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J. Biol. Chem., Vol. 276, Issue 29, 27246-27255, July 20, 2001
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From the
Received for publication, April 11, 2001, and in revised form, May 17, 2001
Although arachidonic acid has been
demonstrated to stimulate a wide variety of cellular functions, the
responsible mechanisms remain poorly defined. We now report that
arachidonic acid stimulated the activity of class Ia
phosphatidylinositol 3-kinase (PI3K) in human umbilical vein
endothelial cells, HL60 cells, and human neutrophils.
Pretreatment of endothelial cells with AG-1478, an inhibitor of the
ErbB receptor family, resulted in the suppression of PI3K activation by
arachidonic acid. The fatty acid enhanced the tyrosine phosphorylation
of ErbB4 but not of ErbB2 or ErbB3. The ability of arachidonic acid to
stimulate PI3K activity in neutrophils was suppressed by indomethacin
and nordihydroguaiaretic acid, inhibitors of the cyclooxygenases and
lipoxygenases, respectively, but not by 17-octadecynoic acid, an
inhibitor of Arachidonic acid
(20:4 In an endeavor to understand further the mechanisms through which
20:4 Materials--
Polyunsaturated fatty acids were purchased from
Sigma-Aldrich or from Cayman (Ann Arbor, MI). 17-octadecynoic
acid was purchased from Cayman. Formyl methionyl leucyl phenylalanine
(fMLP), myelin basic protein, protein A-Sepharose,
phosphatidylinositol, and general reagents for kinase assays were from
Sigma-Aldrich. Lavendustin A and B and AG-1478 were purchased from
Biomol (Plymouth Meeting, PA), and AG-1296 was purchased from
Calbiochem-Novabiochem (Victoria, Australia).
[ Cell Culture--
HL60 cells were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum and antibiotics at up to 1 × 106 cells/ml. Human neutrophils were isolated from the
peripheral blood of healthy volunteers by the rapid single-step method
of Ferrante and Thong (29). The preparations of neutrophils were at
least 98% pure and 99% viable as judged by morphological examination of cytospin preparations and the ability of viable cells to exclude trypan blue. Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase treatment and cultured as described previously (30). Second passage cells (0.25 × 106) were plated
in 10-cm culture dishes and were used after 4 days. Chinese hamster
ovary cells stably expressing ErbB2 or ErbB3 were maintained as
described previously (31). All cells were washed twice with Hanks'
balanced salts solution (HBSS) 30 min before being incubated with
20:4 Preparation of Cellular Extracts--
Incubations were
terminated by either removing the incubation medium and washing the
cells once with HBSS (4 °C) (HUVEC) or adding an equal volume of
cold (4 °C) HBSS into the incubation tubes followed immediately by
centrifugation (3000 × g, 3 min, 4 °C). The cells
were lysed by incubation in buffer A (20 mM Hepes, pH 7.4, 0.5% (v/v) Nonidet P-40, 100 mM NaCl, 1 mM
EDTA, 2 mM Na3VO4, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin, aprotinin, pepstatin A, and benzamidine) for
2 h with constant mixing (4 °C) as described previously (20). After lysis, cell debris was sedimented (12,000 × g,
30 s), and the protein content of the soluble fractions was
determined by the Lowry method. For detection of Akt phosphorylation
and the tyrosine phosphorylation of cellular proteins by Western blot analysis, the incubations were terminated by the removal of the incubation medium, and Laemmli buffer was added. The samples were boiled (5 min) and stored ( Immunoprecipitation--
Lysates containing equal amounts of
protein (0.5-1 mg) were precleared with protein A-Sepharose (15 µl,
20 min, 4 °C) before being incubated with the anti-p85 PI3K Assay--
The activity of PI3K was assayed essentially as
described previously (32). Briefly, MgCl2 (10 µl, stock
concentration of 100 mM) and PtdIns (10 µl, stock
concentration of 4 µg/ml sonicated in 10 mM Tris/HCl
containing 1 mM EDTA, pH 7.6) were added to the above
prepared samples, and the reaction was started by the addition of 15 µCi of [32P]ATP. The tubes were incubated at room
temperature for 10 min with constant mixing. The reaction was
terminated by the addition of 40 µl of 8 M HCl and 320 µl of CHCl3/CH3OH (1:1). After vigorous mixing and centrifugation the aqueous phase was removed, and the chloroform phase was washed (three times) with 500 µl of
CH3OH/H2O (1:1). The lipid samples were applied
to Silica gel 60 thin layer chromatography plates that were developed
with CHCl3/CH3OH/H2O/30% NH4OH (90:70:14.6:5.4 v/v) (32). The PtdIns 3 32P spots were located, and the radioactivity was
determined using an Instant Imager (Packard Instruments).
Western Blotting--
Equal amounts of denatured proteins per
lane were separated on 12% (8% for the analysis of ErbB receptors)
polyacrylamide gels, transferred to nitrocellulose (100 V, 1.5 h),
and immunodetected as described previously (20). Immediately after
transfer, the blots were stained with Ponceau S (0.1% in 5% acetic
acid) to confirm the even transfer of proteins within all the lanes of the gels. The blots that showed dissimilar protein levels between the
lanes were discarded. Anti-ERK2, ACTIVE ERK, p85 Superoxide Production--
Superoxide production was measured by
monitoring the chemiluminescence resulting from the oxidation of
lucigenin (9,9'-bis-N-methyl-acridinium nitrate) (33).
Briefly, neutrophils (1 × 106 cells) were
preincubated with wortmannin or Me2SO (0.1% v/v) for 10 min at 37 °C. 20:4 Statistical Analysis--
Where appropriate, differences were
analyzed by analysis of variance or unpaired Student's t
test and were considered significant at p < 0.05.
The effect of 20:4 Consistent with the above data, incubation of neutrophils with 20:4
Activation of the Phosphatidylinositol 3-Kinase-Akt/Protein
Kinase B Signaling Pathway in Arachidonic Acid-stimulated Human Myeloid
and Endothelial Cells
INVOLVEMENT OF THE ErbB RECEPTOR FAMILY*
§,,
**
Department of Immunopathology, Women's and
Children's Hospital, North Adelaide 5006, South Australia, the
¶ Molecular and Cellular Biology Laboratory, Cooperative Research
Center for Tissue Growth and Repair, Commonwealth Scientific and
Industrial Research Organization, Division of Health Sciences
and Nutrition, Thebarton 5031, South Australia, the Department
of Paediatrics, University of Adelaide, Adelaide 5005, South Australia,
and the ** School of Pharmaceutical, Molecular and Biomedical Sciences,
University of South Australia, Adelaide 5001, South Australia
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylation of arachidonic acid by cytochrome P450
monooxygenases. Consistent with this, the activity of PI3K in
neutrophils was stimulated by 5-hydroxyeicosatetraenoic acid.
Arachidonic acid also transiently stimulated the phosphorylation of Akt
on Thr-308 and Ser-473. Although PI3K was not required for the
activation of the mitogen-activated protein kinases, ERK1, ERK2, and
p38, in arachidonic acid-stimulated neutrophils, the fatty acid acted
via PI3K to stimulate the respiratory burst. These results not
only define a novel mechanism through which some of the actions of
arachidonic acid are mediated but also demonstrate that, in addition to
ErbB1 (epidermal growth factor receptor), ErbB4 can also be
transactivated by a non-epidermal growth factor-like ligand.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6)1 is a second
messenger that is released by the action of phospholipase
A2 in activated cells (1). The liberated 20:46 is either
metabolized within the cells or released into the extracellular space
where it can exert stimulatory or inhibitory effects on cellular
responses. The actions of 20:46 include the stimulation of
neutrophil superoxide production and degranulation (2, 3), insulin
secretion by isolated rat islets of Langerhans (4), induction of the
MAP kinase phosphatase-1 gene in vascular smooth muscle cells (5),
differentiation of HL60 cells (6) and zone chondrocytes (7), adherence
of neutrophils and human breast cancer cells to various substrata (3,
8), and proliferation of certain tumor and normal cell types (9, 10).
On the other hand, 20:46 has been reported to inhibit cell-cell
communication via gap junctions (11, 12), cytokine-induced adhesion
molecule expression in endothelial cells (13), and proliferation of
HL60 cells in conjunction with the induction of differentiation (6). In
cell-free assays, 20:46 and other polyunsaturated fatty acids have
been widely demonstrated to stimulate the activity of protein kinase C
(PKC) (14, 15). The ability of 20:46 to modulate the permeability of
the Na+, K+, and H+ channels
(16-18) and stimulate Ca2+ mobilization (3) has also been
demonstrated. In intact cells, 20:46 has been found to stimulate the
activity of the neutral sphingomyelinase (19), the tyrosine
phosphorylation of a number of cytosolic proteins (20), and the
activities and/or phosphorylation of the extracellular signal-regulated
protein kinase (ERK)-1 and ERK2, p38, c-Jun N-terminal kinase (20-23),
the cytosolic and secretory phospholipase A2 (24)-and MAP
kinase-activated protein kinase 2 (8). On the other hand, 20:46 has
been demonstrated to inhibit tumor necrosis factor-stimulated
activation of NF-
B (13). Although 20:46-derived metabolites are
biologically active, the above actions are mostly unaffected by the
addition of lipoxygenase and cyclooxygenase inhibitors.
6 acts, we investigated whether 20:46 affected the activity
of phosphatidylinositol 3-kinase (PI3K). PI3K is a family of lipid
kinases that phosphorylate inositol-containing phospholipids on the D-3
position of the inositol ring, resulting in the formation of
phosphatidylinositol (PtdIns) 3 P, PtdIns 3,4 P2, and
PtdIns 3,4,5 P3 (25). PI3K is divided into three main
classes based on structural similarity and in vitro
substrate specificity. Class I kinases are heterodimers of a catalytic
and regulatory subunit and are coupled to either tyrosine kinases
(class Ia) or to heterotrimeric G protein-coupled receptors (class Ib)
(25). Within cells, the preferred substrate for class I PI3K is PtdIns
4,5 P2, and only class I PI3Ks have been shown to activate
Akt (also known as PKB), which is encoded by three closely related
genes, PKB
, PKB
and PKB
(25).
Class Ia enzymes include the p110, p110, and p110
catalytic subunits that are complexed to one of seven adaptor (regulatory) proteins derived from three separate genes
(p85, p85, and p55) (25). The
adaptor proteins, via their two Src homology 2 domains, mediate
the interaction of class Ia PI3K with tyrosine-phosphorylated residues.
To date, only one class Ib PI3K, PI3K, has been identified, and this
is composed of a p110 catalytic subunit and a 101-kDa regulatory
subunit (25). Class I PI3Ks are sensitive to inhibition by wortmannin
and LY294002 (25). Mammalian class II PI3K is exemplified by the
190-kDa PI3K-C2 (26). This kinase is refractory to inhibition by
wortmannin and LY294002, and it predominantly uses PtdIns and PtdIns 4P
as substrates in vitro and can phosphorylate PtdIns 4,5 P2 in the presence of phosphatidylserine (26). The third
class of PI3K is structurally related to Saccharomyces
cerevisiae Vps34. This is a PtdIns-specific 3-kinase, and it
associates with p150 for enzymatic activity (27). PI3K has a diverse
range of roles in cell biology including the promotion of cell
survival, mediation of the actions of insulin, and regulation of
neutrophil chemotaxis in response to bacterial peptides (25, 28). We
now report that 20:46 stimulated the activity of PI3K in human
endothelial and myeloid cells. A role for ErbB4 in the activation of
PI3K by 20:46 in the endothelial cells was also established.
Although -hydroxylation of the fatty acid by cytochrome P450
monooxygenases was not required for the stimulation of PI3K activity in
neutrophils, metabolism of 20:46 by cyclooxygenases and
lipoxygenases was required. These data suggest that some of the actions
of 20:46 may be mediated by PI3K.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]ATP (specific activity 4000 Ci/mmol) was
obtained from Geneworks (Adelaide, Australia). Polyclonal anti-p85,
ERK2, ErbB2, ErbB3, ErbB4, and Akt antibodies and the
anti-phosphotyrosine antibody, PY99, were purchased from Santa Cruz
Biotechnology. The anti-phosphotyrosine antibody, PY100, was obtained
from New England Biolabs. Anti-ACTIVE ERK, anti-phospho-Akt (Thr-308)
and anti-phospho-Akt (Ser-473) antibodies were obtained from Promega
(Madison, WI), New England Biolabs, and Upstate Biotechnology (Lake
Placid, NY), respectively. The Western blot recycling kit was obtained
from Alpha Diagnostics (San Antonio, TX). Enhanced chemiluminescence
solutions and reinforced nitrocellulose were from NEN Life Sciences
Products (Boston, MA) and Schleicher and Schuell (Dassel, Germany),
respectively. Arachidonic acid was dissolved in ethanol, and fMLP,
17-octadecynoic acid, and the tyrphostins, AG-1478 and AG-1296, were
dissolved in dimethyl sulfoxide (Me2SO). The final
concentrations of the vehicles were: 0.1% ethanol (v/v) and 0.1%
Me2SO (v/v). Control cells received an equivalent amount of
a vehicle that did not affect cellular responses.
6 or vehicle.
20 °C).
subunit
antibody or PY99 (3 µg/sample). After mixing for 2 h (4 °C),
the immune complexes were precipitated by the addition of protein
A-Sepharose (15 µl). The immunoprecipitates were collected by
centrifugation (16,000 × g, 15 s) and washed once
with buffer A (4 °C). For PI3K activity assays, samples that were
immunoprecipitated with the anti-p85 subunit antibody were washed
once with buffer A, followed by another wash with buffer B (10 mM Tris/HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, and 100 µM
Na2VO4), and resuspended in 50 µl of buffer
B. For Western blot analyses, the samples that were immunoprecipitated with PY99 were mixed with Laemmli buffer and boiled (5 min).
subunit, Akt, and
phospho-Akt antibodies were used to detect ERK2, dual-phosphorylated ERK1 and ERK2, the p85 subunit, Akt, and phosphorylated Akt, respectively. To detect ErbB receptor phosphorylation, samples that
were immunoprecipitated with PY99 were Western blotted first with an
anti-ErbB2 antibody. The blots were stripped using the Western blot
recycling kit and reblotted with the anti-ErbB4 antibody, and the
stripping and reprobing steps were repeated for the detection of ErbB3.
To confirm that tyrosine-phosphorylated proteins remained bound to the
nitrocellulose, the blots were finally probed with the anti-p85
subunit antibody. Immunocomplexes were detected by enhanced
chemiluminescence (20).
6 (20 µM) and lucigenin,
dissolved in HBSS (250 µM v/v), were added, and the
resulting chemiluminescence (mV) was recorded using a luminometer
(Model 1250 or 1251 with MultiUse software, Bio-Orbit Oy, Turku,
Finland). The results are expressed as the maximum rate of superoxide
production achieved (mV).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 on the activity of PI3K was investigated in
cell types that had been shown previously to respond to this fatty acid
(2, 6, 13). Incubation of neutrophils (Fig. 1a), HL60 cells (Fig.
1b), and HUVECs (Fig. 1c) with 20:46 increased the activity of PI3K. Because the anti-p85 antibody that was used to
immunoprecipitate the kinase cross-reacts with all the currently known
forms of the regulatory subunit of class Ia PI3Ks, the data represent
activation of class Ia PI3K. Although the effect of 20:46 on kinase
activity was transient in neutrophils and HL60 cells, peaking at 2 and
5 min, respectively, PI3K activity in HUVECs increased with time over
the 10-min incubation period (Fig. 1). The stimulatory effect of
20:46 on PI3K activity was detectable at 5 µM fatty
acid (Fig. 2). Although PI3K activity in
neutrophils increased in a dose-dependent manner,
increasing the fatty acid concentration above 5 µM did
not produce a greater effect on kinase activity in HL60 cells.
View larger version (25K):
[in a new window]
Fig. 1.
Kinetics of PI3K activation by
20:4 6. Neutrophils (3 × 107 cells in 30 ml of HBSS) (a), HL60 cells
(2 × 107 cells in 20 ml of HBSS) (b), and
HUVECs (6 × 106 cells in 6 ml of HBSS)
(c) were incubated with 20:46 (20 µM) for
the indicated times at 37 °C and lysed. Fractions were assayed for
PI3K activity as described under "Experimental Procedures." To
visualize the PtdIns 3P spots and quantify the radioactivity associated
with the phosphorylated lipid, the thin layer chromatography plates
were loaded onto an Instant Imager. A representative image is shown for
each set of experiments. The histograms (-fold stimulation) represent
means ± S.E. of 3-4 separate experiments. Each experiment with
neutrophils and HUVECs was conducted using cells from a different donor
or cord, respectively. Significance of difference: p < 0.05 by analysis of variance.
View larger version (20K):
[in a new window]
Fig. 2.
Dose-dependent stimulation of
PI3K activity by 20:4 6. Neutrophils
(a) and HL60 cells (b) were incubated with
20:46 at the concentrations indicated for 5 min and lysed. PI3K was
immunoprecipitated, and kinase activity was determined as described
under "Experimental Procedures." The results are means ± S.E.
of three (a) or mean ± range of two (b)
separate experiments. Significance of difference (a):
p < 0.05 by analysis of variance.
6
increased the level of tyrosine phosphorylation of the p85 subunit
of PI3K (Fig. 3). The kinetics for this
change in phosphorylation were similar to those found for the increase
in kinase activity (Fig. 1a). To confirm that tyrosine
phosphorylation was required for the activation of PI3K by 20:46,
neutrophils and HUVECs were preincubated with lavendustin A, a tyrosine
kinase inhibitor. The data in Fig. 3, b and c,
show that pretreatment of HUVECs and neutrophils, respectively, with
lavendustin A for 10 min caused a dose-dependent inhibition
of the ability of 20:46 to stimulate PI3K. Lavendustin B, a negative
control for lavendustin A, did not affect the ability of 20:46 to
stimulate PI3K activity (data not shown). These data demonstrate that
20:46 stimulated the activity of PI3K in a tyrosine
kinase-dependent manner.
View larger version (17K):
[in a new window]
Fig. 3.
Role of tyrosine phosphorylation
in the stimulation of PI3K activity by
20:4 6. Neutrophils (3 × 107 cells in 30 ml of HBSS) were incubated with 20:46
(20 µM) for the indicated times at 37 °C and lysed.
After immunoprecipitation with the anti-phosphotyrosine antibody, PY99,
the samples were subjected to Western blot analysis using the
anti-p85 subunit antibody (a) as described under
"Experimental Procedures." In other experiments, HUVECs
(b) and neutrophils (c) were preincubated with
Me2SO or lavendustin A (0.01 or 0.1 µM) for
10 min before being stimulated with 20:46. After lysis and
immunoprecipitation, kinase activity was determined as described under
"Experimental Procedures." Results (means ± S.E. of three
experiments) are expressed as -fold stimulation over control
(b and c). The numbers in parentheses represent
band density (arbitrary units) (a). Significance of
difference between 20:46 and 20:46 plus inhibitor: *,
p < 0.05; **, p < 0.01.
Lavendustin A was discovered as an inhibitor of the epidermal growth
factor (EGF) receptor (34). Our previous studies in WB rat liver
epithelial cells have demonstrated that 20:46 caused the appearance
of a major tyrosine-phosphorylated band that migrated on
SDS-polyacrylamide gel electrophoresis (12% gels) with a molecular mass that was in excess of 138 kDa (20). This protein was also strongly tyrosine-phosphorylated in cells that were stimulated with
either lysophosphatidic acid or EGF (20). Analyzed on 8% SDS gels,
lysates from 20:46-stimulated HUVECs were found to contain two major
proteins (molecular masses of 180-190 and 85 kDa) that exhibited
enhanced levels of tyrosine phosphorylation compared with those from
control cells (Fig. 4a).
Interestingly, in lysophosphatidic acid-stimulated COS and Vero cells
(35), a tyrosine-phosphorylated protein of 170-180 kDa has been
identified recently as the EGF receptor. It was found that
transactivation of the EGF receptor was necessary for the stimulation
of PI3K by lysophosphatidic acid (35). Although the 85-kDa protein
(Fig. 4a) was likely to be the p85 subunit of PI3K, the
180-190-kDa protein could be a member of the ErbB receptor family.
This receptor family is composed of ErbB1 (the EGF receptor), ErbB2,
ErbB3, and ErbB4 (36), and HUVECs have been reported to express ErbB2, ErbB3, and ErbB4 but not ErbB1 (37). Lysates from HUVECs therefore were
subjected to immunoprecipitation with PY99 and Western blotting with an
antibody against ErbB2, ErbB3, or ErbB4. The data in Fig. 4c
demonstrate that 20:46 selectively enhanced the tyrosine
phosphorylation of ErbB4. This effect was partially reduced by AG-1478,
an EGF receptor-specific inhibitor that also inhibits ErbB2, ErbB3, and ErbB4 (38). 20:46 did not stimulate the tyrosine phosphorylation of
ErbB2 (Fig. 4b) or ErbB3 (Fig. 4d). The ability
of the anti-ErbB2 and anti-ErbB3 antibodies to detect ErbB2 and ErbB3,
respectively, was confirmed in Chinese hamster ovary cells transfected
with ErbB2 or ErbB3 (31) (Fig. 4).
|
To determine whether transactivation of ErbB4 was required for the
stimulation of PI3K activity by 20:46, HUVECs were pretreated with
AG-1478. The data in Fig. 5 demonstrate
that AG-1478 suppressed PI3K activation by 20:46. In contrast,
AG-1296, a specific platelet-derived growth factor receptor inhibitor,
did not affect the ability of 20:46 to stimulate PI3K activity in
HUVECs (data not shown). These data are consistent with transactivation
of ErbB4 being required for the activation of PI3K in HUVECs by
20:46. The data with lavendustin A (Fig. 3c) suggest that
a member of the ErbB receptor family could also be involved in the
activation of PI3K by the fatty acid in neutrophils.
|
Our previous studies have demonstrated that the ability of 20:46 to
stimulate the activity of p38 was not suppressed by inhibitors of the
cyclooxygenases and lipoxygenases (22). To investigate whether
inhibitors of the cyclooxygenases and lipoxygenases affected the
ability of 20:46 to stimulate PI3K activity, neutrophils were
preincubated with indomethacin and nordihydroguaiaretic acid, respectively. The data in Fig.
6a demonstrate that
indomethacin and nordihydroguaiaretic acid each partially inhibited
20:46-stimulated PI3K activity. In contrast, the cytochrome P450
monooxygenase inhibitor, 17-octadecynoic acid, did not affect kinase
activity (data not shown). These data suggest that metabolism of
20:46 by cyclooxygenases and lipoxygenases but not by cytochrome
P450 monooxygenases was required for stimulation of PI3K activity. The
mechanisms by which 20:46 stimulated the activity of PI3K and p38
were therefore distinct. The 5-lipoxygenase is the most prominent
lipoxygenase in neutrophils (3), and activated neutrophils liberate
5-hydroxyeicosatetraenoic acid (5-HETE) into the incubation medium. In
support for a role for the lipoxygenases in the action of 20:46 on
PI3K activity, incubation of neutrophils with 5-HETE resulted in the
activation of class Ia PI3K (Fig. 6b). These data suggest
that 5-HETE may mediate, at least in part, the effect of 20:46 on
PI3K activity in neutrophils.
|
A hallmark of receptor-mediated activation of class Ia PI3K is the
activation of Akt (25). This is mediated through the phosphorylation of
Akt on Thr-308 and Ser-473 by PDK1 and PDK2 (possibly a modified
PDK1) in the presence of PtdIns 3,4 P2 and/or PtdIns 3,4,5 P3 (25). Activation of PI3K is also accompanied by the
activation of p70S6 kinase. Both PDK1 and Akt are implicated in
mediating this response (39). Interestingly, it has been demonstrated
that stimulation of p70S6 kinase activity by the 20:46 metabolites,
8,12-iso-isoprostaneF2
-III and
prostaglandin F2, in ventricular myocytes was not
accompanied by Akt phosphorylation. This is despite the demonstration
that the effects of
8,12-iso-isoprostaneF2-III and prostaglandin
F2 on p70S6 kinase were suppressed by the PI3K
inhibitor, wortmannin (40). Because prostaglandin F2 has
been reported to stimulate PI3K activity in vascular smooth muscle
cells (41), the data from the above study (40) imply that under certain
circumstances activation of PI3K could be uncoupled from Akt
activation. To investigate whether 20:46 was able to activate Akt,
lysates from 20:46-stimulated neutrophils were examined for Akt
phosphorylation. The data in Fig. 7
demonstrate that 20:46 caused the phosphorylation of Akt on Thr-308
(Fig. 7a) and Ser-473 (Fig. 7b). The bacterial
tripeptide fMLP also stimulated the phosphorylation of Akt (Fig.
7a), consistent with its ability to activate class Ib
PI3K (28) and class Ia PI3K (Fig. 7c). Similar to the
kinetics of PI3K activation by 20:46 (Fig. 1), the increase in
Thr-308 phosphorylation was transient although Ser-473 phosphorylation
was maintained for at least 10 min. The presence of approximately equal
amounts of Akt within each lane was confirmed in duplicate blots
stained with an anti-Akt antibody (data not shown). The data suggest a
distinctive effect of 20:46 on the PI3K-Akt signaling pathway, one
that is not observed with cyclooxygenase products of the fatty acid.
The data also argue against of
8,12-iso-isoprostaneF2-III and prostaglandin F2 being responsible for the stimulatory effect of
20:46 on PI3K and Akt activities in neutrophils.
|
PI3K has been reported to be an upstream regulator of the ERK1, ERK2,
and p38 MAP kinase cascades when cells are stimulated by certain
agonists (42, 43). Given that 20:46 stimulates the activities of MAP
kinases (20-23), a possible role for PI3K in the regulation of ERK1
and ERK2 activation by 20:46 was investigated. The data in Fig.
8a demonstrate that the
ability of 20:46 to stimulate ERK dual phosphorylation in
neutrophils was not inhibited by wortmannin, even at a concentration of
100 nM. A duplicate blot stained with the anti-ERK2
antibody confirmed the presence of approximately equal amounts of ERK2
within each lane (data not shown). Although the data show strong ERK2
(p42-phospho-ERK band) phosphorylation in samples prepared from
20:46-stimulated cells, phospho-ERK1 (the p43-phospho-ERK band) was
also detectable, albeit less consistently than phospho-ERK2. However,
upon longer exposure of the nitrocellulose sheet to film, the
phospho-ERK1 bands were readily visible (data not shown). Wortmannin
(1-100 nM) also failed to suppress p38 activation by
20:46 in neutrophils (data not shown). These results reinforce the
notion that different mechanisms were involved in the stimulation of
PI3K and MAP kinase activities by 20:46. To determine whether PI3K
plays a functional role in 20:46-stimulated cells, the effect of
wortmannin on 20:46-stimulated neutrophil respiratory burst was
investigated. The data in Fig. 8b demonstrate that
wortmannin dose-dependently inhibited 20:46-stimulated superoxide production with an IC50 of ~1 nM
(Fig. 8b). Neutrophil viability was not affected as
determined by the trypan blue exclusion test.
|
In addition to 20:46, fatty acids in the 3 and 9 series are
also biologically active. Both docosahexaenoic acid (22:63) and
oleic acid (18:19) are released from membrane phospholipids by the
group II and group V secretory phospholipase A2,
respectively, in activated cells (44, 45). Although some fatty acids
may differ in their actions and their ability to affect cellular
functions, different fatty acids have been demonstrated to mimic each
other with respect to certain actions such as stimulation of the
neutrophil respiratory burst (3, 6, 11). The data in Fig.
9 demonstrate that 22:63 and 18:19,
at equimolar concentrations to 20:46, also stimulated the activity
of PI3K, albeit to a slightly lower degree than 20:46. In contrast,
the saturated fatty acid, stearic acid, which does not affect the
neutrophil respiratory burst (3), did not increase PI3K activity. These
data suggest that the ability of fatty acids to stimulate PI3K activity
is restricted to mono- and polyunsaturated fatty acids.
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DISCUSSION |
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ABSTRACT
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DISCUSSION
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Exogenously applied 20:46 alters cellular functions at
concentrations reported to be present in stimulated cells (46). This is
likely to be because of the ability of 20:46 or metabolites to
stimulate the activities of intracellular signaling molecules such as
PKC, the MAP kinases, and phospholipase A2 or to modulate ion channel activity and cause an increase in intracellular
Ca2+ concentration (14-24). The data in the present study
demonstrate that the addition of exogenous 20:46 stimulated the
activity of class Ia PI3K.
An interesting finding of the present study is that 20:46 activates
ErbB4 in HUVECs and that inhibition of this receptor by AG-1478
suppressed the ability of the fatty acid to stimulate PI3K. Although
neutrophils have not been demonstrated to express ErbB receptors, these
leukocytes produce heparin-binding EGF-like growth factor (47) that
binds ErbB1 or ErbB4 (36). The data with lavendustin A suggest that a
member of the ErbB receptor family could also be involved in
neutrophils. Although our previous studies in WB epithelial cells have
demonstrated that 20:46, lysophosphatidic acid, and EGF each
stimulated the tyrosine phosphorylation of a protein with a molecular
mass in excess of 138 kDa (20), an accurate assessment of the molecular
mass of the protein in the 12% gel was not possible. Nevertheless, our
data (20) hinted at the possibility that the tyrosine-phosphorylated
protein was the EGF receptor. Interestingly, 20:46 has been reported
recently to stimulate the tyrosine phosphorylation of the EGF receptor in renal proximal tubule epithelial cells (48). Our demonstration that
ErbB4 (HUVECs) or another member of the ErbB receptor family (neutrophils) is involved in the activation of PI3K not only provides a
novel mechanism by which 20:46 stimulated the activity of this lipid
kinase but also significantly enhances our understanding of how the
actions of 20:46 are mediated. Although transactivation of the EGF
receptor has been reported for an increasingly large number of
non-EGF-like ligands, our novel results demonstrate that ErbB4 can also
be transactivated. How 20:46 stimulated the tyrosine phosphorylation
of ErbB4 in HUVECs remains to be investigated.
Although PI3K has been reported to be an upstream regulator of MAP
kinases under certain circumstances (42, 43), our data in neutrophils
demonstrate that stimulation of ERK1, ERK2, and p38 activities by
20:46 did not require PI3K. This is in direct contrast to reports
that fMLP- and interleukin 8-stimulated ERK1/ERK2 activation in
neutrophils depends on PI3K (43, 49). Similarly, insulin-stimulated
ERK1/ERK2 activation in rat adipocytes requires PI3K (42). However,
this cross-talk between PI3K and the MAP kinase signaling pathways does
not operate ubiquitously (25) and may depend on a number of factors.
Thus, although antigen receptor activation of the MAP kinase kinase
kinase was inhibited by wortmannin, suggesting a PI3K involvement, EGF
stimulation of MAP kinase kinase kinase was wortmannin-insensitive
(50). Even within the same cell type, the culture condition may affect the dependence of the MAP kinase pathways on PI3K. Thus, insulin-like growth factor 1-stimulated activation of ERK1 and ERK2 in adherent MCF-7 cells was inhibited by the PI3K inhibitor, LY294002, whereas stimulation of ERK1 and ERK2 activity by this growth factor in MCF-7
cells cultured in suspension was not (51). The ability of wortmannin to
inhibit MAP kinase activation may depend on signal strength (52).
Furthermore, because PI3K has been demonstrated to translocate to
different cellular compartments in an agonist-dependent manner (53), it is conceivable that the coupling of PI3K to individual
MAP kinase modules may also depend on the compartment in which members
of the PI3K-Akt signaling pathway are localized. Based on our previous
demonstrations that 20:46 stimulated the translocation of PKC to a
particulate fraction and that depletion of cellular PKC prevented the
activation of ERK1 and ERK2 by 20:46 (20), the evidence supports PKC
but not PI3K being an upstream regulator of ERK1 and ERK2 in
20:46-stimulated cells. This is despite PDK1 being implicated in
phosphorylating the activation or T-loop of AGC Ser/Thr protein kinases
(PKA, PKG, and PKC) including a number of PKC isozymes and MAP kinases
(25).
Although cyclooxygenase and lipoxygenase inhibitors did not suppress
20:46-stimulated activation of p38 in neutrophils (22), stimulation
of PI3K activity in this cell type depended, at least in part, on
products of the cyclooxygenases and lipoxygenases. Metabolism of the
fatty acid by cytochrome P450 CYP4A gene family members was
not required. Despite the demonstration that prostaglandin F2 (41) up-regulated the activity of PI3K and the
implied activation of PI3K by
8,12-iso-isoprostaneF2-III (40), an
involvement of these metabolites in the activation of the PI3K-Akt pathway by 20:46 was excluded. Neither
8,12-iso-isoprostaneF2-III nor prostaglandin
F2 stimulated Akt phosphorylation (40). Recently, the
peptido-leukotriene, leukotriene D4, has also been reported
to stimulate the activity of PI3K in renal mesangial cells (54).
However, it is unlikely that leukotriene D4 was involved in
the stimulation of PI3K activity by 20:46 in neutrophils because
leukotriene D4-stimulated activation of ERK1, ERK2, and p38
was inhibited by wortmannin (54), whereas 20:46-stimulated MAP
kinase activation in neutrophils was not. Moreover, mature myeloid cells do not produce leukotriene D4 (55). In
contrast to our data with 20:46, leukotriene
D4-stimulated PI3K was not affected by AG-1478 but was
suppressed by AG-1296 (54). Further studies will be needed to determine
whether the above-described differences are caused by tangible
differences in action between leukotriene D4 and
prostaglandin F2 on the one hand and 20:46 or a
metabolite(s) of the fatty acid other than leukotriene D4, isoprostanes, and prostaglandins on the other. Our data obtained from
neutrophils suggest that a 5-lipoxygenase product such as 5-HETE may be
involved in mediating the effect of 20:46 on PI3K activity in this
cell type, and this is consistent with the 5-lipoxygenase being the
most prominent lipoxygenase in neutrophils (3).
It is therefore clear that a number of 20:46 metabolites can
stimulate the activity of PI3K, albeit with differences in the ability
of these metabolites to activate Akt, to couple PI3K to MAP kinase
modules, and the differential utilization of the EGF and
platelet-derived growth factor receptor (40, 41, 54). Interestingly, 20:46 metabolites that are capable of inhibiting PI3K
have also been identified. Most notable is lipoxin A4. This metabolite, formed by a transcellular biosynthetic route (56), has been
found to inhibit the ability of leukotriene D4 to stimulate PI3K (54). One of the best characterized actions of lipoxin A4 is its ability to negatively regulate neutrophil and
eosinophil function (56). It is possible that in these cell types, the response to ligands that simultaneously mobilize 20:46 and activate PI3K will by modulated by the balance between the levels of lipoxins and those of the other metabolites in the milieu of the cells.
3 and 9 fatty acids, but not the saturated stearic acid, are also
capable of activating PI3K despite being the sources of vastly
different metabolites. This implies that 20:46-derived metabolites
are not strictly required for the stimulation of PI3K by an unsaturated
fatty acid. In in vitro assays, 20:46 and 18:19 directly activate purified PKC (14), whereas in intact cells, the 3
fatty acids are able to stimulate ERK1 and
ERK2.2 Thus, the ability of
22:63 and 18:19 to stimulate the activity of PI3K is consistent
with their ability to evoke some of the cellular responses observed
with 20:46. These responses include the induction of HL60 cell
differentiation and the stimulation of neutrophil and macrophage
superoxide production, adhesion to plastic, and up-regulation of
CD11b/CD18 (3, 6). In contrast, saturated fatty acids are devoid of
these activities. Although not generally considered to be second
messenger molecules, there is evidence that 22:63 and 18:19 are
released from membrane phospholipids in activated cells by the group II
and group V secretory phospholipase A2, respectively, (44,
45). The levels of the secretory phospholipase A2 are known
to be elevated in tissue fluids under certain pathophysiological
conditions such as in the synovial fluid of rheumatoid arthritis
patients (57). Thus, the presence of these unsaturated fatty acids in
these fluids is a possibility. Given the current interest in diet
manipulation in favor of monounsaturated and 3 fatty acid, our
finding that 3 and 9 fatty acids stimulated PI3K activity may be
physiologically relevant. We speculate that the release of these fatty
acids by the secretory phospholipase A2 in activated cells
will continue to provide the modulation of PI3K by fatty acids, despite
the shift in the membrane phospholipid fatty acid content away from 6 to 3 and 9 species. Our demonstration that PI3K plays an important role in neutrophil-mediated articular cartilage destruction in rheumatoid arthritis (32) highlights the dilemma in separating the
anti-inflammatory properties of 3 fatty acids from their stimulatory
actions on neutrophil respiratory burst (3), a process that depends on
PI3K.
A role for PI3K in 20:46-stimulated superoxide production was
established in this study. Activation of the PI3K-Akt signaling pathway
is known to exert an antiapoptotic effect on certain cell types,
possibly as a consequence of the actions of Akt on components of the
apoptotic machinery such as phosphorylation of BAD (25). The PI3K-Akt
pathway has also been implicated in the promotion of cellular
proliferation (25). Interestingly, 20:46 has been reported to
promote the growth of certain tumor (9) and nontumor cell types (10).
The enhancement of carcinosarcoma cell growth by 20:46 has also been
shown to be accompanied by the suppression of apoptosis (9). Thus, it
is possible that the growth-promoting and apoptosis-suppressing effects
of 20:46 on these cell types could be mediated, at least in part,
via the activation of PI3K and Akt. In HL60 cells, 20:46 and 3
fatty acids have been reported to induce granulocytic differentiation,
which is accompanied by the suppression of proliferation and the
induction of cell death by apoptosis and necrosis (6). This action is
similar to that induced by all-trans-retinoic acid, a known
inducer of granulocytic differentiation (6). Because PI3K is required
for the induction of granulocytic differentiation by retinoic acid
(58), our data offer an explanation for how 20:46 and 3 fatty
acids were able to induce HL60 cell differentiation. The
differentiation program may over-ride the survival signals provided by
the PI3K-Akt axis, thereby giving rise to the cell death that
accompanies differentiation.
We have suggested previously that 20:46 liberated through the
activation of the cytosolic phospholipase A2 could amplify or prolong intracellular signaling initiated by a receptor that is
coupled to the enzyme (20, 22). In this scenario, the binding of an
agonist to its receptor results in the activation, on the one hand, of
signaling molecules such as p21ras, PKC, ERK1, ERK2, and/or
p38, and on the other hand, the activities of other signaling molecules
such as PI3K and Akt. Activated ERK1, ERK2, and/or p38 phosphorylate
the cytosolic phospholipase A2 (59, 60), and if the
intracellular Ca2+ concentration is also elevated after
receptor engagement, phospholipid hydrolysis by the cytosolic
phospholipase A2 occurs. This sets up a signaling loop. The
demonstration that exogenous 20:46 stimulates the activities of PKC,
ERK1, ERK2, p38, the cytosolic and secretory phospholipase
A2, PI3K, and the phosphorylation of Akt (20, 22,
24) is consistent with such a loop. Current evidence, although
incomplete, suggests that such a loop may exist, at least in human
neutrophils. Thus, ligation of the fMLP or Fc (FcRIIa or
FcRIIIb) receptors results in the activation of all the above-listed signaling molecules and liberation of 20:46 (61-63). To
further prove the existence of the loop, it is also essential to
demonstrate that inhibition of phospholipase A2 decreases
the magnitude of receptor-mediated activation of signaling molecules
that are upstream of phospholipase A2 such as the MAP
kinases. Although evidence for this in neutrophils is still lacking,
studies in human monocytic cells have demonstrated that inhibition of
either the activity or the expression of phospholipase A2
resulted in the attenuation of ligand-stimulated activation of MAP
kinases (64). The ability of inhibitors of phospholipase
A2, ERK, or p38 modules to partially inhibit not only fMLP-
(32, 63) but also 20:46- (24, 65) stimulated superoxide production
suggests that this positive feedback loop in neutrophils is functional.
The idea of positive feedback loops in intracellular signaling
involving the PKC-ERK1 and ERK2-phospholipase A2 loop has
recently been the subject of a modeling study (66). Depending on the
strength and duration of the initial signal at the cell surface, the
model predicts that signaling via these loops can become
self-sustaining, and this is supported by our observation that
exogenous 20:46 stimulated the activation of the cytosolic and
secretory phospholipase A2 (24). The model proposes that
the level of MAP kinase phosphatase-1 expression is one mode by which
signaling via the PKC-ERK-phospholipase A2 positive
feedback loop can be terminated (66). Interestingly, 20:46 has been
reported to induce the expression of MAP kinase phosphatase-1 (5).
Because our data in neutrophils show that PI3K is not required for the
activation of MAP kinases by 20:46, it is unlikely that PI3K is a
participant in the loop, at least in neutrophils.
In summary, the present study establishes that unsaturated fatty acids
such as 20:46 are capable of stimulating the class Ia PI3K-Akt
signaling pathway. Although eicosanoid products of 20:46 are
required for the activation of PI3K by 20:46 in myeloid cells, our
data strongly suggest that a member of the ErbB receptor family is also
required. In HUVECs, this was ErbB4. Fig.
10 summarizes the major signaling
pathways that 20:46 has been found to activate in intact cells.
|
| |
ACKNOWLEDGEMENTS |
|---|
We thank Ms. M. Busitill, Ms. L. Marin, and Dr. Y. Q. Li for technical assistance and Professor Yosef Yarden (Weizmann Institute of Science, Israel) for the generous gift of Chinese hamster ovary cells stably expressing either the ErbB2 or ErbB3 receptors.
| |
FOOTNOTES |
|---|
* This work was supported in part by grants from the National Heart Foundation, Channel 7 Children's Research Foundation, and the National Health and Medical Research Council of Australia.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 should be addressed: Dept. of Immunopathology, Women's and Children's Hospital, 72 King William Rd., North Adelaide, South Australia 5006. Tel.: 61-08-8161-6293; Fax: 61-08-8161-6046; E-mail: chii01@mail.staff.adelaide.edu.au.
Published, JBC Papers in Press, May 18, 2001, DOI 10.1074/jbc.M103250200
2 C. S. T. Hii, Z. H. Huang, and A. Ferrante, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
20:46, arachidonic acid;
MAP, mitogen-activated protein;
PKC, protein kinase C;
PKB, protein kinase B;
PI3K, phosphatidylinositol
3-kinase;
PtdIns, phosphatidylinositol;
fMLP, formyl methionyl leucyl
phenylalanine;
HUVEC, human umbilical vein endothelial cell;
HBSS, Hanks' balanced salt solution;
ERK, extracellular
signal-regulated protein kinase;
EGF, epidermal growth factor;
5-HETE, 5-hydroxyeicosatetraenoic acid;
PDK, 3'-phosphoinositide-dependent kinase;
22:63, docosahexaenoic acid;
18:19, oleic acid.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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B.-S. Li, W. Ma, H. Jaffe, Y. Zheng, S. Takahashi, L. Zhang, A. B. Kulkarni, and H. C. Pant Cyclin-dependent Kinase-5 Is Involved in Neuregulin-dependent Activation of Phosphatidylinositol 3-Kinase and Akt Activity Mediating Neuronal Survival J. Biol. Chem., September 12, 2003; 278(37): 35702 - 35709. [Abstract] [Full Text] [PDF]
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J. Liu, Z. Liu, S. Chuai, and X. Shen Phospholipase C and phosphatidylinositol 3-kinase signaling are involved in the exogenous arachidonic acid-stimulated respiratory burst in human neutrophils J. Leukoc. Biol., September 1, 2003; 74(3): 428 - 437. [Abstract] [Full Text] [PDF]
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 N. Moghaddami, M. Costabile, P. K. Grover, H. P. A. Jersmann, Z. H. Huang, C. S. T. Hii, and A. Ferrante Unique Effect of Arachidonic Acid on Human Neutrophil TNF Receptor Expression: Up-Regulation Involving Protein Kinase C, Extracellular Signal-Regulated Kinase, and Phospholipase A2 J. Immunol., September 1, 2003; 171(5): 2616 - 2624. [Abstract] [Full Text] [PDF]
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 L. A. deGraffenried, W. E. Friedrichs, L. Fulcher, G. Fernandes, J. M. Silva, J.-M. Peralba, and M. Hidalgo Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity Ann. Onc., July 1, 2003; 14(7): 1051 - 1056. [Abstract] [Full Text] [PDF]
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S. Grenier, N. Flamand, J. Pelletier, P. H. Naccache, P. Borgeat, and S. G. Bourgoin Arachidonic acid activates phospholipase D in human neutrophils; essential role of endogenous leukotriene B4 and inhibition by adenosine A2A receptor engagement J. Leukoc. Biol., April 1, 2003; 73(4): 530 - 539. [Abstract] [Full Text] [PDF]
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E. Burkert, D. Szellas, O. Radmark, D. Steinhilber, and O. Werz Cell type-dependent activation of 5-lipoxygenase by arachidonic acid J. Leukoc. Biol., January 1, 2003; 73(1): 191 - 200. [Abstract] [Full Text] [PDF]
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 R. Kettritz, M. Choi, W. Butt, M. Rane, S. Rolle, F. C. Luft, and J. B. Klein Phosphatidylinositol 3-Kinase Controls Antineutrophil Cytoplasmic Antibodies--Induced Respiratory Burst in Human Neutrophils J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1740 - 1749. [Abstract] [Full Text] [PDF]
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