Vascular Biology |
Mediated Monocyte Chemoattractant Protein-1 Induction in Vascular Smooth Muscle Cells
From the Division of Cardiology, Emory University School of Medicine, Atlanta, Ga.
Correspondence to Kathy K. Griendling, PhD, Emory University, Division of Cardiology, 1639 Pierce Dr, 319 WMB, Atlanta, GA 30322. E-mail kgriend{at}emory.edu
| Abstract |
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(TNF-
).
However, the precise signaling pathways leading to MCP-1 induction have
not been fully elucidated in vascular smooth muscle cells (VSMCs).
Cytokine signal transduction involves protein kinases as well
as reactive oxygen species (ROS). The relation between these 2 factors
is not clear. In this study, we show that TNF-
induces a parallel
phosphorylation of extracellular signalregulated
kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase
(p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs.
Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of
MCP-1 induction (43±10% inhibition). Incubation of VSMCs with
multiple antioxidants (diphenylene iodonium, liposomal superoxide
dismutase, catalase, N-acetylcysteine,
dimethylthiourea, and pyrrolidine dithiocarbamate) had
no effect on TNF-
mediated MCP-1 upregulation. However,
simultaneous blockade of the ERK1/2 and ROS pathways by
using PD098059 combined with diphenylene iodonium or
N-acetylcysteine potently enhanced the ability of MAPK
kinase inhibitors to abrogate MCP-1 mRNA expression
(100±2% inhibition). Thus, parallel ROS-dependent and
ERK1/2-dependent pathways converge to regulate TNF-
induced MCP-1
gene expression in VSMCs. These data unmask a complex but organized
integration of ROS and protein kinases that mediates
cytokine-induced vascular inflammatory gene
expression.
Key Words: monocyte chemoattractant protein-1 smooth muscle cells reactive oxygen species cytokines tyrosine kinase
| Introduction |
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(TNF-
), in
several cell types.3
The precise signaling pathways leading to MCP-1 induction have not been
fully elucidated. Conflicting reports in the literature implicate
reactive oxygen species (ROS)-mediated nuclear factor
B
(NF-
B)dependent mechanisms as well as tyrosine kinase pathways in
this process.4 5 6 7 8 To date, there has been no unifying
integrated hypothesis to explain these apparently disparate results.
TNF-
activates multiple signaling pathways, most notably,
mitogen-activated protein kinases (MAPKs).9 MAPKs
are serine/threonine kinases that transduce signals from the cell
membrane to the nucleus.10 11 12 Four groups of MAPKs have
been identified in mammalian cells: the extracellular signalregulated
kinases 1 and 2 (ERK1/2, also termed p42/44 MAPK), the c-Jun
NH2-terminal kinases (JNK, also named
stress-activated protein kinase [SAPK]), p38MAPK (also called
CSBP), and big MAPK 1 (BMK1, also termed ERK5).10 12 13
ERK1/2 is stimulated by growth factors and mitogenic
stimuli,10 12 whereas p38MAPK, JNK, and BMK-1 are
primarily activated by cellular stresses, including
proinflammatory cytokines.13 14 Stimulation of
these MAPKs leads to activation of distinct transcription factors,
including C-Fos, c-Jun, and possibly
NF-
B.10 15 Binding sites for each of these
transcription factors are present in the mouse MCP-1
promoter.16
Although TNF-
stimulation of MAPKs has not been demonstrated in
vascular smooth muscle cells (VSMCs), we have previously shown that
TNF-
activates a membrane-associated nonmitochondrial
oxidase that preferentially hydrolyzes NADH to produce
superoxide.17 These findings raise the possibility that
redox-sensitive pathways contribute to TNF-
induced MCP-1
expression in VSMCs. This has been shown to be true in other cell
types,18 and NF-
B and activator protein-1
(AP-1) binding sites16 represent possible targets
for ROS. In addition, ROS can selectively stimulate different MAPKs,
making the relation between NADH/NADPH oxidasemediated pathways and
MAPK-mediated protein phosphorylation events in the
induction of MCP-1 expression by TNF-
unclear. In the present
study, therefore, we have analyzed the redox sensitivity and
the involvement of MAPKs in TNF-
induced MCP-1 gene expression in
VSMCs in an attempt to provide an integrated understanding of their
respective roles in this process. We found that ROS-sensitive and
redox-insensitive MAPK pathways mediate the induction of MCP-1 by
TNF-
in a convergent, nonserial, parallel signaling cascade circuit,
emphasizing the highly organized interactive nature of intracellular
signals leading to gene induction.
| Methods |
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Cell Culture
VSMCs were isolated from rat thoracic aorta by enzymatic
digestion as described previously.19 Cells were grown in
DMEM supplemented with 10% calf serum, 2 mmol/L glutamine, 100
U/mL penicillin, and 100 µmol/L streptomycin, passaged twice a
week by harvesting with trypsin-EDTA, and seeded into
80-cm2 flasks. For experiments, cells between
passages 6 and 20 were seeded into 100-mm dishes, fed every other day,
and used at confluence.
In some experiments, we used VSMCs that had been stably transfected with antisense p22phox.20 In these cells, p22phox mRNA expression is completely inhibited, and protein expression is markedly attenuated.
Northern Blot Analysis
Total RNA was isolated by using TRI reagent. RNA (10 µg) was
separated on 1% denaturing formaldehyde agarose gels, transferred to
Nytran membranes (Schleicher & Schuell) by overnight upward capillary
blotting with 10x SSC, and immobilized by UV cross-linking
as described previously.21 Consistency of
total RNA loading between samples was controlled by densitometric
analysis of 28S RNA ultraviolet fluorescence in the
presence of ethidium bromide. Mouse MCP-1 cDNA was labeled by use of a
random primer labeling kit (Prime-It II) and
[32P]dCTP. Blots were prehybridized at least 2
hours and hybridized overnight at 42°C in the following solution: 1
mol/L NaCl, 50 mmol/L Tris HCl (pH 7.4), 5x Denhardts solution,
50% formamide, 0.5% SDS, and 100 µg/mL sheared and denatured salmon
sperm DNA. Denhardts solution was omitted during hybridization. After
hybridization, the blots were washed 3 times in 1x SSC and 0.1% SDS
at 56°C. The blots were autoradiographed by using Hyperfilm-MP at
-80°C, and the relative density of each band was determined by laser
densitometry. Staining of the 28S band by ethidium bromide after
transfer to the membrane was used for normalization.
Detection of p38MAPK and ERK1/2 by Immunoblotting
VSMCs at 80% to 90% confluence in 100-mm dishes were made
quiescent by incubation with DMEM containing 0.1% calf serum for 48
hours. Cells were stimulated with agonist at 37°C for specified
durations. After treatment, cells were washed 3 times with ice-cold PBS
and put on ice. Cells were lysed with 500 µL ice-cold lysis buffer at
pH 7.4 (mmol/L: HEPES 50, EDTA 5, and NaCl 50) containing 1% Triton
X-100, protease inhibitors (10 µg/mL aprotinin, 1
mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL
leupeptin), and phosphatase inhibitors (mmol/L: sodium
fluoride 50, sodium orthovanadate 1, and sodium pyrophosphate
10). Solubilized proteins were centrifuged at
12 000g in a microfuge (4°C) for 30 minutes, and
supernatants were stored at -80°C. Extracted protein was quantified
by the micro-Bradford assay. Proteins (25 µg) were separated by using
10% SDS-PAGE and transferred to Hybond-ECL nitrocellulose membranes at
100 V for 1 hour. Membranes were blocked overnight at room temperature
with PBS containing 6% nonfat dry milk and 0.1% Tween 20. The blots
were incubated for 1 hour with primary antibodies (rabbit polyclonal
phospho-specific p38, ERK1/2 antibodies that detect MAPK only when
activated by phosphorylation on TXY) at
1:2000 and 1:40 000 in PBS containing 1% nonfat dry milk and 0.1%
Tween 20, respectively. After incubation with secondary antibodies
(horseradish peroxidaseconjugated goat anti-rabbit antibody, 1:1000)
for 1 hour in PBS containing 1% nonfat dry milk and 0.1% Tween 20,
phosphorylated forms of proteins were detected by ECL
chemiluminescence. It has been shown that
phosphorylation of MAPKs is associated with the
activation of MAPKs22 23 24 ; therefore,
phosphorylation was taken as a measure of MAPK
enzymatic activity.
Western Analysis of MCP-1 Protein
VSMCs at 80% to 90% confluence were made quiescent by
incubation with DMEM containing 0.1% calf serum for 48 hours and
stimulated with TNF-
at 37°C for the specified durations. After
treatment, conditioned medium (10 mL) was collected and concentrated to
1 mL with Centricon filters (10 000g for 30 to 40 minutes).
Proteins were separated on 4% to 20% Tris-glycine gels and
transferred to nitrocellulose membranes. Membranes were blocked
overnight at room temperature with PBS containing 5% nonfat dry milk
and 0.1% Tween 20 and were incubated for 1 hour with primary rabbit
MCP-1 antibodies at 1:500. After incubation with secondary antibodies
(horseradish peroxidaseconjugated goat anti-rabbit antibody, 1:1000),
MCP-1 proteins were detected by ECL chemiluminescence.
Statistical Analysis
Comparisons between groups were made by the unpaired Student
t test. Results are expressed as mean±SE.
| Results |
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and ROS
and ROS to induce MCP-1 mRNA
expression in VSMCs, we exposed cells to TNF-
,
H2O2, or xanthine/xanthine
oxidase (to generate H2O2
and superoxide) and measured MCP-1 levels by Northern analysis.
As shown in Figure 1A
(50 to 800
U/mL) dose-dependently increased MCP-1 expression as early as 2 hours,
and levels remained elevated for at least 24 hours (Figure 1B
(400
U/mL) by 13.1±1.6-fold (n=2). ROS were also able to induce MCP-1 mRNA:
a 2-hour incubation of H2O2
upregulated MCP-1 mRNA in a dose-dependent manner (Figure 1B
|
Role of ROS in TNF-
Induced MCP-1 Upregulation
We have previously shown that TNF-
activates a
membrane-bound p22phox-based NADH/NADPH oxidase and induces superoxide
production,17 but the potential role of this
oxidase in MCP-1 induction has not been investigated. To determine
whether ROS are involved in TNF-
induced MCP-1 upregulation, we
examined the effects of various antioxidants on this response. Figure 2A
shows the effect of NADH/NADPH oxidase
inhibition by preincubation with DPI, an inhibitor of
flavin-containing oxidases,25 and stable transfection with
antisense p22phox.20 Previous experiments have shown that
both interventions inhibited TNF-
induced NADH oxidase activity,
without affecting activity in control cells.17 However,
neither DPI nor p22phox antisense transfection influenced the level of
MCP-1 induced by TNF-
, although antisense p22phox decreased the
baseline. Similarly, inhibitors of xanthine oxidase
(allopurinol, 10 mmol/L) and
lipoxygenase/cytochrome P-450 monooxygenase
(5,8,11,14-eicosatetraynoic acid, 50 µmol/L) did not attenuate
TNF-
induced MCP-1 induction (data not shown).
|
To confirm further the lack of involvement of ROS in TNF-
induced
MCP-1 upregulation, we used a series of antioxidant interventions that
inhibit ROS-mediated reactions by different mechanisms. Neither
N-acetylcysteine (NAC, 10 mmol/L),
dimethylthiourea (10 mmol/L), pyrrolidine
dithiocarbamate (100 µmol/L), liposomal superoxide dismutase
(200 U/mL), nor catalase (3000 U/mL) had any effect on MCP-1 mRNA
induction by TNF-
(Figure 2B
). Each of these interventions
has been shown to successfully inhibit ROS-mediated events in VSMCs and
other cell types when used under the circumstances and concentrations
of the present study.18 26 27 28 29 These data suggest that
blocking redox-mediated reactions alone in VSMCs is not sufficient to
inhibit TNF-
mediated MCP-1 gene induction.
Role of MAPK Pathways in TNF-
Induced MCP-1
Upregulation
Activation of MAPKs depends on tyrosine
phosphorylation. As a first approach to assess a
possible involvement of MAPK pathways in TNF-
induced MCP-1
expression, we examined the effect of the nonselective tyrosine kinase
inhibitor genistein. As shown in Figure 3
, genistein dose-dependently reduced
MCP-1 mRNA in both unstimulated and TNF-
stimulated cells. The
extent of inhibition by genistein was greater in cells exposed to
TNF-
, indicating that MCP-1 induction by TNF-
is at least
partially dependent on a tyrosine kinase pathway. These data raise the
possibility that MAPKs may be involved in this response.
|
To assess the ability of TNF-
to activate MAPKs in these
cells, we measured phosphorylation of ERK1/2 and
p38MAPK, which correlates with activation. As shown in Figure 4A
, TNF-
induced a rapid biphasic
increase in ERK1/2 phosphorylation, which was detected
as early as 2 minutes after addition of TNF-
and reached a maximum
after 15 minutes. In contrast, TNF-
activation of p38MAPK (Figure 4B
) was monophasic and peaked at 10 minutes
|
Because TNF-
strongly activated both ERK1/2 and p38MAPKs, we
examined the involvement of these pathways in TNF-
stimulated MCP-1
mRNA expression in VSMCs. PD098059 (100 µmol/L) and U0126
(50 µmol/L) are specific inhibitors of MAPK kinase,
the upstream kinase responsible for activation of ERK1/2. These
compounds completely inhibit ERK1/2 phosphorylation
induced by the agonist.30 31 Similarly, the downstream
effects of p38MAPK, such as ATF-2
phosphorylation,31 can be prevented by
incubation of the cells with 10 µmol/L SB203580 or
PD169316.31 32 As shown in Figure 5
, inhibition of ERK1/2 alone partially
attenuated TNF-
induced upregulation of MCP-1 mRNA (43±10%,
n=16). Inhibition of p38MAPK alone had no effect on this response, nor
did it enhance the ability of ERK1/2 inhibitors to block
MCP-1 induction (P<0.05). This observation suggests that
the ERK1/2 pathway is partly responsible for TNF-
stimulated MCP-1
upregulation.
|
Interaction of ROS and ERK1/2 in MCP-1 Regulation
The inhibition of TNF-
induced MCP-1 mRNA expression by ERK1/2
inhibitors is only partial. In VSMCs, ERK1/2 activation is
largely redox insensitive, and others have shown that ROS can
transactivate MCP-1, raising the possibility that both
ERK1/2 and a redox-sensitive pathway combine to regulate MCP-1
expression. Figure 6
shows that this was
indeed the case. DPI (10 µmol/L) alone did not affect
TNF-
stimulated MCP-1 expression, but in the presence of PD098059,
it completely abolished the effect of TNF-
(100±2% inhibition,
Figure 6
). Similarly, reduction of oxidant stress by incubation
of the cells with NAC (10 mmol/L) in the presence of PD098059
substantially reduced TNF-
induced MCP-1 mRNA upregulation (data
not shown). In contrast, coincubation of DPI and SB203580 had no effect
on TNF-
induced MCP-1 mRNA expression (data not shown). These data
suggest that ROS and ERK1/2 converge to regulate MCP-1.
|
| Discussion |
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induced MCP-1 mRNA expression in VSMCs. ERK1/2 is
necessary, but not sufficient, for MCP-1 induction. An additional
redox-sensitive pathway also appears to be necessary, because
inhibition of ROS generation by DPI or NAC enhanced the effect of
ERK1/2 inhibition. These observations support a potential interaction
of ROS with MAPKs in the regulation of inflammatory gene expression in
VSMCs.
The present literature suggests a critical role for ROS in MCP-1
gene expression. Numerous studies have shown that exogenous ROS can
induce MCP-1 mRNA,6 18 33 and in general, agonist-induced
or mechanical forceinduced MCP-1 expression can be inhibited by
antioxidants.6 34 35 We have previously shown that TNF-
activates a p22phox-based NADH/NADPH oxidase in
VSMCs17 ; thus, our present finding that multiple
classes of antioxidants had no effect on TNF-
induced MCP-1 mRNA
expression in VSMCs was somewhat unexpected. It has been shown,
however, that TNF-
stimulates the binding of several
transactivating factors to the MCP-1 promoter,16
only some of which are redox sensitive, indicating that MCP-1
expression may be the result of integrated activation of multiple
components of a complex regulatory system.
The present observation that genistein partially attenuates
TNF-
stimulated MCP-1 induction suggests that MAPKs or other
tyrosine kinasedependent mechanisms are involved. In VSMCs, TNF-
stimulates ERK1/2 and p38MAPK (Figure 4
and References 13, 36,
and 3713 36 37 ), as well as JNK.38 Furthermore, we have previously
shown that p38MAPK but not ERK1/2 is downstream from agonist-induced
ROS production in these cells.17 31 The role of
MAPK in TNF-
mediated MCP-1 induction, however, has not been
examined. In the present study, we found that 2 chemically distinct
MAPK kinase inhibitors achieved significant partial
attenuation of TNF-
stimulated MCP-1 mRNA expression. This suggests
that ERK1/2 is necessary, but not sufficient, for induction of this
gene. Similar results have been found for angiotensin II
induction of MCP-1 in these cells.39 Interestingly,
blockade of ROS-dependent mechanisms by DPI or NAC enhanced the ability
of inhibitors of the ERK1/2 pathway to abrogate MCP-1
expression. In other systems, p38MAPK has been shown to participate in
TNF-
induced MCP-1 expression,40 41 but this does not
appear to be the case in VSMCs. The TNF-
stimulated ROS-sensitive
pathway that participates in induction of MCP-1 is apparently
independent of p38MAPK, because SB203580 and PD169316 alone had no
effect on MCP-1 expression, nor were they able to substitute for
antioxidants in augmenting the effects of MAPK kinase
inhibitors. Taken together, these data suggest that
ROS-dependent and ERK1/2 pathways converge to regulate MCP-1 mRNA
expression.
The identity of the ROS-sensitive pathway remains to be defined. As
noted above, TNF-
activates JNK in some cell
types,38 and JNK has been shown to be redox
sensitive.42 However, to date, no role for JNK in MCP-1
mRNA upregulation has been demonstrated.40 It is likely
that other, non-MAPK signaling mechanisms also contribute to
agonist-mediated MCP-1 gene expression. In other systems,
phosphatidylinositol 3-kinase has been shown to be involved in
platelet-derived growth factorstimulated MCP-1
upregulation43 and has been postulated to be redox
sensitive.44 Evidence also exists for a role of NF-
B in
MCP-1 expression,45 46 but the redox sensitivity of
NF-
B activation is controversial and may depend on the
agonist.43
As discussed above, MCP-1 expression depends on the interaction of
multiple regulatory factors whose activity is temporally controlled.
Thus, although ERK1/2 phosphorylation is transient,
activation of this signaling pathway may initiate subsequent downstream
events whose effects are more sustained. Alternatively, prolonged
induction of MCP-1 mRNA by TNF-
may at least in part be due to
MAPK-independent pathways (see above). Although inhibition of ERK1/2
does partially attenuate TNF-
induction of MCP-1, the lack of effect
of antioxidants on the MCP-1 response to TNF-
suggests that ROS
alone do not fully activate transcription. However, when MAPK
kinase inhibitors are combined with antioxidants, TNF-
induction of MCP-1 mRNA levels is completely blocked, suggesting an
interaction between the 2 pathways. Interestingly, Ahmad et
al47 have recently shown that TNF-
activates
AP-1associated signaling components that regulate the nuclear
activity of NF-
B in endothelial cells. This raises
the possibility that ERK1/2, via activation of AP-1, may modify NF-
B
or other unidentified ROS-sensitive transcription factors.
Recent work has revealed that many biological processes are sensitive
to the biochemical action of oxidizing metabolites in the cell. In
contrast to previous speculations that ROS have only toxic biological
properties, these studies have demonstrated that cell growth and
hypertrophy,20 25 28
inflammation,29 paracrine communication,48 49
and apoptosis50 can be initiated and/or propagated
by fluctuations in the redox state. Imbalances in production or
scavenging of ROS can lead to pathological processes, including
atherogenesis. These processes have been shown to be mediated by
cytokines, critical determinants of the inflammatory response.
Our recent discovery that TNF-
activates NADH
oxidase,17 together with the present data
demonstrating its involvement in TNF-
mediated proinflammatory gene
induction, underscores the potential importance of NADH/NADPH oxidase
and ROS in vascular disease.
In summary, we showed in the present study that TNF-
activates both ERK1/2 and p38MAPK but that only ERK1/2 is
involved in the upregulation of MCP-1. However, concomitant and
parallel production of ROS is necessary for full induction of
MCP-1. Thus, ROS-dependent and ERK1/2-dependent pathways converge to
modulate TNF-
induced MCP-1 expression. These data provide insight
into the complex, but highly organized, interactions of ROS and protein
kinases that mediate cytokine-induced vascular inflammatory
gene expression.
| Acknowledgments |
|---|
Received July 12, 1999; accepted August 16, 1999.
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activates a
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