JunB Forms the Majority of the AP-1 Complex and Is a Target
for Redox Regulation by Receptor Tyrosine Kinase and G
Protein-coupled Receptor Agonists in Smooth Muscle Cells*
Gadiparthi N.
Rao
§,
Khurshed A.
Katki
,
Nageswara R.
Madamanchi
,
Yaxu
Wu
, and
Michael J.
Birrer¶
From the
Division of Cardiology, University of Texas
Medical Branch, Galveston, Texas 77555 and the ¶ Division of
Cancer Prevention and Control, NCI, National Institutes of Health,
Rockville, Maryland 20850
 |
ABSTRACT |
To understand the role of redox-sensitive
mechanisms in vascular smooth muscle cell (VSMC) growth, we have
studied the effect of N-acetylcysteine (NAC), a thiol
antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase
inhibitor, on serum-, platelet-derived growth factor BB-, and
thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP)
kinase activation; c-Fos, c-Jun, and JunB expression; and DNA
synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1
activity and DNA synthesis in VSMC. On the contrary, these compounds
had differential effects on agonist-induced ERK2, JNK1, and p38 MAP
kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited
agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos,
c-Jun, and JunB expression except for platelet-derived growth factor
BB-induced ERK2 activation. In contrast, DPI only inhibited
agonist-induced p38 MAP kinase activation and c-Fos and JunB
expression. Antibody supershift assays indicated the presence of c-Fos
and JunB in the AP-1 complex formed in response to all three agonists.
In addition, cotransfection of VSMC with expression plasmids for c-Fos
and members of the Jun family along with the AP-1-dependent
reporter gene revealed that AP-1 with c-Fos and JunB composition
exhibited a higher transactivating activity than AP-1 with other
compositions tested. All three agonists significantly stimulated
reactive oxygen species production, and this effect was inhibited by
both NAC and DPI. Together, these results strongly suggest a role for
redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP
kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity;
and DNA synthesis in VSMC. These results also suggest a role for
NADH/NADPH oxidase activity in some subset of early signaling events
such as p38 MAP kinase activation and c-Fos and JunB induction, which
appear to be important in agonist-induced AP-1 activity and DNA
synthesis in VSMC.
 |
INTRODUCTION |
Redox control plays an important role in gene regulation (1-6).
Underlying the importance of redox mechanisms, oxidants have been
implicated in the pathogenesis of cell proliferative diseases such as
atherosclerosis and cancer and in aging (7-9). Although the exact
mechanisms by which oxidant stress influences the pathogenesis of these
diseases are not clear, several laboratories including ours have
demonstrated that oxidants exhibit mitogenic activity in a variety of
cell types including VSMC1 at
nontoxic doses (10-15). In addition, oxidants mimic growth factors in
several aspects: oxidants stimulate phosphorylation of growth factor
receptor and nonreceptor tyrosine kinases (16-18), they activate
extracellular signal-regulated kinases (ERKs) (19, 20), and they induce
expression of several protooncogenes such as c-Fos and c-Jun (21-25).
More importantly, a number of recent studies have demonstrated that
growth factors stimulate production of H2O2 in
a variety of cell types including VSMC (26-31). In addition, a
requirement for H2O2 production in the
mitogenic signaling events of growth factors has been reported
(31).
NADH/NADPH oxidase is a flavin-containing plasma membrane-bound enzyme
that generates superoxide anion upon activation (32). Superoxide anion
can be dismutated enzymatically or nonenzymatically to
H2O2, which is then scavenged by catalase or
peroxidase (33). In an effort to identify the sources of oxidant
generation, several investigators have reported that growth factors
that induce either hyperplasia or hypertrophy stimulate NADH/NADPH
oxidase activity in various cell types (34, 35). It was also reported
that NADH/NADPH oxidase activity can be regulated in a
growth-dependent manner (35). A role for NADH/NADPH oxidase
activity in tumor necrosis factor-
- and fibroblast growth
factor-induced c-Fos expression and tumor necrosis factor-
-induced
Jun-NH2-terminal kinase-1 (JNK1) activation in chondrocytes
has been reported (36, 37). In another study, inhibition of NADH/NADPH
oxidase activity by antisense targeted depletion of its
p22phox component blunted the hypertrophic
effect of angiotensin II in VSMC (38). Because of these findings, a
role for this enzyme in growth factor-induced signaling events has been
suggested. However, the molecular events underlying the
growth-regulating effects of NADH/NADPH oxidase are not clear. The
purpose of the present investigation was to determine the role of
redox-sensitive mechanisms in the modulation of agonist-induced early
response events in VSMC. Here we report the following observations. 1) NAC, a thiol antioxidant, blocks agonist-induced ERK2, JNK1, and p38
MAPK activation, c-Fos, c-Jun, and JunB expression, AP-1 activity, and
DNA synthesis in VSMC. 2) NADH/NADPH oxidase activity plays a role in
some subset of agonist-induced early response events such as induction
of p38 MAPK activation, c-Fos and JunB expression, AP-1 activity, and
DNA synthesis. 3) JunB appeared to be the predominant member of the
AP-1 complex formed in response to both receptor tyrosine kinase and G
protein-coupled receptor agonists. 4) AP-1 with c-Fos and JunB
composition exhibited a higher transcriptional activity than AP-1 with
other compositions tested. Together, these results strongly suggest
that redox-sensitive mechanisms play an important role in the
modulation of AP-1 activity and growth in VSMC.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Aprotinin, ATP, bis-N-methylacridinium
nitrate (lucigenin), bovine myelin basic protein, EGTA,
-glycerophosphate, leupeptin, phenylmethylsulfonyl fluoride , sodium
deoxycholate, sodium fluoride, sodium orthovanadate, sodium
pyrophosphate, and thrombin were obtained from Sigma. PDGF-BB was from
Genzyme (Cambridge, MA). Diphenyleneiodonium was purchased from Toronto
Research Chemicals (Downsview, Ontario). Anti-ERK1 (SC-93), ERK2
(SC-154), c-Fos (SC-052), JunB (SC-46), JunD (SC-74), JNK1 (SC-474),
and p38 MAPK (SC-728 and SC-535) rabbit polyclonal antibodies and
GST-c-Jun (1-79) (SC-4113) recombinant protein were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). Anti-c-Jun rabbit polyclonal
antibody (PC-06) was purchased from Oncogene Science (Uniondale, NY).
AP-1 consensus double-stranded oligonucleotide and T4 polynucleotide
kinase were from Promega (Madison, WI). Phospho-p38 MAPK antibodies
were obtained from New England Biolabs, Inc. (Beverly, MA).
[14C]Chloramphenicol (52 mCi/mmol),
[methyl-3H]thymidine (70 Ci/mmol), and
[
-32P]ATP (8,000 Ci/mmol) were obtained from NEN Life
Sciences, Inc.
Cell Culture--
VSMC were isolated from the thoracic aortas of
200-300-g male Sprague-Dawley rats by enzymatic digestion as described
earlier (13). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at
37 °C in a humidified 95% air, 5% C02 atmosphere.
DNA Synthesis--
VSMC were plated onto 60-mm dishes, allowed
to grow to 70-80% confluence, and then growth arrested by incubation
in Dulbecco's modified Eagle's medium containing 0.1% (v/v) calf
serum for 72 h. Growth-arrested VSMC were exposed to various
agonists in the presence and absence of the indicated inhibitors for
24 h. Cells were pulse labeled with 1 µCi/ml
[3H]thymidine for 2 h just before the end of the
incubation period and harvested by trypsinization followed by
centrifugation. The cell pellet was resuspended in cold 10% (w/v)
trichloroacetic acid and vortexed vigorously to lyse the cells. The
mixture was allowed to remain on ice for 20 min and was then passed
through a GF/F glass microfiber filter. The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% ethanol, dried, and placed in a liquid scintillation vial containing the mixture. The
radioactivity was measured in a liquid scintillation counter (Beckman
LS 3801).
Western Blot Analysis--
Growth-arrested VSMC were treated
with and without the agonists in the presence and absence of
appropriate inhibitors for the indicated time periods at 37 °C.
Medium was aspirated, and cells were rinsed with cold
phosphate-buffered saline (PBS) and frozen immediately in liquid
nitrogen. 250 µl of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, 1 µg/ml leupeptin, 20 mM
-glycerophosphate, 2 mM sodium fluoride, 2 mM sodium pyrophosphate, and 1 mM sodium
orthovanadate (Na3VO4)) was added to the frozen
monolayers, thawed on ice for 15 min, and scraped into 1.5-ml Eppendorf
tubes. The cell lysates were cleared by centrifugation at 12,000 rpm
for 30 min at 4 °C. The protein content of the supernatants was
determined using Bradford reagent (Bio-Rad). Cell lysates containing
equal amounts of protein were resolved by electrophoresis on a 0.1%
SDS and 10% polyacrylamide gels. The protein was transferred
electrophoretically to a nitrocellulose membrane (Hybond, Amersham
Pharmacia Biotech). After blocking in 10 mM Tris-HCl
buffer, pH 8.0, containing 150 mM sodium chloride, 0.1%
Tween 20, and 5% (w/v) non-fat dry milk, the membrane was treated with
appropriate primary antibodies followed by incubation with appropriate
peroxidase-conjugated secondary antibodies. The antigen-antibody
complexes were detected using chemiluminescence reagent kit (Amersham
Pharmacia Biotech).
ERK, JNK, and p38 MAPK Assays--
After appropriate treatments,
cells were washed with cold PBS and solubilized on ice for 15 min in
lysis buffer containing 20 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 50 mM
-glycerophosphate, 1% Triton X-100, 10 units/ml aprotinin, 2 µM leupeptin, 1 mM
Na3VO4, and 400 µM
phenylmethylsulfonyl fluoride. The cell lysates were cleared by
centrifugation at 13,000 rpm for 10 min at 4 °C. Cell lysates
normalized for protein were immunoprecipitated by incubating with
anti-ERK2 and anti-JNK1 rabbit IgG for ERK2 and JNK1 assays, respectively, for 2 h followed by the addition of 40 µl of 50% (w/v) protein A-Sepharose beads for an additional hour. The beads were
washed three times with lysis buffer, three times with wash buffer (100 mM Tris-HCl, pH 7.6, 500 mM lithium chloride,
0.1% Triton X-100, and 1 mM dithiothreitol), and three
times with kinase buffer (12.5 mM Mops, pH 7.5, 12.5 mM
-glycerophosphate, 7.5 mM
MgCl2, 2 mM EGTA, 0.5 mM sodium
fluoride, and 0.5 mM Na3VO4). The
ERK2 activity present in the immunoprecipitates was determined by
resuspending in 30 µl of kinase buffer containing 5 µg of myelin basic protein, 20 µM ATP, and 1 µCi of
[
-32P]ATP per reaction and incubating at 30 °C for
20 min. For the JNK1 assay, incubation with the kinase buffer was the
same as that for the ERK2 assay except that 1 µg of GST-c-Jun was
used instead of myelin basic protein. The reactions were stopped by adding 20 µl of 4 × Laemmli sample buffer. The samples were
heated at 95 °C for 5 min and analyzed by SDS-gel electrophoresis on 12% acrylamide gels. The dried gel was exposed to X-Omat AR x-ray film
and developed. Activation of p38 MAPK was measured by Western blotting
using phospho-p38 MAPK antibodies.
Electrophoretic Mobility Shift Assay--
Growth-arrested VSMC
were treated with and without agonists for the indicated time periods,
and nuclear extracts were prepared as described earlier (39).
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 20 min at 30 °C.
For supershift analysis, antibodies (4 µg) were added to the reaction
mix after the initial 20-min incubation, and incubation continued for
an additional 2 h. Protein-DNA complexes were resolved on a 4%
polyacrylamide gel using 0.25 × TBE buffer (1 × TBE = 50 mM Tris borate, pH 8.3, and 1 mM EDTA).
Double-stranded oligonucleotides (AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3') were labeled with [
-32P]ATP using a T4 polynucleotide
kinase kit per the supplier's protocol (Promega). Unincorporated
nucleotides were removed by chromatography in a G-25 spin column
(Bio-Rad).
Transient Transfection and CAT Assays--
VSMC culture was
split evenly into 100-mm dishes the day before transfection and grown
in Dulbecco's modified Eagle's medium containing 10% calf serum to
80-90% confluence. Cells were transfected with collagenase (coll)-CAT
plasmid DNA (20 µg/100-mm dish) using a calcium phosphate
precipitation method as described by Angel et al. (40).
Cells were washed with PBS 16 h after transfection, incubated in
Dulbecco's modified Eagle's medium containing 0.1% calf serum for
36 h at 37 °C, and then stimulated with and without agonists in
the presence and absence of appropriate inhibitors for 6 h. In the
other set of experiments, VSMC were cotransfected with expression
plasmids (8 µg each) for c-Fos or Jun family members alone or in
various combinations along with AP-1 reporter plasmid, coll-CAT, and
48 h after transfection cells were processed for CAT activity.
Vector DNA was used to normalize the amount of DNA transfected with the
cells. Cells were washed with PBS, scraped in 1 ml of TEN buffer (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 150 mM NaCl) into an Eppendorf tube and pelleted by
centrifugation at 12,000 g for 1 min at 4 °C. The cell pellet was
suspended in 100 µl of cold 250 mM Tris-HCl buffer, pH
7.5, and the cells were lysed by three repeated freeze-thaw cycles.
Cell debris was removed by centrifugation at 12,000 × g for 5 min at 4 °C. The protein concentration of the
supernatant was determined as described above. CAT activity was
measured by the method of Gorman et al. (41). In brief, 50 µg of protein from each condition was incubated with 20 µl of 4 mM acetyl-CoA, 32.5 µl of 1 M Tris-HCl
buffer, pH 7.5, 4 µl of 50 µCi/ml
[14C]chloramphenicol in a total volume of 150 µl
at 37 °C for 2-4 h. Controls without cell extract and/or with
nontransfected cell extracts were incubated simultaneously. Acetylated
and nonacetylated chloramphenicol was extracted with ethyl acetate and
separated by thin layer chromatography on Silica Gel 1B plates using a
chloroform:methanol mixture (19:1) as solvent, and the air-dried silica
plates were subjected to autoradiography.
Superoxide Anion Production Assay--
Superoxide anion
production in growth-arrested VSMC in response to agonists was measured
according to the method described by Griendling et al. (34).
Cells were washed with PBS and pelleted by centifugation at 1,000 rpm
for 5 min at 4 °C. Cells were then resuspended in assay buffer
consisting of 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2,
35 mM phosphoric acid, and 20 mM Hepes, pH 7.4. Superoxide anion production in VSMC in response to agonists was
measured in the darkroom with a AutoLumat LB 953 luminometer. To start
the assay, lucigenin was added to a final concentration of 250 µM to 1 × 106 cells followed by agonist
in a total volume of 1 ml of assay buffer. Wherever appropriate, 20 mM NAC was added to cells 2 h before the assay, and
DPI (12 µM) was added 30 min before the assay.
Photoemission in terms of relative light units was measured every
minute for 5 min.
All of the experiments were repeated at least three to four times with
similar results. All samples were normalized for protein content before
Western blot analysis, immunoprecipitation, electrophoretic mobility
shift assay and CAT assay. Statistical analysis on
[3H]thymidine uptake data was performed using Student's
t test.
 |
RESULTS |
To study the role of redox-sensitive mechanisms in agonist-induced
cell growth, growth-arrested VSMC were treated with and without 10%
serum, 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and
absence of 20 mM NAC or 12 µM DPI for 24 h, and DNA synthesis was measured by [3H]thymidine
uptake. As shown in Fig. 1, A
and B, all three agonists caused a significant increase in
DNA synthesis in VSMC, and this effect was blocked by NAC and DPI.

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Fig. 1.
NAC and DPI inhibit serum-, PDGF-BB-, and
thrombin-induced DNA synthesis. Growth-arrested VSMC were treated
with and without 10% serum, 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin
in the presence and absence of 20 mM NAC (panel
A) or 12 µM DPI (panel B) for 24 h,
and DNA synthesis was measured by [3H]thymidine uptake.
NAC and DPI were added 30 min before the addition of the agonists.
*p < 0.01 versus control;
**p < 0.01 versus agonist alone.
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To investigate the underlying mechanisms of NAC- and DPI-inhibited VSMC
growth, we first determined the time course of agonist-induced expression of AP-1 components, c-Fos, c-Jun, JunB, and JunD. As shown
in Fig. 2, A and B,
serum, PDGF-BB, and thrombin induced c-Fos (12-20-fold), c-Jun
(3-4-fold), and JunB (5-7-fold) expression in a
time-dependent manner. Induction of c-Fos expression in
response to serum, PDGF-BB, and thrombin was observed at 1 h,
reached a near maximal level at 2 h, and declined thereafter. The
maximal expression of c-Jun in response to these agonists was observed at 2 h, whereas JunB levels peaked 3 h poststimulation.
Significant levels of JunD were present in growth-arrested VSMC which
were increased by 2-fold upon treatment with serum. PDGF-BB and
thrombin had no significant effect on JunD levels.

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Fig. 2.
Time course of serum, PDGF-BB, and thrombin
induction of c-Fos, c-Jun, JunB, and JunD expression in VSMC.
Growth-arrested VSMC were treated with and without 10% serum
(CS, panel A), 10 ng/ml PDGF-BB (panel
A), or 0.1 unit/ml thrombin (panel B) for the indicated
times, and cell lysates were prepared. Cell lysates containing equal
amounts of protein from each condition were analyzed by Western
blotting for c-Fos, c-Jun, JunB, and JunD levels using their respective
antibodies.
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To test whether agonist-induced increases in the levels of c-Fos and
members of the Jun family proteins are mediated via the generation of
oxidants, we treated growth-arrested VSMC with serum, PDGF-BB, or
thrombin in the presence and absence of NAC, a thiol antioxidant, and
the protooncogene levels were determined. NAC significantly decreased
c-Fos (70%), c-Jun (50%), and JunB (70%) expression in response to
serum, PDGF-BB, and thrombin (Fig. 3). In
contrast, NAC had no significant effect on JunD levels in either control or agonist-treated VSMC. NADH/NADPH oxidase has been implicated in cytokine- and growth factor-induced c-Fos expression and
cytokine-induced JNK1 activation and c-Jun expression in chondrocytes
(36, 37). To test the role of NADH/NADPH oxidase activity in
agonist-induced expression of c-Fos and members of the Jun family
proteins, growth-arrested VSMC were treated with and without serum,
PDGF-BB, or thrombin in the presence and absence of DPI, and the
protooncogene levels were determined. Expression of c-Fos in response
to these agonists was almost completely (85%) blocked by DPI (Fig.
4). DPI also significantly (70%)
inhibited JunB expression induced by these agonists. DPI alone induced
c-Jun expression by 3-fold, and it exhibited an additive effect with
agonist-induced expression of this protooncogene (Fig. 4). DPI had no
significant effect on JunD levels in either control or agonist-treated
cells.

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Fig. 3.
Effect of NAC on serum-, PDGF-BB-, and
thrombin-induced expression of c-Fos, c-Jun, JunB, and JunD in
VSMC. Growth-arrested VSMC were treated with and without 10%
serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the
presence and absence of 20 mM NAC for 2 h, and cell
lysates were prepared and analyzed for c-Fos, c-Jun, JunB, and JunD
levels as described in the legend of Fig. 2.
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Fig. 4.
Effect of DPI on serum-, PDGF-BB-, and
thrombin-induced expression of c-Fos, c-Jun, JunB, and JunD in
VSMC. Growth-arrested VSMC were treated with and without 10%
serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the
presence and absence of 12 µM DPI for 2 h, and cell
lysates were prepared and analyzed for c-Fos, c-Jun, JunB, and JunD
levels as described in the legend of Fig. 2.
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The Fos and Jun family proteins constitute the transcription factor
AP-1 (42). To investigate the relationship between the induction of
expression of c-Fos and members of the Jun family proteins and
AP-1-dependent transcription, we studied the expression of
AP-1-responsive reporter gene in growth-arrested VSMC stimulated with
serum, PDGF-BB, or thrombin in the presence and absence of NAC or DPI.
VSMC were transiently transfected with a collagenase-CAT (coll-CAT)
reporter plasmid in which CAT expression was driven by collagenase gene
promoter containing a single AP-1 site, growth arrested, and then
treated with and without the agonists in the presence and absence of
NAC or DPI. All three agonists caused induction of the
AP-1-dependent reporter gene expression, and this response
was significantly blocked by both NAC and DPI (Fig. 5, A and B).
Because NAC and DPI inhibited the AP-1 activity despite the
differential effects of these compounds on serum-, PDGF-BB-, and
thrombin-induced c-Jun expression, we next determined the composition
of AP-1 formed in response to these agonists. Gel supershift analysis
showed the presence of c-Fos and JunB in the AP-1 complex formed in
response to serum and thrombin (Fig. 6). Similar results were obtained with PDGF-BB (data not shown). To determine the functional activity of AP-1 with c-Fos and JunB composition, VSMC were cotransfected with expression plasmids for c-Fos
and members of the Jun family in various combinations along with the
AP-1 reporter plasmid coll-CAT, and CAT activity was measured 48 h
after transfection. Consistent with the gel supershift analysis, AP-1
with c-Fos and JunB composition exhibited a higher transcriptional
activity than AP-1 with other combinations tested (Fig.
7).

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Fig. 5.
Effect of NAC and DPI on serum-, PDGF-BB-,
and thrombin-induced AP-1 transactivation activity. VSMC were
transiently transfected with 73/+63 coll-CAT reporter plasmid,
growth-arrested, treated with and without 10% serum (CS),
10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence
of 20 mM NAC (panel A) or 12 µM
DPI (panel B) for 6 h, and cell lysates were prepared.
Cell lysates normalized for protein were assayed for CAT activity as
described under "Experimental Procedures."
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Fig. 6.
JunB is the predominant component of AP-1 in
serum-, PDGF-BB-, and thrombin-treated VSMC. 5 µg of nuclear
protein from control cells and cells treated with 10% serum, 10 ng/ml
PDGF-BB, or 0.1 unit/ml thrombin for 2 h was incubated with
32P-labeled canonical AP-1 oligonucleotide for 20 min, and
the protein-DNA complex was separated on a 4% nondenaturing
polyacrylamide gel. For supershift analysis, antibodies were added to
the nuclear protein-AP-1 oligonucleotide mix and incubation continued
for an additional 2 h before separation on polyacrylamide gel
electrophoresis.
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Fig. 7.
AP-1 with c-Fos/JunB possess higher
transactivating activity than AP-1 with other compositions. VSMC
were cotransfected with expression plasmids for c-Fos and Jun family
members in various combinations along with the AP-1 reporter gene
coll-CAT. 48 h after transfection cell lysates were prepared and
analyzed for CAT activity.
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ERKs are a group of serine/threonine kinases that respond
preferentially to various growth stimulants. These kinases are
implicated in the expression of c-Fos in response to various agonists
(43-46). To understand the possible mechanisms by which NAC and DPI
inhibit c-Fos expression induced by these agonists, we investigated the effects of these compounds on serum-, PDGF-BB-, and thrombin-induced activation of ERK2 in VSMC. All three agonists caused significant increases (8-15-fold) in ERK2 activity as measured by an immunocomplex kinase assay using myelin basic protein as a substrate (Figs. 8 and 9).
Although NAC completely inhibited the ERK2 activity induced by serum
and thrombin, it had no significant effect on PDGF-BB-induced ERK2
activation (Fig. 8). Serum-, PDGF-BB-, and thrombin-induced ERK2
activity, however, was not affected by DPI (Fig. 9). NAC and DPI by
themselves had no significant effect on ERK2 activity in
growth-arrested VSMC.

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Fig. 8.
Effect of NAC on serum, PDGF-BB, and thrombin
activation of ERK2 and JNK1. After treatment of growth-arrested
VSMC for 5 min (for ERK assay) or 10 min (for JNK assay) with and
without 10% serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml
thrombin in the presence and absence of 20 mM NAC, cell
lysates were prepared. Cell lysates containing equal amounts of protein
from each condition were immunoprecipitated with anti-ERK2 or JNK1
antibodies, and the ERK2 and JNK1 activities were measured by
immunocomplex kinase assays using myelin basic protein or GST-c-Jun as
substrates, respectively, as described under "Experimental
Procedures."
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Fig. 9.
Serum-, PDGF-BB-, and thrombin-induced ERK2
and JNK1 activities are insensitive to inhibition by DPI.
Growth-arrested VSMC were treated with and without 10% serum
(CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the
presence and absence of 12 µM DPI for 5 min (for the ERK2
assay) or 10 min (for the JNK1 assay), and cell lysates were prepared
and assayed for ERK2 and JNK1 activities as described in the legend of
Fig. 8.
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JNKs are a group of MAPKs that are related to ERKs but respond
preferentially to cellular stressors such as UV irradiation and agents
that do not primarily cause growth (47-49). A role for JNKs has been
suggested in the induction of expression of both c-Fos and c-Jun (43,
44, 50). To understand the possible mechanisms by which antioxidants
affect the agonist-induced expression of c-Fos and members of the Jun
family proteins, we studied the effects of NAC and DPI on serum-,
PDGF-BB-, and thrombin-induced JNK1 activity. All three agonists
activated JNK1 (10-25-fold) as determined by an immunocomplex kinase
assay with GST-c-Jun (1-79) as a substrate (Figs. 8 and 9). NAC
significantly inhibited (50-80%) the agonist-induced JNK1 activity
(Fig. 8). On the contrary, DPI alone caused an increase (35% over
control) in JNK1 activity, and it exhibited an additive effect with
agonists on the activation of this enzyme (Fig. 9). It was reported
that p38 MAPK cooperates with ERKs in the induction of expression of
c-Fos in response to UV irradiation (51). To find the possible role of
p38 MAPK in agonist-induced c-Fos expression, we tested the effect of
serum, PDGF-BB, and thrombin on p38 MAPK activation. As determined by the phosphorylation of the enzyme, all three agonists activated p38
MAPK, and this effect was sensitive to inhibition by both NAC and DPI
(Fig. 10).

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Fig. 10.
NAC and DPI inhibit the serum-, PDGF-BB-,
and thrombin-induced activation of p38 MAPK in VSMC.
Growth-arrested VSMC were treated with and without 10% serum
(CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the
presence and absence of 20 mM NAC or 12 µM
DPI for 10 min, and cell lysates were prepared. Cell lysates containing
an equal amount of protein from each condition were analyzed by Western
blotting for phospho-p38 MAPK using specific antibodies.
|
|
To gain further evidence for a role of oxidants in receptor tyrosine
kinase and G protein-coupled receptor agonist-induced growth signaling
events, we tested the effect of serum, PDGF-BB, and thrombin on
superoxide anion production in growth-arrested VSMC. As shown in Fig.
11, all three agonists significantly
stimulated superoxide anion production by 5 min, and this effect was
blocked by both NAC and DPI.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of serum, PDGF-BB, and thrombin on
the production of superoxide anion in VSMC. Growth-arrested VSMC
were treated with and without 10% serum, 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 20 mM NAC
or 12 µM DPI, and superoxide anion production was
measured by photoemission as described under "Experimental
Procedures." Superoxide anion production was expressed as relative
light units/1 × 106 cells for 5 min.
|
|
 |
DISCUSSION |
The important finding of the present study is that JunB is the
major constituent of the AP-1 complex formed in response to, and is a
target for redox regulation by both receptor tyrosine kinase and G
protein-coupled receptor agonists in VSMC. The present findings also
show that antioxidant NAC inhibits serum-, PDGF-BB-, and
thrombin-induced JNK1 and p38 MAPK activation, c-Fos, c-Jun, and JunB
expression, and DNA synthesis in VSMC. Although NAC completely blocked
the serum- and thrombin-induced ERK2 activity, it had no significant
effect on PDGF-BB-induced activation of ERK2. These findings suggest a
role for both redox-sensitive and redox-insensitive mechanisms in
agonist-induced ERK2 activation in VSMC. ERKs are implicated in the
induction of expression of c-Fos via phosphorylation and activation of
the transcription factor Elk1 in response to growth factors (44, 52).
Because NAC had no effect on PDGF-BB-induced ERK2 activity, and it
inhibited completely the c-Fos expression and DNA synthesis induced by
PDGF-BB, it is likely that other mechanisms that are independent of
ERKs modulate the induction of expression of c-Fos by PDGF-BB. This
also implies that activation of ERK2 alone may not be sufficient for
growth induction by PDGF-BB. In fact, others have also reported that
activation of ERKs alone is not sufficient for VSMC mitogenesis induced
by PDGF-BB and angiotensin II (53, 54). It was reported that induction
of c-Fos expression by UV requires a cross-talk between p38 MAPK and
ERKs (51). Because all three agonists activated p38 MAPK, and this
effect was inhibited by NAC, one likely mechanism by which NAC inhibits
the PDGF-BB-induced c-Fos expression could be via down-regulating the
p38 MAPK activity. A similar mechanism can be extrapolated to the
inhibitory effect of DPI on agonist-induced c-Fos expression as DPI,
although having no effect on agonist-induced ERKs activation, blocked
p38 MAPK activation.
Reactive oxygen species production by NADH/NADPH oxidase has been
implicated in cytokine- and growth factor-induced c-Fos expression and
cytokine-induced JNK1 activation and c-Jun expression (36, 37). Because
DPI, a potent inhibitor of NADH/NADPH oxidase, blocked only the c-Fos
and JunB expression and p38 MAPK activation but not the c-Jun
expression or ERK2 and JNK1 activation induced by serum, PDGF-BB, and
thrombin, it is likely that NADH/NADPH oxidase activity is required
only for some subset of agonist-stimulated early response events.
Despite the differential effects of NAC and DPI on agonist-induced ERK2
and JNK1 activation and c-Jun expression, both caused a decrease in
agonist-stimulated AP-1 activity and growth. Because c-Fos and JunB
were present in the AP-1 complex formed in response to serum, PDGF-BB,
and thrombin, and the expression of these protooncogenes was inhibited
by both NAC and DPI, it is possible that these two protooncogenes
account for the agonist-induced AP-1 activity and growth in VSMC.
Indeed, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family revealed that AP-1 with c-Fos and JunB composition possess more gene transactivating activity than AP-1 with
the other compositions tested.
Superoxide anion and H2O2 are the frequently
invoked reactive oxygen species implicated in growth factor-induced
cellular signaling (10, 28, 31, 55, 56). In addition, growth factors
have been reported to activate NADH/NADPH oxidase, a major reactive
oxygen species-generating system in a variety of cell types including
VSMC (28, 34, 35). Our results show that both receptor tyrosine kinase
and G protein-coupled receptor agonists stimulate the production of
superoxide anion in VSMC via a mechanism involving NADH/NADPH oxidase
activity. However, although serum-, PDGF-BB-, and thrombin-induced
superoxide anion production was completely blocked by NAC and DPI, the
early growth response events of these agonists exhibited differential
sensitivity of inhibition to these compounds. Specifically, DPI
inhibited agonist-induced p38 MAPK activation and c-Fos and JunB
expression. On the other hand, NAC inhibited ERKs, JNKs, and p38 MAPK
group of MAPK activation and c-Fos, c-Jun, and JunB expression.
Considering these findings, it is clear that besides NADH/NADPH
oxidase, other mechanisms are involved in the redox regulation of these
early response signaling events of receptor tyrosine kinase and G
protein-coupled receptor agonists. It has been reported that agents
such as hemin and HgCl2, which oxidize thiol groups,
activate Ras and cause mitogenesis in lymphocytes (57, 58). NAC
facilitates synthesis of glutathione, a major intracellular thiol
donor, by providing cysteine (59). Glutathione, besides scavenging
reactive oxygen species, modulates the levels of intracellular thiols
(59). Therefore, the broader inhibitory effects of NAC on
agonist-induced early growth response events indicate a role for
thiols. In fact, oxidation/reduction of thiols has been regarded as a
critical regulatory event in the modulation of activities of several
proteins including growth factor receptors, ion channels, and
transcriptional factors (1-6, 60-63).
Another possible molecule that is involved in agonist-induced
NAC-sensitive early response events could be arachidonic acid. Both
receptor tyrosine kinase and G protein-coupled receptor agonists stimulate the release of arachidonic acid in a variety of cell types
including VSMC (64, 65). In addition, a requirement for arachidonic
acid release and its subsequent metabolism via the
lipoxygenase/cytochrome P-450 monoxygenase pathways has been reported
to be required for both receptor tyrosine kinase and G protein-coupled
receptor agonist-induced growth in many cell types including VSMC (64,
66). Furthermore, activation of secretory type phospholipase
A2s (also known as group II phospholipase A2s),
a group of rate-limiting enzymes for arachidonic acid release, requires
disulfide bond formation (67). It was also reported that expression of
group II phospholipase A2 in transgenic mice results in
epidermal hyperplasia (68). Although a role for arachidonic acid in
agonist-induced thiol-sensitive mechanisms of oxidant generation can be
extrapolated from these findings, future studies are required to test
this hypothesis. In any case, the present study provides evidence for a
role of several redox-sensitive mechanisms including NADH/NADPH oxidase
activity in the early signaling events of both receptor tyrosine kinase
and G protein-coupled receptor agonists in VSMC. In addition, the
present study identifies JunB as the major component of the AP-1
complex formed in response to both receptor tyrosine kinase and G
protein-coupled receptor agonists in VSMC and its possible role in
redox-sensitive AP-1-mediated gene regulation.
 |
ACKNOWLEDGEMENTS |
We are thankful to Dr. Michael Karin of the
University of California at San Diego for generously providing the
various expression plasmids used in this study. We are also thankful to
Dr. Allan Brasier of the University of Texas Medical Branch at
Galveston for use of his luminometer.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the American
Heart Association.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: 4.124B Old John Sealy
Hospital, University of Texas Medical Branch, 301 University Blvd.,
Route 0567, Galveston, TX 77555-0567. Tel.: 409-747-1851; Fax:
409-772-1861; E-mail: grao{at}utmb.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
VSMC, vascular
smooth muscle cell(s);
ERKs, extracellular signal-regulated kinases;
JNKs, Jun NH2-terminal kinases;
MAPK, mitogen-activated
protein kinase;
NAC, N-acetylcysteine;
AP-1, activator
protein-1;
PDGF-BB, platelet-derived growth factor-BB;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
Mops, 4-morpholinepropanesulfonic acid;
CAT, chloramphenicol
acetyltransferase;
DPI, diphenyleneiodonium.
 |
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