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Ras/Rac-Dependent Activation of p38 Mitogen-Activated Protein Kinases in Smooth Muscle Cells Stimulated by Cyclic Strain Stress
From the Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria.
Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail qingbo.xu{at}oeaw.ac.at
Abstract
Abstract—p38, a subfamily of the mitogen-activated protein kinases (MAPKs), is a crucial signal transducer between a variety of extracellular stimuli and gene expression in mammalian cells. This kinase is activated in cultured cells stimulated by heat shock, osmotic stress, and proinflammatory cytokines, but a similar activation of p38 MAPKs in vascular smooth muscle cells (SMCs) stimulated by mechanical stress has yet to be studied. We studied signal pathways leading to time- and strength-dependent p38 activation in rat SMCs in response to cyclic strain stress. p38 phosphorylation in stressed SMCs showed maximal activation at 10 minutes. This activation was significantly inhibited by pretreatment of the SMCs with pertussis toxin, a G-protein antagonist, and enhanced by treatment with suramin, a growth factor receptor antagonist, but opposite effects in the activation of extracellular signal–regulated kinases stimulated by mechanical forces were found. p38 activation was markedly reduced in stressed SMCs after protein kinase C depletion. Interestingly, SMC lines stably expressing dominant-negative ras (ras N17) or rac1 (rac1 N17) almost abolished p38 phosphorylation induced by cyclic strain stress. When p38 activation was inhibited by the specific inhibitor SB 202190, SMC migration, determined in a Boyden chamber in response to stimulation with platelet-derived growth factor-BB, and SMC proliferation, stimulated by cyclic strain stress, were abrogated. Thus, we provide the first evidence that cyclic strain stress rapidly activates p38 MAPKs via activation of protein kinase C ras/rac signal pathways, suggesting that p38 MAPKs are important signal transducers mediating the mechanical stress–induced cell responses essential for SMC migration and proliferation.
Key Words: mechanical stress • p38 mitogen-activated protein kinase • G proteins • vascular smooth muscle cells • signal transduction
Altered hemodynamic stress is correlated with cardiovascular diseases, including hypertension, venous bypass graft arteriosclerosis, and atherosclerosis.1 2 3 In vivo, the vessel walls are exposed to 2 main hemodynamic forces: shear stress, the dragging frictional force created by blood flow and mechanical stretch, and tension, a cyclic strain stress created by blood pressure.4 5 Shear stress stimulates endothelial cells to release NO6 and prostacyclin,7 resulting in vessel relaxation, whereas cyclic strain stress influences vascular smooth muscle cells (SMCs), 1 of the major constituents of blood vessel walls that is responsible for the maintenance of vascular tone.8 If the cyclic strain stress is persistent and chronically elevated, SMCs may undergo a supervenient change in structure, beginning with hypertrophy (mainly seen in small vessels) and hyperplasia (in large vessels), leading to gradual thickening of arterial walls and susequent hypertension and arteriosclerosis.1 9 10 11 There is accumulating evidence that mechanical force regulates the synthesis and/or secretion of numerous factors, including endothelin-1,12 platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),13 14 and angiotensin II,15 16 all of which are mitogens that stimulate SMC proliferation and migration. Therefore, mechanical stress plays an important role in the development of cardiovascular diseases such as hypertension and atherosclerosis.17
Mitogen-activated protein kinases (MAPKs) are a family of ubiquitous and well-characterized serine/threonine kinases thought to play a critical role in regulating cellular events required for cell growth, differentiation, and apoptosis in response to a variety of extracellular stimuli.18 19 20 Three major subfamilies of MAPKs have been identified in mammalian cells: the extracellular signal–regulated kinases (ERKs), the c-Jun NH2-terminal protein kinases (JNKs) or stress-activated protein kinases (SAPKs), and the p38 MAPKs.18 19 20 ERKs are characteristically activated by numerous hormones and growth factors and are associated with cell growth and hypertrophy.21 They are highly activated in the arterial wall in response to acute hypertension,22 angioplasty,23 24 25 26 and hypercholesterolemia26A and in cultured SMCs stimulated by mechanical stress.27 28 p38 MAPK, a homologue of the yeast HOG-1 kinase, is strongly activated by environmental stress factors, including UV light,29 30 oxidants,31 32 lipopolysaccharide,33 osmotic stress,34 heat shock,35 and proinflammatory cytokines.36 37 However, no data are available on whether cyclic strain stress can stimulate p38 MAPK activation in SMCs. We evaluated the potential effects of mechanical stress on p38 phosphorylation, possible signaling pathways, and the biological effects on SMCs cultivated on a flexible membrane and subjected to cyclic strain stress. We demonstrated that mechanical stress causes rapid p38 phosphorylation in SMCs, an event that appears to be mediated by protein kinase C (PKC) ras/rac pathways.
Methods
Materials
Monoclonal antibodies against phosphorylated
ERK1/2, JNK1/2, and p38 MAPK; pan-p38; and H-ras were obtained from
Santa Cruz Biotechnology Inc. Suramin, phorbol
12-myristate 13-acetate (PMA), pertussis toxin (PTX), and G418
were obtained from Sigma Chemical Co. SB 202190 and PD 98059 were
purchased from Calbiochem. Plasmids expressing dominant-negative ras
(ras N17), dominant-negative rac1 (rac1 N17), and myc-tagged antibody
were provided by Dr G. Baier (Institute for Medical Biology and Human
Genetics, University of Innsbruck, Innsbruck, Austria). SuperFect
reagent for transfection was purchased from Qiagen.
Cell Culture
SMCs were isolated by enzymatic digestion of rat aortas by using
a modification of the procedure of Ross and Kariya38 as
described previously39 and cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco) supplemented with 20% fetal
calf serum (FCS), penicillin (100 U/mL), and streptomycin (100
µg/mL). Cells were incubated at 37°C in a humidified atmosphere of
5% CO2. The medium was changed every 3 days, and cells
were passaged by treatment with a 0.2% trypsin–0.02% EDTA solution.
Experiments were conducted on SMCs that had just achieved
confluence.
Stable Transfection
Rat SMCs were transfected with vector, ras N17, and rac1 N17
plasmids by using the Superfect kit (Qiagen) according to the
manufacturer’s instructions. After transfection, the cells were
cultured for 24 hours, divided 1:4, and placed in culture medium
supplemented with 20% FCS and 150 µg/mL G418 to select those
carrying a neomycin-resistant gene. When 80% cell death in a
parallel group of normal SMCs was observed, the medium containing 150
µg/mL G418 was changed to a medium containing 50 µg/mL G418 to
maintain selection. After 4 to 8 weeks, individual cell colonies were
transferred for clone expansion and maintained in culture medium
supplemented with 20% FCS and 50 µg/mL G418. Western blotting
analysis with antibodies to H-ras and myc-tagged rac1 proteins
was used to identify ras N17– and rac1 N17–transfected SMCs.
Cyclic Strain Stress
SMCs were plated on silicone elastomer–bottomed culture plates
(Flexcell). Cells achieving 90% confluence were serum-starved for 3
days and subjected to mechanical stress with the cyclic stress unit, a
modification of the unit initially described by Banes et
al,40 consisting of a controlled vacuum unit and a base
plate to hold the culture plates (FX3000 AFC-CTL, Flexcell). Vacuum (15
to 20 kPa) was repeatedly applied to the elastomer-bottomed plates via
the base plate, which was placed in a humidified incubator with 5%
CO2 at 37°C. Cyclic deformation (60 cycles/min) and 5%
to 25% elongation of elastomer-bottomed plates were
used.27
Cell Pretreatment
SMCs grown in silicone elastomer–bottomed culture plates to
80% confluence were serum-starved for 3 days, and suramin (0.3
mmol/L), PMA (1 µmol/L), PTX (0.1 µg/mL), SB 202190 or PD
98059 were added. The plates were incubated for an additional 1 or 24
hours before application of cyclic strain stress.
Protein Extraction and Western Blot Analysis
After strain stress, SMCs were washed twice with cold (4°C)
PBS (pH 7.4) and harvested on ice in buffer A containing 20 mmol/L
HEPES (pH 7.4), 2 mmol/L EDTA, 50 mmol/L
ß-glycerophosphate, 1 mmol/L DTT, 1 mmol/L
Na3VO4, 1% Triton, 10% glycerol, 1 µg/mL
leupeptin, 1 µg/mL aprotinin, and 100 µmol/L PMSF. The
suspension was incubated on ice for 20 minutes with vortexing every 5
minutes. Cellular debris was then pelleted by
centrifugation for 30 minutes at 13 000 rpm (Eppendorf
centrifuge) at 4°C, supernatants were collected, and protein
concentration was measured by the Bio-Rad assay (Bio-Rad
Laboratories).
Membrane protein preparation for ras and rac analysis was similar to that described by Pomerantz et al.41 In brief, SMCs were washed with cold PBS (4°C), scraped in PBS, pelleted, and resuspended in 500 µL of homogenizing buffer (25 mmol/L HEPES, 1.0 mmol/L EDTA, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, and 1.0 mmol/L PMSF). Cells were sonicated for 10 seconds and centrifuged at 2000 rpm for 10 minutes to remove debris. The supernatant was centrifuged at 55 000g for 1 hour at 4°C. The cytosolic supernatant was removed, the membrane pellet was resuspended in 50 µL of homogenizing buffer and sonicated for 10 seconds, and protein concentration was measured. For ras and rac protein analysis, 30 to 100 µg of membrane protein was used under reduced conditions (2.5% SDS and 250 µmol/L DTT for 5 minutes at 90°C). Western blot analysis methods were similar to those described previously.42 In brief, 30 to 100 µg of proteins was separated by electrophoresis through 10% or 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were probed with antibodies against H-ras; myc-tagged rac1; and phosphorylated ERK1/2, JNK1/2, or p38 MAPK; specific antibody-antigen complexes were detected by using the ECL western blot detection kit (Amersham Co). Graphs of blots were obtained in the linear range of detection and were quantified for the level of specific induction by scanning laser densitometry (Power-Look II, UMAX Data System Inc.).
Kinase Assays
For kinase assays, 0.5 mL of supernatant containing 0.5 mg of
proteins was incubated with 10 µL of antibody against mammalian
phospho-p38 MAPK for 2 hours at 4°C with rotation. Subsequently, 40
µL of protein G–agarose suspension (Santa Cruz Biotechnology Inc)
was added, and rotation was continued for 1 hour at 4°C.
Immunocomplexes were precipitated by centrifugation and
washed twice with buffers A, B (500 mmol/L LiCl, 100 mmol/L
Tris, 1 mmol/L DTT, and 0.1% Triton X-100; pH 7.6), and C
(20 mmol/L MOPS, 2 mmol/L EGTA, 10 mmol/L
MgCl2, 1 mmol/L DTT, and 0.1% Triton X-100; pH 7.2).
p38 MAPK activities in the immunocomplexes were measured as described
previously.43 In brief, immunocomplexes were incubated
with myelin basic protein (6 µg, Upstate Biotechnology Inc) and
[
-P32]ATP (5 µCi) for 20 minutes. To stop the
reaction, 15 µL of 4x Laemmli buffer was added, and the mixture was
boiled for 5 minutes. Proteins in the kinase reaction were resolved by
SDS–polyacrylamide gel electrophoresis (15% gel) and
subjected to autoradiography.44
SMC Migration Assay
Migration of SMCs was assayed on polycarbonate filters
(Nucleopore Corp) having 5.0-µm pores in 48-well chemotaxis chambers
(Neuro Probe Inc).45 46 Cultured SMCs were trypsinized
after they had been pretreated with SB 202190 and PD 98059 for 1 hour
and suspended at a concentration of 5.0 x 105
cells/mL in serum-free DMEM with streptomycin, penicillin, SB 202190,
and PD 98059. A volume of 50 µL of SMC suspension was placed in the
upper chamber, and 30 µL of DMEM containing 100 ng/mL PDGF-BB was
placed in the lower chamber. The chambers were incubated at 37°C for
24 hours. After incubation, the filters were removed and the SMCs on
the upper side of the filter were scraped off. SMCs that had migrated
to the lower side of the filter were fixed in methanol, stained with
Diff-Quick staining solution (Baxter), and quantified under a
microscope (x100). Migration activity was expressed as the mean number
of cells that had migrated per x20 field.
[3H]Thymidine Incorporation
SMCs cultured in the flexible plates in medium containing
20% FCS at 37°C for 24 hours were serum-starved for 2 days; SMCs
were treated with SB 202190 and PD 98059 for 1 hour before being
subjected to 15% elongation stress for 30 minutes (60 cycles/min) and
incubated at 37°C for 24 hours. [3H]Thymidine was added
6 hours before cell harvest. Radiation activities were measured.
Statistical Analysis
ANOVA was performed when >2 groups were compared. An unpaired
Student’s t test was used to assess differences between 2
groups. A value of P<0.05 was considered significant.
Results
Effects of Mechanical Stress on MAPK Activation in Rat Aortic
SMCs
As shown in Figure 1A
, strain stress
treatment (60 cycles/min, 15% elongation) resulted in significant
increases in p38 MAPK phosphorylation. Kinetic
analysis indicates that this response occurred as early as 5
minutes, with maximum induction achieved 10 minutes after treatment and
declining thereafter (Figure 1A). Figure 1B shows pan-p38 MAPK proteins
from the corresponding blot, indicating a similar amount of loaded
protein. Similarly, growth-arrested SMCs were exposed to cyclic strain
stress for various times, and protein extracts from control and treated
cells were analyzed for ERK1/2 and JNK/SAPK
phosphorylation. As shown in Figure 1C, cell treatment
with mechanical stress resulted in a time-dependent induction of ERK1/2
phosphorylation that was evident at 8 minutes but that
returned to baseline by 3 hours. JNK2 phosphorylation
was highly induced by mechanical stress, but not JNK1 (Figure 1D).
Figure 1E summarizes data of p38 phosphorylation as
determined by quantification of optical densities from
autoradiograms of 3 experiments.
|
To perform tensile strength–response investigations, SMCs were
stretched for elongations of 5%, 10%, 15%, and 25% of their
original size, and the increase in p38 phosphorylation
corresponded with the increase in stretch stress of 5% to 25% (Figure 2A). Figure 2B shows the amount of total
p38 MAPKs from the corresponding blot. ERK and JNK/SAPK
phosphorylation was also observed (Figures 2C and 2D).
|
Suramin Blocked ERK but Not p38 and JNK/SAPK
Phosphorylation
We recently demonstrated that mechanical stress directly
stimulates PDGF receptor phosphorylation or activation
and that suramin, a broad-spectrum receptor antagonist,
blocked such activation.27 47 It would be interesting to
study whether growth factor receptors are also responsible for p38
activation during cyclic strain stress. Suramin treatment did not block
p38 phosphorylation but rather enhanced the activation
of p38 MAPK (Figures 3A and 3B),
suggesting that the initial signal of p38 activation in mechanically
stressed SMCs is independent of growth factor receptor activation. As
expected, suramin blocked mechanical stress–stimulated ERK
phosphorylation (Figure 3C), but not that of JNK/SAPK
(Figure 3D).
|
Effect of PTX and PKC Depletion on p38 Phosphorylation
As shown in Figure 4A, inactivation
of inhibitory GTP-binding protein (Gi protein) with PTX
(0.1 µg/mL) significantly inhibited p38
phosphorylation (by >50%), but not ERK (Figure 4B),
suggesting that the p38 signal pathway is at least partially dependent
on Gi protein in response to mechanical stress in SMCs. Figure 4C
summarizes data of p38 phosphorylation as determined by
quantification of optical densities from autoradiograms
of 2 experiments. PMA, a cell-permeable activator of PKC,
caused a high level of p38 phosphorylation (Figure 5A), whereas SMCs pretreated with PMA for
24 hours to deplete PKC showed decreased activation of p38 MAPKs
compared with the untreated group (Figure 5B), indicating a
PKC-dependent pathway. Figure 5C shows pan-p38 MAPK from the
corresponding blot. ERK phosphorylation was not
influenced by such treatment (Figure 5D), indicating distinct signal
pathways leading to ERK and p38 MAPK activation. Figure 5E summarizes
data of p38 phosphorylation as determined by
quantification of optical densities from autoradiograms
of 2 experiments; these indicate a significant inhibition of p38
activation by PKC depletion.
|
|
Involvement of ras/rac in p38 Activation
A variety of extracellular stimuli induces ras activation,
resulting in activation of the ras/raf/ERK kinase (MEK)/MAPK
cascade.48 To determine whether ras also mediates p38
phosphorylation in stressed SMCs, they were stably
transfected with plasmid expressing dominant-negative ras (ras N17) or
a vector plasmid as a control. Because plasmids contain the selective
marker gene neo, multiple G418-resistant colonies were selected
and tested for expression of ras protein by Western blotting
analysis with anti–H-ras antibody. As shown in Figure 6A, ras protein was found at lower levels
in vector-transfected controls and at much higher levels in
ras-transfected cells. ras N17 expression almost blocked p38
phosphorylation (Figure 6B). Figure 6C shows pan-p38
MAPK from the corresponding blot. Similarly, ERK activities stimulated
by mechanical stress in ras N17 SMC lines were also reduced (Figure 6D).
|
Rac is a member of the ras superfamily of small GTP-binding proteins.
Increasing evidence indicates that members of rac regulate a diverse
array of cellular events, including control of cell growth,
cytoskeletal reorganization, activation of protein kinases, and cardiac
myocyte hypertrophy.49 To explore the role of
rac in p38 phosphorylation in stress-stimulated SMCs,
we established SMC lines that stably expressed rac 1 that encoded an
myc-tagged form of a dominant-negative rac1 (rac1 N17), which expressed
a high level of this gene product (Figure 7A). We next assessed the effects of
mechanical stress on p38 phosphorylation in the rac1
N17 cell lines. As shown in Figure 7B, p38
phosphorylation in rac1 N17 cell lines was completely
blocked when compared with that in vector-transfected cell lines. The
results suggest that rac plays a key role in the signal transduction
leading to p38 phosphorylation. Pan-p38 MAPKs from the
corresponding blot are shown in Figure 7C. In addition, ERK activation
was also significantly reduced (Figure 7D).
|
Mechanical Strain Stress Activates MEK1/2 and MKK3
Growing evidence shows that growth factors acting through ras
primarily stimulate the raf/MEK1/2/ERK cascade of protein
kinases.48 In contrast, heat shock, proinflammatory
cytokines, and hyperosmolarity induce stress-related signal
pathways, including the MEKK kinase/SEK MAPK kinase
([MKK])4/SAPK(JNK) and/or the MKK3 or MKK6/p38 (hog)
pathways.48 MEK1/2 and MKK3
phosphorylation was assessed to investigate whether
mechanical strain stress activates both signal pathways.
Mechanical strain stress (60 cycles/min, 15% elongation) resulted in a
time-dependent activation of MEK1/2 and MKK3
phosphorylation (Figure 8). Maximal activation was achieved 5
minutes after stress, which was earlier than that seen with p38 and ERK
activation stimulated by stretch stress, indicating that MKK3 and MEK
are upstream activators for p38 and ERK, respectively.
|
p38-Dependent SMC Migration and Proliferation
SMC migration and proliferation are the key events in the
development of restenosis after angioplasty and venous bypass
graft. The mechanisms by which p38 MAPKs are involved in SMC migration
and proliferation remain to be elucidated. We therefore investigated
the role of p38 MAPK activation in SMC migration and proliferation. SB
202190, a specific inhibitor of p38 MAPKs, significantly
inhibited p38 activation in SMCs in response to mechanical strain
stress, as determined by kinase assay (Figure 9A); it also markedly inhibited SMC
migration stimulated by PDGF-BB added to the lower Boyden chamber
(Figure 9B). To explore the relationship between p38 MAPK activation
and SMC proliferation, quiescent SMCs were pretreated with SB 202190
for 1 hour and stretched for 10 minutes, and thymidine incorporation
was measured. As shown in Figure 9C, SB 202190 inhibited DNA synthesis
in SMCs induced by cyclic strain stress. These results indicate that
p38 MAPK pathways mediate SMC migration and proliferation in response
to growth factors and mechanical strain stress, respectively.
Meanwhile, we wondered whether PD 98059, an MEK inhibitor,
might have an effect on DNA synthesis in SMCs induced by cyclic strain
stress and SMC migration stimulated by PDGF-BB. Figure 9D shows that PD
98059 markedly inhibited DNA synthesis in SMCs induced by cyclic strain
stress, whereas PD 98059 failed to block PDGF-induced SMC migration
(data not shown), implicating a role for ERK in mediating SMC
proliferation induced by mechanical stress.
|
Discussion
We have recently demonstrated that cyclic strain stress rapidly activates the PDGF receptor/ERK/activator protein-1 signal pathway.27 In the current study, we provide the first evidence that mechanical stress–induced p38 MAPK activation, independent of growth factor receptors, is mediated by PKC, ras, and rac pathways. We also demonstrate that inhibition of p38 MAPK activity abrogates SMC migration in response to PDGF-BB and SMC proliferation stimulated by mechanical stress, which are key events in the development of arteriosclerosis. Thus, our findings could significantly enhance our understanding of the molecular mechanisms of vascular diseases related to altered hemodynamic stress.
How do SMCs "sense" and transduce the signals in response to
mechanical stress leading to rapid phosphorylation of
p38 MAPKs? We hypothesize that G proteins, including heterotrimeric and
small molecular G proteins, are directly activated as
preliminary targets of mechanical stress, independent of growth
factors. Supporting this hypothesis is the fact that p38 activation in
stretched SMCs was significantly blocked by pretreatment with PTX, a
Gi-protein antagonist, and that dominant-negative ras or
rac–transfected SMCs blocked p38 phosphorylation
stimulated by mechanical stress. Accumulating evidence also
indicates that G proteins act as a molecular switch via the change from
GDP to GTP, transducing extracellular stimuli into intracellular
responses.21 50 51 52 53 It is possible that mechanical stress
elongates and changes cellular morphology, leading to G protein
conformational alteration and subsequent exposure of the binding sites
for the changing of GDP to GTP, resulting in the MKK3/p38 MAPK pathway
activation. Concomitantly, recent reports by Gudi et al53
indicate that G proteins may act as primary mechanosensors in
shear-stressed endothelial cells. Treatment of
endothelial cells with antisense G
q
oligonucleotides inhibited shear stress–induced ras
GTPase activity, whereas "scrambled"
oligonucleotide treatment had no
effect.17 53 G proteins reconstituted into liposomes, in
the absence of receptors, have shown increased activity in response to
shear stress.17 Therefore, further study of the molecular
mechanisms of mechanical stress–activated G proteins could
enhance our knowledge of how a physical force is converted into
biological signals. On the other hand, our previous studies indicated
that suramin could inhibit PDGF receptor
phosphorylation induced by stretch
stress27 and that neointimal hyperplasia in
mouse vein grafts was reduced by locally applied suramin in
vivo.46 In this study, we found that suramin could inhibit
ERK, but not JNK, activation and enhance the p38
phosphorylation in response to mechanical stress. The
possible explanation for this phenomenon is that suramin blocked growth
factor and ERK/activator protein-1 pathways, but not G
protein–coupled receptor signaling, leading to increased p38
phosphorylation after mechanical stress. Thus, 2 signal
pathways, growth factor receptor/ERK/activator protein-1
and G protein/p38 MAPK transcriptional factors, may be required in
parallel for SMC proliferation. When 1 pathway is blocked, a reduced
rate of SMC proliferation can be seen. Support for this notion is the
fact that either the MEK inhibitor PD 98059 or the p38 MAPK
inhibitor SB 202190 significantly reduced the proliferation
rate of SMCs after stimulation by mechanical stress (Figure 9
).
In vivo, many factors or environmental stimuli are believed to be responsible for SMC migration, proliferation, or hypertrophy, which are key events in the development of (spontaneous) atherosclerosis, hypertension-related arteriosclerosis, angioplasty-induced restenosis, and venous bypass graft arteriosclerosis.1 2 3 It has been well established that ERK activation is closely related to cell proliferation in many types of cells.54 55 56 57 58 59 Concerning the influence of ERK activation on cell migration, some contradictory results have been reported. Lundberg et al60 and Xi et al61 reported that PD 98059 could inhibit ERK activity induced by angiotensin II and PDGF-BB that paralleled a partial inhibition of cell migration, whereas Riedy et al62 and Knall et al63 have found that inhibition of ERK activity failed to block cell migration in response to angiotensin II or interleukin-8, findings that were consistent with ours: that PD 98059 had no effect on SMC migration in response to PDGF-BB (data not shown). Whether p38 MAPK activation is associated with SMC proliferation in response to mechanical stress is still undetermined. In this study, we have demonstrated for the first time that p38 MAPK activation is essential for cyclic strain stress–induced DNA synthesis in SMCs. Interestingly, SB 202190, a specific inhibitor of p38 MAPKs, inhibited not only DNA synthesis but also SMC migration in response to stimulation. Concomitantly, Hedges et al64 reported that SB 203580 could completely inhibit SMC migration after their stimulation with PDGF-BB, interleukin-1ß, and transforming growth factor-ß. The mechanism appears to be that SB 203580 inhibited p38 activation, resulting in inhibition of heat shock protein 27 phosphorylation, a direct substrate of p38 MAPKs, which appear to modulate the polymerization of actin and play a role in actin cytoskeleton rearrangement. Ravanti et al65 and other groups66 67 reported that p38 MAPK could mediate matrix metalloproteinase gene expression and secretion in many types of cells, which in turn degrade the basement membrane, resulting in migration and invasion. Thus, we hypothesize that p38 MAPK activation by mechanical stress, together with other MAPK members, might be relevant in maintaining homeostasis of the arterial wall in vivo. Mechanical stress may directly perturb the cell surface or alter receptor or G protein conformation, which activates PKC, ras, or rac/p38 MAPK signal pathways, resulting in heat shock protein 27 phosphorylation or secretion of matrix metalloproteinases. An understanding of the p38 MAPK signal pathways that regulate SMC migration and proliferation in response to mechanical stress may contribute to the development of novel therapeutic strategies to inhibit SMC migration and growth in vascular diseases.
Acknowledgments
This work was supported by grants P12568-MED and P13099-MED (to Q.X.) from the Austrian Science Fund and P7919 (to Q.X.) from the Jubiläumsfonds of the Austrian National Bank. We thank Dr G. Baier (Institute for Medical Biology and Human Genetics, University of Innsbruck, Innsbruck, Austria) for kindly providing the dominant-negative ras N17 and rac1 N17 plasmids and the antibody against myc-tag.
Received August 4, 1999; accepted November 30, 1999.
References
1. Folkow B. Hypertensive structural changes in systemic precapillary resistance vessels: how important are they for in vivo haemodynamics? J Hypertens. 1995;13:1546–1559.[Medline] [Order article via Infotrieve]
2. Zou Y, Hu Y, Metzler B, Xu Q. Signal transduction in arteriosclerosis: mechanical stress-activated MAPKs in vascular smooth muscle cells. Int J Mol Med. 1998;1:827–834.[Medline] [Order article via Infotrieve]
3.
Thubrikar MJ, Robicsek F. Pressure-induced
arterial wall stress and atherosclerosis.
Ann Thorac Surg. 1995;59:1594–1603.
4.
Davies PF. Flow-mediated endothelial
mechanotransduction. Physiol Rev. 1995;75:519–560.
5. Patrick CW Jr, McIntire LV. Shear stress and cyclic strain modulation of gene expression in vascular endothelial cells. Blood Purif. 1995;13:112–124.[Medline] [Order article via Infotrieve]
6.
Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced
release of endothelium-derived relaxing factor.
Am J Physiol. 1986;250:H1145–H1149.
7. Bhagyalakshmi A, Frangos JA. Mechanism of shear-induced prostacyclin production in endothelial cells. Biochem Biophys Res Commun. 1989;158:31–37.[Medline] [Order article via Infotrieve]
8. Vinters HV, Berliner JA. The blood vessel wall as an insulin target tissue. Diabetes Metab. 1987;13:294–300.
9. Henry JP, Grim CE. Psychosocial mechanisms of primary hypertension. J Hypertens. 1990;8:783–793.[Medline] [Order article via Infotrieve]
10. Pickering TG. The study of blood pressure in every day life. In: Elbert T, Vaitl D, eds. Behavioural Medicine in Cardiovascular Disorders. New York, NY: John Wiley and Sons; 1988:27–85.
11.
Dzau VJ, Gibbons GH, Morishita R, Pratt RE. New
perspectives in hypertension research: potentials of vascular biology.
Hypertension. 1994;23:1132–1140.
12.
Kuchan MJ, Frangos JA. Shear stress regulates
endothelin-1 release via protein kinase C and cGMP in cultured
endothelial cells. Am J Physiol. 1993;264:H150–H156.
13.
Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE.
Mechanical strain induces growth of vascular smooth muscle cells via
autocrine action of PDGF. J Cell Biol. 1993;123:741–747.
14.
Cheng GC, Briggs WH, Gerson DS, Libby P, Grodzinsky AJ,
Gray ML, Lee RT. Mechanical strain tightly controls fibroblast growth
factor-2 release from cultured human vascular smooth muscle cells.
Circ Res. 1997;80:28–36.
15.
Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I,
Mizuno T, Takano H, Hiroi Y, Ueki K, Tobe K, et al.
Angiotensin II partly mediates mechanical stress–induced
cardiac hypertrophy. Circ Res. 1995;77:258–265.
16.
Kudoh S, Komuro I, Hiroi Y, Zou Y, Harada K, Sugaya T,
Takekoshi N, Murakami K, Kadowaki T, Yazaki Y. Mechanical stretch
induces hypertrophic responses in cardiac myocytes of
angiotensin II type 1a receptor knockout mice.
J Biol Chem. 1998;273:24037–24043.
17.
Traub O, Berk BC. Laminar shear stress: mechanisms by
which endothelial cells transduce an atheroprotective
force. Arterioscler Thromb Vasc Biol. 1998;18:677–685.
18.
Davis RJ. The mitogen-activated protein kinase
signal transduction pathway. J Biol Chem. 1993;268:14553–14556.
19. Seger R, Krebs EG. The MAPK signaling cascade. FASEB J. 1995;9:726–735.[Abstract]
20. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994;369:156–160.[Medline] [Order article via Infotrieve]
21.
Zou Y, Komuro I, Yamazaki T, Kudoh S, Uozumi H,
Kadowaki T, Yazaki Y. Both Gs and Gi proteins are critically involved
in isoproterenol-induced cardiomyocyte
hypertrophy. J Biol Chem. 1999;274:9760–9770.
22. Xu Q, Liu Y, Gorospe M, Udelsman R, Holbrook NJ. Acute hypertension activates mitogen-activated protein kinases in arterial wall. J Clin Invest. 1996;97:508–514.[Medline] [Order article via Infotrieve]
23.
Hu Y, Cheng L, Hochleitner BW, Xu Q. Activation of
mitogen-activated protein kinases (ERK/JNK) and AP-1
transcription factor in rat carotid arteries after balloon injury.
Arterioscler Thromb Vasc Biol. 1997;17:2808–2816.
24.
Pyles JM, March KL, Franklin M, Mehdi K, Wilensky RL,
Adam LP. Activation of MAP kinase in vivo follows balloon overstretch
injury of porcine coronary and carotid arteries. Circ
Res. 1997;81:904–910.
25. Lille S, Daum G, Clowes MM, Clowes AW. The regulation of p42/p44 mitogen-activated protein kinases in the injured rat carotid artery. J Surg Res. 1997;70:178–186.[Medline] [Order article via Infotrieve]
26.
Koyama H, Olson NE, Dastvan FF, Reidy MA. Cell
replication in the arterial wall: activation of signaling
pathway following in vivo injury. Circ Res. 1998;82:713–721.
26.
Hu Y, Dietrich H, Metzler B, Wick G, Xu Q. Hyperexpression and
activation of extracellular signal-reduced kinases (ERK1/2) in
atherosclerotic lesions of cholesterol-fed rabbits. Arterioscler
Thromb Vasc. Biol.. 2000;20:18–26.
27.
Hu Y, Bock G, Wick G, Xu Q. Activation of PDGF receptor
in vascular smooth muscle cells by mechanical stress. FASEB
J. 1998;12:1135–1142.
28. Reusch HP, Chan G, Ives HE, Nemenoff RA. Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition. Biochem Biophys Res Commun. 1997;237:239–244.[Medline] [Order article via Infotrieve]
29. Iordanov M, Bender K, Ade T, Schmid W, Sachsenmaier C, Engel K, Gaestel M, Rahmsdorf HJ, Herrlich P. CREB is activated by UVC through a p38/HOG-1-dependent protein kinase. EMBO J. 1997;16:1009–1022.[Medline] [Order article via Infotrieve]
30. Price MA, Cruzalegui FH, Treisman R. The p38 and ERK MAPK pathways cooperate to activate ternary complex factors and c-fos transcription in response to UV light. EMBO J. 1996;15:6552–6563.[Medline] [Order article via Infotrieve]
31. Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, Kuboki K, Meier M, Rhodes CJ, King GL. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest. 1999;103:185–195.[Medline] [Order article via Infotrieve]
32.
Ogura M, Kitamura M. Oxidant stress incites spreading
of macrophages via extracellular signal-regulated kinases and
p38 mitogen-activated protein kinase. J
Immunol. 1998;161:3569–3574.
33.
Dean JL, Brook M, Clark AR, Saklatvala J. p38
mitogen-activated protein kinase regulates
cyclooxygenase-2 mRNA stability and transcription
in lipopolysaccharide-treated human monocytes. J
Biol Chem. 1999;274:264–269.
34.
Sheikh-Hamad D, Di Mari J, Suki WN, Safirstein R, Watts
BA III, Rouse D. p38 kinase activity is essential for osmotic induction
of mRNAs for HSP70 and transporter for organic solute betaine in
Madin-Darby canine kidney cells. J Biol Chem. 1998;273:1832–1837.
35. McDowell HE, Eyers PA, Hundal HS. Regulation of system A amino acid transport in L6 rat skeletal muscle cells by insulin, chemical and hyperthermic stress. FEBS Lett. 1998;441:15–19.[Medline] [Order article via Infotrieve]
36.
Awane M, Andres PG, Li DJ, Reinecker HC.
NF-
B-inducing kinase is a common mediator of IL-17-, TNF--, and
IL-1ß-induced chemokine promoter activation in intestinal epithelial
cells. J Immunol. 1999;162:5337–5344.
37.
He Q, LaPointe MC. Interleukin-1ß regulation of the
human brain natriuretic peptide promoter involves Ras-,
Rac-, and p38 kinase-dependent pathways in cardiac myocytes.
Hypertension. 1999;33:283–289.
38. Ross R, Kariya B. Smooth muscle cells in culture. In: Bohr DF, Somlyo AP, Sparks HV, eds. Handbook of Physiology: Circulation, Vascular Smooth Muscle. Bethesda, MD: American Physiological Society; 1980:69–91.
39.
Xu Q, Li DG, Holbrook NJ, Udelsman R. Acute
hypertension induces heat-shock protein 70 gene expression in rat
aorta. Circulation. 1995;92:1223–1229.
40. Banes AJ, Gilbert J, Taylor D, Monbureau O. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J Cell Sci. 1985;75:35–42.[Abstract]
41.
Pomerantz KB, Lander HM, Summers B, Robishaw JD,
Balcueva E, Hajjar DP. G-protein-mediated signaling in
cholesterol-enriched arterial smooth muscle
cells, 1: reduced membrane-associated G-protein content due to
diminished isoprenylation of G- subunits and p21ras.
Biochemistry. 1997;36:9523–9531.[Medline]
[Order article via Infotrieve]
42. Xu Q, Hu Y, Kleindienst R, Wick G. Nitric oxide induces heat-shock protein 70 expression in vascular smooth muscle cells via activation of heat shock factor 1. J Clin Invest. 1997;100:1089–1097.[Medline] [Order article via Infotrieve]
43. Liu Y, Guyton KZ, Gorospe M, Xu Q, Lee JC, Holbrook NJ. Differential activation of ERK, JNK/SAPK and P38/CSBP/RK map kinase family members during the cellular response to arsenite. Free Radic Biol Med. 1996;21:771–781.[Medline] [Order article via Infotrieve]
44.
Hu Y, Metzler B, Xu Q. Discordant activation of
stress-activated protein kinases or c-Jun
NH2-terminal protein kinases in tissues of heat-stressed
mice. J Biol Chem. 1997;272:9113–9119.
45.
Zempo N, Koyama N, Kenagy RD, Lea HJ, Clowes AW.
Regulation of vascular smooth muscle cell migration and proliferation
in vitro and in injured rat arteries by a synthetic matrix
metalloproteinase inhibitor. Arterioscler Thromb Vasc
Biol. 1996;16:28–33.
46.
Hu Y, Zou Y, Dietrich H, Wick G, Xu Q. Inhibition of
neointima hyperplasia of mouse vein grafts by locally
applied suramin. Circulation. 1999;100:861–868.
47. Hu Y, Hochleitner BW, Wick G, Xu Q. Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stress-activated protein kinases, in in vitro propagated endothelial cells. Exp Gerontol. 1998;33:601–613.[Medline] [Order article via Infotrieve]
48. Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol. 1999;11:211–218.[Medline] [Order article via Infotrieve]
49. Pracyk JB, Tanaka K, Hegland DD, Kim KS, Sethi R, Rovira II, Blazina DR, Lee L, Bruder JT, Kovesdi I, Goldshmidt-Clermont PJ, Irani K, Finkel T. A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy. J Clin Invest. 1998;102:929–937.[Medline] [Order article via Infotrieve]
50. Holler C, Freissmuth M, Nanoff C. G proteins as drug targets. Cell Mol Life Sci. 1999;55:257–270.[Medline] [Order article via Infotrieve]
51. Dorn GW II, Brown JH. Gq signaling in cardiac adaptation and maladaptation. Trends Cardiovasc Med. 1999;9:26–34.[Medline] [Order article via Infotrieve]
52.
Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett
SB, Chien KR, Brown JH, Dorn GW II. Enhanced G signaling: a common
pathway mediates cardiac hypertrophy and apoptotic
heart failure. Proc Natl Acad Sci U S A. 1998;95:10140–10145.
53.
Gudi S, Nolan JP, Frangos JA. Modulation of GTPase
activity of G proteins by fluid shear stress and phospholipid
composition. Proc Natl Acad Sci U S A. 1998;95:2515–2519.
54.
Li C, Hu Y, Mayr M, Xu Q. Cyclic strain stress-induced
mitogen-activated protein kinase (MAPK) phosphatase 1
expression in vascular smooth muscle cells is regulated by Ras/Rac-MAPK
pathways. J Biol Chem. 1999;274:25273–25280.
55.
Metzler B, Li C, Hu Y, Sturm G, Ghaffari-Tabrizi N, Xu
Q. LDL stimulates mitogen-activated protein kinase
phosphatase-1 expression, independent of LDL receptors, in vascular
smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999;19:1862–1871.
56. Bornfeldt KE, Krebs EG. Crosstalk between protein kinase A and growth factor receptor signaling pathways in arterial smooth muscle. Cell Signal. 1999;11:465–477.[Medline] [Order article via Infotrieve]
57.
Talarmin H, Rescan C, Cariou S, Glaise D, Zanninelli G,
Bilodeau M, Loyer P, Guguen-Guillouzo C, Baffet G. The
mitogen-activated protein kinase kinase/extracellular
signal-regulated kinase cascade activation is a key signalling pathway
involved in the regulation of G(1) phase progression in proliferating
hepatocytes. Mol Cell Biol. 1999;19:6003–6011.
58. Pintucci G, Steinberg BM, Seghezzi G, Yun J, Apazidis A, Baumann FG, Grossi EA, Colvin SB, Mignatti P, Galloway AC. Mechanical endothelial damage results in basic fibroblast growth factor-mediated activation of extracellular signal-regulated kinases. Surgery. 1999;126:422–427.[Medline] [Order article via Infotrieve]
59. Tanaka K, Abe M, Sato Y. Roles of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in the signal transduction of basic fibroblast growth factor in endothelial cells during angiogenesis. Jpn J Cancer Res. 1999;90:647–654.[Medline] [Order article via Infotrieve]
60. Lundberg MS, Curto KA, Bilato C, Monticone RE, Crow MT. Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways. J Mol Cell Cardiol. 1998;30:2377–2389.[Medline] [Order article via Infotrieve]
61.
Xi XP, Graf K, Goetze S, Fleck E, Hsueh WA, Law
RE. Central role of the MAPK pathway in ang II–mediated DNA synthesis
and migration in rat vascular smooth muscle cells. Arterioscler
Thromb Vasc Biol. 1999;19:73–82.
62. Riedy MC, Brown MC, Molloy CJ, Turner CE. Activin A and TGF-ß stimulate phosphorylation of focal adhesion proteins and cytoskeletal reorganization in rat aortic smooth muscle cells. Exp Cell Res. 1999;251:194–202.[Medline] [Order article via Infotrieve]
63.
Knall C, Worthen GS, Johnson GL. Interleukin
8-stimulated phosphatidylinositol-3-kinase activity regulates the
migration of human neutrophils independent of extracellular
signal-regulated kinase and p38 mitogen-activated protein
kinases. Proc Natl Acad Sci U S A. 1997;94:3052–3057.
64.
Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey
E, Weber LA, Gerthoffer WT. A role for p38(MAPK)/HSP27 pathway in
smooth muscle cell migration. J Biol Chem. 1999;274:24211–24219.
65.
Ravanti L, Heino J, Lopez-Otin C, Kahari VM. Induction
of collagenase-3 (MMP-13) expression in human skin
fibroblasts by three-dimensional collagen is mediated by p38
mitogen-activated protein kinase. J Biol Chem. 1999;274:2446–2455.
66. Westermarck J, Holmstrom T, Ahonen M, Eriksson JE, Kahari VM. Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases Jun N-terminal kinase and p38. Matrix Biol. 1998;17:547–557.[Medline] [Order article via Infotrieve]
67.
Simon C, Goepfert H, Boyd D. Inhibition of the p38
mitogen-activated protein kinase by SB 203580 blocks
PMA-induced Mr 92,000 type IV collagenase secretion and in
vitro invasion. Cancer Res. 1998;58:1135–1139.
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