1Department of Surgery and 2Veterans Affairs Medical Center, Oregon Health Sciences University, Portland, Oregon, 97201; 3Department of Medicine, Howard University, Washington, District of Columbia 20060; and 4Departments of Medicine, Microbiology, Immunology, and Veterans Affairs Medical Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Submitted 1 July 2002 ; accepted in final form 15 February 2003
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ABSTRACT |
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vacA; cagA; picB/cagE; bacteria; signal transduction; fura 2; fluo 4; cell culture; immunofluorescence; thapsigargin; genistein; herbimycin; G protein; stomach
Despite the recent advances in H. pylori-host cell mutagenesis and
transcriptional profiling
(14), little is known about
certain aspects of H. pylori signaling in normal gastric cells such
as the regulation of intracellular Ca2+ concentrations
([Ca2+]i). In other model systems, bacterial
adherence to the host cell has been shown to result in specific
[Ca2+]i changes
(10). For example, the
adherence of certain Escherichia coli strains to intestinal
epithelial cells results in increased [Ca2+]i
and inositol trisphosphate levels
(11). It has also been shown
that Salmonella induces intracellular Ca2+
changes that were linked to the activation of an NF-B-dependent
inflammatory pathway (13). In
this regard, a study using the intracellular Ca2+
chelator BAPTA along with calmodulin inhibitors found that H. pylori
activation of NF-
B and IL-8 signaling in MKN45 human gastric cancer
cells was Ca2+-calmodulin dependent
(32). The reported H.
pylori-induced hummingbird phenotype in gastric cells is similar to the
morphological events seen with hepatocyte growth factor or scatter factor
(HGF/SF) on MDCK cells or hepatocytes
(44). It has also been
reported that HGF/SF can induce changes in
[Ca2+]i, which have been shown to be linked
to changes in cell morphology and proliferation
(1,
19,
31,
34).
However, despite the extensive work on various aspects of H. pylori-induced signaling in gastric cancer cells, the specific mechanism(s) of intracellular Ca2+ mobilization by H. pylori in normal human gastric mucous epithelial cells has not yet been thoroughly examined. Also, many H. pylori signaling studies have used either nongastric or gastric cancer cell lines as a model system, which always introduces a degree of uncertainty as to whether the events observed are applicable to normal gastric cells. As an alternative to nongastric gastric cancer cell lines, several in vitro model systems of normal human gastric mucous epithelial cells have been established (7, 48, 54, 61). The use of nontransformed cell culture models provides a more accurate representation of the environment that H. pylori may encounter in the normal human gastric mucosa. The aim of the present study, therefore, was to examine the effects of H. pylori on intracellular Ca2+ signaling in normal human gastric epithelial cells. We found for the first time that H. pylori produces specific transient [Ca2+]i changes in normal human gastric mucous epithelial cells and that these H. pylori-induced [Ca2+]i changes could also be replicated in a nontransformed gastric mucous epithelial cell line (HFE-145 cells). We also found that a G protein-dependent/PLC pathway primarily regulated H. pylori-induced intracellular Ca2+ release, whereas H. pylori-induced Ca2+ influx was primarily regulated by components of a G protein-, src kinase-, and PLA2-dependent pathway. Finally, we report that mutagenesis of picB/cagE and cagA genes (located within the cag PAI), but not the vacA gene, alters the capacity of H. pylori to produce a full [Ca2+]i response.
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MATERIALS AND METHODS |
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Gastric epithelial cell culture. Human gastric mucous cells were isolated and cultured as previously described (48). The Oregon Health Sciences University (OHSU) Human Studies Subcommittee approved all procedures and handling of human tissue. Briefly, H. pylori-free gastric tissues were obtained from patients undergoing surgical gastrectomy. The surgical specimens were washed twice in serum-free media and pinned down on polymerized Sylgard, and the epithelium was removed by scraping the surface with a glass slide. The scraped tissue pieces were minced by using razor blades then washed three times at 100 g for 3 min in serum-free media. The pellets were then transferred to siliconized 125-ml screw-cap Erlenmeyer flasks containing 20 ml of serum-free culture media with 20 mg/ml of type I collagenase and 0.1% bovine serum albumin. The flasks were then gassed with 95% O2-5% CO2, put into a 37°C shaking water bath, and gyrated at 120 oscillations/min for 45 min. At the end of the incubation period, the collagenase-digested mixture was put into a 50-ml syringe with an attached 15-gauge luer-stub adapter, and the contents were pushed through a 200-µm nylon mesh screen. The mesh-filtered suspension was washed twice in serum-free media and centrifuged at 100 g for 3 min, then the pellet was resuspended in 15 ml of serum-free culture media and a 200-µl aliquot was taken for cell counts in a Coulter Counter. The 15-ml suspension was divided into three 5-ml aliquots in 16 x 125-cm Falcon round-bottom tubes, then 5 ml of isosmotic Percoll was added to each tube. The tubes were centrifuged for 15 min at 100 g at 24°C, and the bottom pellet, containing the gastric mucous epithelial cells, was removed. The pellet was washed three times and centrifuged at 20 g for 3 min in serum-free cell culture media, then the cells were plated on 0.45-µm Falcon porous filters (catalog no. 353180; 12 mm, 0.45-µm pore size), 25 mm round glass coverslips, or 96-well plastic dishes.
The HFE-145 human gastric mucous epithelial cell line (provided by D.
Smoot) were plated and grown under the same experimental conditions as the
above human primary gastric mucous epithelial cells. The HFE-145 cells were
originally developed from normal human gastric epithelial cells by the
transfection of normal cells with SV40 Large T-antigen and human telomerase
vectors (53). The cells have a
doubling time of 24 h and are strongly positive for
cytokeratin-10,11,18 and weakly positive for
cytokeratin-13,16,20, which is almost identical to cytokeratin
staining of the parental cell line. These cells also stain positive for
neutral mucin using periodic acid-Schiff and negative for alcian blue (acidic
mucin), which is consistent with normal gastric epithelial cells. Growth of
these cells was inhibited when cells were placed in soft agar, suggesting that
these cells are not tumorigenic. The cells constitutively express mRNA from
Muc-5ac, Muc-5b, and Muc-6 genes, which is consistent with
normal gastric epithelial cells. Electron microscopy shows that these cells
form tight junctions when grown as monolayers on plastic tissue culture dishes
and on glass slides (53).
H. pylori culture. The H. pylori bacteria used in this study were the wild-type vacA+, cag+ 60190 (ATCC 49503), an isogenic vacA mutant, isogenic cagA mutant, and an isogenic picB/cagE mutant. H. pylori 60190 contains a type s1a/m1 vacA allele (59). The vacA, cagA, and picB-/cagE mutants have been previously described (39, 52, 59). The bacteria were grown on blood agar plates (trypticase soy agar with 5% sheep blood; PML Microbiologicals, Tualatin, OR) under microaerobic conditions using a CampyPak jar (Fisher Scientific) at 36°C. Unless noted otherwise, all bacteria were harvested at 24 h by using a sterile cotton swab and 3 ml of PBS (pH 7.1). The bacterial suspensions were put into 12-ml Falcon round-bottom tubes, and the H. pylori was resuspended by gentle inversion. A 1-ml aliquot of the suspension was put into a cuvette, and the H. pylori concentration was determined by using optical density 600 nm (OD600) where an OD of 1 = 1.2 x 109 colony-forming units (CFU)/ml. All final bacterial suspensions (1 x 1051 x 109 CFU/ml) were adjusted with the appropriate mammalian Ringer solution. The mammalian Ringer solution consisted of (in mM): 137 NaCl, 4 KCl, 25 NaHCO3, 2 KH2PO4, 15 HEPES, 1 MgSO4, 2 CaCl2, and 25 glucose, pH 7.4. Periodically, the bacteria were plated in serial dilutions on agar plates and H. pylori concentrations were checked by counting the bacterial colonies after 3 days of incubation.
H. pylori sonicates were made by growing the bacteria on agar plates for 24 h and then harvesting the bacteria in PBS as indicated above. The bacteria were washed twice in PBS by centrifugation at 10,000 g for 15 min, and then the pellet was resuspended in mammalian Ringer (pH 7.4). The bacterial suspensions were disrupted by sonication (10 30-s pulses), the sonicates were filtered through a 0.2-µm filter, and the protein content was determined by using a Bio-Rad protein assay. Aliquots were frozen and stored at 80°C until needed. For control studies, both live bacteria and bacterial sonicates were heated to 70°C for 30 min to generate heat-inactivated bacteria and sonicates.
H. pylori and gastric cell [Ca2+]i using fura 2. Primary cultures of gastric mucous epithelial cells, the HFE-145 cell line, and AGS gastric cancer cells were grown on either permeable Falcon filters or 25-mm round glass coverslips. After 24 h in serum-free media, the cells were loaded with fura 2-AM according to modifications of previously described techniques (47). Briefly, the cells were loaded with 2.5 µM fura 2-AM in fresh serum-free media for 45 min at 37°C. After fura 2-AM loading, the cells were washed twice with fresh serum-free media, then twice with mammalian Ringer. When extracellular Ca2+-free Ringer solutions were used, the CaCl2 was replaced with NaCl and the solution was characterized as nominally Ca2+-free Ringer. In preliminary experiments, we found that the use of our nominally Ca2+-free Ringer solution did not affect H. pylori adherence. However, the addition of 1 mM EDTA and 1 mM EGTA to the nominally Ca2+-free Ringer solution decreased H. pylori adherence and disrupted monolayer integrity over the experimental time period and therefore could not be used (data not shown). Solutions were oxygenated with 5% CO295% O2, kept warm at 37°C in a heated water bath, and perfused into the chamber by using a variable Millipore pump.
After fura 2 loading, the gastric cultures were transferred to a horizontal open perfusion chamber that had been modified to hold either a permeable Falcon filter or a glass coverslip (46). The chamber was then placed on the stage of a Nikon Diaphot TMD inverted microscope equipped with a fluorescence objective (Nikon Fluor-phase-3DM, numerical aperature 60/0.7, 160-mm working distance). [Ca2+]i measurements were made at 340/380 nm excitation and 510 nm emission wavelengths from an SLM-Aminco spectrophotometer (Rochester, NY). The effects of nonspecific H. pylori fluorescence scatter and cell autofluorescence were determined by placing an unloaded gastric cell monolayer with varying doses of H. pylori (1 x 1051 x 109 CFU/ml) on the microscope stage, then emission ratios were recorded and subtracted from the final fura 2 tracings. In preliminary experiments, we found light scattering by the bacteria and cellular autofluorescence did not exceed 5% of the total fura 2 fluorescence.
In some experiments, the rates of H. pylori-induced intracellular Ca2+ release and influx were estimated by using the "Ca2+ add-back technique" (43). For these experiments, fura 2-loaded gastric cells are initially incubated in the absence of extracellular Ca2+, then H. pylori is added and [Ca2+]i is recorded as intracellular Ca2+ release, then extracellular Ca2+ (2.0 mM) is "added back" and the [Ca2+]i change is measured as Ca2+ influx.
The final calibration of the fura 2 signal was done at the end of each experiment by adding 5 µM ionomycin for 10 min to saturate fura 2 with Ca2+ to obtain maximal fluorescence (Fmax), then 10 mM EGTA and 10 mM EDTA plus 60 mM Tris·HCl, pH 10.5, was added to chelate the Ca2+ from fura 2 to determine the minimal fluorescence (Fmin). Additional points on the calibration curve were determined by using a series of defined Ca2+-calibration solutions (Kit #1, C-3008; Molecular Probes), and [Ca2+]i was calculated (58). The fluorescence tracing analysis and data smoothing were done with software provided by the manufacturer (SLM-Aminco).
H. pylori and gastric cell [Ca2+]i using a fluo 4 96-well assay. In some experiments we wanted a quicker throughput assay to measure the relative change in [Ca2+]i produced by H. pylori with different kinase inhibitors. For these experiments, measurement of [Ca2+]i was done using a modification of a 96-well fluorescence assay (25). Cultures of normal human gastric epithelial cells were grown to confluence in 96-well plates and loaded with 2.5 µM fluo 4-AM for 45 min at 37°C. After being loaded, the cultures were washed twice with mammalian Ringer and then incubated for another 30 min with fresh 37°C Ringer solution. Fluo 4 fluorescence was recorded using a 96-well fluorescence plate reader (FluoStar; BMG Technologies, Durham, NC) equipped with excitation (485 ± 20 nm) and emission (530 ± 20 nm) filters. After the addition of H. pylori or drugs, fluorescence measurements were made every 2 s. The Fmax measurement for the human gastric epithelial cells was obtained by adding a solution of 5 µM ionomycin and 100 µM digitonin to the gastric cells for 15 min. After this, the Fmin measurement was obtained by adding a solution of 10 mM EGTA-10 mM EDTA in 60 mM Tris·HCl, pH 10.5, for 15 min. The values for Fmax and Fmin were graphed, and individual fluorescence values were obtained by using a single wavelength equation for fluo 4 (58).
Statistics. All data are expressed as means ± SE. The differences between means were considered significant when the P value calculated from Students' t-test for paired cultures was <0.05. Multiple cell culture comparisons were analyzed by using ANOVA and Duncan's multiple-range tests. Unless stated otherwise, n represents the total number of different "individual" cell preparations isolated from different surgical specimens. All statistical calculations were made using SigmaStat statistical software (SPSS, San Rafael, CA).
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RESULTS |
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To independently validate the previous results, we tested the effects of H. pylori on [Ca2+]i in the nontumorigenic HFE-145 human gastric mucous epithelial cell line. The HFE-145 cell line has morphological and phenotypic properties similar to those seen in primary gastric mucous epithelial cells (53). Compared with the primary gastric mucous epithelial cell cultures, we found that H. pylori produced similar [Ca2+]i changes in the HFE-145 cell line (Fig. 1B). That is, H. pylori dose-dependently produced the characteristic biphasic peak and plateau changes in [Ca2+]i (Fig. 1B; Table 1). For example, H. pylori (1 x 109 CFU/ml) produced a change in baseline [Ca2+]i from 106 ± 2 nM to a peak of 177 ± 4 nM, which was followed by a decline and then an increase to plateau levels of 140 ± 4 nM (n = 14; Table 1). Although some quantitative differences in H. pylori-induced intracellular Ca2+ signaling existed between the primary human gastric mucous epithelial cell cultures and the HFE-145 cell line, overall the qualitative patterns of H. pylori-induced [Ca2+]i changes were similar in both cell types. It should be noted that after several passages (>25) of the HFE-145 cell line, we began to lose the characteristic H. pylori-induced intracellular Ca2+ response that was observed in early cultures (data not shown).
In addition to live bacteria, we also tested the capacity of H. pylori sonicates (05 µg/ml) to induce changes in [Ca2+]i in normal gastric mucous epithelial cells. However, we found that the H. pylori sonicates, even at the highest concentration used (5 µg/ml), produced only a small, slow, steady change in [Ca2+]i from a baseline level of 103 ± 3 nM to 119 ± 4 nM over a 60-min time course (n = 9). Because the bacterial sonicates did not reproduce the [Ca2+]i changes observed with live intact H. pylori, they were not used for the remainder of our experiments. Compared with the untreated control cultures, we also found that heat-killed H. pylori produced no significant change (P > 0.05) in [Ca2+]i over the 60-min time period (control = 106 ± 3 nM; heat-killed bacteria = 108 ± 4 nM; n = 9; Fig. 1). We also found that the pretreatment of the gastric mucous epithelial cell cultures with the Ca2+ chelator BAPTA completely abolished the wild-type H. pylori-induced [Ca2+]i changes over 60 min (control = 106 ± 3 nM; H. pylori-treated = 107 ± 3 nM; n = 7). These data confirm that intact live H. pylori are able to induce a specific [Ca2+]i change in normal gastric mucous epithelial cells. In addition, the H. pylori-induced [Ca2+]i change is characterized by an initial transient peak [Ca2+]i increase followed by a sustained plateau [Ca2+]i phase that can be specifically blocked by the Ca2+ chelator BAPTA.
The role of vacA, cagA, and picB/cagE genes in H. pylori-induced [Ca2+]i changes. It is well established that the VacA toxin secreted by H. pylori is an important virulence factor in the pathogenesis of peptic ulcer disease (36). In addition, the CagA protein encoded by the cagA gene within the H. pylori cag PAI is involved in gastric host cell cytoskeletal responses (5, 16, 4951). Also, residing within the cag PAI is the picB/cagE gene, which encodes a structural component of the type IV secretion system that is important in translocating the CagA protein as well as activation of signaling mechanisms involved in immune responses and cell growth and apoptosis (37). We therefore were interested in testing the role of the H. pylori vacA, cagA, and picB/cagE genes on intracellular Ca2+ signaling. Using a vacA isogenic mutant strain on primary cultures of human gastric mucous epithelial cells, we found that the vacA isogenic mutant produced peak and plateau [Ca2+]i changes that were nearly identical to those seen in the wild-type 60190 strain (Fig. 2A; Table 2). The heat-killed H. pylori vacA isogenic mutant strain produced no change in [Ca2+]i in the gastric cells (Fig. 2A). In contrast to the wild-type and vacA isogenic mutant strains, the cagA and picB/cagE isogenic strains produced markedly attenuated [Ca2+]i changes (Fig. 2B). That is, the wild-type strain (1 x 109 CFU/ml) produced a peak [Ca2+]i response of 184 ± 4 nM compared with the significantly (P < 0.05) smaller peak responses of 157 ± 4 and 143 ± 5 nM, respectively, from the cagA and picB/cagE isogenic mutants (Table 2). Of the two mutant strains, the picB/cagE-induced [Ca2+]i peak response of 143 ± 5 nM was also found to be significantly (P < 0.05) lower than the cagA-induced [Ca2+]i peak response of 157 ± 4 nM (Table 2). We also found that after treating the gastric cells for 15 min with either the cagA or picB/cagE mutant strains we could no longer detect a [Ca2+]i change over the remainder of the 60-min time course (Fig. 2B; Table 2). That is, the cagA and picB/cagE isogenic mutants did not generate the typical prolonged plateau phase as seen with the H. pylori wild-type strain or vacA isogenic mutant (compare Fig. 2, B to A; Table 2). We have also found this same attenuated [Ca2+]i response using other independent picB/cagE and cagA mutant strains derived from wild-type 60190 strain (M. J. Rutten and T. L. Cover, unpublished data). These data suggest that the expressed products of the cagA and picB/cagE genes (but not vacA) contribute to H. pylori-induced [Ca2+]i changes.
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H. pylori-induced [Ca2+]i: intracellular Ca2+ release vs. Ca2+ influx. One of the main intracellular Ca2+ stores within most cells is the thapsigargin-sensitive sarcoendoplasmic reticular (SERCA) Ca2+ store (43, 58). Thapsigargin inhibits the Ca2+-ATPase on the SERCA membrane, which then leads to the release of Ca2+ and depletion of the intracellular Ca2+ store (43). As a first step in identifying the source(s) of the H. pylori-induced [Ca2+]i change, fura 2-loaded gastric mucous epithelial cells grown on glass slides were pretreated with 500 nM thapsigargin, and then H. pylori (1 x 109 CFU/ml) was added and [Ca2+]i was measured. In thapsigargin-pretreated gastric cells, H. pylori wild-type and vacA, cagA, and picB/cagE mutant strains all had reduced [Ca2+]i peak levels [from 184 ± 4, 177 ± 5, 157 ± 4, and 143 ± 3 nM to 117 ± 4, 115 ± 3, 112 ± 4, and 110 ± 3 nM, respectively (n = 9; Fig. 3A)]. The H. pylori-induced wild-type and vacA [Ca2+]i plateau change was only slightly diminished by the thapsigargin pretreatment (Fig. 3A). Because the cagA and picB/cagE H. pylori strains were previously found to produce no [Ca2+]i plateau change (see Fig. 2), the thapsigargin pretreatment of the gastric cells was without effect on this portion of the [Ca2+]i response (Fig. 3B).
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In addition to the release of intracellular Ca2+ stores, the opening of Ca2+ channels in the plasma cell membrane can also contribute to the change in [Ca2+]i (43). A general experimental approach in examining the contribution of Ca2+ channels to the total agonistinduced [Ca2+]i change is to use the Ca2+ add-back technique (see MATERIALS AND METHODS; Ref. 43). For these experiments, fura 2-loaded primary gastric mucous epithelial cells were first pretreated with extracellular Ca2+-free Ringer solution, H. pylori was added, and [Ca2+]i was recorded, followed 30 min later by the readdition of extracellular Ca2+ to the Ringer. We found that there was only a small, nonsignificant (P > 0.05) reduction in the wild-type and vacA H. pylori-induced [Ca2+]i peak responses using extracellular Ca2+-free buffer (Fig. 4A). However, the extracellular Ca2+ treatment greatly reduced the wild-type and vacA H. pylori-induced [Ca2+]i plateau response from control levels of 145 ± 4 and 141 ± 3 nM to 103 ± 4 and 100 ± 4 nM, respectively (n = 10; Fig. 4A). When extracellular Ca2+ was added back to the Ringer solution, we found that the [Ca2+]i in the H. pylori wild-type and vacA-treated gastric cultures rose to near control plateau levels (Fig. 4A). The extracellular Ca2+ treatment also did not significantly (P > 0.05) reduce the cagA- and picB/cagE-induced [Ca2+]i peak response from control levels of 157 ± 4 and 151 ± 4 nM to 143 ± 5 and 138 ± 4 nM, respectively (n = 10; Fig. 4B). Because the cagA or picB/cagE mutant strains do not produce an [Ca2+]i plateau change (see Fig. 2), the effect of readdition of extracellular Ca2+ to the Ringer solution to detect Ca2+ influx was without effect on this portion of the [Ca2+]i response (Fig. 4B). Overall, these experiments suggest that the H. pylori-induced peak [Ca2+]i change is dependent primarily on release of intracellular thapsigargin-sensitive Ca2+ stores, whereas the H. pylori-induced plateau [Ca2+]i change is primarily dependent on extracellular Ca2+ influx.
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H. pylori-induced Ca2+ release is regulated by a PLC-dependent mechanism. Several agonists as well as certain bacterial pathogens have been shown to induce Ca2+ release from intracellular stores through a PLC-mediated process (42). In the next series of experiments, we used the PLC inhibitor U-73122 to test whether PLC activation is involved in H. pylori-induced [Ca2+]i changes. In the wild-type and vacA isogenic strains, the U-73122 pretreatment considerably reduced the [Ca2+]i peak increase with less of an effect on the [Ca2+]i plateau change (Fig. 5, A and B). Compared with untreated controls, the U-73122 also significantly reduced the cagA or picB/cagE peak change in [Ca2+]i (Fig. 5, C and D). Because the cagA and picB/cagE isogenic strains do not generate a plateau [Ca2+]i change, U-73122 was without effect on this portion of the [Ca2+]i response (Fig. 5, C and D). As a control, cultures were pretreated with the structurally related but ineffective PLC drug U-73343, and it did not alter any of the [Ca2+]i changes produced by the wild-type, vacA, cagA, and picB/cagE strains on the gastric cells (data not shown). However, it should be noted that the control compound U-73343 used at concentrations >2.5 µM actually inhibited H. pylori-induced Ca2+ signaling, indicating that at >2.5 µM both U-73122 and U-73343 exhibit nonspecific inhibitory effects (data not shown). These data suggest that H. pylori-induced intracellular Ca2+ release (but not Ca2+ influx) is under the control of a PLC-dependent mechanism.
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H. pylori-induced [Ca2+]i changes are regulated by src kinases. It has now been well documented that the src kinases play an important signaling role in the phosphorylation of the translocated CagA protein as well as participation of H. pylori-induced cytoskeletal changes (2, 16, 50, 55). It has also been shown in other cell types that src kinases play a modulatory role in agonist-induced [Ca2+]i changes (9). We therefore wanted to examine the role of src kinases in H. pylori-induced intracellular Ca2+ signaling in gastric cells where src kinase activity was inhibited by using PP2. Pretreatment of the primary human gastric mucous epithelial cell cultures with PP2 produced a dose-dependent differential decrease in H. pylori wild-type and vacA isogenic [Ca2+]i peak and plateau changes (Fig. 6). That is, PP2 pretreatment (0.55.0 µM) of the gastric cells was most effective in reducing the H. pylori wild-type and vacA mutant [Ca2+]i plateau response (Fig. 6, A and B). Only at higher concentrations of PP2 (15 µM) did we observe a reduction in both the H. pylori wild-type strain and vacA mutant [Ca2+]i peak and plateau changes to baseline levels (Fig. 6, A and B). We also found that only at the highest PP2 concentration used (15 µM) was the cagA and picB/cagE [Ca2+]i peak change reduced to baseline levels (Fig. 6, C and D). Because the cagA and picB/cagE isogenic strains do not generate an [Ca2+]i plateau change (see Fig. 2), the PP2 was without effect on this portion of the [Ca2+]i response (Fig. 6, C and D). Overall, these results with the various concentrations of PP2 suggest that the src kinases are more likely to have a regulatory role in controlling the H. pylori wild-type and vacA mutant-induced [Ca2+]i "plateau" (Ca2+ release) than the [Ca2+]i "influx" change.
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H. pylori-induced Ca2+ influx is regulated by G proteins and a PLA2-dependent mechanism. In several cell types, agonist-induced changes in [Ca2+]i can be altered by PTX-sensitive G proteins as well as PLA2 activity (23, 28). In addition, it has been reported that H. pylori-induced arachidonic acid release from the human cervical adenocarcinoma HeLa cell line could be abolished by PTX and the PLA2 inhibitor MAFP (40). For the next series of experiments, we were therefore interested in determining the effects of PTX treatment on H. pylori-induced Ca2+ release and Ca2+ influx in cultures of normal human gastric mucous epithelial cells. We found that PTX pretreatment of the gastric cells caused a reduction in both the wild-type and vacA H. pylori-[Ca2+]i peak and plateau changes to baseline [Ca2+]i levels (Fig. 7, A and B). The effect of PTX pretreatment also caused a reduction of the cagA and picB/cagE mutant [Ca2+]i peak change to near baseline levels (Fig. 7, C and D). Because the cagA and picB/cagE isogenic strains do not generate a [Ca2+]i plateau change, the PTX pretreatment had no effect on this portion of the [Ca2+]i response (Fig. 7, C and D).
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We next investigated the effects of inhibiting cPLA2 on H. pylori-induced intracellular Ca2+ signaling in primary cultures of normal human gastric mucous epithelial cells. We found no significant effect (P > 0.05) of MAFP on the wild-type and vacA H. pylori-induced [Ca2+]i peak change, whereas MAFP had a significant effect (P < 0.05) on the wild-type and vacA H. pylori-induced [Ca2+]i plateau change (Fig. 8 A and B). Also, MAFP had no significant effect (P > 0.05) on the cagA or picB/cagE-induced [Ca2+]i peak change (Fig. 8, C and D). Because the cagA and picB/cagE isogenic strains do not generate a [Ca2+]i plateau change, MAFP was without effect on this portion of the [Ca2+]i response (Fig. 8, C and D). Overall, these results suggest that the H. pylori-induced plateau phase (Ca2+ influx) is under the regulation of a G protein/cPLA2-dependent pathway.
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DISCUSSION |
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In the present study, we also examined a role for the H. pylori vacA, cagA, and picB/cagE genes on intracellular Ca2+ signaling. The H. pylori VacA toxin has been shown to produce several membrane permeability events in gastric cells, and it is also an important virulence factor in the pathogenesis of peptic ulcer disease (36). However, from our studies we conclude that the H. pylori VacA toxin has no direct role in mediating H. pylori-induced [Ca2+]i changes. That is, we found no difference between our H. pylori wild-type strain and a vacA isogenic mutant in their abilities to produce an [Ca2+]i change. This finding is in contrast to other pathogens in which extracellular toxins have been shown to have a role in host cell Ca2+ signaling. For example, the pore-forming toxin aerolysin, from Aeromonas hydrophila, has been shown to activate G protein-dependent intracellular Ca2+ release in human granulocytes (24). In contrast to the vacA mutant, we found that the [Ca2+]i response was greatly reduced when the gastric cells were treated with either a cagA or picB/cagE isogenic mutant strain. Specifically, we found that the [Ca2+]i peak change was markedly reduced with the cagA and picB/cagE isogenic mutants, and these mutant stains did not generate the typical prolonged plateau phase as seen with the H. pylori wild-type strain or vacA mutant. In addition, the picB/cagE-induced [Ca2+]i peak response was found to be significantly lower than the cagA-induced [Ca2+]i peak response.
Role of signaling intermediates on H. pylori-induced
[Ca2+]i
changes. After identifying the initial H. pylori-induced
[Ca2+]i response, the mechanistic components
for each of the H. pylori-induced
[Ca2+]i phases were examined by using
different kinase or drug inhibitors. We found, for example, that the
pretreatment of the gastric cells with the PLC inhibitor U-73122, and not the
structural control U-73343 analog, attenuated the wild-type H.
pylori-induced [Ca2+]i peak phase to
near control levels. Even more effective was the Gi protein
inhibitor PTX, which completely reduced the H. pylori-induced
[Ca2+]i peak phase to baseline control levels
in all of the H. pylori wild-type and mutant strains tested. PTX
pretreatment was also effective in reducing the wild-type and
vacA mutant H. pylori-induced
[Ca2+]i plateau (Ca2+
influx) phase. However, because the cagA and
picB/cagE mutants
produced no [Ca2+]i plateau phase, the use of
PTX pretreatment with these mutants on
[Ca2+]i plateau changes were redundant. The
src kinase inhibitor PP2 was found to produce dose-dependent
differential effects on H. pylori-induced
[Ca2+]i changes. That is, low concentrations
of PP2 were most effective in attenuating wild-type and
vacA mutant H. pylori-induced
[Ca2+]i plateau changes, with only high
concentrations reducing H. pylori-induced
[Ca2+]i peak changes. These data suggest that
a range of src kinase activity is likely to control the final H.
pylori-induced intracellular Ca2+ signal. We also
found that pretreatment of gastric cells with the cytoplasmic PLA2
(cPLA2) inhibitor MAFP was able to reduce (but not to control
levels) the H. pylori-induced
[Ca2+]i plateau response in the gastric
cells. In this regard, it has been shown the activity of cPLA2 is
Ca2+ dependent and that the increase in cPLA2
activity will increase arachidonic acid, which can modulate
Ca2+ influx
(23). It is possible that the
first H. pylori-induced phase of intracellular
Ca2+ mobilization will secondarily activate
cPLA2, which in turn could generate arachidonic acid and modulate
Ca2+ influx (Fig.
9).
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H. pylori, Ca2+ signaling, and host cell pathogenesis. It is now known that H. pylori can initiate multiple signaling pathways within the host gastric cell by using a variety of effector stimuli, ranging from small extracellular molecules, such as urease-generated ammonia, to the VacA toxin or the use of specialized injected molecules such as the CagA protein (57). As a focus for our own study, we chose to look at what role the VacA toxin, the CagA protein, and a functional type IV injection system may have on intracellular Ca2+ signaling in normal human gastric mucous epithelial cells. We also chose to use a buffered urea-free Ringer solution (pH 7.4) to help minimize the potential effects that H. pylori-generated urease activity or ammonia might have on the overall H. pylori-induced [Ca2+]i change (4). However, we cannot rule out in our study the contribution of other H. pylori-secreted/shed factors that may contribute to the overall [Ca2+]i response (29). In addition, some of the H. pylori extracellular shed/secreted proteins, such as HP0305, have been reported to have sequence homology to regulators of G protein signaling, which makes it possible that these proteins could modulate or contribute to the overall H. pylori host cell [Ca2+]i response (22). However, because H. pylori was able to increase [Ca2+]i within minutes after the addition of the bacteria, we believe that soon after bacteria adherence, certain signaling intermediates are immediately activated, such as G proteins and PLC, which were both found to be important in the H. pylori-induced [Ca2+]i peak change (Fig. 9). In other cell types, it is well established that agonists or pathogens can quickly increase PLC activity and the formation of inositol trisphosphate, which releases Ca2+ from intracellular Ca2+ stores (3). Our time-course and inhibitor studies suggest that H. pylori is likely to involve a similar PLC pathway that produced the characteristic rise and fall of [Ca2+]i observed within the first 15 min after the addition of bacteria. It should be emphasized again that our cagA isogenic mutant produced only a small [Ca2+]i peak response (and no [Ca2+]i plateau change) and that this cagA-induced peak [Ca2+]i change was further reduced to baseline [Ca2+]i levels by the PLC inhibitor U-73122. These data indicate that there may be CagA-dependent and CagA-independent pathways for PLC activation, but overall, increases in PLC activity along with the physical translocation of the CagA protein are likely to be the major contributing factors to the initial H. pylori-induced [Ca2+]i change. It is noteworthy that other studies (2) have shown that phosphorylated CagA protein can be detected as early as 15 min after the addition of H. pylori to gastric cells, which is well within the time course of our [Ca2+]i peak response.
Another facet of intracellular Ca2+ signaling is the potential for cross-talk between different receptor systems (9). For example, it has been reported that H. pylori can transactivate the EGFR (21, 62). Although the role of H. pylori transactivation of the EGFR and Ca2+ signaling was not examined in our study, it has been reported that Salmonella can transactivate the EGFR to produce an increase in [Ca2+]i that was important for bacterial entry (35). H. pylori can also activate adenylate cyclase and increase intracellular cAMP within AGS gastric cancer cells, and the H. pylori-induced cAMP increase is independent of the vacA, cagA, and cag PAI genes (63). In this regard, intracellular Ca2+ release has also been shown to be regulated by cAMP and PKA (3), suggesting that H. pylori is capable of activating multiple receptor systems that are in turn capable of coordinating host cell Ca2+ signaling.
Our study also suggests that the translocated CagA protein may be necessary for the continuation of the Ca2+ signal for the induction of a [Ca2+]i plateau (Ca2+ influx) change. That is, in addition to the inability of our cagA mutant to produce the [Ca2+]i peak phase, we also found that the src kinase inhibitor PP2 (which has been shown to block CagA protein phosphorylation; Ref. 50) was also capable of inhibiting the H. pylori-induced [Ca2+]i peak phase. It has been suggested that the ability of the CagA protein to perturb host cell functions is dependent on the number and sequences of tyrosine sites that are phosphorylated (15, 41). At this time we do not know to what degree the CagA protein has to be phosphorylated to induce the H. pylori [Ca2+]i changes.
Several studies have also implicated a link between an H. pylori-induced [Ca2+]i change and a biological response. That is, pretreatment of MKN45 gastric cancer cells with the intracellular Ca2+ chelator BAPTA was shown to completely block wild-type H. pylori-induced IL-8 secretion (32). It has also been reported that BAPTA was able to block H. pylori-induced arachidonic acid release that is involved in the production of prostaglandin E2 (40). Of interest, however, was the fact that the expression of the CagA protein was not important for the above-mentioned Ca2+-dependent H. pylori-induced IL-8 release (32), whereas the CagA protein was necessary for Ca2+-dependent H. pylori-induced arachidonic acid synthesis (40). One might propose that, depending on the final H. pylori-induced [Ca2+]i change, different signaling pathways could be activated based on a specific [Ca2+]i threshold. That is, even in the presence of a cagA mutant, which we have shown generates only a small [Ca2+]i peak change, this small [Ca2+]i response may be sufficient enough to release IL-8, but a larger [Ca2+]i threshold and the CagA protein are both needed for activation of the arachidonic acid/prostaglandin signaling pathway. In this context, it is also possible that the various components of the H. pylori-induced biphasic [Ca2+]i signal, i.e., the peak and plateau phases, may be utilized differently depending on the nature of the Ca2+-dependent signaling molecule within the host gastric cell. It also appears that differential signaling by H. pylori may hold true for other gastric host cell responses. That is, H. pylori-induced MAP kinase activity has also been reported to be induced in a "biphasic" manner over several hours (27). In addition, recent microarray transcriptional studies of H. pylori-treated AGS gastric cancer cells found that many H. pylori-induced signaling genes are transiently expressed within 1 h (14). It is also highly likely that there are other genes inside or outside the H. pylori cag PAI, as well certain structural components from H. pylori itself, that could participate in the H. pylori-induced [Ca2+]i response. For example, it has been suggested that the direct binding of the defective picB/cagE type IV injection apparatus to the plasma cell membrane itself is enough to activate other receptors, as well as translocate other unknown molecules through the type IV injection apparatus that can participate in host cell responses (51).
In summary, we found that H. pylori produces specific transient [Ca2+]i changes in normal human gastric mucous epithelial cells and that these H. pylori-induced [Ca2+]i changes could also be replicated in a nontransformed gastric mucous epithelial cell line (HFE-145 cells). A G protein/PLC-dependent pathway primarily regulated the H. pylori-induced intracellular Ca2+ release, whereas H. pylori-induced Ca2+ influx was under the control of a G protein-, src kinase-, and PLA2-dependent pathway (Fig. 9). Finally, we report that mutagenesis of picB/cagE and cagA genes (located within the cag PAI), but not the vacA gene, alters the capacity of H. pylori to produce a full [Ca2+]i response. For future studies, it will be important to look at other genes inside and outside the cag PAI to determine their effects on H. pylori-induced [Ca2+]i changes.
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ACKNOWLEDGMENTS |
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Portions of these data were presented in abstract form at Digestive Disease Week, May 2023, 2001, Atlanta, GA.
These studies were supported in part by the Medical Research Service of the Department of Veterans Affairs (to M. J. Rutten and C. W. Deveney) and by National Institutes of Health Grants R01-AI-39657 and DK-53623 (to T. L. Cover).
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FOOTNOTES |
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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.
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