From the 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
Department of Pharmacology, University of North Carolina,
Chapel Hill, North Carolina 27599
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ABSTRACT |
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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 ,
I, and
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.
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INTRODUCTION |
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Arachidonic acid
(20:46)1 is a second
messenger that is released by the action of phospholipase
A2 in activated cells (1). In in vitro assays,
20:4
6 and other polyunsaturated fatty acids have been demonstrated
to stimulate the activity of protein kinase C (PKC) (2, 3). When added
exogenously, 20:4
6 is biologically active in a wide spectrum of
cells. For example, 20:4
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 20:4
6 are
mediated are poorly understood.
We have previously demonstrated that 20:46 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 20:4
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 20:4
6 affects
the activity of p38. We now report the novel finding that 20:4
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 20:4
6 also stimulated the phosphorylation/activity of ERK
but not of JNK, although the activity of JNK was weakly stimulated by
20:4
6 in Jurkat cells. 20:4
6 also stimulated the translocation of
a number of PKC isozymes to a particulate fraction in neutrophils. The effect of 20:4
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 20:4
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 20:4
6.
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EXPERIMENTAL PROCEDURES |
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Materials
Fatty acids, 20:46, 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. [
-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 20:4
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 20:46 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
-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
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
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.
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RESULTS |
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Incubation of HeLa cells, HL60 cells, and human neutrophils with
20:46 (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 20:4
6. Kinase activity assays in neutrophils
confirmed this (Fig. 1). In contrast to the above data, 20:4
6 did
not enhance p38 dual phosphorylation in Jurkat cells (data not shown)
that express p38 (26).
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Our previous studies in human neutrophils have demonstrated that
20:46 stimulated superoxide production, degranulation, and adherence
to plastic surfaces and increased the expression of CD11b/CD18 (5-7).
To elucidate the mechanisms through which 20:4
6 exerted these
actions, the effects of 20:4
6 on MAP kinases were studied in more
detail in the neutrophils. 20:4
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 20:4
6, and phosphorylation
increased with increasing concentrations of 20:4
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
20:4
6 (Fig. 2b). Very little dual-phosphorylated p38 was
left at 10 min after the addition of 20:4
6. The ability of 20:4
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 20:4
6 in the presence of nordihydroguaiaretic acid or
indomethacin.
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We next examined whether 20:46 also stimulated the activity of JNK
in human neutrophils. Although 20:4
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, 20:4
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).
3 fatty acids also stimulated JNK activity in Jurkat cells (data
not shown).
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Although we have previously demonstrated that 20:46 stimulated the
activity of ERK in WB rat liver cells (12), the effect of 20:4
6 on
ERK activity in neutrophils has not been reported. Because the above
data demonstrate that 20:4
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 20:4
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 20:4
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 20:4
6-stimulated cells (Fig.
5b). Kinase assays demonstrate that the enhancement of ERK
activity by 20:4
6 was concentration-dependent. An
increase in kinase activity was detectable at 5 µM
20:4
6, the lowest concentration tested (Fig.
6a). The effect of 20:4
6
peaked at around 15 min after the addition of 20:4
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 20:4
6 and declined rapidly thereafter.
20:4
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
3 fatty acids, eicosapentaenoic acid (20:5
3) and docosahexaenoic acid (22:6
3) (data not shown).
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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 20:46
in neutrophils. Neutrophils contain PKC
,
I,
II,
, and
(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 GTP
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
II was detected in a particulate fraction, and this was increased
after incubation with 20:4
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). 20:4
6 also caused a small amount of PKC
and
I to
associate with the particulate fraction (Fig. 7). Neither PKC
nor
was detected in the particulate fraction (data not shown). An
involvement of PKC in the activation of MAP kinases by 20:4
6 was
confirmed by the observation that GF109203X, an inhibitor of classical
PKC isozymes (34), attenuated the stimulatory effect of 20:4
6 on ERK
activity by >85% (Fig. 8a). GF109203X also caused a modest reduction in the ability of 20:4
6 to
stimulate the appearance of dual-phosphorylated p38 (Fig.
8b).
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DISCUSSION |
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20:46 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 20:4
6 in ligand-stimulated responses has been
adequately demonstrated using inhibitors of cytosolic phospholipase
A2 and antisense technology (1). The non-esterified 20:4
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 20:4
6 in the
extracellular medium as a measure of phospholipase A2
activation (1, 35, 36). Although cell-associated 20:4
6 can directly serve as an endogenous second messenger and is a substrate for the
lipoxygenases and cyclooxygenases, 20:4
6 that is released into the
extracellular fluid has the potential to exert autocrine and paracrine
effects. Consistent with this suggestion, exogenously added 20:4
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 20:4
6,
and in isolated islets of Langerhans, glucose was found to increase
cell-associated nonesterified 20:4
6 by up to 75 µM
(37, 38). However, the mechanisms through which 20:4
6 act are still
poorly understood.
The present study demonstrates that exogenous 20:46 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 20:4
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 20:4
6 in the neutrophils. However, 20:4
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 20:4
6 (19, 20). The ability of 20:4
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, 20:4
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 20:4
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 20:46 on p38 and ERK activation.
Thus, 20:4
6 not only stimulated the translocation of PKC
,
I,
and
II to a particulate fraction in neutrophils, but the effects of
20:4
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 p38
(also
known as p38, CSBP, RK), p38
, p38
(ERK6/SAPK3), and p38
(SAPK4) (41, 42). PMA selectively stimulated the activity of p38
and
p38
without significantly affecting the activity of p38
or p38
(42), indicating that the
and
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 p38
and/or p38
. 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. 20:46 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 20:4
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-1 (rsk-2) and p70 S6
kinase (47). However, the effects of GF109203X on 20:4
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 20:46 to stimulate JNK activity in neutrophils is
in direct contrast to the observations in stromal (20) and proximal
tubular epithelial cells (19). Although 20:4
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 20:4
6 requires activation of the NADPH oxidase (19).
Given that 20:4
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
20:46, 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 20:46
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, 20:4
6 did not increase the amount of
dual-phosphorylated p38 in Jurkat T cells. Our studies also demonstrate
that 20:4
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
20:4
6. The MAP kinase species that is recruited will depend on the
cell type.
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ACKNOWLEDGEMENT |
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We thank Dr. S. L. Pelech, University of British Columbia, Canada, for the gift of anti-ERK antibody, R2.
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FOOTNOTES |
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* 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: 20:4-6,
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; GTP
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.
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