Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Clinical studies and in vitro data from isolated parietal
cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by
H+-K+-ATPase, an integral protein of parietal
cell apical membranes. To test the hypothesis that H. pylori
downregulates H+-K+-ATPase -subunit (HK
)
gene expression and to identify potential intracellular signaling
pathways mediating such regulation, we transfected human gastric
adenocarcinoma (AGS) cells with human and rat HK
5'-flanking
DNA fused to a luciferase reporter plasmid. Histamine caused
dose-dependent, cimetidine-sensitive (10
4
M) increases in cAMP, free intracellular Ca2+, and
HK
promoter activation in AGS cells. H. pylori infection of
transfected AGS cells dose dependently inhibited basal and histamine-stimulated HK
promoter activity by 80% and 66%,
respectively. H. pylori dose dependently inhibited phorbol
myristate acetate-induced (10
7 M) and staurosporine-
(10
7 M) and calphostin C-sensitive (5 × 10
8 M) activation of HK
promoter. Also, H. pylori inhibited epidermal growth factor (EGF)
(10
8 M), genistein-sensitive (5 × 10
5 M) activation of HK
promoter, reducing
activity to 60% of basal level. These data suggest that H. pylori
inhibits HK
gene expression via intracellular pathways involving
protein kinases A and C and protein tyrosine kinase, AGS cells have
functional histamine H2 and EGF receptors, and transiently
transfected AGS cells are a useful model for studying regulation of
HK
gene expression.
gastric adenocarcinoma cells; luciferase; promoter regulation; acid secretion; H2 receptor
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INTRODUCTION |
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THIS STUDY TESTS THE HYPOTHESIS that Helicobacter
pylori infection of human gastric epithelial cells inhibits gene
expression regulated by the 5'-flanking region of the
H+-K+-ATPase -subunit (HK
) gene. H. pylori is a spiral, microaerophilic, Gram-negative bacterium that
colonizes the gastric mucosa in 25-50% of the population in
developed countries and 70-90% in developing countries (52).
H. pylori is a causative agent of peptic ulcer disease, gastric
adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma
(18). In addition, disturbances of normal acid secretory mechanisms
accompany H. pylori gastric infection, resulting in either
hypo- or hyperchlorhydria, depending on the clinical setting. Thus in
human studies, acute H. pylori infection has been associated
with hypochlorhydria (25, 37-39, 41); in animal studies, dogs (23)
and ferrets (34) were rendered achlorhydric after infection with H. felis and H. mustelae, respectively. Chronic H. pylori infection either stimulates acid secretion, decreasing gastric pH and predisposing to duodenal ulcer (20), or
causes impaired acid secretion with increased risk for gastric cancer (19, 33, 43).
Studies (8, 32) of gastric mucosal cells infected in vitro with H. pylori have demonstrated inhibition of acid secretory function. Thus in rabbit (8) and guinea pig gastric cell isolates (32), [14C]aminopyrine accumulation (measuring parietal cell intracellular acidification) was reduced after H. pylori infection. A nonhuman-infecting Helicobacter species (H. felis) also inhibited acid secretion in rabbit parietal cells (54). In human parietal cells, H. pylori infection inhibited histamine-, carbachol-, and dibutyryl cAMP-stimulated acid secretion (28, 29). Sonicates of H. pylori also reduced acid secretion in rabbit parietal cells (8), and vacuolating toxin in supernatants of H. pylori had similar inhibitory effects on guinea pig parietal cells (32). Other putative acid-inhibitory factors associated with H. pylori are acid inhibitory factor 2, present in an organic solvent extract of H. pylori (7), and a 92-kDa protein (27). In contrast, an H. pylori fatty acid (cis-9,10-methylene-octadecanoic acid) has been reported to stimulate acid secretion in isolated guinea pig parietal cells (4).
Gastric acid secretion is mediated by an Mg2+-dependent,
K+-stimulated, H3O+-transporting,
P-type ATPase (H+-K+-ATPase, EC 3.6.1.36) (21,
44). The -subunit of the enzyme (HK
, Mr,
~94,000) is a polytopic integral protein of tubulovesicular and
secretory canalicular membranes in acid-secreting gastric parietal
cells. Close interaction of HK
with an integral monotopic glycosylated
-subunit (HK
, Mr,
~60,000-80,000) is required for functional electroneutral
exchange of luminal K+ for cytoplasmic protons (9). The
acid secretagogues histamine, gastrin, and carbachol induce HK
gene
transcription in enriched canine parietal cell preparations (5).
Pretreatment of the cells with omeprazole, a specific, irreversible,
covalently bound inhibitor of H+-K+-ATPase,
also induced HK
gene transcription (6). Several HK
promoter
response elements have been associated with induction of HK
gene
transcription. A 5'-flanking sequence motif (GATA) of rat HK
and HK
genes transfected into HeLa cells binds the parietal
cell-specific transcription factors GATA-GT1, 2, and 3, with
concomitant transcription of both genes (42, 51). Deletional analysis
of canine HK
5'-flanking sequences transfected into canine
parietal cells showed that binding of the transcription factor Sp1 to a
site between bases
54 and
45 activated constitutive HK
transcription (40). A similar approach showed that epidermal growth
factor (EGF)-induced transcriptional activation of HK
gene was
accompanied by protein binding to a 5'-flanking sequence between
bases
162 and
156 (30). Most recently, a preliminary study (41) showed HK
promoter activation when p53, a transcription factor involved in cell cycle regulation and apoptosis, interacts with
an HK
5'-flanking segment between bases
162 and
144. The repercussions of H. pylori gastric infection on
HK
gene transcription have not been reported.
To study the mechanisms whereby H. pylori gastric infection may
affect expression levels of the H+-K+-ATPase
gene, we cloned human and rat HK 5'-flanking sequences into a
mammalian expression vector upstream of firefly luciferase coding
sequence. Transfection of human gastric adenocarcinoma cells (AGS
cells) with these vectors allowed luminometric measurement of transient
luciferase expression as a measure of HK
5'-flanking sequence
activity in response to acid secretagogues and H. pylori. Transfection studies in this AGS cell model revealed specific regulation of HK
promoter activity by histamine, EGF, and H. pylori and implicated protein kinases A (PKA) and C (PKC) and protein tyrosine kinase (PTK) as mediators of such regulation.
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MATERIALS AND METHODS |
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Materials. Ham's F-12, HEPES, antibiotic-antimycotic solution (10,000 U/ml penicillin G, 25 g/ml amphotericin B, and 10,000 mg/ml streptomycin), and Hanks' balanced salt solution (HBSS) without phenol red were acquired from Cellgro Mediatech (Herndon, VA). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA). Calphostin C, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and phorbol-12-myristate-13-acetate (PMA) were purchased from Calbiochem-Novabiochem (La Jolla, CA). Histamine (free base), cimetidine, genistein, staurosporine, EGF, IBMX, and EGTA were purchased from Sigma Chemical (St. Louis, MO). Restriction enzymes were obtained from Promega (Madison, WI), and Cytodex-3 microcarrier beads were from Amersham Pharmacia Biotech (Piscataway, NJ). All other reagents were of molecular biology grade or the highest grade of purity available.
Cells and bacteria.
Human AGS cells (ATCC CRL 1739) 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 was obtained from the American Type
Culture Collection (Rockville, MD) (ATCC no. 49603). H. pylori
were cultured on 5% horse blood agar plates (Remel, Lenexa, KS),
which were incubated in a BBL Campy Pouch microaerophilic system
(Becton Dickinson, Cockeysville, MD) at 37°C. Cultures were
routinely screened for urease activity and discarded unless positive.
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 OD600nm = 2.4 × 108 colony-forming units/ml) (31). For experiments
using heat-killed H. pylori, bacteria were suspended in sterile
PBS and heated at 80°C for 30 min. Esherichia coli (strain
DH5, Life Technologies, Grand Island, NY) were seeded onto agar
plates and incubated at 37°C overnight. A single colony was
inoculated into 10 ml LB broth without antibiotics and cultured
overnight in a shaking incubator (225 rpm) at 37°C. The bacterial
suspension was centrifuged at 4°C at 1,000 g, and
sedimented bacteria were washed twice with sterile PBS. E. coli
were resuspended in PBS for infection of AGS cells.
HK promoter-reporter gene constructs.
Genomic DNAs encompassing the 5'-flanking regions of both human
and rat gastric H+-K+-ATPase genes were kindly
provided by Dr. Gary Shull, University of Cincinnati. The human DNA was
precipitated and dissolved in TE buffer (Tris · HCl
and EDTA). A 2.2-kbp segment of the promoter sequence
(ending at the translation start site) was amplified by PCR (Gene-Amp
XL-PCR kit, Perkin-Elmer, Norwalk, CT) using forward
(5'-AATATGGTACCTCGACTCGA-TCCGTCCACCTCA-3') and reverse (5'-AATATAAGCTTGCCTGTGCTCCCACCCA-ACA-3') oligonucleotide
primers that add Kpn I and Hind III restriction sites
to the 5' and 3' ends, respectively, of the 2.2-kbp
promoter sequence. Approximately 1 ng of DNA was used as a
template, and the reaction proceeded at 94°C for 1 min and 61°C
for 8 min, for 30 cycles. After digestion with Kpn I and
Hind III, the PCR product was ligated into the Kpn I
and Hind III cloning sites of luciferase expression vector pGL2-Basic (Promega). The rat HK
5'-flanking region was
excised from the donor plasmid with Nhe I and Bgl II,
yielding a 2.2-kbp segment that was ligated into the corresponding
cloning sites of pGL2-Basic. All plasmid constructs were purified by
CsCl double banding. At least three separate preparations of each
plasmid construct were used to acquire experimental data from AGS cell transfections.
Transient transfection and luciferase assay.
AGS cells were plated into six-well plates (300,000 cells/well) and
incubated for 18 h at 37°C in 5% CO2-95% air. The
cells were washed once with 2 ml Opti-MEM I serum-reduced medium (Life Technologies). An aliquot (2 µg) of either rat or human HK
promoter-luciferase plasmid DNA was mixed with 100 µl Opti-MEM I
(solution A). Lipofectamine reagent (10 µl, Life
Technologies) was mixed with 90 µl Opti-MEM I (solution B).
Solutions A and B were mixed, incubated at room temperature for 30 min, and layered onto the cells. The pGL2-Control plasmid, containing SV40 promoter and enhancer, was used as a positive
control; a negative control was provided by transfection with
pGL2-Basic plasmid, containing neither promoter nor enhancer. Cells
were then incubated for 5 h at 37°C in 5% CO2-95%
air. After cell treatments as specified below, the cells were lysed
using passive lysis buffer (Promega) according to the manufacurer's suggested protocol. The luciferase-catalysed cellular light emission (relative light units; RLU) reflecting HK
promoter activity was measured for 30 s (AutoLumat LB 953, Wallac, Gaithersburg, MD), and
luciferase activity was expressed as a percentage of unstimulated control. As a control for interassay variability, control wells in each
experiment were cotransfected with an aliquot (0.04 µg) of
Renilla pRL-TK plasmid DNA (Promega). The ratio of basal HK
promoter-driven light emission to that driven by the Renilla
pRL-TK plasmid DNA was relatively constant in the control wells
from experiment to experiment. Calculated interassay variability was minimal (CV% = 6.3%), reflecting the high reproducibility of the lipofectamine transfection protocol.
AGS cell treatments.
Transfected AGS cells were placed in fresh serum-free Ham's F-12
medium without antibiotic and incubated for 30 min at 37°C with
cimetidine (106 to 10
4 M) or
vehicle, followed by a further 5 h incubation at 37°C with histamine or vehicle. Transfected AGS cells were incubated for 24 h
with H. pylori (0.6-24 × 107
bacteria/ml) at multiplicities of infection (MOI) ranging from 20 to
800 or with E. coli (0.6-6 × 107
bacteria/ml) at MOI ranging from 20 to 200. Viability of cells after
H. pylori infection was 97%, measured by trypan blue exclusion and the LIVE/DEAD viability kit using the manufacturer's suggested protocol (Molecular Probes). Probes of intracellular signaling pathways, such as EGF, PMA, staurosporine, calphostin C, OAG, and
genistein, were incubated with transfected cells at concentrations and
times as specified in RESULTS.
cAMP measurement.
AGS cells were grown to confluence in six-well culture plates. AGS
medium was replaced with fresh, serum-free AGS medium containing 0.4 mM
IBMX without or with histamine (106 to 3 × 10
4 M) or with histamine (10
4 M)
and cimetidine (10
4 M) for 30 min at 37°C.
Medium was then aspirated from the wells and replaced with 2 ml
ice-cold ethanol (66% vol/vol). Cell suspensions recovered from wells
were centrifuged for 10 min at 2,000 g, and the
supernatants were lyophilized and stored at
70°C until
measurement. Cellular cAMP content was determined using a competitive
cAMP enzyme immunoassay kit (Signal Transduction Products, San
Clemente, CA).
Free intracellular Ca2+
measurement.
AGS cell free intracellular Ca2+ concentration
([Ca2+]i) was measured by a
high-throughput optical screening system for cell-based fluorometric
assays (47) [fluorometric imaging plate reader system (FLIPR),
Molecular Devices, Sunnyvale, CA]. The instrument simultaneously
reads all 96 wells of a test microplate with kinetic updates in the
subsecond range and includes a 6-W argon ion laser (Coherent, Santa
Clara, CA), an optical scanning system, an integrated 96-tip pipettor
to transfer reagents from two 96-well addition trays to the test
microplate, temperature control (37 ± 0.1°C), and a
charge-coupled device camera. AGS cells were seeded (~50,000 cells/well) into a 96-well clear-bottom black test microplate (Corning
Costar, Cambridge, MA) and placed overnight in a 5%
CO2-95% air incubator at 37°C. Cells were dye loaded
with 4 µM fluo 3-AM ester (excitation at 488 nm, emission at 540 nm;
Molecular Probes) in a loading buffer (1 × HBSS, pH 7.4, with 20 mM HEPES and 2.5 mM probenecid) for 1 h at 37°C. Probenecid avoids
measurement of artifactually elevated Ca2+ signals by
blocking anion transporter-mediated accumulation of fluo 3 free acid in
intracellular Ca2+ storage compartments. The test
microplate was washed four times with loading buffer and loaded into
the FLIPR instrument. Cimetidine, vehicle, or EGTA was then added from
the first addition tray to specified wells of the test microplate.
Emission intensities in each well of the test microplate were measured
simultaneously at 6-s intervals for 10 min. Histamine and EGTA, or
vehicle, was then added from the second addition tray to specified
wells of the test microplate. Emission intensities in each well of the test microplate were then measured simultaneously at 1-s intervals for
6 min. Differences in fluo 3 emission intensities between vehicle-treated wells and cimetidine-, histamine-, or EGTA-treated wells, respectively, were calculated by FLIPR software and displayed as
time courses of emission intensity for each condition, expressed as the
percentage of maximal emission intensity elicited by
103 M histamine.
Scanning electron microscopy. AGS cells (2 × 106) and Cytodex-3 microcarrier beads (8 × 104, cell-to-bead ratio = 25) were suspended in 10 ml AGS medium in a rotating high-aspect-ratio culture vessel (Synthecon, Houston, TX), and the cells were grown to confluency (3 days) at 37°C in a 5% CO2-95% air incubator. Aliquots of the bead/cell suspension were placed on Thermonox glass coverslips in six-well plates, and H. pylori were added to the cells at an estimated MOI of 10. After 24 h, the growth medium was removed, and beads with adherent cells were fixed for 30 min in 2% glutaraldehyde/cacodylate buffer at room temperature, rinsed in 0.1 M cacodylate buffer with 7% sucrose, and postfixed in 2% aqueous osmium tetroxide for 2 h. Gold-coated samples were examined in the JEOL LV5410 scanning electron microscope.
Interleukin-8 assay.
Samples of medium from transfected, H. pylori-infected AGS
cells were removed from the wells after 24 h and stored at
70°C. Interleukin-8 (IL-8) concentrations of the samples
were determined by ELISA (human IL-8 Duo-Set ELISA development system,
R&D Systems, Minneapolis, MN).
Statistical analysis. Comparisons between treatment groups were made by using unpaired Student's t-tests and ANOVA. Findings of P < 0.05 were taken to indicate statistical significance. The Statistica software package (Statsoft, Tulsa, OK) was used for this purpose.
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RESULTS |
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Analysis of modulation of HK promoter activity in gastric epithelial
cells was achieved by transfecting human AGS cells with a plasmid
(pGL2-Basic) containing 2.2 kbp of the 5'-flanking region of the
human or rat HK
gene fused to a reporter (firefly luciferase) gene
plasmid. The activation status of the HK
promoter in response to
gastric secretory agonists, antagonists, and H. pylori was then
measured by exposing transfected cells to luciferin and expressing the
resulting light emission intensity in RLU, using the light emission of
transfected but untreated cells as the basal level.
As a first step in validating transfected AGS cells as an appropriate
experimental system for studies of alteration of HK gene expression
by H. pylori infection, we sought to establish the
responsiveness of the cells to the acid secretagogue histamine, in
terms of increases in cAMP levels (22). AGS cells were incubated with
histamine alone or with histamine and cimetidine (an
H2-receptor antagonist), and then cAMP concentrations in a
cytoplasmic extract of the cells were measured using a
competitive enzyme-linked immunoassay. As shown in Fig.
1, histamine elicited dose-dependent
increases in AGS cell cAMP concentration, with the greatest increase
(39% over basal cAMP concentration) occurring at
10
4 M histamine. Coincubation of the cells with
10
4 M cimetidine completely suppressed elevation of
AGS cell cAMP by histamine.
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To further substantiate the functionality of the putative
H2 receptor on AGS cells, we measured
[Ca2+]i in AGS cells treated with
histamine. Gastric parietal cells have been shown to respond to
histamine stimulation with transient increases in
[Ca2+]i (36). AGS cells were loaded
with the nonratiometric fluorescent Ca2+ probe fluo 3, and
relative changes in [Ca2+]i in AGS
cells in response to histamine, EGTA, and cimetidine were measured as
changes in fluorescence emission intensities (540 nm) of intracellular
fluo 3. As shown in Fig. 2, histamine elicited
dose-dependent increases in AGS cell
[Ca2+]i. In the presence of
extracellular EGTA (1.5 mM), the increase in
[Ca2+]i caused by 1 mM and 300 µM
histamine was ~20% of the EGTA-free response (Fig. 2, A and
B), and no response was observed with 100 µM histamine (Fig.
2C). In the presence of cimetidine (103 M),
the increase in [Ca2+]i caused by 1 mM and 200 µM histamine was ~20% of the cimetidine-free response
(Fig. 2, D and E). Persistence of some
histamine-stimulated [Ca2+]i
elevation in the presence of 10
3 M cimetidine
suggests that AGS cells may express H2 receptor subpopulations with differing cimetidine sensitivity. Nevertheless, the
data indicate that histamine-stimulated transient increases in AGS cell
[Ca2+]i are mediated at least in
part by endogenous histamine H2 receptors coupled to a
plasma membrane Ca2+ influx pathway. The mechanism of such
coupling in AGS cells remains to be clarified.
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Because histamine is known to induce HK gene transcription by a
receptor-mediated signaling pathway in isolated canine gastric parietal
cells (5), we measured the activation status of HK
promoter in AGS
cells transfected with the pGL2 HK
-reporter plasmid. Figure
3 shows the dose-dependent stimulation of
HK
promoter activity by histamine treatment of transfected AGS
cells. At a concentration of 10
4 M, histamine
stimulated the human HK
promoter sequence activity by 103 ± 8%
(n = 7). Preincubation of transfected AGS cells with 10
4 M cimetidine, an H2 receptor
antagonist, restricted histamine-dependent activation of HK
promoter
sequence to 38 ± 2.2% (n = 7); cimetidine alone
(10
4 M) exerted no significant activation of HK
promoter sequence.
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Having demonstrated histamine-dependent activation of the HK
promoter in transfected AGS cells, we sought to identify components of
intracellular signaling pathways that might be involved in this
response. In view of several inconsistent reports of PKC involvement in
acid secretory signal transduction in parietal cells (10, 11, 40), we
first studied the effect on the human HK
promoter of activation of
PKC by PMA. Transfected AGS cells were preincubated for 1 h with either
calphostin C (5 × 10
8 M), a highly specific
PKC inhibitor, or staurosporine (10
7 M), a
relatively nonspecific PKC inhibitor, and then incubated for 5 h with
or without PMA. Figure 4A shows
that PMA stimulated the HK
promoter sequence in a dose-responsive
manner, with maximal (2.7-fold) stimulation elicited at
10
7 M PMA. Concurrent incubation of the cells with
PMA and calphostin C restricted HK
promoter activation to 1.7-fold
of basal, whereas staurosporine eliminated the PMA response completely.
Because PMA specificity is not restricted to activation of PKC, we
measured the effect of the diacylglycerol analog OAG on PKC-mediated
HK
promoter activation. Figure 4B shows that OAG exerted a
dose-responsive stimulation of human HK
promoter transfected into
AGS cells and that this stimulation was preempted by treatment of the
cells with calphostin C (5 × 10
8
M). These data indicate a role for PKC in regulation of
HK
gene transcription initiation in gastric epithelial cells.
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Because PTK have also been implicated in regulation of parietal cell
acid secretion (3, 12, 35) and indeed may also activate PKC with acid
secretory sequelae (55), we next studied the effect on the human HK
promoter of activation of PTK by EGF. Figure
5 shows that EGF stimulated the human HK
promoter in a dose-responsive manner, with maximal stimulation elicited
at 10
8 M EGF. Concurrent incubation of the cells
with EGF and the PTK inhibitor genistein (5 × 10
5 M) eliminated the EGF response; genistein alone
had no significant effect on HK
promoter activity.
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Having shown responsiveness of an exogenous HK promoter sequence in
AGS cells to physiologically relevant stimuli, including histamine,
PMA, OAG, and EGF, we then asked whether H. pylori infection of
the cells would impact on promoter activation by these stimuli, thereby
providing potential molecular mechanisms at the level of
H+-K+-ATPase gene expression for the observed
clinical pathophysiology of H. pylori infection. To exclude the
possibility that any observed changes in HK
promoter activity after
H. pylori infection were simply artifacts arising from reduced
AGS cell viability, we measured infected cell viability by both trypan
blue exclusion and a fluorescence-based assay. Over a range of H. pylori MOI (20-400), AGS cells showed >95% viability 24 h
after H. pylori infection (data not shown). Although plasma
membrane integrity was clearly unaffected by H. pylori, at
least by these criteria, scanning electron microscopy revealed
significant differences between uninfected and infected cells. Normal,
uninfected AGS cells growing on the surface of Cytodex 3 microcarrier
beads displayed dense surface arrays of microvilli and microplicae
(Fig. 6A). In contrast,
after 24 h incubation with H. pylori, AGS cell surface
microvilli and microplicae were greatly reduced in number, and numerous
plasma membrane blebs were in evidence (Fig. 6B).
In this photomicrograph, three H. pylori organisms are seen
attached to cell plasma membranes at the interface between adjacent AGS
cells. To verify that AGS cells, having undergone reporter plasmid
transfection and H. pylori infection, nonetheless retained
functional physiological responses in spite of these morphological
changes, we tested the ability of the cells to mount an IL-8 secretory
response to H. pylori infection. Figure 7 shows that at H. pylori MOI of up
to 100, transfected AGS cells exhibited a robust secretion of IL-8,
raising the medium IL-8 concentration from the lower limit of ELISA
detection (~30 pg/ml) to over 800 pg/ml. The specificity of this
response for viable H. pylori was shown by the significantly
attenuated IL-8 response to infection with heat-killed H. pylori.
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Infection of transfected AGS cells for 24 h with H. pylori was
accompanied by marked inhibition of both human and rat HK 5'-flanking sequence basal activation of luciferase gene
transcription/translation (Fig.
8). Significantly, human HK
promoter
was far more sensitive to H. pylori infection than rat HK
promoter; half-maximal inhibition of human HK
promoter occurred at
an H. pylori MOI of ~65, while equivalent inhibition
of rat HK
promoter required an MOI of 650. Heat-killed H. pylori at an MOI of 65 had no inhibitory effect on the human HK
promoter, again demonstrating the specificity of the inhibition for
viable H. pylori. As a further test of specificity, the effect
of another Gram-negative bacterium, E. coli DH5
, on human
HK
promoter status was measured. Transfected AGS cells were
incubated for 24 h at 37°C with E. coli at MOI between 20 and 200; no inhibition of human HK
promoter activity was detected at
even the higher MOI tested (data not shown). Clearly, the differential inhibition of basal activation of human and rat HK
promoter by H. pylori is consistent with the specificity of the organism
for human hosts.
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Given the sensitivity of basal HK promoter activation to H. pylori infection, at least in the setting of the AGS cell line, we
then assessed the extent to which acid secretagogue-induced promoter
activation was impacted by H. pylori infection of the cells.
Aliquots of transfected AGS cells were incubated with histamine (10
4 M), PMA (10
7 M), or EGF
(10
8 M), in the presence or absence of H. pylori (at MOI = 50 or 100), and HK
promoter activation was
measured 24 h later. As noted before, histamine alone caused a 2.4-fold
activation of HK
promoter; this stimulation was reduced almost to
basal levels by H. pylori infection at MOI of 50 and to 80% of
basal level by MOI of 100 (Fig.
9A). Incubation of HK
promoter-transfected AGS cells with PMA caused a 10-fold stimulation of
promoter activity; when the cells were infected with H. pylori
at an MOI of 50, PMA caused only 4.3-fold stimulation of HK
promoter activity, and infection at MOI of 100 allowed only 2.2-fold
stimulation (Fig. 9B). Finally, incubation of HK
promoter-transfected AGS cells with EGF (10
8 M)
caused a twofold stimulation of promoter activity (Fig. 9C). However, H. pylori infection of the cells at an MOI of 50 and 100 reduced HK
promoter activity below the basal level, to 75% and
60%, respectively. The marked differences in promoter activity measured in this set of experiments, compared with activities shown in
Figs. 4A and 5, reflect the longer secretagogue incubation times here (24 h instead of 5 h). At MOI of 50 and 100, H. pylori infection alone inhibited basal HK
promoter
activity by 42% and 56%, respectively (Fig. 8). Together, these data
are consistent with downregulation of HK
promoter activity by H. pylori infection being mediated at least in part by modulation of
PKA and PKC and PTK activities.
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DISCUSSION |
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In this study, we tested the hypothesis that H. pylori
infection of human gastric epithelial cells mobilizes intracellular signaling pathways that inhibit H+-K+-ATPase
gene expression. Human and rat H+-K+-ATPase
5'-flanking sequences fused to a luciferase reporter gene were
transiently transfected into human AGS, and responsiveness of the HK
promoter sequence to acid secretory agonists and antagonists and to
H. pylori infection was measured.
The gastric epithelial origin of AGS cells makes this cell line
particularly useful for in vitro studies of H. pylori
pathophysiology. For example, in response to H. pylori
infection, AGS cells have been shown (14) to secrete the cytokine
IL-8, a neutrophil chemotactic factor increased in H. pylori-infected patients with active gastritis. This process is
initiated by H. pylori attachment to AGS cells, which promotes
reorganization of actin and associated cellular proteins such as
vasodilator-stimulated phosphoprotein (46) and has also been shown to
induce tyrosine phosphorylation of 145-kDa host protein (45). The
subsequent activation of nuclear factor-B and its translocation into
the nucleus where interaction with IL-8 promoter response elements
stimulates IL-8 gene expression were also analyzed in AGS cells (31,
48).
However, the derivation of AGS cells from transformed gastric
adenocarcinoma tissue raises the possibility that AGS cell responses to
H. pylori may be different from normal gastric mucosal cell responses, particularly after the distinctly nonphysiological lipofectamine-mediated transfection of the cells with HK-reporter gene plasmids. In this context, our demonstration of high AGS cell
viability, H. pylori-mediated effacement of surface microvilli [reported in primary human gastric epithelial cells from patient gastric biopsies (49)], robust secretion of IL-8 in response to
H. pylori infection, and apparently functional histamine
H2-receptor signal transduction pathways suggests that the
cell line retains significant physiological parallels with normal
gastric epithelial cells. IL-8, an activator of neutrophils, T cells,
and basophil histamine degranulation, plays a prominent role in
mediating gastric inflammation and epithelial cell degeneration and as
such may have significant effects on acid output in vivo. In this in
vitro study using only epithelial cells, downregulation of HK
promoter activity cannot be attributed to IL-8 secreted by AGS cells.
We observed that E. coli DH5
infection of AGS cells did not
inhibit HK
promoter activity, even though E. coli DH5
induces robust IL-8 secretion in AGS cells (48).
Another particularly significant parallel with gastric cells emerges
from our measurements of the relative susceptibility of human and rat
HK promoter to inhibition by H. pylori. Because we
transfected both rat and human HK
promoters into the same host
(human AGS cells), the differential susceptibility of promoter downregulation to H. pylori infection must originate in
specific sequence differences between the 2.2-kbp 5'-flanking
segments of the two HK
genes. These sequence differences are guiding
our continued study of promoter regulation by H. pylori.
Clearly, the human promoter's far greater sensitivity to
downregulation by H. pylori is entirely consistent with the
fact that, with the exception of nonhuman primates (17), the human
stomach is the only substantial reservoir of H. pylori.
As part of our validation of the AGS cell line as an appropriate model
for studies of HK promoter regulation, we sought to establish that
functional histamine H2 receptors were present on the cell
surface. Our measurements of histamine-stimulated, cimetidine-sensitive
elevations of [Ca2+]i and cAMP in
AGS cells provide strong evidence for the presence of H2
receptors on these cells functionally coupled to an adenylate cyclase
pathway (cAMP generation) and to another pathway promoting transient
elevation of free intracellar Ca2+. Induction of
[Ca2+]i pulses in AGS cells by
100-300 µM histamine (Fig. 2) is consistent with
[Ca2+]i oscillations induced by
10
4 M histamine in ~50% of rabbit parietal cells
(36), suggesting that H2 receptor coupling to intracellular
signaling pathways may be similar in AGS and parietal cells. Although
10
5 and 10
6 M histamine also
elicited [Ca2+]i pulses in rabbit
parietal cells (36), those measurements derived from observations of
single cells and show considerable heterogeneity in
[Ca2+]i responsiveness to histamine
among cells in the same preparation. In contrast,
10
4 M histamine induced only minimal elevations
of [Ca2+]i in <10% of canine
parietal cells (15). Paradoxically, histamine stimulation
(10
4 M) of rat hepatoma cells transfected with
canine histamine H2 receptor caused concurrent transient
elevations of [Ca2+]i and cAMP
(16). Our finding that maximal elevation of AGS cell cAMP was elicited
by 10
4 M histamine is consistent both with the
H2 receptor-transfected hepatoma cell study (16) and with
the original observations in isolated rabbit gastric glands of maximal
cAMP elevation by 10
4 M histamine (10). In the
latter study, 10
4 M histamine stimulated a
200% increase in cAMP concentration over basal levels, whereas in the
present study, AGS cell stimulation at the same histamine concentration
yielded cAMP elevation of 50%. The difference in responsiveness may
reflect fewer H2 receptors on AGS cells or subclasses of
H2 receptors with differing pharmacological profiles.
Together, our cAMP and Ca2+ data clearly point to
expression of functional histamine H2 receptors on AGS
cells, a property of this cell line that has not previously been described.
Our demonstration of HK promoter activation in response to histamine
treatment of AGS cells recapitulates histamine-induced HK
gene
transcription in isolated canine parietal cells (5) and is yet another
line of evidence that functional histamine receptors are present on AGS
cells. Interestingly, histamine H2 receptors have been
shown to be expressed on MKN-45 gastric carcinoma cells (2). Further
studies of histamine-dependent signal transduction pathways in AGS
cells should facilitate the dissection of molecular mechanisms of HK
gene regulation.
The inhibitory effects of H. pylori infection on basal as well
as histamine, PMA, OAG, and EGF-induced activation of the human HK
promoter transfected into AGS cells are possibly reflective of the
transient hypochlorhydria accompanying acute H. pylori infection in humans (25, 37-39, 43). The in vitro data acquired in
the present study are consistent with clinical studies showing that
normal acid secretion is restored in patients with hypochlorhydria when
their H. pylori infection is eradicated (19) and that
H+-K+-ATPase mRNA levels in such patients are
increased after H. pylori eradication (24). The data can thus
be interpreted in terms of H. pylori attachment to gastric
epithelial cells causing downregulation of HK
gene transcription,
resulting in fewer functional proton pumps being synthesized, and a
consequent increase in gastric pH that would favor mucosal colonization
by the organism.
The molecular mechanisms by which H. pylori shuts down
H+-K+-ATPase gene expression appear to require
the participation of at least two intracellular signaling pathways.
Inhibition of OAG-induced activation of HK promoter by calphostin C
and of PMA-induced HK
activation by staurosporine and H. pylori implicate PKC as a common intermediary in both up- and
downregulation of H+-K+-ATPase gene expression.
The physiological role of PKC in cytoplasmic rather than nuclear
regulation of acid secretion is somewhat controversial, phorbol esters
having been shown to both inhibit and activate acid secretion (53),
depending on the state of activation of the adenylate cyclase pathway.
In addition, several different PKC isoforms have been identified in
parietal cells, one or more of which are involved in cytoplasmic
tubulovesicular transformations leading to secretory canalicular
activation of H+-K+-ATPase (12). At the nuclear
level, by activating PKC, phorbol esters such as PMA have been shown to
induce the protooncogenes c-fos and c-jun, whose
expression leads to formation of the heterodimeric transcription factor
activator protein-1 (1). More in-depth studies of mobilization of AGS
cell transcription factors in the presence and absence of H. pylori will clarify the coupling of intracellular kinases to HK
promoter activation.
Our data showing stimulation of human HK promoter activity by EGF,
and inhibition of that stimulation both by genistein and by H. pylori, may point to a role for extracellular
signal-regulated protein kinases (ERKs) as mediators of H. pylori effects on HK
gene expression. Genistein is a
broad-spectrum inhibitor of protein kinases; however, the concentration
we used in this study (50 µM) is well below the genistein
IC50 for PKC and PKA (350 µM). EGF exerts its properties
of growth promotion, regulation of endocrine and exocrine secretion,
and intestinal electrolyte transport by receptor binding and subsequent
induction of ERKs and the early response gene c-fos (26). In
isolated parietal cells, acute administration of EGF inhibits acid
secretion (11), whereas chronic EGF increases acid secretion (11, 30).
The reversal of these effects by PTK inhibitors (30), which decrease
phosphorylation of at least one ERK isoform (11), and the induction of
acid secretion by prolonged activation of ERKs (50), have been
interpreted in terms of HK
promoter response elements receiving
input from ERK- and c-fos-dependent pathways (50). As we have
shown, human HK
5'-flanking sequence transfected into AGS
cells is responsive to EGF receptor stimulation; whether the response
is mediated by mobilization of an endogenous ERK pathway remains to be
clarified, as does the mechanism by which H. pylori overcomes
EGF-stimulated HK
promoter activation.
The significant finding of this study was that H. pylori
inhibits basal and agonist-stimulated activation of human
H+-K+-ATPase 5'-flanking sequence
transfected into AGS cells. In addition, histamine, EGF, PMA, and OAG
activated the promoter in an antagonist-sensitive manner, indicating
that AGS cells possess functional histamine and EGF receptors and that
both PTK and PKC and PKA signaling pathways play a role in promoter
regulation. Also significant was the localization of H. pylori
species specificity with respect to proton pump regulation to the
5'-flanking sequence of the HK gene. The downregulation of
human HK
promoter by H. pylori, measured in this study in
the transfected AGS cell model, may represent the in vitro correlate of
the hypochlorhydria reported in clinical studies of acute H. pylori
infection. Further studies are required to establish whether
H+-K+-ATPase mRNA levels and
H+-K+-ATPase expression itself are reduced as a
consequence of acute H. pylori infection. In addition,
identification of the H. pylori-induced factor(s) responsible
for downregulation of HK
promoter activity is clearly a high priority.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Gary Shull for generous provision of plasmids containing rat and human H+-K+-ATPase 5'-flanking DNA, Drs. John Raymond and Spencer Shorte for discussions and assistance with FLIPR (Public Health Service shared equipment Grant S10-RR-13005), Drs. Steve Frawley and Scott Willard for discussions and assistance with luminometry, and Dr. Tom Gettys for discussion of cAMP assays. We acknowledge the expert assistance of the Oligonucleotide Synthesis Facility, the Biomolecular Computing Resource Facility, and the Electron Microscopy Facility at the Medical University of South Carolina.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43138 and National Aeronautics and Space Administration Grant NAG8-1385 (A. J. Smolka).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. J. Smolka, Dept. of Medicine, Medical Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: smolkaaj{at}musc.edu).
Received 17 August 1999; accepted in final form 4 January 2000.
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