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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e1-e9

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e1.)
© 2000 American Heart Association, Inc.

ATVB Electronic Pages

Ras/Rac-Dependent Activation of p38 Mitogen-Activated Protein Kinases in Smooth Muscle Cells Stimulated by Cyclic Strain Stress

Chaohong Li; Yanhua Hu; Gertraud Sturm; Georg Wick; Qingbo Xu

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 [{gamma}-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 1ADown, 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 1ADown). Figure 1BDown 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 1CDown, 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 1DDown). Figure 1EDown summarizes data of p38 phosphorylation as determined by quantification of optical densities from autoradiograms of 3 experiments.



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  		Figure 1. Time course of p38 MAPK phosphorylation in SMCs exposed to mechanical stress. SMCs were serum-starved for 3 days, treated with cyclic strain stress (60 cycles/min, 15% elongation) for the indicated times, and then harvested. Proteins were extracted, electrophoresed on 10% SDS-polyacrylamide gels, transferred to mitrocellulose membranes, and probed by using antibodies to phosphorylated (P) p38 (A) and pan-p38 MAPKs (B). C and D, Results of the Western blot analysis for phosphorylated (P) ERK1/2 and JNK1/2. E, Statistical data of phosphorylated (P) p38 protein induction from 3 independent experiments. *Significant difference from untreated controls at P<0.05.

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 2ADown). Figure 2BDown shows the amount of total p38 MAPKs from the corresponding blot. ERK and JNK/SAPK phosphorylation was also observed (Figures 2CDown and 2DDown).



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  		Figure 2. Effects of mechanical strain stress on p38 MAPK activation. SMCs were serum-starved for 3 days and treated with strain stress (60 cycles/min, 5% to 25% elongation) for 10 minutes. Western blot analysis indicated phosphorylation of p38 (A), pan-p38 (B), phospho (P)-ERK (C), and JNK (D) as described in the legend to Figure 1Up, and in Methods.

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 3ADown and 3BDown), 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 3CDown), but not that of JNK/SAPK (Figure 3DDown).



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  		Figure 3. Effects of suramin on p38 MAPK phosphorylation. Quiescent SMCs pretreated with suramin (0.3 mmol/L) for 30 minutes were stressed for 10 minutes and harvested for protein extracts. The results of Western blot analysis are shown for phosphorylated (P) p38 (A), ERK1/2 (C), and JNK1/2 (D). B, Statistical data of phosphorylated (P) p38 protein levels. Data represent similar results from 3 independent experiments. S indicates serum treatment as a positive control. *Significant difference from strain-stressed group at P<0.05.

Effect of PTX and PKC Depletion on p38 Phosphorylation
As shown in Figure 4ADown, inactivation of inhibitory GTP-binding protein (Gi protein) with PTX (0.1 µg/mL) significantly inhibited p38 phosphorylation (by >50%), but not ERK (Figure 4BDown), suggesting that the p38 signal pathway is at least partially dependent on Gi protein in response to mechanical stress in SMCs. Figure 4CDown 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 5ADown), whereas SMCs pretreated with PMA for 24 hours to deplete PKC showed decreased activation of p38 MAPKs compared with the untreated group (Figure 5BDown), indicating a PKC-dependent pathway. Figure 5CDown shows pan-p38 MAPK from the corresponding blot. ERK phosphorylation was not influenced by such treatment (Figure 5DDown), indicating distinct signal pathways leading to ERK and p38 MAPK activation. Figure 5EDown 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.



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  		Figure 4. Effect of PTX on p38 MAPK phosphorylation. Quiescent SMCs pretreated with PTX (0.1 µg/mL) for 24 hours were stressed for 10 minutes and harvested for protein extracts. The results of Western blot analysis are shown for phosphorylated (P) p38 (A) and ERK1/2 (B). C, Statistical data of phosphorylated (P) p38 protein levels. S indicates serum treatment as positive control. *Significant difference from strain-stressed group at P<0.05.



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  		Figure 5. Effect of PMA on p38 phosphorylation. Quiescent SMCs pretreated with PMA (1 µmol/L) for 10 minutes (A) or 24 hours (B), were treated with cyclic strain stress for 10 minutes. The results of Western blot analysis are shown for phosphorylated (P) p38 (A and B) and ERK1/2 (D). C, Total p38 proteins labeled with anti–pan-p38 antibodies for each sample tested. E, Statistical data of phosphorylated (P) p38 protein levels. S indicates serum treatment as positive control. *Significant difference from strain-stressed group at P<0.05.

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 6ADown, 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 6BDown). Figure 6CDown shows pan-p38 MAPK from the corresponding blot. Similarly, ERK activities stimulated by mechanical stress in ras N17 SMC lines were also reduced (Figure 6DDown).



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  		Figure 6. Dominant-negative ras overexpression inhibited p38 activation. Rat SMCs were stably transfected with constructs expressing dominant-negative ras (pEF–ras N17; R) or vector (pEF-neo; N) by using the SuperFect reagent in a 1:2 ratio (wt/wt). The transfected cells were cultured overnight, divided 1:4, and placed in culture medium supplemented with 150 µg/mL G418. SMC lines expressing ras N17 were identified with an anti–H-ras antibody, as determined by Western blotting (A). SMC lines were treated with mechanical stress for the indicated times (60 cycles/min, 15% elongation; B). Western blot analysis was performed with anti–phosphorylated (P) p38 (B), pan-p38 (C), and anti-phosphorylated ERK1/2 (D) antibodies. Data represent similar results from 3 independent experiments.

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 7ADown). We next assessed the effects of mechanical stress on p38 phosphorylation in the rac1 N17 cell lines. As shown in Figure 7BDown, 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 7CDown. In addition, ERK activation was also significantly reduced (Figure 7DDown).



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  		Figure 7. Dominant-negative rac1 overexpression blocked p38 phosphorylation. The procedures for establishing stably expressing dominant-negative rac1 (pEF–rac1 N17) cell lines are similar to those described in the legend to Figure 6Up and in Methods. A, Results of Western blot analysis with an anti–myc-tagged rac1 antibody. SMC lines were treated with mechanical stress for the indicated times (60 cycles/min, 15% elongation; B). Western blot analysis was performed with anti–phosphorylated (P) p38 (B), pan-p38 (C), and anti–phosphorylated ERK1/2 (D) antibodies. Data represent similar results from 3 independent experiments.

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 8Down). 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.



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  		Figure 8. MKK3- and MEK-phosphorylation (P). Quiescent SMCs were stressed for 10 minutes and harvested for protein extracts. The results of Western blot analysis are shown for phosphorylated MEK1/2 (A) and MKK3 (B). The procedures are similar to those described in the legend to Figure 1Up. Data represent similar results from 2 independent experiments.

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 9ADown); it also markedly inhibited SMC migration stimulated by PDGF-BB added to the lower Boyden chamber (Figure 9BDown). 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 9CDown, 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 9DDown 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.



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  		Figure 9. Effect of SB 202190 on p38 activation and SMC migration/proliferation. Quiescent SMCs were treated with SB 202190 for 1 hour before being stressed (10 minutes). p38 proteins were immunoprecipitated from protein extracts, and their kinase activities were measured on the basis of phosphorylation of myelin basic protein (MBP) substrate (A). To assess migration, SMCs were stimulated with 100 ng/mL PDGF-BB, and the effect of SB 202190 on SMC migration was determined in a Boyden chamber as described in Methods. Migrating cell numbers in the absence and presence of SB 202190 are shown in B. Results are expressed as a percentage of PDGF-stimulated migration (mean±SD). C and D, Statistical results of DNA synthesis in SMCs pretreated with SB 202190 or PD 98059 that were subjected to strain stress. *Significant difference from control at P<0.05.

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{alpha} 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 9Up).

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.

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