this study were toxigenic, while the cag- strains did not produce a functional cytotoxin. Isogenic H. pylori mutants lacking the picB or cagA genes were also studied together with their parental cag+, toxigenic, wild-type strain (no. 60190) (28). The H. pylori clinical isolates, strain 60190, and the picB- and cagA- mutants were obtained from the culture collection of the Vanderbilt University Campylobacter and Helicobacter Laboratory (Nashville, TN) and have been described previously (16, 28).
H. pylori filtrate was prepared by suspending the bacteria in antibiotic-free medium for 30 min at room temperature, pelleting the bacteria at 4000 x g for 10 min and then filtering the medium through a 0.2-µm pore size filter (Acrodisc, Gelman, Ann Arbor, MI). In some experiments H. pylori were heat treated for 30 min by boiling or were treated with 2 or 20 µg/ml chloroamphenicol (Amersham, Arlington Heights, IL) for 60 min at 37°C.
Western blot analysis of phospho-specific MAP kinase activation
AGS cells were grown to confluence on twelve-well plates and maintained in serum-free medium for 24 h before the experiment. Cells were fed with fresh serum-free medium 2 h before stimulation. At the end of the experiment the monolayers were washed three times with PBS and lysed with SDS buffer (containing 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol, 50 mM DTT, 0.1% (w/v) bromophenol blue). Samples were then sonicated, heated to 100°C for 5 min, and loaded onto a 10% SDS-PAGE gel. After running the gel, the proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked for 3 h at room temperature with a 5% (w/v) solution of dried milk in Tris-buffered saline (pH 7.4). This was followed by an overnight incubation at 4°C with the phospho-specific MAP kinase Abs diluted 1/1000 in blocking buffer. The membranes were then washed three times with Tris-buffered saline and incubated at room temperature for 1 h with peroxidase-conjugated goat anti-rabbit IgG (1/2000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA). A SuperSignal chemiluminescent substrate (Pierce, Rockford, IL) was used for detection.
Phospho-specific p44/p42 MAP kinase Ab was used to detect ERK1/2. This Ab detects p44 and p42 MAP kinase (ERK1 and ERK2) only when they are catalytically activated by phosphorylation at Thr202 and Tyr204. Phospho-specific p38 MAP kinase Ab was used to detect p38 activated by phosphorylation at Thr180 and Tyr182. Phospho-specific p54/p46 MAP kinase Ab was used to detect JNK. This Ab detects p54 and p46 MAP kinase only when they are activated by phosphorylation at Thr183 and Tyr185. All three Abs were obtained from New England Biolabs (Beverly, MA). Nonphosphospecific Abs to ERK, p38, and JNK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Controls for these Western blot experiments consisted of AGS cells harvested at the zero time point for kinetic experiments or cultured in the absence of H. pylori and harvested at the same time as the test cells (usually after 1 h of incubation) for all other experiments.
In vitro kinase assay
Confluent monolayers of AGS cells were lysed in 1 ml of lysis
buffer (150 mmol NaCl, 1% Nonidet P-40, 0.8 mmol
MgCl2, 5 mmol EGTA, 1 mmol
Na3VO4, 15 µg/ml
leupeptin, 1 mmol PMSF, and 50 mmol HEPES, pH 7.5). ERK2 (p42), p38, or
JNK1 (p46) MAP kinases were precipitated with 2 µg of rabbit specific
IgG (Santa Cruz Biotechnology). After incubation for 2 h at 4°C,
20 µl of protein G-Sepharose (Santa Cruz Biotechnology) were added,
and samples were further incubated for 1 h. Immunopellets were
washed twice in lysis buffer, twice in kinase buffer (30 mM NaCl, 0.1
mM Na3VO4, 2 mM DTT, 20 mM
MgCl2, and 30 mmol HEPES, pH 7.5.) and then
resuspended in 40 µl of kinase buffer. The kinase reaction was
started by addition of 20 µM ATP, 100 µCi/ml
[
-32P]ATP (DuPont-NEN, Boston, MA), and 10
µg myelin basic protein (Sigma) as substrate for ERK and p38, and 2
µg of GST c-Jun1–79 (Stratagene, La Jolla, CA)
as substrate for JNK1. Samples were subjected to SDS-PAGE (12%) and
analyzed by autoradiography.
Treatment of AGS cells with specific MAP kinase inhibitors
In some experiments AGS cells were treated with the specific p38 MAP kinase inhibitor SB203580 (10 µM; Calbiochem, La Jolla, CA) for 30 min before exposure to H. pylori and during the incubation period of the experiment (37). A similar protocol was used for experiments using the MAP kinase inhibitor PD98059 (25 µM; Calbiochem). PD98059 blocks ERK1/2 activation by specifically inhibiting MEK1, the kinase that catalyzes ERK1/2 phosphorylation (38).
IL-8 protein levels in AGS cell-conditioned medium were measured by ELISA as previously described (15, 35, 39). Statistical analyses were performed using SigmaStat for Windows version 2.0 (Jandel Scientific Software, San Rafael, CA). Unless stated otherwise, ANOVA followed by protected t tests were used for intergroup comparisons.
Western blot analysis of IB degradation
AGS cells were pretreated with the specific MAP kinase
inhibitors PD98059 (25 µM) and/or SB203580 (10 µM) for 30 min
preceding infection with H. pylori
(108 bacteria/ml). Incubation of AGS cells with
H. pylori was maintained for 1 h in the presence of the
inhibitors, after which time the cells were washed three times with PBS
and lysed. Equal amounts of protein were loaded onto a 10% SDS-PAGE,
the gel was run, and the proteins were then transferred to
nitrocellulose membranes. The blots were blocked for 1 h in a 5%
(w/v) solution of dried milk in Tris-buffered saline solution
containing 0.1% Tween-20, followed by immunoblotting with a rabbit
polyclonal Ab against IB (Santa Cruz Biotechnology) at a 1/1000
dilution. After washing the blot three times with Tris-buffered saline,
a peroxidase-conjugated goat anti-rabbit Ab (Jackson ImmunoResearch
Laboratories) at a 1/2000 dilution was applied. Immunoreactive bands of
the 36-kDa IB protein were detected using SuperSignal
chemiluminescent substrate.
EMSA for NF-B activation
AGS cells were pretreated for 30 min with the MAP kinase inhibitors PD98059 (25 µM) and/or SB203580 (10 µM). The AGS monolayers were then infected with H. pylori for 1 h. To prepare nuclear extracts AGS cells were washed three times with ice-cold PBS and scraped in ice-cold TNE buffer (40 mmol/L Tris-HCl (pH 7.5), 0.15 mol/L NaCl, and 1 mmol/L EDTA). Cells were pelleted by centrifugation and then resuspended in 400 µL of buffer A (10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L ethylene glycol-bis(B-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, and 0.5 mmol/L PMSF), and incubated on ice for 10 min. Nonidet P-40 (23 µl of a 10% solution) was added, and the cells were incubated for an additional 2 min on ice. After centrifugation at 12,000 x g for 5 s, the nuclear pellet was resuspended in 85 µl of buffer B (20 mmol/L HEPES (pH 7.9), 0.42 mol/L KCl, 1 mmol/L EDTA, 1 mmol/L ethylene glycol-bis(B-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, and 0.1 mmol/L PMSF) and incubated on ice for 30 min. After a further centrifugation at 12,000 x g, for 2 min, the supernatants were recovered as nuclear extracts that were immediately frozen on dry ice and stored at -80°C.
Single-stranded complementary oligonucleotides bearing the human IL-8
gene NF-B site were prepared by custom oligonucleotide synthesis
(Genosys Biotechnologies, The Woodlands, TX). After annealing, 100 ng
of the double-stranded oligonucleotide was labeled in a Klenow fill-in
reaction in the presence of [-32P]dCTP. The
probe was then purified on a Sephadex G-25 spin column (Boehringer
Mannheim, Indianapolis, IN) and diluted to yield approximately 15,000
cpm and 0.1 ng of DNA/µL. Binding reactions (20 µl) contained 0.1
ng (
15,000 cpm) of double-stranded probe, 5–12 µg of extracted
protein (the protein concentration of the samples were adjusted to
ensure equal loading), 2 µg of poly(dI-dC) (Pharmacia, Piscataway,
NJ), 10 mmol/L 2-ME, and 1% Ficoll. After first incubating the protein
extracts for 10 min at room temperature, the radiolabeled probe was
added. After an additional 30 min at room temperature, the reaction
mixtures were then loaded on a nondenaturing 6% polyacrylamide gel in
0.2 mol/L glycine, 25 mmol/L Tris-HCl, and 1 mmol/L EDTA. The gel was
run, dried, and exposed to autoradiography film for 6–18 h at -80°C
with an intensifying screen.
Supershift assays with anti-NF-B p50 (sc-114), and
anti-NF-B p65 (sc-109; both from Santa Cruz Biotechnology) were
performed to confirm the identity of the complexes binding the IL-8
promoter B probe. Each Ab (2 µg) was added to the DNA probe at the
start of the 30-min incubation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Confluent monolayers of AGS gastric epithelial cells were infected
with H. pylori strain 43504 at bacterial densities ranging
from 106-108/ml. After
1 h cell lysates were prepared and examined by Western blot
analysis using phospho-specific Abs to ERK1/2, p38, and JNK isoforms
p54 and p46. Control cells showed low or undetectable levels of
activated MAP kinases (Fig. 1
A). Contact with H.
pylori resulted in a marked and dose-dependent increase in the
phosphorylation of all three MAP kinases. In the case of ERK, two
immunoreactive bands are evident representing phosphorylated p44 (ERK1,
upper band) and p42 (ERK2, lower band) MAP
kinases. For JNK two immunoreactive bands are again evident
representing phosphorylated p54 (upper band) and p46
(lower band) MAP kinases.
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B
the kinase activities of ERK2, p38, and JNK1 in cell extracts were each
markedly increased within 30 min of H. pylori inoculation of
AGS monolayers. Intact H. pylori are required to activate MAP kinases in AGS cells
We and others have reported that contact with intact bacteria is
required for H. pylori-mediated activation of the
transcription factor NF-B in gastric epithelial cells (10, 15, 18, 20, 21). We next examined whether contact with H.
pylori was also required for epithelial cell MAP kinase
activation. As shown in Fig. 2 H.
pylori that were killed by heat treatment for 30 min at 100°C
were no longer capable of MAP kinase induction. Soluble factors
contained in a cell-free H. pylori filtrate also failed to
activate epithelial cell MAP kinases. Pretreatment of H.
pylori with chloramphenicol, a bacteriostatic agent that inhibits
bacterial protein synthesis, had no apparent inhibitory effect on MAP
kinase phosphorylation. These data demonstrate that MAP kinase
activation requires live, intact bacteria and does not result from
soluble factors present in bacterial culture filtrate. However, de novo
bacterial protein synthesis is not required.
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Fig. 3A illustrates the
time course of MAP kinase activation following gastric epithelial cell
contact with H. pylori. Phosphorylation of ERK1/2 and p38
are clearly evident within 5 min of H. pylori inoculation.
Both JNK isoforms are activated within 30 min. Phosphorylation of
ERK1/2, p38, and JNK appears to be maximal at 30–60 min.
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A incubation
of AGS cells with the cag+ H.
pylori strain 43504 resulted in marked activation of ERK1/2, p38,
and JNK. Incubation with the same concentration of a
cag- H. pylori (strain J68)
again resulted in the rapid activation of ERK1/2 (Fig. 3B).
However, the intensity of ERK1/2 phosphorylation was less than that
observed with the cag+ strain. The
cag- isolate also induced far less
phosphorylation of p38 and very little phosphorylation of JNK compared
with the cag+ strain.
The differences in MAP kinase activation induced by strains 43504 and
J68 led us to study a panel of cag+ and
cag- H. pylori clinical isolates. As
illustrated in Fig. 4 all three of the
cag+ H. pylori isolates induced
a marked activation of the MAP kinases ERK1/2, p38 and JNK. Each of the
three cag- H. pylori isolates
was less potent in inducing MAP kinase activation. Furthermore, a
consistent pattern of differential MAP kinase phosphorylation was
observed with the three cag- strains;
ERK1/2 phosphorylation was somewhat less intense than with the
cag+ strains, p38 phosphorylation was
substantially less, and there was minimal phosphorylation of
JNK.
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Having found distinct patterns of MAP kinase activation by
cag+ and cag-
H. pylori clinical isolates, we next examined the
participation of specific cag pathogenicity island gene
products in this process. The picB region of the
cag pathogenicity island is known to be involved in gastric
epithelial cell NF-B activation and IL-8 up-regulation (12, 14, 22, 28). As shown in Fig. 5
the cag+ reference strain 60190 (wild type)
induced the phosphorylation of ERK1/2, p38, and JNK in AGS gastric
epithelial cells as observed with other
cag+ H. pylori. The
cagA- isogenic mutant was similar to the
wild type in terms of its ability to activate epithelial cell MAP
kinases apart from a possible reduction in ERK1/2 phosphorylation.
However, the picB- isogenic mutant was
less potent in inducing epithelial cell MAP kinase activation. Compared
with the parental wild-type strain, ERK1/2 phosphorylation was only
slightly reduced, p38 phosphorylation was moderately reduced, and JNK
phosphorylation was markedly reduced. Thus, disruption of
picB in this cag+ strain
resulted in a pattern of MAP kinase activation similar to that seen
with cag- strains (see Fig. 4).
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H. pylori infection is known to stimulate gastric epithelial cell IL-8 production, and MAP kinases have been reported to regulate the upstream signaling events that control cytokine transcription. We asked, therefore, whether MAP kinase activation might be involved in H. pylori-induced IL-8 production by gastric epithelial cells. In these experiments we used the specific MAP kinase inhibitors SB203580, which blocks p38 kinase activity, and PD98059, which blocks MEK1 kinase activity, thereby preventing ERK1/2 phosphorylation. In control experiments, SB203580 (10 µM) inhibited p38 MAP kinase activity in H. pylori-infected (strain 43504; 108 bacteria/ml) AGS cells as demonstrated using the in vitro p38 kinase assay (data not shown). Similarly, the ability of PD98059 (25 µM) to block MEK1 kinase activity in H. pylori-infected AGS cells was confirmed by Western blotting using the phosphospecific ERK1/2 Ab (data not shown).
AGS cells were pretreated for 30 min with the MAP kinase inhibitors used singly or in combination. The gastric epithelial cell monolayers were then inoculated with H. pylori (strain 43504; 108 bacteria/ml). IL-8 levels were measured in the conditioned medium harvested 7 h later.
H. pylori induced a 36-fold increase in AGS cell IL-8
production (Fig. 6). The p38 inhibitor
SB303580 reduced epithelial cell IL-8 production by 82%
(p < 0.001), the MEK1 inhibitor PD98059
reduced IL-8 production by 78% (p < 0.001),
and a combination of the two inhibitors completely abrogated H.
pylori stimulation of IL-8 production (42% of control IL-8 level;
p < 0.0001).
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IB degradation and NF-B activation are not altered by the
MAP kinase inhibitors PD98059 and SB203580 in H.
pylori-infected AGS gastric epithelial cells
Previous studies have demonstrated that
cag+ H. pylori activate gastric
epithelial cell NF-B, leading to up-regulation of IL-8 mRNA
transcription and protein production (12, 14, 15, 22, 28).
We now find that cag+ bacteria also
activate MAP kinases, leading to an increase in IL-8 production by
gastric epithelial cell lines. In view of these parallel findings we
next asked whether MAP kinase activation was part of the upstream
signaling pathway leading to IB kinase activation, IB
degradation, and the subsequent activation and nuclear translocation of
NF-B.
As shown in Fig. 7A detectable
levels of IB were evident in control AGS gastric epithelial cells
by Western blot analysis. Exposure of the gastric epithelial cell to
H. pylori resulted in a marked reduction in IB levels
consistent with IB kinase activation leading to IB
phosphorylation and degradation. This marked reduction in IB
levels was not prevented by pretreatment of epithelial cells with the
MAP kinase inhibitors PD98059 and/or SB203580.
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B shows an EMSA of nuclear extracts from AGS gastric
epithelial cells examining NF-B binding to an oligonucleotide probe
containing the IL-8 promoter B binding sequence. Nuclear extracts
from control cells contained little activated NF-B
(lane 1). NF-B binding to the IL-8
promoter site was markedly increased following H. pylori
infection (lane 2). Activation and nuclear
translocation of NF-B did not appear to be influenced by the MAP
kinase inhibitors PD98059 and/or SB203580 (lanes
3–5). EMSA supershift studies using Abs to p50 and p65 B
proteins confirmed that p65-containing NF-B is the predominant form
binding to the IL-8 promoter site. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation.
H. pylori has adapted to interact specifically with
gastric-type epithelial cells. H. pylori infection is
limited to areas of the gastrointestinal tract that are lined by
gastric epithelium, and the bacterium is known to activate several
gastric epithelial cell signaling events. Previous studies have shown
that adherence of H. pylori to AGS gastric epithelial cells
induces tyrosine phosphorylation of host proteins, cytoskeletal
reorganization, NF-B activation, and up-regulation of expression of
a variety of inflammatory response genes including IL-8 (10, 13, 15, 21, 41). We now report that H. pylori also
induces the phosphorylation of ERK, JNK, and p38 MAP kinase family
members. Threonine and tyrosine phosphorylation of MAP kinases was
evident within 5 min of H. pylori inoculation. Contact with
intact bacteria appears to be required for MAP kinase activation. A
similar requirement for bacterial contact or adherence was described
previously for gastric epithelial cell NF-B activation by H.
pylori (15). Our experiments using the bacterial
protein synthesis inhibitor chloramphenicol indicate that MAP kinase
activation does not require de novo bacterial protein production.
Instead, epithelial cell activation appears to result from contact with
preformed bacterial factors, which is consistent with the observed
rapid onset of MAP kinase activation.
We did not examine specifically whether H. pylori LPS
activates MAP kinases in AGS cells. However, in a previous study we
were unable to demonstrate AGS cell NF-B activation (or IL-8
production) in response to LPS (15). Furthermore, soluble
factors present in bacterial culture filtrate did not activate AGS cell
MAP kinases. These findings suggest that H. pylori LPS is
not responsible for MAP kinase activation in gastric epithelial cells.
This is in contrast to human monocytic cells, which are activated by
H. pylori culture filtrate and by purified H.
pylori LPS as shown by other investigators and by us (35, 42, 43, 44).
We find that H. pylori cag+ strains are
more potent than cag- strains in inducing
gastric epithelial cell MAP kinase phosphorylation. A consistent
pattern of differential MAP kinase activation was observed with the
panel of cag+ and
cag- isolates examined in this study. The
most striking differences between the cag+
and cag- strains were seen for JNK
phosphorylation, which was minimal after exposure to the
cag- bacteria. p38 MAP kinase activation
was also far less evident with the cag-
strains. In previous studies, disruption of the picB region
of the cag pathogenicity island resulted in a marked
reduction in the ability of H. pylori to activate epithelial
cell NF-B and up-regulate IL-8 protein production (22, 28). We now find that a picB-
mutant also shows reduced potency in MAP kinase activation compared
with its cag+ parental strain. Again, an
almost complete loss of JNK activation and a markedly reduced potency
of p38 activation were evident. Thus, the pattern of MAP kinase
activation induced by the picB- mutant
recapitulates the pattern observed using the
cag- clinical isolates. These findings
indicate that gene products of the cag pathogenicity island
are necessary for the observed differences in MAP kinase induction by
H. pylori cag+ and
cag- strains.
H. pylori vacuolating cytotoxin has been reported to inhibit EGF-mediated signal transduction and ERK2 activation in Kato III gastric epithelial cells (45, 46). Broth culture supernatants from a vacA+ H. pylori strain, but not its isogenic vacA- mutant, inhibited epithelial cell EGF receptor activation, and ERK2 phosphorylation in response to EGF. We now find that H. pylori bacteria, but not culture filtrate, activate ERK1 and ERK2 in gastric epithelial cells. Furthermore, ERK activation is more pronounced following exposure to cag+ strains compared with cag- strains that secrete less vacuolating cytotoxin. Thus, vacuolating cytotoxin produced by cag+ bacterial strains in our experimental system is unable to prevent H. pylori-induced ERK activation. The interplay between bacterial activation of gastric epithelial cell MAP kinases and the blockade of related signaling pathways by vacuolating cytotoxin requires further investigation.
MAP kinase signaling regulates the expression of many proinflammatory cytokines, including IL-8. This led to our experiments using the specific MAP kinase inhibitors SB203580, which blocks p38 kinase activity, and PD98059, which blocks MEK1 activity, thereby preventing ERK1/2 phosphorylation. Unfortunately, a suitable JNK inhibitor was not available for use in our studies. Both SB203580 and PD98059 substantially reduced IL-8 production by H. pylori-stimulated AGS cells. Thus, activation of both the p38 and MEK1-ERK1/2 pathways are required for a maximal gastric epithelial cell IL-8 response to H. pylori. When both pathways were blocked by combining the two inhibitors, H. pylori-induced IL-8 production was abolished.
Cross-talk between the MAP kinase and NF-B pathways has been
demonstrated in a number of recent studies. For example, the MAP kinase
family members MAP kinase kinase kinase (MEKK1) and NF-B-inducing
kinase (NIK) can each directly activate the IB kinase signalsome,
resulting in IB phosphorylation and release of activated NF-B
(29, 30, 31, 47, 48, 49). Thus, we examined whether MAP kinase
activation by H. pylori was an upstream signaling event in
the pathway leading to NF-B activation and, hence, IL-8 gene
transcription. Although the MAP kinase inhibitors SB203580 and PD98059
were both effective in blocking IL-8 production, neither had any
apparent effect on H. pylori-induced IB degradation or
on NF-B activation and binding to the IL-8 promoter B site. These
data suggest that p38 and MEK1 MAP kinases are not required for
H. pylori-mediated IB degradation or NF-B
activation. Thus, the MAP kinase and NF-B pathways may exert
independent regulatory effects on gastric epithelial cell IL-8
production following H. pylori infection. In other systems,
p38 MAP kinases and NF-B have been shown to regulate cytokine gene
expression by independent pathways (32, 50, 51). However,
the mechanism by which p38 regulates cytokine production without
altering NF-B activation and DNA binding is not known.
Bacterial adherence to or at least contact with the gastric epithelial cell appears to be necessary for H. pylori-induced MAP kinase activation. However, the adhesins or other bacterial factors, host receptors, and intermediary host signaling molecules that are engaged to activate MAP kinase family members are unknown. Further studies are needed to elucidation these upstream events and define the specific bacterial and host factors that interact to activate the epithelial cell signaling pathways. The potential downstream effects of epithelial cell MAP kinase activation are multiple and varied. In this study we examined MAP kinase-mediated activation of IL-8 production. MAP kinases regulate cell proliferation, differentiation, and programmed death, in addition to stress and inflammatory responses. Hence, activation of gastric epithelial cell MAP kinases by H. pylori may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia. The differential activation of MAP kinases by cag+ and cag- strains may be an important determinant of strain-specific differences in the host response to H. pylori infection of the gastric mucosa.
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Footnotes |
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2 Address correspondence and reprint requests to Sarah Keates, Dana 601, Gastroenterology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail address:
3 Abbreviations used in this paper: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinases; MEKK1, MAP kinase kinase kinase; NIK, NF-B-inducing kinase; EGF, epidermal growth factor; MEK-1, MAP kinase kinase.
Received for publication April 21, 1999. Accepted for publication September 7, 1999.
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References |
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B activation. Infect. Immun. 66:2346.B in gastric epithelial cells. Gastroenterology 113:1099.[Medline]
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B and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J. Biol. Chem. 272:12422.This article has been cited by other articles:
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 J. Zhang, H. Li, J. Wang, Z. Dong, S. Mian, and F.-S. X. Yu Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa-Infected Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2569 - 2576. [Abstract] [Full Text] [PDF]
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M. Nakayama, M. Kimura, A. Wada, K. Yahiro, K.-i. Ogushi, T. Niidome, A. Fujikawa, D. Shirasaka, N. Aoyama, H. Kurazono, et al. Helicobacter pylori VacA Activates the p38/Activating Transcription Factor 2-mediated Signal Pathway in AZ-521 Cells J. Biol. Chem., February 20, 2004; 279(8): 7024 - 7028. [Abstract] [Full Text] [PDF]
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D. J. Mitchell, H. Q. Huynh, P. J. M. Ceponis, N. L. Jones, and P. M. Sherman Helicobacter pylori Disrupts STAT1-Mediated Gamma Interferon-Induced Signal Transduction in Epithelial Cells Infect. Immun., January 1, 2004; 72(1): 537 - 545. [Abstract] [Full Text] [PDF]
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 S. D. Savkovic, A. Koutsouris, and G. Hecht PKC{zeta} participates in activation of inflammatory response induced by enteropathogenic E. coli Am J Physiol Cell Physiol, September 1, 2003; 285(3): C512 - C521. [Abstract] [Full Text] [PDF]
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P. J. M. Ceponis, D. M. McKay, R. J. Menaker, E. Galindo-Mata, and N. L. Jones Helicobacter pylori Infection Interferes with Epithelial Stat6-Mediated Interleukin-4 Signal Transduction Independent of cagA, cagE, or VacA J. Immunol., August 15, 2003; 171(4): 2035 - 2041. [Abstract] [Full Text] [PDF]
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K. L. Marlink, K. D. Bacon, B. C. Sheppard, H. Ashktorab, D. T. Smoot, T. L. Cover, C. W. Deveney, and M. J. Rutten Effects of Helicobacter pylori on intracellular Ca2+ signaling in normal human gastric mucous epithelial cells Am J Physiol Gastrointest Liver Physiol, June 9, 2003; 285(1): G163 - G176. [Abstract] [Full Text] [PDF]
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 Y. Churin, L. Al-Ghoul, O. Kepp, T. F. Meyer, W. Birchmeier, and M. Naumann Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response J. Cell Biol., April 28, 2003; 161(2): 249 - 255. [Abstract] [Full Text] [PDF]
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F. Betis, P. Brest, V. Hofman, J. Guignot, M.-F. Bernet-Camard, B. Rossi, A. Servin, and P. Hofman The Afa/Dr Adhesins of Diffusely Adhering Escherichia coli Stimulate Interleukin-8 Secretion, Activate Mitogen-Activated Protein Kinases, and Promote Polymorphonuclear Transepithelial Migration in T84 Polarized Epithelial Cells Infect. Immun., March 1, 2003; 71(3): 1068 - 1074. [Abstract] [Full Text] [PDF]
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J Berkes, V K Viswanathan, S D Savkovic, and G Hecht Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation Gut, March 1, 2003; 52(3): 439 - 451. [Abstract] [Full Text] [PDF]
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I. J. Choi, J. S. Kim, J. M. Kim, H. C. Jung, and I. S. Song Effect of Inhibition of Extracellular Signal-Regulated Kinase 1 and 2 Pathway on Apoptosis and bcl-2 Expression in Helicobacter pylori-Infected AGS Cells Infect. Immun., February 1, 2003; 71(2): 830 - 837. [Abstract] [Full Text] [PDF]
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 S. Suerbaum and P. Michetti Helicobacter pylori Infection N. Engl. J. Med., October 10, 2002; 347(15): 1175 - 1186. [Full Text] [PDF]
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B. Neu, P. Randlkofer, M. Neuhofer, P. Voland, A. Mayerhofer, M. Gerhard, W. Schepp, and C. Prinz Helicobacter pylori induces apoptosis of rat gastric parietal cells Am J Physiol Gastrointest Liver Physiol, August 1, 2002; 283(2): G309 - G318. [Abstract] [Full Text] [PDF]
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S. Keates, S. Sougioultzis, A. C. Keates, D. Zhao, R. M. Peek Jr., L. M. Shaw, and C. P. Kelly cag+ Helicobacter pylori Induce Transactivation of the Epidermal Growth Factor Receptor in AGS Gastric Epithelial Cells J. Biol. Chem., December 14, 2001; 276(51): 48127 - 48134. [Abstract] [Full Text] [PDF]
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J. M. Cox, C. L. Clayton, T. Tomita, D. M. Wallace, P. A. Robinson, and J. E. Crabtree cDNA Array Analysis of cag Pathogenicity Island-Associated Helicobacter pylori Epithelial Cell Response Genes Infect. Immun., November 1, 2001; 69(11): 6970 - 6980. [Abstract] [Full Text] [PDF]
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S. D. Savkovic, A. Ramaswamy, A. Koutsouris, and G. Hecht EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G890 - G898. [Abstract] [Full Text] [PDF]
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G. Le'Negrate, V. Ricci, V. Hofman, B. Mograbi, P. Hofman, and B. Rossi Epithelial Intestinal Cell Apoptosis Induced by Helicobacter pylori Depends on Expression of the cag Pathogenicity Island Phenotype Infect. Immun., August 1, 2001; 69(8): 5001 - 5009. [Abstract] [Full Text] [PDF]
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 A. Masamune, M. Yoshida, Y. Sakai, and T. Shimosegawa Rebamipide Inhibits Ceramide-Induced Interleukin-8 Production in Kato III Human Gastric Cancer Cells J. Pharmacol. Exp. Ther., August 1, 2001; 298(2): 485 - 492. [Abstract] [Full Text] [PDF]
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A. Banan, J. Z. Fields, Y. Zhang, and A. Keshavarzian Phospholipase C-{gamma} inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants Am J Physiol Gastrointest Liver Physiol, August 1, 2001; 281(2): G412 - G423. [Abstract] [Full Text] [PDF]
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Y. Hirata, S. Maeda, Y. Mitsuno, M. Akanuma, Y. Yamaji, K. Ogura, H. Yoshida, Y. Shiratori, and M. Omata Helicobacter pylori Activates the Cyclin D1 Gene through Mitogen- Activated Protein Kinase Pathway in Gastric Cancer Cells Infect. Immun., June 1, 2001; 69(6): 3965 - 3971. [Abstract] [Full Text] [PDF]
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R. M. Peek Jr. Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions: IV. Helicobacter pylori strain-specific activation of signal transduction cascades related to gastric inflammation Am J Physiol Gastrointest Liver Physiol, April 1, 2001; 280(4): G525 - G530. [Abstract] [Full Text] [PDF]
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C. Audibert, C. Burucoa, B. Janvier, and J. L. Fauchere Implication of the Structure of the Helicobacter pylori cag Pathogenicity Island in Induction of Interleukin-8 Secretion Infect. Immun., March 1, 2001; 69(3): 1625 - 1629. [Abstract] [Full Text] [PDF]
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S. Takahashi, Y. Keto, T. Fujita, T. Uchiyama, and A. Yamamoto FR167653, a p38 Mitogen-Activated Protein Kinase Inhibitor, Prevents Helicobacter pylori-Induced Gastritis in Mongolian Gerbils J. Pharmacol. Exp. Ther., January 1, 2001; 296(1): 48 - 56. [Abstract] [Full Text]
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T. Pomorski, T. F. Meyer, and M. Naumann Helicobacter pylori-induced Prostaglandin E2 Synthesis Involves Activation of Cytosolic Phospholipase A2 in Epithelial Cells J. Biol. Chem., January 5, 2001; 276(1): 804 - 810. [Abstract] [Full Text] [PDF]
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R. A. Gupta, D. B. Polk, U. Krishna, D. A. Israel, F. Yan, R. N. DuBois, and R. M. Peek Jr. Activation of Peroxisome Proliferator-activated Receptor gamma Suppresses Nuclear Factor kappa B-mediated Apoptosis Induced by Helicobacter pylori in Gastric Epithelial Cells J. Biol. Chem., August 10, 2001; 276(33): 31059 - 31066. [Abstract] [Full Text] [PDF]
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D. Zhao, A. C. Keates, S. Kuhnt-Moore, M. P. Moyer, C. P. Kelly, and C. Pothoulakis Signal Transduction Pathways Mediating Neurotensin-stimulated Interleukin-8 Expression in Human Colonocytes J. Biol. Chem., November 21, 2001; 276(48): 44464 - 44471. [Abstract] [Full Text] [PDF]
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J.-R. Basque, P. Chailler, and D. Menard Laminins and TGF-beta maintain cell polarity and functionality of human gastric glandular epithelium Am J Physiol Cell Physiol, April 1, 2002; 282(4): C873 - C884. [Abstract] [Full Text] [PDF]
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