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(Circulation Research. 1996;79:310-316.)
© 1996 American Heart Association, Inc.

Articles

MAP Kinase Activation by Flow in Endothelial Cells

Role of ß1 Integrins and Tyrosine Kinases

Takafumi Ishida, Timothy E. Peterson, Nicholas L. Kovach, Bradford C. Berk

the Department of Medicine, Cardiology and Hematology (N.L.K.) Divisions, University of Washington, Seattle. This manuscript was sent to Francois M. Abboud, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Correspondence to Bradford C. Berk, MD, PhD, Cardiology Division, Box 357710, University of Washington, Seattle, WA 98195. E-mail bcberk@u.washington.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activated protein (MAP) kinase is rapidly stimulated by flow in bovine aortic endothelial cells. Integrin receptors may act as mechanotransducers, as suggested by rapid remodeling of focal adhesion complexes in response to flow. To study the role of integrins in flow-mediated MAP kinase activation, we compared the effects of ß1 integrin activation (with 8A2 antibody) and flow in cultured human umbilical vein endothelial cells (HUVECs). Both 8A2 (3 µg/mL) and flow (shear stress, 12 dynes/cm²) stimulated MAP kinase, although the flow response was faster and greater. To characterize flow-activated tyrosine kinases, tyrosine-phosphorylated proteins were immunoprecipitated and identified by Western blot. There was a time-dependent increase in phosphotyrosine content in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD proteins. A 125-kD protein was identified as focal adhesion kinase (FAK), suggesting that flow activates integrins. In comparison with flow, 8A2 caused less tyrosine phosphorylation of fewer proteins, although FAK was tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 and flow caused additive increases in MAP kinase. Antibody 8A2 increased binding of the ß1 affinity–sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a ß1-activating antibody and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that "costimulatory" events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.

Key Words: MAP kinase • signal transduction • endothelial cells

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fluid shear stress is an important hemodynamic force recognized by endothelial cells that modulates vessel function and structure. An important question concerns the sensing mechanisms by which endothelial cells respond to fluid shear stress. It is unclear whether the same shear stress receptor transduces rapid endothelial cell signals, such as production of nitric oxide, and slower signals, such as changes in cell morphology and proliferation.¹ We have previously shown² that fluid shear stress rapidly stimulates MAP kinase (peak activation at 10 minutes) in bovine aortic endothelial cells. Stimulation of MAP kinase was time and force dependent, independent of calcium, and required protein kinase C. Many studies (see Reference 3) indicate that MAP kinase is a convergent regulatory kinase for signals transduced by many different kinds of receptors, including tyrosine kinase receptors,^{4 5} G protein–coupled receptors,^{6 7} and extracellular matrix–coupled receptors such as integrins.^{8 9 10 11} The upstream kinases responsible for MAP kinase activation by these different receptors are unique on the basis of their coupling to individual receptors and on tissue distribution. Nonetheless, it has been possible to identify these upstream kinases by use of specific pharmacological inhibitors and dominant negative kinases.^{12 13} Many of these kinases associate with plasma membrane receptors and linker proteins such as Grb2.¹⁴ Coimmunoprecipitation of receptors with associated regulatory proteins has been a useful approach to identifying signal-transduction mechanisms used by specific receptors.^{15 16} Thus, understanding the nature of the kinases activated by flow in endothelial cells may provide insight into the identity of the shear stress receptor.

As an initial approach to identifying kinases activated by fluid shear stress, we have chosen to study tyrosine-phosphorylation events stimulated by flow in HUVECs. In the present study we compared tyrosine-phosphorylation events mediated by flow and by ß1 integrin activation. A role for integrin-mediated signal-transduction events in the endothelial cell response to flow is suggested by several studies. (1) Previous investigators have shown that there is rapid remodeling of focal adhesion contacts in response to flow, suggesting that these sites of cell attachment may be important in mechanotransduction.^{17 18} Focal adhesion contacts are integrin-rich complexes that may mediate signal transduction as well as cell attachment. Among the integrins present in HUVECs, the ß1-containing integrins are predominant.¹⁹ Integrin-mediated signal transduction requires activation of the integrin receptor (eg, ₂ß₁) by interactions with extracellular matrix ligands (eg, fibronectin) and recruitment of intracellular kinases.^{20 21 22 23 24} Integrin activation leads to phosphorylation of a 125-kD tyrosine kinase shown to be localized to focal adhesions²⁵ and termed FAK. Phosphorylation of FAK on tyrosine generates a site for binding of SH2-domain–containing proteins such as Grb2¹¹ and leads to recruitment and activation of Src-like protein kinases including Src itself, Fyn, and Csk,^{24 26 27} which then phosphorylate another focal adhesion protein, paxillin.^{28 29 30} (2) Paxillin has been shown to change its alignment in response to flow.³¹ In addition, it was reported that when endothelial cells migrated into a wounded area on tissue-culture plastic, there was an increase in FAK phosphorylation.²² Thus, FAK and paxillin (and by inference integrins present in focal adhesion contacts) may be involved in the endothelial cell response to flow. (3) MAP kinase, which we have shown to be activated by fluid shear stress in endothelial cells,² is also stimulated by integrin activation.^{8 9 10 11}

To investigate the tyrosine-phosphorylation events stimulated in endothelial cells by flow, we immunoprecipitated phosphotyrosine-containing proteins after exposure of cultured HUVECs to physiological fluid shear stress. The role of integrins in mediating tyrosine phosphorylation was then studied by comparing the phosphotyrosine profiles of flow and the ß1-activating antibody, 8A2. The results indicate that flow stimulates signals typical of integrin receptor activation, such as phosphorylation of FAK, but it appears that ß1 integrin activation accounts for only a portion of the flow-mediated response in HUVECs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
HUVECs were obtained from umbilical veins as previously described.³² Cells at passages between 1 and 3 were grown in RPMI 1640 supplemented with 20% FBS and used at confluence. Rat aortic smooth muscle cells were obtained from 10- to 12-week-old male Sprague-Dawley rats as previously described.³³

Preparation of Flow Plates
Flow plates were coated with 2.5% gelatin, except in the experiment for Fig 8. In that experiment, flow plates were coated with antibodies (10 µg/mL) at 4°C overnight, blocked with 1% BSA for 1 hour, and washed three times with PBS.

View larger version (28K): [in this window] [in a new window]   Figure 8. MAP kinase activation by flow on various anti–ß1 integrin antibodies. HUVECs were allowed to attach to plates coated with 8A2, LM534, or 4B5 for 50 minutes. Cells were then exposed to flow (shear stress, 12 dynes/cm2) for 10 minutes (+flow) or maintained in static culture for 10 minutes. MAP kinase activity was determined by an in-gel–kinase assay.

Flow System
Cells were grown on 2x4-cm slides of tissue-culture plastic, which were cut from the bottom of tissue-culture dishes. Before the experiment, cells were rinsed free of culture media with HEPES-buffered saline solution (containing, in mmol/L, NaCl 130, KCl 5, CaCl₂ 1.5, MgCl₂ 1.0, HEPES 20; pH 7.4), with 10 mmol/L glucose and either maintained in static condition or exposed to fluid shear stress in a parallel-plate chamber at 37°C exactly as described previously.² After varying times of exposure to fluid shear stress, cells were washed gently with ice-cold PBS (containing, in mmol/L, NaCl 137, KCl 2.7, Na₂HPO₄ 4.3, KH₂PO₄ 1.4; pH 7.3), and cell lysates were prepared as described below.

Immunoprecipitation and Western Blot Analysis
After treatment, the cells were washed with PBS, and 0.5 mL of RIPA lysis buffer (containing, in mmol/L, HEPES [pH 7.4] 10, EDTA 5, sodium pyrophosphate 50, NaF 50, NaCl 50, plus 100 µmol/L Na₃VO₄, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, fresh 0.5 mmol/L PMSF, and 10 µg/mL leupeptin) was added. Cell lysates were prepared by scraping, sonication, and centrifugation for 5 minutes at 14 000 rpm in a microfuge (4°C). The lysates were immunoprecipitated, and immune complexes were recovered by the addition of protein G–agarose (GIBCO-BRL), incubation for 3 hours at 4°C, and centrifugation. The beads were washed four times with lysis buffer. For Western blot analysis, cell lysates or immunoprecipitates were subjected to SDS-PAGE under reducing conditions, and proteins were then transferred to nitrocellulose (Hybond-ECL, Amersham) as previously described.² The membrane was blocked for 2 hours at room temperature with a commercial blocking buffer from GIBCO. The blots were incubated for 1 hour at room temperature with primary antibodies (anti-ERK1, anti-ERK2, and anti-FAK antibodies from Santa Cruz Biotechnology; monoclonal anti-paxillin antibody from Zymed Laboratories Inc; and anti-phosphotyrosine 4G10 from Upstate Biotechnology Inc), followed by incubation for 1 to 2 hours with secondary antibody (conjugated horseradish peroxidase). Immunoreactive bands were visualized by using chemiluminescence (ECL, Amersham Life Science Inc).

MAP Kinase Assays
An in-gel–kinase assay to measure MAP kinase phosphotransferase activity was performed on cell lysates exactly as previously described.³³ MAP kinase activity was measured by densitometry of autoradiograms (in the linear range of film exposure) using NIH Image 1.49.

Measurement of ß1 Activation State
The activation state of ß1 integrins was measured by FACS analysis using the 15/7 antibody as previously described.³⁴

Materials
All chemicals were obtained from Sigma Chemical Company unless otherwise indicated.

Statistical Analysis
All experiments were performed at least three times, and data are presented as mean±SEM. Significant differences were determined by Student's t test (P<.05). For comparison of MAP kinase activation by shear stress and 8A2 (Fig 6), the sign test (Systat) was used.

View larger version (41K): [in this window] [in a new window]   Figure 6. Flow- and mAb 8A2–mediated "additive" activation of MAP kinase in HUVECs. Cells were exposed to flow for 10 minutes at the indicated shear stress (flow alone, 2 dynes/cm2 to 12 dynes/cm2) or to 3 µg/mL 8A2 for 5 minutes and then to the indicated shear stress for an additional 10 minutes (flow+8A2). For cells pretreated with 8A2, the medium was removed after 5 minutes and fresh medium added for the exposure to flow. As negative controls, cells were maintained in static culture for 15 minutes (control) or treated with 8A2 for 5 minutes, washed, and maintained in fresh medium for 10 minutes (control+8A2). Cell lysates were prepared, and an in-gel–kinase assay for MAP kinase was then performed, using 10 µg cell protein per lane. The arrow indicates the position of the bands identified as MAP kinase. Results are representative of three experiments. To analyze the significance of these observations, the autoradiographic densities from three experiments were quantified, as described in "Materials and Methods." The results for each experiment were normalized to the density of the control sample, which was arbitrarily adjusted to 1.0. To compare results from different experiments, the response to 1 µmol/L PMA, which was always the maximum, was normalized to 100. The maximal response to 8A2 and flow+8A2 was then determined on a relative basis. The results were plotted on a log-log scale and differences analyzed by the sign test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fluid Shear Stress and 8A2 Stimulate MAP Kinase in HUVECs
We previously found that flow stimulates a time- and force-dependent activation of MAP kinase (ERK-1 and ERK-2) in bovine aortic endothelial cells.² The time course for flow-mediated stimulation of MAP kinase in HUVECs is shown in Fig 1A and 1B. MAP kinase activity, measured by in-gel–kinase assay, peaked at 10 minutes, with return to baseline activity at 90 minutes after exposure to flow (Fig 1A). Similar results were obtained when MAP kinase activation was assayed by the "band shift" on Western blots using anti–MAP kinase antibodies (Fig 1B). Of interest, the p44-kD MAP kinase was not expressed to the same extent as p42-kD MAP kinase, unlike bovine aortic endothelial cells, in which equal expression was observed. Analysis of six experiments indicated that MAP kinase was first activated at 5 minutes (range 2 to 10 minutes), with peak at 15 minutes (range 10 to 30 minutes).

View larger version (29K): [in this window] [in a new window]   Figure 1. Time course for MAP kinase activation by flow and mAb 8A2 in HUVECs. A, Cells were exposed to flow (shear stress, 12 dynes/cm2) for the indicated times and cell lysates prepared. An in-gel–kinase assay for MAP kinase was then performed using 10 µg cell protein per lane. The arrow indicates the positions of the band identified as MAP kinase. B, Cells were exposed to flow (shear stress, 12 dynes/cm2) for the indicated times and cell lysates prepared. Western blot analysis for MAP kinase was then performed using 20 µg cell protein per lane. C, Cells were exposed to 3 µg/mL 8A2 for the indicated times and cell lysates prepared for in-gel–kinase assay. As a positive control, 200 nmol/L PMA was added for 10 minutes. As negative controls, two ß1 antibodies (LM534 and 4B5) were added (3 µg/mL for 30 minutes). D, Cells were exposed to 8A2 for the indicated times and cell lysates prepared for Western blot analysis. Arrows indicate the position of the phosphorylated 42-kD (p42) and 44-kD (p44) bands identified as activated MAP kinase. Results are representative of three to six experiments.

The mAb 8A2 specifically activates ß1 integrins and induces a change in ß1 integrin affinity for its ligand.³⁵ In HUVECs treated with 3 µg/mL 8A2, there was a time-dependent increase in MAP kinase activity, as assessed by in-gel–kinase assay (Fig 1C). To investigate the specificity of 8A2, we compared MAP kinase activation by other ß1 antibodies; two control ß1 antibodies, LM534 and 4B5, were unable to stimulate MAP kinase in HUVECs (Fig 1C). A similar time course for 8A2 stimulation of MAP kinase activation was observed when MAP kinase was measured by band shift on Western blot, as shown in Fig 1D. It should be noted that MAP kinase activation in HUVECs in response to 8A2 was quite variable. Comparing different preparations of HUVECs and different preparations of 8A2, MAP kinase was first activated at 15 minutes (range 5 to 20 minutes), with peak activation at 35 minutes (range 20 to 60 minutes). The concentration of 8A2 required for maximal activation varied from 3 µg/mL to 10 µg/mL.

Flow Is a More Rapid and Potent Activator of MAP Kinase Than 8A2 in HUVECs
Two important differences were observed when MAP kinase activation by flow was compared with activation by 8A2. First, flow was a much more powerful activator of MAP kinase than was 8A2. We used 200 nmol/L PMA as a maximal activator of MAP kinase in HUVECs. As shown in Fig 2, the maximal increase in MAP kinase activity stimulated by flow (12 dynes/cm² at peak time) was not significantly different from that stimulated by PMA (86±17%, n=6, P>.05), while the increase stimulated by 8A2 (3 µg/mL at peak time) was significantly smaller than that stimulated by PMA (42±5%, n=5, P<.01) or flow (P<.01). Second, in cells prepared identically, peak activation of MAP kinase by 8A2 was always slower than peak activation by flow, with an average difference of 20 minutes to reach peak activity.

View larger version (10K): [in this window] [in a new window]   Figure 2. Summary of MAP kinase activation by flow and mAb 8A2 in HUVECs. The results from all in-gel–kinase experiments that included 8A2, flow, and PMA were analyzed, and the increase in MAP kinase was measured by gel densitometry. The autoradiographic densities were quantified as described in "Materials and Methods." The results for each experiment were normalized to the density of the control sample, which was arbitrarily adjusted to 1.0. To compare results from different experiments, the response to 200 nmol/L PMA, which was always the maximum, was normalized to 100. The maximal response to 8A2 and flow was then determined on a relative basis. Results are the mean±SEM of five to six determinations. *P<.01 vs flow or PMA.

Fluid Shear Stress and 8A2 Stimulate Protein Tyrosine Phosphorylation in HUVECs
To determine the identity of the tyrosine kinases that may be involved in flow- and ß1 integrin–mediated MAP kinase activation, tyrosine-phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibody, and Western blot analysis was performed with anti-phosphotyrosine antibody after exposing HUVECs to flow (12 dynes/cm²) and 8A2 (3 µg/mL) for various times. As shown in Fig 3A, flow stimulated a time-dependent increase in multiple tyrosine-phosphorylated proteins of 60 to 80 kD, 110 kD, 125 to 150 kD, and 180 to 190 kD. The mAb 8A2 stimulated tyrosine phosphorylation of proteins of 50 kD, 110 kD, and 125 to 150 kD (Fig 3B). Thus, 8A2 and flow demonstrated similar but not identical patterns of tyrosine phosphorylation. Two important differences were that protein tyrosine phosphorylation stimulated by flow was more rapid and involved many more proteins (eg, compare 60- to 80-kD and 180- to 190-kD proteins at 30 minutes) than 8A2.

View larger version (48K): [in this window] [in a new window]   Figure 3. Effect of flow and mAb 8A2 on tyrosine phosphorylation in HUVECs. Cells were exposed to flow (A, shear stress, 12 dynes/cm2) and 8A2 (B, 3 µg/mL) for the indicated times. Additional cells were exposed to 10 U/mL -thrombin for 2 minutes. Cell lysates were prepared and tyrosine-phosphorylated proteins immunoprecipitated from 200 µg cell protein, using 4G10 antibody, as described in "Materials and Methods." Equal amounts of immunoprecipitated protein were then analyzed for phosphotyrosine content on Western blots, using 4G10 antibody. Results are representative of three experiments.

Fluid Shear Stress and 8A2 Stimulate Tyrosine Phosphorylation of FAK but Not Paxillin
To identify tyrosine-phosphorylated proteins, we immunoprecipitated specific proteins after flow and assayed their phosphotyrosine content on Western blots with anti-phosphotyrosine antibody. As shown in Fig 4A, flow stimulated a time-dependent increase in FAK phosphotyrosine content, first apparent at 30 minutes, with peak at 120 minutes. The magnitude of the increase in FAK phosphorylation in response to flow was as great as that obtained with thrombin (10 U/mL for 2 minutes) in HUVECs. The tyrosine-phosphorylated proteins demonstrated at 125 to 150 kD in Fig 3A showed increases in tyrosine phosphorylation within 2 minutes. The time for this increase in phosphorylation is more rapid than the increase in FAK phosphorylation (Fig 4A), which suggests that other proteins (besides FAK) may be tyrosine phosphorylated in response to flow. As shown in Fig 4B, 8A2 (3 µg/mL) also stimulated FAK phosphorylation, with a time course that was similar to that observed for flow.

View larger version (22K): [in this window] [in a new window]   Figure 4. Flow- and mAb 8A2–stimulated FAK tyrosine phosphorylation in HUVECs. Cells were exposed to flow (A, shear stress, 12 dynes/cm2) and 8A2 (B, 3 µg/mL) for the indicated times. Additional cells were exposed to 10 U/mL -thrombin for 2 minutes. Cell lysates were prepared and FAK was immunoprecipitated from 200 µg cell protein, using anti-FAK antibody as described in "Materials and Methods." Results are representative of three experiments.

To determine whether the 60- to 80-kD tyrosine-phosphorylated proteins included paxillin, a substrate for FAK, we immunoprecipitated paxillin at various times after stimulation of HUVECs by flow. As shown in Fig 5, there was no significant increase in tyrosine phosphorylation of paxillin. In contrast, angiotensin II stimulated a significant increase in tyrosine phosphorylation of paxillin in rat smooth muscle cells. Equal amounts of protein were immunoprecipitated, as confirmed by Western blot analysis with anti-paxillin antibody (not shown).

View larger version (26K): [in this window] [in a new window]   Figure 5. Lack of stimulation of paxillin tyrosine phosphorylation by flow in HUVECs. Cells were exposed to flow (shear stress, 12 dynes/cm2) for the indicated times. As a positive control, rat smooth muscle cells (RASM) were exposed to 100 nmol/L angiotensin II (Ang II) for 5 minutes. Cell lysates were prepared and paxillin was immunoprecipitated from 200 µg cell protein, using anti-paxillin antibody, as described in "Materials and Methods." Results are representative of three experiments.

8A2 Treatment Enhances Flow-Mediated Activation of MAP Kinase
To examine the interaction between ß1 integrin activation and flow stimulation of HUVECs, cells were pretreated with 3 µg/mL 8A2 or vehicle for 5 minutes prior to initiation of flow. Exposure to flow alone (shear stress of 2, 4, 8, and 12 dynes/cm² for 10 minutes) caused a force-dependent increase in MAP kinase activity (Fig 6, left). Maximal activation occurred at 8 dynes/cm². Pretreatment with 8A2 for 5 minutes, followed by 10 minutes' incubation under no-flow conditions, caused no activation of MAP kinase (control, +8A2). When cells were preexposed to 8A2 for 5 minutes and then exposed to flow (Fig 6, right), there was a significant shift in the force dependence for flow-mediated activation of MAP kinase, with maximal activation at 2 to 4 dynes/cm² (P<.05). These results show that there is an apparent additive effect of 8A2 and flow to stimulate MAP kinase in HUVECs.

8A2, but Not Fluid Shear Stress, Activates ß1 Integrins
To verify that 8A2 was activating ß1 integrins, we measured the increase in binding of mAb 15/7. The 15/7 recognizes an activation-dependent epitope of ß1 integrins. The mechanism by which mAb 8A2 induces this epitope is thought to be direct induction of a conformational change.³⁶ The activation-dependent epitope can also be induced by "inside-out" signaling, such as that observed with stem cell factor treatment of some hematopoietic cell lines that increases cell adhesion with a time course that parallels expression of mAb 15/7.³⁴ In response to 8A2 treatment of HUVECs (3 µg/mL for 10 minutes), there was a dramatic increase in 15/7 binding, from 1.0 (normalized) to 48.0±12 (n=2), measured by FACS analysis (Fig 7). In contrast, flow (12 dynes/cm² for 10 minutes) did not increase 15/7 binding (0.3±0.2, n=2).

View larger version (13K): [in this window] [in a new window]   Figure 7. Effect of flow and 8A2 on 15/7 binding in HUVECs. HUVECs were exposed to flow (shear stress, 12 dynes/cm2) and 3 µg/mL 8A2 for 10 minutes. Cells were harvested and 15/7 binding was determined by FACS analysis, as described in "Materials and Methods." The results for each experiment were normalized to the fluorescence value of the control sample, which was arbitrarily adjusted to 1.0. Results are the mean±SEM of two experiments.

Anti–ß1 Integrin Antibodies Allow MAP Kinase Activation
To determine further the role of ß1 integrins in flow-induced signal events, we compared MAP kinase activation by flow when HUVECs were plated on anti–ß1 integrin antibodies in the absence of other matrix molecules. Cells were attached to flow plates coated with 8A2, LM534, and 4B5 for 50 minutes and then exposed to flow (12 dynes/cm²) for 10 minutes. MAP kinase was minimally activated after 50 minutes' adhesion to the antibodies alone (Fig 8). In response to flow, MAP kinase was rapidly stimulated, and the extent of activation was comparable among the antibodies. These data suggest that anchorage to ß1 integrins alone is sufficient for MAP kinase activation by flow.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of this study are that both flow and ß1 integrin activation stimulate protein tyrosine phosphorylation, MAP kinase, and FAK in HUVECs. However, ß1 integrin activation mediates only a portion of the events stimulated by flow. The evidence for activation of mechanisms in addition to ß1 integrins by flow is supported by several findings: (1) activation of MAP kinase in response to flow was more rapid and of greater magnitude than that in response to 8A2; (2) flow stimulated tyrosine phosphorylation of many more proteins than 8A2; and (3) flow and 8A2 were additive for MAP kinase activation. These results have several implications for our understanding of the role of integrins in endothelial cell mechanotransduction.

The initial hypothesis of the present study was that ß1 integrins might act as the endothelial cell "shear stress receptor." This hypothesis was based on previous work demonstrating rapid alterations in focal adhesion contacts in endothelial cells exposed to flow^{17 18} and the similarity in signal-transduction events stimulated by flow and integrin activation. Shared signals potentially include increases in intracellular calcium,^{37 38 39 40 41} protein kinase C activation,^{2 21 42} MAP kinase activation,^{2 8 9 10 11} remodeling of actin stress fibers,^{8 31 43 44 45} changes in cell growth rates,^{46 47} and changes in gene expression.⁴⁸ On the basis of our findings, it appears unlikely that ß1 integrins are solely responsible for the early responses stimulated by flow. However, the present results do not rule out the possibility that other integrins (or combinations of several integrins) may be required for the early events activated by flow.

The results obtained using the 15/7 antibody to measure the effect of flow and 8A2 on ß1 integrins must be interpreted with caution. As discussed above, the mechanism by which 8A2 induces the epitope for 15/7 is thought to be via a conformational change. Although it is possible that this epitope can also be induced by inside-out signaling in response to other stimuli, such as stem cell factor,³⁴ there is no evidence that 8A2 induces 15/7 binding via an intracellular mechanism in HUVECs. However, we show here that binding of 8A2 can induce cellular signaling processes (MAP kinase and tyrosine phosphorylation) as well as proliferation of HUVECs in serum-free conditions (Kovach et al, unpublished observation, 1996). Recent findings by Akiyama et al⁴⁹ clearly show that stimulation of tyrosine phosphorylation by integrins is mediated by cytoplasmic portions of ß1 integrins. This activation of integrins can occur regardless of which extracellular (or even transmembrane) domain is present. Thus, events that result in activation of MAP kinase may occur independent of events that regulate the conformation and structure of extracellular domains of the integrins. The data in Fig 7 therefore suggest that the "outside-in" signaling activated by 8A2 is different from that potentially activated by flow. The difference in signaling stimulated by 8A2 and by flow is further demonstrated by the results of Fig 8, in which flow-mediated MAP kinase activation of cells plated on 8A2 was not different from that of cells plated on the non–ß1-activating antibodies. These data suggest that flow-induced MAP kinase activation does not require preactivated ß1 integrins and that adhesion to a substratum via ß1 integrins is sufficient. The current data do not prove that flow does not activate ß1 integrins or that ß1 integrins are not required for MAP kinase activation by flow. In fact, the recent study by Miyamoto et al⁵⁰ demonstrating clustering of MAP kinase near activated integrins suggests that this is an early event in integrin signal transduction. Thus, it is still quite possible that flow may modulate focal adhesion complexes by outside-in signaling, because both FAK and MAP kinase are activated by flow. Future studies to elucidate the mechanism of the alteration in extracellular ß1 integrins induced by flow and 8A2 may provide insight into novel signal events activated by flow.

Previous investigators have demonstrated that integrin-matrix interactions stimulate MAP kinase.^{8 9 10 11} This has been shown by experiments involving cell adhesion to integrin receptor ligands (such as fibronectin) and immobilized antibodies and by use of activating antibodies. In the study by Schlaepfer et al,¹¹ binding of fibroblasts to fibronectin was shown to promote association of Grb2 and Src kinase with FAK. Because Src kinase has been suggested to be upstream of MAP kinase by Gupta et al,⁵¹ these investigators proposed a signal-transduction pathway from FAK to MAP kinase via Grb2 and Src kinase. The results of the present study suggest that the FAK may be involved in MAP kinase activation by flow and ß1 integrins. Our data are strongest for ß1 integrin–mediated activation, as shown by the close correlation in the time course for 8A2-mediated FAK tyrosine phosphorylation (a marker for FAK activation) and MAP kinase activity (Figs 1 and 4). Future experiments using microinjection or electroporated anti-FAK antibodies to inhibit FAK function⁵² may provide direct evidence for the role of FAK in flow- and 8A2-mediated MAP kinase activation.

The present study provides several novel insights into the nature of the endothelial cell shear stress receptor. First, we have shown that a tyrosine kinase is likely involved, based on stimulation of multiple tyrosine-phosphorylation events, including activation of FAK, a tyrosine kinase. In addition, because Src kinase may be important in FAK-mediated signal transduction,¹¹ it is possible that Src family kinases may be potential mediators of flow-mediated signal transduction. Second, the inability of ß1 integrin activation alone by 8A2 to reproduce fully the effects of flow is not surprising. Shattil and colleagues⁴⁵ described similar findings regarding FAK phosphorylation by platelet binding to fibrinogen or activation with an anti-ß3 antibody (LIBS6). They showed that FAK phosphorylation required both integrin activation and "costimulatory" events such as calcium mobilization and protein kinase C activation by epinephrine. Mobilization of intracellular calcium and stimulation of tyrosine phosphorylation by interleukin-1 in human fibroblasts were also shown to require substrate attachment, clustering of IL-1 receptors, and activation of FAK.⁵² Combinations of signal events such as calcium mobilization, cell-matrix interaction, and PKC activation likely occur in endothelial cells in response to flow.^{2 37 38 42} Thus, it appears possible that integrins may play an important modulatory role in flow-mediated signal transduction, as shown by the enhanced activation of MAP kinase when HUVECs were stimulated with 8A2 prior to exposure to flow. In summary, both ß1 integrins and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways in HUVECs. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that costimulatory events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.

	Selected Abbreviations and Acronyms

 

FACS = fluorescence-activated cell sorter FAK = focal adhesion kinase HUVEC(s) = human umbilical vein endothelial cell(s) mAb = monoclonal antibody MAP kinase = mitogen-activated protein kinase PMA = phorbol 12,13 myristate acetate

	Acknowledgments

 
This work was supported by grants from the American Heart Association and the National Institutes of Health. Dr Berk is an Established Investigator of the American Heart Association. We thank Mari Ishida, Masatoshi Kusuhara, and Masafumi Takahashi for invaluable advice; John Harlan for helpful discussions; and Tom Eunson for supplying HUVECs.

Received October 30, 1995; accepted May 1, 1996.

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				O. Traub, B. P. Monia, N. M. Dean, and B. C. Berk PKC-epsilon Is Required for Mechano-sensitive Activation of ERK1/2 in Endothelial Cells J. Biol. Chem., December 12, 1997; 272(50): 31251 - 31257. [Abstract] [Full Text] [PDF]

				S. Li, M. Kim, Y.-L. Hu, S. Jalali, D. D. Schlaepfer, T. Hunter, S. Chien, and J. Y-J. Shyy Fluid Shear Stress Activation of Focal Adhesion Kinase. LINKING TO MITOGEN-ACTIVATED PROTEIN KINASES J. Biol. Chem., November 28, 1997; 272(48): 30455 - 30462. [Abstract] [Full Text] [PDF]

				J. K. Miyashiro, V. Poppa, and B. C. Berk Flow-Induced Vascular Remodeling in the Rat Carotid Artery Diminishes With Age Circ. Res., September 19, 1997; 81(3): 311 - 319. [Abstract] [Full Text]

				B. S. Wung, J. J. Cheng, H. J. Hsieh, Y. J. Shyy, and D. L. Wang Cyclic Strain–Induced Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells Involves Reactive Oxygen Species Activation of Activator Protein 1 Circ. Res., July 19, 1997; 81(1): 1 - 7. [Abstract] [Full Text]

				J. M. Muller, W. M. Chilian, and M. J. Davis Integrin Signaling Transduces Shear Stress–Dependent Vasodilation of Coronary Arterioles Circ. Res., March 1, 1997; 80(3): 320 - 326. [Abstract] [Full Text]

				H. Jo, K. Sipos, Y.-M. Go, R. Law, J. Rong, and J. M. McDonald Differential Effect of Shear Stress on Extracellular Signal-regulated Kinase and N-terminal Jun Kinase in Endothelial Cells. Gi2- AND Gbeta /gamma -DEPENDENT SIGNALING PATHWAYS J. Biol. Chem., January 10, 1997; 272(2): 1395 - 1401. [Abstract] [Full Text] [PDF]

				J. Surapisitchat, R. J. Hoefen, X. Pi, M. Yoshizumi, C. Yan, and B. C. Berk Fluid shear stress inhibits TNF-alpha activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members PNAS, May 22, 2001; 98(11): 6476 - 6481. [Abstract] [Full Text] [PDF]

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