Stimulation of p38 Phosphorylation and Activity by Arachidonic Acid in HeLa Cells, HL60 Promyelocytic Leukemic Cells, and Human Neutrophils
EVIDENCE FOR CELL TYPE-SPECIFIC ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES*

Charles S. T. HiiDagger §, Zhi H. HuangDagger , Andrea Bilney, Maurizio CostabileDagger , Andrew W. Murray, Deborah A. RathjenDagger , Channing J Derparallel , and Antonio FerranteDagger

From the Dagger  Department of Immunopathology, Women's and Children's Hospital, North Adelaide, South Australia 5006 and  School of Biological Sciences, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia and parallel  Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Although it is well appreciated that arachidonic acid, a second messenger molecule that is released by ligand-stimulated phospholipase A2, stimulates a wide range of cell types, the mechanisms that mediate the actions of arachidonic acid are still poorly understood. We now report that arachidonic acid stimulated the appearance of dual-phosphorylated (active) p38 mitogen-activated protein kinase as detected by Western blotting in HeLa cells, HL60 cells, human neutrophils, and human umbilical vein endothelial cells but not Jurkat cells. An increase in p38 kinase activity caused by arachidonic acid was also observed. Further studies with neutrophils show that the stimulation of p38 dual phosphorylation by arachidonic acid was transient, peaking at 5 min, and was concentration-dependent. The effect of arachidonic acid was not affected by either nordihydroguaiaretic acid, an inhibitor of the 5-, 12-, and 15-lipoxygenases or by indomethacin, an inhibitor of cyclooxygenase. Arachidonic acid also stimulated the phosphorylation and/or activity of the extracellular signal-regulated protein kinase and of c-jun N-terminal kinase in a cell-type-specific manner. An examination of the mechanisms through which arachidonic acid stimulated the phosphorylation/activity of p38 and extracellular signal-regulated protein kinase in neutrophils revealed an involvement of protein kinase C. Thus, arachidonic acid stimulated the translocation of protein kinase C alpha , beta I, and beta II to a particulate fraction, and the effects of arachidonic acid on mitogen-activated protein kinase phosphorylation/activity were partially inhibited by GF109203X, an inhibitor of protein kinase C. This study is the first to demonstrate that a polyunsaturated fatty acid causes the dual phosphorylation and activation of p38.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Arachidonic acid (20:4omega 6)1 is a second messenger that is released by the action of phospholipase A2 in activated cells (1). In in vitro assays, 20:4omega 6 and other polyunsaturated fatty acids have been demonstrated to stimulate the activity of protein kinase C (PKC) (2, 3). When added exogenously, 20:4omega 6 is biologically active in a wide spectrum of cells. For example, 20:4omega 6 has been reported to inhibit gap junctional permeability between adherent cells (4); stimulate superoxide production and degranulation and increase the expression of CD11b/CD18 in human neutrophils (5-7), prime macrophages, and monocytes for enhanced respiratory burst (8); stimulate insulin secretion from isolated islets of Langerhans (9); modulate the permeability of K+, Na+, and H+ channels in a variety of cell types (10-12); stimulate gene transcription (13); and cause differentiation and death (14). However, the molecular mechanisms through which the actions of 20:4omega 6 are mediated are poorly understood.

We have previously demonstrated that 20:4omega 6 and other polyunsaturated fatty acids stimulate the activity of the extracellular signal-regulated protein kinase (ERK) in WB rat liver epithelial cells (15), suggesting that ERK may mediate some of the biological actions of polyunsaturated fatty acids. Others have reported that arachidonic acid and its metabolites stimulate ERK activity in smooth muscle cells (16). ERK and the closely related p38 and jun N-terminal kinase (JNK) are members of the mitogen-activated protein (MAP) kinase family of kinases (17). These kinases are activated when cells are exposed to growth factors, cytokines, and/or various forms of stress (17, 18). Activation of ERK, JNK, and p38 MAP kinases are achieved through the dual phosphorylation of threonine and tyrosine residues in the TXY motif by upstream MAP kinase kinases. MAP kinases have been proposed to regulate a diverse range of biological functions, including cytokine production and cell growth, differentiation, and death (17, 18). Although 20:4omega 6 has recently been reported to stimulate the activity of JNK in proximal tubular epithelial cells and in stromal cells (19, 20), we are not aware of any studies that have investigated whether 20:4omega 6 affects the activity of p38. We now report the novel finding that 20:4omega 6 stimulated the dual phosphorylation of p38 in HeLa cells, HL60 cells, human umbilical vein endothelial cells, and human neutrophils but not in Jurkat T cells. Further characterization in neutrophils demonstrated that 20:4omega 6 also stimulated the phosphorylation/activity of ERK but not of JNK, although the activity of JNK was weakly stimulated by 20:4omega 6 in Jurkat cells. 20:4omega 6 also stimulated the translocation of a number of PKC isozymes to a particulate fraction in neutrophils. The effect of 20:4omega 6 on p38 and ERK dual phosphorylation/activity was partially blocked by GF109203X, a specific inhibitor of PKC. These data demonstrate that the ability of 20:4omega 6 to stimulate the activity of p38 and JNK is cell type-dependent and suggest that p38, ERK, JNK, and PKC are potential mediators of the biological actions of 20:4omega 6.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Fatty acids, 20:4omega 6, formyl-methionyl-leucyl-phenylalanine, phorbol 12-myristate 13-acetate (PMA), myelin basic protein, kinase A peptide inhibitor, protein A-Sepharose, and general reagents for kinase assays were from Sigma. [gamma -32P]ATP (specific activity 4000 Ci/mmol) was obtained from Bresatec, Adelaide, Australia. The anti-ERK antibody, R2, was a kind gift from Dr. S. Pelech, University of British Columbia, or was purchased from Upstate Biotechnology, Inc., Lake Placid, NY. Rabbit anti-p38 and anti-JNK1 antibodies were obtained from Santa Cruz Biotech. The anti-ACTIVETM ERK and p38 antibodies were obtained from Promega Inc., Santa Cruz, CA. Enhanced chemiluminescence (ECL) solutions and reinforced nitrocellulose were from NEN Life Science Products and Schleicher and Schuell, respectively. Glutathione-Sepharose beads was from Pharmacia Biotech, Australia. Fatty acids, PMA, and formyl-methionyl-leucyl-phenylalanine were dissolved in ethanol, dimethyl sulfoxide (Me2SO), and Me2SO, respectively. The final concentrations of the vehicles were: ethanol, 0.1% (v/v) and Me2SO, 0.1% (v/v). Control cells received vehicle(s) alone.

Cell Culture

HeLa cells were maintained in Dulbeco's modified Eagle's medium in the presence of fetal calf serum (10%) and antibiotics as described previously (21). Cells (0.25 × 106) were plated in 10-cm culture dishes and were used after 4 days. HL60 and Jurkat T cells were maintained in RPMI 1640 supplemented fetal calf serum with and antibiotics at <= 1 × 106/ml. All cells were washed once with Hanks' balanced salts solution (HBSS) 30 min before being incubated with 20:4omega 6 or vehicle.

Isolation and Incubation of Neutrophils

Human neutrophils were isolated from the peripheral blood of healthy volunteers by the rapid single-step method of Ferrante and Thong (22). The preparation of neutrophils was of >98% purity and >99% viability as judged by morphological examination of cytospin preparations and the ability of viable cells to exclude trypan blue. Cells in HBSS were incubated in the presence of 20:4omega 6 for the times indicated.

Preparation of Cellular Extracts

Incubations were terminated by removing the incubation medium and washing the cells once with HBSS (4 °C).

p38 and JNK-- For p38 and JNK assays, cells were lysed in 150 µl of buffer A (20 mM Hepes, pH 7.4, 0.5% (v/v) Nonidet P-40, 100 mM NaCl, 1 mM EDTA, 2 mM, 2 mM Na3VO4, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin, aprotinin, pepstatin A, and benzamidine) for 2 h at 4 °C. After centrifugation (16,000 × g, 5 min), the supernatants were collected for kinase assay or for Western blotting as described below.

ERK-- Activation of ERK was determined by a kinase assay and by Western blotting. Pelleted cells were sonicated (3 × 10 s, output of 2 units, Soniprobe) in buffer B (25 mM Tris-HCl, pH 7.5, 2 mM EGTA, 25 mM NaCl, 1 mM Na3VO4, 38 mM p-nitrophenylphosphate, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) and centrifuged (100,000 × g, 20 min), and the supernatants (termed cytosolic fractions) were collected for Western blotting as described below or for kinase assay. Before the kinase assay, cytosolic fractions were batch-adsorbed onto phenyl-Sepharose CL4B. After washing the beads with 10% (2×) and 35% (2×) ethylene glycol in buffer A (v/v), ERK was eluted with 60% ethylene glycol (23). Previous studies have demonstrated that phenyl-Sepharose-adsorbed ERK1 and ERK2 are eluted between 35 and 60% ethylene glycol (23). The activity of ERK was assayed as described below.

PKC-- To study PKC translocation, neutrophils were sonicated in buffer C (25 mM Tris-HCl, pH 7.5, containing 1 mM dithiothreitol, 5 mM EGTA, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin, aprotinin, pepstatin A, and benzamidine). After incubating on ice for 30 min, samples were centrifuged (100,000 × g, 30 min), and the pellets were resuspended in Buffer C containing 2% Triton X-100. After a 30-min incubation on ice, samples were centrifuged (100,000 × g, 30 min), the supernatant was mixed with Laemmli buffer, and PKC isozymes were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with isozyme-specific anti-PKC antibodies as described below.

Kinase Assays

p38-- p38 was immunoprecipitated before determination of kinase activity. Briefly, lysates (700 µg of protein) were precleared with protein A-Sepharose (15 µl/sample). Anti-p38 antibody (3 µg/sample) was added, and tubes were incubated with constant mixing for 90 min at 4 °C. The antigen-antibody complexes were precipitated by the addition of protein A-Sepharose (20 µl/sample). The immunoprecipitates were washed once with buffer A and once with assay buffer (20 mM Hepes, pH 7.2, 20 mM beta -glycerophosphate, 3.8 mM p-nitrophenyl phosphate, 10 mM MgCl2, 1 mM dithiothreitol, 50 µM Na3VO4, and 20 µM ATP) at 4 °C. The assay was started by adding 30 µl of assay buffer (30 °C) containing 10 µCi of gamma 32P-ATP, 3.8 mM p-nitrophenylphosphate, and 15 µg of myelin basic protein. After 15 min, the assay was terminated by the addition of Laemmli buffer and boiling the samples for 5 min at 100 °C. Phosphorylated myelin basic protein was resolved by 12% SDS-polyacrylamide gel electrophoresis and was detected and quantitated using an Instant Imager (Packard Instruments).

JNK-- A solid phase assay was used to assay JNK activity as described previously (24). Briefly, glutathione S-transferase-jun (1-79) fusion protein was purified from bacterial lysates using glutathione-Sepharose beads at 4 °C with gentle rocking. 1 mg of lysate protein, 15 mM MgCl2, and 10 µM ATP were added to 25 µl (packed volume) of glutathione S-transferase-jun (1-79) coupled to glutathione-Sepharose beads. The mixtures were incubated for 2 h at 4 °C with gentle rocking. After centrifugation (16,000 × g, 5 min), the beads were washed once with buffer A, once with wash buffer (10 mM Pipes, pH 7, 100 mM NaCl, and the protease inhibitors, which were added to buffer A) and once with assay buffer (see p38 above). The assay was started by adding 35 µl assay buffer containing 8 µCi of gamma 32P-ATP and bringing the temperature to 30 °C. After 20 min, the assay was terminated by the addition of Laemmli buffer and boiling the samples for 5 min at 100 °C. Samples were resolved by 10% SDS-polyacrylamide gel electrophoresis, and detection and quantitation of phosphorylated glutathione S-transferase-jun (1-79) was as described above.

ERK-- ERK activity was assayed as described previously (12, 23) by monitoring the incorporation of 32Pi into myelin basic protein in the presence of EGTA and protein kinase A peptide inhibitor. The assay mixture did not contain added phospholipids. Assays were terminated by spotting aliquots of the reaction mixture onto P81 filter paper. After 3 washes (5 min each) with 75 mM orthophosphoric acid, radioactivity associated with the paper was determined by liquid scintillation spectrometry. There was no detectable protein kinase A activity in phenyl-Sepharose-purified fractions, since omission of the protein kinase A peptide inhibitor from the assay mixture did not result in increased phosphorylation of myelin basic protein (Ref. 23 and data not shown). Active p38, if present in the fractions, was unlikely to contribute to any significant degree toward the total myelin basic protein kinase activity because the time course of ERK activity did not correlate with the appearance of dual-phosphorylated p38 (see "Results"). Consequently, it is unlikely that PKC, Ca2+/calmodulin-dependent kinases, p38, or protein kinase A were responsible for phosphorylating myelin basic protein in these samples.

Western Blotting

Denatured proteins were separated on either 10 (ERK and p38) or 12% (PKC) polyacrylamide gels and transferred to nitrocellulose (100 V, 1.5 h), and immunoreaction and detection were carried out as described earlier (25). Immediately after transfer, blots were stained with Ponceau S (0.1% in 5% acetic acid) to confirm equal loading of all lanes of the gels. Affinity-purified polyclonal anti-ERK antibody, R2, anti-ACTIVETM ERK, or anti-ACTIVETM p38 antibody and anti-PKC isozyme-specific antibodies were used to detect ERK isoforms, dual-phosphorylated ERK, dual phosphorylated p38, and PKC isozymes, respectively. Immunocomplexes were detected by enhanced chemiluminescence (25).

Statistical Analysis

Where appropriate, differences were analyzed by analysis of variance or unpaired Student's t test and were considered significant when p < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Incubation of HeLa cells, HL60 cells, and human neutrophils with 20:4omega 6 (20 µM) for 4 min resulted in the dual phosphorylation of p38 as detected by Western blotting (Fig. 1). Similar results were obtained in human umbilical vein endothelial cells (data not shown). Ponceau S staining confirmed that, within a particular experiment, the individual lanes were loaded with equal amounts of proteins (data not shown). Since the anti-ACTIVETM p38 antibody only detects p38 that had been dual phosphorylated on the TGY activation motif, these results indicate activation of p38 by 20:4omega 6. Kinase activity assays in neutrophils confirmed this (Fig. 1). In contrast to the above data, 20:4omega 6 did not enhance p38 dual phosphorylation in Jurkat cells (data not shown) that express p38 (26).


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Fig. 1.   Stimulation of p38 dual phosphorylation and activity by 20:4omega 6. HeLa cells (4 × 106 cells), HL60 cells (2 × 107 cells), and neutrophils (3 × 107 cells) were incubated with 20:4omega 6 (20 µM) for 4 min at 37 °C and lysed. Fractions were prepared and Western blotted with anti-ACTIVETM p38 antibody (a). In some experiments with neutrophils, kinase activity was also determined using myelin basic protein as a substrate (b), as described under "Experimental Procedures." Results are representative of 3-4 separate experiments.

Our previous studies in human neutrophils have demonstrated that 20:4omega 6 stimulated superoxide production, degranulation, and adherence to plastic surfaces and increased the expression of CD11b/CD18 (5-7). To elucidate the mechanisms through which 20:4omega 6 exerted these actions, the effects of 20:4omega 6 on MAP kinases were studied in more detail in the neutrophils. 20:4omega 6 stimulated the dual phosphorylation of p38 in a concentration- and time-dependent manner (Fig. 2). Thus, dual-phosphorylated p38 could be detected at 5 µM 20:4omega 6, and phosphorylation increased with increasing concentrations of 20:4omega 6 up to 20 µM, the maximum concentration tested (Fig. 2a). Stimulation of p38 dual phosphorylation, detectable at less than 2 min, was transient, peaking at 5 min after exposure to 20:4omega 6 (Fig. 2b). Very little dual-phosphorylated p38 was left at 10 min after the addition of 20:4omega 6. The ability of 20:4omega 6 to stimulate dual phosphorylation of p38 was not diminished by either nordihydroguaiaretic acid, a broad spectrum inhibitor of the 5-, 12-, and 15-lipoxygenase or by indomethacin, an inhibitor of cyclooxygenase (Fig. 3). A small amount of dual-phosphorylated p38 was detected in neutrophils that had been exposed to either nordihydroguaiaretic acid or indomethacin per se. This was likely to be due to the accumulation of low levels of endogenous 20:4omega 6 in the presence of nordihydroguaiaretic acid or indomethacin.


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Fig. 2.   Characteristics of 20:4omega 6-stimulated p38 dual phosphorylation. Neutrophils were incubated with 20:4omega 6 at the concentrations indicated for 4 min (a) or with 20:4omega 6 (20 µM) for up to 10 min (b). Cells were lysed and the fractions were Western-blotted as described under "Experimental Procedures." The results are representative of at least three experiments.


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Fig. 3.   Lack of effect of nordihydroguaiaretic acid or indomethacin on the appearance of dual phosphorylated p38. Neutrophils were preincubated for 10 min with either nordihydroguaiaretic acid (NDGA) (10 µM) or indomethacin (100 µM) before being exposed to 20:4omega 6. Samples were prepared and Western blotted as described under "Experimental Procedures." The results are representative of three experiments.

We next examined whether 20:4omega 6 also stimulated the activity of JNK in human neutrophils. Although 20:4omega 6 has been reported to stimulate the activity of JNK in stromal and rabbit proximal tubular epithelial cells (19, 20), the fatty acid did not stimulate JNK activity in neutrophils (data not shown). This was not because neutrophils do not express JNK, because the presence of JNK1 was detected in neutrophils (data not shown). However, 20:4omega 6 stimulated the activity of JNK in Jurkat T cells (Fig. 4), although the degree of activation was less than that observed with A23187/PMA (Fig. 4). omega 3 fatty acids also stimulated JNK activity in Jurkat cells (data not shown).


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Fig. 4.   20:4omega 6 stimulated the activity of JNK in Jurkat cells. Jurkat cells were stimulated with 0-20 µM 20:4omega 6 (a), with 0-10 µM A23178 and 35 nM PMA (b) for 30 min, or with 20:4omega 6 (10 µM) or A23187 (1 µM)/PMA (35 nM) for up to 120 min (c). The cells were lysed, and JNK activity was assayed as described under "Experimental Procedures." Results of the time course experiments (c) are expressed as cpm 32Pi incorporated into glutathione S-transferase jun (1-79). Results are representative of three separate experiments.

Although we have previously demonstrated that 20:4omega 6 stimulated the activity of ERK in WB rat liver cells (12), the effect of 20:4omega 6 on ERK activity in neutrophils has not been reported. Because the above data demonstrate that 20:4omega 6 stimulated the dual phosphorylation of p38 and the activity of JNK in a cell type-specific manner, it is, therefore, important to determine whether the activity of ERK in neutrophils is affected by 20:4omega 6. Activated ERK isoforms display reduced electrophoretic mobility in SDS-polyacrylamide gels (12, 21, 25, 27) because of the dual phosphorylation of ERK on the TEY activation motif. Incubation of neutrophils with 20:4omega 6 caused a retardation in the electrophoretic mobility of the 42- and 43-kDa forms of ERK to give apparent Mr values of 43- and 44-kDa (Fig. 5a), consistent with their activation by phosphorylation. When the fractions were Western-blotted with anti-ACTIVETM ERK antibody that detects active, dual-phosphorylated ERK, two bands with Mr values of approximately 43-and 44-kDa were detected predominantly in samples from 20:4omega 6-stimulated cells (Fig. 5b). Kinase assays demonstrate that the enhancement of ERK activity by 20:4omega 6 was concentration-dependent. An increase in kinase activity was detectable at 5 µM 20:4omega 6, the lowest concentration tested (Fig. 6a). The effect of 20:4omega 6 peaked at around 15 min after the addition of 20:4omega 6 (Fig. 6b) and was longer lasting than stimulation of p38 dual phosphorylation or activity (Fig. 2b). This argues strongly against the possibility that contaminating p38, which also phosphorylates myelin basic protein (Fig. 1b), was responsible for phosphorylating myelin basic protein in these fractions, since the appearance of dual-phosphorylated p38 peaked at 5 min after the addition of 20:4omega 6 and declined rapidly thereafter. 20:4omega 6 also stimulated the activity of ERK in human umbilical vein endothelial cells, human mesangial cells, Jurkat cells, HL60 cells, and human monocytes but not in PC12 pheochromocytoma cells (data not shown). The activity of ERK in these cells was also stimulated by the omega 3 fatty acids, eicosapentaenoic acid (20:5omega 3) and docosahexaenoic acid (22:6omega 3) (data not shown).


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Fig. 5.   Stimulation of ERK phosphorylation by 20:4omega 6 in human neutrophils. Neutrophils (3 × 107 cells in 30 ml of HBSS) were stimulated with 20:4omega 6 (20 µM, 10 min), formyl-methionyl-leucyl-phenylalanine (50 nM, 5 min), or PMA (100 nM, 5 min). Cytosolic fractions were prepared and Western-blotted with the anti-ERK antibody, R2 (a), or with the anti-ACTIVETM ERK antibody (b) as described under "Experimental Procedures." a: lane 1, control; lane 2, formyl-methionyl-leucyl-phenylalanine; lane 3, 20:4omega 6; lane 4, PMA. b: lane 1, control; lane 2, 20:4omega 6. Results are representative of at least three experiments.


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Fig. 6.   The activity of ERK in neutrophils was stimulated in a concentration- and time-dependent manner by 20:4omega 6. Neutrophils were incubated with 20:4omega 6 at the concentrations indicated for 10 min (a) or for the times indicated (b, control (open circle ); 20 µM 20:4omega 6 (bullet )). ERK in cytosolic fractions was partially purified, and the activity was assayed as described under "Experimental Procedures." Results are the mean ± S.E. of three determinations (a) or the mean of duplicate determinations (b). Results are representative of at least three experiments.

Since polyunsaturated fatty acids have been demonstrated to activate PKC in vitro (2, 3) and to stimulate the translocation of PKC in WB rat liver epithelial cells (12), we investigated whether PKC was involved in the activation of the p38 and ERK cascades by 20:4omega 6 in neutrophils. Neutrophils contain PKC alpha , beta I, beta II, delta , and zeta  (28). Although polyunsaturated fatty acids have been reported to stimulate Ca2+ mobilization (29), activate the neutral sphingomyelinase (30), and amplify H+ ion channel conductance (31) in intact neutrophils and to stimulate GTPgamma S loading of the heterotrimeric G-proteins in neutrophil membrane fractions (32), the effect of fatty acids on PKC translocation in neutrophils has not been reported. In unstimulated neutrophils, a substantial amount of PKC beta II was detected in a particulate fraction, and this was increased after incubation with 20:4omega 6 (Fig. 7). The existence of particulate fraction-associated PKC in unstimulated cells has also been observed in the T lymphocyte cell line, CTLL-2 (33). 20:4omega 6 also caused a small amount of PKC alpha  and beta I to associate with the particulate fraction (Fig. 7). Neither PKC delta  nor zeta  was detected in the particulate fraction (data not shown). An involvement of PKC in the activation of MAP kinases by 20:4omega 6 was confirmed by the observation that GF109203X, an inhibitor of classical PKC isozymes (34), attenuated the stimulatory effect of 20:4omega 6 on ERK activity by >85% (Fig. 8a). GF109203X also caused a modest reduction in the ability of 20:4omega 6 to stimulate the appearance of dual-phosphorylated p38 (Fig. 8b).


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Fig. 7.   20:4omega 6 stimulated the translocation of PKCalpha , beta I, and beta II. Neutrophils were stimulated with 20:4omega 6 (20 µM) for 3 min, and PKC fractions were prepared and Western-blotted as described under "Experimental Procedures." The results are representative of three experiments.


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Fig. 8.   Inhibition of ERK activity and p38 phosphorylation by GF109203X. Neutrophils (1 × 107 in 10 ml HBSS for ERK activity assays, 3 × 107 in 30 ml HBSS for p38 phosphorylation) were preincubated with GF109203X (0.5 µM, filled bars) or Me2SO (0.1% v/v, open bars) for 10 min. The cells were then incubated with 20:4omega 6 (20 µM) for 10 (ERK activity assays) or 4 (for p38 phosphorylation) min. Determination of ERK activity (a) and p38 dual phosphorylation (b) were carried out as described under "Experimental Procedures." Data on ERK activity are means ± S.E. of three determinations. Results are representative of three experiments. Significance of difference: * p < 0.05.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

20:4omega 6 is a second messenger molecule that is liberated from the sn-2 position of membrane phospholipids by ligand-stimulated activation of the cytosolic phospholipase A2 (1). The importance of endogenously generated 20:4omega 6 in ligand-stimulated responses has been adequately demonstrated using inhibitors of cytosolic phospholipase A2 and antisense technology (1). The non-esterified 20:4omega 6 that is released has been found to be cell-associated as well as being released into the extracellular medium. Thus, many studies have assayed for the appearance of radiolabeled 20:4omega 6 in the extracellular medium as a measure of phospholipase A2 activation (1, 35, 36). Although cell-associated 20:4omega 6 can directly serve as an endogenous second messenger and is a substrate for the lipoxygenases and cyclooxygenases, 20:4omega 6 that is released into the extracellular fluid has the potential to exert autocrine and paracrine effects. Consistent with this suggestion, exogenously added 20:4omega 6 has been shown to be biologically active to a wide spectrum of cell types at concentrations (1-20 µM) that have been reported to be present in stimulated cells. Thus, neutrophils have been reported to contain 100-2,200 pmol/107 cells of 20:4omega 6, and in isolated islets of Langerhans, glucose was found to increase cell-associated nonesterified 20:4omega 6 by up to 75 µM (37, 38). However, the mechanisms through which 20:4omega 6 act are still poorly understood.

The present study demonstrates that exogenous 20:4omega 6 stimulated the dual phosphorylation of p38 in HeLa cells, HL60 cells, human neutrophils, and human umbilical vein endothelial cells but not in Jurkat cells. We demonstrate in neutrophils that this increase in p38 dual phosphorylation was accompanied by an increase in p38 kinase activity. Stimulation of p38 dual phosphorylation by 20:4omega 6 in neutrophils was independent of its metabolism by either lipoxygenase or cyclooxygenase since the effect was not affected by nordihydroguaiaretic acid, a broad spectrum inhibitor of the 5-, 12-, and 15-lipoxygenases or by indomethacin, an inhibitor of cyclooxygenase. The fatty acid also stimulated the dual phosphorylation and activity of ERK but not of JNK in neutrophils. This, therefore, excludes an involvement of JNK in the actions of 20:4omega 6 in the neutrophils. However, 20:4omega 6 stimulated the activity of JNK in Jurkat T cells, an observation that is consistent with reports in proximal tubular epithelial cells and stromal cells that the activity of JNK was stimulated by 20:4omega 6 (19, 20). The ability of 20:4omega 6 to stimulate the activity of ERK in human neutrophils and a number of other primary cell types and cell lines is consistent with our previous observations in WB rat liver epithelial cells (15). However, 20:4omega 6 did not affect the activity of ERK in PC12 cells, although ERK activity in these cells was strongly stimulated by PMA.2 These data, therefore, demonstrate that 20:4omega 6 stimulated the activity of MAP kinases in a cell type/line-specific manner.

The present study demonstrates that PKC may be involved, at least in part, in mediating the effects of 20:4omega 6 on p38 and ERK activation. Thus, 20:4omega 6 not only stimulated the translocation of PKC alpha , beta I, and beta II to a particulate fraction in neutrophils, but the effects of 20:4omega 6 on p38 dual phosphorylation and ERK activity were attenuated by the PKC inhibitor, GF109203X. Consistent with a possible involvement of PKC in the p38 and ERK cascades, PMA, a direct activator of PKC, stimulates the activity of ERK in all cell types examined (18) and of p38 in some cell types (39, 40). Recent studies have revealed that at least four members of the p38 family exist. These are p38alpha (also known as p38, CSBP, RK), p38beta , p38gamma (ERK6/SAPK3), and p38delta (SAPK4) (41, 42). PMA selectively stimulated the activity of p38gamma and p38delta without significantly affecting the activity of p38alpha or p38beta (42), indicating that the gamma  and delta  forms of p38 are regulated by PKC. It remains to be determined whether the PMA-responsive p38 in neutrophils (39) and U937 cells (40) are p38gamma and/or p38delta . Although PKC may regulate the ERK cascade by direct phosphorylation of raf-1 (43) or via Shc/Ras (44), it is currently not known how PKC may regulate the p38 cascade.

In contrast to its effect on ERK activity, the effect of GF109203X on p38 dual phosphorylation was partial. This could imply that PKC is not the sole upstream regulator of the p38 cascade. 20:4omega 6 has been reported to stimulate the release of rho-GDI from its complex with rac2. guanine nucleotide dissociation inhibitor (45), and constitutively active rac has been found to stimulate the activity of p38 via p21-activated kinase (46). Hence, the fatty acid may also activate p38 via modulation of rac2 and p21-activated kinase, independently of PKC. Alternatively, the partial inhibition could suggest the possibility that neutrophils express both PKC-dependent and independent p38 forms. Until specific antibodies to p38 subtypes become available commercially, it is not possible to determine which p38 form(s) is activated by 20:4omega 6.

Although GF109203X has been generally regarded as a specific PKC inhibitor, a recent study has found that GF109203X also inhibited the activity of MAP kinase activated protein kinase-1beta (rsk-2) and p70 S6 kinase (47). However, the effects of GF109203X on 20:4omega 6-stimulated ERK activity and p38 dual phosphorylation were unlikely to be due to inhibition of rsk-2 or p70 S6 kinase, since neither of these kinases are upstream regulators of ERK or p38.

The inability of 20:4omega 6 to stimulate JNK activity in neutrophils is in direct contrast to the observations in stromal (20) and proximal tubular epithelial cells (19). Although 20:4omega 6 stimulated the activity of JNK in Jurkat cells, this effect was weak compared with that caused by A21387 and PMA. Our failure to detect JNK activity was not because of a lack of JNK expression in neutrophils. Studies in proximal tubular epithelial cells have shown that stimulation of JNK activity by 20:4omega 6 requires activation of the NADPH oxidase (19). Given that 20:4omega 6 strongly stimulates the NADPH oxidase in neutrophils, it is, therefore, surprising that the fatty acid failed to stimulate the activity of JNK in neutrophils. This result clearly demonstrates that generation of oxygen radicals per se is insufficient to stimulate the JNK cascade.

Many ligands that stimulate the activity of MAP kinases also stimulate the activity of cytosolic phospholipase A2. It has been widely reported that ERK or p38 directly phosphorylates cytosolic phospholipase A2 in activated cells and in in vitro assays (48, 49) and, with the exception of thrombin-stimulated platelets (50), ERK or p38 has been found to directly regulate the enzymatic activity of cytosolic phospholipase A2. Our study, therefore, suggests that fatty acids such as 20:4omega 6, which are liberated by ligand-stimulated cytosolic phospholipase A2, may participate in sustaining/amplifying MAP kinase activity and the activity of cytosolic phospholipase A2. Consistent with this, exogenously added polyunsaturated fatty acids have been found to stimulate the activity of cytosolic phospholipase A2 in intact neutrophils.3

It is currently not clear how fatty acids are taken up into cells and exert their effects. There is evidence that indicates that fatty acids enter cells by simple diffusion (51) and/or via a carrier-mediated process. Proteins that function as fatty acid transporters have been reported to exist on the plasma membrane of a number of cell types (52, 53). It is possible that these mechanisms are not mutually exclusive. Clearly, partititioning of a fatty acid into the plasma membrane per se is insufficient to exert a biological action (54, 55). A detergent-like action of polyunsaturated fatty acids on the neutrophils has been excluded at concentrations that were used in this study (56). It is also unlikely that the biological activities of a fatty acid are dependent on esterification into membrane phospholipids, since the effects of fatty acids are reversed after the addition of delipidated serum albumin (4, 29) too rapidly to support an esterification-based mechanism of action. A direct agonist-like fatty acid action is therefore likely.

The present study establishes for the first time that 20:4omega 6 stimulates the dual phosphorylation of p38 MAP kinase and that this stimulation is cell type-specific. Although this effect was observed in HeLa cells, HL60 cells, human umbilical vein endothelial cells, and human neutrophils, 20:4omega 6 did not increase the amount of dual-phosphorylated p38 in Jurkat T cells. Our studies also demonstrate that 20:4omega 6 stimulates the activity of ERK and JNK. Again, this effect was cell type-specific. Our data, therefore, suggest that ERK, p38, JNK, and PKC are potential mediators of the biological actions of 20:4omega 6. The MAP kinase species that is recruited will depend on the cell type.

    ACKNOWLEDGEMENT

We thank Dr. S. L. Pelech, University of British Columbia, Canada, for the gift of anti-ERK antibody, R2.

    FOOTNOTES

* This work was supported in part by the National Heart Foundation and the National Health and Medical Research Council. Portions of this work were presented at the 5th International Conference on Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation, and Other Related Diseases in La Jolla, California in September, 1997.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.

§ To whom correspondence and reprint requests should be addressed. Tel.: 08-204-6293; Fax: 08-204-6046; E-mail: chii{at}medicine.adelaide.edu.au.

1 The abbreviations used are: 20:4omega -6, arachidonic acid; ERK, extracellular signal-regulated protein kinase; MAP kinase, mitogen-activated protein kinase; JNK, jun N-terminal kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; HBSS, Hanks' balanced salt solution; Pipes, 1,4-piperazinediethanesulfonic acid; GTPgamma S, guanosine 5'-O-(thiotriphosphate).

2 C. S. T. Hii and A. Ferrante, unpublished data.

3 B. S. Robinson, C. S. T. Hii, and A. Ferrante, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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