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Originally published In Press as doi:10.1074/jbc.M408877200 on September 30, 2004 Originally published In Press as doi:10.1074/jbc.M408877200 on August 27, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46621-46630, November 5, 2004
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p21-activated Kinase Regulates Endothelial Permeability through Modulation of Contractility*

Rebecca A. Stockton{ddagger}, Erik Schaefer§, and Martin Alexander Schwartz{ddagger}||

From the {ddagger}Cardiovascular Research Center, University of Virginia Health System, Charlottesville, Virginia 22908, §BioSource International, Hopkinton, Massachusetts 01748, Departments of Microbiology and Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908

Received for publication, August 4, 2004 , and in revised form, August 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cells lining the vasculature have close cell-cell associations that maintain separation of the blood fluid compartment from surrounding tissues. Permeability is regulated by a variety of growth factors and cytokines and plays a role in numerous physiological and pathological processes. We examined a potential role for the p21-activated kinase (PAK) in the regulation of vascular permeability. In both bovine aortic and human umbilical vein endothelial cells, PAK is phosphorylated on Ser141 during the activation downstream of Rac, and the phosphorylated subfraction translocates to endothelial cell-cell junctions in response to serum, VEGF, bFGF, TNF{alpha}, histamine, and thrombin. Blocking PAK activation or translocation prevents the increase in permeability across the cell monolayer in response to these factors. Permeability correlates with myosin phosphorylation, formation of actin stress fibers, and the appearance of paracellular pores. Inhibition of myosin phosphorylation blocks the increase in permeability. These data suggest that PAK is a central regulator of endothelial permeability induced by multiple growth factors and cytokines via an effect on cell contractility. PAK may therefore be a suitable drug target for the treatment of pathological conditions where vascular leak is a contributing factor, such as ischemia and inflammation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The endothelial cell monolayer lining the vasculature forms a barrier that maintains the integrity of the blood fluid compartment but permits passage of soluble factors and leukocytes in a regulated manner. Dysregulation of this process produces vascular leakage into underlying tissues, which accompanies the inflammation associated with pathological conditions involving edema (reviewed in Refs. 1–3). Edema associated with vascular permeability also occurs in ischemic injury due to the secretion of vascular endothelial growth factor (VEGF)1 by hypoxic tissues, which increases tissue damage in animal models of stroke and myocardial infarction (4, 5). Vascular permeability is characterized by altered cell-cell contacts and the appearance of paracellular pores between adjacent cells. Integrity of the endothelial barrier is regulated in part by opposing roles of the actin cytoskeleton in which cortical F-actin stabilizes cell-cell contacts, whereas intracellular stress fibers exert tension to induce permeability (reviewed in Refs. 6 and 7).

The small GTPase Rac regulates formation and function of cell-cell adhesions in a number of systems. In epithelial and endothelial cell types, Rac is important for both the assembly of adherens and tight junctions and for their disruption during cell scattering or in response to agonists that trigger permeability (8–12). These complex effects suggest that different Rac effector pathways may differentially regulate cell-cell junctions. Precise temporal and spatial regulation of Rac and its effector pathways are likely to be critical for determining the balance between strengthening and disrupting cell-cell adhesions. However, the downstream pathways that govern these effects are poorly understood.

The p21-activated kinases (PAKs) are serine/threonine kinases activated downstream of Rac and Cdc42 that participate in multiple cellular functions, including motility, morphogenesis, and angiogenesis (13). GTP-bound Rac and Cdc42 bind to inactive PAK, releasing steric constraints imposed by a PAK autoinhibitory domain and permitting PAK auto-phosphorylation and activation. Numerous autophosphorylation sites have been identified that serve as markers for activated PAK (14–16). Prominent PAK downstream targets include LIM kinase, which regulates actin polymerization through its effect on cofilin (17), and myosin light chain (MLC). PAK2 catalyzes monophosphorylation of MLC at Ser19 to increase contractility and trigger cell retraction (18–20). However, PAK can also inhibit MLC kinase and thereby limit MLC phosphorylation and retraction (21, 22). In endothelial cells, expression of catalytically active PAK1 increased MLC phosphorylation and cell contractility, whereas inhibiting PAK reduced cell contractility (23). Thus, in these cells, the dominant effect of PAK appears to be the promotion of contractility.

In this study, the observation that PAK phosphorylated on Ser141 strongly localized to cell-cell junctions prompted us to examine a possible effect on monolayer permeability. We found that PAK plays a key role in the induction of permeability by a wide variety of growth factors and cytokines and that cell contractility mediates these effects.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Culture—Bovine aortic endothelial cells (BAEC) (a generous gift from Dr. Helene Sage, Hope Heart Institute, Seattle WA) were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (Atlanta Biologicals, Atlanta, GA), 100 µg/ml dihydrostreptomycin, and 60 units/ml penicillin (Sigma) in a humidified 37 °C incubator with 5% CO2. Stock cells were passaged 2–3 times/week and used between passages 9–14. Prior to the experiments, the cells were incubated in medium with 0.5% serum for 18 h. Human umbilical vein endothelial cells (HUVECs) were a gift from Dr. Brett Blackman (University of Virginia Cardiovascular Research Center) and were grown in EGM-2 medium (Clonetics) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and used at passages 3–10.

Reagents—VEGF was from Genentech. Active Rac expression constructs pcDNA3-V12Rac and pEGFP-V12Rac, inactive Rac pcDNA3-N17Rac, and pEGFP-N17Rac were described previously (24, 25). Vectors for wild-type PAK1 (pcDNA3-PAK1), active PAK1 (pCMV6-T423E PAK), and dominant negative PAK1 (pCMV6-H83R/H86R/H299R) were described by Kiosses et al. (23). The PAK autoinhibitory domain (AID), comprising residues 83–149, was cloned into the pDsRed1-N1 vector (Clontech) using KpnI and BamHI sites. The PAK function-blocking peptide (26) comprises PAK1 residues 1–13, which bind the Nck SH3 domain linked to the human immunodeficiency virus Tat polybasic sequence in order to facilitate membrane permeability. The control peptide contains mutations in two prolines that are required for SH3 domain binding. The peptides were synthesized by the Scripps Research Institute protein synthesis facility.

Transfections—Cells were grown to confluence on 22-mm glass coverslips, tissue culture plates, or Transwell filters, all coated with fibronectin (FN) at 10 µg/ml in phosphate-buffered saline (PBS). Plasmid DNA was transfected using Effectene (Invitrogen) in growth medium according to the manufacturer's protocols. The control cells were transfected with pEGFP-C1, pCMV6, or pcDNA3 as vector-only controls. The transfected cells were grown 24 h in Dulbecco's modified Eagle's medium 10% serum and then transferred to starvation medium 18 h before use.

Fluorescence Imaging—Cells were grown to confluence on FN-coated glass coverslips in medium with 10% serum and then serum-starved for 18 h. Treated cells were washed once with PBS, fixed for 20 min in 3.5% formaldehyde, and permeabilized for 10 min with 0.1% Triton X-100 in PBS. Coverslips were blocked for 30 min with 10% goat serum in PBS and then incubated for 12 h at 4 °C with rabbit anti-phospho-PAK Ser141 (BioSource International), goat anti-PAK1/2/3 (BioSource International), mouse anti-{beta}-catenin (Santa Cruz Biotechnology) at the ratio of 1:500, or rabbit anti-phospho-MLC (BioSource International) at the ratio 1:500. Control coverslips, for comparison with the anti-phospho antibody treatments, were probed with the same antibody dilution mixed 1:1 with the antigenic phosphopeptide. Coverslips were washed with PBS and incubated with species-specific Alexa 488, 568, or Cy5-labeled anti-IgG at 1:1,000 for 2 h at room temperature. Some coverslips were washed again and incubated for 30 min in 0.66 µM Alexa-conjugated phalloidin (Molecular Probes). Coverslips were mounted on glass slides with Immunofluore mounting medium (ICN Immunobiologicals). For epifluorescence microphotography, images were acquired using a Nikon Diaphot inverted fluorescence microscope with a Roper charge-coupled device camera. For confocal microscopy, images were acquired using either a Bio-Rad Radiance 2100 coupled to a Nikon scope, or an Olympus Fluoview 2-laser confocal microscope. Digitized images were processed using either Innovision ISEE or Adobe PhotoShop software.

Western Blots—Confluent cells were starved in 0.5% serum for 18 h, treated as described in the legends to Figs. 1, 2, 3 and 6, washed with cold PBS, and then harvested by scraping in cold lysis buffer (25 mM Tris, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 137 mM NaCl, 50 µM EDTA, 10 µg/ml each aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged for 10 min at 13,000 x g. The supernatants were diluted to equivalent protein concentrations and separated on a 10% SDS-polyacrylamide gel. Protein was transferred to polyvinylidene difluoride membrane, and blots were probed with primary antibodies (described under "Fluoresence Imaging") followed by horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Inc.). Rabbit anti-phospho-PAK Thr423 antibody was from BioSource International. Blots were washed and developed using ECL (Amersham Biosciences).



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  		FIG. 1.
Phosphorylation and localization of PAK in endothelial cells. A, confluent endothelial cells were starved for 18 h in medium with 0.5% serum and then left untreated or treated with 10% serum for 2 h. The cells were harvested and PAK phosphorylation analyzed by Western blotting. Upper panels, anti-phospho-PAK Ser141 (pPAK (S141)); anti-total PAK1/2/3 (tot PAK); anti-phospho-PAK Ser141 plus antigenic phosphopeptide (pPAK S141+AP) as a competitive inhibitor. Lower panels, BAEC were probed with anti-phospho-PAK Thr423 antibody (pPAK (T423)), anti-total PAK (tot PAK), and pPAK T423 antibody plus antigenic peptide (pPAK T423+AP). Similar results were obtained in three experiments. B, cells on FN-coated glass coverslips were treated as described in A, either with (+) or without (–) 10% serum. The cells were fixed and stained with anti-phospho-PAK Ser141 (pPAK) or anti-total PAK1/2/3 (tot PAK) as indicated. As a control, cells were stained with anti-phospho-PAK plus competitive phosphopeptide (+serum, Ab+AP). Scale bar = 50 µ. Similar results were obtained in three experiments.

 



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  		FIG. 2.
PAK activation is downstream of Rac. Confluent BAEC were transiently transfected with vector only (vector), wt PAK1 (PAK1), constitutively active T423E PAK (acPAK), dominant negative (H83R/H86R/H299R) PAK (dnPAK), constitutively active Rac (acRac), or dominant negative inactive Rac (dnRac). Cultures were serum-starved (–) or in 10% serum (+); cells were lysed and analyzed by Western blotting. Upper row, anti-phospho-PAK Ser141 (pPAK); middle row, anti-total PAK1/2/3 (tot PAK); and lower row, anti-phospho-PAK plus competitive phospho-peptide (pPAK+AP). Similar results were obtained in four experiments

 



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  		FIG. 3.
PAK inhibitory peptide prevents PAK junctional translocation but not phosphorylation. A, confluent endothelial cells were serum-starved (0.5%) for 18 h and treated without (–) or with (+) 10% serum. All were treated for 60 min with 20 µg/ml of PAK-blocking peptide (+PAK pep) or control peptide (+con pep) as indicated. The cell lysates were analyzed by Western blotting with anti-phospho-PAK (pPAK), total PAK (tot PAK) or anti-phospho-PAK antibody plus antigenic phosphopeptide (pPAK+AP). B, cells on coverslips were treated as described in A, fixed, and stained with anti-phospho-PAK Ser141 antibody, alone or with antigenic phosphopeptide (+serum, Ab+AP). Scale bar = 50 µ.

 



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  		FIG. 6.
Regulation of myosin light chain (MLC) phosphorylation by PAK. A, confluent BAEC on FN were treated with vehicle only (+vehicle), 20 µg/ml control peptide (+con pep), 20 µg/ml PAK-blocking peptide (+PAK pep), 5 µM ML7 (+ML7) with (+) or without (–) 25 ng/ml VEGF. Cells were harvested at the indicated times post-addition of VEGF and analyzed by Western blotting for phosphorylated MLC (upper four rows), total MLC (bottom lower row, tot MLC), or phospho-MLC antibody plus antigenic peptide (top lower strip, pMLC+AP) to demonstrate antibody specificity. B, as described in A, BAEC were pretreated with 20 µg/ml control peptide (+con pep) or PAK-blocking peptide (+PAK pep) followed by 25 ng/ml of VEGF, 25 ng/ml bFGF, 10 µM histamine, 0.1 units/ml thrombin, or 10 ng/ml of TNF{alpha}. Upper row, anti-phospho-MLC (pMLC); middle row, total MLC (total MLC); and lower row, pMLC plus an antigenic phospho-peptide (pMLC+AP). C, BAEC on coverslips were incubated as described in B with control peptide (con pep) or the PAK peptide (PAK pep) followed by 30 min of cytokines as indicated. The cells were then fixed and stained for phospho-MLC. D, BAEC on coverslips were transiently transfected to express the PAK AID and then treated with 25 ng/ml VEGF for 30 min, fixed, and stained for {beta}-catenin. Cells expressing the AID construct (asterisk) tended to be extremely enlarged, whereas adjacent nonexpressing cells remained smaller and unable to detach. E, confluent BAEC on Transwell 3.0-µm filters were starved in medium with 0.5% serum for 18 h and then treated without (white bars) or with 25 ng/ml VEGF (black bars) and with vehicle only (vehicle) or with 1.0, 2.5, or 5.0 µM ML7 for 60 min. Permeability was assayed as described under "Experimental Procedures."

 
Monolayer Permeability and Paracellular Pore Formation Assays— Cells were plated onto FN-coated polycarbonate membranes (3.0-µ pore size; Transwell, Costar) at 6 x 104 cells/cm2 and cultured for 4 days with medium changes every other day until confluence was reached. Where indicated, cells in normal growth medium with 10% serum were transfected 36 h before the assays. For experiments using a PAK-blocking or control peptide, medium in the upper chamber was replaced with medium containing peptides for 60 min before the experiment; control cell chambers received replacement medium containing vehicle only. In experiments using growth factors or other stimulants, medium containing those additions was added to the upper chambers for 30–60 min, as indicated. Horseradish peroxidase (HRP, Sigma) was then added to the upper chambers at a final concentration of 1.5 µg/ml. After 20 min, the filters were removed and immediately fixed in 3.5% paraformaldehyde for 30 min. Medium was harvested from the lower chamber, and HRP activity was determined colorimetrically by absorbance at 490 nm to detect the O-phenylenediamine reaction product after 15 min of incubation with 0.5 mM guaiacol, 50 mM Na2HPO4, and 0.6 mM H2O2. The mean HRP concentration in the lower chamber medium from cells in variously treated wells was normalized to the HRP concentration in the control wells transfected with vector only or treated with vehicle only, and the results were graphed as means ± S.E. of 4–7 experiments. To quantify paracellular pore formation and correlate to permeability, the fixed cell monolayer-covered filters were stained for 2 h with Coomassie Blue (0.25% in 1:1:2 of methanol:glacial acetic acid:double distilled (dd)H2O), destained in the same solution minus dye, rinsed in ddH2O, and air-dried. Stained cell monolayers were examined by phase-contrast microscopy and the number of pores/mm2 of filter surface counted in eight randomly selected fields per filter. These values were normalized to the number of pores in the control cells.

Statistical Analysis—For all quantitative assays, data from four to eight experiments were analyzed for statistical significance by analysis of variance (ANOVA) or Student's t test, as appropriate, using SigmaStat analytical software (Jandel Scientific) and are shown graphically as means ± S.E. Photographic or blot images shown are representative of results seen consistently in multiple experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PAK Phosphorylation and Translocation to Cell-Cell Junctions—PAK1, -2, and -3 are held in an inactive conformation via an interaction of the kinase domain with a sequence in the regulatory N terminus named the AID (13). Binding of activated Rac or Cdc42 to PAK leads to autophosphorylation of several sites that confer sustained increases in PAK kinase activity (16, 27). One of these sites, Ser141 in PAK2 (which corresponds to Ser144 in PAK1), is within the AID and its phosphorylation contributes to activation by blocking the interaction of the AID with the kinase domain. To localize activated PAK in endothelial cells, we therefore utilized an antibody that specifically recognizes the phosphorylated Ser141 site.

To initially evaluate PAK phosphorylation in endothelial cells in response to serum, confluent bovine aortic and human umbilical vein endothelial cells (BAEC and HUVEC, respectively) were serum-starved (0.5% serum) for 18 h and then stimulated with 10% serum. Western blotting with anti-phospho-PAK Ser141 antibody showed that serum stimulation increased PAK phosphorylation in both cell types (Fig. 1A, upper panels), consistent with previous studies that demonstrated increased PAK kinase activity in endothelial cells treated with serum or growth factors (28). Western blots of BAEC lysates using an antibody specific for PAK phosphorylated at Thr423 confirmed the results seen with the phospho-Ser141 antibody (Fig. 1A, lower panels). Fluorescence staining of similarly treated cells with anti-phospho-PAK Ser141 (pPAK) also showed an increase in PAK phosphorylation in response to serum, with the activated fraction of the protein localized mainly to cell-cell junctions (Fig. 1B). Staining with an antibody that recognizes the total pool of PAK1, -2, and -3 (Fig. 1B, tot PAK) also showed an increase in the staining at cell-cell borders plus a large amount of diffuse cytoplasmic fluorescence that presumably represents the unphosphorylated fraction. The phosphorylated PAK antigenic peptide blocked staining by the phospho-PAK antibody (Fig. 1, +serum, Ab+AP), demonstrating that the pattern is specific. Taken together, these data show that PAK is autophosphorylated on Ser141 in response to serum and that the phosphorylated fraction localizes specifically to cell-cell contacts.

Rac Regulation of PAK Phosphorylation—To confirm these results and evaluate the involvement of Rac upstream of PAK activation, BAEC were transiently transfected to overexpress wild-type PAK1 (Fig. 2, PAK1), constitutively active T423E PAK1 (acPAK), dominant negative (H83R/H86R/H299R) PAK1 (dnPAK), constitutively active V12Rac (acRac), or a dominant negative N17Rac (dnRac). Green fluorescent protein vector was included in all transfections, and transfection efficiency for these experiments was ~30–35%, when green fluorescent protein expressors were scored under a low power objective (although this value is probably an underestimate because lower expressors may be missed). Transfected cells were serum-starved for 18 h and then treated with 10% serum for 2 h or left untreated. Western blots of cell lysates were probed with the phospho-PAK Ser141 antibody (pPAK), total PAK antibody (tot PAK), or phospho-PAK antibody in the presence of the antigenic phosphopeptide (pPAK+AP) (Fig. 2). Cells expressing wtPAK1 had increased levels of Ser141 phosphorylation, which was further increased after serum stimulation. The active PAK mutant showed higher phosphorylation, which was not further affected by serum. DnPAK transfection reduced levels of phospho-PAK even in the presence of serum. Active Rac also increased PAK phosphorylation both with and without serum, whereas dnRac suppressed PAK phosphorylation. Efficient blocking by the antigenic phosphopeptide demonstrates the specificity of the phospho-PAK antibody. These results suggest that Rac is the major upstream regulator of PAK activation under these conditions.

Effect of a PAK Function-blocking Peptide—Previous work mapped the inhibitory sequences responsible for the dominant negative effects of kinase-dead PAK in endothelial cells to the first proline-rich repeat that mediates binding to the SH3 domain of Nck (23). A synthetic peptide in which this sequence was fused to the polybasic sequence from the human immunodeficiency virus Tat protein to facilitate cell entry had a very similar effect, reducing endothelial cell motility and contractility (26). The peptide did not appear to inhibit PAK kinase activity but did prevent its translocation to sites of action, which, in those studies with cells at low density, were actin stress fibers. We therefore asked whether this peptide might also limit pPAK translocation to cell-cell borders. BAECs and HUVECs were incubated with either the PAK-blocking peptide or the control peptide in which two critical prolines are mutated to prevent binding to SH3 domains. Western blots of lysates from these cells (Fig. 3A) showed that neither peptide altered PAK phosphorylation with or without serum, consistent with published data (26). Staining of similarly treated BAECs (Fig. 3B) showed that the PAK-blocking peptide (+PAK pep) inhibited phospho-PAK translocation to cell-cell junctions in response to serum. Instead, greater phospho-PAK staining was observed in the cytosol and nucleus. The control peptide (+con pep) had no effect, and similar results were obtained with HUVECs (not shown). The PAK peptide therefore abrogates localization of phospho-PAK to cell-cell contacts in endothelial cells.

PAK Regulates Monolayer Permeability—Junctional localization of phosphorylated and presumably activated PAK suggested a possible role in regulation of permeability across the endothelial monolayer. To test this idea, BAECs and HUVECs grown on filters with 3.0-µm pores were transiently transfected with wtPAK1, active or dnPAK, or active or dnRac. Movement of HRP across the filters was then measured. Overexpression of wtPAK1 increased HRP movement >2-fold in both cell types, whereas both active PAK and active Rac increased movement by greater amounts (Fig. 4A). The PAK-blocking peptide (Fig. 4A, black bars) strongly inhibited this increase in permeability, whereas the control peptide (gray bars) had no statistically significant effects. These data identify a role for PAK in the regulation of junctional permeability in endothelial cells.



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  		FIG. 4.
PAK controls permeability. A, BAEC (left graph) and HUVEC (right graph) were grown to confluence on FN-coated 3.0-µ pore Boyden chamber filters in normal growth medium with 10% serum and then transiently transfected with vector only (vector), wt PAK1 (PAK1), active PAK1 (acPAK), dominant negative PAK1 (dnPAK), active Rac (acRac) or dominant negative Rac (dnRac). Cells were incubated in medium with 0.5% serum for 18 h, and then the upper chamber medium was replaced with medium containing vehicle only (white bars), 20 µg/ml of eithercontrol peptide (gray bars) or PAK-blocking peptide (black bars) for 60 min. Monolayer permeability was assayed by peroxidase leak assay as described under "Experimental Procedures." Asterisks within the bars indicate significant difference compared with control vector-only, vehicle-only cells. Asterisks above the bars indicate significant difference compared with vector-only plus control peptide cells of each transfected type. B, BAEC (left graph) or HUVEC (right graph) on FN-coated filters were treated with vehicle only (white bars), control peptide (black bars), or PAK peptide (gray bars) as described in A and then were treated as indicated with 25 ng/ml VEGF (+VEGF), 25 ng/ml bFGF (+bFGF), 10 ng/ml TNF{alpha}, 10 µM histamine, or 0.1 units/ml thrombin for 30 min. C, cells grown as described in A and B were mock-transfected (no vector, white bars) or transiently transfected with vector-only (black bars) or the PAK autoinhibitory domain (PAK AID, gray bars). The cells were treated with vehicle only, VEGF, bFGF, or histamine as described above for 30 min and then permeability assayed as described under "Experimental Procedures."

 
Growth Factor and Cytokine-stimulated Permeability— Vascular permeability is regulated by a variety of soluble cytokines and factors. VEGF is one such factor (4, 5); similarly, bFGF, histamine, thrombin, and TNF{alpha} also increase vascular permeability. We therefore examined the effect of the PAK inhibitory peptide on permeability induced by these agents. The addition of VEGF induced a 2–3-fold increase in movement of HRP across the endothelial monolayer, which was almost completely blocked by the PAK peptide (gray bars) in BAEC and HUVEC (Fig. 4B). The control peptide (black bars) had no detectable effect. These effects correlated with the accumulation of phospho-S141 PAK at cell-cell contacts (Fig. 5). To confirm this result, cells were transiently transfected with a construct coding for AID from PAK1 (Fig. 4C). Although control empty vector (black bars) had no significant effect, the AID construct (gray bars) efficiently blocked VEGF-induced permeability. The other cytokines tested had similar effects on permeability, which were also blocked by the inhibitory PAK peptide and the AID (Fig. 4, B and C). These results show that PAK activity is required for induction of vascular leak by VEGF and other factors.



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  		FIG. 5.
Effects of PAK on remodeling of cell-cell junctions and the actin cytoskeleton. A, confluent BAECs on coverslips were serum-starved for 18 h and then treated with 25 ng/ml VEGF. The cells were fixed and stained for phospho-PAK and F-actin. Images show phospho-PAK (pPAK) and F-actin (F-actin) at the indicated times after the addition of VEGF. White arrows indicate pores between cells. Scale bar = 50 µ. B, phospho-PAK (pPAK) and F-actin (F-actin) are shown in cells prepared and treated as described in A after 60 min with VEGF withpeptide additions. The cells were treated with 20 µg/ml control peptide (+con pep) or 20 µg/ml PAK-blocking peptide (+PAK pep) with or without 25 ng/ml of VEGF as indicated. Scale bar = 50 µ. C, cells on filters were transfected with vector only (vector only), wtPAK1 (+PAK1), constitutively active PAK (+acPAK), or the PAK-AID (+PAK AID). Serum-starved cells were treated with vehicle only (white bars), PAK peptide only (striped bars), VEGF only (black bars), or VEGF plus PAK peptide (gray bars), as described in B. Values are the means + S.E. of four experiments normalized to the control treatment for that experiment. Asterisks within the bars indicate significant difference compared with control vector-only, vehicle-only cells. Asterisks above the bars indicate significant difference compared with vehicle-only treatments within each type of transfected cell.

 
Role of Contractility—The results with the PAK AID were surprising in that transfection efficiencies were below 50%, and yet the increase in permeability was almost completely inhibited. In considering possible explanations for this result, we recalled that myosin-dependent contractility is well known to regulate cell-cell junctions and permeability across endothelial or epithelial monolayers (29–32). Although both negative or positive effects of PAK on myosin light chain phosphorylation have been reported (18, 19, 33–35), in endothelial cells, PAK clearly promotes myosin phosphorylation and cell contractility (23, 26). Staining of treated cells with rhodamine-phalloidin to visualize F-actin showed first that VEGF addition to BAEC monolayers induced rapid formation of spaces or pores between the cells within 15 min, followed at later times by more general separation of adjacent cells (Fig. 5A). Both localization of phospho-PAK to cell-cell contacts and formation of intercellular spaces were largely blocked by PAK peptide but not control peptide (Fig. 5B). When the formation of pores between adjacent cells was examined, BAEC transfected with active PAK induced pores in the absence of VEGF (Fig. 5C), consistent with results from permeability assays, whereas the PAK AID and the PAK peptide inhibited pore formation. These results show alterations in monolayer organization associated with permeability.

Visualization of the actin cytoskeleton in BAEC treated with VEGF showed that increased permeability and pore formation correlated with increases in the intensity of staining for actin bundles both at cell-cell borders and spanning the cell (Fig. 5A), as described previously (36–39). Treatment with the PAK-blocking peptide (Fig. 5B) did not dramatically affect the number or intensity of actin stress fibers, but this result is consistent with previous studies that showed a decrease in contractility despite persistence of bundled actin cables (23). Because PAK is known to increase endothelial cell contractility, we assayed myosin phosphorylation in this system. Western blotting with an anti-phospho-MLC antibody showed that MLC phosphorylation induced by VEGF was inhibited by the PAK peptide nearly as well as the MLC kinase inhibitor ML7 (Fig. 6A). PAK peptide also inhibited MLC phosphorylation stimulated by bFGF, histamine, thrombin, and TNF{alpha} (Fig. 6B). Staining for phospho-MLC in monolayer cells treated with these factors also demonstrated phospho-MLC localization to cell-cell junctions, which was abrogated by the PAK inhibitory peptide (Fig. 6C). We also observed, as has been noted previously (32, 40), that anti-phospho-MLC stained actin stress fibers at the basal surface (data not shown); however, these structures were much less evident at higher focal planes where junctional staining was most prominent (Fig. 6C).

These results provide a possible explanation for the dominant effect of the PAK AID in transiently transfected cells. Decreases in tension in a fraction of the population may be sufficient to prevent breakup of cell-cell junctions where tension must be exerted from both sides of a junction. When one-half of the junctional pair is relaxed because of a decrease in myosin phosphorylation, it may undergo an increase in cell spreading when its neighbors contract. Indeed, we often noted large, well spread PAK AID-positive cells (Fig. 6D, white asterisk) surrounded by smaller untransfected cells in the culture (Fig. 6D). To test the importance of myosin phosphorylation, cells were treated with the myosin light chain kinase inhibitor ML7. We observed a dose-dependent decrease in both baseline and VEGF-stimulated permeability (Fig. 6E). The highly efficient inhibition by transient transfection with PAK AID is therefore consistent with a myosin-dependent mechanism.

Effects on Adherens Junctions—Because permeability is controlled in part by adherens junctions, we also examined {beta}-catenin distribution in VEGF-treated cells (Fig. 7). The addition of the PAK-blocking peptide to otherwise untreated cells appeared to thicken junctional staining of this marker compared with cells treated with the inactive control PAK peptide. The addition of VEGF decreased junctional integrity, inducing a shift to more broken staining along the cell-cell borders, as has been described (36, 41, 42). The PAK peptide largely prevented this VEGF-induced rearrangement, whereas the control peptide had no effect. Thus, changes in permeability correlate with effects on a junctional marker and support the notion that decreased tension in a subpopulation of cells can be transmitted to a wider region of the monolayer.



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  		FIG. 7.
Stabilization of adherens junctions. Confluent HUVEC on FN-coated coverslips were starved in medium with 0.5% serum for 18 h, preincubated in 20 µg/ml control peptide (+con pep) or PAK-blocking peptide (+PAK pep) and then treated with (+VEGF) or without (no VEGF) 25 ng/ml VEGF for 60 min. The cells were fixed and stained for {beta}-catenin. Scale bar = 50 µ.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Serine 141 on PAK2 is a site within the AID sequence that is phosphorylated during activation of the kinase. Phosphorylation of this site contributes to activation by blocking interaction of the AID with the kinase domain to relieve autoinhibition. Using a newly developed antibody specific for the phosphorylated state, we observed that the majority of phospho-Ser141 PAK localized to areas of cell-cell contact. This observation prompted us to test the role of PAK in the regulation of junctional permeability. We found that either expression of activated PAK1 or activation of endogenous PAK by expression of Rac1 was sufficient to increase permeability across endothelial monolayers. Conversely, blocking PAK function with a peptide that displaces active PAK from cell-cell contacts or expression of the AID to inhibit PAK kinase activity, prevented increases of vascular permeability stimulated by VEGF, bFGF, histamine, TNF{alpha}, and thrombin. The very similar responses in HUVECs and BAECs, which represent endothelial cells from different species and different vessels, suggest that the role of PAK in regulating junctional integrity is likely to be highly conserved throughout the vasculature.

For VEGF, the effects of PAK on permeability were mediated at least in part by changes in myosin phosphorylation and cellular contractility. This idea is consistent with a number of previous studies indicating that PAK can positively regulate myosin phosphorylation (18, 19, 35, 43). They are also fit well with a large body of data implicating myosin phosphorylation and contractility in regulating endothelial barrier function (reviewed in Ref. 44). Rho-mediated myosin phosphorylation and cellular contractility have also been implicated in endothelial junction stability and vascular permeability (45); whether Rho and PAK pathways interact or represent parallel mechanisms remains to be investigated. It should, however, be kept in mind that endothelial permeability can be altered without changes in myosin phosphorylation or contractile forces, (reviewed in Ref. 46). Thus, the data do not exclude additional myosin-independent effects of PAK on junctional components.

These data also help elucidate the biphasic role of Rac in the formation and turnover of intercellular junctions. Rac contributes to junctional formation and stabilization (8–10) but is also required for cell scattering, which involves the breakdown of cell-cell junctions, a dual role that has been difficult to understand. Our data suggest that modulation of the effector pathways downstream of Rac is likely to be critical for the decision to scatter versus remaining as a well organized epithelial or endothelial tissue. IQGAP is implicated in stabilizing adherens junctions downstream of Rac and Cdc42 (47). Previous studies have shown that Rac activated downstream of specific nucleotide exchange factors preferentially stimulates different effector pathways (48–50). Thus, conditions that favor interaction of small GTPases with PAK would lead to junctional disruption, whereas those that favor IQGAP would lead to junctional stabilization. Investigation of the mechanisms by which specific downstream effectors are selectively activated will be an important direction for future work.

Vascular permeability is a precisely regulated function that can contribute positively to immune responses and wound healing; however, leakage of fluid and immune cells into tissues can have serious and life-threatening consequences in a variety of diseases. Fluid accumulation in the lungs because of increased permeability of the pulmonary vasculature leading to respiratory insufficiency is a key element in acute respiratory distress syndrome (51). Vascular leak after stroke or myocardial infarction due to the release of VEGF by hypoxic tissues substantially increases tissue injury after these events (4, 5). Vascular leak and tissue edema contribute to organ failure in sepsis (52). The present study identifies PAK as a key mediator of vascular permeability in response to a wide variety of mediators. PAK may therefore be a promising pharmacological target for treatment of these diseases.


   FOOTNOTES
 
* This research was supported by National Institutes of Health (NIH) Training Grant 5T32 HL-007284-27 through the University of Virginia Cardiovascular Research Center and NIH Grant RO1-GM47214 (to M. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Cardiovascular Research Ctr., 415 Lane Rd., MR5 Rm. G-111, Charlottesville, VA 22908. Tel.: 434-243-4813; Fax: 434-924-2828; E-mail: maschwartz{at}virginia.edu.

1 The abbreviations used are: VEGF, vascular endothelial growth factor; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; TNF{alpha}, tumor necrosis factor {alpha}; bFGF, basic fibroblast growth factor; FN, fibronectin; PAK, p21-activated kinase; MLC, myosin light chain; AID, autoinhibitory domain; PBS, phosphate-buffered saline; HRP, horseradish peroxidase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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