Divisions of 1 Gastroenterology and Hepatology and 2 Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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To test the hypothesis that Helicobacter pylori regulates gastric cell secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), culture media from infected and uninfected human gastric adenocarcinoma (AGS) cells were analyzed by zymography, MMP activity assays, and immunoblotting. AGS cells secreted gelatinolytic (prominently 90 kDa) and caseinolytic (110 kDa) activity together with MMP-1, MMP-3, and TIMP-1, TIMP-2, and TIMP-3 isoforms. H. pylori secreted caseinolytic activity (60 kDa), MMP-3-like enzyme activity, and TIMP-3 immunoreactivity. H. pylori infection increased the 110-kDa caseinolytic activity and induced new gelatinolytic (~35 kDa) and caseinolytic (22 kDa) activities. Infection also increased both basal secretion and activation of MMP-1 and MMP-3, enhanced TIMP-3 secretion, and increased the formation of MMP-3/TIMP-3 complexes. TIMP-1 and TIMP-2 secretion were unchanged. Normal AGS cells showed a pancellular distribution of TIMP-3, with redistribution of immunoreactivity toward sites of bacterial attachment after H. pylori infection. The data indicate that MMP and TIMP secretion by AGS cells is modulated by H. pylori infection and that host MMP-3 and a TIMP-3 homolog expressed by H. pylori mediate at least part of the host cell response to infection.
AGS cells; Helicobacter pylori; host response; metalloproteinase; tissue inhibitor of metalloproteinases
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INTRODUCTION |
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HELICOBACTER PYLORI
IS A SPIRAL, microaerophilic, neutralophilic gram-negative
bacterium that colonizes the gastric mucosa in 25-50% of the
population in developed countries and 70-90% in developing
countries (15). H. pylori is a causative agent of peptic ulcer disease, gastric adenocarcinoma, and gastric
mucosa-associated lymphoid tissue lymphoma (4).
Gastroduodenal diseases caused by H. pylori are associated
with infiltration of the gastric mucosa by neutrophils, lymphocytes,
monocytes, and plasma cells. Mobilization of inflammatory cells is a
host-response mechanism induced by H. pylori-stimulated
gastric epithelial cell secretion of an array of cytokines, most
prominently interleukin-1 and -8 (9). Another host-response mechanism may involve secretion of the antibiotic peptide
-defensin. mRNA coding for
-defensin was reported to be induced
by in vitro H. pylori infection of human gastric epithelial cells (18). An analogous host-response mechanism was
recently identified in mouse small intestine, where Escherichia
coli infection stimulated generation of the antibiotic peptide
-defensin by host metalloproteinase-catalyzed hydrolysis of an
inactive
-defensin proform (19).
The identities and mechanisms of action of virulence factors secreted by H. pylori that induce such host responses are poorly understood. Genomic DNA of virulent H. pylori strains includes a 40-kb segment of DNA (pathogenicity island) containing up to 29 open reading frames, two of which encode a vacuolating toxin (VacA) and cytotoxin-associated immunodominant protein (CagA) (29). Other pathogenicity island genes encode membrane-associated proteins similar to those comprising bacterial type IV secretion systems (20). Most recently, H. pylori type IV secretion system proteins have been shown to promote migration of CagA protein into gastric epithelial cells, where it is tyrosine phosphorylated and subsequently modulates phosphorylation of host proteins (10).
Inflammatory cell mucosal infiltration and antibiotic peptide activation both involve metalloproteinase activity. Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that degrade extracellular matrix proteins during tissue morphogenesis and remodeling in wound healing and are associated with tumor angiogenesis, invasion, and metastasis, arthritis, and atherosclerosis (8, 13, 23). MMP expression is regulated transcriptionally by growth factors, hormones, cytokines, and cell-cell matrix interactions. Cell surface (membrane-type) and extracellular MMPs secreted by epithelial and stromal cells are activated by proteolytic cleavage of their NH2 terminal domains and are inhibited by noncovalent 1:1 stoichiometric interaction with tissue inhibitors of metalloproteinases (TIMPs). These constitute a family of four homologous 20- to 30-kDa proteins (TIMP-1, -2, -3, and -4) with 125-amino acid NH2 terminal domains and 65-amino acid COOH terminal domains, each stabilized by three disulfide bonds (1).
Recent studies have addressed gastric MMPs and TIMPs and their role in gastric pathophysiology. MMP-1 (interstitial type I and type III-specific collagenase), the type IV-specific collagenases MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B), and TIMP-2 have been localized immunochemically in parietal cells, surface cells, and foveolar epithelial cells of normal human and rabbit gastric mucosa (14). In contrast, enhanced levels of mRNAs encoding MMP-1, MMP-3, MMP-7, and TIMP-1 were detected by in situ hybridization assays of human gastric mucosal samples representing peptic ulcers, Crohn's disease, and ulcerative colitis, and their involvement in tissue remodeling and epithelial regeneration was suggested (12). Overexpression of different MMPs has been reported in gastric cancer and appears to be correlated with increased metastatic potential (7).
Bacterial pathogens themselves synthesize and secrete a wide array of proteinases, of which the most common are metalloproteinases, particularly membrane-bound forms, which possess additional functional domains compared with the secreted forms (17). The functions of bacterial metalloproteinases are currently the focus of intense investigation and appear to include disruption of host defenses against invasive bacteria. Potential targets for these enzymes are host proteinase cascades, cytokine networks, extracellular matrix components, and host enzyme inhibitor inactivation (7). Analysis of the complete H. pylori genome revealed the presence of a nucleotide sequence encoding a putative zinc metalloprotease (16). At the same time, a 200-kDa zinc-dependent endoproteinase was isolated from H. pylori and found to be stably expressed on the surface of the bacterial outer membrane and also to be secreted into the culture medium, raising the possibility of metalloproteinase involvement in degradation of host proteins (21).
We recently investigated the effects of H. pylori infection
on H+-K+-ATPase -subunit promoter activity
in human adenocarcinoma (AGS) cells (5). The
responsiveness of AGS cells to the acid secretagogues histamine,
epidermal growth factor, and phorbol ester, in terms of elevation of
free intracellular Ca2+ and cAMP concentrations, suggested
that AGS cells were an appropriate model in which to study the effect
of H. pylori on gastric epithelial MMPs and TIMPs. In the
present study, we tested the hypothesis that H. pylori
infection of AGS cells modulates MMP and/or TIMP expression levels
and activities. The results indicate that AGS cells secrete MMP-1 and
MMP-3, as well as TIMP-1, -2, and -3, and that H. pylori
secretes both MMP-3-like enzyme and TIMP-3-like protein. H. pylori infection of AGS cells also enhances activation of host
MMP-1 and MMP-3.
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MATERIALS AND METHODS |
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Materials. Ham's F-12, HEPES, and antibiotic-antimycotic solution (10,000 U/ml penicillin G, 25 mg/ml amphotericin B, and 10,000 mg/ml streptomycin) were acquired from Cellgro Mediatech (Herndon, VA). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA). MMP-1 [Antibody (Ab)-1], TIMP-1 (Ab-1), TIMP-2 (Ab-1), and MMP-3 (Ab-2) monoclonal antibodies were purchased from Oncogene (Cambridge, MA). TIMP-3 (AB802) polyclonal antibody and MMP-3 (MAB 1339) monoclonal antibody were purchased from Chemicon (Temecula, CA). Control MMPs and TIMPs prepared from human skin fibroblast-conditioned media were obtained from Sigma (St. Louis, MO). Secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA) and Jackson Immunoresearch Laboratories (West Grove, PA). Fluorogenic substrates for measurement of MMP enzyme activity were purchased from Bachem (Torrance, CA). All other reagents were of molecular biology grade or the highest grade of purity available.
Cells and bacteria.
AGS cells [CRL 1739; American Type Culture Collection (ATCC)] were
maintained in AGS medium (Ham's F-12, 10% fetal bovine serum, 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, and 100 µg/ml
streptomycin) at 37°C in a 5% CO2-95% air incubator and used between passages 42 and 56. A strain of
H. pylori positive for vacuolating cytotoxin
(vacA+), cytotoxin-associated protein
(cagA+), and urease (ATCC number 49603) was
cultured on 5% horse blood agar plates (Remel, Lenexa, KS) incubated
at 37°C in sealed microaerophilic pouches (BBL Campy Pouch; Becton
Dickinson, Cockeysville, MD). Cultures were routinely screened for
urease activity. For AGS cell infections, H. pylori were
harvested between 48 and 72 h after inoculation of agar plates,
resuspended in sterile PBS, and enumerated by absorbance at 600 nm [1
optical density (OD)600nm = 2.4 × 108 colony-forming units/ml]. AGS cells (2.5 × 106) were seeded into T-75 flasks and infected at 90%
confluence with H. pylori at a multiplicity of infection
(MOI) of 50 in either serum-free Ham's F-12 or in fresh Ham's F-12
containing 10% serum. Infected AGS cells, uninfected AGS cells, and
H. pylori alone (in the same amounts as used for infection)
were cultured in T-75 flasks for 4, 6, 24, or 48 h as required.
Conditioned media were centrifuged for 10 min at 4,000 g to
remove cells and bacteria, and serum-free media were concentrated
10-200× by ammonium sulfate precipitation. Unconditioned
serum-free medium was concentrated and used as a negative
control. Media aliquots were stored at 70°C until used.
Zymography. Concentrated media were mixed with SDS-PAGE sample buffer (10% glycerol, 2% SDS, and 63 mM Tris, pH 7.0) without reducing agent and applied to nonreducing 10% acrylamide gels containing 0.1% gelatin or to nonreducing 4-16% acrylamide gels containing 0.1% casein (NuPAGE; Novex, Encinitas, CA). Electrophoresis was carried out for 90 min at 125 V at room temperature, and resolved proteins were renatured in situ by immersing the gels in 2.7% (wt/vol) Triton X-100 for 30 min at room temperature. The gels were then rinsed in zymogram developing buffer (Novex) for 30 min and incubated overnight at 37°C in the same buffer. Gelatinolytic or caseinolytic activity in the gels was visualized as negative staining with Coomassie brilliant blue. Metalloenzyme activity was stimulated by addition of 2 mM aminophenylmercuric acetate (APMA) to samples before electrophoresis. Metalloenzyme activity was inhibited similarly by using 20 mM EDTA and 2 mM 1,10-phenanthroline. Zymograms shown in this study are representative replicates selected from at least three experiments.
Enzyme activity assay. MMP activity of unconcentrated and 10-130× concentrated serum-free conditioned media from infected and uninfected AGS cells, as well as from H. pylori cultures, was measured using the fluorogenic MMP-1- and MMP-3-specific synthetic peptide substrates M-1905 and M2110 [Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2 and Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2, respectively]. In all cases, specificity of enzyme activity was shown by enzyme inhibition with 2 mM 1,10-phenanthroline. For studying the interaction between AGS cells and bacterial MMPs and TIMPs, equal amounts of AGS-conditioned and H. pylori-conditioned concentrated media (with or without heat treatment) were combined and incubated at 37°C for 30 min. Heat treatment of the samples was carried out at 65°C for 20 min. Substrate fluorescence evoked by metalloproteinase activity was measured for 200 s in an Aminco-SLM DW2 spectrofluorimeter at an emission wavelength of 399 nm with a slit width of 1 mm and sensitivity of 800 V.
Western blotting. Aliquots of concentrated media from H. pylori-infected or uninfected AGS cell cultures, or of medium from H. pylori alone, were mixed with SDS-PAGE sample buffer with or without reducing agent (dithiothreitol) and heated at 95°C for 5 min. Electrophoresis was carried out on 4-12% or on 10% acrylamide Bis-Tris gels (NuPAGE; Novex) for 35 min at 200 V. Resolved proteins were transferred to 0.2-µm nitrocellulose membranes (Osmonics, Westborough, MA) and blocked with 5% nonfat dry milk for 2 h. Protein replicas were washed three times in TTBS (20 mM Tris, 0.5M NaCl, pH 7.5, and 0.05% Tween 20) and incubated overnight at room temperature in the appropriate dilutions of MMP or TIMP antibodies. The replicas were then washed three times in TTBS and incubated for 1 h at room temperature in appropriate dilutions of secondary antibodies. Immunoreactive protein bands were visualized by using enhanced chemiluminescence and recorded on ECL Hyperfilm (ECL kit; Amersham Pharmacia, Piscataway, NJ).
Immunocytochemistry. Cells were seeded onto glass coverslips, cultured in 24-well plates to 90% confluence, and then incubated with H. pylori at an MOI of 50 for 3 h. H. pylori was also seeded alone and then concentrated onto glass coverslips by centrifugation (CytoSpin). Adherent cells on the coverslips were fixed with acetone for 6 min and washed several times with PBS (pH 7.4). Coverslips were blocked in 4% normal goat serum at 37°C for 30 min, washed in PBS, and then incubated for 1 h in TIMP-3-specific polyclonal antiserum diluted 1:50 in 1% BSA in PBS. After three washes in PBS, coverslips were incubated for 30 min in 1:100 dilutions in 1% BSA in PBS of FITC-labeled goat anti-rabbit IgG, and then washed three times in PBS. Negative controls included replacement of primary antibodies with nonimmune rabbit serum. Images of immunostained cells were recorded under epifluorescent illumination (490 nm) in a Zeiss Axiovert 35 microscope equipped with a digital camera (CCD-100, Dage-MTI) and ImagePro 3.0 Plus software.
Protein measurement. Culture medium protein concentrations, measured with Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA), were as follows. The protein concentration of serum-free unconditioned medium was 0.38 ± 0.08 mg/ml, that of AGS-conditioned medium was 0.75 ± 0.17 mg/ml, that of infected AGS-conditioned medium was 1.96 ± 0.29 mg/ml, and that of H. pylori-conditioned medium was 0.82 ± 0.21 mg/ml. Data are expressed as means ± SE (n = 6). Five-microliter culture medium aliquots were used for enzyme activity assays. For zymographic and immunoblot assays, 18-µl culture medium aliquots were applied to each lane.
Data analysis.
Densitometric analysis of zymogram gels was carried out using Scion
Image scanning software. Data are expressed as percentage of control
activity (the densitometric intensity of uninfected AGS
cell-conditioned medium was set to 100%) and are shown next to the
gels. Enzyme activity data were analyzed by linear regression and were
expressed as initial reaction velocity (fluorescence unit/s). Data comparisons were made by Student's
t-test and ANOVA, with P < 0.05 regarded as
significant. All experiments were carried out at least three times.
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RESULTS |
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As a first step toward the identification and characterization of
putative gastric epithelial cell secretory MMPs, gelatinolytic and
caseinolytic activities in AGS cell-conditioned culture media were
assessed by zymography. Aliquots of 50-200× concentrated serum-free culture medium were electrophoresed on denaturing, nonreducing, gelatin-containing gels, which were subsequently stained
with Coomassie blue; resolved gelatinolytic proteins were detected as
unstained bands. The zymogram in Fig. 1
shows traces of gelatinolytic activity in unconditioned medium,
and AGS cell-conditioned medium shows multiple bands of activity from
>250 to <30 kDa, with gelatinolytic activity being most prominent at
~90 kDa. Pretreatment of AGS cell-conditioned medium for 30 min at
37°C with APMA increased activity at ~90 kDa. APMA treatment also
increased activity at ~35 kDa at the expense of activity at ~160
and 60 kDa. Addition of either 20 mM EDTA or 2 mM 1,10-phenanthroline
to AGS cell-conditioned medium inhibited activity at ~160, ~90, and
~60 kDa. EDTA treatment also inhibited activity at 120 kDa. These
data indicate that AGS cells secrete a spectrum of gelatinases, a
subset of which are Ca2+- and Zn2+-dependent
metalloenzymes and are activated by APMA.
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As shown in the zymogram in Fig. 2,
coculture of AGS cells with H. pylori increased
gelatinolytic activity at ~90 kDa and at lower molecular masses
ranging from 25 to 40 kDa compared with the uninfected control. No
gelatinolytic activity was present in the concentrated serum-free
medium or in medium conditioned by H. pylori alone. H. pylori-induced changes in three secreted gelatinolytic activities
are expressed in Fig. 2 as percent densitometric intensity compared
with uninfected AGS cell control (100%).
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In complementary experiments, the presence of metalloproteinases of the
stromelysin family (MMP-3, MMP-10, and MMP-11) in AGS cell-conditioned
media was assessed by electrophoresis in casein-containing gels. As
shown in the zymogram in Fig. 3,
APMA-inducible caseinolytic activity at ~110 and ~22 kDa was
detected in AGS cell-conditioned medium. H. pylori infection
of AGS cells at an MOI of 50 resulted in increased APMA-inducible
caseinolytic activity being detected in the cell-conditioned medium. As
in Fig. 2, changes in caseinolytic activity following APMA treatment
and/or H. pylori infection are expressed in the figure as
percent densitometric intensity compared with uninfected AGS cell
control (100%). Serum-free AGS cell medium conditioned by growth of
H. pylori alone showed the presence of weak caseinolytic
activity at ~60 kDa (Fig. 4). This
activity may represent an activated form of the previously-reported 200-kDa H. pylori metalloproteinase (21).
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To characterize further the metalloproteinase activities detected by
zymography, we assessed the immunoreactivity of AGS cell-conditioned media by immunoblotting with a panel of MMP antibodies. Serum-free AGS
cell culture medium was used to avoid detection of matrix metalloenzymes present in serum. Under serum-free conditions, the rate
of AGS cell protein secretion is decreased, and therefore culture media
were concentrated by ammonium sulfate precipitation. Concentrated
(50-200×) AGS cell-conditioned media resolved on reducing
4-12% or 10% acrylamide SDS-PAGE gels showed immunoreactivity with both MMP-1 and MMP-3 antibodies at ~50 kDa (Fig.
5). MMP-3 antibody (MAB 1339) also
detected immunoreactivity at ~60 kDa (Fig. 5B), and
another MMP-3 antibody (Ab-2) revealed the presence of 25-, 35-, and
45-kDa immunoreactive species corresponding to active forms of
stromelysin-1 (Fig. 5C). H. pylori infection of the AGS cells increased MMP-1 immunoreactivity (Fig. 5A) and
increased the intensity of MMP-3-immunoreactive bands representing both latent (~60 kDa) and active (~50, 45, and 25 kDa) forms of the enzyme (Fig. 5, B and C). No MMP-1 or MMP-3
immunoreactivity was detected in serum-free AGS cell medium conditioned
by H. pylori alone (data not shown). These data indicate
that AGS cells secrete the metalloproteinases MMP-1 and MMP-3 and that
H. pylori infection stimulates both MMP-1 and MMP-3
secretion and activates preformed MMP-3.
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Further details of the functional properties of these AGS cell MMPs
were acquired in synthetic peptidolytic activity assays. AGS cell or
H. pylori-conditioned media (10× concentrated) were incubated with fluorogenic MMP-1- or MMP-3-specific substrates whose
hydrolysis was measured by fluorometry as changes in emitted fluorescence as a function of time (fluorescence units/s).
MMP-1-specific protease activity was undetectable in AGS
cell-conditioned media and amounted to 0.0083 ± 0.0008 fluorescence units/s in H. pylori-conditioned media
(Fig. 6A). In contrast,
MMP-3-specific protease activities in AGS cell-conditioned media were
0.047 ± 0.0008 compared with 0.657 ± 0.001 fluorescence
units/s in H. pylori-conditioned media. To determine whether
H. pylori proteases activate AGS cell MMP-3, media
conditioned by cocultured H. pylori and AGS cells, by AGS cells alone, or by H. pylori alone were combined before and
after heat treatment, and the resulting MMP-3-specific protease
activities were measured by fluorometry. Medium aliquots were
concentrated 130 times to optimize detection of enzyme activity. As
shown in Fig. 6B, conditioned media from cocultured H. pylori and AGS cells had 15-fold more MMP-3 specific protease
activity than AGS cell-conditioned media and 4.5-fold more than in
H. pylori-conditioned media. MMP-3-specific protease
activity in conditioned media from AGS cells alone, H. pylori alone, and AGS-H. pylori cocultures was
inactivated by heat treatment (Fig. 6B). As shown in Fig.
6C, MMP-3 activity of combined media was unaffected by heat
treatment of AGS cell medium; however, heat treatment of the H. pylori-conditioned medium significantly reduced the MMP-3 activity
of the combined media, and heat treatment of both AGS cell-conditioned
and H. pylori-conditioned media eliminated all MMP-3
activity. These data indicate that AGS cells secrete MMP-3 activity and
that H. pylori secretes both MMP-1- and MMP-3-like protease
activity. Combined MMP-3-like activity is significantly potentiated by
coculture of the bacteria with AGS cells. The persistence of low levels
of MMP-3 activity in heat-treated H. pylori-conditioned
media indicates that H. pylori MMP-3-like activity is more
heat stable than its mammalian counterpart. The data show that host
cell-bacterial interactions significantly affect MMP-3 activity and
that bacterial factors are required for increased host MMP-3 production
and activation, as suggested by immunoblot data (Fig. 5).
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Reasoning that TIMPs are a possible source of MMP inhibition, we probed
reducing SDS-PAGE replicas of conditioned media with antibodies
directed against TIMP-1, TIMP-2, or TIMP-3. Positive controls for
antibody specificity were provided by human skin fibroblast extracts
(MMP-Control-1 and -2; Sigma). Figure
7A shows a time-dependent
increase in TIMP-1 signal at ~29 kDa in both infected and uninfected
AGS cell-conditioned media. H. pylori infection appeared not
to affect the amount of secreted TIMP-1. No TIMP-1 immunoreactivity was
evident in H. pylori-conditioned media sampled after 24- or
48-h culture. With TIMP-2-specific antibody, ~21 kDa TIMP-2
immunoreactivity was evident at 24 and 48 h in H. pylori-infected and uninfected AGS cell-conditioned media (Fig.
7B); TIMP-2 signal strength was unaffected by H. pylori infection, and no TIMP-2 immunoreactivity was evident in
H. pylori-conditioned media harvested after 24- or 48-h
incubation. TIMP-3-specific antibody detected a prominent
immunoreactive band at 70 kDa and two less reactive bands at 88 and 52 kDa in AGS cell-conditioned medium (Fig. 7C). H. pylori-infected AGS cell-conditioned medium also showed the 88- and 70-kDa bands, a prominent 24-kDa band, and two less reactive TIMP-3
bands at 45 and 35 kDa (Fig. 7C). Significantly, both the
70- and 24-kDa TIMP-3-like immunoreactive bands were present in
H. pylori-conditioned medium (Fig. 7C).
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Further insights into TIMP-3 interaction with bacterial and host cell
MMPs were acquired from nonreducing SDS-PAGE immunoblot analyses of
conditioned media. As shown in Fig. 8,
both the TIMP-3 and the MMP-3 blots detected immunoreactivity at ~98
kDa. In addition to the monomeric 24-kDa TIMP-3 detected on reducing
blots (Fig. 7C), the nonreduced TIMP-3 immunoblot also
detected 60- and 45-kDa bands in media conditioned by AGS cells,
H. pylori, or cocultured AGS cells and H. pylori.
The prominent lower band at ~52 kDa in the MMP-3 immunoblot (Fig. 8)
represents the noncomplexed form of MMP-3. The MMP-3-specific antibody
(MAB 1339) showed higher immunoreactivity with nonreduced MMP-3 than
with the fully-reduced form shown in Fig. 5B, in which the
same amount of protein was applied to the gel. These data indicate that
heteromeric TIMP-3/MMP-3 complexes are formed in H. pylori-conditioned and in AGS cell-conditioned media and that
H. pylori infection of AGS cells augments the formation of
TIMP-3/MMP-3 complexes.
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Direct visualization of TIMP-3 immunoreactivity in AGS cells and in
H. pylori was accomplished by immunocytochemistry with TIMP-3 polyclonal antibody. In Fig. 9,
A and B, a phase-contrast micrograph of
immunostained AGS cells is compared with a fluorescent image of the
same field showing a granular pancellular distribution of TIMP-3
immunoreactivity. The same antibody gave intense immunoreactivity with
H. pylori alone (Fig. 9C), but immunostaining of
H. pylori-infected AGS cells showed that the previously
pancellular granular distribution of TIMP-3 immunoreactivity was now
aggregated and polarized toward the side of the cells closest to the
highest extracellular concentrations of H. pylori (Fig.
9D). These data suggest that H. pylori
interaction with gastric epithelial cell surfaces activates cellular
redistribution and exocytotic secretion of TIMP-3.
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DISCUSSION |
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In this study, we tested the hypothesis that H. pylori infection of gastric epithelial cells modulates bacterial and host cell MMP and/or TIMP expression levels. The gelatinolytic and caseinolytic data from AGS cell culture or H. pylori coculture supernatants indicate that AGS cells express comparable levels of at least two different types of MMPs by immunoblot analysis: interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3). However, depending on the antibody used, the immunoblot data show more (antibody MAB 1339) or less (antibody Ab-2) MMP-3 than MMP-1, and activity data using MMP-1- and MMP-3-specific fluorogenic substrates indicates that stromelysin-1 contributes the majority of secreted protease activity in both AGS cells and H. pylori. Our observation of increased MMP-1 and MMP-3 activity in H. pylori-cocultured AGS cell supernatants is consistent with H. pylori proteinase activation of these host cell metalloproteinases. A potential candidate for this role is the ~60-kDa caseinolytic proteinase we detected in H. pylori culture medium. This enzyme has lower molecular mass than the previously described H. pylori metalloenzyme (21), which could indicate differences among the H. pylori strains or could represent an enzymatically active proteolytic fragment of the larger enzyme. The fact that the H. pylori ~60-kDa caseinolytic proteinase has similar substrate specificity to human stromelysin-1 (MMP-3) indicates that H. pylori expresses an MMP-3-like metalloproteinase.
Sequence comparison between human stromelysin-1 (MMP-3) gene and the H. pylori genome further supports expression of MMP-3-like enzymes in H. pylori. Six open reading frames in the H. pylori genome have between 26 and 44% sequence similarity to a homologous 104-amino acid domain in the human MMP-3 gene sequence. Since the estimated molecular mass of these proteins is 25-44 kDa, the H. pylori-derived enzymatic activity detected at higher molecular masses on nondenaturing caseinolytic gels may represent complexes of bacterial MMPs with one another or with bacterial TIMPs.
Increased MMP activity is known to be followed by enhanced secretion of TIMPs (1). To date, four human TIMP isoforms have been identified (1). These TIMPS are two-domain proteins, with the NH2 terminal domain forming inhibitory complexes with MMPs. The COOH terminal domains of vertebrate TIMPs mediate formation of noninhibitory complexes with MMPs, exemplified by the TIMP-2-proMMP-2 complex (6). The TIMP-3 COOH terminal domain anchors the protein to the extracellular matrix by virtue of specific molecular interactions with extracellular matrix constituents (11). TIMPs also have other biological activities, for example, cell growth-promoting and erythroid-potentiating properties that are independent from their MMP-inhibitory activity. Genomic and cDNA sequence comparisons have identified putative homologous TIMPs in Drosophila and Caenorhabditis elegans (1), although functional or structural properties of nonmammalian TIMPs have not been reported.
In the present study, immunoblot analysis of H. pylori-conditioned media, AGS cell-conditioned media, and H. pylori-AGS cell coculture supernatants showed that TIMP-3 isoforms are expressed independently by H. pylori and AGS cells. Furthermore, nonreducing immunoblots indicated that host and bacterial TIMP-3 isoforms participate in heteromeric complex formation with AGS cell MMP-3, a finding that is consistent with decreased MMP-3 activity in H. pylori-AGS cell coculture supernatants as measured by fluorogenic substrate enzyme assays. Reducing and nonreducing immunoblots also revealed the presence of other SDS-stable TIMP-3-MMP complexes. Previous studies have reported inhibition of MMP activities by SDS-stable TIMP interactions with the enzymes (3, 22). Thus recombinant TIMP-2 was reported to inhibit rabbit fibroblast interstitial collagenase by formation of 1:1 molar ratio stoichiometric complexes with the 52-kDa procollagenase and the 46-kDa inactive intermediate of the enzyme. Both complexes and a third complex of TIMP-2 with the active 42-kDa collagenase were all stable in SDS (3). Human fibroblast collagenase and stromelysin-1 have also been reported to form SDS-stable inhibitory complexes with TIMP-1 (22).
Another characteristic of TIMPs is their spatially localized region of activity. The activity of TIMP-3, which appears to bind strongly to extracellular matrix components (11), is limited to areas close to its site of synthesis (1). Our immunocytochemical data are consistent with this localization, showing distribution of TIMP-3 immunoreactivity in infected cells close to the site of H. pylori attachment. We propose that H. pylori infection of AGS cells activates AGS cell TIMP-3, a process that may be mediated by the H. pylori MMP-3-like proteinase identified in this study, and that this locally-acting AGS cell TIMP-3 is then responsible for inhibition of both bacterial proteinases and activated host MMPs.
The TIMP-3 polyclonal antibody used in this study for immunoblot detection and immunocytochemical localization of TIMP-3 is directed against the COOH terminal domain of TIMP-3. Immunoreactivity of an H. pylori-secreted protein with this antibody suggests that H. pylori TIMP-3-like protein shares sequence similarity with human TIMP-3 COOH terminal amino acids. Sequence comparisons of human TIMP-3 gene with the H. pylori genomic sequence revealed seven H. pylori open reading frames with 22-53% similarity to 71 COOH terminal amino acids of human TIMP-3. At this time, functional roles have not been ascribed to putative H. pylori TIMP-like protein. Clearly, as inhibitors of host cell metalloenzymes involved in facilitating neutrophil and macrophage infiltration of the gastric mucosa, bacterial TIMPs could have important roles in promoting bacterial colonization of host epithelial surfaces.
Detection of secreted MMPs and TIMP in culture media conditioned only
by H. pylori suggests an additional novel functional role
for the type IV secretion system of H. pylori.
Macromolecular type IV secretory assemblies of gram-negative bacteria
have been implicated in conjugative transfer of DNA in E. coli, transfer of plasmid DNA from Agrobacterium
tumefaciens to plant cell nuclei, Bordetella pertussis
secretion of pertussis toxin, migration of Rickettsia,
Legionella, and Brucella signaling factors to host cell
cytoplasm and vacuolar spaces, and pedestal formation by H. pylori contact with epithelial cell plasma membranes
(2). Recently, the type IV secretion system of H. pylori was shown to mediate transfer of the bacterial CagA protein
into gastric epithelial cells, where its tyrosine phosphorylation
modulated tyrosine phosphorylation of a spectrum of host cell proteins
(10). Whether the type IV secretion system of H. pylori is also responsible for secretion of MMPs and TIMP remains
to be determined. Delivery of these MMPs and TIMP into the
extracellular matrix encompassing gastric epithelial cells would allow
bacteria to modulate host cell MMP and TIMP mobilization, which could
culminate in generation of -defensins and other antibiotic peptides
(18).
In summary, the significant results of this study are that gastric epithelial cells secrete MMP-1 and MMP-3 and that H. pylori infection increases secretion of both metalloproteinases. H. pylori also secretes MMP-1- and MMP-3-like metalloproteinases, the latter accounting for the majority of H. pylori-secreted proteinase activity. Gastric epithelial cells and H. pylori both express TIMP-3, and H. pylori infection of the cells induces cellular redistribution of TIMP-3 to the site of H. pylori attachment to the cells. We propose that the outcome of gastric mucosal infection by H. pylori depends in part on interactions between epithelial and bacterial MMPs and TIMPs identified in this study. To the extent that H. pylori MMPs and TIMP-3-like proteins promote bacterial colonization of gastric epithelium, these MMPs and their inhibitors may be considered a novel class of H. pylori virulence factors.
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ACKNOWLEDGEMENTS |
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We acknowledge the expert technical assistance of Charles Hammond and Kellie Larsen. We thank the MUSC Spectroscopy Core Facility at the Medical University of South Carolina.
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FOOTNOTES |
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This work was supported by NASA (NAG8-1385) and National Institute of Diabetes and Digestive and Kidney Diseases (DK-43138) grants to A. Smolka.
Address for reprint requests and other correspondence: A. J. Smolka, Dept. of Medicine CSB 921E, Medical Univ. of South Carolina, 96 Jonathan Lucas St., Charleston, SC 29425 (E-mail: smolkaaj{at}musc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 November 2000; accepted in final form 9 May 2001.
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