Secretory Phospholipase A2 Mediates Cooperative Prostaglandin Generation by Growth Factor and Cytokine Independently of Preceding Cytosolic Phospholipase A2 Expression in Rat Gastric Epithelial Cells*

Satoshi AkibaDagger, Ryo Hatazawa, Kyoko Ono, Kazuyuki Kitatani, Misako Hayama, and Takashi Sato

From the Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan

Received for publication, November 9, 2000, and in revised form, March 9, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A2s (cPLA2 and sPLA2) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA2 and cyclooxygenase (COX)-2 markedly, PGE2 slightly, and type IIA sPLA2 and COX-1 not at all, whereas IL-1beta increased sPLA2 only. Both stimuli synergistically increased PGE2, sPLA2, and the two COXs but not cPLA2. The onset of the PGE2 generation paralleled the sPLA2 release but was apparently preceded by increases in cPLA2 and the two COXs. The increase in PGE2 was impaired by inhibitors for sPLA2 and COX-2 but not COX-1. cPLA2 inhibitors suppressed PGE2 generation by TGF-alpha alone but not augmentation of PGE2 generation or sPLA2 release by IL-1beta in combination with TGF-alpha . Furthermore, despite an increase in cPLA2 including its phosphorylated form (phosphoserine), A23187-induced arachidonic acid liberation was impaired in the TGF-alpha /IL-1beta -stimulated cells, in which p11, a putative cPLA2 inhibitory molecule, was also increased and co-immunoprecipitated with cPLA2. These results suggest that synergistic stimulation of sPLA2 and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE2. Presumably, the preceding cPLA2 expression is not involved in the PGE2 generation, because of impairment of its hydrolytic activity in the stimulated cells.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)-alpha and epidermal growth factor (EGF) (17). TGF-alpha (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-alpha , 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-alpha induces cell proliferation and morphogenesis through a COX-2-dependent mechanism in RGM1 cells (25). On the other hand, the expression of interleukin (IL)-1beta , 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-1beta 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-1beta 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-alpha and IL-1beta are implicated in the healing of gastric lesions through, at least in part, COX-2-dependent PGE2 generation. However, the role of TGF-alpha and IL-1beta in the activation of the PLA2 isozyme(s) contributing to the generation during the healing remains to be elucidated.

Recently, we demonstrated that TGF-alpha 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-alpha 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-alpha -stimulated PGE2 generation occurs under the control of inducible p11, which negatively regulates the hydrolytic activity of cPLA2. Considering the possibility that IL-1beta is also involved in the healing of gastric lesions through generation of PG, TGF-alpha may cooperate with IL-1beta to stimulate efficiently the generation in gastric epithelial cells. This appears likely, because a combination of EGF and IL-1beta has been shown to induce synergistically COX-2 expression and PGE2 generation in human gingival fibroblasts (33). IL-1beta 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-alpha and IL-1beta . 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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Recombinant human TGF-alpha was obtained from PeproTech (London, UK). IL-1beta 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-alpha , IL-1beta , 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-alpha , IL-1beta , 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-alpha and IL-1beta 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-alpha , IL-1beta , 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 beta -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-alpha , IL-1beta , 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-alpha , IL-1beta , 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-alpha , IL-1beta , 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-alpha and IL-1beta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Increase in PGE2 Preceded by Expression of COXs in Response to TGF-alpha and IL-1beta -- As shown in Fig. 1A, stimulation of RGM1 cells with 50 ng/ml TGF-alpha for 24 h slightly increased PGE2 in a time-dependent manner, whereas 5 ng/ml IL-1beta had no effect on the basal level of PGE2. However, a combination of 50 ng/ml TGF-alpha and 5 ng/ml IL-1beta 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-1beta or TGF-alpha (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-alpha but not 5 or 10 ng/ml IL-1beta 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-alpha or 5 ng/ml IL-1beta under our experimental conditions (Fig. 2A). However, the combination of TGF-alpha and IL-1beta 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-alpha and 5 ng/ml IL-1beta 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-alpha /IL-1beta -induced expression of COX-2 or COX-1 (result not shown).


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Fig. 1.   Synergistic PGE2 generation induced by TGF-alpha and IL-1beta . A, the [3H]AA-labeled cells were stimulated with (circles) or without (squares) 5 ng/ml IL-1beta for the indicated periods in the presence (closed symbols) or absence (open symbols) of 50 ng/ml TGF-alpha . B and C, the labeled cells were stimulated with (closed symbols) or without (open symbols) 50 ng/ml TGF-alpha (B) or 5 ng/ml IL-1beta (C) for 24 h in the presence of various concentrations of IL-1beta (B) or TGF-alpha (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-alpha /IL-1beta -stimulated cells. A, cells were stimulated with or without TGF-alpha and/or IL-1beta at the indicated concentrations for 24 h. B, cells were stimulated with 50 ng/ml TGF-alpha and 5 ng/ml IL-1beta 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-alpha and 5 ng/ml IL-1beta for 24 h. The amount of PGE2 generated was measured. The results shown in A and B are representative of three separate experiments.

cPLA2 Expression and Delayed sPLA2 Release in Response to TGF-alpha and IL-1beta -- To identify the PLA2 isozyme(s) contributing to the synergistic effect of TGF-alpha and IL-1beta on PGE2 generation, the change in cPLA2 upon stimulation was examined (Fig. 3). Stimulation with 50 ng/ml TGF-alpha for 24 h increased cPLA2 protein and activity, whereas 10 ng/ml IL-1beta did not affect the basal levels (Fig. 3, A and B). In contrast to the stimulatory effect of IL-1beta on TGF-alpha -induced PGE2 generation (Fig. 1B), 50 ng/ml TGF-alpha -increased cPLA2 protein or activity was not augmented in the presence of 2-10 ng/ml IL-1beta (Fig. 3, A and B). Furthermore, the combination of 50 ng/ml TGF-alpha and 5 ng/ml IL-1beta 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-alpha and IL-1beta . A, cells were stimulated with or without TGF-alpha and/or IL-1beta 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-alpha for 24 h in the presence of various concentrations of IL-1beta . cPLA2 activity in lysate of the cells was determined. C, cells were stimulated with 50 ng/ml TGF-alpha and 5 ng/ml IL-1beta for the indicated periods, and then cPLA2 protein was analyzed. The results shown in A and C are representative of three separate experiments.

We further determined the change in sPLA2 activity in the extracellular medium. The result shown in Fig. 4A indicates that IL-1beta (2-10 ng/ml) dose-dependently increased sPLA2 activity, which was enhanced in the presence of 50 ng/ml TGF-alpha , although TGF-alpha 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-1beta alone or in combination with 50 ng/ml TGF-alpha , 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-alpha and IL-1beta (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-1beta for 24 h induced type IIA sPLA2 mRNA expression, whereas 50 ng/ml TGF-alpha did not affect the mRNA, which was undetectable in unstimulated cells. Consistent with the change in sPLA2 activity, TGF-alpha augmented the mRNA expression of type IIA sPLA2 induced by IL-1beta , 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-alpha and IL-1beta . A, cells were stimulated with (closed symbols) or without (open symbols) 50 ng/ml TGF-alpha for 24 h in the presence of various concentrations of IL-1beta . B, cells were stimulated with (circles) or without (squares) 5 ng/ml IL-1beta for the indicated periods in the presence (closed symbols) or absence (open symbols) of 50 ng/ml TGF-alpha . A and B, sPLA2 activity in the medium of the cells was determined. C, cells were stimulated with or without (-) 50 ng/ml TGF-alpha , 5 ng/ml IL-1beta , 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.

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-alpha and IL-1beta 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-alpha and/or 5 ng/ml IL-1beta for 24 h (data not shown).

Effects of PLA2 Inhibitors on TGF-alpha /IL-1beta -stimulated PGE2 Generation-- To examine the involvement of cPLA2 in the synergistic PGE2 generation by TGF-alpha and IL-1beta , 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-alpha and 5 ng/ml IL-1beta , 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-alpha alone was also inhibited, whereas synergistic PGE2 generation induced by both TGF-alpha and IL-1beta was not affected (Fig. 5B), indicating that the cPLA2 inhibitors had no effect on the augmentation by IL-1beta of TGF-alpha -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-alpha and IL-1beta . 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-alpha /IL-1beta -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-alpha and 5 ng/ml IL-1beta 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-alpha alone or in combination with 5 ng/ml IL-1beta for 24 h. The amount of PGE2 generated was measured.

Fig. 6 illustrates the effects of indoxam, a specific sPLA2 inhibitor (45, 46), on increases in sPLA2 activity and PGE2 in response to TGF-alpha and IL-1beta . When RGM1 cells were stimulated with both 50 ng/ml TGF-alpha and 5 ng/ml IL-1beta 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-alpha /IL-1beta -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-alpha /IL-1beta -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-alpha /IL-1beta -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-alpha and 5 ng/ml IL-1beta 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-alpha and 5 ng/ml IL-1beta 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.

Effects of TGF-alpha and IL-1beta 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-alpha for 3-24 h inhibited A23187-induced AA liberation in parallel with p11 expression (18). In the present study, we further examined whether TGF-alpha exhibits similar effects in the presence of IL-1beta . The results shown in Fig. 7A indicate that when RGM1 cells, which had been stimulated with 50 ng/ml TGF-alpha for 24 h in the presence or absence of 5 ng/ml IL-1beta , were washed and further stimulated with 1 µM A23187 for 10 min, TGF-alpha diminished A23187-induced AA liberation as compared with TGF-alpha -unstimulated cells even in the presence of IL-1beta . Furthermore, 50 ng/ml TGF-alpha induced an increase in p11 irrespective of the presence of 5 ng/ml IL-1beta (Fig. 7B). However, IL-1beta 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-alpha alone or in combination with 5 ng/ml IL-1beta . 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-alpha on A23187-induced AA liberation occurs in parallel with p11 expression but independently of IL-1beta -elicited signaling.


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Fig. 7.   Effects of TGF-alpha and IL-1beta 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-alpha , 5 ng/ml IL-1beta , 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-alpha , 5 ng/ml IL-1beta , or both for 24 h, and then p11 protein was analyzed. C, cells were stimulated with or without (Control) 50 ng/ml TGF-alpha alone or in combination with 5 ng/ml IL-1beta 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-1beta 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.

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-1beta (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-1beta -enhanced AA liberation, the effects of MAFP and indoxam were determined (Fig. 7D). Treatment of IL-1beta -stimulated cells with 10 µM MAFP markedly inhibited the IL-1beta /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-1beta -primed RGM1 cells are stimulated with Ca2+-mobilizing agonists.

Comparison of the Effects of TGF-alpha 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-alpha /IL-1beta -stimulated RGM1 cells, effects of dibutyryl cAMP, a protein kinase A activator, were compared with those of TGF-alpha . As shown in Fig. 8A, 0.5 mM dibutyryl cAMP as well as 50 ng/ml TGF-alpha augmented the increase in sPLA2 activity induced by 5 ng/ml IL-1beta , although it did not increase sPLA2 activity by itself. The stimulatory effect of dibutyryl cAMP or TGF-alpha 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-alpha of IL-1beta -induced sPLA2 release. However, unlike TGF-alpha , the combination of 0.5 mM dibutyryl cAMP and 5 ng/ml IL-1beta did not sufficiently induce PGE2 generation or COX-2 expression as compared with those by the combination of TGF-alpha and IL-1beta (Fig. 8, B and C).


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Fig. 8.   Effects of dibutyryl cAMP and TGF-alpha on sPLA2 release, PGE2 generation, and COX-2 expression in IL-1beta -stimulated cells. A, cells were stimulated with (+) or without (-) 0.5 mM dibutyryl cAMP (DB), 5 ng/ml IL-1beta (IL), or a combination of IL-1beta with dibutyryl cAMP or 50 ng/ml TGF-alpha (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-1beta (IL) with 50 ng/ml TGF-alpha (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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several growth factors and proinflammatory cytokines including TGF-alpha and IL-1beta are implicated in the healing of gastric lesions. TGF-alpha and IL-1beta 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-alpha -induced proliferation and morphogenesis of RGM1 cells (25) and IL-1beta -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-alpha and IL-1beta of the repair of gastric injury is mediated through, at least in part, generation of PG. We demonstrated here that whereas TGF-alpha or IL-1beta 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-alpha and IL-1beta 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-alpha /IL-1beta -stimulated RGM1 cells.

The onset of the synergistic increase in PGE2 induced by both TGF-alpha and IL-1beta 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-alpha and IL-1beta 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-alpha 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-1beta dose-dependently enhanced TGF-alpha -induced PGE2 generation (Fig. 1B), it did not augment increases in cPLA2 protein and activity in response to TGF-alpha at the same concentration ranges (Fig. 3, A and B). The time-dependent increase in cPLA2 induced by both TGF-alpha and IL-1beta apparently preceded the onset of the synergistic PGE2 generation (Figs. 1A and 3C). Furthermore, the augmentation by IL-1beta of TGF-alpha -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-1beta and TGF-alpha .

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-1beta in the presence of TGF-alpha was well consistent with the augmentation by IL-1beta of the TGF-alpha -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-alpha and IL-1beta . 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-alpha /IL-1beta -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-1beta -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-1beta -primed RGM1 cells are activated by Ca2+-mobilizing agonists. However, TGF-alpha alone did not stimulate sPLA2 expression despite an increase in cPLA2 (Figs. 3 and 4). Furthermore, under conditions where TGF-alpha and IL-1beta 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-alpha /IL-1beta -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-alpha 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-alpha and IL-1beta 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-1beta , TGF-alpha 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-alpha /IL-1beta -stimulated cells, cPLA2 may be unable to contribute to the sPLA2-mediated PGE2 generation.

IL-1beta 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-1beta alone, however, PGE2 generation was not observed despite an increase in sPLA2 (Figs. 1 and 4). This inability of IL-1beta may be due to the small increase in COX-2 in response to IL-1beta alone (Fig. 2A). Indeed, under conditions where COX-2 was increased by TGF-alpha , IL-1beta was able to induce sPLA2-mediated PGE2 generation (Figs. 1, 2, and 6). Although TGF-alpha increased COX-2, it augmented IL-1beta -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-1beta and dibutyryl cAMP did not sufficiently increase PGE2 or COX-2 as compared with those by the combination of IL-1beta and TGF-alpha (Fig. 8, B and C). Taken together, these observations suggest that the synergistic PGE2 generation induced by IL-1beta and TGF-alpha is mediated through cooperative action of sPLA2 with COX-2.

In summary, based on the results obtained here, we suggest that TGF-alpha and IL-1beta 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-alpha on the hydrolytic activity of cPLA2, the negative regulation of which presumably occurs in parallel with p11 expression.

    ACKNOWLEDGEMENT

We thank Dr. K. Hanasaki (Shionogi Research Laboratories, Shionogi & Co., Ltd.) for kindly providing indoxam, an sPLA2 inhibitor.

    FOOTNOTES

* This work was supported by a grant-in-aid for scientific research and the Frontier Research Program of the Ministry of Education, Science, Sports and Culture of Japan.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.

Dagger To whom correspondence should be addressed: Dept. of Pathological Biochemistry, Kyoto Pharmaceutical University Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Fax: 81-75-595-4759; E-mail: akiba@mb.kyoto-phu.ac.jp.

Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M010201200

    ABBREVIATIONS

The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; sPLA2, secretory PLA2; AA, arachidonic acid; COX, cyclooxygenase; EGF, epidermal growth factor; IL, interleukin; PG, prostaglandin; TGF, transforming growth factor; MAFP, methylarachidonyl fluorophosphonate; AACOCF3, arachidonyltrifluoromethyl ketone; DMEM, Dulbecco's modified Eagle's medium.

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ABSTRACT
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RESULTS
DISCUSSION
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