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Am J Physiol Heart Circ Physiol 280: H189-H197, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 1, H189-H197, January 2001

Role of p38 MAP kinase in endothelial cell alignment induced by fluid shear stress

Nobuyoshi Azuma1,2, Nobuyuki Akasaka1,2, Hiroyuki Kito1, Masataka Ikeda1, Vivian Gahtan1, Tadahiro Sasajima2, and Bauer E. Sumpio1

1 Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06510; and 2 First Department of Surgery, Asahikawa Medical College, Asahikawa, Japan 078-8510


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p38/mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MAPKAP kinase 2)/heat shock protein (HSP)25/27 pathway is thought to play a critical role in actin dynamics. In the present study, we examined whether p38 was involved in the morphological changes seen in endothelial cells (EC) exposed to shear stress. Cultured bovine aortic EC were subjected to 14 dyn/cm2 laminar steady shear stress. Peak activation of p38, MAPKAP kinase 2, and HSP25 were sixfold at 5 min, sixfold at 5 min, and threefold at 30 min compared with static control, respectively. SB-203580 (1 µM), a specific inhibitor of p38, abolished the activation of MAPKAP kinase 2 and HSP25 as well as EC elongation and alignment in the direction of flow elicited by shear stress. The mean orientation angle of cells subjected to shear without SB-203580, with SB-203580, or static control were 17, 50, and 43°, respectively (P < 0.05). EC transfected with the dominant negative mutant of p38-alpha aligned randomly with no stress fiber formation despite exposure to shear stress. These data suggests that the pathway of p38/MAPKAP kinase 2/HSP25/27 is activated in response to shear stress, and this pathway plays an important role in morphological changes induced by shear stress.

mitogen-activated protein kinase; heat shock protein; actin; reorientation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR ENDOTHELIAL CELLS (EC) line the luminal surface of blood vessels and are exposed to forces of the circulation such as fluid shear stress, circumferential distention, and blood pressure. Shear stress, the tangential hemodynamic force, has been implicated in regulating EC function and vascular remodeling. For example, shear stress modulates the secretion of nitric oxide and other vasoactive substances and the expression of genes encoding coagulation molecules, growth factors, and adhesion molecules (2, 23, 45). EC also align in the direction of flow in response to shear stress (26, 30). Regional differences in shear in the vessel wall are thought to play a role in atherogenesis (9). Atherosclerotic lesions are preferentially located in disturbed flow regions, such as the bifurcation of the carotid artery and iliac artery, and EC at these regions are activated and align at random (5, 38, 50).

Long-term exposure of cultured EC to shear stress leads to the reorientation of EC. Cells are elongated along the direction of flow with stress fiber formation in response to shear stress in vitro (5, 12, 31). It has been shown that activation of tyrosine kinase, intracellular calcium, and an intact microtubule network are necessary for EC reorientation (36). However, the molecular mechanism of reorientation is poorly understood.

A number of studies have demonstrated the relationship between shear stress and mitogen-activated protein (MAP) kinase activation (21, 34, 40, 46). These studies have indicated that shear stress activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal protein kinase (JNK), which in turn regulate several transcriptional factors, but there is no evidence showing that these are responsible for the morphological changes induced by shear stress.

p38, another member of the MAP kinase family, has been implicated in cell shape changes and migration (16, 43). In particular, the p38/MAP kinase-activated protein kinase 2 (MAPKAP kinase 2)/heat shock protein (HSP)25/27 pathway is thought to play a critical role in actin dynamics. We hypothesized that p38 might be involved in the morphological events elicited by shear stress.

The purpose of the present study was to clarify the downstream pathways of p38 and to examine whether p38 was involved in EC reorientation by use of SB-203580, a specific inhibitor of p38, as well as transfection experiments using a dominant negative mutant of p38-alpha .


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Plasmids, the wild-type and dominant negative mutant of p38-alpha (p38 AGF) (6) cloned by pCMV5, were generous gifts from Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center, MA). Antibodies anti-phospho-specific HSP27 and anti-HSP27 were generous gifts from Dr. Colleen M. Brophy (Medical College of Georgia, Augusta, GA) and Dr. Michael Welsh (University of Michigan Medical Center, Ann Arbor, MI), respectively. Anti-HSP25 (StressGen Biotechnologies, Victoria, Canada), anti-phospho-specific p38 (New England BioLabs, Beverly, MA), anti-p38 (Santa Cruz Biotechnology, Santa Cruz, CA), horseradish peroxidase-conjugated secondary antibody (New England BioLabs), anti-MAPKAP kinase 2 (sheep polyclonal antibody for immunoprecipitation; Upstate Biotechnology, Lake Placid, NY), anti-MAPKAP kinase 2 (goat polyclonal antibody for immunoblot, Biotechnology), and anti-von Willebrand factor (Dako, Carpinteria, CA) were obtained. Proteins and chemicals: recombinant HSP25 (StressGen Biotechnologies).

SB-203580 (Calbiochem, San Diego, CA), rhodamine phalloidin (Molecular Probes, Engene, OR), and fetal calf serum (FCS, Hyclone Laboratories, Logan, UT) were purchased. Dulbecco's modified Eagle's medium F-12, antibiotics, LipofectAmine Plus, and Opti-MEN were obtained from GIBCO-BRL (Gaithersburg, MD). Anti-Flag M2 monoclonal, FITC-conjugated anti-mouse IgG whole molecule, and other chemicals were purchased from Sigma (St. Louis, MO).

Cell culture. Bovine aortic EC were obtained by scraping the intimal surface of aortas obtained from freshly killed calves at a local slaughterhouse (1). Cells were maintained in Dulbecco's modified Eagle's medium F-12, supplemented with 10% heat-inactivated FCS, 5 µg/ml deoxycytidine/thymidine, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B) and grown at 37°C in a humidified 5% CO2 incubator. EC were identified by their typical cobblestone appearance and indirect immunofluorescence staining for factor VIII antigen. Cells used in this study were between passages 3 and 6.

Flow experiment. The flow system that we used creates a steady laminar shear stress, as described by Frangos et al. (8). In brief, EC were seeded on collagen I-coated glass slides (75 × 38 mm, Fisher Scientific, Pittsburgh, PA). After attaining confluence, EC on the glass slide were subjected to 14 dyn/cm2 of shear stress using a parallel flow chamber (Strite Industries, Cambridge, Canada). The channel of the flow chamber is 250 µm in height, 2.2 cm in width, and 5.5 cm in length. The shear stress was generated by a hydrostatic pressure difference between an upper and a lower reservoir. The medium circulating in flow apparatus was kept at 37°C with 5% CO2. Cells that were not subjected to shear stress were kept in the same incubator and served as a static control.

In some experiments, a specific inhibitor of p38, SB-203580, was used. SB-203580 was dissolved in DMSO and then diluted with serum-free medium to 1, 5, or 10 µM SB-203580 in final concentration. Serum-starved EC were treated with each concentration of SB-203580 or DMSO as vehicle 1 h before flow experiment. EC were then subjected to shear stress with serum-free medium containing SB-203580 or vehicle. Our pilot studies utilizing 20 µM SB-203580 indicated that, although morphological damage of cells with vesicle formation in the cytosol was present, visible cell damage was absent with 10 µM SB-203580.

Immunoblot for phospho-p38 and phospho-HSP25. Activation of the p38 MAP kinase was assessed by determining phosphorylation of p38 by immunoblotting with a phospho-specific antibody. In brief, after exposure to shear stress, cells were washed in ice-cold PBS twice and scraped in lysis buffer containing 25 mM HEPES (pH 7.4), 0.5 M NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 5 mM EDTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were centrifuged for 15,000 g for 10 min, and supernatants were collected. Protein content was measured by Bio-Rad protein assay system (Bio-Rad, Hercules, CA). Laemmli sample buffer was added to equal amounts of each sample, and samples were boiled for 5 min. Samples were resolved on 10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham, Arlington Heights, IL). The membrane was probed with phospho-specific p38 antibody as a primary antibody and horseradish peroxidase-conjugated secondary antibody with washing, as suggested by the manufacturer. Immunodetection was carried out by ECL reagents (Amersham) and quantitated with a phosphoimager densitometer (Molecular Dynamics, Sunnyvale, CA). Densitometric data were expressed as the fold induction compared with the static control level. To ensure equal loading of protein, the membrane was stripped and reprobed with anti-p38 antibody.

The activation of HSP25 was also assessed by determining the degree of phosphorylation of HSP25 using an anti-phospho-specific HSP27 antibody as the primary antibody. For detection of total HSP25, an anti-HSP27 antibody as well as an anti-HSP25 antibody was used.

In vitro kinase assay for MAPKAP kinase 2. For determining activation of MAPKAP kinase 2, cell lysates were immunoprecipitated with anti-MAPKAP kinase 2 antibody, followed by in vitro kinase assay using recombinant HSP25 as a substrate. In brief, aliquots of the collected protein (250 µg) were incubated with 4 µg of anti-MAPKAP kinase 2 antibody for 1 h at 4°C and then incubated with protein G-sepharose beads for 1 h at 4°C. The immunoprecipitates were washed and incubated with kinase buffer [25 mM HEPES (pH 7.4), 20 mM MgCl2, 20 mM beta -glycerolphosphate, 1 mM PMSF, 1 mM Na3VO4, 1 mM dithiothreitol, and 10 µg/ml leupeptin], 10 µCi of [gamma -32P]ATP, 25 µM ATP, and 200 µg/ml of recombinant HSP25 for 20 min at 30°C. The reaction was terminated by adding sample buffer containing 2% beta -mercaptoethanol and boiling for 5 min. The supernatants were resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was subjected to autoradiography and quantitated with by densitometry. The densitometric value of phospho-recombinant HSP25 was expressed as the fold induction compared with the static control level. The membrane was also probed with the anti-MAPKAP kinase 2 antibody as a primary antibody and horseradish peroxidase-conjugated secondary antibody, followed by immunodetection to ensure equal loading of protein.

Morphological study. EC were subjected to 14 dyn/cm2 of shear stress for 24 h and then fixed with 3.7% formaldehyde followed by staining for F-actin using rhodamine phalloidin. In some experiments, EC were exposed to SB-203580 or vehicle 1 h before flow and then subjected to shear stress for 24 h with medium containing SB-203580 or vehicle.

At least four identical fields for each specimen were photographed with ×50 magnification. To assess the morphological changes, the photographs were analyzed with a public software "Image" (NIH Research Service Branch, Bethesda, MD). The cell orientation angle between the principal cell axis and the direction of flow and the perimeter and area of cells were measured, and the cell shape index [defined as (4pi  × cell area)/(cell perimeter)2] was calculated. If the cell completely aligned in the direction of flow, the orientation angle would be 0°. If all cells aligned randomly, the orientation angle would be 45°. The shape index = 1 when the cell is a perfect circle. If the cell is elongated, the value of the shape index approaches zero. Three separate experiments were performed, and at least 160 cells in each group were measured.

Transfection. EC were transfected with either a Flag-tagged wild-type or dominant negative mutant of p38-alpha . In brief, EC were cultured on a collagen I-coated glass slides. After attaining 70% confluence, EC were washed with Opti-MEN and then incubated with 7 µg/ml of LipofectAmine Plus and 2 µg of plasmids per glass slide for 6 h at 37°C. Medium containing 10% serum was added, and EC were cultured to confluence. Approximately 48 h after plasmid transfection, EC were subjected to 14 dyn/cm2 shear stress for 24 h. Cells were then fixed with 3.7% formaldehyde and double-stained with anti-Flag antibody and rhodamine phalloidin. Cells stained with anti-Flag antibody were considered transfected.

Immunostaining Cells on glass slides were fixed with 3.7% formaldehyde for 10 min followed by permeabilization with 0.1% Triton X-100 for 5 min. After being washed with PBS, the cells were incubated with 1% bovine serum albumin for 20 min to block any nonspecific reaction and then incubated with mouse anti-Flag antibody for 1 h. After being washed with PBS three times, rhodamine phalloidin and FITC-conjugated anti-mouse IgG were applied as a secondary antibody for 1 h. All procedures were done at room temperature. After the addition of anti-fade fixative, cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan), and photographs were taken. To evaluate the morphology of the transfected cells, 36 consecutive fields of each specimen were photographed. The orientation angle and shape index of the transfected cells and nontransfected cells surrounding the transfected cells were measured.

Statistical analysis. Data are presented as means ± SE. Statistical significance for the densitometric data was determined by one-way ANOVA followed by a multiple comparison procedure. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time course of p38, MAPKAP kinase 2, and HSP25 activation. Shear stress activated p38 in a time-dependent manner. As shown in Fig. 1A, p38 was activated as early as 2 min, peaked at 5 min, and was sustained for at least 2 h. The densitometric data of three independent experiments showed that the peak activation of p38 was sixfold at 5 min (P < 0.05 compared with static control), and the activation at 2 h was still twofold compared with that of static control (Fig. 1C).


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  		Fig. 1.   Time course of p38 phosphorylation in response to shear stress. Serum-starved bovine aortic endothelial cells (EC) were kept static (represented by time = 0 min) or subjected to 14 dyn/cm2 of steady laminar shear stress for 2, 5, 10, 30, 60, and 120 min. Cell lysates were resolved on 10% SDS-PAGE followed by transfer to nitrocellulose membrane electrophoretically. Immunoblot was carried out with specific antibodies, and each band was detected by ECL reagent. A: representative immunoblot with anti-phospho-specific p38. B: the membrane was stripped and then reprobed with anti-p38 antibody, showing equal loading. C: densitometric data of phosphorylated p38 bands from 3 separate experiments. Data were expressed as the fold induction compared with static control. Bar graphs represent means ± SE. *P < 0.05 compared with static control.

MAPKAP kinase 2 was activated with a similar temporal pattern as p38 (Fig. 2A). The densitometric data of three separate experiments indicated a sixfold induction of MAPKAP kinase 2 with a peak at 5 min (P < 0.05 compared with static control). Activation at 2 h was still twofold compared with the basal static level (Fig. 2C).


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  		Fig. 2.   Time course of mitogen activated protein (MAP) kinase-activated protein (MAPKAP) kinase 2 activation in response to shear stress. Serum-starved bovine aortic EC were kept static (time = 0 min) or subjected to 14 dyn/cm2 of steady laminar shear stress for 2, 5, 10, 30, 60, and 120 min. Cell lysates were immunoprecipitated with anti-MAPKAP kinase 2 antibody, followed by in vitro kinase assay using recombinant heat shock protein (HSP)25 as a substrate with [gamma -32P]ATP. The immunocomplex was resolved on 12% SDS-PAGE followed by transfer to nitrocellulose membrane electrophoretically. A: representative autoradiogram of an in vitro kinase assay for MAPKAP kinase 2. B: immunoblot with anti-MAPKAP kinase 2 antibody, showing equal loading. C: densitometric data of phosphorylated HSP25 bands from 3 independent experiments. Data were expressed as the fold induction compared with static control. Bar graphs represent means ± SE. *P < 0.05 compared with static control.

As shown in Fig. 3, shear stress induced a threefold activation of HSP25 with a peak at 30 min (P <=  0.05 compared with static control). The onset and peak of activation of HSP25 were relatively delayed compared with the activation of p38 and MAPKAP kinase 2. The activity of HSP25 was sustained and still 2.3-fold at 120 min compared with static control. The anti-phospho-specific HSP27 antibody that we used in this study was created against a synthetic peptide,VAAPAYSRALSRQLS. This antibody cross reacts with bovine HSP25. To confirm that the band detected by this phospho-specific antibody was truly phosphorylated HSP25, we stripped the membrane and reprobed with anti-HSP27 (from Dr. Welsh) as well as anti-HSP25 (StressGen). Bands detected by both anti-HSP27 and anti-HSP25 antibody corresponded exactly to the bands detected with anti-phospho-specific HSP27.


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  		Fig. 3.   Time course of HSP25 phosphorylation in response to shear stress. Serum-starved bovine aortic EC were kept static (time = 0 min) or subjected to 14 dyn/cm2 of shear stress for 2, 5, 10, 30, 60, and 120 min. Cell lysates were resolved on 12% SDS-PAGE then transferred to nitrocellulose membrane. Immunoblot was carried out and bands were detected by ECL reagent. A: immunoblot with anti-phospho-specific HSP27. B: immunoblot with anti-HSP27 antibody. C: densitometric data of phosphorylated HSP25 bands. Data were expressed as the fold induction compared with static control. Bar graphs represent means ± SE from 4 separate experiments. *Significant difference statistically compared with static control (P <=  0.05).

SB-203580 inhibited activation of MAPKAP kinase 2 and HSP25. To confirm that MAPKAP kinase 2 and HSP25 are downstream of p38, we employed a specific inhibitor of p38 activity, SB-203580. An in vitro kinase assay for MAPKAP kinase 2 of EC subjected to shear stress for 5 min or kept static (Fig. 4A) indicated that shear stress induces a robust activation of MAPKAP kinase 2. Treatment with SB-203580 at even the lowest concentration (1 µM) inhibited this activation. Densitometric data of three separate experiments indicated that the activation of MAPKAP kinase 2 in the absence of SB-203580 was statistically significant compared with that of static control (P < 0.05) (Fig. 4C). There was no significant difference between static control and any of the SB-203580 treated groups.


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  		Fig. 4.   Effects of SB-203580 on MAPKAP kinase 2 activation induced by shear stress. Serum-starved bovine aortic EC were incubated with SB-203580 (1, 5, and 10 µM) or vehicle (0 µM) 1 h before experimentation. Cells were subsequently subjected to shear stress for 5 min or kept static. Cell lysates were immunoprecipitated with anti-MAPKAP kinase 2 antibody followed by an in vitro kinase assay using recombinant HSP25 as a substrate as described in Fig. 2. A: autoradiogram of a typical in vitro kinase assay for MAPKAP kinase 2. B: representative immunoblot with anti-MAPKAP kinase 2 antibody. C: densitometric data of phosphorylated HSP25 bands. Data are expressed as the fold induction compared with static control. Bar graphs represent means ± SE from 3 separate experiments. *Significant difference compared with sheared cells without SB-203580 (P < 0.05) .

A typical immunoblot with anti-phospho-specific HSP25 antibody is shown in Fig. 5, demonstrating the effect of SB-203580 on HSP25 phosphorylation. Again, 1 µM SB-203580 was sufficient to abolish HSP25 phosphorylation induced by shear stress. The densitometric data confirmed that the effect of SB-203580 was dose dependent.


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  		Fig. 5.   Effects of SB-203580 on HSP25 phosphorylation induced by shear stress. Serum-starved bovine aortic EC were incubated with SB-203580 (1, 5, and 10 µM) or vehicle (represented by 0 µM) 1 h before experimentation. Cells were subsequently subjected to shear stress or kept static for 30 min. Immunoblot was carried out as described in Fig. 3. A: immunoblot with anti-phospho-specific HSP27. B: immunoblot with anti-HSP antibody. C: densitometric data of phosphorylated HSP25 bands. Bar graphs represent the result of a typical experiment. A duplicate experiment had similar results.

Both MAPKAP kinase 2 and HSP25 were inhibited by the specific inhibitor of p38 in a dose-dependent manner, confirming that MAPKAP kinase 2 and HSP25 were downstream of p38 in EC exposed to shear stress.

Morphological studies with SB-203580 and p38-alpha transfection. The SB-203580 data above suggests that HSP25 is a downstream substrate for p38. Because HSP25 is thought to be involved in the regulation of cell morphology through its interaction with actin filaments, we investigated the effect of treatment of EC exposed to shear stress with SB-203580. EC maintained in a static environment demonstrated dense actin fibers around the cell membrane, the so-called dense peripheral bands (Fig. 6A). Cells subjected to shear stress lost these peripheral bands; central actin fibers that aligned in the direction of flow (stress fibers) were visualized. In addition, the cells were elongated and aligned in the direction of flow (Fig. 6B). SB-203580 (10 µM) completely inhibited the morphological changes induced by shear stress; EC still maintained the dense peripheral band and were randomly oriented without stress fiber formation (Fig. 6C). Because 1 µM SB-203580 abolished the activation of MAPKAP kinase 2 and HSP25 induced by shear stress, we also tested the effects of this low concentration on cell morphology. Figure 6D demonstrates that even 1 µM SB-203580 inhibited the morphological changes induced by shear stress.


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  		Fig. 6.   Effects of SB-203580 on the morphological changes induced by shear stress. Bovine aortic EC were incubated with SB-203580 or vehicle 1 h before experimentation and then subjected to shear stress or kept static for 24 h. Cells were fixed with 3.7% formaldehyde and then permeabilized with Triton X-100, followed by staining F-actin with rhodamine phalloidin. Photographs were taken by use of fluorescence microscope. Original magnification ×100. A: cells kept static without SB-203580. B: cells subjected to shear stress without SB-203580. C: cells subjected to shear stress with 1 µM SB-203580. D: cells subjected to shear stress with 10 µM SB-203580. Arrows indicate the direction of flow.

Figure 7 depicts the morphometric analysis of EC orientation with shear stress. The average orientation angle of the static group was 43°, whereas the angle for the shear stress group was 17°. EC exposed to shear stress in the presence of SB-203580 did not orient in any specific manner. There was no significant difference in the orientation angles of the static control and SB-203580-treated group by ANOVA. In addition, the shape index of sheared cells in the absence of SB-203580 was 0.3, which was much less than static control (0.6), whereas the shape index of sheared cells treated with SB-203580 was similar to the control group.


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  		Fig. 7.   Morphometric analysis for the SB-203580 study. Bovine aortic EC were incubated with SB-203580 (1, 5, and 10 µM) or vehicle (0 µM) 1 h before experimentation and then subjected to shear stress with SB-203580 (0, 1, 5, and 10 µM) or kept static for 24 h. Cells were fixed, stained, and photographed as described in Fig. 6. Photographs were analyzed by computer with the software "Image" to measure the principal axis, perimeter, and area of cells. A: cell orientation angle. B: shape index (see MATERIALS AND METHODS for detail). Bar graph represents means ± SE from 3 independent experiments. *Significant difference compared with sheared cells without SB-203580 (P < 0.05).

To confirm the effects of SB-203580 and to define whether the alpha -subtype of p38 is involved in the morphological events induced by shear stress, we employed a dominant negative mutant of p38-alpha . EC transfected with wild-type p38-alpha and exposed to shear stress aligned in the direction of flow with stress fiber formation (Fig. 8A), whereas cells transfected with the dominant negative p38-alpha aligned randomly. Furthermore, the latter cells were devoid of central stress fibers (Fig. 8B). Morphometric analysis of cells transfected with a wild-type or dominant negative mutant of p38-alpha confirmed that cells transfected with the dominant negative p38-alpha were elongated and their orientation angle was random compared with cells transfected with the wild-type p38-alpha (P <=  0.001; Fig. 9). Because SB-203580 inhibits only the alpha - and beta -subtype of p38, our results suggest that at least the alpha -subtype of p38 is implicated in the morphological events induced by shear stress.


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  		Fig. 8.   Effects of dominant negative mutant of p38-alpha on morphological changes induced by shear stress. Bovine aortic EC cultured on glass slides were transfected with Flag-tagged wild-type or dominant negative mutant of p38-alpha by use of LipofectAmine Plus reagents. Approximately 48 h after transfection, cells were subjected to shear stress for 24 h. Cells were then fixed, permeabilized, and stained with mouse anti-Flag antibody followed by FITC-conjugated anti-mouse IgG as well as rhodamine phalloidin. A: anti-Flag antibody staining for cells transfected with wild-type p38-alpha . Transfected cell stained with fluorescein. B: cells at the same field as A were stained by rhodamine phalloidin. Arrow indicates the direction of flow. Arrow head represents the transfected cell. C: anti-Flag antibody staining for cells transfected with dominant negative mutant of p38-alpha . Transfected cells are stained with fluorescein. D: cells at the same field as C were stained by rhodamine phalloidin. Arrow indicates the direction of flow. Arrow heads represent the transfected cells. Original magnification is ×100.



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  		Fig. 9.   Morphometric analysis of the orientation angle and shape index of EC transfected with wild-type or dominant negative muntant of p38-alpha . Cells transfected with wild-type or dominant negative muntant of p38-alpha were subjected to 14 dyn/cm2 of shear stress. Consecutive 36 fields of the each specimen were then photographed. On each photograph, the orientation angle and shape index of transfected cells and nontransfected cells were measured. Number of measured cells of transfected with wild-type (WT) p38, nontransfected cells surrounding WT cells (WnT), cells transfected with dominant negative mutant of p38-alpha (DT), and nontransfected cells surrounding DT cells (DnT) were 39, 168, 42, and 155, respectively. All cells were subjected to shear stress. A: orientation angle. The orientation angle of DT cells was significantly higher than that of WT cells, WnT cells, and DnT cells (* P <=  0.001). B: shape index. The shape index of DT cells was significantly larger that that of WT, WnT, and DnT cells (* P <=  0.001). The shape index of WT cells was significantly smaller than that of WnT and DnT cells (** P <=  0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

p38 is activated by stress, such as osmotic and heat shock and ultraviolet radiation, and by chemical mediators, such as lipopolysacharides, tumor necrosis factor-alpha , and interleukin-1 (10, 15, 29, 42). Five submembers of the p38 family, p38 (p38-alpha ), p38-beta [stress-activated protein kinase (SAPK) 2b, p38-beta 1] (19), p38-beta 2 (7), p38-gamma (ERK6, SAPK3) (35), and p38-delta (SAPK4) (20, 47), have been reported to date. A number of downstream effector pathways, such as MAPKAP kinase 2 (42), MAPKAP kinase 3 (3), MAP kinase-interacting protein kinase 1 and 2 (11, 49), as well as the transcriptional factors ATF2 (19) and CHOP (48), have been implicated. Furthermore, activation of MAPKAP kinase 2 results in activation of the transcriptional factor CREB (44) as well as HSP27 in human cells and HSP25 in bovine and murine cells (4, 17).

In the present study, we show that 1) shear stress activates the p38/MAPKAP kinase 2/HSP25/27 pathway and 2) p38 plays an important role in morphological changes induced by shear stress by use of specific inhibitor of p38, SB-203580, and transfection with the dominant negative mutant of p38-alpha .

The pyridinyl imidazole SB-203580 is widely used as the specific inhibitor of p38 and has been shown not to inhibit other MAP kinase members even at a concentration of 100 µM (4). It has been demonstrated that SB-203580 blocks activation of only the alpha - and beta -subtypes of p38 and not the other subtypes (13, 24). Our results indicate that SB-203580 inhibited both MAPKAP kinase 2 activation and HSP25 phosphorylation induced by shear stress in a dose-dependent manner. This is consistent with the notion that MAPKAP kinase 2 and HSP25 are downstream of p38. Whereas the peak phosphorylation of p38 (at 5 min in 2 experiments and at 2 min in 1 experiment) was similar to that of MAPKAP kinase 2 activation (at 5 min), peak phosphorylation of HSP25 occurred at 30 min. Li et al. (33) had previously reported HSP were activated by shear stress, including HSP25. They demonstrated the time course of HSP25 activation by use of in vivo 32P labeling (33). Although we employed a different method to evaluate the activation of HSP25, our time course of HSP25 activation data is very similar to their study.

HSP27/20 has been shown to regulate cellular morphology by acting as an uncapping protein of actin (25). Thus we hypothesized that p38 might be involved in the morphological changes, such as elongation and reorientation, induced by shear stress. Our results show that even the lowest concentration of SB-203580, which inhibits both MAPKAP kinase 2 activation and HSP25 phosphorylation (1 µM), inhibits EC elongation and reorientation induced by shear stress almost completely. This finding was confirmed by transfection studies using the dominant negative mutant of p38-alpha . The cells transfected with wild-type p38-alpha were reoriented in the direction of the shear stress, whereas cells transfected with dominant negative p38-alpha remained randomly aligned with no stress fiber formation. Taken together, these results strongly suggest that p38, and at least the p38-alpha subtype, plays an important role in reorientation of EC induced by shear stress. Although the mechanism by which p38 regulates these morphological events is not completely elucidated, our transfection studies indicate that p38-alpha may influence stress fiber formation.

Because we did not block HSP25 directly, we cannot conclude that HSP25 is involved in the morphological changes induced by shear stress. However, there are a number of recent studies that address the relationship between HSP25 and morphological events. Lavoie et al. (27, 28) have shown that phosphorylation of HSP27 is implicated in pinocytosis with F-actin accumulation in response to growth factors as well as stabilization of actin filaments in response to heat shock. It has also been shown that HSP27 is involved in migration of EC and smooth muscle cells (16, 41). These data are consistent with the hypothesis that p38 regulates cell morphology through HSP25/27. Further studies using a dominant negative mutant of HSP25/27 would clarify this issue. Likewise, our results do not explain why the morphological changes take 24 h to manifest, whereas the peak phosphorylation of HSP25 occurs after 30 min of exposure to shear stress. The process of EC reorientation is complex, and many elements, such as actin-binding proteins, myosin-related proteins, and proteins related to the focal adhesion complex, have been implicated. In addition, Malek et al. (36) have demonstrated that shear stress induces actin mRNA expression, and acromycin, which inhibits de novo protein synthesis, blocks EC orientation with shear. These reports lead to the hypothesis that p38 activates not only MAPKAP kinase 2 but other downstream proteins, including transcriptional factors, which are capable of inducing de novo synthesis of actin or other proteins involved in regulation of cell morphology.

The upstream events of p38 are also not well defined. Members of the Rho family, Rho, Rac, and Cdc42, have been investigated as potential upstream regulators of p38 because these small GTP-binding proteins are important in the regulation of cell morphology. However, studies in different cell lines provide contradictory information. For example, Rac is upstream of p38 in some cell types (37, 51) but not others (14). We (2) recently reported that Rho is involved in EC reorientation induced by shear stress. Rho activates the Rho-associated coiled-coil-forming protein kinase (ROCK) p160ROCK (18), which has been shown to regulate myosin light-chain kinase, which in turn regulates myosin light chain (22). This finding and the results of our present study lead to two alternate hypotheses: 1) that myosin is regulated by Rho through p160ROCK, and actin is regulated by p38 through HSP25/27; or 2) Rho regulates both p160ROCK and p38 directly. The p160ROCK pathway and p38-MAPKAP kinase 2/HSP25/27 pathway might work together to regulate actinomyosin contractility and their redistribution, resulting in cell movement and morphological changes. Further studies to investigate how the cell regulates both p38 and Rho are needed. In this regard, Nobes and Hall (39) demonstrated that Rho, Rac, and Cdc42 are important for cell movement. They postulate that Rho is the driving force for movement, whereas Rac determines the polarity of the movement (39). Others have shown that the p38/MAPKAP kinase 2/HSP25/27 pathway is also important for cell movement (16, 41, 43). Thus the investigation of the signaling pathways associated with p38 and Rho is important not only for understanding the morphological changes of the cells but also for cell migration.

The mechanosensor resulting in p38 activation remains unknown. Rousseau et al. (43) demonstrated that vascular endothelial growth factor (VEGF) stimulated p38 activation, which causes EC migration. On the other hand, Shyy has shown that the VEGF receptor is tyrosine phosphorylated in response to shear stress in a time-dependent manner (Prof. J. Y. J. Shyy, University of California at Riverside, Riverside, CA, personal communication). These reports suggest that the VEGF receptor might be a potential candidate for a mechanotransducer leading to p38 activation.

In conclusion, we demonstrate that p38, especially the p38-alpha subtype, regulates EC reorientation, elongation, and stress fiber formation in response to shear stress through, most likely, MAPKAP kinase 2 and HSP25 activation.


    ACKNOWLEDGEMENTS

We thank Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center, MA) for providing wild-type and dominant negative mutant of p38-alpha and Dr. Michael Welsh (University of Michigan Medical Center, Ann Arbor, MI) for providing anti-HSP27. We also thank Dr. Colleen M. Brophy (Medical College of Georgia, Augusta, GA) for providing anti-phospho-specific HSP27 and for valuable discussion.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant R01 HL-47345 to B. E. Sumpio and the V. A. Merit Review Board and American Heart Association National.

Address for reprint requests and other correspondence: B. E. Sumpio, Dept. of Surgery, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06510.

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 11 February 2000; accepted in final form 18 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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