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J Appl Physiol 89: 2391-2400, 2000;
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Vol. 89, Issue 6, 2391-2400, December 2000

Role of mitogen-activated protein kinases in pulmonary endothelial cells exposed to cyclic strain

Hiroyuki Kito1,2, Emery L. Chen2, Xiujie Wang2, Masataka Ikeda2, Nobuyoshi Azuma1, Nobuyuki Nakajima2, Vivian Gahtan2, and Bauer E. Sumpio2

1 First Department of Surgery, Chiba University School of Medicine, Chiba 260, Japan; and 2 Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to examine the role of mitogen-activated protein kinases (MAPKs) activation in bovine pulmonary arterial endothelial cells (EC) exposed to cyclic strain. EC were subjected to 10% average strain at 60 cycles/min. Cyclic strain induced activation of extracellular signal-regulated kinase (ERK; 1.5-fold), c-Jun NH2-terminal protein kinase (JNK; 1.9-fold), and p38 (1.5-fold) with a peak at 30 min. To investigate the functional role of the activated MAPKs, we analyzed cells after treatment with PD-98059, a specific ERK kinase inhibitor, or SB-203580, a catalytic inhibitor for p38, and after transient transfection with JNK(K-R), and MEKK(K-M) the respective catalytically inactive mutants of JNK1 and MAPK kinase kinase-1. Cyclic strain increased activator protein-1 (AP-1) binding activity, which was blocked by PD-98059 and SB-203580. Activity of AP-1-dependent luciferase reporter driven by 12-O-tetradecanoyl-phorbol-13-acetate-responsive element (TRE) was induced by cyclic strain, and this was attenuated by PD-98059, MEKK(K-M), JNK(K-R), and SB-203580. PD-98059 and SB-203850 did not inhibit cell alignment and migration induced by cyclic strain. MEKK(K-M) and JNK(K-R) transfection did not block cyclic strain-induced cell alignment. In conclusion, cyclic strain activates ERK, JNK, and p38, and their activation plays a role in transcriptional activation of AP-1/TRE but not in cell alignment and migration changes in bovine pulmonary arterial EC.

activator protein-1; mechanical stress


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR ENDOTHELIAL CELLS (EC) line the inner surface of blood vessels and are constantly exposed to physical forces induced by the repetitive cardiac cycle. These mechanical forces, including cyclic strain, pulsatile pressure, and shear stress, have been shown to regulate vascular tone, thrombogenicity and vascular remodeling (11, 24, 25, 32). Recent studies on nuclear events that are involved in strain-induced EC changes have indicated that cyclic strain can regulate expressions of platelet-derived growth factor, tissue plasminogen activator, endothelial nitric oxide synthase, and monocyte chemotactic protein-1 genes (1, 25, 26, 29) through specific promoter binding sites. Moreover, transcription factors [cAMP response element binding protein, nuclear factor-kappa B, and activator protein 1 (AP-1)] and immediate-early response genes (c-fos, fosB and c-jun) are also regulated by cyclic strain in EC (6, 27). Although EC play an important role in cyclic strain-dependent vascular changes, the intracellular mechanisms by which signals are transmitted from putative mechanoreceptor(s) on EC that sense cyclic strain and couple them to nuclear events, remain largely unknown.

Mitogen-activated protein kinases (MAPKs) are induced in response to extracellular stimuli and have been implicated in the signal transduction from the cell membrane to the nucleus in EC. The family of MAPK consists of at least three subgroups, including extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 signaling pathways. Recent reports demonstrate that shear stress activates ERK, JNK, and p38 (2, 14, 17, 28) and that cyclic strain activates ERK (13, 31) in cultured aortic EC. However, the functional role of each MAPK subfamily activated by hemodynamic forces has not been fully examined. Because MAPK has been implicated in the regulation of cell shape and migration (10, 15, 19, 22), we hypothesized that strain-induced changes in EC shape (12) and migration (32) are dependent on MAPK activation. In this study, we investigated the role of cyclic strain-induced ERK, JNK, and p38 activation in bovine pulmonary arterial EC. We show here that cyclic strain activates ERK, JNK, and p38 and that these have an important role in AP-1/TRE (12-O-tetradecanoyl-phorbol-13-acetate-responsive element) transcription activity but not in the cell morphological changes and migration induced by cyclic strain.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vitro application of tensional deformation. Bovine pulmonary arterial EC were isolated similar to the technique of bovine aortic EC (33) and were seeded on flexible-bottomed six-well culture plates coated with collagen I (Flex I, Flexcell International, McKeesport, PA). After reaching subconfluence, cells were serum starved for 16 h and subjected to mechanical deformation as previously described (32). In these experiments, the membrane bottoms were subjected to 37.5-, 75-, and 150-mmHg vacuum, which produces an average strain of 2.5, 5, and 10%, respectively, on attached cells (7), at a rate of 60 cycles/min. Static control experiments were performed on cells on stretch plates not exposed to cyclic strain. In some experiments, cells were treated with either PD-98059 (Calbiochem, San Diego, CA) for 2 h or SB-203580 (Calbiochem) for 30 min before exposure to cyclic strain.

Transient transfection and reporter gene assay. Plasmids JNK(K-R), MEKK(K-M), hemagglutinin (HA)-tagged JNK1, and the luciferase reporters driven by 4 copies of TRE consensus (4×TRE-Pl-Luc) were gifts from Dr. John Y-J Shyy and were previously described (5, 17, 18, 23). Transfection was performed at 70% confluence using lipofectamine (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's protocol. For the JNK assays, MEKK(K-M) was cotransfected with HA-JNK1 and pCMV-beta gal, the beta -galactosidase reporter vector driven by the human cytomegalovirus promoter. In reporter gene assays, cells were cotransfected with either MEKK(K-M) or JNK(K-R) along with 4×TRE-Pl-Luc and pCMV-beta gal. After 5 h of incubation, the transfected cells were incubated in fresh complete medium overnight. The cells were then subcultured onto the stretch plates and used for experimentation. In general, 30-40% of the cells were transfected, as determined by X-gal staining.

Luciferase and beta -galactosidase activities in harvested cells were determined by a luminometer (Berthold) and beta -galactosidase system (Promega, Madison, WI) using o-nitrohenyl-beta -D-galactopyranoside as a substrate, respectively.

Assessment of MAPK activation. Cells were lysed in a lysis buffer (25 mmol/l HEPES, pH 7.4, 500 mmol/l NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 5 mmol/l EDTA, 50 mmol/l sodium fluoride, 1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1 mmol/l sodium orthovanadate). Further procedure was performed as previously described (13). To detect the phosphorylated form of each MAPK, anti-active MAPK antibody (Promega), phosphospecific JNK antibody (New England Biolabs, Beverly, MA), and phosphospecific p38 antibody (New England Biolabs) were used according to company protocols. To verify each total protein level, membranes were reprobed with appropriate antibodies (anti-ERK2, anti-JNK1, and anti-p38 antibodies; Santa-Cruz Biotechnology, Santa Cruz, CA).

To assay endogenous MAPK activities, cell proteins were incubated with anti-ERK2, anti-JNK1, or anti-p38 antibodies for 1 h at 4°C and then incubated with protein A-Sepharose (Amersham Pharmacia Biothec, Uppsala, Sweden) for 1 h at 4°C. The immune complex beads were washed twice with the lysis buffer and twice with the kinase buffer (25 mmol/l HEPES, pH 7.4, 20 mmol/l MgCl2, 20 mmol/l beta -glycerophosphate, 2 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1 mmol/l sodium orthovanadate). The kinase reaction was initiated by suspending the immune complex beads in 20 µl of the kinase buffer containing 1 µCi [gamma -32P]ATP, 25 µmol/l00 ATP, 2 µg myelin basic protein (MBP) for ERK, 2 µg GST-c-Jun(1-79) fusion protein (Stratagene, La Jolla, CA) for JNK, or 5 µg GST-ATF-2 fusion protein (New England Biolabs) for p38 as substrates. The reaction mixture was incubated for 20 min at 30°C and terminated by the addition of 5× SDS sample buffer. After boiling for 5 min, the proteins were resolved by either 10 or 15% SDS-polyacrylamide gel electrophoresis followed by autoradiography. To assay exogenous JNK1 (HA-JNK1) kinase activity, HA-JNK1 was immunoprecipitated with anti-HA monoclonal antibody (Boehringer Mannheim, Indianapolis, IN) and protein A-Sepharose beads, followed by in vitro kinase assay [GST-c-Jun(1-79) fusion protein as a substrate] using the same procedure as described above.

Electrophoretic mobility shift assay. Nuclear extract preparation and electrophoretic mobility shift assay were performed as described previously (6). An AP-1 consensus oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3') was obtained from Promega. For specific competition, 100 pmol/l of unlabeled AP-1 oligonucleotides were used; for nonspecific competition, 100 pmol/l of double-stranded SP-1 oligonucleotide (5'-ATTCGATCGGGGCGGGGCGAGC-3') were used. For supershift assays, 1 µg of the polyclonal anti-c-Jun antibody (Santa Cruz Biotechnology) was added to the nuclear extract 15 min before the addition of the labeled oligonucleotide.

Cell morphology and migration. To study cell morphology, cells were exposed to cyclic strain for 24 h in the presence of DMSO, PD-98059, or SB-203580. Cells were stained with 0.1% crystal violet (Sigma Chemical, St. Louis, MO). Cells transfected with beta -galactosidase were developed using X-gal as a substrate according to the manufacturer's recommendation (Promega).

The migration study was performed using the "fence method" as described previously (33). To inhibit cell proliferation, cells were treated with 10 µg/ml mitomycin C 2 h before exposure to cyclic strain (3). Then cells were subjected to cyclic strain in the presence of reagents for 7 days.

Statistical analysis. Data are presented as means ± SE. Either Student's unpaired t-test or analysis of variance with post hoc testing was utilized as appropriate. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cyclic strain phosphorylates and activates ERK, JNK, and p38. Ten percent average cyclic strain induced phosphorylation of ERK1/2, JNK1/2, and p38 in a time-dependent manner with a peak at 30 min (Fig. 1). This was confirmed by activation studies because exposure to cyclic strain induced phosphorylation of MBP, GST-c-Jun(1-79), and GST-ATF2, substrates for ERK, JNK, and p38, respectively, with similar patterns (Fig. 2). By 5 min after the initiation of the strain, substrate phosphorylation occurred. At 30 min, substrate phosphorylation peaked and decreased afterward. By 240 min, phosphorylation of the various substrates returned to basal levels.


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  		Fig. 1.   Time course of mitogen-activated protein kinase (MAPK) phosphorylation by cyclic strain. Bovine pulmonary arterial endothelial cells (EC) were exposed to 10% average strain for the indicated time. MAPK phosphorylation was analyzed by immunoblot, using phosphospecific MAPK antibodies. Values are means ± SE for at least 4 experiments. Shown are representative immunoblots and densitometric data showing forms of MAPK. A: ERK1 and ERK2, extracellular signal-related kinase-1 and -2, respectively; p-ERK1 and p-ERK2, phosphorylated ERK1 and ERK2, respectively. B: JNK1 and JNK2, c-Jun NH2-terminal protein kinase-1 and -2, respectively; p-JNK1 and p-JNK2, phosphorylated JNK1 and JNK2, respectively. C: p-p38, phosphorylated p38. * Significant difference (P < 0.05) between each time point and time 0 (static control).



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  		Fig. 2.   Time course of MAPK activation by cyclic strain. Kinase activities were determined by in vitro kinase assays with myelin basic protein (MBP; A), glutathione S-transferase-c-Jun (GST-c-Jun; B), and GST-activating transcription factor-2 (GST-ATF-2; C) as substrates for ERK, JNK, and p38, respectively. Shown are the representative autoradiograms and densitometric analysis of MAPK activation. Values are means ± SE from 4 separate experiments. * Significant difference (P < 0.05) between each time point and time 0 (static control).

Cyclic strain stimulates MAPK in a strain amplitude-dependent manner. Cells were exposed to varying cyclic strain (0, 2.5, 5, and 10% average strain) for 30 min. For ERK, MBP phosphorylation peaked at 5% average strain and did not significantly change further when average strain increased to 10% (Fig. 3A). JNK activation was detected at 2.5% average strain and increased with strain amplitude (Fig. 3B). Activation of p38 was not detected at 2.5% average strain but was observed at higher strain amplitudes (Fig. 3C). Taken together, our data suggest that ERK, JNK, and p38 are activated by cyclic strain in an amplitude-dependent manner and maximum activation occurred after exposure to 10% average stain.


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  		Fig. 3.   Strain dependence of MAPK activation by cyclic strain. EC were exposed to cyclic strain at indicated amplitudes for 30 min. MAPK activation was analyzed by in vitro kinase assays. Shown are representative autoradiograms of ERK (MBP; A), JNK (GST-c-Jun; B), and p38 (GST-ATF-2; C) and the representative densitometric data of kinase activities at various strain amplitudes relative to those at 0% strain (static control). Values are means ± SE from 3 separate experiments. * Significant increase (P < 0.05) compared with 0% strain (static control).

Inhibition of MAPK. Cyclic strain increased kinase activity of ERK, JNK, and p38 in cells that were treated with DMSO as a solvent control (compare first and second lanes in Fig. 4A). Two-hour incubation with 20 µM PD-98059, a specific inhibitor of ERK kinase activation, completely attenuated cyclic strain-induced ERK activity (Fig. 4, A and B). In contrast, 20 µM PD-98059 did not affect JNK and p38 activities (Fig. 4A). Treatment with 40 µM PD-98059 decreased cyclic strain-induced kinase activities of both ERK and JNK (data not shown). This indicates that the specificity of the inhibitory effect of PD-98059 for ERK was achieved at the concentration of 20 µM.


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  		Fig. 4.   Inhibition of MAPK activation by PD-98059, MAPK kinase kinase [MEKK(K-M)], JNK(K-R), or SB-203580. A: EC were treated with 0.03% DMSO for 2 h, 20 µM PD-98059 for 2 h, or 5 µM SB-203580 for 30 min, followed by being exposed to 10% average strain for 30 min or being kept as static controls. MAPK activities were analyzed by in vitro kinase assays. Shown are representative autoradiograms of ERK, JNK, and p38 activities. B and C: quantification of ERK and p38 activities, respectively, stimulated by 10% average strain in the presence of respective inhibitor. Values are means ± SE from 3 separate experiments expressed as the fold induction compared with DMSO-treated static controls. D: epitope-tagged hemagglutinin (HA)-JNK1 was cotransfected with pcDNA3 empty vector or MEKK(K-M) into EC, followed by being subjected to 10% average strain for 30 min. The cell lysates were immunoprecipitated with anti-HA monoclonal antibody for an in vitro kinase assay using GST-c-Jun(1-79) as a substrate. Top film, representative autoradiogram showing HA-JNK1 activity. Bottom film, immunoblot with anti-HA antibody showing HA-JNK1 protein level. Bar graph, quantification of HA-JNK1 activity in various samples relative to those in the cDNA3-transfected static control. * Significant difference (P < 0.05) between groups.

When cells were treated with 5 µM SB-203580, a catalytic inhibitor for p38, cyclic strain-induced p38 activity was blocked; however, ERK and JNK activities were not affected, as determined by in vitro kinase assays (Fig. 4, A and C). Treatment with 10 µM SB-203580 eliminated cyclic strain-induced kinase activities of both p38 and JNK (data not shown). This indicates that specificity of the inhibitory effect of SB-203580 for p38 was achieved at the concentration of 5 µM.

Inhibition of JNK was achieved by overexpression of MEKK(K-M), a catalytically inactive enzyme construct of MAPK kinase kinase-1 (MEKK1). Cyclic strain activated exogenous HA-JNK1 in pcDNA3-transfected cells (compare first and second lanes in Fig. 4D). Cyclic strain-induced HA-JNK1 activity was reduced by the transfection with MEKK(K-M) when cells were exposed to cyclic strain for 30 min (Fig. 4D).

ERK and p38 critically contribute to transcription factor AP-1 binding activity. AP-1 binding activity increased 2 and 4 h after exposure to cyclic strain and returned to the basal level at 8 h (Fig. 5A). Preincubation of unlabeled AP-1 oligonucleotide with nuclear proteins harvested from cells exposed to cyclic strain (4 h) effectively competed for binding to the factor, whereas the SP-1 unlabeled oligonucleotide for a nonspecific competitor did not. These data indicate that the strain-induced increase in binding activity was specific for the AP-1. Addition of the anti-c-Jun antibody to the binding reaction resulted in a shift of the binding complexes to a slow migration (Fig. 5B), indicating the presence of c-Jun protein in the DNA binding complexes. To test that ERK and p38 signaling pathways were involved in cyclic strain-induced AP-1 activity, cells were treated with 20 µM PD-98059 or 5 µM SB-203580 and subjected to cyclic strain for 4 h, respectively. Blockage of ERK and p38 by PD-98059 and SB-203580 eliminated cyclic strain-induced AP-1 binding activity (Fig. 5C). These data suggest that AP-1 binding activity induced by cyclic strain is, at least, mediated through the ERK and p38 signaling pathways.


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  		Fig. 5.   Activator protein-1 (AP-1) binding activity by cyclic strain. A: EC were exposed to 10% average strain for indicated time. AP-1 binding activity of nuclear protein was analyzed by electrophoretic mobility shift assay using an oligonucleotide containing an AP-1 binding site. B: in competitor or supershift studies, protein extracts obtained from EC exposed to 10% average strained cells for 4 h were preincubated with no addition (N), unlabeled AP-1 oligonucleotide (AP-1 Oligo), unlabeled SP-1 oligonucleotide (SP-1 Oligo), or anti-c-Jun antibody (S) for 15 min before incubation with radiolabeled AP-1 oligonucleotide. Arrow indicates "supershift." C: EC were treated with 0.03% DMSO for 2 h, 20 µM PD-98059 for 2 h, or 5 µM SB-203580 for 30 min, followed by being subjected to 10% average strain for 4 h or being kept as static controls. AP-1 binding activities were analyzed by electrophoretic mobility shift assay. Film, representative autoradiogram. Bar graph, quantification of AP-1 binding activity in various samples relative to those in DMSO-treated, static controls. Values are means ± SE from 4 separate experiments. * Significant difference (P < 0.05) between groups.

Cyclic strain activates TRE promoter activity. In cells treated with DMSO, cyclic strain caused a significant increase (1.4-fold) in induction of 4×TRE-Pl-Luc (23), compared with the static control (Fig. 6A). Treatment with 20 µM PD-98059 or 5 µM SB-203580 to block the ERK and p38 signaling pathways attenuated cyclic strain-induced TRE promoter activity (Fig. 6A). To block the JNK signaling pathway, JNK(K-R) and MEKK(K-M) were used. In pcDNA3 transfected cells, cyclic strain caused 1.5-fold induction of 4×TRE-Pl-Luc activity. The cotransfection of JNK(K-R) or MEKK(K-M) eliminated cyclic strain-induced luciferase activities (Fig. 6B).


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  		Fig. 6.   Effect of PD-98059, MEKK(K-M), JNK(K-R), and SB-203580 on the luciferase reporters driven by 4 copies of 12-O-tetradecanoyl-phorbol-13-acetate-responsive element (TRE) consensus (4×TRE-Pl-Luc) expression induced by cyclic strain. A: EC were transfected with 4×TRE-Pl-Luc together with phosphorylated cytomegalovirus-beta -galactosidase used as an internal control for transfection. Transfected cells were treated with 0.03% DMSO, 20 µM PD-98059, or 5 µM SB-203580, followed by either no application (static) or application (strain) of 10% average strain for 6 h, and then subjected to luciferase and beta -galactosidase activity assays. B: 4×TRE-Pl-Luc was cotransfected with pcDNA3 vector, MEKK(K-M), or JNK(K-R), together with beta -galactosidase, into cells. Each bar graph represents 1 of 4 separate experiments with similar results. Each experiment was carried out at least 4 times. Normalized luciferase activity was obtained by luciferase activity normalized for transfection efficiency on the basis of beta -galactosidase activity. Values are means ± SE and are expressed as the fold induction of normalized luciferase activity compared with those in DMSO-treated, static controls (A) or pcDNA3-transfected, static controls (B).

MAPKs are not involved in cyclic strain-induced cell orientation and migration changes. Bovine pulmonary arterial EC grown on the periphery of membranes that were subjected to higher strain for 24 h were elongated and aligned their long axes perpendicular to the force vector (11, 12). The presence of DMSO did not impair cell elongation and alignment to the force vector by cyclic strain (Fig. 7B), although static control cells maintained their polygonal shapes and random orientation (Fig. 7A). After 24-h exposure to cyclic strain in the presence of 20 µM PD-98059 (Fig. 7C) or 5 µM SB-203580 (Fig. 7D), cells still retained the cyclic strain-induced cell orientation. To block the JNK signaling pathway, cells were cotransfected either MEKK(K-M) or JNK(K-R) with beta -galactosidase plasmids and stained with X-gal to detect transfected cells. Both transfected and nontransfected cells were still elongated and aligned perpendicular to the force vector (Fig. 7, E and F). Moreover, when we used 20 µM PD-98059 combined with expression of JNK(K-R) and/or 5 µM SB-203580 treatment to block two or all three MAPK signaling pathways, none of the inhibitor combination blocked cyclic strain-induced cell orientation. These data indicate that cell orientation with cyclic strain is independent of MAPK signaling pathways.


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  		Fig. 7.   Morphological changes induced by cyclic strain. EC were subjected to cyclic strain for 24 h in the presence of 0.03% DMSO (B), 20 µM PD-98059 (C), or 5 µM SB-203580 (D). Cells kept in stationary condition in the presence of 0.03% DMSO were static controls (A). On completion of cyclic strain, cells were stained with crystal violet. Cells, cotransfected with MEKK(K-M) (E) or JNK(K-R) (F) together with beta -galactosidase, were subjected to cyclic strain for 24 h and then stained with X-gal. Cells in the periphery of membranes were observed with phase-contrast microscopy. Edge of membranes is shown at top left, and the force vector is perpendicular to the corner. Bars, 100 µm.

After 7 days of cyclic strain, migration of EC was observed (Fig. 8). In the static control, there was no difference between migration distance of the cells in the center of the membrane and those in the periphery (Fig. 8A). When cells were exposed to cyclic strain in the presence of DMSO, EC in the periphery of the membrane, where cells were exposed to higher strain, had a greater migration distance than in the center of the membrane, where cells were exposed to lower strain (Fig. 8B). Treatment with PD-98059 (Fig. 8C) or SB-203850 (Fig. 8D) did not change the cyclic strain-induced migration. These data suggest that migration with cyclic strain seems to be independent of the ERK and p38 signaling pathways.


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  		Fig. 8.   Effect of PD-98059 and SB-203580 on cyclic strain-induced migration. EC were seeded on one-half of a side of a membrane using the "fence method." After reaching subconfluence, cells were treated with 10 µg/ml mitomycin C for 2 h. Fences were removed to permit cells migrate to other half of the membranes. Cells kept in stationary condition in the presence of 0.03% DMSO were static controls (A). After exposure to cyclic strain in the presence of DMSO (B), PD-98059 (C), and SB-203580 (D) for 7 days, cells were stained with crystal violet. black-down-triangle , Levels of the cell edge when the fence was removed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we provide several lines of evidence that cyclic strain induces different MAPK activation, which leads to the regulation of gene transcriptional events but is not directly responsible for the morphological and migration changes. First, cyclic strain induced rapid and transient activation of ERK, JNK, and p38. Peak activation occurred by 30 min of exposure to cyclic strain. Second, cyclic strain increased AP-1 binding activity, which was blocked by the inhibitors for the ERK and p38 signaling pathways. Third, cyclic strain increased reporter activity of 4×TRE-Pl-Luc, which was attenuated by the blockage of the ERK, JNK, and p38 signaling pathways. Finally, PD-98059 and SB-203850 did not inhibit cell alignment and migration induced by cyclic strain. Dominant negative mutant constructs of MEKK1 and JNK1, when expressed in cells, also did not block cell alignment by cyclic strain. Together, these findings indicate that cyclic strain induces ERK, JNK, and p38 and that their activation plays a critical role in transcriptional activation of AP-1/TRE but they are not involved in cell alignment or migration changes in bovine pulmonary arterial EC. The latter finding was surprising because there have been numerous reports on the importance of MAPK in influencing cell shape and migration (10, 15, 19, 22).

Although the mechanoreceptors that sense cyclic strain have not been identified, growth factor receptors, integrins, and cell-cell junctions are thought to be candidates. When exposed to cyclic strain, EC receive stimuli from several sites, including 1) the luminal side, 2) adjacent EC, and 3) the abluminal side. We have observed that exposure of EC to cyclic strain causes tyrosine phosphorylation of Elk-1 (J. Smith and B. E. Sumpio, unpublished data, 1998) and platelet endothelial cell adhesion molecule-1 (4) and also induces reorganization of integrin alpha 5beta 1 and alpha 2beta 1 (33) in EC. We postulate that activation of one or all of these receptors transduce mechanical stimuli into intracellular signals in EC. Because each MAPK pathway translates specific extracellular signals into discrete physiological responses, signals subsequent to activation of multiple receptors influence the duration and magnitude of MAPK activation. It is reported that fluid shear stress also activates ERK and JNK but with a different pattern (14, 17). Maximum activation of ERK occurs 10 min after applying shear stress (17, 28) and after 30 min for JNK (17). P38 is also activated by shear stress, peaking at 5 min after application of shear stress (2). To explain these different patterns of MAPK activation by shear stress, differential regulation of ERK and JNK have been hypothesized, including guanine nucleotide binding protein (14), phosphatidylinositol 3-kinase (9), and focal adhesion kinase (pp125FAK) (16). In response to cyclic strain, MAPK activation was detected by 5 min (rapid) but returned to the basal level by 240 min (transient) (Fig. 1B). Although activation of JNK (1.9-fold greater than static controls) is greater than that of ERK (1.4-fold) and p38 (1.4-fold), ERK, JNK, and p38 have their peak activation at 30 min after exposure to cyclic strain. This suggests that integrated signals from mechanoreceptors may have an effect on magnitude but not on peak time of each MAPK activation. More studies will be required to test this hypothesis.

Further complicating the analysis is the fact that not only different cell types, but even the same cell type harvested from different locations, may respond to hemodynamic forces in a variable fashion (11). Our laboratory recently reported on ERK activation in bovine aortic cells subjected to a strain regimen similar to that used in the present study (13). We noted peak activation of ERK in bovine aortic cells by 10 min that was sustained to 30 min. Thus both pulmonary arterial and aortic EC respond to cyclic strain with ERK activation, albeit with different time courses.

To test the functional role in MAPK activation in pulmonary arterial EC by cyclic strain, we first found the optimal dose of inhibitors to block cyclic strain-induced ERK and p38 activation. We showed higher concentrations of PD-98059 and SB-203580 caused an unspecific inhibition of other pathways, resulting in the loss of specificity to the respect pathway. This is supported by the previous report that high concentration (>10 µM) SB-203580 inhibits particular recombinant JNK (JNK1alpha 1, JNK1alpha 2, and JNK2) kinase activities in vitro (30). Because there is no chemical inhibitor for the JNK signaling pathway, we performed transient transfection studies using two dominant negative mutant constructs; JNK(K-R) and MEKK(K-M) (17, 18). Because MEKK1 is a MAPK kinase kinase and preferentially activates the JNK cascade, we used MEKK(K-M) to block the JNK signaling pathway for its functional study. Our present study showed that MEKK(K-M) decreased JNK1 activation (Fig. 3D), which indicates a potential MEKK1-JNK pathway in EC exposed to cyclic strain.

The present study showed that AP-1 binding activity significantly increased after 4-h exposure to cyclic strain in bovine pulmonary arterial EC (Fig. 4A). AP-1 is composed of members of the Jun and Fos families. ERK, JNK, and p38 phosphorylate and activate Elk-1, c-Jun, or ATF-2, resulting in enhanced c-fos and c-jun gene expression (5, 8, 21). Thus MAPK signaling pathways influence AP-1 activity by both increasing the abundance of AP-1 components and by stimulating their activity directly. In this regard, our laboratory previously reported that cyclic strain induces c-fos and c-jun gene expression associated with the increase in AP-1 binding activity in human umbilical vein EC (27). This study showed that ERK and p38 signaling pathways are of critical importance for AP-1 binding activity in bovine pulmonary arterial EC exposed to cyclic strain, because blockage of ERK and p38 activity by PD-98059 and SB-203580, respectively, attenuated cyclic strain-induced AP-1 binding activity.

Because AP-1 binds to the TRE in gene promoter regions, we measured activation of an AP-1-dependent reporter gene, 4×TRE-Pl-Luc (23). Previous reports indicated that the transcription factor that mediates 4×TRE-Pl-Luc activation is AP-1, because transactivation assays using c-Jun or c-Jun-Fos expression plasmids induce 4×TRE-Pl-Luc (17, 23). Cyclic strain increased reporter activity of 4×TRE-Pl-Luc (Fig. 5). Taken together with the results that cyclic strain induces AP-1 binding activity (Fig. 4A), our data suggest that cyclic strain induces AP-1/TRE transcription activity, which leads to immediate early gene expression. The ERK and p38 signaling pathways play an important role in cyclic strain-induced AP-1/TRE activity, because blockage of each MAPK signaling pathway, using respective inhibitors, attenuated cyclic strain-induced TRE activity (Fig. 5B). Moreover, MEKK(K-M) and JNK(K-R) attenuated 4×TRE-Pl-Luc reporter activity induced by cyclic strain, further implicating a potential link of MEKK1-JNK pathway to AP-1/TRE transcription in response to cyclic strain. Li et al. (17) reported that JNK pathway but not ERK pathway is essential for the shear-induced activation of AP-1/TRE in bovine aortic EC. Wung et al. (31) also reported that cyclic strain-induced early growth response-1 expression is mediated mainly via the Ras-ERK pathway, not the Ras-Rac-JNK pathway, in bovine aortic EC. Our present study shows that, in contrast to these two reports, all three MAPK pathways play a pivotal role in the induction of AP-1/TRE in bovine pulmonary arterial EC exposed to cyclic strain. Although each MAPK signaling pathway is critical to induce AP-1/TRE, the individual role of each MAPK signaling pathway warrants further study.

Cyclic strain modulates cell morphology and migration. Long-term exposure of EC to cyclic strain causes the elongation and alignment of their long axes perpendicular to the force vector (11) and a greater migration of EC exposed to higher strain than those exposed to lower strain (32). It is generally accepted that the dynamic reorganization of the actin cytoskeleton affects cell morphology and migration (19). Forces generating movement in the actin cytoskeleton include actin-filament polymerization and motor protein such as myosin. In addition to actin reorganization, other cellular activities such as changes in gene transcription need to be coordinate. The ERK signaling pathway has been reported to directly activate myosin light chain kinase (15). The p38/MAPK-activated protein kinase-2/heat shock protein 27 pathway has been shown to be capable of modulating cell morphology and migration through actin reorganization (10, 22). Thus MAPK may function in regulating morphology and migration in both transcription-dependent and -independent manners. The present study showed that the blockage of individual MAPK signaling pathways failed to inhibit morphological changes or migration by cyclic strain (Fig. 7 and 8). When treated with a combination of PD-98059, JNK(K-R), and SB-203580, cells still aligned. Together, these data indicate that MAPK activation has no or minimal influence on cyclic strain-induced morphological changes and migration. It is likely that cyclic strain-induced morphological changes are predominantly regulated by different pathways. Our laboratory previously reported that tyrosine phosphorylation, especially by pp125FAK and paxillin and also by rho p21, plays a critical role in cyclic strain-induced morphology and migration (32, 34). Moreover, pp125FAK and paxillin are downstream of rho p21 activated by cyclic strain (34). Although it was reported that pp125FAK is upstream of the Ras-MAPK pathway (16), the present study shows that MAPK is not involved in cyclic strain-induced morphological changes and migration. This suggests that MAPK is not a downstream target of pp125FAK signaling, which regulates cell morphology and migration due to cyclic strain. There are other potentially important targets of rho p21 and pp125FAK activated by cyclic strain. For example, rho has been reported to enhance myosin light chain phosphorylation by inactivation of myosin phosphatase (20).

In summary, our data indicate that ERK, JNK, and p38 signaling pathways are activated in bovine pulmonary arterial EC exposed to cyclic strain. These MAPK isoforms are functionally critical for AP-1/TRE transcriptional events but are not at all or are only minimally involved in the cell morphology and migration changes induced by cyclic strain. Further studies on the upstream regulators of MAPK, which control ERK, JNK, and p38, would be helpful in aiding our understanding of the molecular mechanism of cyclic strain-induced morphological changes and migration.


    ACKNOWLEDGEMENTS

We thank Dr. J. Y-J Shyy for helpful suggestions on transfection experiments, Dr. Bernhart Schmidt for technical guidance with protein assays, Dr. Yoshiko Yano for help with the migration study, and Drs. Changhua Ji and Michael Centrella for advice on the reporter gene assays.


    FOOTNOTES

This work was supported by grants to B. E. Sumpio from the National Heart, Lung, and Blood Institute (ROI HL-54732), the Veterans Affairs Merit Review, and the American Heart Association (National Affiliate).

Original submission in response to a special call for papers on "Cellular Responses to Mechanical Stress."

Address for reprint requests and other correspondence: B. E. Sumpio, Dept. of Surgery, Yale Univ. School of Medicine, 333 Cedar St., FMB 137, New Haven, CT 06510 (E-mail: bauer.sumpio{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 May 2000; accepted in final form 27 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 89(6):2391-2400
8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society


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