J Biol Chem, Vol. 275, Issue 2, 883-889, January 14, 2000
Regulation of Vascular Smooth Muscle Cell Proliferation by
Nuclear Factor-
B and Its Inhibitor, I-B*
Sachi
Hoshi
,
Masaki
Goto,
Noriyuki
Koyama,
Ken-ichi
Nomoto, and
Hiroshi
Tanaka
From the Eisai Co. Ltd., Tsukuba Research Laboratories,
Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan
 |
ABSTRACT |
Proliferation of vascular smooth muscle cells
(SMC) is a crucial event in the formation of atherosclerotic tissues
and is regulated by nuclear transcriptional factors including nuclear factor-
B (NF-B). We constructed a reporter gene assay to measure NF-B-dependent transcriptional activity in SMC. Thrombin
receptor-activating peptide (TRAP) and basic fibroblast growth factor
(bFGF) stimulated SMC proliferation and rapidly enhanced the NF-B
transcriptional activity in a dose-dependent manner.
4-Cyano-5,5-bis-(methoxyphenyl)4-pentenoic acid (E5510) significantly
inhibited SMC proliferation and also suppressed NF-B transcription
stimulated by TRAP and bFGF. In contrast, although tumor necrosis
factor (TNF)-
activated NF-B transcription, E5510 had no effect
on TNF--induced activation. NF-B was activated after the
stimulation of TRAP, bFGF, and TNF- in electrophoretic mobility
shift assay, and E5510 suppressed the NF-B activation induced by
TRAP and bFGF but not the activation by TNF-. Western blot analysis
of I-B and I-B
, inhibitors of NF-B, indicated that
I-B degradation, rather than I-B degradation, was important
in NF-B activation after the stimulation of TRAP and bFGF. PD98059,
an inhibitor of extracellular signal-regulated kinase (ERK) kinase,
suppressed NF-B transcriptional activity and SMC proliferation. The
phosphorylation of ERK1/2 was rapidly induced by TRAP and bFGF but not
by TNF-. These results indicate that TRAP and bFGF induced I-B
degradation and NF-B activation through a distinct pathway from
TNF- and that ERK1/2 may play an important role in NF-B
activation induced by TRAP and bFGF.
| |
INTRODUCTION |
NF-B1 has been
reported to play a pivotal role in regulating gene expression
controlling inflammation, cell differentiation, apoptosis, and
proliferation (1). The NF-B family, which shares the Rel homology
domain, consists of p50, p52, p65, c-Rel, and RelB. Immunohistochemical
studies indicated that human atherosclerotic tissues expressed NF-B
proteins (2). The expression of NF-B is enhanced in vascular tissue
during SMC proliferation after lumen injury (3, 4). In culture, NF-B
is activated by growth stimulants and cytokines in SMC (5-7).
Antisense of p65 of NF-B inhibited SMC proliferation and intimal
thickening of injured arteries in the rat (8, 9). These studies
indicate that NF-B is involved in SMC proliferation in
vitro and in vivo. Proliferation of SMC plays an
important role in the formation of intimal thickening in animals and
human with several vascular diseases, such as restenosis after
percutaneous transluminal coronary angioplasty and atherosclerosis (10,
11). However, intracellular signals leading to NF-B activation in
SMC proliferation is still unclear.
I-B is an inhibitory protein of NF-B. The I-B family includes
I-B, I-B, I-B
, p100, p105, and Bcl-3. After cell
stimulation, I-B is phosphorylated and degraded by ubiquitination
and cleavage by proteasome enzymes. As a result, NF-B is released as
an active form, is localized into nuclei, and transmits signals through binding to DNA (1). I-B degrades immediately after injury in
vascular walls (4). Microinjection or liposomal delivery of I-B
blocks NF-B activation and inhibits SMC proliferation (12, 13).
Immunohistochemical study of human atherosclerotic tissues, using the
antibody recognizing I-B-binding site of p65 of NF-B, indicated
that p65 of NF-B was activated in SMC of atherosclerotic lesions,
but not in normal vascular tissues (2). These findings suggest that
I-B plays a key role to regulate the activation of NF-B in SMC
proliferation on vascular walls.
Proliferation of vascular SMC may be regulated by growth factors and
cytokines in the formation of atherosclerotic lesions. A number of
factors to regulate SMC proliferation have been identified, such as
thrombin, basic fibroblast growth factor (bFGF), and tumor necrosis
factor- (TNF-). The response of thrombin is mimicked by thrombin
receptor-activating peptide (TRAP). These factors have been reported to
stimulate NF-B activation in various cells. On the other hand, a
previous paper indicated that
4-cyano-5,5-bis-(methoxyphenyl)4-pentenoic acid (E5510) suppressed
NF-B activation induced by thrombin in SMC (14). In this study, we
first focused on NF-B activation and I-B degradation to be
regulated by bFGF, TRAP, TNF-, and E5510. We have reported in this
paper that TRAP and bFGF induced I-B degradation and NF-B
activation through an E5510-sensitive pathway and TNF- through an
E5510-insensitive pathway. Extracellular-regulated kinase (ERK) 1/2
pathway is activated in SMC proliferation in vivo and
in vitro (15, 16) and plays a key role in NF-B activation in monocytes (17, 18). Here we have also reported the role of ERK1/2 in
NF-B activation induced by TRAP and bFGF but not by TNF-.
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MATERIALS AND METHODS |
Reagents--
TRAP (SFLLRN) was purchased from Peninsula
Laboratories; bFGF was from Amersham Pharmacia Biotech, and TNF- was from Genzyme. PD98059 was purchased from Sigma. E5510
(Fig. 1) was prepared in Eisai, according
to the method previously reported (19).
Cell Culture--
Rat vascular SMC were isolated by the explant
method (20). Briefly, aortic explants were obtained from the thoracic
aorta of rats weighing 200 g, and the tissue explants cultured in
Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.)
were supplemented with 10% fetal bovine serum. After 2 weeks, the
cells that had migrated out of the explant were removed by
trypsinization and seeded in T-75 flasks. Confluent SMC at the 2nd
passage were subcultured successively at a 1:2 split ratio. SMC were
used up to the 10th passage.
SMC Proliferation Assay--
Proliferation activity was
determined to measure [3H]thymidine incorporation into
SMC (21). Confluent SMC were trypsinized, suspended in DMEM
supplemented with 10% fetal bovine serum, and seeded at 2.5 × 104 cells per well into 24-well flasks (Corning Glass).
After 24 h, the cells were washed with serum-free DMEM and starved
with DMEM containing 0.1% bovine serum albumin for 24 h. Then the
cells were incubated with growth factors at indicated concentrations for 18 h. In experiments to determine the effect of E5510, the cells were pretreated with E5510 for 2 h. Cells were incubated with [3H]thymidine (Amersham Pharmacia Biotech) for
6 h, and [3H]thymidine incorporation into the cells
was counted by a liquid scintillation counter.
To determine the change in cell number, the growth activity was
measured by a colorimetric assay using MTT reagent,
3-(4,5-dimethythiazol-2-yl)2,5-diphenyltetrazolium bromide (Sigma)
(22). In brief, after SMC were starved for 20 h, growth factors
were added to each well. At an indicated time after stimulation, cells
were incubated with 0.6 mg/ml MTT reagent for 4 h. The reagent was
reduced by living cells to form insoluble blue formazan product. The
cells were washed, solubilized with 10% sodium dodecyl sulfate, and
quantified as the absorbance at 570 nm.
Assay for NF-B Activity--
NF-B dependent
transcriptional activity in SMC was measured using placental alkaline
phosphatase (PLAP) reporter gene combined with
NF-B-dependent human immunodeficiency virus-1-long
terminal repeat (HIV-1-LTR) sequence (23, 24). SMC were transiently transfected with it using DEAE-dextran (Amersham Pharmacia Biotech). At
12 h after transfection, cells were starved with DMEM containing 0.1% bovine serum albumin for 24 h, and stimulants at indicated concentrations were added to the cells for 24 h. In some
experiments, cells were pretreated with E5510 for 2 h before
stimulation. Culture supernatant was collected, and alkaline
phosphatase activity was measured with a microplate luminometer (EG & G
Berthold, Germany).
DNA binding activity of NF-B was studied using electrophoretic
mobility shift assay (EMSA). SMC were activated with stimulants for the
indicated times, and the cells were collected with a cell scraper.
Nuclear extracts were prepared and applied to gel shift assay as
described previously (24, 25). Briefly, 2 µg of nuclear extracts were
incubated with a 35-base pair double-stranded 32P-labeled
probe encoding the B consensus sequence (5'-GGC TAC AAG GGA CTT TCC
GCT GGG GAC TTT CCA GC-3') in the binding buffer containing 10 mM Tris-HCl, 40 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40,
1% deoxycholate, 3 µg/ml polydeoxyinosinic-deoxycytidylic acid at
room temperature for 30 min. Then samples were applied to native 5%
polyacrylamide gels and analyzed on BAS 2000 (Fuji Photo Film Co.,
Japan). For competition assay, 20- or 40-fold molar excess unlabeled
consensus oligonucleotide was added at 30 min prior to addition of the
labeled probe. Components of NF-B proteins were identified by
supershift assay using antibodies against p50, p52, p65, c-Rel, and
RelB antibodies (Santa Cruz Biotechnology, Inc.). For control, DNA activity of AP-1 was studied using AP-1-specific probes in EMSA and
anti-c-Jun and anti-NF-ATc2 antibodies (Santa Cruz Biotechnology, Inc.)
in supershift assay (24, 25).
Western Blotting--
Serum-starved SMC in 100-mm dishes were
stimulated with bFGF, TRAP, or TNF- for 15 min and solubilized with
ice-cold buffer (pH 7.8) containing 20 mM HEPES, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na3VO4, 1 mM NaF. The cells were collected with a cell scraper in the
ice-cold buffer and then homogenized with 60 strokes of a Dounce
homogenizer at 4 °C. The homogenates were centrifuged at 4600 × g for 10 min, and 20 µg of the cytosolic fraction was
subjected to electrophoresis on 10% SDS gels. Protein was transferred
to polyvinylidene difluoride membranes and detected with anti-I-B
and anti-I-B antibodies (Santa Cruz Biotechnology, Inc.) and
horseradish peroxidase-linked donkey anti-rabbit IgG antiserum.
Detection was quantitated by a chemiluminescence technique using ECL
reagents and ECL hyperfilms (Amersham Pharmacia Biotech).
Assay for ERK1/2 Activity--
ERK1/2 activity was measured by
MAPK enzyme assay system (Amersham Pharmacia Biotech). Briefly, SMC
were starved with DMEM containing 0.1% bovine serum albumin for
24 h. After growth stimulation at the indicated times, cell
stimulation was terminated by a rapid aspiration of the medium and
addition of 500 µl of ice-cold lysis buffer containing 25 mM Tris-HCl (pH 7.4), 25 mM sodium phosphate, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1% Triton X-100. Cell lysates
were incubated on ice for 20 min and centrifuged for 20 min at 4 °C.
MAPK activity was assayed by addition of kinase buffer containing 1.0 µCi of [-32P]ATP and a substrate peptide that
contains Thr-699 phosphorylation site of epidermal growth factor
receptor followed by incubation at 30 °C for 30 min. Reaction was
terminated by spotting the samples onto binding papers. The papers were
immediately washed with distilled water 3 times and placed into
scintillation vials, and radioactivity was counted.
Statistical Analysis--
Data were analyzed by analysis of
variance. When a significant difference was detected, the data were
further analyzed by Dunnett's multiple range test. Statistical
significance was assumed for p < 0.05 and
p < 0.01.
| |
RESULTS |
Proliferation of vascular SMC may be regulated by growth factors
and cytokines in the formation of atherosclerotic lesions. A number of
factors to regulate SMC proliferation have been identified, such as
thrombin, bFGF, and TNF-. The response of thrombin is mimicked by
TRAP. Nuclear transcriptional factors such as NF-B regulate SMC
proliferation. We first determined the effect of growth factors and
cytokine TNF- on DNA synthesis in cultured SMC by
[3H]thymidine incorporation assay. TRAP and bFGF induced
[3H]thymidine incorporation into SMC in a
dose-dependent manner (Fig.
2A). TRAP at 10 µM and bFGF at 1 ng/ml increased the activity up to 3 and
6.5 times, respectively, to the unstimulated activity. TNF- did not
affect the activity even at concentrations up to 10 ng/ml. Since
previous papers indicated that E5510 inhibited SMC proliferation in
intimal thickening in canine vascular tissue (26, 27), we determined
the effect of E5510 on cultured SMC proliferation. E5510 inhibited
[3H]thymidine incorporation induced by TRAP and bFGF
(Fig. 2B). E5510 attenuated the activity of TRAP to the
basal level at 500 µM. E5510 decreased the activity of
bFGF in a dose-dependent manner, and the inhibition was
significant at 0.5 µM and higher concentrations. E5510
had no effect on [3H]thymidine incorporation into
untreated and TNF--treated cells.
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Fig. 2.
Effect of TRAP, bFGF, and
TNF- on [3H]thymidine
incorporation into cultured rat SMC. A, quiescent SMC
were incubated with TRAP, bFGF, and TNF- at indicated concentrations
for 24 h and with [3H]thymidine for the last 6 h. [3H]Thymidine incorporation into the cells was counted
by a liquid scintillation counter. Results were shown as average value
and S.E. Statistical analysis was done with Dunnett's multiple range
test. *, p < 0.05; **, p < 0.01 versus control (without stimulants). n = 5 for each point. B, cells were pretreated with E5510 for
2 h and TRAP (10 µM), bFGF (10 ng/ml), and TNF-
(3 ng/ml) were added. *, p < 0.05; **,
p < 0.01 versus control (without E5510).
n = 5 for each point.
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To determine the effect of E5510 on the increase in cell number, we
utilized MTT assay. TRAP and bFGF increased cell number in a
time-dependent manner. During 6 days incubation, TRAP
increased cell number 3.3 times and bFGF 4.5 times. TNF- induced the
slight increase in cell number at 1 day but no significant increase at 3 and 6 days (Fig. 3A). E5510
suppressed the increase in cell number induced by TRAP and bFGF. E5510
diminished the activity of TRAP at 300 µM and the
activity of bFGF at 100 µM to the unstimulated level.
E5510 had no effect on cell number of untreated and TNF--treated cells (Fig. 3B). These results show that TRAP and bFGF were
potent stimulants for SMC proliferation, whereas TNF- was less
potent and that E5510 inhibited DNA synthesis and proliferation of SMC after the stimulation of TRAP and bFGF.
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Fig. 3.
Effect of TRAP, bFGF, and
TNF- on rat SMC proliferation.
A, quiescent SMC were incubated with TRAP, bFGF, and TNF-
for indicated days, and cell numbers were evaluated with MTT assay.
Results were shown as average value and S.E. Statistical analysis was
done with Dunnett's multiple range test. *, p < 0.05;
**, p < 0.01 versus control (without
stimulants). n = 5 for each point. B, cells
were pretreated with E5510 for 2 h and TRAP (10 µM),
bFGF (10 ng/ml), and TNF- (3 ng/ml) were added for 6 days. *,
p < 0.05; **, p < 0.01 versus control (without E5510). n = 5 for
each point.
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NF-B has been reported to be important in SMC proliferation.
Components of NF-B, p65 and p50, were expressed in vascular walls,
and their expression was induced during cultured SMC proliferation (3-7). To measure the NF-B-dependent transcriptional
activity, SMC were transfected with HIV-1-LTR PLAP. Tandem NF-B
sequence exists in the LTR of HIV-1, and NF-B positively regulates
transcription of this gene (23). TRAP, bFGF, and TNF- stimulated the
transcriptional activity in a dose-dependent manner (Fig.
4A). TRAP enhanced the activity significantly at 3 µM and more. bFGF and TNF-
increased the transcriptional activity at 3 and 10 ng/ml. E5510 at 100 µM suppressed the activity stimulated by TRAP and bFGF
(Fig. 4B). In contrast, E5510 did not affect the
TNF--induced transcriptional activity even at 500 µM.
These results showed that the NF-B-dependent transcriptional activity was induced by TRAP and bFGF through a
distinct pathway from that of TNF- in SMC.
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Fig. 4.
Effect of TRAP, bFGF, and
TNF- on
NF-B-dependent transcriptional
activity. A, quiescent SMC transfected with
NF-B-dependent HIV-1-LTR PLAP construct were incubated
with TRAP, bFGF, and TNF- at indicated concentrations for 24 h.
PLAP activity was determined as shown under "Materials and
Methods." Results were shown as average value and S.E. Statistical
analysis was done with Dunnett's multiple range test. *,
p < 0.05; **, p < 0.01 versus control (without stimulants). n = 5 for each point. B, cells were pretreated without (open
columns) or with E5510 at 100 µM (hatched
columns) or 500 µM (closed columns) for
2 h, and TRAP (10 µM), bFGF (10 ng/ml), or TNF-
(3 ng/ml) were added. *, p < 0.05; **,
p < 0.01 versus control (without E5510).
n = 4-6 for each point.
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Transcriptional activation of genes by NF-B requires its binding to
DNA. To determine NF-B activity to bind to DNA after SMC
stimulation, the nuclear extracts were analyzed in EMSA using an
oligonucleotide containing NF-B consensus sequence (Fig.
5). EMSA showed the two bands
corresponding to NF-B, p50/p50 homodimer complex, and p50/p65
heterodimer complex. After the treatment with TRAP, bFGF, and TNF-,
NF-B activation in binding to DNA was induced, which was in parallel
with the NF-B transcriptional activity in the reporter gene assay.
When E5510 was added to the culture, the agent suppressed the NF-B
activation in the stimulation by TRAP and bFGF. In contrast, E5510 at
the same dose had no effect on the activation by TNF-. Supershift
assay was done using antibodies against p50, p52, p65, c-Rel, and RelB.
For control, anti-c-Jun and anti-NF-ATc2 antibodies were used. Anti-p50
and p65 antibodies induced the dramatic shift of the bands, and
anti-c-Rel induced the shift of the minor portion of the band. Other
antibodies did not affect the migration of the band. 20- or 40-fold
molar excess of unlabeled consensus oligonucleotide diminished NF-B
band (data not shown). These data showed that NF-B complexes
contained p50 and p65 predominantly and c-Rel in minor portion of the
band. To study the specificity of NF-B signals, we next determined AP-1 activity in SMC.
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Fig. 5.
Effect of E5510 on
NF-B activity in EMSA. A, SMC
were pretreated with E5510 (500 µM) for 2 h and then
incubated with TRAP (10 µM), bFGF (10 ng/ml), and TNF-
(3 ng/ml) for 60 min. Nuclear extracts from the cells were evaluated in
EMSA. B, NF-B components were identified by a supershift
assay. The nuclear extracts were subjected to EMSA in the presence of
the indicated antibodies.
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We evaluated the same nuclear extract in EMSA using AP-1-specific
probes and confirmed that E5510 had no effect on AP-1 activity in the
treatment of TRAP, bFGF, and TNF- (data not shown). The results of
NF-B activity in EMSA was quantitated in BAS2000 and compared with
the results of AP-1 activity (Fig. 6).
E5510 suppressed the NF-B activation induced by TRAP by 79.5% and
the activation by bFGF by 73.0%. In contrast, E5510 at the same dose
had no effect on the activation by TNF-. E5510 did not decrease the
DNA binding activity of AP-1. These results suggest that E5510
suppressed NF-B DNA binding activity induced by TRAP and bFGF but
not the activity by TNF- and that the effect was selective to
NF-B but not to AP-1.
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Fig. 6.
Inhibition of NF-B
activity but not AP-1 activity by E5510 in EMSA. NF-B activity
(closed columns) and AP-1 activity (hatched
columns) were quantitated as radioactivity and indicated as a
relative value to the control (without E5510). The data were the
average value of two independent experiments.
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NF-B activity is regulated by I-B proteins, and the degradation
of I-B results in NF-B activation. Cytosolic extracts were
isolated from SMC treated with TRAP, bFGF, and TNF-, and the
degradation of I-B and I-B proteins was determined by Western blot analysis (Fig. 7). I-B
was degraded 60 min after stimulation by TRAP and bFGF. After 180 min,
the amount of the protein was recovered. In contrast, TNF- did not
promote the I-B degradation in SMC. On the other hand, the amount
of I-B was once increased 15 and 30 min after stimulation by any
of TRAP, bFGF, and TNF- and then degraded after 60 and 180 min.
These data suggest that I-B degradation was regulated in a
different way from I-B degradation in SMC and that I-B
degradation, rather than I-B degradation, might play a role in
NF-B activation induced by TRAP and bFGF.
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Fig. 7.
Time-dependent change of
I-B and
I-B proteins after
stimulation of TRAP, bFGF, and TNF-. SMC
were incubated with TRAP (10 µM), bFGF (10 ng/ml), and
TNF- (3 ng/ml). I-B and I-B in cytosolic extracts were
determined by Western blot analysis.
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To study the mechanism how E5510 suppressed NF-B activation, the
effects of E5510 on the degradation of I-B and I-B proteins were determined by Western blot analysis (Fig.
8). Compared with vehicle-treated cells,
E5510-treated cells showed the suppressed degradation of I-B in
the activation by TRAP and bFGF. E5510 had no effect on I-B
degradation induced by TNF-. On the other hand, E5510 suppressed
I-B degradation in the activation by bFGF and TNF-. But E5510
had no effect on I-B degradation induced by TRAP. These results
suggest that E5510 was potent to suppress the degradation of I-B
proteins. The degradation of I-B, rather than I-B, was
important in NF-B activation after the stimulation of TRAP and bFGF
in SMC.
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Fig. 8.
Inhibition of
I-B and
I-B degradation by
E5510. Cells were pretreated with E5510 (300 µM) for
2 h, and TRAP (10 µM), bFGF (10 ng/ml), and TNF-
(3 ng/ml) were added. IB and IB in cytosolic extracts at 60 min were determined by Western blot analysis.
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MAPK is a key component in cell proliferation. Among the MAPK family,
ERK1/2 plays a role in stimulating SMC proliferation, and ERK1/2
activity is dramatically induced during SMC proliferation in injured
arteries in the rat (16). Therefore, we determined the possibility that
ERK1/2 regulated NF-B activity in SMC using an NF-B reporter gene
assay and an ERK kinase inhibitor PD98059 (Fig.
9A). PD98059 significantly
decreased the NF-B transcriptional activity induced by TRAP and
bFGF. At 10 µM PD98059, the NF-B transcriptional
activity of TRAP and bFGF was reduced to the control level. PD98059 at
the same dose had no effect on vehicle-treated culture. In addition,
the effect of PD98059 on SMC proliferation was determined in MTT assay
(Fig. 9B). PD98059 suppressed SMC proliferation induced by
TRAP and bFGF. PD98059 decreased the proliferation induced by TRAP and
bFGF to 27.5 and 19.8% of the control, respectively. These data
indicate that ERK1/2 was important in NF-B activation and SMC
proliferation.
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Fig. 9.
Inhibition of NF-B
activity and growth activity by an ERK kinase inhibitor, PD98059.
SMC were pretreated with PD98059 at 10 µM for 2 h,
and bFGF (10 ng/ml) and TRAP (10 µM) were added.
A, NF-B-dependent PLAP activity was
determined at 24 h by PLAP assay as shown under "Materials and
Methods." n = 9-11 for each point. B,
growth activity at 6 days was evaluated by MTT assay. n = 5 for each point. Results were shown as average value and S.E.
Statistical analysis was done with Dunnett's multiple range test. *,
p < 0.05; **, p < 0.01 versus control (without PD98059).
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Finally, the effect of E5510 on ERK1/2 activity in SMC was determined
(Fig. 10). ERK1/2-dependent
phosphorylation assay clearly indicated that TRAP and bFGF stimulated
ERK1/2 activation. TRAP significantly activated ERK1/2 at 15 and 30 min
after the stimulation, and the activity was returned to the level of
unstimulated cells at 60 min. bFGF induced the activation at 15 min,
and the activity decreased rapidly at 30 and 60 min. In contrast,
TNF- had no effect on ERK1/2 activity at concentrations of 3-10
ng/ml, although TNF- at these doses activated NF-B. Then we
determined the effect of E5510 on ERK1/2 activity. It was remarkable,
however, that E5510 did not affect the ERK1/2 activity stimulated by
TRAP and bFGF. Since ERK1/2 was important in NF-B activation and SMC
proliferation induced by TRAP and bFGF, these results indicated that
E5510 suppressed a signal in the downstream of ERK1/2 activation
leading to NF-B activation in SMC proliferation.
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Fig. 10.
Effect of bFGF, TRAP,
TNF-, and E5510 on ERK1/2 activity in
SMC. A, quiescent SMC were incubated with bFGF (10 ng/ml), TRAP (10 µM), and TNF- (3 ng/ml). ERK1/2
activity was determined at 15, 30, and 60 min. B, cells were
incubated in the absence ( ) or presence (+) of E5510 at 300 µM for 2 h, and bFGF (10 ng/ml), TRAP (10 µM), and TNF- (3 ng/ml) were added. ERK1/2 activity
was determined at 15 min. Results were shown as average value of two
independent experiments.
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| |
DISCUSSION |
Proliferation of vascular SMC is a crucial event in the formation
of atherosclerotic tissues and is regulated by growth factors. We
studied the effects of growth factors on cultured rat SMC
proliferation. TRAP and bFGF, but not TNF-, stimulated cell
proliferation in [3H]thymidine incorporation assay and
increased cell number. E5510 dose-dependently inhibited SMC
proliferation induced by TRAP and bFGF. In animal experiments, E5510
inhibited SMC proliferation in intimal thickening in canine vein graft
and Teflon graft models (26, 27). These reports indicate that E5510 is
a potent inhibitor of SMC proliferation in culture and in
vivo.
TRAP and bFGF activated the NF-B-dependent transcription
in SMC. E5510 suppressed NF-B activation of TRAP and bFGF and also blocked TRAP- and bFGF-inducible degradation of I-B. These
findings indicate that the inhibitory effect of E5510 on NF-B
activation was due to the suppression of I-B degradation. We
propose the signal cascade of NF-B activation and SMC proliferation,
as discussed below (Fig. 11).
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Fig. 11.
Proposed signal pathways leading to
NF-B activation. TRAP and bFGF activate
ERK1/2, I-B degradation, and NF-B activation. TNF- activates
I-B degradation through a distinct pathway (×).
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The mechanism has not been identified how E5510 inhibited the
degradation of I-B and I-B. E5510 was originally prepared as an anti-platelet agent. In previous studies of ours and others (19,
28, 29) using human platelets, E5510 had a potent inhibitory effect on
platelet aggregation and subsequent release of mitogens. The effect was
found to be dependent on the inhibition of cyclooxygenase (COX).
Inhibition of COX-1 and -2 activities by E5510 resulted in blocking
collagen- and arachidonic acid-induced platelet aggregation, like a COX
inhibitor indomethacin. Salicylates, anti-inflammatory agents with an
inhibitory activity of COX, inhibit lipopolysaccharide-induced NF-B
activation and tissue factor expression through blocking I-B
degradation in monocytic THP-1 cells (30). Furthermore, salicylates
suppress TNF--induced I-B phosphorylation, degradation, NF-B activation, and adhesion molecules expression (31). However, indomethacin has no effect on the I-B degradation in the same model (31). COXs are induced in SMC and other cells by growth factors
and cytokines (32, 33). COXs produce prostaglandins and activate
cAMP-dependent kinase that suppresses SMC proliferation (15, 34). As a consequence, the suppression of COX activity does not
inhibit but instead enhances SMC proliferation (32, 35). A recent paper
has indicated that COX-2 expression is unrelated to NF-B activity in
rat SMC (36). Therefore, the inhibitory effect of E5510 on COX activity
may not contribute to the suppression of I-B degradation and NF-B activation.
On the other hand, E5510 is a potent inhibitor of phosphodiesterase
(PDE) activity, and it may inhibit SMC proliferation through cAMP
generation. Inhibition of PDE activities by E5510 results in blocking
thrombin-induced platelet aggregation. In the experiments with purified
PDE proteins, E5510 suppresses the activity of PDE types II, III, and V
and increased cAMP and cGMP levels in platelets (29). The suppression
of PDE type III elevates cAMP levels and inhibits SMC proliferation
(37). The increase of cAMP and the activation of
cAMP-dependent kinase suppress NF-B activation in
monocytic THP-1 cells and endothelial cells (38).
NF-B-dependent tissue factor gene expression is
inhibited by elevated cAMP and by overexpression of catalytic subunit
of cAMP-dependent kinase, whereas cAMP does not prevent
nuclear translocation of NF-B (38). cAMP-dependent
kinase mediated the phosphorylation of cAMP response element-binding
protein and the subsequent recruitment of the transcriptional
coactivator cAMP response element-binding protein, which inhibits
NF-B activity (39). cAMP blocks Raf-1 leading to the activation of
ERK kinases (40). Thrombin-induced ERK1/2 activation in airway SMC is
suppressed by forskolin which increases cAMP (41). Forskolin also
inhibits PDGF-induced ERK activity in aortic SMC (42). But in contrast
to cAMP, E5510 did not inhibit ERK activation by TRAP and bFGF. Another
possibility should be considered that E5510 blocked NF-B activation
in a cAMP-independent manner.
It is important in this study that E5510 suppressed the I-B
degradation and NF-B activation by TRAP and bFGF but not the activation by TNF- in SMC. These data indicate that the I-B degradation and NF-B activation by TRAP and bFGF is mediated through
a distinct pathway from TNF-. So far, a number of agents have been
identified to block NF-B activation through the inhibition of I-B
degradation, just like E5510. Sanguinarine, a benzophenanthridine alkaloid, inhibited I-B degradation and NF-B activation induced by TNF-, interleukin-1, phorbol ester, and okadaic acid but not the
activation by hydrogen peroxide and ceramide (43). Tissue factor
expression in endothelial cells (EC) is dependent upon NF-B
activation, and dilazep, another anti-platelet agent, inhibited the
expression of tissue factor mRNA and protein induced by thrombin and phorbol ester but not the expression induced by TNF- (44). The
pathway to NF-B activation may be different in the signals by
different inducers.
Protein kinase C (PKC), MAPK/ERK kinase, and MAPK are important to
regulate NF-B activation (45, 46). Conventional, novel, and atypical
PKC isotypes are involved in ERK1/2 activation during phorbol
ester-induced proliferation of Swiss 3T3 cells (47). When SMC are
cultured in high glucose condition, NF-B activation is induced
through a PKC-dependent pathway (48). Thrombin activates PKC and NF-B in EC (49, 50). Enforced expression of MAPK/ERK kinase
kinase-1, -2, and -3 induces I-B degradation in HeLa cells (51). Our
data that an ERK inhibitor PD98059 attenuated NF-B activation
suggest the important role of ERK kinase for NF-B activation in SMC.
Since E5510 suppressed the degradation of I-B proteins but not
ERK1/2 activity, signals in the downstream of ERK1/2 may be a target
that E5510 blocks.
This paper provides the first evidence that the degradation of
I-B, rather than I-B, was important in NF-B activation for SMC proliferation after the stimulation of TRAP and bFGF. E5510-treated cells showed the suppressed degradation of I-B in
the activation by TRAP and bFGF, whereas E5510 had no effect on
I-B degradation induced by TNF-. On the other hand, E5510 suppressed I-B degradation in the activation by bFGF and TNF- without effect on I-B degradation induced by TRAP. There has been
accumulating evidence that kinases activate NF-B. Protein tyrosine
kinases induce I-B degradation and NF-B activation (52). The
serine/threonine phosphatase inhibitors induce the rapid degradation of
I-B (53, 54). I-B phosphorylates by casein kinase II (55). I-B
is phosphorylated and degraded by I- kinase (IKK)- and -, and
overexpression of NF-B-inducible kinase stimulates the kinase
activities of IKK- and -, whereas overexpression of MEKK1
preferentially stimulates the kinase activity of IKK- (56). The
activation of IKK-, but not IKK-, is stimulated by the
overexpression of PKC 
(57). Nuclear expression of PKC increases
during SMC and EC proliferation (58, 59) and thrombin activates PKC
, whereas TNF fails to activate it (58, 60, 61). It is possible that
the inhibition of these processes is a target of E5510. However, the
mechanism of E5510 to block I-B degradation and NF-B activation
needs further studies.
In contrast to TRAP and bFGF, TNF- was a weak stimulant for SMC
proliferation in this study, although TNF- was potent to activate
NF-B in SMC. The reason is unclear but TNF- may exert bidirectory
signals to promote and suppress proliferation. As discussed above,
TNF- induces the production of COXs and prostaglandins that suppress
SMC proliferation (32). Alternatively, NF-B activation may be
important but not sufficient to induce certain biological responses
such as cell proliferation. In bovine coronary SMC, the proliferation
is induced by thrombin but not by TRAP, although both stimulate NF-B
activation (62). In the presence of other mitogens such as serum,
TNF- enhances SMC proliferation in culture (63). Matrix
metalloproteinase expression is synergistically activated by the
combination of TNF- and bFGF through two transcriptional factors
NF-B and AP-1. NF-B is induced by TNF- and AP-1 by bFGF (64).
Thrombin activates PKC and NF-B, whereas TNF- activated NF-B
but not PKC in EC (49, 50). Thrombin and TRAP potentiate TNF--induced E-selectin expression in EC (65). Synergistic signal
pathways may be necessary for cell proliferation in addition to NF-B activation.
In conclusion, TRAP and bFGF induce I-B degradation and NF-B
activation through a distinct pathway from TNF-, and ERK1/2 may play
an important role in NF-B activation leading to SMC proliferation.
Since the suppression of SMC proliferation is the beneficial approach
to prevent vascular diseases, E5510 is useful as a therapeutic agent
and a good tool to study NF-B-dependent signals for SMC proliferation.
| |
ACKNOWLEDGEMENT |
We thank Dr. Kouichi Katayama for helpful discussions.
| |
FOOTNOTES |
*
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: Tel: 81-298-47-5809;
Fax: 81-298-47-2037; E-mail: m-hoshi@hhc.eisai.co.jp.
| |
ABBREVIATIONS |
The abbreviations used are:
NF-B, nuclear
factor-B;
DMEM, Dulbecco's modified Eagle's medium;
SMC, smooth
muscle cells;
TRAP, thrombin receptor activating peptide;
bFGF, basic
fibroblast growth factor;
TNF, tumor necrosis factor;
ERK, extracellular signal-regulated kinase;
MTT, 3-(4,5-dimethythiazol-2-yl)2,5-diphenyltetrazolium bromide;
PLAP, placental alkaline phosphatase;
E5510, 4-cyano-5,5-bis-(methoxyphenyl)4-pentenoic acid;
HIV-1, human
immunodeficiency virus type 1;
LTR, long terminal repeat;
EMSA, electrophoretic mobility shift assay;
IKK, I- kinase;
PKC, protein
kinase C;
EC, endothelial cells;
PDE, phosphodiesterase;
COX, cyclooxygenase;
MAPK, mitogen-activated protein kinase.
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TOP
ABSTRACT
INTRODUCTION
