Cyclic Strain Stress-induced Mitogen-activated Protein Kinase (MAPK) Phosphatase 1 Expression in Vascular Smooth Muscle Cells Is Regulated by Ras/Rac-MAPK Pathways*

Recently, we demonstrated that mechanical stress results in rapid phosphorylation or activation of platelet-derived growth factor receptors in vascular smooth muscle cells (VSMCs) followed by activation of mitogen-activated protein kinases (MAPKs) and AP-1 transcription factors (Hu, Y., Bock, G., Wick, G., and Xu, Q. (1998) FASEB J. 12, 1135–1142). Herein, we provide evidence that VSMC responses to mechanical stress also include induction of MAPK phosphatase-1 (MKP-1), which may serve as a negative regulator of MAPK signaling pathways. When rat VSMCs cultivated on a flexible membrane were subjected to cyclic strain stress (60 cycles/min, 5–30% elongation), induction of MKP-1 proteins and mRNA was observed in time- and strength-dependent manners. Concomitantly, mechanical forces evoked rapid and transient activation of all three members of MAPKs, i.e.extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal protein kinases (JNKs), or stress-activated protein kinases (SAPKs), and p38 MAPKs. Suramin, a growth factor receptor antagonist, completely abolished ERK activation, significantly blocked MKP-1 expression, but not JNK/SAPK and p38 MAPK activation, in response to mechanical stress. Interestingly, VSMC lines stably expressing dominant negative Ras (Ras N17) or Rac (Rac N17) exhibited a marked decrease in MKP-1 expression; the inhibition of ERK kinases (MEK1/2) by PD 98059 or of p38 MAPKs by SB 202190 resulted in a down-regulation of MKP-1 induction. Furthermore, overexpressing MKP-1 in VSMCs led to the dephosphorylation and inactivation of ERKs, JNKs/SAPKs, and p38 MAPKs and inhibition of DNA synthesis. Taken together, our findings demonstrate that mechanical stress induces MKP-1 expression regulated by two signal pathways, including growth factor receptor-Ras-ERK and Rac-JNK/SAPK or p38 MAPK, and that MKP-1 inhibits VSMC proliferation via MAPK inactivation. These results suggest that MKP-1 plays a crucial role in mechanical stress-stimulated signaling leading to VSMC growth and differentiation.

Intracellular signaling stimulated by growth factors, cytokines, osmotic shock, and stress involves the initiation of one or more phosphorylation cascades leading to the rapid and revers-ible activation of mitogen-activated protein kinases (MAPKs), 1 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 (1)(2)(3). Three major subfamilies of MAPKs have been identified, including the extracellular signal-regulated kinases (ERKs), c-Jun NH 2 -terminal protein kinases (JNKs) or stress-activated protein kinases (SAPKs), and p38 MAPKs (1)(2)(3). They are strongly activated in the arterial wall in response to angioplasty (4 -7), hypertension (8), and hypercholesterolemia, 2 which are risk factors for vascular diseases.
In vivo, vessel walls are exposed to three main hemodynamic forces: shear stress, the dragging frictional force created by blood flow; transmural pressure, created by the hydrostatic forces of blood within the blood vessel; and mechanical stretch or tension, a cyclic strain stress created by blood pressure (19,20). VSMCs are one of the major constituents of blood vessel wall responsible for the maintenance of vascular tone (21). Factors ranging from physical exertion to psychological stress lead to a transient rise in blood pressure (22,23), and if the factors are persistent and chronic, the arteriole walls gradually thicken, resulting in hypertension (22)(23)(24)(25). In humans, atherosclerotic lesions occur preferentially at bifurcations and curvatures (26), where hemodynamic force is disturbed (27). There is growing evidence that mechanical force initiates intracellular signaling and regulates the synthesis and/or secretion of numerous factors, including NO (28), prostacyclin (29), endothelin-1 (30), platelet-derived growth factor, fibroblast growth factor (31,32), and angiotensin II (33,34), which are crucial factors in maintaining the homeostasis of the vessel wall. Thus, mechanical stress plays an important role in the development of hypertension and atherosclerosis (35).
Xu et al. (36) have previously shown that acute hypertension induces a rapid and transient expression of MKP-1 mRNA followed by elevated MKP-1 protein in rat aorta. The MKP-1 induction is blocked by prevention of elevation in blood pressure, i.e. administration of the vasodilator agent sodium nitroprusside. However, it is not known whether MKP-1 production is initiated by hemodynamic force per se or by hormones, cytokines in vivo. In the present study, we evaluated potential effects of mechanical stress on MKP-1 induction in VSMCs cultivated on a flexible membrane and subjected to cyclic strain stress. We demonstrated that mechanical stress causes rapid MKP-1 expression in VSMCs, which appears to be mediated by Ras-and Rac-MAPK pathways, respectively.
Cell Culture-VSMCs were isolated by enzymatic digestion of rat aortas using a modification of the procedure of Ross and Kariya (37), as described previously (38), and cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 20% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 g/ml). Cells were incubated at 37°C in a humidified atmosphere of 5% CO 2 . The medium was changed every 3 days, and cells were passaged by treatment with 0.2% trypsin, 0.02% EDTA solution. Experiments were conducted on VSMCs that had just achieved confluence.
Stable Transfection-Rat VSMCs were transfected with Ras N17, Rac N17, and MKP-1 plasmids, respectively, by using SuperFect Kit according to the manufacture's instructions. After transfection, the cells were cultured for 24 h, divided 1 to 4, and placed in culture medium supplemented with 20% fetal calf serum and 150 g/ml G418 to select those carrying a neomycin-resistant plasmid. When 80% cell death in a parallel group of normal VSMCs was observed, the medium containing 150 g/ml G418 was changed into medium containing 50 g/ml G418 to maintain selection. After 4 -8 weeks, individual cell colonies were transferred for clone expansion and maintained in culture medium supplemented with 20% fetal calf serum and 50 g/ml G418. Ras N17-, Rac N17-, and MKP-1-transfected VSMCs were identified by Western blotting analysis with antibodies to Ha-Ras, myc-tagged, and MKP-1 proteins.
Cyclic Strain Stress-VSMCs were plated on silicone elastomer-bottomed culture plates (Flexcell, Meckeesport, PA). 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. (39) consisting of a controlled vacuum unit and a base plate to hold the culture plates (FX3000 AFC-CTL, Flexcell). A vacuum (15 to 20 kilopascals) was repeatedly applied to the elastomerbottomed plates via the base plate, which was placed in a humidified incubator with 5% CO 2 at 37°C. Cyclic deformation (60 cycles/min) and 5 to 30% elongation of elastomer-bottomed plates were used (40).
Cell Pretreatment-VSMCs, growing in silicone elastomer-bottomed culture plates to 90% confluence, were serum-starved for 3 days, and suramin, PD 98059, or SB 202190 was added and incubated for 1 h before application of cyclic strain stress.
Membrane protein preparation for Ras and Rac analysis was similar to that described by Pomerantz et al. (41). Briefly, VSMCs were washed with cold phosphate-buffered saline (4°C), scraped in phosphate-buffered saline, pelleted, and resuspended in 500 l of homogenizing buffer (25 mM Hepes, 1.0 mM EDTA, 1 g/ml leupeptin, 1 g/ml pepstatin A, and 100 M phenylmethylsulfonyl fluoride). Cells were sonicated for 10 s and centrifuged at 2,000 rpm for 10 min to remove debris. The supernatant was centrifuged at 55,000 ϫ g for 1 h at 4°C. The cytosolic supernatant was removed, the membrane pellet was resuspended in 50 l of homogenizing buffer and sonicated for 10 s, and protein concentration was measured. For total protein extract, cells were washed with cold phosphate-buffered saline (4°C), pelleted, resuspended in 100 l of buffer A, sonicated for 10 s, and centrifuged at 2,000 rpm for 10 min before supernatant harvest. For Ras and Rac protein analysis, 30 -100 g of membrane protein was used in reduced conditions (2.5% SDS, 250 M dithiothreitol for 5 min at 90°C). The procedure used for Western blot analysis was similar to that described previously (42). In short, 30 -100 g of proteins were separated by electrophoresis through a 10% or 12% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. The blots were probed with antibodies against MKP-1, Ha-Ras, myc-tagged Rac1, phosphorylated-ERK1/2, -JNK1/2, or -p38 MAPK, and specific antibody-antigen complexes were detected using the ECL Western blot Detection Kit (Amersham Pharmacia Biotech). 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., Hsinchu, Taiwan) of graphs.
RNA Isolation and Northern Blot-Total RNA was isolated as described elsewhere (36). RNA (10 g/lane) was denatured with formaldehyde (Merck), electrophoresed in a 1% agarose gel, transferred onto nylon membrane (Zeta Probe, Bio-Rad), and UV-cross-linked in a UV Stratalinker (Stratagene Inc., La Jolla, CA). Hybridizations were performed using a fluorescein-labeled (Amersham Pharmacia Biotech) cDNA probe for MKP-1, as described previously (36). The membranes were then washed, detected with antifluorescein alkaline phosphatase conjugate (1:5,000) (Amersham Pharmacia Biotech), and exposed to ECL films. Graphs of blots were obtained in the linear range of detection. Accuracy of loading and transfer as well as RNA integrity was confirmed by quantitative analysis of the 28 S and 18 S RNA.
ERK2 or p38 activities in the immunocomplexes were measured as described previously (43,44). Briefly, immunocomplexes were incubated with myelin basic protein (6 g; Upstate Biotechnology, Inc., Lake Placid, NY) and [␥-32 P]ATP (5 Ci) for 20 min. To stop the reaction, 15 l of 4ϫ Laemmli buffer was added, and the mixture was boiled for 5 min. Proteins in the kinase reaction were resolved by SDS-polyacrylamide gel electrophoresis (15% gel) and subjected to autoradiography.
The assay for JNK/SAPK activity was performed as described above using GST-c-Jun as a substrate (the plasmid was provided by Dr. J. Woodgett) produced in Escherichia coli and isolated using glutathione Sepharose 4B RediPack columns (Amersham Pharmacia Biotech) per manufacturer's protocol. Proteins in the kinase reaction were resolved by SDS-polyacrylamide gel electrophoresis (12% gel) and subjected to autoradiography (3,43,44).
[ 3 H]Thymidine Incorporation-Transfected VSMCs cultured in the flexible plates in medium containing 20% fetal calf serum at 37°C for 24 h were serum-starved for 4 days. VSMCs were stressed by elongation of 15% for 30 min (60 cycles/min) and incubated at 37°C for 24 h. Statistical Analysis-Analysis of variance was performed when more than two groups were compared. An unpaired Student's t test was used to assess differences between two groups. A p value less than 0.05 was considered significant.

RESULTS
Cyclic Strain Stress Induced MKP-1 Expression-To explore the possibility that the expression of MKP-1 is altered after mechanical stress, MKP-1 mRNA in stressed VSMCs were determined by Northern blot analysis. As shown in Fig. 1A, strain stress treatment (60 cycles/min, 15% elongation) resulted in significant increases in MKP-1 mRNA. Kinetic anal-ysis indicates that this response occurred as early as 8 min with maximum induction achieved 30 min after treatment and declining thereafter (Fig. 1A). Fig. 1B showed 18 S and 28 S RNA from the corresponding blot, indicating a similar amount of RNA loaded. Likewise, growth-arrested VSMCs were exposed to cyclic strain stress for various times, and protein extracts from control and treated cells were analyzed for MKP-1 induction. As shown in Fig. 1C, cells treated with mechanical stress resulted in a time-dependent induction of MKP-1 proteins that was evident at 8 min, peaked at 30 min, and returned to basal line by 4 h. Fig. 1D summarizes data of MKP-1 protein induction as determined by quantification of optical densities from autoradiograms of three experiments. A significant increase in MKP-1 proteins of VSMCs was observed between 30 min and 3 h.
To further establish the relationship between mechanical strain stress and MKP-1 expression, a tensile strength response analysis of mechanical stress-induced MKP-1 mRNA accumulation was performed. As shown in Fig. 2A, VSMCs were stretched for elongation of 5, 10, 15, 20, 25, and 30% of original size, respectively, and the increase of MKP-1 mRNA amounts corresponded with the increased magnitudes of stretch stress of 5-20%. Fig. 2B shows the amount of 18 S and 28 S RNA from the corresponding blot. Similar results were observed at the level of MKP-1 proteins (Fig. 2C). Fig. 2D shows statistical data from three experiments. A significant induction was found in stressed VSMCs elongated between 10 and 30%. Our results provided the first evidence that MKP-1 is induced by mechanical stress in VSMCs.
There is evidence that mechanical force rapidly activates ERK and JNK/SAPK in VSMCs (45,46), but no data exists as to whether p38 MAPK is also activated in response to cyclic strain stress. Phosphorylation of p38 MAPK, ERK1/2, and JNK1/2 was determined by Western blot analysis using antiphosphorylated-p38 MAPK, -ERK1/2, and -JNK1/2 antibodies, respectively. Fig. 3A shows phosphorylation of p38 MAPK in VSMCs treated with cyclic strain stress for various times. The highest level of p38 phosphorylation was obtained between 8 and 30 min and declined thereafter. Similarly, ERK 1/2 phosphorylation was evident, but ERK proteins did not alter in response to strain stress (Fig. 3, B and C). ERK activity was measured for immunoprecipitated ERK2 to phosphorylate myelin basic protein, indicating that mechanical strain stress induced marked ERK activation (Fig. 3D). Strain stress also resulted in a rapid activation of JNK/SAPK (Fig. 3, E and F) in VSMCs. The activities of three MAPKs declined between 30 and 120 min in response to mechanical stress (Fig. 3), whereas higher levels of MKP-1 proteins persisted (Fig. 1C), suggesting a role of MKP-1 on inactivation of MAPKs.
Suramin Partially Blocked MKP-1 Expression-We recently demonstrated that mechanical stress directly stimulates platelet-derived growth factor receptor phosphorylation or activa- tion and that suramin, a broad spectrum receptor antagonist, blocked such activation (40). It was not known if growth factor receptors were also responsible for MKP-1 induction during mechanical stress. Suramin treatment markedly blocked MKP-1 expression (Fig. 4A), suggesting that the initial signal in mechanical stressed-VSMCs is, at least in part, generated on the plasma membrane via growth factor receptor activation. As expected, suramin completely blocked mechanical stress-stimulated phosphorylation of ERK (Fig. 4B) but not JNK/SAPK (Fig. 4C). Surprisingly, it apparently enhanced the activation of p38 MAPK (Fig. 4D). These results suggest that suraminblocked MKP-1 expression may be due to inhibition of the growth factor receptor-ERK pathway, independent of the JNK/ SAPK and P38 MAPK pathways.
Involvement of Ras/Rac in MKP-1 Expression-A large number of extracellular signals stimulates Ras activation, resulting in Ras-dependent MAPK activation. Recent data reported by Gudi et al. (47) indicate that Ras protein was activated in response to shear stress in endothelial cells. It would be inter-esting to determine whether Ras also mediates MKP-1 expression in stressed VSMCs. VSMCs were stably transfected with plasmid-expressing dominant negative Ras (Ras N17) or a vector plasmid as control. Since 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-Ha-Ras antibody. As shown in Fig.  5A, Ras protein was at a lower level in vector-transfected controls and much higher in Ras-transfected cells. Ras N17 expression reduced ERK activation (Fig. 5B) and significantly blocked MKP-1 induction (Fig. 5, C and D), indicating Ras-ERK-dependent MKP-1 induction. Interestingly, stronger stress (25% elongation)-induced MKP-1 expression can only be partially blocked by Ras N17, suggesting that other signal pathways may be also involved.
Rac is a member of the Ras superfamily of small GTPbinding proteins. Increasing evidence indicates that members of Rac regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization and the activation of protein kinases, and cardiac myocyte hypertrophy (48). To explore the role of Rac in MKP-1 expression in stressstimulated VSMCs, we established VSMC lines stably expressing Rac1, encoding a myc-tagged form of a dominant negative Rac1 (Rac1 N17) that expressed a high level of this gene product (Fig. 6A). Surprisingly, overexpression of Rac1 N17 markedly inhibited ERK1/2 phosphorylation (Fig. 6B), indicating Rac-dependent ERK activation in response to mechanical stress. We next assessed the effects on MKP-1 expression in the Rac1 N17 cell lines treated with mechanical stress. As seen in  ERK-and p38-dependent MKP-1 Induction-As shown in Figs. 1 to 3, ERK, JNK, and p38 MAPK activation by mechanical stress proceeded to MKP-1 expression. To investigate whether these kinases are involved in strain stress-stimulated MKP-1 expression, ERK, JNK, and p38 MAPK phosphorylation and MKP-1 expression were simultaneously determined in stressed VSMCs treated with PD 98059, a specific MAPK kinase (MEK1/2) inhibitor. Mechanical stress-induced ERK1/2 phosphorylation was completely abolished by PD 98059 pretreatment (Fig. 7A) but not JNKs/SAPKs (Fig. 7B) and p38 MAPKs (Fig. 7C). MKP-1 induction was evidently inhibited (Fig. 7D). These results support the notion that mechanical stress-induced MKP-1 expression is partially dependent on ERK activation. Furthermore, p38 MAPK activity was abrogated by SB 202190, a specific inhibitor for p38 MAPKs (Fig.  8A). It was unexpected that mechanical stress-induced MKP-1 expression was also inhibited, at least in part (Fig. 8B). These observations implicate the involvement of p38 MAPKs in the MKP-1 induction.
MKP-1 Inhibited MAPK Activation and VSMC Proliferation-To study the effects of MKP-1 on MAPK activation and on VSMC proliferation, we established VSMC lines overexpressing MKP-1 and determined ERK, JNK/SAPK, and p38 MAPK phosphorylation and DNA synthesis in response to mechanical stress. Transfected clones stably expressing MKP-1 were identified by Western blot analysis. Although our antibody could not differentiate endogenous rat MKP-1 from overexpressed rMKP-1 proteins, the cells under low growth conditions (2% calf serum or 5% stretch elongation) minimized endogenous MKP-1 to an undetectable level, as seen in the Western blot (Fig. 9A). Cells transfected with MKP-1-expressing plasmids showed a high level of MKP-1 (Fig. 9A). Compared with vector-transfected cells, a 50 -80% reduction in ERK, JNK/SAPK, and p38 MAPK phosphorylation was observed in MKP-1-expressing VSMCs when stimulated with mechanical stress (Fig. 9, B-D). To determine the effects of MKP-1 on DNA synthesis induced by mechanical stress, rMKP-1 or vectortransfected cell lines were stimulated by mechanical stress for 30 min. As shown in Fig. 9E, [ 3 H]thymidine incorporation in rMKP-1-expressing VSMCs was significantly lower than vector-transfected cells. These results suggest that MKP-1 inhibits VSMC growth via inactivation or dephosphorylation of three MAPKs. DISCUSSION Recent evidence indicates that mechanical stress is an important extracellular stimulus and regulates gene expression, protein synthesis, and growth and differentiation of cardiovascular cells (28 -34, 45-55). The present study demonstrates for the first time that mechanical stress causes MKP-1 expression, which is crucial in regulation of MAPK activities in VSMCs. Mechanical stress also induces activation of ERK1/2, JNKs/ SAPKs, and p38 MAPKs. This process involves small molecular GTP-binding proteins, Ras and Rac. These results have several implications. First, understanding that strain stress induces MKP-1 expression may strengthen the notion that mechanical stress plays an important role in regulating VSMC growth. Although strain stress stimulates ERK, JNK/SAPK, and p38 MAPK activation, which appears to be a component common to signaling pathways initiated by a wide range of growth-stimulating factors, including mitogens and hormones (1-3, 56 -61), MKP-1 serves as a negative regulator, controlling cell growth via inactivation of three MAPKs. Second, mechanical stress plays a crucial role in the regulation of VSMC tone caused by autocrine and paracrine vasoconstrictors (28 -30, 51), such as angiotensin II and endothelin-1, which result in MKP-1 expression. In this process, MKP-1 might be involved in inactivation of numerous kinases that influence cell tone. Finally, stressinduced MKP-1 expression is not only mediated by Ras but also by Rac proteins, suggesting a complicated network of stressinduced signaling in VSMCs. Thus, our findings could significantly advance our understanding of the role of MKP-1 in VSMC proliferation, which is a key event in the pathogenesis of hypertension-related arteriosclerosis, angioplasty-induced restenosis, and vein graft arteriosclerosis.
How do the VSMCs sense and transduce the signals in response to mechanical stress, leading to MKP-1 expression? One possibility is that growth factor receptor activation is partially responsible for mechanical stress-induced MKP-1 expression. This concept is supported by our previous observation that mechanical stress could induce rapid phosphorylation of platelet-derived growth factor receptors in VSMCs (40). Suramin, a growth factor receptor antagonist, inhibited phosphorylation of platelet-derived growth factor receptor (40) and could also significantly inhibit stress-induced MKP-1 expression (Fig. 4). As we previously hypothesized (40), mechanical stress elongates and changes cellular morphology, leading to receptor conformation, resulting in exposure of the kinase domain and subsequent autophosphorylation. Thus, the mechanism of mechanical stress-induced MKP-1 expression is similar to growth factor-stimulated MKP-1 induction. Since suramin could not completely block MKP-1 expression, another possible primary mechanosensor candidate may be G proteins. Recent reports by Gudi et al. (47) 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. Treatment of endothelial cells with pertussis toxin prevented shear stressmediated activation of ERK1/2 (35,47). G proteins reconstituted in liposomes, in the absence of receptors, show an increase in activity in response to shear stress (35). This assumption is supported by our results (Figs. 5 and 6) that expression of dominant negative Ras and Rac in VSMCs exposed to mechanical stress could significantly inhibit MKP-1 expression. These observations suggest that increases in the elongational and transitional mobility in cell membranes activate membrane-bound G proteins by facilitating exchange of GDP to GTP, subsequently leading to MAPK activation and MKP-1 expression. Our hypothesis is schematically depicted in Fig. 10. In our study system, we demonstrated that MAPKs play an important role in mediating mechanical stress-induced MKP-1 expression, because both PD 98059 (MEK inhibitor) and SB 202190 (p38 MAPK inhibitor) could inhibit MKP-1 expression. The time course of phosphorylation of three MAPKs appears simultaneously. Therefore, mechanical stress-induced MKP-1 expression is dependent on ERK, p38 MAPK, and possibly JNK/SAPK activation. On the other hand, MKP-1 as a negative regulator for dephosphorylation and inactivation of MAPKs has been implicated in other types of cells (9 -12, 17, 18). Herein, we provided direct evidence that MKP-1 dephosphorylated three members of MAPKs in stressed VSMCs. Overexpression of MKP-1 could significantly inhibit phosphorylation and activation of MAPKs and DNA synthesis (Fig. 9), indicating important roles of MKP-1 in regulating MAPK activation and cell proliferation.
All tissues in the body are subjected to physical forces originating either from tension, created by cells themselves, or from the environment (8,35,(62)(63)(64). The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized. Physical stimuli must be sensed by cells and transmitted through intracellular signal transduction pathways to the nucleus, resulting in physiological responses or pathological conditions. Growth and proliferation of VSMCs have been shown to be associated with numerous vascular disease states, including medial hypertrophy in hypertension, intimal thickening in atherosclerosis, and restenosis after angioplasty, and are believed to be related with a sustained mechanical stress (21,27,35). Thus, we postulate that the balance between MKP-1 and MAPK levels induced by mechanical stress in VSMCs is critical to maintain homeostasis of the arterial wall. From a therapeutic point of view, our understanding of the molecular mechanisms regulating MAPK and MAPK phosphatase activities by mechanical stress could lead to new strategies for the effective prevention and control of vascular disorders.