From the Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin, Germany
Received for publication, May 4, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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Helicobacter pylori initiates an
inflammatory response and gastric diseases, which are more common in
patients infected with H. pylori strains carrying the
pathogenicity island, by colonizing the gastric epithelium. In the
present study we investigated the mechanism of prostaglandin
E2 (PGE2) synthesis in response to H. pylori infection. We demonstrate that H. pylori
induces the synthesis of PGE2 via release of arachidonic
acid predominately from phosphatidylinositol. In contrast to H. pylori wild type, an isogenic H. pylori strain with a
mutation in the pathogenicity island exerts only weak arachidonic acid
and PGE2 synthesis. The H. pylori-induced
arachidonic acid release was abolished by phospholipase A2
(PLA2) inhibitors and by pertussis toxin (affects the
activity of G The Helicobacter pylori infection induces the release
of a number of proinflammatory cytokines and chemokines from the
gastric epithelium (1) and plays a critical role in the development of
gastritis, peptic ulcer disease, and rarely, in gastric carcinoma and
B-cell mucosa-associated lymphoid tissue-associated carcinoma (2, 3). Evidence has been presented that an increase of prostaglandins
(PGs)1 in gastric tissue from
patients may play a crucial role in H. pylori infection (4).
In gastrointestinal epithelia, PGE2 is implicated in
maintaining the normal function and structure of the gastric mucosa by
modulating diverse cellular functions such as secretion of fluid
and electrolytes, mucosal blood flow, and cell proliferation
(5, 6).
Studies have shown that H. pylori strains differ in their
virulence and in their ability to trigger the induction of inflammatory mediators in gastric epithelial cell lines (1). The response is more
intense to strains carrying the cagA gene. The analysis of
the genomic region containing the cagA gene revealed a
40-kilobase DNA region, which represents a pathogenicity island (PAI)
and codes for 31 genes (7). Upon contact with the gastric epithelium, PAI-encoded components contribute in a specialized type IV secretion machinery that translocates the CagA protein into the eukaryotic target
cell where it is phosphorylated on tyrosine residues (8-12). H. pylori infection triggers by unknown bacterial factors multiple biochemical pathways in host cells including activation of
transcription factors NF- One of the mechanisms for increased PG production in response to
H. pylori infection is an induction of COX-2 expression (20, 21). Another rate-limiting step in the control of PG production is the
release of AA from membrane phospholipids, which is known to occur via
a number of different pathways. One involves the activation of
phospholipase A2 (PLA2), others involve the
action of PLC or phospholipase D (PLD) (22-24). In this report, we
studied the control of PGE2 and AA production in response
to H. pylori infection of epithelial cells after specific
labeling of potential phospholipid precursors and selective inhibition
of enzymes involved in the pathways of AA production. The presented
results provide evidence that colonization of epithelial cells by
H. pylori induces a release of PGE2 and AA by
activation of the cytosolic PLA2 (cPLA2) via
pertussis toxin-sensitive heterotrimeric
G Bacteria--
The isogenic H. pylori strains
P12 wild type, cagA (mutation affect
cagA with a probable polar effect in the PAI), and
vacA (25) were used for colonization of human epithelial
cell lines. For cultivation, the bacteria were resuspended in brain
heart infusion (Difco) medium. 103 bacteria were seeded on
agar plates containing 10% horse serum and cultured for 48-72 h at
37 °C in a microaerophilic atmosphere (generated by Campy Gen,
Oxoid, Basingstoke, UK). For stock cultures, H. pylori was
resuspended in brain heart infusion medium supplemented with 10% fetal
calf serum (FCS, Life Technologies, Inc.) and 20% glycerol, and
maintained at Cell Culture and H. pylori Infection--
Gastric epithelial
cells (AGS) and HeLa cells were grown in RPMI 1640 (Life Technologies,
Inc.) supplemented with 4 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin (Biochrom KG, Berlin,
Germany), and 10% FCS at 37 °C in a humidified atmosphere of 95%
air and 5% CO2. The cells were seeded into 35-mm-diameter Petri dishes or 6-well plates for 48 h before infection. 24 h before infection, the medium was replaced by fresh RPMI 1640 medium supplemented with 0.1% FCS. For the infection, the bacteria were harvested in PBS (pH 7.4) using sterile cotton swabs and diluted corresponding to the multiplicity of infection (MOI) as described in
the figure legends. Cells were incubated for different periods of time
in the absence (controls) or presence of the bacteria. To enhance the
bacteria host cell interaction, bacteria were centrifuged onto the
epithelial cell monolayer for 2 min at 500 × g.
Infection with H. pylori was routinely monitored by light
microscopy. Stimulation of the cells with 125 ng/ml melittin (Sigma) or
100 nM 12-O-tetradecanoylphorbol-13-acetate, (Sigma) was performed for the indicated periods of time.
Cell Labeling--
Cells in 35-mm Petri dishes were
metabolically labeled with 0.2 µCi of 14C-labeled AA
(specific activity 55 mCi/mmol), 0.5 µCi of
[methyl-14C]choline chloride (55 mCi/mmol), or
0.5 µCi of [14C]palmitic acid (57 mCi/mmol) (Amersham
Pharmacia Biotech) in 2 ml of RPMI 1640 medium containing 0.1% FCS for
24 h. Approximately 95% of the total AA radioactivity, 35% of
the total choline radioactivity, and 90% of the total palmitic acid
radioactivity added to the medium was incorporated into the cells
during this time. Lysophosphatidylinositol (lysoPI) formation was
studied in cells prelabeled with 2 µCi of
myo-[3H]inositol (16 Ci/mmol) (ICN
Biomedicals, Eschwege, Germany) in 1 ml of RPMI 1640 medium containing
0.1% FCS for 24 h. Before infection, the medium was removed, and
the cells were washed three times with nonradioactive RPMI 1640 medium
containing 0.1% bovine serum albumin and incubated in the same medium
for 30 min. In some experiments, various inhibitors of kinases or
lipid-metabolizing enzymes were added to the cells during this incubation.
Lipid Analysis--
After the incubations, the medium was
carefully removed for analysis, and lipids were extracted from the
cells by the addition of chloroform, methanol, 20 mM acetic
acid (50:220:10, v/v) to the dishes. In the case of the incubation
media, chloroform/methanol (1:2.2, v/v) was added to give a single
phase. The phase were split according to the method of Bligh and Dyer
(26). The chloroform phase was dried, and lipids were applied on high
performance thin layer chromatography (TLC, Merck) plates. For
determination of lysoPI, the cells and media were extracted twice with
chloroform/methanol (1:2, v/v). The organic extracts were dried and
then subjected to butanol/water partition. The radiolabeled lipids were
recovered in the butanol phases, which were washed with water, dried,
and analyzed by TLC. For phospholipid analysis, the plates were first developed in chloroform, methanol, 30% aqueous ammonium hydroxide, water (90:54:5.5:5.5, v/v) followed by
chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v) for
the second direction. For improved separation of phosphatidylinositol
(PI) and phosphatidylserine (PS), the plates were first developed in
chloroform/methanol/acetone/acetic acid/water (60:12:24:18:6, v/v)
followed by 1-butanol/acetic acid/water (80:26:26, v/v) for the second
direction. The separation of lysolipids was obtained by one-dimensional
thin layer chromatography using chloroform, methanol, 4 M
ammonium hydroxide (9:7:2, v/v). The 14C-containing
radiolabeled spots were imaged for 2 h on a
14C-sensitive screen and quantified on a Fuji Imaging
System imager (FUJIFILM BAS-1000, Raytest, Straubenhardt,
Germany). The 3H-containing radioactive lipids were
detected by fluorography. The TLC plates were dipped in 0.4%
2,5-diphenyloxazol dissolved in 2-methylnaphthalene supplemented
with 10% xylene (27) and exposed to Kodak X-Omat S films at
Determination of [3H]Inositol Phosphates--
For
analysis of the formation of inositol phosphates, cells in 6-well
plates were incubated for 24 h in 1 ml of RPMI 1640 medium
containing 0.1% FCS and 2 µCi of
myo[3H]inositol (ICN Biomedicals, Eschwege,
Germany). Cells were washed two times with Hanks' balanced salt
solution and preincubated with 10 mM LiCl (in this
solution) for 10 min at 37 °C. In some experiments, PLC inhibitor
was added to the cells during this incubation. Cells were then
incubated with the bacteria for 120 min at 37 °C or stimulated
either with lysophosphatidic acid (Sigma) or 0.1 mM ATP
(Roche Molecular Biochemicals) as positive controls. The incubation was
terminated by aspiration of the medium and the addition of 0.1 M NaOH to the dishes. Neutralization of the extracts was
performed with 0.2 M formic acid. Total inositol phosphates
were determined as described in Schulz et al. (29).
Immunoblot and Inhibitors--
For the analysis of p38
activation, the cells were infected with H. pylori for the
indicated periods of time, and the lysed cells were analyzed in an
Immunoblot using a p38 phospho-specific antibody (sc-7973, Santa Cruz).
The stripped blot (ECL kit, Amersham Pharmacia Biotech) was incubated
with an anti-p38 antibody (sc-535, Santa Cruz) to indicate equivalent
protein amounts in all lanes. For cPLA2 detection an
anti-PLA2 antibody (sc-454, Santa Cruz) was used.
The inhibitors used in this study were as follows: mepacrine, sodium
arsenite (Sigma);
1-(6-(17 H. pylori Increases AA Release from Epithelial Cells--
To
investigate whether H. pylori might elicit release of AA for
PGE2 formation (PGE2 release was induced in
HeLa and AGS cells infected with H. pylori wild type and an
isogenic vacA mutant but not by an isogenic cagA
mutant (data not shown)), 14C-labeled AA-labeled AGS and
HeLa cells were incubated for different periods of time in the absence
or presence of different H. pylori strains. To trap the
released radioactivity (free AA and its metabolites) in the
extracellular medium, incubation was performed in the presence of 0.1%
bovine serum albumin. Lipids were extracted from cells and their medium
and analyzed by two-dimensional chromatography. As shown in Fig.
1, colonization of AGS (Fig.
1A) and HeLa cells (Fig. 1B) by the
P12 wild-type strain increased the intracellular accumulation and extracellular release of free radiolabeled AA as
compared with controls. Typically, a 3-6-fold increase of free AA
(intra- and extracellular 14C-labeled AA) over the basal
release was observed within 60 min at a MOI of 200 (Fig.
1C), which did not increase further, suggesting reincorporation and/or metabolism of the released AA. Incubation of
cells with isogenic vacA mutant induced a comparable
increase in AA release like the wild-type strain, whereas incubation
with the isogenic cagA mutant caused a significant lower
increase in AA release. Melittin, known to stimulate AA release by
direct activation of PLA2 (30), was used as a positive
control and typically induced a 7-10-fold increase of AA over basal
release.
The analysis of 14C radioactivity in the cellular
phospholipids revealed that the AA release induced by H. pylori (P12) was accompanied by a substantial decrease
in the percentage of the total 14C-labeled AA content in
the PS/PI pool (Table I). The magnitude of the decrease ranged from 30 to 50% within the experiments. In terms
of total counts, the amount of 14C-labeled AA that
disappeared from the phospholipid pool in H. pylori-colonized cells matched approximately the amount of
radioactivity released. Two-dimensional phospholipid analysis, which
separates PS from PI, revealed that PI was the exclusive source of AA
in the PS/PI spot. The percentage of
[14C]phosphatidylcholine (PC) decreased slightly,
although these changes were only significant after longer time points
(12% after 180 min). At longer time points, H. pylori
caused also a small increase (14% after 180 min) in the percentage of
[14C]phosphatidylethanolamine (PE) presumably by
interconversion of intact phospholipids and reincorporation of released
AA into PE specifically. In melittin-treated cells (positive control), the phospholipids whose radiolabeled content decreased were PC and PI.
Exposure of cells to P12 wild-type strain or treatment with
melittin resulted also in a small but significant increase in
14C-labeled AA-labeled PA and probably reflects the
production of diacylglycerol (DAG) via activation of PLC (see below)
and its phosphorylation by DAG kinase (31).
Involvement of Cytosolic Phospholipase A2 in H. pylori-induced AA Release--
As shown in Fig.
2, H. pylori
(P12)-induced release of 14C-labeled AA was
significantly inhibited by both mepacrine (30 and 100 µM), a nonspecific inhibitor of the PLA2
(32), and MAFP (10 and 50 µM), a potent inhibitor of
cPLA2 and Ca2+-independent PLA2
(iPLA2) (33). In parallel with inhibition of AA release,
both inhibitors abolished the decrease in the radiolabeled content of
the PI pool while not affecting that of PC (data not shown), indicating
that this lipid may serve as a major source for AA. To further
delineate the type of PLA2 that is involved in the H. pylori-induced AA release, the effect of the
iPLA2-specific inhibitor HELSS (33) and the secretory
PLA2 inhibitor aristolochic acid (34) was tested. H. pylori-induced AA release from the cells was not affected by
aristolochic acid (50 µM) (data not shown). Likewise,
HELSS at 10 µM did not affect the AA release, whereas
higher concentrations (50 µM) inhibited both basal and H. pylori-induced AA release (data not shown), probably by
inhibiting other important effectors in the signal transduction (35).
These findings support a role of cPLA2 in the AA
release.
H. pylori induces an increase of cytosolic Ca2+ when
colonizing epithelial cells (19), and intracellular Ca2+
regulates cPLA2 activity; therefore, we studied the role of
Ca2+ in AA release. Chelating intracellular calcium by
preincubation of cells with BAPTA/AM (200 and 400 µM)
abolished release of AA in response to H. pylori (Fig.
2C). Taken together, these results strongly suggest that AA
release stimulated by H. pylori is
Ca2+-dependent.
The release of AA from membrane phospholipids could also occur through
PLC activation followed by the action of the DAG lipase (23). However,
H. pylori-induced AA release from the cells was insensitive
to inhibition by U73122 (1 and 10 µM) (Fig.
2D), an inhibitor of the PLC (36). Unexpectedly, U73122
itself caused an increase in basal AA release, thereby increasing the AA formation in H. pylori-infected cells. Under the same
experimental conditions, H. pylori caused a 1.2- and 2-fold
increase of total inositol phosphates (mono-, bis-, and trisphosphate)
in myo-[3H]inositol-labeled HeLa and AGS
cells, respectively, which was prevented by the PLC inhibitor U73122
(results not shown), demonstrating that the failure of the inhibitor to
block H. pylori-induced AA release is not the result of
U73122 failing to inhibit PLC. RHC80267 (40 and 80 µM),
an inhibitor of the DAG lipase (37), slightly inhibited the basal
release of AA from cells by about 30% but had no effect on H. pylori-induced AA release (Fig. 2E). Therefore, PLC and
DAG lipase signaling pathways are not involved in the H. pylori-induced AA release.
Another potential pathway of AA release involves activation of PLD
(24). To test this hypothesis, the release of water-soluble reaction
products into the medium was measured in HeLa cells that had been
labeled with [14C]choline. Of the radioactivity
incorporated into lipids, about 90 and 10% was found in PC and
sphingomyelin, respectively. The addition of
12-O-tetradecanoylphorbol-13-acetate (100 nM),
known to activate PLD in HeLa cells (38), resulted in a sustained release of radioactivity into the medium (about 2-fold over control), whereas incubation of cells with H. pylori up to 180 min did
not increase the release of choline metabolites as compared with
control cells (Table II). In addition, no
significant decrease of [14C]PC in cells incubated with
H. pylori (P12) was observed. These results
indicate that H. pylori cells do not activate hydrolysis of
PC by PLD. To gain further evidence that H. pylori does not affect the PLD activity, the effect of wortmannin, known to inhibit activation of PLD (39), was tested. At a concentration of 100 nM, wortmannin had no effect on H. pylori-induced AA release (data not shown).
Furthermore, PEt accumulation was measured in response to H. pylori in HeLa cells pretreated with 0.5% ethanol for 10 min (data not shown). In the presence of ethanol PLD catalyzed a
transphosphatidylation reaction, resulting in the production of PEt and
a consequent decrease in PA formation. As a positive control, HeLa
cells prelabeled with 14C-labeled AA or
[14C]palmitic acid were incubated for 60 min with
12-O-tetradecanoylphorbol-13-acetate in the presence of
0.5% ethanol. This substance caused an ethanol-dependent accumulation of [14C]PEt and a decrease of PA production.
In contrast, incubation of cells with H. pylori failed to
stimulate the formation of PEt, and PA formation was only marginally
decreased, indicating that H. pylori cells do not activate
PLD. Moreover, ethanol treatment did not prevent H. pylori-induced AA release, thereby conclusively ruling out the
involvement of PLD in H. pylori-induced AA release.
H. pylori Induces Generation of lysoPI in Epithelial Cells--
To
further validate that activation of cPLA2 is involved in
H. pylori-induced AA release, the generation of lysolipids
was monitored in HeLa cells prelabeled with [3H]inositol
or [14C]choline (Fig. 3).
When prelabeled cells were treated with melittin (positive control),
formation of both lysoPI and lysophosphatidylcholine was observed,
consistent with preferential release of AA from PI and PC via the
PLA2 pathway (see above). When
[3H]inositol-prelabeled cells were incubated with
P12 wild-type strain, the 3H radioactivity
associated with lysoPI increased noticeably. Under the same
experimental conditions, H. pylori (P12) did not
stimulate the formation of lysophosphatidylcholine in
[14C]choline-prelabeled cells. The predominant formation
of lysoPI in the presence of H. pylori (P12) is
in agreement with the substantial decrease in the percentage of the
total 14C-labeled AA content in the PI pool (Table I) when
14C-AA-labeled cells where infected with H. pylori.
H. pylori-induced AA Release Is PTX-sensitive and Involves the
Activity of p38 Kinase--
To explore the role of G-proteins in
modulating the H. pylori (P12) effects on AA
release, HeLa cells were pretreated with 1 µg/ml PTX for 24 h.
PTX alone did not have any effect on basal AA release, but PTX
treatment significantly reduced the H. pylori-induced AA
release (Fig. 4A). These
results suggest that PTX-sensitive G-proteins are involved in mediating
the stimulatory effect of H. pylori on AA release.
Next, we investigated whether protein kinase C and/or mitogen-activated
protein kinases are involved in the H. pylori-induced AA
release. Before colonization with H. pylori
(P12), cells were pretreated with various inhibitors of
protein kinase C: staurosporine (63), BIM (40), and K-252a
(41). As shown in Fig. 4B, staurosporine (0.1 µM), BIM (2.5 µM), and K252a (0.1 µM) failed to affect the AA release induced by H. pylori. Higher concentrations of BIM (10 µM) had no
effect on H. pylori-induced AA release, whereas staurosporine (1 µM) and K252a (1 µM)
increased the basal AA release and thereby the AA formation in H. pylori-infected cells (data not shown). Preincubation with a
specific inhibitor of the extracellular signal-regulated
kinase-activating pathway, PD98059 (42), had no significant effect on
the AA release induced by H. pylori (P12) (Fig.
4C). We have shown previously that 50 µM
PD98059 completely blocked the activation of the extracellular
signal-regulated kinase pathway in response to H. pylori
(17). In contrast, pretreatment of cells with SB202190 (10 and 20 µM), a specific inhibitor of the p38 stress-activated
kinase (43), significantly decreased the release of AA induced by
H. pylori (P12) (Fig. 4D). In
agreement with the inhibition of the AA release by SB202190, H. pylori-induced activation (phosphorylation) of p38 kinase (Fig.
4E) and phosphorylation of cPLA2 (Fig.
4F) were blocked in the presence of the inhibitor. As a
positive control, cells were treated with arsenite (0.5 mM) known to increase AA release through phosphorylation of cPLA2 via p38
kinase (44).
Infection of epithelial cells by H. pylori induced a
rapid generation of AA and the production of PGE2. The
separation of cell-associated lipids by TLC demonstrated that the
release of AA induced by H. pylori was accompanied by a
substantial decrease in the PI pool in preference to that of PC,
suggesting that PI could serve as a major source for AA in the
PGE2 synthesis. Consistent with this conclusion, lysoPI,
which is characteristic for the hydrolysis of PI by PLA2
(22, 45), was formed when the cells were incubated with H. pylori. Among the various types of mammalian PLA2, the
cPLA2 has a key role in the release of AA from cell membranes to serve as substrate for the production of PGs. This enzyme
has a high specificity for AA at the sn-2 position of
phospholipids and requires for activation both elevation of the
intracellular concentration of Ca2+ and a phosphorylation
step (22, 46). We found, consistent with cPLA2 involvement,
that pretreatment of cells with the cPLA2 inhibitor MAFP,
which, although it also inhibits iPLA2, is selective for
cPLA2 among known Ca2+-dependent
phospholipases (33), blocked the release of AA. In contrast, the
H. pylori-induced AA release was not affected by HELSS or
aristolochic acid, indicating that iPLA2 and secretory PLA2 are not involved. The inhibitory effect of the
intracellular calcium chelator BAPTA confirmed the involvement of a
Ca2+-dependent PLA2 in the AA
release. In addition, exposure of cells to H. pylori
resulted in a decrease in the electrophoretic mobility of
cPLA2, a finding consistent with cPLA2
phosphorylation (46), which is known to increase the catalytic activity
of cPLA2 in vitro (22).
Several evidences were presented in this study ruling out the
involvement of additional pathways in the generation of AA. First,
although exposure of HeLa and AGS cells to H. pylori induced generation of myo-[3H]inositol phosphates,
confirming previous findings (18), inhibition of inositol phosphate
production by the PI-PLC inhibitor U71322 had no effect on AA release,
indicating that PI-PLC is not involved. Second, no significant release
of choline metabolites or a decrease of PC was detected when
[14C]choline-labeled cells were exposed to H. pylori, ruling out the activation of PLD by H. pylori.
This conclusion was supported by the observation that in the presence
of ethanol, which substitutes for water in the transphosphatidylation
reaction catalyzed by PLD, formation of PEt was not detectable in
H. pylori-infected cells. Furthermore, formation of PEt
would decrease the amount of PA, thereby inhibiting the AA release, if
PA is the source for AA. However, inhibition of H. pylori-induced AA release was not observed for cells treated with
ethanol. Moreover, wortmannin, known to inhibit PLD activation in a
number of cell types (39), failed to affect the AA release induced by
H. pylori. Finally, in the presence of the DAG lipase
inhibitor RHC80267, H. pylori-induced AA release was unimpeded.
The H. pylori-induced predominant hydrolysis of PI, which is
exclusively located in the inner leaflet of the plasma membrane (47),
suggests that the host epithelial cell membrane was not damaged by the
H. pylori phospholipases A1, A2, or
C (48-50) or sphingomyelinase (51). Moreover, by using the isogenic
PAI mutant strain, which does not induce cPLA2 activation,
we exclude the possibility that the bacterial phospholipases are able
to activate host cell AA release from the epithelial cells, as has been
shown in the case of the Clostridium perfringens In a number of studies including ours, activation of cPLA2
has been shown to be PTX-sensitive, implying that members of the heterotrimeric G In conclusion, in this study we have shown that colonization of
epithelial cells by H. pylori promotes a rapid release of AA
predominately from PI for PGE2 production via activation of cPLA2. The signaling pathway requires a pertussis
toxin-sensitive G-protein and p38 stress-activated protein kinase but
not the activation of PLC, PLD, protein kinase C, and extracellular
signal-regulated kinase. The H. pylori-induced release of AA
and PGE2 from epithelial cells and their role in the
support or prevention of physiological and/or inflammatory reactions in
the stomach remain to be elucidated. Release of AA for PG production by
activation of cPLA2 could play an important role in mucosal
defense to the bacterial infection (5, 62) since gastric epithelial
cells are the first site of contact with H. pylori. On the
other hand, prolonged activation of cPLA2 is likely to be
damaging to the gastric epithelia by excessive degradation of membrane
phospholipids releasing AA and lysophospholipids.
i/G
o). The role of
phospholipase C, diacylglycerol lipase, or phospholipase D was excluded
by using specific inhibitors. An inhibitor of the stress-activated p38
kinase (SB202190), but neither inhibitors of protein kinase C nor an
inhibitor of the extracellular-regulated kinase pathway (PD98059),
decreased the H. pylori-induced arachidonic acid release.
H. pylori-induced phosphorylation of p38 kinase and
cytosolic PLA2 was blocked by SB202190. These results
indicate that H. pylori induces the release of
PGE2 from epithelial cells by cytosolic PLA2
activation via G
i/G
o proteins and the p38
kinase pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and AP-1 (13-17), phospholipase C (PLC)
(18), and the increase of the cytosolic free calcium concentration as
well as the generation of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate (19). The activation of the
PGE2 signaling pathway in H. pylori-colonized
gastric cells has not been studied so far.
i/G
o proteins and the p38
stress-activated kinase cascade. This process does not seem to involve
PLC or PLD pathways. The identification of H. pylori-specific signaling pathways leading to the induction of
putative anti-inflammatory immune response mediators is of substantial
interest for therapeutic intervention to overcome H. pylori-induced diseases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
80 °C. The individual lipids were identified by comparison with
commercial standards (Sigma). Phosphatidylethanol (PEt) standard was
prepared as described in Huang and Cabot (28); LysoPI was prepared by
PLA2 (Sigma) treatment of PI. Lipids and standards were
visualized with common lipid-locating agents such as iodine or
molybdenum blue spray for phosphate on the same plates. When analysis
of the choline release was required, radioactivity released into the
medium and associated with the cells was measured in aliquots of the
media and the cell extracts before phase splitting using a Wallac 409
-counter (Berthold-Wallac, Bad Wildbad, Germany). The content of
PGE2 in the medium was measured by enzyme-linked
immunosorbent assay (ELISA) kit according to the procedure indicated by
the manufacturer (DRG Instruments GmbH, Marburg, Germany).
-3-methoxyestra-1,3,5-(10)trien-17-yl)amino/hexyl)-1H-pyrrole-2,5-dione) (U73122), 1,6-bis-(cyclohexyloximinocarbonylamino) hexane
(RHC80267), methyl arachidonylfluorophosphonate (MAFP) (Biomol,
Hamburg, Germany); aristolochic acid, haloenol lactone suicide
substrate (HELSS), 1,2-bis(o-aminophenoxy)ethane-N,
N,N',N'-tetraacetic acid
tetra(acetoxymethyl)ester (BAPTA/AM), pertussis toxin (PTX),
2-(2-amino-3-methoxyphenyl)oxanaphthalen-4-one (PD98059),
4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) imidazole
(SB202190), staurosporine, bisindolylmaleimide I (BIM), K-252a
(Calbiochem). Stock solutions of U73122 and RHC80267 were prepared in
ethanol and stored at
20 °C; mepacrine, aristolochic acid, HELSS,
BAPTA/AM, PD98059, SB202190, BIM, and K-252a were prepared in dimethyl
sulfoxide; PTX was prepared in double-distilled water. For all
experiments, the effect of the appropriate vehicle was also determined.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of H. pylori on AA
release and phospholipid hydrolysis in AGS and HeLa cells prelabeled
with 14C-labeled AA. AGS and HeLa cells in
35-mm-diameter dishes were grown for 24 h in 2 ml of medium
containing 0.2 µCi of 14C-labeled AA and washed three
times with nonradioactive medium containing 1 mg/ml bovine serum
albumin before incubation with different H. pylori strains
at a MOI of 200. At various times up to 180 min the supernatant was
removed and cleared of detached cells by centrifugation. The lipids
were extracted separately from labeled cells and their medium and
analyzed by two-dimensional TLC. As a control, cells were stimulated
with the PLA2 activator melittin (125 ng/ml). A,
AGS cells; B, HeLa cells. Representative
two-dimensional TLC plates of total lipid extracts from untreated cells
(control (C)), cells exposed to H. pylori
(P12) for 60 min, and cells treated with melittin for 60 min
are shown. The location of individual species was verified using lipid
standards. NL, neutral lipids. Note that stimulation of
cells with melittin for 60 min resulted in lysis of some cells, as
determined by trypan blue-staining and, consequently, in the appearance
of lipids in the supernatant (asterisks). C, time
course of 14C-labeled AA formation (sum of intracellular
accumulation and extracellular release) by cells incubated with the
H. pylori strains P12 (wild type),
vacA, and the PAI mutant cagA at a MOI of 200 and
cells treated with melittin. Data are expressed as the percentage of
control cells and represent the means ± S.E. of at least three
independent experiments.
Effect of H. pylori on AA release and phospholipid hydrolysis in
14C-AA-labeled HeLa cells
View larger version (20K):
[in a new window]
Fig. 2.
Effect of phospholipase inhibitors on
H. pylori-induced AA release. HeLa cells were
labeled as described in the legend to Fig. 1 and then reincubated in
nonradioactive medium containing 1 mg/ml bovine serum albumin.
Mepacrine (A), MAFP (B), BAPTA/AM (C),
U71322 (D), or RHC80267 (E) were added for 30 min. Cells were then exposed to H. pylori (P12)
at a MOI of 200 or left untreated for another 60 min. The lipids were
extracted from cells and their medium and analyzed by two-dimensional
H. pylori TLC. Results are expressed as the percentage of AA
formation in vehicle-treated cells (control) incubated under the same
conditions and represent the means ± S.E. of three independent
experiments (panels A, B, and E);
results in panel C are the means ± S.E. from
triplicate determinations in a representative experiment. The
asterisks denote a significant difference compared with the
respective response to H. pylori without drug pretreatment
(p < 0.05).
Effect of H. pylori on the release of [14C]choline
metabolites in HeLa cells
View larger version (35K):
[in a new window]
Fig. 3.
Effect of H. pylori on
lysophospholipid formation in HeLa cells. HeLa cells in
35-mm-diameter dishes were grown for 24 h in 1 ml of medium
containing 2 µCi of myo-[3H]inositol or 0.5 µCi of [methyl-14C]choline and washed three
times with nonradioactive medium containing 1 mg/ml bovine serum
albumin before incubation with H. pylori (P12) at
a MOI of 200. As a control, cells were stimulated with the
PLA2 activator melittin (125 ng/ml). After 60 min the
supernatant was removed and cleared of detached cells by
centrifugation. The lipids were extracted together from cells and their
medium and analyzed by one-dimensional TLC. Representative TLC plates
of total lipid extracts from untreated cells (control), cells exposed
to H. pylori (P12) for 60 min, and cells treated
with melittin for 60 min are shown. Lysolipid formation was confirmed
by chromatography of PLA2-treated total lipid extracts
isolated from the labeled cells. The location of individual species was
verified using lipid standards. SM, sphingomyelin.
View larger version (23K):
[in a new window]
Fig. 4.
H. pylori-induced AA release is
PTX-sensitive and involves the activity of p38 kinase. HeLa cells
were labeled for 24 h as described in the legend to Fig. 1 and
pretreated with PTX (1 µg/ml, included during labeling)
(A), with inhibitors of the protein kinase C (staurosporine,
BIM, and K-252a (all for 30 min)) (B), with an inhibitor of
the extracellular signal-regulated kinase-activating pathway (PD98059)
(30 min) (C), or with a specific inhibitor of the p38
stress-activated protein kinase (SB202190) (30 min) (D).
Thereafter, H. pylori (P12) was added at a MOI of
200, and incubation was continued for another 60 min. The lipids were
extracted from cells and their medium and analyzed by two-dimensional
H. pylori TLC. Data are expressed as a percentage of AA
formation in control cells without drug pretreatment incubated under
the same conditions. In panels A and C, data represent the
means ± S.E. from three independent experiments. The
asterisks denote a significant difference compared with the
respective response to H. pylori without drug pretreatment
(p < 0.05). In panel B, results shown are
the mean of two experiments plus or minus the difference from the mean.
E, HeLa cells were pretreated with SB202190 (20 µM, 30 min) and infected with H. pylori
(P12) at a MOI of 200 for the indicated periods of time.
Phosphorylation of p38 kinase was monitored from whole cell lysates
using a p38 phospho-specific antibody (upper panel). As a
loading control, the same blot was probed with an anti-p38 antibody
(lower panel). F, the shift in electrophoretic
mobility of cPLA2, indicating cPLA2
phosphorylation was monitored in an immunoblot using an
anti-cPLA2 antibody. Lanes 1 and 4,
cell lysates prepared from nonstimulated; lanes 2 and
5, arsenite (Ars.)-stimulated (0.5 mM, 15 min); lanes 3 and 6, H. pylori (P12)-infected cells (MOI 200, 60 min).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-toxin
(52).
i/G
o proteins are
involved in its regulation (53-56). Although the underlying mechanism
for the regulatory role of G
i/G
o proteins
on cPLA2 activation is still unclear, it has been proposed
that the
subunits released from
G
i/G
o proteins can stimulate
cPLA2 activity and AA release (57). Furthermore, it has
been shown that mitogen-activated protein kinases are involved in
cPLA2 activation (22, 46). Our studies, using specific inhibitors of protein kinase C and the extracellular signal-regulated kinase cascade, clearly demonstrated that the ability of H. pylori to cause a release of AA from epithelial cells does not
depend on the activation of these kinases. This conclusion is supported by the observation that H. pylori induces the activation of
the extracellular signal-regulated kinase cascade in a PAI-independent manner (14, 17), whereas in marked contrast to that, AA release induced
by H. pylori is PAI-dependent. Recently,
evidence for the involvement of p38 kinase in cPLA2
activation has been presented (44, 58-60). In H. pylori-stimulated cells, activation of p38 kinase, the mobility
shift of cPLA2, and AA release were clearly abolished after
pretreatment of cells with the p38 kinase inhibitor SB202190. These
findings indicate the involvement of the p38 kinase in the signaling
cascade leading to cPLA2 phosphorylation and AA release.
Notably, SB202190 has been used to inhibit p38 kinase activated by
various stimuli (60, 61) and thereby shown to reduce phosphorylation of
cPLA2 in HeLa cells (59) as well as to block AA release in
thrombin-stimulated platelets (61). Our findings are also consistent
with the observation that activation of the p38 kinase by H. pylori in epithelial cells is strictly PAI-dependent
(14). The release of AA was sensitive to both PTX and an inhibitor of
p38 kinase, indicating that PTX-sensitive G
i/G
o proteins could be involved in p38
kinase activation. Hence, additional work is required to elucidate the
signaling upstream of p38 kinase involved in the activation of
cPLA2 by H. pylori.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Schöneberg and A. Schulz for their invaluable help in measuring the formation of inositol
phosphates and for the generous supply of PTX, Dr. Gerry T. Snoek for
valuable discussions regarding inositol lipids analysis, and Dr.
A. G. Börsch-Haubold for comments on cPLA2
detection. We also gratefully acknowledge and appreciate the support of
C. Bartsch, S. Weler, and B. Wieland in these studies.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Fonds der Chemischen Industrie (to M. N. and T. F. M.).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.
Present address: University of Amsterdam, Dept. of Cell Biology
and Histology, Academic Medical Center L3, Meibergdreef 15, 1105 AZ
Amsterdam, The Netherlands.
§ To whom correspondence should be addressed: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, Schumannstrasse 21/22, 10117 Berlin, Germany. Tel.: 49-30-28460410; Fax: 49-30-28460401; E-mail: naumann@mpiib-berlin.mpg.de.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M003819200
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ABBREVIATIONS |
---|
The abbreviations used are: PG, prostaglandin; PAI, pathogenicity island; AA, arachidonic acid; BIM, bisindolylmaleimide I; DAG, diacylglycerol; HELSS, haloenol lactone suicide substrate; MAFP, methyl arachidonylfluorophosphonate; MOI, multiplicity of infection; PA, phosphatidic acid; PC, phosphatidylcholine; PD98059, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one; PE, phosphatidylethanolamine; PEt, phosphatidylethanol; PI, phosphatidylinositol; PLA2, phospholipase A2; iPLA2, Ca2+-independent PLA2; cPLA2, cytosolic PLA2; PLC, phospholipase C; PLD, phospholipase D; PS, phosphatidylserine; SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) imidazole; SM, sphingomyelin; PTX, pertussis toxin.
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