p21-activated Kinase Regulates Endothelial Permeability through Modulation of Contractility

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, TNFα, 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. 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, TNFα, 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. 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. 1Suzuki Y. Ruiz-Ortega M. Lorenzo O. Ruperez M. Esteban V. Egido J. Int. J. Biochem. Cell Biol. 2003; 35: 881-900Crossref PubMed Scopus (579) Google Scholar, 2Dudek S.M. Garcia J.G. J. Appl. Physiol. 2001; 91: 1487-1500Crossref PubMed Google Scholar, 3van Nieuw Amerongen G. van Hinsbergh V. Vascul. Pharmacol. 2002; 39: 257-272Crossref PubMed Scopus (134) Google Scholar). Edema associated with vascular permeability also occurs in ischemic injury due to the secretion of vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; TNFα, tumor necrosis factor α; 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. by hypoxic tissues, which increases tissue damage in animal models of stroke and myocardial infarction (4Paul R. Zhang Z. Eliceiri B. Jiang Q. Boccia A. Zhang R. Chopp M. Cheresh D. Nat. Med. 2001; 7: 222-227Crossref PubMed Scopus (305) Google Scholar, 5Weis S. Shintani S. Weber A. Kirchmair R. Wood M. Cravens A. McSharry H. Iwakura A. Yoon Y.-S. Himes N. Burstein D. Doukas J. Soll R. Losordo D. Cheresh D. J. Clin. Investig. 2004; 113: 885-894Crossref PubMed Scopus (296) Google Scholar). 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. 6Bogatcheva N. Garcia J. Verin A. Biochemistry (Mosc.). 2002; 67: 75-84Crossref PubMed Scopus (167) Google Scholar and 7Lee T. Gotlieb A. Microsc. Res. Tech. 2003; 60: 115-127Crossref PubMed Scopus (82) Google Scholar). 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 (8Takaishi K. Sasaki T. Kotani H. Nishioka H. Takai Y. J. Cell Biol. 1997; 139: 1047-1059Crossref PubMed Scopus (474) Google Scholar, 9Ridley A. Comoglio P. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar, 10Hordijk P.L. ten Klooster J.P. van der Kammen R.A. Michiels F. Oomen L.C. Collard J.G. Science. 1997; 278: 1464-1466Crossref PubMed Scopus (393) Google Scholar, 11Wojciak-Stothard B. Ridley A. Vascul. Pharmacol. 2002; 39: 187-199Crossref PubMed Scopus (397) Google Scholar, 12Eriksson A. Cao R. Roy J. Tritsaris K. Wahlestedt C. Dissing S. Thyberg J. Cao Y. Circulation. 2003; 107: 1532-1538Crossref PubMed Scopus (102) Google Scholar). 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 (13Bokoch G. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (885) Google Scholar). 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 (14Zenke F.T. King C.C. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 32565-32573Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 15Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (134) Google Scholar, 16Gatti A. Huang Z. Tuazon P.T. Traugh J.A. J. Biol. Chem. 1999; 274: 8022-8028Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Prominent PAK downstream targets include LIM kinase, which regulates actin polymerization through its effect on cofilin (17Edwards D. Sanders L. Bokoch G. Gill G. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (849) Google Scholar), and myosin light chain (MLC). PAK2 catalyzes monophosphorylation of MLC at Ser19 to increase contractility and trigger cell retraction (18Chew T. Masaracchia R. Goeckeler Z. Wysolmerski R. J. Muscle Res. Cell Motil. 1998; 19: 839-854Crossref PubMed Scopus (168) Google Scholar, 19Goeckeler Z.M. Masaracchia R.A. Zeng Q. Chew T.-L. Gallagher P. Wysolmerski R.B. J. Biol. Chem. 2000; 275: 18366-18374Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 20Zeng Q. Lagunoff D. Masaracchia R. Goeckeler Z. Cote G. Wysolmerski R. J. Cell Sci. 2000; 113: 471-482Crossref PubMed Google Scholar). However, PAK can also inhibit MLC kinase and thereby limit MLC phosphorylation and retraction (21Sanders L.C. Matsumura F. Bokoch G.M. de Lanerolle P. Science. 1999; 283: 2083-2085Crossref PubMed Scopus (505) Google Scholar, 22Wirth A. Schroeter M. Kock-Hauser C. Manser E. Chalovich J.M. de Lanerolle P. Pfitzer G. J. Physiol. (Lond). 2003; 549: 489-500Crossref Scopus (61) Google Scholar). In endothelial cells, expression of catalytically active PAK1 increased MLC phosphorylation and cell contractility, whereas inhibiting PAK reduced cell contractility (23Kiosses W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus (244) Google Scholar). 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. 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 and were A. J. W.B. M.A. R. S. Schwartz M.A. J. Cell Biol. 2002; PubMed Scopus Google Scholar, M.A. Schwartz M.A. J. 2000; 19: PubMed Scopus Google Scholar). for PAK1 active PAK1 and dominant PAK1 were by (23Kiosses W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus (244) Google Scholar). The PAK autoinhibitory domain was into the and The PAK W.B. J. S. Cheresh N. Schwartz M.A. Res. 2002; PubMed Scopus Google PAK1 which bind the domain to the human in to permeability. The in that are for domain The were by the Research were grown to on tissue or with at μg/ml in phosphate-buffered was in growth medium to the The cells were with or as The cells were grown in Dulbecco's modified Eagle's medium 10% serum and to medium 18 were grown to on in medium with 10% serum and for 18 h. cells were with PBS, for in and for with in were for with 10% serum in and incubated for at °C with Ser141 at the of or at the for with the were with the with the were with and incubated with or at for at were and incubated for in were on with medium were a with a were a to a or an were or cells were in 0.5% serum for 18 as in the to and with PBS, and by in 0.5% μg/ml and were for at The were to and on a 10% was to and were with by horseradish was from were and activation is downstream of were with PAK1 active PAK dominant PAK active Rac or dominant inactive Rac were or in 10% serum cells were and by Ser141 and were in prevents PAK translocation but endothelial cells were for 18 and or with 10% were for 60 with μg/ml of or as The cell were by with PAK or cells on were as in and with Ser141 or with of myosin light chain phosphorylation by on were with μg/ml μg/ml with or were at the of and by for phosphorylated MLC MLC or to as in were with μg/ml or by of VEGF, bFGF, histamine, units/ml or of MLC and an on were incubated as in with or the PAK by of cytokines as The cells were and for on were to the PAK and with for and for the to be whereas adjacent cells and to on were in medium with 0.5% serum for 18 and or with and with or with or for 60 Permeability was as Permeability and were at and for with medium was cells in growth medium with 10% serum were the a or medium in the was with medium for 60 the cell medium In growth factors or medium was to the for as was to the at a of the were and in for was from the and was by at to the of with and The in the medium from cells in was to the in the with or with and the were as of paracellular formation and to the cell were for with in of in the in and cell were examined by and the number of of in These were to the number of pores in the cells. data from to were for by of or as and are as or are of in multiple PAK and to and are in an inactive via an of the kinase domain with a in the the (13Bokoch G. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (885) Google Scholar). of activated Rac or Cdc42 to PAK to autophosphorylation of sites that increases in PAK kinase A. Huang Z. Tuazon P.T. Traugh J.A. J. Biol. Chem. 1999; 274: 8022-8028Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, C. L. L. Manser E. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of these Ser141 in PAK2 to in is the and its phosphorylation to activation by the of the with the kinase activated PAK in endothelial cells, therefore an that the phosphorylated Ser141 PAK phosphorylation in endothelial cells in response to serum, bovine aortic and human umbilical vein endothelial cells and HUVEC, were for 18 and with 10% with Ser141 that serum increased PAK phosphorylation in both cell with that increased PAK kinase in endothelial cells with serum or growth factors R. W.B. Schwartz M.A. Cheresh J. Cell Biol. 2003; PubMed Scopus Google Scholar). of an for PAK phosphorylated at the with the of cells with Ser141 also an increase in PAK phosphorylation in response to serum, with the activated of the localized to cell-cell junctions with an that the of and also an increase in the at cell-cell a of that the The phosphorylated PAK by the that the is these data that PAK is on Ser141 in response to serum and that the phosphorylated to cell-cell Rac of PAK these and the of Rac of PAK were to PAK1 active PAK1 dominant PAK1 active or a dominant was in and for these was were a this is an may be cells were for 18 and with 10% serum for or of cell were with the Ser141 PAK or in the of the increased of Ser141 phosphorylation, which was increased serum The active PAK phosphorylation, which was by reduced of in the of Active Rac also increased PAK phosphorylation both with and serum, whereas PAK by the the of the These suggest that Rac is the regulator of PAK activation these of a PAK the for the dominant effects of PAK in endothelial cells to the that mediates to the domain of (23Kiosses W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus (244) Google Scholar). in which this was to the from the human to cell a endothelial cell and contractility W.B. J. S. Cheresh N. Schwartz M.A. Res. 2002; PubMed Scopus Google Scholar). The to inhibit PAK kinase but its translocation to sites of in with cells at were actin stress We therefore this also limit translocation to cell-cell and were incubated with the or the in which critical are to to of from these cells that altered PAK phosphorylation with or serum, with data W.B. J. S. Cheresh N. Schwartz M.A. Res. 2002; PubMed Scopus Google Scholar). of that the translocation to cell-cell junctions in response to was in the and The and were with The PAK therefore of to cell-cell contacts in endothelial cells. PAK of phosphorylated and activated PAK a possible role in regulation of permeability across the endothelial this and grown on with pores were with active or or active or of across the was of increased in both cell types, whereas both active PAK and active Rac increased by The strongly this increase in whereas the effects. These data a role for PAK in the regulation of permeability in endothelial cells. and Vascular permeability is regulated by a variety of soluble cytokines and factors. is such factor (4Paul R. Zhang Z. Eliceiri B. Jiang Q. Boccia A. Zhang R. Chopp M. Cheresh D. Nat. Med. 2001; 7: 222-227Crossref PubMed Scopus (305) Google Scholar, 5Weis S. Shintani S. Weber A. Kirchmair R. Wood M. Cravens A. McSharry H. Iwakura A. Yoon Y.-S. Himes N. Burstein D. Doukas J. Soll R. Losordo D. Cheresh D. J. Clin. Investig. 2004; 113: 885-894Crossref PubMed Scopus (296) Google bFGF, histamine, and also increase vascular permeability. We therefore examined the effect of the PAK on permeability induced by these The of induced a increase in of across the endothelial which was by the PAK in and The These effects with the of PAK at cell-cell contacts this cells were with a for from PAK1 the permeability. The cytokines effects on which were also by the PAK and the and These that PAK is for induction of vascular leak by and factors. of with the PAK were in that were and the increase in permeability was In possible for this that contractility is to regulate cell-cell junctions and permeability across endothelial or epithelial J. H. C. J. Cell. Physiol. 1995; PubMed Scopus Google Scholar, J. A. 2003; PubMed Scopus Google Scholar, H. R. Zhang Y. H. J. M. M. H. R. J. 1998; PubMed Scopus Google Scholar, M. de Lanerolle P. Res. 2002; PubMed Scopus Google Scholar). both or effects of PAK on myosin light chain phosphorylation have been (18Chew T. Masaracchia R. Goeckeler Z. Wysolmerski R. J. Muscle Res. 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Schwartz M.A. Res. 2002; PubMed Scopus Google Scholar). of cells with to F-actin that to induced formation of or pores between the cells at by separation of adjacent cells of to cell-cell contacts and formation of were by PAK but the formation of pores between adjacent cells was with active PAK induced pores in the of with from permeability whereas the PAK and the PAK These in monolayer associated with permeability. of the actin cytoskeleton in with that increased permeability and formation with increases in the of for actin both at cell-cell and the cell as S. M. E. J. Cell Sci. 1998; PubMed Google Scholar, S.M. Verin N. Garcia J.G. J. Cell Mol. Biol. 2002; PubMed Scopus Google Scholar, A. F. G. F. M. R. M. V. E. Mol. Biol. Cell. 2002; PubMed Scopus Google Scholar, Verin M.A. F. A. Garcia J. Physiol. 2001; PubMed Google Scholar). with the the number or of actin stress fibers, but this is with that a in contractility of actin (23Kiosses W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus (244) Google Scholar). PAK is to increase endothelial cell contractility, myosin phosphorylation in this with an that MLC phosphorylation induced by was by the PAK as as the MLC kinase PAK also MLC phosphorylation by bFGF, histamine, and for in monolayer cells with these factors also to cell-cell which was by the PAK We also as been M. de Lanerolle P. Res. 2002; PubMed Scopus Google Scholar, W.B. Daniels R.H. Otey C. Bokoch G.M. Schwartz M.A. J. Cell Biol. 1999; 147: 831-844Crossref PubMed Scopus Google Scholar), that actin stress fibers at the these were at where was These a possible for the dominant effect of the PAK in cells. in tension in a of the may be to of cell-cell junctions where tension be from both of a of the is of a in myosin phosphorylation, may an increase in cell its PAK cells by cells in the the of myosin phosphorylation, cells were with the myosin light chain kinase We a in both and permeability The by with PAK is therefore with a on permeability is in part by adherens also examined in cells The of the to cells to of this with cells with the inactive PAK The of a to the cell-cell as been S. M. E. J. Cell Sci. 1998; PubMed Google Scholar, J. Physiol. 2001; PubMed Google Scholar, J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The PAK this whereas the Thus, in permeability with effects on a and the that tension in a of cells can be to a of the on PAK2 is a the that is phosphorylated during activation of the of this to activation by of the with the kinase domain to a for the phosphorylated that the of PAK localized to of cell-cell observation prompted us to the role of PAK in the regulation of permeability. We found that expression of activated PAK1 or activation of PAK by expression of was to increase permeability across endothelial PAK function with a that active PAK from cell-cell contacts or expression of the to inhibit PAK kinase increases of vascular permeability by VEGF, bFGF, histamine, TNFα, and thrombin. The in and which endothelial cells from different and different suggest that the role of PAK in integrity is likely to be the VEGF, the effects of PAK on permeability were at in part by in myosin phosphorylation and cellular contractility. is with a number of that PAK can regulate myosin phosphorylation (18Chew T. Masaracchia R. Goeckeler Z. Wysolmerski R. J. Muscle Res. Cell Motil. 1998; 19: 839-854Crossref PubMed Scopus (168) Google Scholar, 19Goeckeler Z.M. Masaracchia R.A. Zeng Q. Chew T.-L. Gallagher P. Wysolmerski R.B. J. Biol. Chem. 2000; 275: 18366-18374Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, N. N. L. S. A. T. Biol. 2003; PubMed Scopus Google Scholar, M.A. Chernoff J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). are also with a of data myosin phosphorylation and contractility in endothelial barrier function (reviewed in S.M. Garcia J.G. J. Appl. Physiol. 2001; 91: 1487-1500Crossref PubMed Scopus Google Scholar). myosin phosphorylation and cellular contractility have also been in endothelial and vascular permeability M. M. K. Weber M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and PAK pathways or to be be in that endothelial permeability can be altered in myosin phosphorylation or (reviewed in T. Garcia J.G. J. J. Physiol. 2000; PubMed Google Scholar). Thus, the data effects of PAK on These data also the role of Rac in the formation and of junctions. Rac to formation and (8Takaishi K. Sasaki T. Kotani H. Nishioka H. Takai Y. J. Cell Biol. 1997; 139: 1047-1059Crossref PubMed Scopus (474) Google Scholar, 9Ridley A. Comoglio P. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar, 10Hordijk P.L. ten Klooster J.P. van der Kammen R.A. Michiels F. Oomen L.C. Collard J.G. Science. 1997; 278: 1464-1466Crossref PubMed Scopus (393) Google but is also for cell which the of cell-cell a role that been to data suggest that of the effector pathways downstream of Rac is likely to be critical for the to as a epithelial or endothelial is in adherens junctions downstream of Rac and Cdc42 J. M. K. M. T. N. S. Y. K. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar). have that Rac activated downstream of factors different effector pathways K. Y. N. Collard J. Bokoch G.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, conditions that of small with PAK to whereas that to of the by which downstream are activated be an important for Vascular permeability is a regulated function that can to and leakage of fluid and cells into can have and in a variety of in the of increased permeability of the vasculature to is a key in A. Vascul. Pharmacol. 2002; 39: PubMed Scopus Google Scholar). Vascular leak stroke or myocardial infarction due to the of by hypoxic increases tissue injury these (4Paul R. Zhang Z. Eliceiri B. Jiang Q. Boccia A. Zhang R. Chopp M. Cheresh D. Nat. Med. 2001; 7: 222-227Crossref PubMed Scopus (305) Google Scholar, 5Weis S. Shintani S. Weber A. Kirchmair R. Wood M. Cravens A. McSharry H. Iwakura A. Yoon Y.-S. Himes N. Burstein D. Doukas J. Soll R. Losordo D. Cheresh D. J. Clin. Investig. 2004; 113: 885-894Crossref PubMed Scopus (296) Google Scholar). Vascular leak and tissue edema to in C. P. 2000; 7: PubMed Google Scholar). The PAK as a key of vascular permeability in response to a wide variety of PAK may therefore be a target for treatment of these