 |
INTRODUCTION |
Activation of phospholipase A2
(PLA2)1 in a
variety of cells including inflammatory cells leads to the liberation
of arachidonic acid (AA), which is, in turn, converted to several types
of prostaglandins (PGs) by the sequential action of cyclooxygenase
(COX) and PG synthases, this being the initial step in the generation
of PG (1). Among numerous types of PLA2s identified in
mammalian cells and tissues, Ca2+-dependent
cytosolic PLA2 (cPLA2, type IV) and
non-pancreatic secretory PLA2 (sPLA2, types
IIA, V, and X) are responsible for stimulus-induced AA liberation and
subsequent PG generation (2-7). Recent reports showed that activation
of cPLA2 is required for the onset of
sPLA2-catalyzed hydrolysis of membrane phospholipids (8) or
for induction of sPLA2 expression (9, 10) in mouse P388D1 macrophages and rat 3Y1 fibroblasts stimulated with
proinflammatory stimuli. Conversely, exogenous sPLA2
stimulates cPLA2 activation through a receptor-mediated
mechanism in human 1321N1 astrocytoma cells (11) or lysophospholipid
formation resulting from hydrolysis of membrane phospholipids in rat
mesangial cells (12). Thus, the existence of cross-talk between the two
PLA2s has been proposed. On the other hand,
stimulus-induced AA liberation in platelets having the two
PLA2s is mainly mediated by cPLA2 (13), because sPLA2 is unable to hydrolyze membrane phospholipids of
platelets unless the membrane undergoes certain modifications, such as
lipid peroxidation (14). Therefore, the isozyme(s) of PLA2s
contributing to the generation of PG varies with the types of cells
and/or signaling pathways elicited by agonists.
It is generally accepted that PG plays an important role in the
homeostasis or progression of a variety of diseases with inflammation. The generation of PG in gastric cells including epithelial cells and
fibroblasts is involved in the healing of gastric lesions and in the
maintenance of gastric mucosal integrity (15, 16). In an animal model
of stress-induced gastric ulcers, generation of PGE2 and
expression of two COX isoforms, COX-1 and COX-2, are accelerated during
the healing of the lesions in parallel with increases in transforming
growth factor (TGF)-
and epidermal growth factor (EGF) (17). TGF-
(18) and EGF (19) have been shown to stimulate PGE2
generation and COX-2 expression in rat gastric epithelial RGM1 cells
and guinea pig gastric mucosal cells. TGF-
, which is produced by
gastric epithelial cells (20), exhibits numerous biological activities
including inhibition of gastric acid secretion and stimulation of
migration and proliferation of gastric epithelial cells (21, 22)
through binding to EGF receptor (23, 24). A recent study showed that
TGF-
induces cell proliferation and morphogenesis through a
COX-2-dependent mechanism in RGM1 cells (25). On the other
hand, the expression of interleukin (IL)-1
, a proinflammatory
cytokine, as well as COX-2 is also accelerated during the healing of
ischemia/reperfusion-induced gastric lesions in an animal model (26).
In human gastric fibroblasts, stimulation with IL-1
induces
COX-2-dependent PGE2 generation (27), leading
to the expression of hepatocyte growth factor (27, 28), which
stimulates proliferation of rabbit gastric epithelial cells (28, 29).
Furthermore, cytoprotection by IL-1
against ethanol-induced gastric
injury was shown to be dependent on the generation of PGE2
in an animal model (30). Consequently, it is conceivable that TGF-
and IL-1
are implicated in the healing of gastric lesions through,
at least in part, COX-2-dependent PGE2
generation. However, the role of TGF-
and IL-1
in the activation of the PLA2 isozyme(s) contributing to the generation
during the healing remains to be elucidated.
Recently, we demonstrated that TGF-
induces cPLA2
expression as well as PGE2 generation with no change in
sPLA2 activity in RGM1 cells, although the increase in
PGE2 was slight (18). Furthermore, TGF-
was found to
inhibit Ca2+ ionophore-induced cPLA2 activation
in parallel with an increase in p11 (18), known as a putative
cPLA2 inhibitory molecule (31, 32). These findings suggest
that TGF-
-stimulated PGE2 generation occurs under the
control of inducible p11, which negatively regulates the hydrolytic
activity of cPLA2. Considering the possibility that IL-1
is also involved in the healing of gastric lesions through generation
of PG, TGF-
may cooperate with IL-1
to stimulate efficiently the
generation in gastric epithelial cells. This appears likely, because a
combination of EGF and IL-1
has been shown to induce synergistically
COX-2 expression and PGE2 generation in human gingival
fibroblasts (33). IL-1
is well known to induce sPLA2
expression in a variety of cells (34-36). However, several growth
factors including EGF have been reported to inhibit cytokine-induced sPLA2 release (37, 38) but enhance PG generation (37) in rat calvarial osteoblasts and astrocytes. Thus, little is known about
the PLA2 isozymes acting upon stimulation with both growth factors and cytokines.
The present study was undertaken to identify the isozyme(s) of
PLA2s contributing to the generation of PGE2 in
rat gastric epithelial RGM1 cells stimulated with a combination of
TGF-
and IL-1
. For this purpose, we examined changes in the two
PLA2 isozymes, cPLA2 and sPLA2, in
comparison to an increase in PGE2 upon the stimulation,
taking into account the possible existence of cross-talk between the
PLA2s and selective cooperation with certain COXs.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human TGF-
was obtained from
PeproTech (London, UK). IL-1
was from Collaborative Biomedical
Products (Bedford, MA). A23187 and NS-398
(N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide) were from Calbiochem. Valeryl salicylate, methylarachidonyl
fluorophosphonate (MAFP), and arachidonyltrifluoromethyl ketone
(AACOCF3) were from Cayman Chemical (Ann Arbor, MI).
Indoxam was donated by Dr. K. Hanasaki (Shionogi Research Laboratories,
Shionogi & Co., Ltd Osaka, Japan). Antibodies against COX-1, COX-2, and
cPLA2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). The anti-p11 antibody and anti-phosphoserine antibody were from
Transduction Laboratories (Lexington, KY) and NanoTools
Antikörpertechnik (Germany), respectively. Nucleotides, as
probes, were from Greiner Japan (Tokyo, Japan).
1-Palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine
(55 mCi/mmol) was from Amersham Pharmacia Biotech. [3H]AA
(100 Ci/mmol) and
1-stearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine
(160 Ci/mmol) were from PerkinElmer Life Sciences. Other reagents were
obtained from Wako Pure Chemical Industries (Osaka, Japan) or Sigma.
Cell Culture and Preparation of [3H]AA-labeled
Cells--
RGM1, a non-transformed epithelial cell line derived from
normal rat gastric mucosa (39), was purchased from Riken Cell Bank
(Tsukuba, Japan). RGM1 cells were maintained in Dulbecco's modified
Eagle's medium/Ham's F-12 medium (DMEM/F-12; 1:1) supplemented with
20% heat-inactivated fetal calf serum, 100 units/ml penicillin, and
100 µg/ml streptomycin at 37 °C under humidified air containing 5% CO2. Cells were plated in 35-mm culture dishes at
1 × 106 cells in DMEM/F-12 containing 0.01% bovine
serum albumin and incubated for 24 h in the presence or absence of
[3H]AA (1 µCi/ml). The labeled or unlabeled cells were
washed three times with DMEM/F-12 containing 0.01% bovine serum
albumin and a further two times with DMEM/F-12. The cells were placed
in 1 ml of DMEM/F-12 for the following experiments.
PGE2 Generation--
The
[3H]AA-labeled RGM1 cells were treated with various
reagents and then stimulated with TGF-
, IL-1
, or both as
described in the figure legends. Lipids in the medium and cells were
extracted and separated by thin layer chromatography on a Silica Gel G
plate using an upper phase of ethyl acetate/isooctane/acetic
acid/H2O (9:5:2:10, v/v) as the development system. The
area corresponding to PGE2 was scraped off, and the
radioactivity was determined by liquid scintillation counting.
AA Liberation Induced by A23187--
The
[3H]AA-labeled RGM1 cells were stimulated with TGF-
,
IL-1
, or both for 24 h, washed, and placed in 1 ml of fresh
DMEM/F-12. The cells were treated with various reagents in the presence
of 10 µM BW755C (a COX and lipoxygenase inhibitor) for 30 min and further stimulated with 1 µM A23187 for 10 min.
Lipids in the medium and cells were extracted and separated by thin
layer chromatography on a Silica Gel G plate using petroleum
ether/diethyl ether/acetic acid (40:40:1, v/v) as the development
system (40). The area corresponding to free fatty acid was scraped off,
and the radioactivity was determined by liquid scintillation counting.
Conversion of Exogenous AA to PGE2--
RGM1 cells
were stimulated with TGF-
and IL-1
in the presence of indoxam as
described in the figure legends, washed, and placed in 0.5 ml of fresh
DMEM/F-12. The cells were further treated with indoxam for 30 min and
incubated with a mixture of [3H]AA and the unlabeled
compound (50 Ci/mol, 2 µM) for 30 min. After lipids in
the medium and cells were extracted, the radioactivity of
[3H]PGE2 was determined as described above.
Immunoblot Analysis for COXs, cPLA2, and
p11--
After stimulation of RGM1 cells with TGF-
, IL-1
, or
both as described in the figure legends, the cells were washed and
scraped off in buffer A (100 mM NaCl, 2 mM
EGTA, 100 µM p-(amidinophenyl)methanesulfonyl fluoride, 100 µM leupeptin, 20 mM
-glycerophosphate, 1 mM Na3VO4, and 10 mM Tris-HCl, pH 7.4) containing 0.05% Triton X-100.
Samples (10 µg of protein for p11 and 20 µg of protein for
cPLA2 and COXs) were solubilized and subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5 (for
cPLA2 and COXs) or 15% (for p11) gel. The proteins were
transferred to a nitrocellulose membrane, and then antibodies against
COXs, cPLA2, or p11 were applied. The bound antibodies were
visualized using peroxidase-conjugated secondary antibodies and
enhanced chemiluminescence Western blotting detection reagents
(Amersham Pharmacia Biotech).
Assay for cPLA2 Activity--
RGM1 cells were
treated with or without MAFP or AACOCF3 and stimulated with
TGF-
, IL-1
, or both, as described in the figure legends. After
the medium was removed, the cells were scraped off and sonicated in
buffer A containing 0.05% Triton X-100. The protein concentrations in
the lysate were adjusted to 1 mg/ml, and the lysate was treated with 5 mM dithiothreitol at 37 °C for 15 min to inhibit
sPLA2 activity. The sample containing 10 µl of the lysate
was incubated with a mixture of
1-stearoyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine
and the unlabeled compound (250 Ci/mol, 2 µM) at 37 °C
for 1 h in the presence of 5 mM CaCl2 and
50 mM Tris-HCl, pH 8.5, in a final volume of 200 µl.
After lipid extraction, the [3H]AA liberated was analyzed
as described above, and the enzyme activity was calculated.
Assay for sPLA2 Activity--
RGM1 cells, placed in
0.5 ml of DMEM/F-12 containing 0.1 mg/ml heparin, were stimulated with
TGF-
, IL-1
, or both as described in the figure legends. After the
medium was centrifuged, the supernatant (20 µl), as an enzyme source,
was incubated with
1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine
(2 µM) at 37 °C for 15 min in the presence of 5 mM CaCl2, 1 mg/ml bovine serum albumin, and 50 mM Tris-HCl, pH 8.5, in a final volume of 200 µl. After lipid extraction, the [14C]linoleic acid liberated was
analyzed as described above, and the enzyme activity was calculated.
RNA Blotting--
RGM1 cells (8 × 106) in
100-mm dishes were stimulated with TGF-
, IL-1
, or both for
24 h. Total RNA (30 µg) was extracted from the cells using
TRIzol reagent, subjected to formaldehyde-agarose gel electrophoresis
on a 1% gel, and transferred to a nylon membrane. The membrane was
hybridized with probes for rat type IIA (5'-GTG CCA CAT CCA CGT TTC TCC
AGA CGG TTG), V (5'-TCA TGG ACT TCA GTT CTA GCA AGC CCC CTG), or X
(5'-TAT CGG TAT AGC TTG GGG CTG CAG CCG GCA) sPLA2, which
had been labeled with alkaline phosphatase for 12 h using a
commercial labeling kit (Amersham Pharmacia Biotech). The bound probes
were visualized using CDP-Star detection reagent (Amersham Pharmacia
Biotech). After the probes were stripped off, the membrane was
rehybridized with alkaline phosphatase-labeled probe for
glyceraldehyde-3-phosphate dehydrogenase (5'-GAG GGA GTT GTC ATA TTT
CTC GTG GTT CAC).
Immunoprecipitation--
RGM1 cells (8 × 106)
in 100-mm dishes were stimulated with TGF-
and IL-1
for 24 h, sonicated in buffer B (100 mM KCl, 1 mM EGTA, 100 µM leupeptin, 100 µM
p-(amidinophenyl)methanesulfonyl fluoride, 1.1 mM CaCl2, and 20 mM Tris-HCl, pH
7.4), and subjected to immunoprecipitation of cPLA2.
Briefly, the lysate (0.4 mg of protein) was incubated with protein
A-agarose at 4 °C for 30 min, as a pre-clearing step (41), and
centrifuged at 10,000 × g for 3 min. The supernatant
was incubated with anti-cPLA2 antibodies overnight at
4 °C and further with protein A-agarose for 2 h. After
centrifugation, the pellet obtained was washed three times with buffer
B and solubilized. The sample was subjected to immunoblot analysis with
antibodies against cPLA2, phosphoserine, or p11 as
described above.
Statistical Analysis--
Values are expressed as the mean ± S.E. of three or four separate experiments. Data were analyzed by
one-way analysis of variance followed by Bonferroni's test.
p < 0.05 was considered statistically significant.
 |
RESULTS |
Increase in PGE2 Preceded by Expression of COXs in
Response to TGF-
and IL-1
--
As shown in Fig.
1A, stimulation of RGM1 cells
with 50 ng/ml TGF-
for 24 h slightly increased PGE2
in a time-dependent manner, whereas 5 ng/ml IL-1
had no
effect on the basal level of PGE2. However, a combination
of 50 ng/ml TGF-
and 5 ng/ml IL-1
was found to stimulate
synergistically the generation of PGE2 with a marked
increase observed 18 h after the stimulation. The synergistic effect on PGE2 generation was dose-dependent of
IL-1
or TGF-
(Fig. 1, B and C). Under
similar experimental conditions, we determined changes in COX-1 and
COX-2 proteins (Fig. 2). Fig.
2A shows that 50 ng/ml TGF-
but not 5 or 10 ng/ml IL-1
significantly increased COX-2, whereas in combination they
synergistically induced COX-2 expression. However, in contrast to the
time course of PGE2 generation (Fig. 1A), Fig.
2B revealed that COX-2 expression induced by the combination
was apparently increased 3-6 h after the stimulation. On the other
hand, COX-1 protein was not detectable in RGM1 cells even when the
cells were stimulated with 50 ng/ml TGF-
or 5 ng/ml IL-1
under
our experimental conditions (Fig. 2A). However, the combination of TGF-
and IL-1
was found to increase markedly COX-1
protein. The increase in COX-1 induced by the combination was also
observed 3-6 h after the stimulation, as shown in Fig. 2B.
To examine the involvement of the two COXs in the synergistic effect on
PGE2 generation, the effects of COX inhibitors on the generation of PGE2 were determined. The results shown in
Fig. 2C indicate that generation of PGE2 in
response to both 50 ng/ml TGF-
and 5 ng/ml IL-1
was suppressed
markedly by pretreatment with 0.1 or 1 µM NS-398, a COX-2
inhibitor (42), but not by 2 or 5 µM valeryl salicylate,
a COX-1 inhibitor (43). Under those conditions, 0.1 µM
NS-398 did not affect TGF-
/IL-1
-induced expression of COX-2 or
COX-1 (result not shown).

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Fig. 1.
Synergistic PGE2 generation
induced by TGF- and
IL-1 . A, the
[3H]AA-labeled cells were stimulated with
(circles) or without (squares) 5 ng/ml IL-1
for the indicated periods in the presence (closed symbols)
or absence (open symbols) of 50 ng/ml TGF- . B
and C, the labeled cells were stimulated with (closed
symbols) or without (open symbols) 50 ng/ml TGF-
(B) or 5 ng/ml IL-1 (C) for 24 h in the
presence of various concentrations of IL-1 (B) or TGF-
(C). The amount of PGE2 generated was measured.
*, p < 0.001; **, p < 0.05, relative
to the corresponding response of the unstimulated cells. Each point
shown in B and C represents the mean of two
separate experiments.
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Fig. 2.
Increases in COX-1 and COX-2 and effects of
COX inhibitors on PGE2 generation in
TGF- /IL-1 -stimulated
cells. A, cells were stimulated with or without TGF-
and/or IL-1 at the indicated concentrations for 24 h.
B, cells were stimulated with 50 ng/ml TGF- and 5 ng/ml
IL-1 for the indicated periods. A and B, COX-1
and COX-2 proteins were analyzed by immunoblotting. C, the
[3H]AA-labeled cells were treated with or without ( )
NS-398 (0.1 or 1 µM; NS) or valeryl salicylate
(2 or 5 µM; VS) for 30 min and then stimulated
with or without (Control) 50 ng/ml TGF- and 5 ng/ml
IL-1 for 24 h. The amount of PGE2 generated was
measured. The results shown in A and B are
representative of three separate experiments.
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|
cPLA2 Expression and Delayed sPLA2 Release
in Response to TGF-
and IL-1
--
To identify the
PLA2 isozyme(s) contributing to the synergistic effect of
TGF-
and IL-1
on PGE2 generation, the change in cPLA2 upon stimulation was examined (Fig.
3). Stimulation with 50 ng/ml TGF-
for
24 h increased cPLA2 protein and activity, whereas 10 ng/ml IL-1
did not affect the basal levels (Fig. 3, A and
B). In contrast to the stimulatory effect of IL-1
on
TGF-
-induced PGE2 generation (Fig. 1B), 50 ng/ml TGF-
-increased cPLA2 protein or activity was not
augmented in the presence of 2-10 ng/ml IL-1
(Fig. 3,
A and B). Furthermore, the combination of
50 ng/ml TGF-
and 5 ng/ml IL-1
time-dependently
increased cPLA2 protein with an apparent increase observed
6 h after the stimulation (Fig. 3C), indicating that
the time-dependent expression of cPLA2 preceded the onset of the synergistic increase in PGE2 (Fig.
1A).

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Fig. 3.
Changes in cPLA2 protein and
activity in response to TGF- and
IL-1 . A, cells were stimulated
with or without TGF- and/or IL-1 at the indicated concentrations
for 24 h. cPLA2 protein was analyzed by
immunoblotting. B, cells were stimulated with (closed
symbols) or without (open symbols) 50 ng/ml TGF- for
24 h in the presence of various concentrations of IL-1 .
cPLA2 activity in lysate of the cells was determined.
C, cells were stimulated with 50 ng/ml TGF- and 5 ng/ml
IL-1 for the indicated periods, and then cPLA2 protein
was analyzed. The results shown in A and C are
representative of three separate experiments.
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We further determined the change in sPLA2 activity in the
extracellular medium. The result shown in Fig.
4A indicates that IL-1
(2-10 ng/ml) dose-dependently increased sPLA2
activity, which was enhanced in the presence of 50 ng/ml TGF-
,
although TGF-
did not increase the activity by itself. As shown in
Fig. 4B, a marked increase in sPLA2 activity was
observed 18 h after stimulation with 5 ng/ml IL-1
alone or in
combination with 50 ng/ml TGF-
, indicating that the synergistic
effect on PGE2 generation (Fig. 1A) occurred in
parallel with an increase in sPLA2 activity. Pretreatment
of cells with 1 µM cycloheximide or 1 µM
actinomycin D inhibited the increase in sPLA2 activity
induced by both TGF-
and IL-1
(data not shown). We further
determined the change in mRNA for types IIA, V, and X
sPLA2s, as shown in Fig. 4C. Stimulation with 5 ng/ml IL-1
for 24 h induced type IIA sPLA2 mRNA
expression, whereas 50 ng/ml TGF-
did not affect the mRNA, which
was undetectable in unstimulated cells. Consistent with the change in
sPLA2 activity, TGF-
augmented the mRNA expression
of type IIA sPLA2 induced by IL-1
, whereas mRNA for
type V or X sPLA2 was not detectable under our experimental
conditions.

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Fig. 4.
sPLA2 release and expression
induced by TGF- and
IL-1 . A, cells were stimulated
with (closed symbols) or without (open symbols)
50 ng/ml TGF- for 24 h in the presence of various
concentrations of IL-1 . B, cells were stimulated with
(circles) or without (squares) 5 ng/ml IL-1
for the indicated periods in the presence (closed symbols)
or absence (open symbols) of 50 ng/ml TGF- . A
and B, sPLA2 activity in the medium of the cells
was determined. C, cells were stimulated with or without
( ) 50 ng/ml TGF- , 5 ng/ml IL-1 , or both for 24 h, and then
mRNA for type IIA, V, or X sPLA2 or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
analyzed. The results are representative of three separate
experiments.
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Recently, we reported the possible involvement of type VI
Ca2+-independent PLA2 in zymosan-stimulated
liberation of AA in P388D1 cells, in which the
PLA2 translocates to membranes upon the stimulation (44).
We further examined the effect of TGF-
and IL-1
on type VI
PLA2 activity in RGM1 cells. However, the activity in the
lysate or membrane fraction of unstimulated RGM1 cells was not affected by stimulation with 50 ng/ml TGF-
and/or 5 ng/ml IL-1
for 24 h (data not shown).
Effects of PLA2 Inhibitors on
TGF-
/IL-1
-stimulated PGE2 Generation--
To examine
the involvement of cPLA2 in the synergistic
PGE2 generation by TGF-
and IL-1
, we tested the
effects of MAFP and AACOCF3, cPLA2 inhibitors,
on cPLA2 activity and PGE2 generation upon the
stimulation. As shown in Fig.
5A, when RGM1 cells pretreated with 2 µM MAFP or 5 µM AACOCF3
were stimulated with both 50 ng/ml TGF-
and 5 ng/ml IL-1
, the
cPLA2 activity in lysate of the stimulated cells as well as
basal activity in unstimulated cells were significantly suppressed by
the inhibitors. Under these conditions, PGE2 generation induced by TGF-
alone was also inhibited, whereas synergistic PGE2 generation induced by both TGF-
and IL-1
was not
affected (Fig. 5B), indicating that the cPLA2
inhibitors had no effect on the augmentation by IL-1
of
TGF-
-induced PGE2 generation. Recently, MAFP and
AACOCF3 were reported to inhibit stimulus-induced sPLA2 release (9, 10, 36), suggesting a requirement of cPLA2 for sPLA2 expression. As shown in Fig.
5A, however, the cPLA2 inhibitors did not affect
an increase in sPLA2 activity induced by both TGF-
and
IL-1
. Under our experimental conditions, 2 µM MAFP or
5 µM AACOCF3 had no effect on cell viability
(92 or 90%, respectively; control, 96%; the mean of two
determinations), but more than 10 µM MAFP or
AACOCF3 decreased it to less than 70 or 80%, respectively,
as estimated by trypan blue dye exclusion.

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Fig. 5.
Effects of MAFP and AACOCF3 on
cPLA2 activity, sPLA2 release, and
PGE2 generation in
TGF- /IL-1 -stimulated
cells. A, cells were treated with (+) or without ( ) 2 µM MAFP or 5 µM AACOCF3 for 30 min and then stimulated with or without (Control) 50 ng/ml
TGF- and 5 ng/ml IL-1 for 24 h. cPLA2 activity
in the cell lysate (open columns) and sPLA2
activity in the medium (closed columns) were determined.
B, the [3H]AA-labeled cells were treated with
MAFP or AACOCF3 as in A and then stimulated with
or without (Control) 50 ng/ml TGF- alone or in
combination with 5 ng/ml IL-1 for 24 h. The amount of
PGE2 generated was measured.
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|
Fig. 6 illustrates the effects of
indoxam, a specific sPLA2 inhibitor (45, 46), on increases
in sPLA2 activity and PGE2 in response to
TGF-
and IL-1
. When RGM1 cells were stimulated with both 50 ng/ml
TGF-
and 5 ng/ml IL-1
for 12 h and further incubated for
12 h in the presence of indoxam (5-100 nM), the increased sPLA2 activity in the medium was inhibited by
indoxam in a dose-dependent manner (Fig. 6A).
Under these conditions, the sPLA2 inhibitor significantly
suppressed TGF-
/IL-1
-stimulated PGE2 generation at
the same concentration ranges (Fig. 6B). However, 100 or 200 nM indoxam did not affect cPLA2 activity or
conversion of exogenous AA to PGE2 in
TGF-
/IL-1
-stimulated cells (Fig. 6C).

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Fig. 6.
Effects of indoxam on sPLA2
release, PGE2 generation, cPLA2 activity, and
conversion of exogenous AA to PGE2 in
TGF- /IL-1 -stimulated
cells. A and B, the
[3H]AA-labeled or unlabeled cells were stimulated with
(closed symbols) or without (open symbols) 50 ng/ml TGF- and 5 ng/ml IL-1 for 12 h and further incubated
for 12 h in the presence of various concentrations of indoxam.
sPLA2 activity in the medium (A) and the amount
of PGE2 generated (B) were determined.
C, cells were stimulated with or without
(Control) 50 ng/ml TGF- and 5 ng/ml IL-1 for 12 h
and further incubated for 12 h in the presence of 100 or 200 nM indoxam. For cPLA2 activity (closed
columns), after being washed, lysate of the cells was further
treated with same concentration of indoxam for 30 min, and
cPLA2 activity in the lysate was determined. For conversion
of exogenous AA to PGE2 (open columns), after
being washed, the cells were further treated with same concentration of
indoxam for 30 min and incubated with 2 µM
[3H]AA for 30 min. The amount of PGE2
generated was measured. The result shown in C represents the
mean of two separate experiments.
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Effects of TGF-
and IL-1
on A23187-induced AA
Liberation--
It was reported that p11, known as annexin II light
chain, directly inhibits cPLA2 activity through binding to
the enzyme (31), and its overexpression results in suppression of
Ca2+ ionophore A23187-induced AA liberation (32). Recently,
we showed that incubation of RGM1 cells with TGF-
for 3-24 h
inhibited A23187-induced AA liberation in parallel with p11 expression (18). In the present study, we further examined whether TGF-
exhibits similar effects in the presence of IL-1
. The results shown
in Fig. 7A indicate that when
RGM1 cells, which had been stimulated with 50 ng/ml TGF-
for 24 h in the presence or absence of 5 ng/ml IL-1
, were washed and
further stimulated with 1 µM A23187 for 10 min, TGF-
diminished A23187-induced AA liberation as compared with
TGF-
-unstimulated cells even in the presence of IL-1
.
Furthermore, 50 ng/ml TGF-
induced an increase in p11 irrespective
of the presence of 5 ng/ml IL-1
(Fig. 7B). However, IL-1
alone did not inhibit, but rather enhanced, A23187-induced AA
liberation without influencing the amount of p11. Moreover, as shown in
Fig. 7C, when cPLA2 in RGM1 cells was
immunoprecipitated using anti-cPLA2 antibodies, amounts of
cPLA2 protein including its phosphorylated form
(phosphoserine) in the precipitate were increased by stimulation with
50 ng/ml TGF-
alone or in combination with 5 ng/ml IL-1
. Under
these conditions, p11 protein was also precipitated; its amounts in the
precipitate were increased with an increase in cPLA2
protein (Fig. 7C). These results suggest that the inhibitory
effect of TGF-
on A23187-induced AA liberation occurs in parallel
with p11 expression but independently of IL-1
-elicited signaling.

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Fig. 7.
Effects of TGF- and
IL-1 on AA liberation induced by A23187 and
amounts of phosphorylated cPLA2 and p11. A,
the [3H]AA-labeled cells were stimulated with or without
(Control) 50 ng/ml TGF- , 5 ng/ml IL-1 , or both for
24 h. After being washed, the cells were treated with 10 µM BW755C for 30 min and then stimulated with
(closed columns) or without (open columns) 1 µM A23187 for 10 min. The amount of AA liberated was
determined. *, p < 0.01, relative to the response of
A23187 alone. B, cells were stimulated with or without
(Control) 50 ng/ml TGF- , 5 ng/ml IL-1 , or both for
24 h, and then p11 protein was analyzed. C, cells were
stimulated with or without (Control) 50 ng/ml TGF- alone
or in combination with 5 ng/ml IL-1 for 24 h. The lysate
prepared from the cells was subjected to immunoprecipitation of
cPLA2, and then the precipitate obtained was analyzed by
immunoblotting with antibodies against cPLA2,
phosphoserine, or p11. D, the [3H]AA-labeled
cells were stimulated with or without (Control) 5 ng/ml
IL-1 for 24 h and washed. The cells were treated with (+) or
without ( ) 10 µM MAFP or 50 nM indoxam
(INDX) for 30 min in the presence of 10 µM
BW755C, and then stimulated with (closed columns) or without
(open columns) 1 µM A23187 for 10 min. The
amount of AA liberated was determined. **, p < 0.05. The results shown in B and C are representative
of three separate experiments.
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It has been shown that overexpression of sPLA2 enhances
A23187-induced AA liberation, and the augmented liberation is
suppressed by the cPLA2 inhibitor MAFP (47), suggesting
that cPLA2 is involved in the sPLA2-catalyzed
liberation of AA. The present study showed that 1 µM
A23187-induced AA liberation was enhanced by preincubation with 5 ng/ml
IL-1
(Fig. 7A), which increased sPLA2 (Fig.
4) in RGM1 cells having constitutive cPLA2 (Fig. 3). To
examine the contribution of cPLA2 and sPLA2 to
the IL-1
-enhanced AA liberation, the effects of MAFP and indoxam
were determined (Fig. 7D). Treatment of IL-1
-stimulated
cells with 10 µM MAFP markedly inhibited the IL-1
/A23187-induced AA liberation, whereas 50 nM indoxam
diminished the enhanced liberation to the level of AA liberation
induced by A23187 alone. We confirmed that MAFP but not indoxam almost completely suppressed AA liberation induced by A23187 alone (Fig.
7D). Incubation with 10 µM MAFP within 1 h had no effect on cell viability (data not shown). These findings
suggest that cPLA2 is involved in the hydrolytic action of
sPLA2 when IL-1
-primed RGM1 cells are stimulated with
Ca2+-mobilizing agonists.
Comparison of the Effects of TGF-
with Those of Dibutyryl
cAMP--
It has been suggested that the EGF receptor mediates the
activation of type I protein kinase A through a direct binding of the
Grb2 adaptor protein to the regulatory subunit of the kinase in
EGF-stimulated human mammary epithelial MCF-10A cells (reviewed in Ref.
48). To examine the role of protein kinase A in the sPLA2
release in TGF-
/IL-1
-stimulated RGM1 cells, effects of dibutyryl
cAMP, a protein kinase A activator, were compared with those of
TGF-
. As shown in Fig. 8A,
0.5 mM dibutyryl cAMP as well as 50 ng/ml TGF-
augmented
the increase in sPLA2 activity induced by 5 ng/ml IL-1
,
although it did not increase sPLA2 activity by itself. The
stimulatory effect of dibutyryl cAMP or TGF-
was almost completely
suppressed by pretreatment with 100 nM H-89, a protein
kinase A inhibitor (data not shown). These results suggest that protein
kinase A may be involved in the augmentation by TGF-
of
IL-1
-induced sPLA2 release. However, unlike TGF-
, the
combination of 0.5 mM dibutyryl cAMP and 5 ng/ml IL-1
did not sufficiently induce PGE2 generation or COX-2
expression as compared with those by the combination of TGF-
and
IL-1
(Fig. 8, B and C).

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Fig. 8.
Effects of dibutyryl cAMP and
TGF- on sPLA2 release,
PGE2 generation, and COX-2 expression in
IL-1 -stimulated cells. A,
cells were stimulated with (+) or without ( ) 0.5 mM
dibutyryl cAMP (DB), 5 ng/ml IL-1 (IL), or a
combination of IL-1 with dibutyryl cAMP or 50 ng/ml TGF-
(TGF) for 24 h. sPLA2 activity in the
medium was determined. B, the [3H]AA-labeled
cells were stimulated with (+) or without ( ) 0.5 mM
dibutyryl cAMP (DB) or a combination of 5 ng/ml IL-1
(IL) with 50 ng/ml TGF- (TGF) or dibutyryl
cAMP for 24 h. The amount of PGE2 generated was
determined. C, cells were stimulated as in B, and
then COX-2 protein was analyzed. A representative result of three
separate experiments is shown.
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DISCUSSION |
Several growth factors and proinflammatory cytokines including
TGF-
and IL-1
are implicated in the healing of gastric lesions. TGF-
and IL-1
have been shown to stimulate the generation of PG
by rat gastric epithelial RGM1 cells (18, 25) and human gastric
fibroblasts (27), respectively. Furthermore, a COX inhibitor prevents
TGF-
-induced proliferation and morphogenesis of RGM1 cells (25) and
IL-1
-induced expression of hepatocyte growth factor in human gastric
fibroblasts (27), which stimulates proliferation of rabbit gastric
epithelial cells (28, 29). These observations suggest that acceleration
by TGF-
and IL-1
of the repair of gastric injury is mediated
through, at least in part, generation of PG. We demonstrated here that
whereas TGF-
or IL-1
alone had little or no effect, in
combination they synergistically stimulated the generation of
PGE2 by RGM1 cells (Fig. 1), suggesting that the
cooperative action of TGF-
and IL-1
also is involved in PG
generation during the healing of gastric mucosal lesions. In the
present study, we further examined the contribution of
cPLA2 and sPLA2 to the generation of
PGE2 via a certain COX isoform(s), and the possible
interaction between the two PLA2s in the
TGF-
/IL-1
-stimulated RGM1 cells.
The onset of the synergistic increase in PGE2 induced by
both TGF-
and IL-1
was observed 18 h after the stimulation
(Fig. 1A). Under these conditions, both stimuli
synergistically increased levels of COX-1 and COX-2 proteins with a
significant change observed 3-6 h after the stimulation (Fig. 2,
A and B), indicating that the increases in the
two COXs preceded the synergistic effect on PGE2
generation. Although COX-1 is known as a constitutive enzyme (49), our
result showing an increase in COX-1 is consistent with the recent
finding that the expression of COX-1 as well as COX-2 is stimulated
during the healing of gastric ulcers in an animal model (17).
However, the synergistic PGE2 generation was suppressed by
the COX-2 inhibitor NS-398 but not the COX-1 inhibitor valeryl
salicylate (Fig. 2C), suggesting that PGE2
generation in the stimulated cells is mediated by COX-2 rather than
COX-1. The preference for COX-2 over COX-1 may be due to a difference in ability to utilize AA (50) and/or selective coupling of COX-2 with
PGE2 synthase (51). Collectively, our findings suggest that
an increase in COX-2 is required for the synergistic PGE2 generation induced by both TGF-
and IL-1
but is not critical for
the onset of the generation.
The generation of PG in the stomach plays an important role in the
healing of gastric lesions and in the maintenance of gastric mucosal
integrity (15, 16). Therefore, a number of studies have focused on the
regulation of COX isoforms, as shown in gastric epithelial cells (19,
25, 52, 53), gastric fibroblasts (27), and animal models of gastric
ulcers (17, 26, 54). However, little is known about the regulation of
PLA2 isozyme(s) in gastric cells, despite an important role
for PLA2 as a key enzyme providing AA, a precursor for PGs.
Recently, we reported that stimulation of RGM1 cells with TGF-
alone
induces an increase in cPLA2 but not sPLA2 in
parallel with a slight and gradual increment of PGE2 (18),
suggesting that cPLA2 is involved in the generation of
PGE2. In the present study, although IL-1
dose-dependently enhanced TGF-
-induced PGE2
generation (Fig. 1B), it did not augment increases in
cPLA2 protein and activity in response to TGF-
at the
same concentration ranges (Fig. 3, A and B). The
time-dependent increase in cPLA2 induced by
both TGF-
and IL-1
apparently preceded the onset of the
synergistic PGE2 generation (Figs. 1A and
3C). Furthermore, the augmentation by IL-1
of
TGF-
-stimulated PGE2 generation was not affected by the
cPLA2 inhibitors MAFP or AACOCF3 (Fig.
5B). These results suggest, therefore, that the preceding increase in cPLA2 is not involved in the synergistic
PGE2 generation induced by both IL-1
and TGF-
.
Among several types of sPLA2s identified in mammalian cells
and tissues (2), types IIA, V, and X sPLA2s are implicated in stimulus-induced PG generation in a variety of cells (5-12). However, the role of sPLA2 in PG generation by gastric
cells remains unknown. In the present study, we demonstrated that a
dose- and time-dependent increase in sPLA2
activity evoked by IL-1
in the presence of TGF-
was well
consistent with the augmentation by IL-1
of the TGF-
-induced
PGE2 generation (Figs. 1 and 4). Furthermore, suppression
of the augmented PGE2 generation by the specific
sPLA2 inhibitor indoxam was observed in parallel with
inhibition of the activity of sPLA2 released at the same
concentration ranges (Fig. 6). These findings suggest that an increase
in sPLA2 is involved in the synergistic PGE2
generation induced by both TGF-
and IL-1
. Considering the result
that mRNA for type IIA sPLA2 but not for types V or X
sPLA2 was increased upon the stimulation (Fig.
4C), the increased sPLA2 activity may be due to,
at least in part, induction of type IIA sPLA2 expression.
Although type IIA sPLA2 has been suggested to stimulate
liberation of AA through hydrolysis of membrane phospholipids (12, 55,
56), certain changes in membrane properties are required for an
increase in the susceptibility of membrane phospholipids to
sPLA2 (7, 14). At present, the mechanism(s) responsible for
the change in cellular susceptibility to sPLA2 in
TGF-
/IL-1
-stimulated RGM1 cells is unclear.
A number of studies have suggested the existence of interaction between
sPLA2 and cPLA2. As the mechanism responsible
for the interaction, exogenous sPLA2 has been shown to
activate p42 mitogen-activated protein kinase, inducing activation of
cPLA2 and liberation of AA, in human 1321N1 astrocytoma
cells (11) and rat mesangial cells (12). Conversely, stimulus-induced
cPLA2 activation accelerates sPLA2-catalyzed AA
liberation in P388D1 macrophages (8) and
sPLA2-overexpressing cells (47). Furthermore, cPLA2 inhibitors (MAFP and AACOCF3) suppress
expression of sPLA2 (9, 10, 36) and subsequent
sPLA2-mediated generation of PG (9, 10) induced by
cytokines or lipopolysaccharide in several types of cells. Thus, the
mechanisms underlying the cross-talk between the two PLA2s
vary with the types of cells and/or stimulation systems. In the present
study, we showed that A23187-induced AA liberation was potentiated in
IL-1
-stimulated RGM1 cells (Fig. 7A), in which
constitutive cPLA2 and inducible sPLA2
co-existed (Figs. 3 and 4). The augmentation was markedly prevented by
MAFP in addition to the sPLA2 inhibitor indoxam (Fig.
7D). These findings also suggest that cPLA2 may
contribute to sPLA2-catalyzed AA liberation when
IL-1
-primed RGM1 cells are activated by Ca2+-mobilizing
agonists. However, TGF-
alone did not stimulate sPLA2 expression despite an increase in cPLA2 (Figs. 3 and 4).
Furthermore, under conditions where TGF-
and IL-1
synergistically
stimulated PGE2 generation with preceding cPLA2
expression and concomitant sPLA2 release (Figs. 1, 3 and
4), indoxam suppressed the PGE2 generation, whereas
cPLA2 inhibitors had no effect on the increase in
sPLA2 or PGE2 (Figs. 5 and 6). Accordingly, our
findings suggest that cPLA2 is not involved in the
sPLA2 release and subsequent PGE2 generation in
the TGF-
/IL-1
-stimulated cells.
It has been shown that p11, known as annexin II light chain, directly
inhibits cPLA2 activity through binding to the enzyme (31),
and further that a decrease in constitutive p11 results in a further
increase in basal levels of free AA as well as A23187-induced AA
liberation (32), suggesting that the hydrolytic activity of
cPLA2 is ordinarily regulated by p11 in cells. We recently reported that priming of RGM1 cells with TGF-
alone for 3-24 h
inhibited A23187-induced, cPLA2-catalyzed AA liberation, and concurrently increased p11 but not annexin II heavy chain (18).
Suppression of the p11 expression by a tyrosine kinase inhibitor
restored the inhibited AA liberation in response to A23187 (18). The
present study showed that priming with both TGF-
and IL-1
also
resulted in inhibition of cPLA2-catalyzed AA liberation
despite an increase in cPLA2 protein, including its
phosphorylated form, and further that even in the presence of IL-1
,
TGF-
increased p11, which was co-immunoprecipitated with
cPLA2 (Fig. 7). Based on these observations, we speculate that p11 expression might be involved in the impairment of the hydrolytic activity of cPLA2, although further study is
needed to clarify whether p11 actually associates with
cPLA2 within the intracellular environment. It is possible,
therefore, that because of the inhibitory effect on cPLA2
in the TGF-
/IL-1
-stimulated cells, cPLA2 may be
unable to contribute to the sPLA2-mediated PGE2 generation.
IL-1
has been shown to stimulate the expression of sPLA2
(34, 35) and COX-2 (27, 57) as well as the generation of PG in a
variety of cells. In RGM1 cells stimulated with IL-1
alone, however,
PGE2 generation was not observed despite an increase in
sPLA2 (Figs. 1 and 4). This inability of IL-1
may be due
to the small increase in COX-2 in response to IL-1
alone (Fig.
2A). Indeed, under conditions where COX-2 was increased by
TGF-
, IL-1
was able to induce sPLA2-mediated
PGE2 generation (Figs. 1, 2, and 6). Although TGF-
increased COX-2, it augmented IL-1
-induced sPLA2 release
(Fig. 4). Furthermore, dibutyryl cAMP, a protein kinase A activator,
also exhibited similar stimulatory effect on the sPLA2
release (Fig. 8A). However, the combination of IL-1
and
dibutyryl cAMP did not sufficiently increase PGE2 or COX-2 as compared with those by the combination of IL-1
and TGF-
(Fig. 8, B and C). Taken together, these observations
suggest that the synergistic PGE2 generation induced by
IL-1
and TGF-
is mediated through cooperative action of
sPLA2 with COX-2.
In summary, based on the results obtained here, we suggest that TGF-
and IL-1
synergistically stimulate the expression of sPLA2 and COX-2, therefore inducing an increase in
PGE2 generation, which is independent of the expression of
cPLA2 and COX-1, in rat gastric epithelial RGM1 cells.
Furthermore, no contribution of cPLA2 to the
PGE2 generation may be ascribed in part to the inhibitory
effect of TGF-
on the hydrolytic activity of cPLA2, the
negative regulation of which presumably occurs in parallel with p11 expression.