Activation of mitogen-activated protein kinase by fumonisin B1 stimulates cPLA2 phosphorylation, the arachidonic acid cascade and cAMP production
Eric Pinelli,
Nathalie Poux,
Liliane Garren1,
Bernard Pipy2,
Marcel Castegnaro1,
David J. Miller3 and
Annie Pfohl-Leszkowicz4
ENSAT, Laboratoire de Toxicologie et Sécurité Alimentaire, 1 avenue Agrobiopole, BP 107, 31326 Auzeville-Tolosane,
1 Unit of GeneEnvironment Interactions, IARC, 150 cours Albert Thomas, 69008 Lyon,
2 CHU Rangueil, Laboratoire d'Etude du Macrophage, avenue Jean Poulhies, 31400 Toulouse, France and
3 Department of Chemistry, 228 Steacie Building, Carleton University, Ottawa, Ontario K1S 5B6, Canada
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Abstract
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Activation of mitogen-activated protein kinase (MAPK) results in pleiotropic effects such as modulation of the transcription and activation of enzymes involved in signal transduction. One such enzyme is the cytoplasmic phospholipase A2 (cPLA2), which releases arachidonic acid (AA). AA is the precursor of prostaglandins and leukotrienes, two inflammatory mediators, which regulate gene expression and protein kinase (PK) activity. Fumonisin B1 (FB1) was shown to increase PKC translocation and stimulate MAPK. We have investigated the effect of FB1 on the AA cascade in a human epithelial cell line and the signal transduction pathway regulating PLA2 activation. We observed that FB1 stimulated cPLA2 activity and increased AA release by a mechanism independent of PKC activation and that the activation of cPLA2 is a two-step process: the first is phosphorylation of cPLA2 by MAPK; the second is a consequence of the increase in sphingosine inside and outside the cells after 2 h, which is known to induce a rise in intracellular free calcium. Overall, this suggests that the effect of FB1 on cells is partially dependent on the action of FB1 on the enzymes involved in the cell cycle, such as MAPK and PKA, and on bioactive fatty acids, such as the prostaglandins and leukotrienes, and also on disruption of sphingolipid metabolism. In addition, we have observed down-regulation of cPLA2 activity and AA metabolism by a mechanism involving prostaglandin production, cAMP synthesis and PKA activation.
Abbreviations: AA, arachidonic acid; cAMP, cyclic adenosine 3',5'-monophosphate; cPLA2, cytoplasmic phospholipase A2; EMEM, Eagle's minimal essential medium; FB1, fumonisin B1; H89, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide; HETE, hydroxyeicosatetraenoic acid; LT, leukotriene; MAPK, mitogen-activated protein kinase; OPA, o-phthaldialdehyde; PBS, phosphate-buffered saline; PG, prostaglandin; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; Sa, sphinganine; So, sphingosine.
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Introduction
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Fumonisins are a group of naturally occurring metabolites produced by several fungal species, in particular Fusarium moniliforme, which contaminates corn (1,2). Fumonisins induce several animal toxicoses, including equine leukoencephalomalacia (3) and porcine pulmonary oedema (4,5). Gelderblom et al. (6) have demonstrated that FB1 induces hepatocarcinomas in rats. Exposure to fumonisin B1 (FB1) through dietary consumption of Fusarium-contaminated corn products has been ecologically associated with increased rates of human oesophageal cancer (1)
A number of studies have examined the effects of FB1 on the growth of cells, with the aim of determining the cellular basis for the diseases associated with FB1 (for a review see ref. 7). FB1 stimulates DNA synthesis in cultures of Swiss 3T3 fibroblasts (8), but inhibits the proliferation of renal epithelial cells, keratinocytes, hepatocytes and hepatoma cells (9,10) and induces apoptosis (10). Gelderblom et al. have suggested that the inhibitory effect on cell proliferation is a key event in fumonisin-induced cancer promotion (11). Several studies have indicated that FB1, which is a structural analogue of sphingosine (So) and sphinganine (Sa), inhibits sphingolipid biosynthesis by interfering with the sphinganine N-acyltransferase pathway (8,12). More recently, it has been suggested that FB1 induces mitogen-activated protein kinase (MAPK) activity through a mechanism independent of the accumulation of So or Sa (13). Activation of MAPK induced some effects, including transcription, modulation and activation of enzymes involved in cell signal transduction (14). One such enzyme is cytoplasmic phospholipase A2 (cPLA2), which releases arachidonic acid (AA) from the Sn-2 position of phospholipids (15). This activation of cPLA2 by MAPK is caused by phosphorylation of the cPLA2 on Ser505 (15). AA is the rate-limiting precursor for synthesis of prostaglandins (PGs) and leukotrienes (LTs), two classes of potent inflammatory mediator (16) which are also involved in cell regulation. Metabolites of AA regulate protein activation and gene expression (1720). PGE2 increases cAMP production and activates cAMP-dependent protein kinase (PKA) in numerous cell types (18,19). In turn, cAMP and PKA inhibit MAPK activation (21,22) and PKA regulates cPLA2 activity (23).
In order to elucidate how FB1 affects cell regulation, we have investigated the effects of FB1 on the AA cascade in human bronchial epithelial cells. First, we analysed the release of AA, PGs and LTs, reflecting cPLA2, cyclooxygenase and lipoxygenase activity, respectively. Second, the mechanism involved in cPLA2 activation was studied by use of specific inhibitors: calphostin as an inhibitor of PKC (24), N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H89) as an inhibitor of PKA (25) and PD098059 as an inhibitor of the MAPK cascade (26). We have also examined the mechanism of MAPK activation of cPLA2 in relation to sphingolipid turnover and cAMP formation by FB1.
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Materials and methods
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Materials
Radiolabelled PG and LT standards and the Enhanced Chemiluminescence (ECL) detection kit for western blotting were purchased from Amersham Life Science (Les Ulis, France). C-20 Sa (C20) was a generous gift from Dr A.H. Merrill Jr (Emory University School of Medicine, Atlanta, GA). Sa, So, mercaptoethanol, o-phthaldialdehyde (OPA), boric acid, indomethacin, PD098059, calphostin, forskolin, 1-isobutyl-3-methylxanthine, the serine-phosphorylated antibody, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, mercaptoethanol, Nonidet-P40 and sodium orthovanadate were purchased from Sigma (St Louis, MO). Labelled [5,6,8,9,11,12,14,15-3H]arachidonic acid (166 Ci/mmol) was obtained from CEA France. H89 was obtained from France Biochem. (Meudon, France). The culture media, phosphate-buffered saline (PBS), Eagle's minimum essential medium (EMEM) and foetal bovine serum were obtained from Gibco (Cergy Pontoise, France). cAMP analysis reagents were purchased from Immunotech (Marseille, France). The MAP kinase antibody was purchased from Promega (Charbonnière, France), cPLA2 antibody and protein Aagarose were purchased from Santa Cruz Biotechnology (Le Perray en Yvelines, France).
Cell culture conditions
Human bronchial epithelial cells (WI26 VA) obtained from ECACC (Salisbury, Wiltshire, UK) were cultivated in 200 ml flasks in EMEM containing 44 mM NaHCO3, 10% foetal bovine serum, 2% vitamins and 2% non-essential amino acids for 48 h at 37°C under 5% CO2. After trypsin digestion, the cells were resuspended in this medium to obtain 1x106 cells/ml and a volume of 1 ml was distributed in 6-well Falcon plates (Grenoble, France) for analysis of AA metabolites and assays for cAMP and distributed in 200 ml Falcon flasks for western blot analysis, immunoprecipitation and shingolipid turnover assay.
Analysis of released cyclooxygenase and lipooxygenase metabolites
The cells were incubated for 24 h with labelled AA (2 µCi/well) under the same conditions as described above in a final volume of 2 ml. The labelled cells were then washed twice with EMEM. The cells were treated for 2 h with various concentrations of FB1 (0.1, 1 or 10 µM). In some cases, cells were incubated for 30 min in the presence of metabolic inhibitors: the PKC inhibitor calphostin (0.5 µM), the MAPK inhibitor PD098059 (10 µM) or the PKA inhibitor H89 (0.1 µM) before treatment for 1 h with FB1 (10 µM). After incubation, the culture medium (2 ml) was collected and the various labelled AA oxygenation products released by cells were extracted and evaluated by thin layer radiochromatography as previously described (27). Supernatants were acidified to pH 5 with HCl and extracted by chromatography on SEP-PAK C18 columns (Fisher, Elancourt, France). AA metabolites were eluted with 5 ml methanol. After evaporation to dryness under nitrogen, the residues were dissolved in 80 µl methanol and applied to previously activated (1 h at 100°C) thin layer silica gel LK-6-DF (Merck, Darmstadt, Germany). AA metabolites were separated by chromatography in ethylacetate:water:isooctane:acetic acid (110:100:50:20 v/v/v/v). The radioactive metabolites were identified with a Berthold TLC scanner against the corresponding standards. Results were expressed as d.p.m. of [3H]AA.
Western blot and immunoprecipitation analysis
Bronchial epithelial cells (2x106/flask) were cultivated for 48 h. Confluent cells were then washed once with incubation buffer (EMEM with 1% bovine serum albumin) and incubated at 37°C with FB1 (10 µM) for different times in the absence or presence of PD098059 (10 µM) added to the cell culture medium 30 min before FB1 stimulation. After removal of the culture medium, the cells were washed twice with cold PBS and lysed in 10 mM TrisHCl, pH 7.4, 200 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 0.5 mM dithio-threitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and 10 µg/ml aprotinin at 4°C. Cellular debris was then precipitated at 25 000 g for 10 min. The supernatant containing the cytosolic protein was collected. For western blot analysis, an equal amount of lysate (50 µg of total protein) was boiled in SDS buffer, resolved by SDSPAGE and immunoblotted with anti-activated MAPK antibody. Bound proteins were detected by enhanced chemiluminescence. For immunoprecipitation assay, cell lysates were incubated for 1 h with 1 µg of monoclonal cPLA2 antibody and then mixed with 20 µl of mouse IgGprotein Aagarose beads on a rotating plate for 12 h at 4°C. Immunoprecipitates were collected by centrifugation for 5 min at 2500 r.p.m. and the pellets washed three times with cold PBS and resuspended in 20 µl electrophoresis sample buffer, boiled and resolved by SDSPAGE. The proteins were immunoblotted with an anti-phosphoserine antibody. Bound proteins were detected by enhanced chemiluminescence. The quantification was performed with a Bio-Rad scanner and Molecular Analyst Software (Bio-Rad Laboratories, Richmond, CA). Histogram values are the means of three separate analyses.
Determination of So and Sa
Bronchial epithelial cells (2x106/flask) were cultivated for 48 h as above. Confluent cells were then washed once with incubation buffer (EMEM with 1% bovine serum albumin) and incubated at 37°C with FB1 (10 µM) for different periods of time (5, 15 and 30 min and 1, 2, 4, 8 and 24 h). So and Sa analyses were performed as previously described by Castegnaro et al. (28). Briefly, after incubation, the cell culture medium was separated from the cells and both were collected for sphingolipid extraction. Fifty microlitres of potassium hydroxide solution (1 M) and C20 Sa as internal standard were added to 2 ml of culture medium or resuspended cells in water. Sphingolipids were extracted by addition of 2 ml ethylacetate and stirred on a vortex for 2 min. After extraction the solution was centrifuged for 5 min at 700 g and the ethylacetate (upper) phase was collected. A second ethylacetate extraction was performed and the two ethylacetate extracts were pooled. The ethylacetate was then evaporated to dryness in a heating block at 60°C under a stream of nitrogen. Derivatization of the samples was performed for at least 30 min after dissolution of each dried sample in 275 µl of a 0.07 M K2HPO4/methanol (1/9) solution and addition of 25 µl of OPA mixture (12.5 mg of OPA diluted in 250 µl of ethanol, 12.5 µl of mercaptoethanol and a 3% boric acid solution adjusted to pH 10.5 with KOH to obtain a final volume of 12.5 ml). Separation and quantification of sphingolipids were done by HPLC on a 250x4.6 mm, 5 µm Kromasil C18 column, with a four-step phosphate buffermethanol gradient (at T = 0, 100% 0.07 M K2HPO4/methanol (1/9) (A); at T = 30 min, 70% A and 30% methanol (B); at T = 35 min, 0% A and 100% B). Fluorescence was monitored at 340 nm excitation, 455 nm emission and retention times for So, Sa and C-20 Sa were ~11, 14 and 32 min, respectively.
cAMP analysis
Bronchial epithelial cells (1x106/well) were seeded into 6-well culture plates. After 24 h incubation, confluent cells were washed once with incubation buffer (EMEM, 1% bovine serum albumin, 0.5 mM 1-isobutyl-3-methylxanthine) and incubated at 37°C with FB1 for 20 min. Some cells were also incubated in the presence of indomethacin (0.1 µM), a cyclooxygenase inhibitor, or the MAPK inhibitor PD098059 (10 µM) 30 min before FB1 stimulation. Forskolin (20 µM) was used as a positive control. To stop the reaction, the medium was discarded and the cells were lysed with 500 µl of 65% cold ethanol (20°C). After an incubation of 5 min at 0°C, the lysate was removed, lyophilized and finally dissolved in a radioimmunoassay system from Immunotech (Marseille, France). Briefly, each sample or standard was incubated with 125I-labeled cAMP (111 kBq/vial) in antibody-coated tubes. After incubation for 18 h at 4°C the contents of the test tube were aspirated and bound radioactivity was counted in a gamma counter. A standard curve was prepared with six standards ranging from 0 to 50 nM. The values were obtained from this curve by interpolation and expressed as nanomolar.
Statistical analysis
Results are expressed as means ± SD. Data were analysed by one-way analysis of variance, and multiple comparisons of each treatment were calculated applying the Tukey method (29).
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Results
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Effect of FB1 on release of AA metabolites
Figure 1
shows the ability of FB1 to induce activation of AA metabolism. The amounts of PGs [6-K-PGF1
, PGF2
, thromboxane B2, PGE2, PGD2 and PGA2] and LTs [LTC4-D4, LTB4 and hydroxyeicosatetraenoic acids (HETEs)] produced by bronchial epithelial cells increased proportionally to the FB1 concentration. At 0.1 µM FB1, no effect on the AA cascade was observed. At 1 µM FB1, formation of LTC4-D4, LTB4 and HETEs was enhanced, indicating a significant increase in the lipoxygenase pathway (P < 0.05). Prostacyclin (6-K-PGF1
) synthesis was also significantly stimulated (P < 0.05). At the highest concentration (10 µM), production of all the metabolites of the AA cascade was stimulated (P < 0.01).
Roles of PKC, MAPK and PKA in activation of PGs and LTs release induced by FB1
As PLA2 is regulated directly by phosphorylation via PKC and MAPK, we tested their implications using specific inhibitors. In addition, since cPLA2 activity is indirectly affected by PKA activity, we also tested the role of this kinase with a specific inhibitor. Figure 2
shows the percentage variation in release of AA metabolites when cells were pretreated with PKC, MAPK and PKA inhibitors before FB1 activation, in comparison with treatment with FB1 alone. When cells where pretreated with calphostin, which is a PKC inhibitor, no difference in PLA2 stimulation by FB1 was observed. Pretreatment of cells with the MAPK cascade inhibitor PD098059 prevented the stimulatory effect of FB1; a significant decrease in all the metabolites of the AA cascade was measured (P < 0.01). In contrast, pretreatment of cells with the PKA inhibitor H89 synergistically enhanced activation by FB1 of the AA cascade. All the metabolites of the AA cascade were significantly increased.
Effect of FB1 on MAPK activation
By use of an antibody that detects only the phosphorylated forms of MAPK subtypes (ERK1 and ERK2) we have investigated the phosphorylation of both kinases after stimulation of the cells by FB1 in the presence or absence of PD089059. As depicted in Figure 3
, FB1 (10 µM) time dependently increased ERK1 and ERK2 phosphorylation in these cells. Phosphorylation started 5 min after FB1 stimulation, was maximal at 1 h and decreased slowly until 24 h. As shown in Figure 3
, preincubation of the cells for 30 min in the presence of the MAPK cascade inhibitor PD098059 (10 µM) reduced basal and FB1-induced ERK1 and ERK2 phosphorylation.

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Fig. 3. Time-dependent FB1-induced ERK1 and ERK2 phosphorylation in human epithelial lung cells. Confluent cells were cultivated with 10 µM FB1 for different periods of time (5, 15 and 30 min and 1, 2, 4, 8 and 24 h) in the absence or presence of 10 µM PD098059. Phosphorylation of ERKs was determined by western blot analysis using an anti-phosphorylated ERK antibody. For each experiment, one representative western blot of three separate independent experiments is presented.
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Time-related accumulation of sphingolipids in FB1-treated cells
To correlate the effects of FB1 on kinase activity and sphingolipid metabolism, we have investigated the effect of FB1 on sphingolipid turnover over a short period. Figure 4A
shows the effect of FB1 (10 µM) on So and Sa accumulation in human epithelial cell culture as a function of time of exposure. The results demonstrate that the So concentration in the cells remained constant for 1 h, increased significantly (P < 0.01) at 2 h and decreased slowly until 24 h. In contrast, the Sa concentration increased significantly at 2 h after FB1 treatment and was time dependent until 24 h. Figure 4B
shows the time effect of FB1 (10 µM) on So and Sa production by cells and their transfer into the extracellular medium. So production increased significantly (P < 0.01) after 2 h of FB1 treatment and remained constant until 24 h. In the same way as in the cells, extracellular Sa increased significantly after 2 h of FB1 treatment and the effect increased until 24 h. These results demonstrate that more than 1 h was necessary for FB1 to efficiently inhibit ceramide synthase and therefore to lead to an increase in Sa and, to a lesser extent, in So.
Time effect of FB1 on cPLA2 Ser phosphorylation
The activation of cPLA2 by MAP kinase is caused by phosphorylation of cPLA2 on Ser505. Analysis of cPLA2 phosphorylation was performed on cell lysates by immunoprecipitation, as described in Materials and methods. As shown in Figure 5A
, FB1 induced serine phosphorylation of cPLA2 time dependently. Figure 5B
shows the quantitative densitometric analysis of cPLA2 phosphorylation in the absence of PD098052. The FB1 effect appeared at 5 min, was increased 3-fold at 2 h and reached a maximal effect (11-fold) at 4 h. cPLA2 phosphorylation induced by FB1 was completely inhibited by the MAPK inhibitor PD098052 (Figure 5A
).

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Fig. 5. Time effect of FB1 on cPLA2 serine phosphorylation. Analysis of cPLA2 phosphorylation was performed on cell lysate by immunoprecipitation as described in Materials and methods. (A) cPLA2 phosphorylation induced by FB1 in the presence or absence of the MAPK inhibitor PD098052. (B) Quantitative densitometric analysis of cPLA2 phosphorylation (arbitrary units). Histograms represent the means ± SD of two separate experiments.
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Effect of FB1 on cAMP production and role of PGs
Figure 6
shows that FB1 increased cAMP production 3-fold. This increase is less than that observed in the presence of forskolin, which is a well-known adenylate cyclase activator and was used as a positive control. The elevation in cAMP induced by FB1 was completely abolished when cells were treated with indomethacin, an inhibitor of cyclooxygenase (30). Similarly, the MAPK cascade inhibitor PD089059, which had no effect alone on basal cAMP production, markedly inhibited FB1 stimulation (P < 0.01).

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Fig. 6. Effect of FB1 on cAMP production and role of cyclooxygenase. Cells were incubated in the presence or absence of FB1 (10 µM) for 15 min, in the absence or presence of 1 µM indomethacin (INDO) or 10 µM PD098059 (PD) 30 min before stimulation of cells by FB1. As a positive control, cells were treated for 10 min with Forskolin (20 µM). Each bar represents the mean ± SD of three determinations. **Value significantly different from control: P < 0.01.
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Discussion
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The synthesis of lipid mediators of cell regulation, such as PGs and LTs, results from stimulation of cPLA2, which cleaves membrane phospholipids to give rise to free acids from which second messengers are generated. More specifically, cPLA2 generates the precursor for the eicosanoids (PGs and LTs) when the cleaved fatty acid is AA (16). In this study we have demonstrated that FB1 causes an increase in AA metabolism in a dose-dependent manner, as we observed an enhancement of production of PGs and LTs. This indicates that cPLA2 activity was increased. This result is particularly important as these metabolites are involved in inflammation, immunomodulation, regulation of transcription factors, Ca2+ mobilization and protein kinase activation (16). Thus modulation of PG and LT production by FB1 could explain some of the deleterious effects of this toxin. cPLA2 activation by FB1 is independent of PKC activation, because the PKC inhibitor calphostin was without effect on cPLA2 activity (Figure 2
), whereas it is strongly dependent on MAPK activation, because the MAPK inhibitor PD098059 completely blocked both formation of the metabolites from the AA cascade (Figure 2
) and cPLA2 phosphorylation on Ser505 (Figure 5
). In addition, we observed rapid phosphorylation of the MAPK enzyme subtypes ERK1 and ERK2 5 min after treatment of cells with FB1, with a maximum effect at 1 h (Figure 3
). Previously, Wattenberg et al. (13) observed activation of MAPK when cells were treated with 25 µM FB1 and speculated that this activation was independent of the inhibitory effect of FB1 on sphingolipid biosynthesis. In this study we confirm that FB1 activates MAPK, even at 10 µM. Moreover, we demonstrate that this induction is independent of the inhibition by FB1 of sphingolipid turnover, as FB1 induced MAPK phosphorylation as soon as 5 min after treatment, while 2 h were necessary for a significant increase in Sa and So. We also observed that MAPK activation decreased after 4 h, while Sa and the Sa:So ratio increased until 24 h and the So level remained nearly constant. These results highlight a new signal transduction pathway for FB1 on cell activation, as explained below.
Previously, it was shown that full activation of cPLA2 requires both phosphorylation of cPLA2 on Ser505 by serine/threonine protein kinase MAPK and a rise in intracellular calcium concentration (15). For example, phorbol esters activate MAPK (15) and slightly induce AA release in different cell types (15,33,34) but do not modify intracellular calcium (31,32). For full activation of cPLA2 cells must be treated with phorbol esters in combination with molecules which increase intracellular calcium concentration (15,31). In view of these results, our findings suggest that two steps are also involved in the mechanism of FB1-induced full activation of cPLA2. The first step (between 5 min and 2 h) is phosphorylation of cPLA2 by MAPK, which appears early (at 5 min), at a time when no rise in intracellular free calcium level could be observed (data not shown). The second step, reflected in dramatic phosphorylation of cPLA2, could be a consequence of an increase of So in and outside the cells from 2 h (So is known to induce a rise in intracellular free calcium; 35). These two events can explain the full activation of cPLA2 and the increase in AA metabolites induced by FB1.
In the second part of this paper we highlighted a down-regulation pathway involving adenylate cyclase and cAMP, as a synergistic effect of the PKA inhibitor H89 on induction of the AA cascade by FB1 was observed (Figure 2
). Intracellular cAMP production is stimulated by FB1 through mechanisms depending on: (i) MAPK activation, because inhibition of MAPK activity by PD089059 significantly inhibits cAMP production (Figure 6
); (ii) cyclooxygenase, because indomethacin, an inhibitor of this pathway, prevents induction by FB1 of cAMP secretion (Figure 6
). These results suggest that FB1-induced cAMP production in these cells was controlled by PGs. Several publications have reported that PGs have a modulatory effect on adenylate cyclase and cAMP secretion (18,19,36). In particular, PGE2 increased cAMP formation (19,36). cAMP and PKA inhibition of MAPK activation has been also demonstrated (21,22). Thus, these data are consistent with a synergistic effect of H89 on the AA cascade. The precise role of cAMP in regulating cell proliferation remains controversial. In some cells, such as 3T3 fibroblasts and thymocytes, cAMP is a mitogenic messenger (37). In contrast, cAMP inhibits the proliferation of many cell lines, including fibroblasts, T cells and neuroblastoma cells (21,38). Many of these controversial effects could be correlated with cell type-specific effects of cAMP in regulation of the MAPK pathway (39). In some cells cAMP increases MAPK activity, in others cAMP decreases it and in others it causes no change. These actions are dictated in part by expression of the Ras related G proteins Rap 1 (40) and B Raf (39). It is known that MAPK is activated by sequential activation of the proto-oncogenes Ras and Raf and MEK. This pathway has been denoted the Ras/Raf/MEK protein kinases cascade (41,42). Nevertheless, MEK can also be activated, independently of Raf, by MEK kinase via heterotrimeric G protein-coupled receptors (14,41,42). As it has recently been demonstrated that FB1 interacts with G protein-coupled receptors (43), this could be a possible mechanism for activation of MAPK.
In conclusion, we have described a specific effect of FB1 on activation of the AA cascade through a MAPK-dependent pathway. In addition, we have observed down-regulation of cPLA2 activity and AA metabolism by a mechanism involving PG production, cAMP synthesis and PKA activation. Overall, this suggests that the effect of FB1 on cells is dependent on the action of FB1 on the enzymes involved in the cell cycle, such as MAPK and PKA, and on bioactive fatty acids, such as the PGs and LTs, and also on disruption of sphingolipid metabolism.
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Acknowledgments
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The authors thank Dr J.Cheney (IARC) for editing this manuscript. This work was supported by grants from Midi-Pyrénées nos 9408014, 9600487 and 9609624, Ministère de l'Enseignement Supérieure et de la Recherche Scientifique, Action Spécifique no. 97-1477, Ministère des Affaires Étrangères, Coopération Afrique du Sud no. 97780171000576 and INSERM EN98-28.
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Notes
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4 To whom correspondence should be addressedEmail: leszkowicz{at}ensat.fr 
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Received December 23, 1998;
revised April 27, 1999;
accepted May 27, 1999.