Cytosolic Phospholipase A2 Is an Effector of Jak/STAT
Signaling and Is Involved in Platelet-derived Growth Factor BB-induced
Growth in Vascular Smooth Muscle Cells*
Chandrahasa R.
Yellaturu
and
Gadiparthi N.
Rao
§¶
From the
Department of Physiology and the
§ Center for Vascular Biology, University of Tennessee
Health Science Center, Memphis, Tennessee 38163
Received for publication, November 4, 2002, and in revised form, January 3, 2003
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ABSTRACT |
Platelet-derived growth factor-BB (PDGF-BB) is a
potent mitogen and chemoattractant for vascular smooth muscle cells
(VSMC). To understand its mitogenic and chemotactic signaling events, we studied the role of cytosolic phospholipase A2
(cPLA2) and the Jak/STAT pathway. PDGF-BB induced the
expression and activity of cPLA2 in a
time-dependent manner in VSMC. Arachidonyl trifluoromethyl ketone, a potent and specific inhibitor of cPLA2,
significantly reduced PDGF-BB-induced arachidonic acid release and DNA
synthesis. PDGF-BB stimulated tyrosine phosphorylation of Jak-2 in a
time-dependent manner. In addition, PDGF-BB activated
STAT-3 as determined by its tyrosine phosphorylation, DNA-binding
activity, and reporter gene expression, and these responses were
suppressed by AG490, a selective inhibitor of Jak-2. AG490 and a
dominant-negative mutant of STAT-3 also attenuated PDGF-BB-induced
expression of cPLA2, arachidonic acid release, and DNA
synthesis in VSMC. Together, these results suggest that induction of
expression of cPLA2 and arachidonic acid release are
involved in VSMC growth in response to PDGF-BB and that these events
are mediated by Jak-2-dependent STAT-3 activation.
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INTRODUCTION |
Increased vascular smooth muscle cell
(VSMC)1 growth is one of the
major components in the thickening of the arterial wall in the
pathogenesis of atherosclerosis and restenosis (1). A variety of
molecules (including peptide growth factors, hormones, eicosanoids, and
oxidants) that are generated at the site of arterial injury and/or
inflammation can influence the growth and migration of VSMC (1-6).
Indeed, increased levels of growth factors such as platelet-derived
growth factor, eicosanoids such as hydroxyeicosatetraenoic acids, and
oxidants such as oxidized low density lipoprotein have been reported in
atheromatous arteries compared with normal arteries (3-9). As these
molecules utilize divergent early mitogenic signaling events in the
induction of VSMC growth (10, 11), targeting inhibition of the activity
of a single mitogen might not be able to suppress VSMC growth and
lesion formation. However, identifying the mechanisms that are less
redundant and that are involved in the mitogenic and chemotactic
activities of many of these molecules may advance therapeutic
developments that mitigate VSMC growth and vessel wall lesions.
Arachidonic acid, a polyunsaturated fatty acid, is an important
component of membrane phospholipids and is released acutely in response
to a variety of agonists, including growth factors, cytokines,
hormones, and oxidants (12-16). Upon release, it is either metabolized
via the cyclooxygenase, lipoxygenase, or cytochrome P450 monooxygenase
pathway, producing prostaglandins, hydroperoxyeicosatetraenoic acids,
or epoxyeicosatrienoic acids, respectively, or is reincorporated into
membrane phospholipids via esterification involving arachidonoyl-CoA synthase and arachidonoyl lysophospholipid transferase (12, 17).
Arachidonic acid and its oxygenative metabolites, known as eicosanoids,
are involved in the regulation of a variety of biological processes,
including vascular tone (17, 18). In addition, these lipid molecules
have been reported to mediate intracellular signaling events in
response to a number of stimuli (19-24). A large body of data also
suggest that arachidonic acid and its eicosanoid metabolites play an
important role in cell survival and growth (25-29). Among the members
of the large phospholipase A2 family characterized thus
far, cytosolic phospholipase A2 (cPLA2) appears
to be the major source of eicosanoid production in response to some
agonists in certain cell types (30-32).
Janus-activated kinases (Jak) are a group of non-receptor tyrosine
kinases that, via phosphorylation, modulate the activities of a group
of transcription factors, viz. signal transducers and activators of transcription (STAT) (33, 34). STAT proteins have
been reported to be involved in the regulation of cell growth and
differentiation (35-38). To understand the molecular events of
platelet-derived growth factor-BB (PDGF-BB)-induced growth and survival
in VSMC, we have studied the role of cPLA2 and the Jak/STAT
pathway. Here, we report for the first time that PDGF-BB induces the
expression of cPLA2 in VSMC in a sustained manner and that
this response requires Jak-2-dependent activation of STAT-3. In addition, we show that Jak-2/STAT-3-dependent
induction of expression of cPLA2 is required for
PDGF-BB-induced arachidonic acid release and growth in VSMC.
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MATERIALS AND METHODS |
Reagents--
Aprotinin, dithiothreitol, phenylmethylsulfonyl
fluoride, sodium orthovanadate, sodium deoxycholate, leupeptin, and
HEPES were purchased from Sigma. AG490 was obtained from Calbiochem. Arachidonic acid (AA) and a cPLA2 assay kit were bought
from Cayman Chemical Co., Inc. (Ann Arbor, MI). Anti-cPLA2
(2832) antibodies and phospho-specific anti-STAT-3 (9131S)
antibodies were procured from Cell Signaling Technology (Beverly, MA).
Phospho-specific anti-Jak-2 (44-426Z) antibodies were from Upstate
Biotechnology, Inc. (Lake Placid, NY). Anti-Jak-2 (sc-294) and
anti-STAT-3 (sc-482) antibodies and consensus oligonucleotides for
STAT-3 (5'-GATCCTTCTGGGAATTCCTAGATC-3') (sc-2571) were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). T4 polynucleotide
kinase was purchased from Invitrogen. [3H]AA (98 Ci/mmol), [
-32P]ATP (3000 Ci/mmol), and
[3H]thymidine (20 Ci/mmol) were obtained from PerkinElmer
Life Sciences.
Cell Culture--
VSMC were isolated from the thoracic aortas of
200-300-g male Sprague-Dawley rats by enzymatic dissociation as
described earlier (39). Cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated
fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cultures were maintained at 37 °C in a humidified 95%
air and 5% CO2 atmosphere. Cells were growth-arrested by
incubation in DMEM containing 0.1% calf serum for 72 h and used
to perform the experiments unless otherwise stated.
[3H]AA Release--
VSMC were labeled with
[3H]AA (0.3 µCi/ml) while growing exponentially; and at
90% confluence, cells were quiesced in DMEM containing 0.1%
calf serum and 0.2 µCi/ml [3H]AA for 72 h at
37 °C. Cells were then rinsed several times with DMEM. After
rinsing, cells were added to 2 ml of DMEM containing 0.1%
bovine serum albumin and treated with and without PDGF-BB (20 ng/ml) in
the presence and absence of the indicated pharmacological inhibitors
for 1 h, and [3H]AA release into the medium was
measured as described previously (15). In the case of testing the
effect of a dominant-negative STAT-3 mutant on [3H]AA
release, cells were transfected first with expression plasmid DNA for
the dominant-negative STAT-3 mutant, followed by labeling with
[3H]AA.
DNA Synthesis--
VSMC with and without appropriate treatments
were pulse-labeled with 1 µCi/ml [3H]thymidine for the
indicated times. After labeling, cells were washed with cold
phosphate-buffered saline, trypsinized, and collected by
centrifugation. The cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 20 min, the cell lysate mixture was passed through
a Whatman GF/C glass-fiber filter. The filter was washed once with cold
5% trichloroacetic acid and once with cold 70% (v/v) ethanol. The
filter was dried and placed in a liquid scintillation vial containing
the scintillant fluid, and the radioactivity was measured in a Beckman
LS 5000TA liquid scintillation counter.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from treated or untreated VSMC as described
previously (39). The protein content of the nuclear extracts was
determined using a Micro BCATM protein assay reagent kit
(Pierce). Protein·DNA complexes were formed by incubating 5 µg of
nuclear protein in a total volume of 20 µl consisting of 15 mM HEPES (pH 7.9), 3 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 µg of bovine serum albumin, 2 µg of poly(dI-dC), 15% glycerol, and
100,000 cpm of 32P-labeled oligonucleotide probe for 30 min
on ice. In some experiments, nuclear extracts were preincubated with
anti-STAT-3 antibodies for 3 h prior to protein-DNA binding assay.
The protein·DNA complexes were resolved by electrophoresis on a 4%
polyacrylamide gel using 1× Tris/glycine/EDTA buffer (25 mM Tris-HCl (pH 8.5), 200 mM glycine, and 0.1 mM EDTA). Double-stranded oligonucleotides were labeled with [
-32P]ATP using the T4 polynucleotide kinase kit
(Invitrogen) following the supplier's protocol.
Western Blot Analysis--
After appropriate treatments, VSMC
were rinsed with cold phosphate-buffered saline and frozen immediately
in liquid nitrogen. Cells were lysed by thawing in 250 µl of lysis
buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium
orthovanadate) and scraped into 1.5-ml Eppendorf tubes. After standing
on ice for 20 min, the cell lysates were cleared by centrifugation at
12,000 rpm for 20 min at 4 °C. Cell lysates containing an equal
amount of protein were resolved by electrophoresis on 0.1% SDS and
10% polyacrylamide gels. The proteins were transferred
electrophoretically to a nitrocellulose membrane (Hybond, Amersham
Biosciences). After blocking in 10 mM Tris-HCl (pH 8.0)
containing 150 mM sodium chloride, 0.1% Tween 20, and 5%
(w/v) nonfat dry milk, the membrane was treated with appropriate
primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences).
Transient Transfection and Chloramphenicol Acetyltransferase
(CAT) Assay--
VSMC were plated evenly onto 100-mm dishes and grown
in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. At
50-80% confluence, the medium was replaced with DMEM containing 0.1% calf serum, and cells were transfected with the pSIE-CAT plasmid using
LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. Thirty hours after transfection, VSMC
were treated with and without PDGF-BB (20 ng/ml) in the presence and
absence of AG490 (25 µM) for 4 h, and cell extracts
were prepared. VSMC lysates were normalized for protein and assayed for
CAT activity using [14C]chloramphenicol and acetyl
coenzyme A as substrates. In parallel experiments, VSMC were plated
evenly onto 60-mm dishes and grown in DMEM supplemented with 10% (v/v)
heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells at 90% confluence were transfected with
dominant-negative STAT-3 mutant plasmid (pFS3DM) DNA (5 µg/60-mm dish) using LipofectAMINE Plus reagent. Cells were washed
with phosphate-buffered saline 16 h after transfection and
incubated in DMEM containing 0.1% calf serum for 36 h at
37 °C. Cells were then treated with and without PDGF-BB (20 ng/ml)
for 36 h, and DNA synthesis was measured by pulse labeling cells
with 1 µCi/ml [3H]thymidine for the last 24 h of
the 36-h treatment period as described above.
Statistics--
All experiments were repeated three times with
similar results. Data on AA release, cPLA2 activity, and
DNA synthesis are presented as means ± S.D. The treatment effects
were analyzed by Student's t test. p values
<0.05 were considered to be statistically significant. In the case of
CAT activity, EMSA, and Western blot analyses, one representative set
of data is shown.
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RESULTS AND DISCUSSION |
To understand the mitogenic signaling events of PDGF-BB in VSMC,
we have studied the role of cPLA2. Quiescent VSMC were
treated with and without PDGF-BB (20 ng/ml) for various times, and cell extracts were prepared. Equal amounts of protein from control and
PDGF-BB-treated cells were analyzed by Western blotting for cPLA2 using its specific antibodies. PDGF-BB induced
cPLA2 expression in a time-dependent manner,
with 2- and 3-fold increases at 8 and 16 h of treatment,
respectively (Fig. 1A). The
increase in cPLA2 expression induced by PDGF-BB also
resulted in an increase in its activity as measured by hydrolysis of
arachidonoyl thiophosphatidylcholine using a commercially
available kit (Fig. 1B). Earlier studies from other
laboratories have reported that cPLA2 plays a role in AA
release and growth in response to some agonists (15, 40, 41). To test
the role of cPLA2 in receptor tyrosine kinase
agonist-induced AA release and growth, we studied the effect of
arachidonyl trifluoromethyl ketone (AACOCF3), a specific
inhibitor cPLA2 (42), on PDGF-BB-induced AA release and
growth. Quiescent VSMC that were prelabeled with [3H]AA
were treated with and without PDGF-BB (20 ng/ml) in the presence and
absence of AACOCF3 (10 µM) for 1 h, and
[3H]AA release was measured. PDGF-BB stimulated
[3H]AA release by ~6-fold, and this effect was
significantly inhibited by AACOCF3 (Fig.
2A). To understand the role of
cPLA2 in PDGF-BB-induced growth, quiescent VSMC were
treated with and without PDGF-BB (20 ng/ml) in the presence and absence
of AACOCF3 (10 µM) for 24 h, and growth
was measured by pulse labeling cells with 1 µCi/ml [3H]thymidine for the last 12 h of the 24-h
incubation period and determining the trichloroacetic acid-precipitable
counts/min. PDGF-BB stimulated [3H]thymidine
incorporation by 9-fold, and this response was completely inhibited by
AACOCF3 (Fig. 2B). AACOCF3 alone had
no toxic effects in VSMC, at least for 72 h as determined by
trypan blue dye exclusion assay.

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Fig. 1.
PDGF-BB induces the expression of
cPLA2 and its activity in VSMC. Quiescent VSMC were
treated with and without PDGF-BB (20 ng/ml) for the indicated times,
and cell extracts were prepared. An equal amount of protein from the
control and each treatment was analyzed by Western blotting for
cPLA2 using its specific antibodies (A) or
assayed for cPLA2 activity using a commercially available
kit (B). For a lane loading control, the same blot in
A was reprobed with anti-STAT-3 antibodies. *,
p < 0.01 versus control. PC,
phosphatidylcholine.
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Fig. 2.
AACOCF3, a selective inhibitor of
cPLA2, reduces PDGF-BB-induced AA release and DNA synthesis
in VSMC. A, VSMC were prelabeled with
[3H]AA, quiesced, and treated with and without PDGF-BB
(20 ng/ml) in the presence and absence of AACOCF3 (10 µM) for 1 h, and [3H]AA release was
measured. B, quiescent VSMC were treated with and without
PDGF-BB (20 ng/ml) in the presence and absence of AACOCF3
(10 µM) for 24 h, and DNA synthesis was measured by
pulse labeling cells with 1 µCi/ml [3H]thymidine for
the last 12 h of the 24-h treatment period. *, p < 0.01 versus control; **, p < 0.01 versus PDGF-BB treatment alone.
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To understand the signaling events underlying PDGF-BB-induced
expression of cPLA2 and AA release, we studied the role of
the Jak/STAT pathway. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for various times, and cell extracts were prepared. An equal amount of protein from the control and each treatment was
analyzed by Western blotting for tyrosine phosphorylation of Jak-2 and
STAT-3 using their phospho-specific antibodies. PDGF-BB stimulated
tyrosine phosphorylation of both Jak-2 and STAT-3 in a
time-dependent manner, with a
maximum effect of 5-15-fold at 10 min
and reaching basal levels by 4 h (Fig. 3A). Jak
proteins phosphorylate STAT proteins at tyrosine residues and activate them, although other mechanisms were also reported to be involved in
the activation of these transcription factors (43, 44). To determine
whether PDGF-BB-stimulated STAT-3 tyrosine phosphorylation is mediated
by Jak-2, we tested the effect of AG490, a potent and specific
inhibitor of Jak-2 (45). AG490 (25 µM) significantly inhibited PDGF-BB-stimulated tyrosine phosphorylation of STAT-3 (Fig.
3B). The inhibition of PDGF-BB-stimulated tyrosine
phosphorylation of STAT-3 by AG490 was not due to its toxic effects in
VSMC, as this compound did not affect the viability of these cells over a period of 72 h as measured by trypan blue dye exclusion assay. Because earlier studies have reported that PDGF-BB phosphorylates STAT-3 independent of Jak-2 and involving the PDGFR, we also tested whether AG490 affects PDGFR tyrosine phosphorylation. AG490 had no
effect on tyrosine phosphorylation of PDGFR induced by PDGF-BB, a
finding that suggests that the inhibitory effect of AG490 on STAT-3
tyrosine phosphorylation is specific.

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Fig. 3.
A, PDGF-BB stimulates tyrosine
phosphorylation of Jak-2 and STAT-3 in a time-dependent
manner in VSMC. Quiescent VSMC were treated with and without PDGF-BB
(20 ng/ml) for the indicated times, and cell extracts were prepared. An
equal amount of protein (40 µg) from the control and each treatment
was analyzed by Western blotting for phospho-Jak-2 (pJak-2)
and phospho-STAT-3 (pSTAT-3) using their phospho-specific
antibodies. B, PDGF-BB-stimulated tyrosine phosphorylation
of STAT-3 is sensitive to inhibition by the Jak-2 inhibitor AG490.
Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the
presence and absence of AG490 (25 µM) for 30 min, and
cell extracts were prepared. An equal amount of protein (40 µg) from
the control and each treatment was analyzed by Western blotting for
phospho-STAT-3 using its phospho-specific antibodies. As a loading
control, the blots were reprobed with anti-STAT-3 antibodies. In the
case of PDGFR tyrosine phosphorylation analysis, an equal amount of
protein from the control and each treatment was immunoprecipitated with
anti-PDGFR antibodies, and the resulting immunocomplexes were analyzed
by Western blotting using antibody PY20. pPDGF-R,
phospho-PDGFR
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Upon tyrosine phosphorylation, STAT proteins undergo either homo- or
heterodimerization and translocate to the nucleus, where they bind (in
this case, STAT-3) to their consensus DNA-binding sequence present in
the promoter regions of genes and induce transcription (33, 46).
To test whether STAT-3, upon its tyrosine phosphorylation induced by PDGF-BB, translocates to the nucleus, quiescent cells were
treated with and without PDGF-BB (20 ng/ml) for 30 min, and the
cytoplasmic and nuclear extracts were prepared. An equal amount of
protein from the cytoplasmic and nuclear extracts of control and
PDGF-BB-treated cells was analyzed by Western blotting for the levels
of tyrosine-phosphorylated STAT-3. Tyrosine-phosphorylated STAT-3
levels were detected only in the nuclear fraction of PDGF-BB-treated cells, and AG490 reduced these levels (Fig.
4). To determine whether translocation of
tyrosine-phosphorylated STAT-3 correlates with increased transcription
activation, STAT-3 DNA-binding activity was measured. Quiescent VSMC
were treated with and without PDGF-BB (20 ng/ml) for 2 h, and
nuclear extracts were prepared. An equal amount of nuclear protein from
the control and each treatment was analyzed by EMSA for STAT-3
DNA-binding activity using 32P-labeled STAT-3 consensus
oligonucleotide as a probe. PDGF-BB increased STAT-3 DNA-binding
activity by 6-fold, and it was inhibited by AG490 (Fig.
5A). Preincubation of nuclear
extracts with anti-STAT-3 antibodies also significantly reduced
PDGF-BB-induced protein·DNA complex formation (Fig. 5A).
This result suggests that PDGF-BB-induced protein·DNA complexes
contain STAT-3 either as homo- or heterodimers with other members of
the STAT transcription factor family. To confirm that increased STAT-3
DNA-binding activity leads to increased transactivation activity, cells
were transiently transfected with a STAT-3-dependent
reporter plasmid (pSIE-CAT), quiesced, and treated with and without
PDGF-BB (20 ng/ml) for 4 h, and cell extracts were prepared. Cell
extracts normalized for protein were assayed for CAT activity. PDGF-BB
induced STAT-3-dependent CAT activity by 4-fold, and AG490
substantially inhibited this response (Fig. 5B). To
understand whether the Jak/STAT pathway plays a role in PDGF-BB-induced
expression of cPLA2, we next studied the effect of AG490 on
this event. AG490 completely inhibited PDGF-BB-induced cPLA2 expression (Fig.
6A). Consistent with this
observation, AG490 also inhibited PDGF-BB-induced cPLA2
activity (Fig. 6B). To obtain additional evidence on the
role of the Jak/STAT pathway in PDGF-BB-induced
cPLA2 expression, we tested the effect of a dominant-negative STAT-3 mutant, FS3DM (38). As shown in Fig. 6C, forced expression of FS3DM blocked PDGF-BB-induced
expression of cPLA2. Next, we examined whether the
inhibition of cPLA2 expression by AG490 and the
dominant-negative STAT-3 mutant leads to decreased AA release. VSMC
that were prelabeled with [3H]AA and quiesced were
treated with and without PDGF-BB (20 ng/ml) in the presence or absence
of AG490 and/or forced expression of the dominant-negative STAT-3
mutant for 1 h, and [3H]AA release was measured.
Both AG490 and the dominant-negative STAT-3 mutant substantially
reduced PDGF-BB-induced [3H]AA release (Fig.
7A). To investigate whether
the Jak/STAT-dependent induction of expression of
cPLA2 and AA release are required for PDGF-BB-induced VSMC
growth, the effects of AG490 and the dominant-negative STAT-3 mutant on
PDGF-BB-stimulated DNA synthesis were studied. As shown in Fig.
7B, PDGF-BB-induced DNA synthesis was significantly reduced
by both AG490 and FS3DM. To validate the effects of FS3DM on
PDGF-BB-induced cPLA2 expression and DNA synthesis, its
effect on endogenous STAT-3 DNA-binding activity was tested. Forced
expression of FS3DM substantially reduced PDGF-BB-induced endogenous
STAT-3·DNA complexes in VSMC (Fig. 7C).

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Fig. 4.
AG490, a potent inhibitor of Jak-2, reduces
PDGF-BB-induced translocation of tyrosine-phosphorylated STAT-3 from
the cytoplasm to the nucleus. Quiescent VSMC were treated with and
without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 µM) for 30 min, and cytoplasmic and nuclear extracts were
prepared. An equal amount of protein (40 µg) from the cytoplasmic and
nuclear extracts of the control and PDGF-BB-treated cells was analyzed
by Western blotting for phospho-STAT-3 (pSTAT-3) using its
phospho-specific antibodies. As a loading control, the blot was
reprobed with anti-STAT-3 antibodies.
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Fig. 5.
AG490 reduces PDGF-BB-induced STAT-3
DNA-binding activity and STAT-3-dependent reporter gene
expression in VSMC. A, growth-arrested VSMC were
treated with and without PDGF-BB (20 ng/ml) in the presence and absence
of AG490 (25 µM) for 2 h, and nuclear extracts were
prepared. Five micrograms of nuclear protein from the control and each
treatment were incubated with 100,000 cpm of 32P-labeled
STAT-3 consensus oligonucleotide probe, and the protein·DNA complexes
were separated by EMSA and subjected to autoradiography. In some
protein-DNA binding reactions, nuclear extracts were preincubated with
anti-STAT-3 antibodies (Ab) for 3 h. B, VSMC
that were transfected with a STAT-3-dependent reporter
plasmid (pSIE-CAT) were quiesced and treated with and without PDGF-BB
(20 ng/ml) in the presence and absence of AG490 (25 µM)
for 4 h, and cell extracts were prepared. Cell extracts containing
an equal amount of protein from the control and each treatment were
analyzed for CAT activity using [14C]chloramphenicol and
acetyl coenzyme A as substrates. The substrate and products were
extracted with ethyl acetate, separated by TLC, and subjected to
autoradiography.
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Fig. 6.
A and B, AG490 inhibits
PDGF-BB-induced expression of cPLA2 and its activity.
Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the
presence and absence of AG490 (25 µM) for the indicated
times, and cell extracts were prepared. An equal amount of protein from
the control and each treatment was analyzed either for
cPLA2 expression by Western blotting using its specific
antibodies (A) or for cPLA2 activity using a
commercially available kit (B). PC,
phosphatidylcholine. C, the dominant-negative STAT-3 mutant
inhibits PDGF-BB-induced expression of cPLA2. VSMC were
transfected with and without the dominant-negative STAT-3 mutant
plasmid (pFS3DM), quiesced, and treated with and without PDGF-BB
(20 ng/ml) for 16 h, and cell extracts were prepared and analyzed
for cPLA2 levels as described for A. *,
p < 0.01 versus control; **,
p < 0.01 versus PDGF-BB treatment
alone.
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Fig. 7.
AG490 and a dominant-negative mutant of
STAT-3 inhibit PDGF-BB-induced [3H]AA release and DNA
synthesis in VSMC. A, VSMC that were prelabeled with
[3H]AA were treated with and without PDGF-BB (20 ng/ml)
in the presence and absence of AG490 (25 µM) and/or
forced expression of the dominant-negative STAT-3 mutant for 1 h,
and [3H]AA release was measured. B, quiescent
VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence
and absence of AG490 (25 µM) and/or forced expression of
the dominant-negative STAT-3 mutant for 24 h, and DNA synthesis
was measured by pulse labeling cells with 1 µCi/ml
[3H]thymidine for the last 12 h of the 24-h
treatment period. *, p < 0.01 versus
control; **, p < 0.01 versus PDGF-BB
treatment alone. C, VSMC were transfected with and without
the dominant-negative STAT-3 mutant plasmid (pFS3DM), quiesced,
and treated with and without PDGF-BB (20 ng/ml) for 2 h, and nuclear extracts were prepared. Five micrograms of nuclear
protein from the control and each treatment were incubated with 100,000 cpm of 32P-labeled STAT-3 consensus oligonucleotide probe,
and the protein·DNA complexes were separated by EMSA.
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The important findings of this study are as follows. 1) PDGF-BB, a
receptor tyrosine kinase agonist and a potent VSMC mitogen, induced the
expression of cPLA2 in a sustained manner in VSMC. 2)
PDGF-BB-induced expression of cPLA2 also resulted in an
increase in cPLA2 activity. 3) AACOCF3, a
selective inhibitor of cPLA2, attenuated PDGF-BB-induced AA
release and growth in VSMC. 4) Jak-2-dependent STAT-3
activation mediated PDGF-BB-induced cPLA2 expression and growth in VSMC. A number of PLA2 enzymes, particularly
group V secretory PLA2 and group IV cPLA2
enzymes, are involved in agonist-induced AA release (31, 32, 47, 48).
Interestingly, cross-activation between secretory PLA2 and
cPLA2 enzymes has been observed in acute and delayed
production of eicosanoids in many cell types, including human
neutrophils and murine macrophages and mast cells (30, 47, 48). Acute
activation of cPLA2 in response to a number of agonists
that are coupled to Ca2+ mobilization, particularly G
protein-coupled receptor agonists, cytokines, and phorbol esters, is
mediated by the mitogen-activated protein kinase cascade and/or protein
kinase C (49, 50). In this study, we have shown for the first time that
PDGF-BB stimulated sustained cPLA2 activity via induction
of its expression. Furthermore, the sustained expression and activity
of cPLA2 appeared to be mediated by and involved in
Jak/STAT-dependent PDGF-BB-induced growth in VSMC. This
conclusion is supported by the findings that AG490, a selective
inhibitor of Jak-2, and a dominant-negative mutant of STAT-3
substantially reduced the expression and activity of cPLA2
and DNA synthesis induced by PDGF-BB. Studies from other laboratories
also indicate that cPLA2 plays a role in serum-induced growth in human coronary artery smooth muscle cells (40). A potential
role for the Jak/STAT pathway in the regulation of cell growth,
differentiation, and survival activities in many cell types, including
hematopoietic cells, has been demonstrated (35-38, 51). Based on these
findings and the present observations, it is likely that
cPLA2 is one of the effector molecules that are involved in
Jak/STAT signaling leading to induction of growth in VSMC by PDGF-BB.
Some STAT transcription factors such as STAT-1 have also been reported
to be involved in the induction of expression of cell cycle inhibitory
molecules such as p21waf1/cip1 and pro-apoptotic enzymes
such as caspase-1 and thereby in apoptosis (52, 53). In this regard, it
is noteworthy that cPLA2-dependent AA release
mediates oxidant-induced apoptosis in some cell types (54). In view of
these findings, it can be speculated that cPLA2 is distal
in the path of Jak/STAT signaling to cell proliferation and/or
apoptosis. Future studies are required to test whether the
responsiveness of cPLA2 to various agonists of cell growth and apoptosis is dependent on activation of different members of the
STAT transcription factor family.
In summary, we have reported for the first time that the receptor
tyrosine kinase agonist PDGF-BB induces the expression of cPLA2 in a manner that is dependent on activation of the
Jak/STAT pathway. In addition, we have shown that
Jak/STAT-dependent induction of expression of
cPLA2 and AA release are involved in PDGF-BB-induced growth
in VSMC.
 |
ACKNOWLEDGEMENTS |
We are thankful to Drs. Ralph A. Bradshaw and
Howard Young for providing the pFS3DM and pSIE-CAT plasmids,
respectively. We are also grateful to Karunkumar Gadiparthi for typing
the manuscript and Peggy McKnight for editorial assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant RO1 HL65165 (to G. N. R.).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
Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-7321; Fax: 901-448-7126; E-mail:
grao@ physio1.utmem.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M211276200
 |
ABBREVIATIONS |
The abbreviations used are:
VSMC, vascular
smooth muscle cell(s);
cPLA2, cytosolic phospholipase
A2;
Jak, Janus-activated kinase;
STAT, signal transducers
and activators of transcription;
PDGF-BB, platelet-derived growth
factor-BB;
PDGFR, platelet-derived growth factor receptor;
AA, arachidonic acid;
DMEM, Dulbecco's modified Eagle's medium;
EMSA, electrophoretic mobility shift assay;
CAT, chloramphenicol
acetyltransferase;
AACOCF3, arachidonyl trifluoromethyl
ketone.
 |
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