1 Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK
2 Department of Medicine, University of Liverpool, Liverpool L69 3BX, UK
3 Department of Medical Microbiology, University of Liverpool, Liverpool L69
3BX, UK
4 Department of Pathology, University of Liverpool, Liverpool L69 3BX, UK
* Author for correspondence (e-mail: avarro{at}liverpool.ac.uk)
Accepted 26 March 2003
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Summary |
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Key words: MMP-7, Migration, Helicobacter pylori, Gastric epithelium, Matrix metalloproteinase
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Introduction |
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The MMPs comprise a family of over 25 members implicated in the proteolysis
of extracellular matrix, growth factors, cytokines, adhesion molecules and
protease inhibitors such as the serpins, resulting in both gain and loss of
function (Nagase and Woessner,
1999; Seiki,
2002
). It is now widely appreciated that induction of distinct
profiles of MMPs, or of their inhibitors, the tissue inhibitors of
metalloproteinases (TIMPs), is a feature of the tissue remodelling that occurs
in many normal and disease processes (e.g. in development, wound healing,
cancer and a wide variety of inflammatory conditions)
(Murphy and Gavrilovic, 1999
).
In the gastrointestinal tract, epithelial cells are often not the source of
the increased MMP production that occurs in response to disease. Instead, it
is the sub-epithelial (i.e. stromal or mesenchymal) cells that produce the
MMPs that play a role in mucosal responses, at least in part by influencing
the environment at the epithelial basolateral membrane. However, an exception
is MMP-7, which is characteristically produced by epithelial cells and plays a
role in a variety of epithelial responses. Thus, for example, in murine small
intestinal Paneth cells, MMP-7 may act as a processing enzyme to convert
prodefensins to their active form, thereby playing a role in the innate
defence system of the gut (Wilson et al.,
1999
).
The epithelial cells of the gastrointestinal tract are protected from
ingested bacteria in part by the barrier presented by gastric acid secretion
that generates an initial environment unfavourable to the survival of
microoganisms. However, unusually, the gastric pathogen H. pylori
(Tomb et al., 1997) colonizes
the microenvironment generated between the surface of gastric epithelial cells
and the overlying mucus glycoprotein gel. In many subjects, infection with
H. pylori is clinically silent; but in some patients it is associated
with duodenal ulcer, and in others it is associated with a progression through
chronic atrophic gastritis to gastric adenocarcinoma
(Fox and Wang, 2001
;
Uemura et al., 2001
). The
cellular and molecular mechanisms that determine the precise constellation of
epithelial cell responses to infection are far from clear. However, this is a
relatively attractive system for studies of the way that bacterial infection
might control MMP-7 expression because: (1) it is relatively straightforward
to obtain primary gastric epithelial cells from H. pylori positive
and control subjects; (2) there is a clear link between H. pylori and
gastric cancer; and (3) MMP-7 expression is known to be increased in gastric
cancer (Honda et al., 1996
).
We report here increased expression of MMP-7 in response to H.
pylori, the consequences for epithelial cell migration, and the relevant
signalling pathways.
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Materials and Methods |
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Bacterial infection of AGS cells and gastric glands in vitro
We routinely used H. pylori strain 60190 (ATCC). In a few
experiments, we also used two gastric isolates, strain E5
(cag+, vacA-) and E6
(cag+, vacA+).
Bacteria were grown in a microaerophilic atmosphere at 37°C on fresh
chocolatized Columbia blood agar for 39 days (Oxoid, Basingstoke, UK).
Unless otherwise stated, H. pylori was added to human gastric glands
or AGS cells at a multiplicity of infection (MOI) of 100. In some experiments,
bacteria were added to 0.2 µm Anapore filter inserts (Life Technologies)
that were suspended above AGS cells. Two such systems were studied: either
bacteria was added directly to the filters, or to AGS cells cultured on
filters that were then inserted over AGS cells transfected with MMP-7-luc (see
below). In other experiments, H. pylori were sonicated for 20 minutes
and added directly to AGS cells.
Patients
Endoscopic pinch biopsies of the gastric corpus and antrum were obtained
from patients attending routine gastroscopy for investigation of dyspepsia.
Most patients had no macroscopic gastroduodenal pathology; none of them had
neoplastic disease, and a total of four H. pylori-positive patients
had peptic ulcer (3 duodenal, 1 antral). H. pylori status was
initially assessed by a rapid urease test (Prontodry, Medical Instruments
Corporation, Solothurn, Switzerland), and subsequently confirmed by histology.
On routine histology, H. pylori-positive biopsies typically exhibited
active chronic gastritis as expected. The present report is based on studies
of 41 patients (25 female, mean age 53.5±1.6 years) who were H.
pylori positive, and 45 who were H. pylori negative (29 female,
mean age 50.9±1.8 years) and were used as controls. Samples were
assigned randomly within the two groups either to extraction for western
blotting or to gastric gland culture. The study was approved by the Ethics
Committee of Royal Liverpool and Broadgreen University Hospitals NHS Trust.
All patients gave informed consent.
Gastrin radioimmunoassay (RIA)
The concentration of amidated gastrins in plasma was determined by RIA
using antibody L2 specific for the C-terminal amide sequence of gastrin, as
described previously (Varro et al.,
1997).
Western blotting
Protein extracts were prepared in RIPA or lysis buffer and western blotting
was performed as previously described
(Varro et al., 2002) using a
rabbit anti-MMP-7 antibody (Chemicon International, Temecula, CA) or
anti-NF-
B (p65, p50) antibodies (Santa Cruz Biotechnology, Santa Cruz,
MA). Samples were reprobed with a goat anti-ß-actin antibody (Santa Cruz
Biotechnology).
Immunohistochemistry
For immunohistochemistry (IHC), a goat anti-MMP-7 antibody (Santa Cruz
Biotechnology) was used with a fluorescein isothiocyanate (FITC)-conjugated
donkey anti-goat IgG (Jackson IR, West Grove, PA) as previously described
(Wroblewski et al., 2002). In
colocalization studies, the following antibodies were used: mouse anti-gastric
mucin (Sigma), rabbit anti-pepsinogen (gift from Mike Samloff, Center for
Ulcer Research, Los Angeles, CA), rabbit
anti-H+/K+ATP-ase (Calbiochem), rabbit anti-somatostatin
(Santa Cruz Biotechnology), rabbit anti-chromogranin A and guinea-pig
anti-gastrin (Hussain et al.,
1999
), mouse anti-vimentin and
-smooth muscle actin
(Research Diagnostics, Flanders, NJ). Texas-Red-labelled donkey anti-mouse,
anti-rabbit or anti-guinea-pig IgG (Jackson IR) were used as appropriate. For
studies of the localization of MMP-7 in gastric biopsies, formalin-fixed
paraffin sections were treated with 0.9% hydrogen peroxide in methanol to
block endogenous peroxide, and antigen was retrieved by heating in 10 mM EDTA,
pH 7.0 for 3 minutes at full pressure in a pressure cooker. Primary rabbit
anti-MMP-7 (Chemicon) was employed with detection by the ChemMate EnVision
HRP/DAB system (DakoCytomation, Ely, UK).
Human primary gland culture
Human biopsies were sliced into 2 mm2 segments using a razor
blade and washed in three changes of Hank's Balanced Salt Solution (HBSS).
Tissue was incubated in 5 ml 1 mM dithiothreitol for 15 minutes, with
continuous gassing with 5% CO2/95% O2 at 37°C, and
shaking at 100 cycles per minute. Tissue was then washed in HBSS (three
times), incubated in 0.5 mg ml-1 collagenase for 30 minutes (Roche,
Lewes, UK), washed again in HBSS (three times) and incubated for a further 30
minutes in collagenase (0.5 mg ml-1). The tissue was then
triturated using a wide mouthed pipette and larger fragments allowed to settle
under gravity for 45 seconds. The supernatant containing isolated glands was
removed and transferred to a clean tube, and shaken vigorously to release
additional glands that were allowed to sediment for 45 minutes. The
supernatant (which contained individual cells and debris) was carefully
removed and discarded. The isolated glands were routinely cultured up to 72
hours in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS
and 1% antibiotic-antimycotic solution (Sigma), at 37°C in 5%
CO2/95% O2, and the medium was changed every 24
hours.
Spreading of human gastric glands
Human gastric glands were cultured in 6-well dishes. In experiments where
AS oligonucleotides were used, either MMP-7 AS oligonucleotide (2 µM) or
negative control oligonucleotides (2 µM) were added at the start of
culture. Medium and oligonucleotides were replaced daily. Intracellular
accumulation of oligonucleotides was confirmed using FITC-labelled MMP-7 AS
oligonucleotide (2 µM) viewed live under a Leica DMIRE2 microscope 17 hours
after addition. Routinely, gastric colonies were cultured for 17 hours and
then mounted on a motorized stage fitted on a Leica DMIRE2 microscope in a
heated, humidified chamber (Solent Scientific, Portsmouth, UK) and images
captured with a Hamamatsu Orca ER camera (Hamamatsu Photonics, Hamamatsu City,
Japan) controlled by Kinetic Imaging AQM-2001 software (Kinetic Imaging,
Liverpool, UK). Serial images were analysed using Lucida 4.0:Analyse software
(Kinetic Imaging), and gland cell spreading was calculated as the mean area of
a gland normalized for cell number determined by Hoechst 33342 (Molecular
Probes, Eugene, OR) nuclear staining.
AGS cell migration and invasion assays
Transwell migration and invasion assays were performed using AGS cells
cultured in 24-well plates containing either 8 µm pore Biocoat® control
inserts (migration assays) or Matrigel-coated inserts (invasion assays),
according to the manufacturer's instructions (Becton Dickinson, Bedford, MA).
Briefly, 2.5x104 AGS cells were diluted in 0.5 ml serum free
(SF) medium and placed in the insert immersed in 0.75 ml SF medium with or
without H. pylori and a mouse monoclonal MMP-7 neutralizing antibody
(Chemicon) for 1522 hours. After incubation, non-invading cells were
removed from the upper surface of the membrane by scrubbing, and invading
cells on the lower surface of the membrane were stained with Diff-Quik reagent
(Dade Behring, Dudingen, Switzerland). Membranes were then removed, mounted in
immersion oil and invading cells counted using a Zeiss 25 Axiovert microscope
(Carl Zeiss, Welwyn Garden City, UK) and Intellicam software (Matrox, Stoke
Poges, UK).
Transient transfection and luciferase assay
Cells (2x105) were plated in 6-well plates in full medium
(FM). The following day, medium was removed and cells were transfected using
TransFast (Promega, Madison, WI) in SF medium for 1 hour. Routinely, MMP-7-luc
was used at 0.51.0 µg well-1. After transfection, 2 ml FM
was added and cells incubated for 2024 hours. Media was then replaced
with 2 ml SF medium and cells were incubated with H. pylori and other
compounds as indicated for 6 hours. Luciferase activity was measured with
Bright-GloTM or Dual GloTM (Promega) using a LumiCount Platereader
(Packard BioScience, Pangbourne, UK) according the manufacturer's protocol.
Results are presented as fold increase over unstimulated control, so 1.0
signifies no change in luciferase activity. Protein concentration was
determined when appropriate using Lowry protein assay kit (Sigma) to monitor
plating efficiency and cell death.
Transcription factor activation
To identify the transcription factors that might bind cis-acting DNA
elements in nuclear extracts in response to H. pylori, we used
MercuryTM Transfactor Profiling Kit-Inflammation 1 (BD Biosciences
Clontech, Palo Alto, CA) according to the manufacturer's protocol. Nuclear
extracts were prepared from control cells, and cells were incubated with
H. pylori using NE-PERTM (Pierce Biotechnology, Rockford,
IL).
Statistics
Results are presented as means±s.e.m.; comparisons were made using a
t test, and were considered significant at P<0.05.
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Results |
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|
Cellular localization of MMP-7 in H. pylori-positive
subjects
The localization of MMP-7 was determined in both paraffin sections of
gastric biopsies and cultured gastric glands. In paraffin sections, MMP-7
immunoreactivity was identified in surface epithelial cells where there was
cytoplasmic localization that tended to be subnuclear (i.e. on the basolateral
side); there was also staining deep in the gastric gland compatible with
localization to chief cells (Fig.
2). The staining in samples from H. pylori-positive
subjects was more intense than in those from H. pylori-negative
subjects, but the cellular distribution was similar. Cultured gastric glands
from the corpus and antrum of normal and H. pylori-positive subjects
contained the major cell types found in vivo. Thus, in corpus cultures,
antibody to H+/K+ATPase identified approximately 10% of
the total cells as parietal cells, and antibodies to pepsinogen and mucus
glycoprotein identified chief and mucus cells (approximately 25 and 35%)
respectively; enterochromaffin-like (ECL) cells, revealed by chromogranin A
immunoreactivity, were typically <5% total. In antral cultures, parietal
cells were absent, and there were abundant mucus (>50% total) and
pepsinogen (approximately 40% total) cells, but G- and D-cells revealed by
gastrin and somatostatin immunoreactivity, respectively, were typically <5%
of total. MMP-7 immunoreactivity was found in many cells that were also
stained with antibodies to mucus glycoprotein
(Fig. 2), or to pepsinogen,
compatible with localization to mucus and chief cells, respectively. In both
cases, only a subset of the two cell types expressed MMP-7. By contrast, in
the corpus, neither parietal cells nor ECL cells exhibited MMP-7
immunoreactivity (not shown). Moreover, in the gastric antrum, MMP-7
immunoreactivity was not identified in G- or D-cells (not shown). The pattern
of localization in different cell types did not differ between H.
pylori-positive and -negative subjects.
|
The majority of cultured glands did not contain cells staining with
antibodies to -smooth muscle actin, or to vimentin (not shown). In
those glands in which positive staining was encountered with these antibodies,
the stained cells never accounted for more than 3% of the total. Thus, the
cultured gastric glands were largely epithelial with little or no
contamination by myofibroblasts or fibroblasts. Moreover, the localization of
MMP-7 in cultured epithelial cells was compatible with that seen in tissue
fixed immediately after sampling.
In cultured glands, MMP-7 was typically sequestered in vesicles that were distinct from those reacting with either mucin or pepsinogen antibodies (Fig. 2). Interestingly, there was a clear localization of MMP-7 to the plasma membrane of cells at the periphery (i.e. the migrating edge) of cultured gastric glands. This pattern was seen in both H. pylori-positive and -negative subjects. When H. pylori was added to control gastric gland cultures, the peripheral cells exhibited a tendency to extend lamellipodia, and MMP-7 immunoreactivity was identified in these structures (Fig. 2).
Role of MMP-7 in gastric gland spreading
We then examined the spreading of cultured gastric glands of both H.
pylori-positive and -negative subjects. Over periods of up to 48 hours in
culture, cells migrated from isolated gastric glands to form monolayer
islands, or colonies, of cells. During this process, cell-cell contacts were
maintained and, although time-lapse video-microscopy revealed cellular
movements indicative of remodelling of these contacts, cells
characteristically did not migrate away from the colony. Careful tracking of
individual cells provided no evidence for mitosis of gland cells, and in
separate studies we also found no evidence of proliferation indicated by
nuclear PCNA in cultured glands (S. Kennedy and A. Varro, unpublished). The
progressive increase in area covered by individual glands therefore represents
both migration and spreading, but not proliferation. Importantly, the
expansion of these islands of gastric gland cells from H.
pylori-positive subjects was significantly greater than in controls
(Fig. 3). In order to determine
whether there was a role for MMP-7 in this response, we examined the effect of
application of MMP-7 AS oligonucleotides. In validation experiments, the
latter were shown to suppress MMP-7 induction by H. pylori in AGS
cells (data not shown). We then showed that MMP-7 AS oligonucleotides
inhibited migration of cultured glands from H. pylori-positive
subjects (Fig. 3). By contrast,
application of MMP-7 AS oligonucleotides had no effect on the spreading of
gland cells from control subjects. In addition, scrambled oligonucleotides
used as a control had no effect on the gland cell spreading.
|
H. pylori stimulation of MMP-7: invasion and migration of a
gastric cancer cell line
We explored the induction of MMP-7 by H. pylori in a gastric
epithelial cell line (AGS cells). Application of H. pylori to AGS
cells for 16 hours increased the abundance of the active 19 kDa form of MMP-7
determined by western blot (Fig.
4). The functional significance of this induction was then
examined using migration and invasion assays. In this model, H.
pylori increased migration of AGS cells through 8 µm Transwell
filters, and stimulated invasion through Matrigel-coated filters compared with
control cells (Fig. 4). A role
for MMP-7 in both migration and invasion was shown by the inhibition of H.
pylori-stimulated migration and invasion by addition of neutralizing
MMP-7 antibody (Fig. 4). Application of sonicates of H. pylori had no effect on AGS cell
migration or invasion, suggesting that a soluble mediator was not
involved.
|
Putative bacterial and host mediators of increased MMP-7 in response
to H. pylori
In order to study the mechanism of H. pylori-induced expression of
MMP-7, we first showed that addition of H. pylori to cultured AGS
cells increased the expression of a construct consisting of 2.3 kb of the
human MMP-7 promoter coupled to a luciferase (i.e. MMP-7-luc) reporter. The
induction of MMP-7-luc in AGS cells was similar for two strains that were
cag+ and vacA+ (E6 and 60190). A third
strain that was vacA- (E5) also produced similar
responses. Thus, although the secreted cytotoxin VacA has been suggested to
activate the small GTPase Rac (Hotchin et
al., 2000), and studies described below implicate Rac in the
induction of MMP-7, the data suggest this toxin was not required for the AGS
cell responses studied here (Fig.
5). In addition, sonicates of H. pylori had no
stimulatory effect on MMP-7-luc (not shown). Moreover, when H. pylori
was applied to 0.2 µm filters cultured over AGS cells transfected with
MMP-7-luc, there was again no increase in MMP-7-luc expression
(Fig. 5). Together, these lines
of evidence suggest that release of a soluble mediator by the bacterium is
unlikely to be the main mechanism involved in MMP-7 induction.
|
It is known that H. pylori increases shedding of HB-EGF and
stimulates production of proinflammatory cytokines (IL-6, TNF-) and
chemokines (IL-8) (Crabtree et al.,
1994
; Moss et al.,
1994
; Romano et al.,
1998
; Wallasch et al.,
2002
). In AGS cells, EGF-family members, IL-8, TNF-
and
IL-6 were all found to increase MMP-7-luc expression
(Fig. 5). We therefore examined
the possibility that H. pylori increased MMP-7 expression in AGS
cells via a paracrine mediator. AGS cells were transfected with MMP-7-luc, and
then cocultured with untransfected AGS cells grown on 0.2 µm pore filters
suspended above the transfected cells. Addition of H. pylori to AGS
cells grown on 0.2 µm Transwell filters had no effect on MMP-7-luc
expression in the cocultured cells. Putative paracrine signals may of course
be diluted in this system, but in the same experimental system using
luciferase expression driven by a promoter sequence of plasminogen activator
inhibitor-2 (Varro et al.,
2002
) there was induction of luciferase by H. pylori up
to 35 fold. Thus, the data support the view that a direct effect due to
adherence of the bacteria was responsible for stimulating MMP-7-luc expression
(Fig. 5).
Putative transcription factors mediating the effect of H. pylorion MMP-7
Previous studies have implicated NF-B and AP-1 in H.
pylori-stimulated transcriptional responses
(Meyer-ter-Vehn et al., 2000
;
Munzenmaier et al., 1997
). We
first confirmed that addition of H. pylori to AGS cells increased p50
and p65 NF-
B translocation to the nucleus by western blotting, and that
binding of p50 and p65 NF-
B to a consensus NF-
B cis-element was
increased in H. pylori-stimulated AGS cell nuclear extracts
(Fig. 6). The functional
significance of these findings for MMP-7 expression was then demonstrated by
showing that an inhibitor of I
B degradation (BAY11-7082) reduced the
MMP-7-luc response to H. pylori to control levels. In addition, an
increase in c-fos binding to a consensus AP-1 cis-element was demonstrated in
nuclear extracts, and cotransfection of AGS cells with MMP-7-luc and a vector
encoding c-fos increased expression 23 fold
(Fig. 6). It is known that
H. pylori increases AP-1 activity via the Erk1/2 pathway
(Meyer-ter-Vehn et al., 2000
)
and, consistent with the involvement of this pathway in the present studies,
we found that PD98059 (which inhibits the Erk1/2 kinase, MEK) inhibited the
response to H. pylori (Fig.
6).
|
Role of RhoA and Rac in mediating the effect of H. pylori on
MMP-7-luc expression
H. pylori is reported to activate small GTPases of the Rho family
(Palovuori et al., 2000). We
therefore screened three representatives of this GTPase family (RhoA, Rac1,
cdc42) for the capacity to stimulate MMP-7-luc expression. Cotransfection of
AGS cells with a vector encoding a constitutively active form of RhoA
(L63RhoA) strongly stimulated MMP-7-luc; there was a moderate response to a
constitutively active form of Rac (L61Rac), whereas a constitutively active
form of cdc42 (L61cdc42) had little effect
(Fig. 7). We then showed that
induction of MMP-7-luc by H. pylori was mediated by RhoA and Rac
because cotransfection of MMP-7-luc and a DN-RhoA vector (N19RhoA), or DN-Rac
(N17Rac), reduced the H. pylori-increased expression of MMP-7-luc
(Fig. 7). In addition,
application of the RhoA inhibitory toxin, C3-transferase, reduced MMP-7-luc
responses to H. pylori by 76.9±4.4%. The MMP-7-luc responses
to L63RhoA and L61Rac were inhibited by BAY11-7082, indicating that
NF-
B was downstream of both RhoA and Rac
(Fig. 7). Interestingly,
cotransfection of MMP-7-luc and L63RhoA with a DN-jun vector resulted in
significant inhibition of MMP-7-luc expression, compatible with activation of
AP-1 downstream of RhoA. By contrast, the DN-jun vector had no effect on Rac
activation of MMP-7-luc expression (Fig.
7).
|
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Discussion |
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Recent reports have noted that MMP-7 expression is induced by bacteria in
several different epithelia, including colon, bladder and airways, but the
cellular mechanisms responsible for this response are unclear
(Lopez-Boado et al., 2000;
Lopez-Boado et al., 2001
;
Parks et al., 2001
). In mouse
small intestine, MMP-7 is expressed in Paneth cells, where it is proposed to
function as a processing enzyme for mediators of innate immunity, the
-defensins (Wilson et al.,
1999
). However, this function seems to be species specific since,
in human small intestine, trypsin has been reported to function as a
prodefensin-processing enzyme (Ghosh et
al., 2002
). Moreover, in normal human small and large intestine,
MMP-7 was reported to be absent (Ghosh et
al., 2002
), although expression may increase in ulcerative colitis
in association with dysplasia (Newell et
al., 2002
). By contrast, we found detectable expression of MMP-7
in epithelial cells in both antral and corpus regions of normal human stomach,
and in both cases expression was increased in the presence of the gastric
pathogen H. pylori.
In mucus and pepsinogen-secreting cells, which were the main MMP-7-containing cells, MMP-7 was found in vesicles distinct from those containing the primary exocrine secretory product. Interestingly, MMP-7 was found at the leading edge of migrating cells, including lamellipodia. By using time-lapse video-microscopy of isolated gastric gland fragments, we showed that cells progressively migrated to form islands, or monolayer colonies of cells, over 23 days through a combination of cell migration and cell spreading. This process was accelerated in H. pylori-positive biopsies, and the response was reversed in the presence of MMP-7 AS oligonucleotides. Moreover, in a cancer cell line (AGS cells) H. pylori stimulated both migration and invasion through artificial basement membrane, and in both cases this was inhibited by neutralization of MMP-7. Together, these data support the idea that H. pylori induction of MMP-7 is a functionally important host cell response mediating cell migration in the stomach.
The stimulation of gastric epithelial cell migration in response to H.
pylori occurs in otherwise normal gastric gland cells. Related processes
have also been described in airways
(Dunsmore et al., 1998;
Parks et al., 2001
). These
phenomena may well be protective and serve to maintain epithelial integrity in
the face of epithelial damage and infection. However, in stomach, the
induction of MMP-7 may also play a role in initiating events that predispose
towards malignancy. Thus, there is increased expression of MMP-7 in gastric
cancer, particularly at the migrating front of tumours
(Adachi et al., 1998
;
Ajisaka et al., 2001
;
Honda et al., 1996
), and AS
inhibition of MMP-7 expression suppressed tumour invasion but not
proliferation (Yonemura et al.,
2001
). In addition, MMP-7 degrades pro-apoptotic factors and
proinflammatory cytokines (e.g. TNF-
and Fas ligand). Decreased
availability of the latter would tend to suppress apoptosis and may therefore
serve to preserve cells after DNA damage and so contribute to tumorigenesis
(Mitsiades et al., 2001
;
Vargo-Gogola et al.,
2002
).
Previous work has shown that MMP-7 expression is regulated by the PEA3 Ets
transcription factor, which appears to enhance transcriptional responses to
both ß-catenin and AP-1 transcription factors
(Crawford et al., 1999;
Crawford et al., 2001
). Our
data suggest that activation of AP-1 by H. pylori
(Naumann et al., 1999
) is one
component of the MMP-7 transcriptional response. But, in addition, we found
that H. pylori stimulation of NF-
B activity contributed to the
induction of MMP-7. The activation of NF-
B by H. pylori has
been described by several groups and has been linked to induction of IL-8
(Gupta et al., 2001
;
Keates et al., 1997
;
Munzenmaier et al., 1997
;
Wada et al., 2001
). The
present data linking NF-
B activation to induction of MMP-7 suggest a
wider range of responsive genes than previously supposed. Moreover, although
IL-8 increased MMP-7 expression, the present data suggest that this is not an
important component of the response to H. pylori. Similarly, the data
suggest that other possible endocrine, paracrine, cytokine or bacterial
mediators are unlikely to mediate the MMP-7 response to H. pylori.
Instead, we suggest that cell adhesion of the bacterium and activation of
small GTPases of the Rho family (Churin et
al., 2001
; Hotchin et al.,
2000
; Palovuori et al.,
2000
) are responsible for induction of MMP-7. In particular, our
data suggest that induction of MMP-7 via NF-
B occurs as a consequence
of activation of Rho and Rac (Montaner et
al., 1999
). Interestingly, there was differential activation of
AP-1 and NF-
B, since both Rho and Rac activated the latter, but only
Rho activated AP-1.
Infection with H. pylori affects approximately 50% of the western
population; some patients exhibit a progression through chronic gastric
atrophy to cancer, others develop peptic ulcer, but most do not exhibit either
disease (Blaser and Berg, 2001;
Peek and Blaser, 2002
;
Uemura et al., 2001
). The
different outcomes from infection are thought to reflect host, pathogen and
environmental variables (Blaser and Berg,
2001
; Fox and Wang,
2001
). The ability of epithelial cells to migrate after damage is
usually considered to be an important component of the host defence mechanism.
In this context, we suggest that H. pylori induction of MMP-7 can be
considered part of a protective host response. In addition, though, we also
suggest that, over longer periods, the prolonged induction of MMP-7
accelerates an oncogenic progression via disruption of epithelial organization
and increased invasion. The identification of increased MMP-7 expression as a
consequence of H. pylori infection should provide a basis for
detailed study of the links between bacterial infection and cancer.
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Acknowledgments |
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References |
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