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Am J Physiol Lung Cell Mol Physiol 289: L999-L1010, 2005. First published July 22, 2005; doi:10.1152/ajplung.00211.2005
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Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesion kinase and adherens junction proteins

Peter V. Usatyuk and Viswanathan Natarajan

Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois

Submitted 10 May 2005 ; accepted in final form 18 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidants, generated by activated neutrophils, have been implicated in the pathophysiology of vascular disorders and lung injury; however, mechanisms of oxidant-mediated endothelial barrier dysfunction are unclear. Here, we have investigated the role of focal adhesion kinase (FAK) in regulating hydrogen peroxide (H2O2)-mediated tyrosine phosphorylation of intercellular adhesion proteins and barrier function in endothelium. Treatment of bovine pulmonary artery endothelial cells (BPAECs) with H2O2 increased tyrosine phosphorylation of FAK, paxillin, {beta}-catenin, and vascular endothelial (VE)-cadherin and decreased transendothelial electrical resistance (TER), an index of cell-cell adhesion and/or cell-matrix adhesion. To study the role of FAK in H2O2-induced TER changes, BPAECs were transfected with vector or FAK wild-type or FAK-related non-kinase (FRNK) plasmids. Overexpression of FRNK reduced FAK expression and attenuated H2O2-mediated tyrosine phosphorylation of FAK, paxillin, {beta}-catenin, and VE-cadherin and cell-cell adhesion. Additionally, FRNK prevented H2O2-induced distribution of FAK, paxillin, {beta}-catenin, or VE-cadherin toward focal adhesions and cell-cell adhesions but not actin stress fiber formation. These results suggest that activation of FAK by H2O2 is an important event in oxidant-mediated VE barrier function regulated by cell-cell and cell-matrix contacts.

focal adhesion kinase-related non-kinase; vascular endothelial-cadherin; {beta}-catenin; hydrogen peroxide; transendothelial electrical resistance

ENDOTHELIAL CELLS (ECs), lining the blood vessels, serve as a semipermeable barrier to circulating cells, plasma albumin, macromolecules, and bioactive agents. Maintenance of EC barrier integrity is critical for vessel wall homeostasis and normal organ function. Among various circulating edemic agents, reactive oxygen species (ROS), generated at sites of inflammation and injury by activated polymorphonuclear leukocytes or ECs, play an important role in the disruption of barrier function (27, 31, 47). There is considerable evidence for the role of cytoskeletal, focal adhesion, and adherens junction proteins in regulating EC barrier function (2, 3, 11, 39). In previous studies, we and others have demonstrated that exposure of ECs to ROS or edemic agents induces actin stress fibers, enhances protein tyrosine phosphorylation of Src, focal adhesion, and adherens junction proteins, and alters EC permeability (6, 8, 9, 13, 19, 23–25, 29, 33, 52). Whereas the role of intracellular Ca2+, Src family kinases, myosin light chain kinase, and protein kinase C in mediating EC barrier dysfunction has been described (14, 15, 22, 25, 40, 49, 55), the involvement of focal adhesions and adherens junctions in barrier regulation is inadequately understood.

Focal adhesions, sites of close contact between cell-cell and cell-extracellular matrix, are essential for normal cell growth, differentiation, inter- and intracellular communication, and tissue integrity (1, 7, 18, 35, 37, 41, 43). Among the protein complexes associated with focal adhesions, focal adhesion kinase (FAK) and paxillin play an important role in the transmission of integrin-induced cytoplasmic signals and in the reorganization of actin cytoskeleton (42, 45, 46, 48). FAK, an ~125-kDa tyrosine kinase, is activated primarily through integrin-mediated cell adhesion to extracellular matrix and to a lesser extent by growth factors, bioactive lipids, neuropeptides, and ROS (1, 3, 7, 9, 35). Autophosphorylation of FAK at Y397 induced by its localization to focal adhesions is central to agonist-dependent phosphorylation on other tyrosine residues of FAK and FAK-dependent cellular responses (7, 37). FAK comprises a central catalytic tyrosine kinase domain flanked by NH2- and COOH-terminal regions (10, 12, 21, 43). A specific sequence encompassing 904–1,012 residues within the COOH-terminal domain of FAK, termed as focal adhesion targeting sequence, localizes FAK to focal adhesions (3, 21, 35, 38). In several tissues, including lung and intestine, the COOH-terminal noncatalytic domain of FAK is separately expressed as FAK-related non-kinase (FRNK) (26, 30, 37, 60). Overexpression of FRNK decreased the rate of cell spreading and cell migration (28) and induced loss of cell adhesion and cell death. These effects of FRNK reflect its ability to compete with FAK for common binding partners and function as a dominant negative inhibitor of FAK (1, 35, 43). In addition to FRNK, expression of a truncated form of deleted FAK blunted the hyperosmolar-induced barrier strengthening and increased activity of FAK and E-cadherin accumulation at EC periphery (36). Similarly, use of FAK antisense oligonucleotides to inhibit FAK expression augmented and prolonged thrombin-induced increase in EC permeability, indicating a negative feedback role of FAK in barrier regulation (29). These data suggest a role of FAK in endothelial permeability; however, specific mechanisms of FAK-mediated regulation of EC barrier function remain undefined.

In the present study, we examined the role of FAK in H2O2-induced transendothelial electrical resistance (TER) changes in bovine pulmonary artery endothelial cells (BPAECs) by overexpressing FRNK, the dominant negative modulator of FAK. Here we report that: 1) overexpression of FRNK decreased the level of FAK protein expression; 2) FRNK attenuated H2O2-induced changes in cell-cell adhesion contacts (Rb)-TER, an index of cell-cell adhesion; 3) FRNK prevented H2O2-dependent phosphorylation of FAK, {beta}-catenin, paxillin, and VE-cadherin; and 4) FRNK blocked H2O2-mediated redistribution of focal adhesions and adherens junction proteins. This study provides direct evidence for the involvement of FAK in the regulation of H2O2-induced phosphorylation of adherens junction proteins and barrier function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Most laboratory reagents, including tissue culture media, were purchased from Sigma-Aldrich. Nonessential amino acids and PBS were obtained from Biofluids. Gold electrodes 8W10E were purchased from Applied Biophysics, and glass coverslips were from Fisher Scientific. Immunobilon-P, 0.45 mm, was from Millipore.

Antibodies. Antibodies for FAK (cat. no. 610087), paxillin (cat. no. 610619), and {beta}-catenin (cat. no. 610153) (all monoclonal) were from BD Transduction Laboratories. VE-cadherin antibody (polyclonal, cat. no. 160840) was from Cayman Chemical. Agarose conjugated phospho-tyrosine (monoclonal, cat. no. sc-5267 AC), protein A/G plus-agarose (cat. no. sc-2003) immunoprecipitation reagents, and bovine serum albumin (cat. no. sc-2323) were from Santa Cruz Biotechnology. Hemagglutinin (HA) antibody (monoclonal, cat. no. 1583816) was obtained from Roche. Anti-phosphotyrosine (monoclonal, cat. no. 05-321) and anti-phospho FAK (Tyr397; polyclonal, cat. no. 07-012) antibodies were from Upstate Biotechnology. Secondary anti-rabbit (cat. no. 170-6515) or anti-mouse IgG (cat. no. 170-6516) (H+L) horseradish peroxidase conjugates were obtained from Bio-Rad. Fluorescence antibodies were purchased from Molecular Probes.

Cell culture. BPAECs (American Type Culture Collection) cultured in MEM were maintained at 37°C in a humidified atmosphere of 5% CO2-95% air and grown to contact-inhibited monolayers with typical cobblestone morphology (49). Cells were cultured on 100-mm dishes for immunoprecipitation or Western blotting (50), on gold electrodes for electrical resistance determinations (16, 17), or on glass coverslips for immunocytochemistry (49).

Plasmids and transfection. Focal adhesive kinase cDNA plasmids (pKH3 vector, pKH3 FAK wild type, dominant negative FAK, and pKH3 FRNK) were kindly provided by Dr. J. L. Guan (Cornell Univ., Ithaca, NY). BPAECs, grown to ~50% confluence on 100-mm culture dishes, on gold electrodes, or on glass coverslips were transfected with 0.2–1.0 µg/ml of FAK cDNA using FuGENE-6 (cat. no. 11815091001, Roche) reagent according to the manufacturer's protocol. Protein expression was determined with Western blotting and immunocytochemistry of FAK or FRNK with the triple HA epitope tag.

Measurement of TER. TER was measured in an electrical cell-substrate impedance sensing system (Applied Biophysics). The total electrical resistance measured dynamically across the endothelial monolayer was determined as the combined resistance between the basal surface of the cell and the electrode (17), reflecting alterations in cell-cell adhesion and/or cell-matrix adhesion. To estimate differences between cell-cell and cell-matrix components, total TER was resolved into values reflecting resistance to current flow beneath the cell layer ({alpha}, cell-matrix adhesion contacts) and resistance to current flow between adjacent cells (Rb), utilizing the method of Giaever and Keese (16), which models the endothelial monolayer mathematically.

Preparation of cell lysates, Western blotting, and immunoprecipitation. ECs were lysed in modified radioimmunoprecipitation assay buffer (cat. no. 9803, Cell Signaling), and protein concentrations were determined with a Pierce protein assay kit. Proteins (10–30 µg) were separated on 6–10% gels by SDS-Page, transferred to polyvinylidene difluoride membranes, and subjected to immunoblotting. Cell lysates (0.5–1 mg protein per ml) were subjected to immunoprecipitation with anti-phosphotyrosine antibody or VE-cadherin antibody at 4°C for 18 h. Protein A/G (20 µl) was added and incubated for an additional 2 h at 4°C. The antibody complex was pelleted and washed three times in ice-cold PBS plus 1 mM vanadate, pellets were dissociated by boiling in SDS sample buffer for 5 min, and samples were analyzed by Western blots on 6 or 10% SDS-PAGE.

Immunofluorescence microscopy. After treatment, as indicated in the figures, ECs were rinsed twice with PBS and treated with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were then rinsed three times with PBS and permeabilized for 2 min with 0.25% Triton X-100 prepared in Tris-buffered saline containing 0.01% Tween 20 (TBST). After being washed with TBST (3 x 5 min), cells were incubated for 1 h at room temperature in blocking buffer containing 1% BSA. Focal adhesion protein localization was stained with primary antibodies for FAK, {beta}-catenin, and paxillin (1:200), and with VE-cadherin (1:100) and Alexa Fluor 488 (monoclonal for FAK, {beta}-catenin, and paxillin, cat. no. A-11029 or polyclonal for VE-cadherin, cat. no. A-11055) as secondary antibodies. FAK wild type and FRNK transfection efficiency was estimated by staining ECs with anti-HA probe as a primary antibody (1:500) and Alexa Fluor 488 (monoclonal, cat. no. A-11029) as a secondary antibody. The amount of transfected cells was estimated as a % of cells that got transfected (green color) to total cells in the field based on nucleus staining with 4',6-diamidino-2-phenylindole (DAPI). Actin stress fibers were determined by staining of cells with Alexa Fluor phalloidin 568 (cat. no. A-12380). Slides were prepared with mounting medium containing DAPI (cat. no. H-1200, Vector Laboratories) to stain the nucleus and examined by a Nikon Eclipse TE 2000-S immunofluorescence microscope with a Hamamatsu digital camera (Japan) using a Nikon Plan Apo 60XA/1.40 oil-immersion objective. Pictures were prepared using MetaVue software (Universal Imaging).

Statistics. ANOVA with Student-Newman-Keuls test was used to compare clearance rates of two or more different treatment groups. The level of significance was taken to be P < 0.05 unless otherwise stated. Data are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H2O2 modulates EC permeability and cell-cell adhesion. Exposure of isolated lungs to H2O2 results in pulmonary edema and vascular leak. Prior studies have described the ability of ROS such as H2O2, diperoxovanadate, and 4-hydroxynonenal to stimulate mitogen-activated protein kinases and Src family of nonreceptor tyrosine kinase and to induce actin remodeling in ECs (15, 23, 24, 49, 50, 52). However, the role of FAK and adherens junction proteins leading to barrier regulation in ECs remains unclear. To assess the effect of H2O2 on EC barrier function, BPAECs were challenged with varying concentrations of H2O2 (1–250 µM), and changes in EC permeability were measured as TER across EC monolayers. As shown in Fig. 1A, H2O2 in a time-dependent manner decreased TER. The decrease in TER mediated by H2O2 was dose dependent (control, 1,100 + 45 {Omega}; 50 µM H2O2, 960 + 38 {Omega}; 100 µM H2O2, 675 + 74 {Omega}; 250 µM H2O2, 605 + 28 {Omega}). To resolve the relative contributions of intercellular junctions and focal adhesions in H2O2-induced barrier function, we resolved TER to Rb and {alpha} (Fig. 1B) using the mathematical model of Giaever and Keese (16). When ECs were challenged with H2O2, the major alteration in TER was due to changes in Rb through increased gap formation mediated by intercellular adhesion proteins (Fig. 1C). In contrast to Rb, very little change to {alpha} that is related to the distance between the endothelial plasma membrane and the surface of the electrode was observed, suggesting very negligible contribution of the state of cell-matrix adhesion in changes in TER. Interestingly, treatment of cells with 50 µM H2O2 decreased Rb followed by recovery of TER to near basal level after 2–3 h. These results indicate that exposure of ECs to H2O2 modulates barrier function through the changes in cell-cell adhesion interactions.



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  		Fig. 1. H2O2 induces endothelial barrier function. A: bovine pulmonary artery endothelial cells (BPAECs) were challenged with different concentrations of H2O2 (1–250 µM), and transendothelial electrical resistance (TER) was measured as described in MATERIALS AND METHODS. B: cell-cell and cell-matrix components were resolved into TER values, cell-cell adhesion contacts (Rb) and cell-matrix adhesion contacts ({alpha}) (C) as described in MATERIALS AND METHODS (n = 3). Veh, vehicle; EC, endothelial cell; Norm. El, normalized electrical.

 
Activation of FAK by H2O2. As modulation of EC barrier function involves modulation of tyrosine phosphorylation of focal adhesion and adherens junction proteins (8, 9, 23–25, 51, 52) and changes in cell-cell adhesion, we investigated the effect of ROS on FAK. Exposure of BPAECs to H2O2 (100 µM) increased tyrosine phosphorylation of total proteins as well as FAK with maximal phosphorylation (~7-fold over control) observed at 30 min followed by a slight decline (~5.5-fold) at 60 min of exposure (Fig. 2, A and B). In our next experiments, we choose to use 100 µM H2O2 and 30-min treatment, which was the most effective concentration to affect cell-cell adhesion and FAK activation.



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  		Fig. 2. Time dependency of H2O2-mediated protein tyrosine phosphorylation. BPAECs were challenged with H2O2 (100 µM) for different time periods, and cell lysates were subjected to 10% SDS-PAGE and Western blotted with anti-phospho-tyrosine (A), anti-focal adhesion kinase (anti-FAK), or anti-phospho-FAK (B) antibodies. *Significantly different from control (P < 0.05; n = 4). p-Tyr, phosphorylated tyrosine; IB, immunoblotting.

 
Expression of HA-tagged FAK wild type, dominant negative FAK, and FRNK in ECs. Next we investigated the role of FAK on ROS-induced EC cell-cell adhesion by overexpressing FAK DNA plasmids (59). BPAECs were transfected with HA-tagged plasmids for 48 h, and cell lysates were subjected to SDS-PAGE and probed with anti-FAK or anti-HA antibodies. As shown in Fig. 3, A and B, transfection with FAK wild type and FRNK resulted in a significant expression of the proteins. On the basis of anti-HA and DAPI staining, transfection efficiency was ~16.0 ± 3.5% for FAK wild type and ~18.0 ± 4.6% for FRNK. Immunolocalization studies revealed that the overexpressed FAK and FRNK were localized both in the cytoplasm and plasma membrane (Fig. 3C). Furthermore, transfection of ECs with FRNK (0.2 µg/µl) for 24, 48, and 72 h resulted in a significant reduction of total FAK protein. On the basis of these results, all the experiments related to FRNK overexpression were for 24 or 48 h with 0.2 µg/ml of the plasmid cDNA concentration.



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  		Fig. 3. Expression of FAK and FAK-related non-kinase (FRNK) in ECs. BPAECs grown on 100-mm dishes or glass coverslips were transiently transfected with FAK cDNA as described in MATERIALS AND METHODS. Cell lysates (20 µg of protein) were subjected to 10% SDS-PAGE and probed with anti-FAK (A) or anti-hemagglutinin (HA) (B) antibodies (n = 4–5). C: cells grown and transfected on glass coverslips as indicated were stained with anti-HA primary antibody and Alexa Fluor 488 as a secondary antibody and examined by immunofluorescence microscopy using a x60 oil objective. Two populations of cells are depicted, one transfected (green) and one not transfected (no color), colocalized with nucleus stained with 4',6-diamidino-2-phenylindole (DAPI). D: time course of total FAK expression in control (a), vector (V)-(b), FAK wild type (Wt)-(c), and FRNK (d)-transfected ECs determined by Western blotting. *Significantly different from control (P < 0.05).

 
FAK regulates H2O2-mediated TER. To assess the role of FAK in H2O2-mediated changes in TER, we next examined changes in electrical resistance in ECs transiently overexpressing either FAK wild type, FAK dominant negative, or FRNK and compared the results to vector control. As shown in Fig. 4A, overexpression of FRNK attenuated H2O2-mediated TER compared with vector-transfected cells or cells overexpressing FAK wild type. The basal electrical resistance of ECs was not impaired by transfection with FRNK compared with vector control or FAK wild type (vector, 1,140 ± 58; wild-type FAK, 1,259 ± 75; FRNK, 986 ± 83). Similar to FRNK, overexpression of dominant negative FAK also partially prevented H2O2-mediated decrease in TER (Fig. 4B). These results provide strong evidence that overexpression of FRNK or dominant negative FAK blocks ROS-induced EC TER. Whereas vector control and FAK wild type had no significant effect on H2O2-induced changes in Rb or {alpha}, overexpression of FRNK resulted in blocking H2O2-dependent changes in Rb (Fig. 4C). However, in FRNK-transfected cells, prolonged treatment with H2O2 (3–4 h) resulted in a decrease in Rb and {alpha} (Fig. 4C). These results provide strong evidence for FAK-dependent changes in TER related to cell-cell adhesion.




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  		Fig. 4. Effect of FAK expression on H2O2-mediated TER. BPAECs grown on gold electrodes were transfected for 48 h with expression vectors encoding vector, FAK Wt, or FRNK (A) or vector and FAK Y397 (Dn) (B) and were then challenged with H2O2 (100 µM). TER was measured as described in MATERIALS AND METHODS. Shown is a representative tracing from 6 independent experiments in duplicate. Total changes in TER after H2O2 treatment were calculated from A and B, respectively. *Significantly different from control (P < 0.05). **Significantly different from H2O2 treatment in vector. (P < 0.01). C: cell-cell (Rb) and cell-matrix ({alpha}) components were resolved into TER values from A as described in MATERIALS AND METHODS.

 
FRNK overexpression attenuates H2O2-mediated phosphorylation of focal adhesion and adherens junction proteins. Having established a role for FAK in EC TER, we next investigated the effect of FRNK on H2O2-mediated phosphorylation of focal adhesion and adherens junction proteins. Analysis of the immunoprecipitates by Western blotting with anti-FAK, anti-paxillin, anti-{beta}-catenin, or anti-phosphotyrosine antibodies revealed that FRNK overexpression attenuated the H2O2-mediated (100 µM, 30 min) phosphorylation of focal adhesion and adherens junction proteins (Fig. 5). These results suggest that activation of FAK by H2O2 regulates tyrosine phosphorylation of focal adhesion and adherens junction proteins in ECs.



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  		Fig. 5. FRNK attenuates H2O2-mediated phosphorylation of focal adhesion proteins. BPAECs grown on 100-mm dishes were transiently transfected for 48 h with expression vectors encoding vector (control), FAK Wt, or FRNK as described in MATERIALS AND METHODS and challenged with H2O2 (100 µM) for 30 min. Immunoprecipitates (IP) were subjected to 10% SDS-PAGE and Western blot with anti-FAK (A), anti-{beta}-catenin (B), anti-paxillin (C), or anti-phospho-Tyr and anti-vascular endothelial (VE)-cadherin (D) antibodies. Changes in protein phosphorylation were quantified by image analysis of the Western blots. Values are means ± SE (n = 3–4). *Significantly different from control (P < 0.05).

 
FRNK attenuates H2O2-mediated redistribution of focal adhesion and adherens junction proteins. As FRNK blocked H2O2-induced tyrosine phosphorylation of FAK, paxillin, {beta}-catenin, and VE-cadherin, we investigated redistribution of focal adhesion and adherens junction proteins in control, FAK wild type, and FRNK-overexpressing cells by immunofluorescence microscopy. Cells grown on glass coverslips were treated with H2O2 (100 µM) for 30 min and were then triple stained to visualize FAK, paxillin, {beta}-catenin, or VE-cadherin (green color) with anti-HA antibody to visualize cells that were transfected (red color) and with DAPI to visualize the nucleus (blue color). At least two pairs of cells were selected in each field, one that was transfected and the other that was not transfected, as visualized with anti-HA antibody. As shown in Fig. 6, A and B, control cells that were exposed to medium alone showed a diffused localization pattern of FAK and paxillin; however, exposure of ECs to H2O2 promoted reorganization of FAK and paxillin toward cell periphery and areas of focal contacts. Overexpression of FRNK mitigated the H2O2-mediated redistribution of FAK and paxillin. Similarly, H2O2 increased redistribution of {beta}-catenin and VE-cadherin (Fig. 6, C and D) at cell periphery that was a punctuated characteristic of VE-cadherin aggregation and membrane ruffle formation (Fig. 6E). These results provide strong evidence for FAK-dependent redistribution of focal adhesion and adherens junction proteins by H2O2 in ECs.




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  		Fig. 6. FRNK attenuates H2O2-mediated reorganization of focal adhesion and adherens junction proteins toward cell periphery. In the images, at least 1 pair of cells has been presented in each field where 1 cell got transfected, confirmed by staining with anti-HA-probe (red color), or no transfection was seen in neighbor. Vector control shows no transfection according to absence of tagged protein. Stimulation of ECs with H2O2 results in FAK (A), paxillin (B), {beta}-catenin (C), and VE-cadherin (D) redistribution to the focal adhesion periphery in vector, Wt, both transfected and nontransfected cells, and in nontransfected FRNK cells, whereas no H2O2-mediated reorganization of focal adhesion and adherens junction proteins was found in FRNK-transfected cells. E: treatment of ECs with H2O2 promotes reorganization of VE-cadherin toward the plasma membrane and cell periphery in a punctuated pattern with characteristics of aggregation and ruffles formation. Shown is a representative image from 4 independent experiments. Bars, 20 µm.

 
Effect of FRNK on H2O2-mediated actin stress fibers. The actin microfilaments play a critical role in EC barrier regulation through their interaction with myosin, focal adhesion proteins, and adherens junction proteins (11, 60). As FRNK attenuates redistribution of FAK, paxillin, {beta}-catenin, and VE-cadherin mediated by H2O2, we examined the effect of FRNK on actin stress fiber formation, myosin light chain phosphorylation, and vinculin organization. As shown in Fig. 7, H2O2-induced reorganization of actin stress fibers in vector- and FAK wild-type-transfected cells and overexpression of FRNK had no effect on H2O2-mediated actin stress fiber formation (Fig. 7). To further confirm that H2O2-induced actin stress fiber is not involved in changes in TER, we tested the effect of latrinculin (29), an agent that blocks actin polymerization, on barrier function. Pretreatment of BPAECs with latrinculin (50 nm) had no effect on changes in TER. Additionally, overexpression of FRNK had no effect on H2O2-mediated myosin light chain phosphorylation or vinculin redistribution (data not shown). These results further confirm that reorganization of focal adhesion and adherens junction proteins, but not actin cytoskeleton, is an important event in oxidant-mediated TER changes.



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  		Fig. 7. Effect of FAK expression on H2O2-mediated actin stress fibers in ECs. BPAECs were grown, transfected, and treated as described in Fig. 6. After treatment, cells were fixed, probed by anti-HA primary antibodies, and stained with Alexa Fluor 488 secondary antibody. Actin stress fibers were stained with Alexa Fluor phalloidin 568, and the nucleus was stained with DAPI. Slides were examined by immunofluorescent microscopy using a x60 oil objective. Shown is a representative image from 3 independent experiments. Bars, 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The onset and progression of acute lung injury in acute respiratory distress syndrome and chronic obstructive pulmonary disease have been linked to neutrophil diapedesis, release of proteases/inflammatory mediators, and ROS reactive nitrogen intermediate (RNI) in pulmonary microcirculation (27, 31). There is a large body of data that implicates a role for ROS/RNI in modification of cellular DNA, proteins, and lipids causing cell injury (47). However, recent studies suggest that ROS/RNI, generated under normal and pathological conditions, modulate signaling pathways involved in the reorganization of focal adhesions, cytoskeleton, and adherens junction proteins (3, 47). The focal adhesion proteins, FAK and paxillin, are important components of signal transduction downstream of integrins that regulate critical cellular events such as cell motility, proliferation, survival, and barrier integrity (1, 7, 35, 37). Whereas integrins localized in the focal adhesion complex are essential in mediating cell-matrix interactions, tight adherens junctions are key determinants in the maintenance of cell-cell contacts (3, 58). However, the role of FAK and adherens junction proteins in regulating EC barrier function remains unclear. The present study focuses on redistribution of adherens junctions proteins and increased focal adhesion in regulating H2O2-mediated barrier function in ECs. Our findings are summarized as: 1) overexpression of FRNK and dominant negative FAK prevented H2O2-induced decrease in Rb TER, an index of cell-cell adhesion; 2) overexpression of FRNK reduced the level of expression of FAK as well as H2O2-dependent tyrosine phosphorylation of FAK, paxillin, {beta}-catenin, and VE-cadherin; and 3) overexpression of FRNK mitigated H2O2-mediated redistribution of FAK, paxillin, {beta}-catenin, and VE-cadherin. We conclude that H2O2-induced phosphorylation of FAK and adherens junction proteins leads to disruption of intercellular tight junctions and redistribution of focal adhesions resulting in a decrease in TER or an increase in permeability.

Endothelial barrier is regulated by competing and tethering forces (11). The changes in barrier function due to H2O2 challenge were preceded by enhanced protein tyrosine phosphorylation of focal adhesion proteins (FAK, paxillin), adherens junction proteins ({beta}-catenin and VE-cadherin), and enhanced focal adhesion formation. These responses indicate that focal adhesions are formed following H2O2-induced cell retraction, probably as a result of decreased cell-matrix interaction. To resolve the relative contributions of intercellular junctions and focal adhesions in H2O2-induced barrier function, using the model by Giaever and Keese (16), we have mathematically resolved H2O2-induced TER to Rb (cell-cell adhesion) and {alpha} (cell-matrix). When the endothelium was challenged with H2O2, the major alteration in TER was due to changes in Rb through increased gap formation mediated by intercellular adhesion proteins. In contrast to Rb, very little change to {alpha} that is related to the distance between the endothelial plasma membrane and the surface of the electrode was observed, suggesting no large contribution of the state of cell-matrix adhesion in changes in TER due to H2O2 treatment.

The role of FAK in H2O2-induced barrier dysfunction was determined using FRNK that is autonomously expressed in mammalian cells (1, 26, 37, 42). FRNK functions as a negative regulation of FAK signaling (37, 38) and thus provides a tool to dissect the role of FAK in cell function such as migration, proliferation, and barrier function. A number of studies have demonstrated that overexpression of FRNK inhibited tyrosine phosphorylation of FAK and paxillin and blocked cell spreading and migration via disruption of focal adhesion assembly (5, 26, 28, 30). In the present study, overexpression of FRNK in BPAECs attenuated H2O2-induced tyrosine phosphorylation of FAK and paxillin, reduced redistribution of FAK in focal adhesions, and prevented changes in permeability, indicating that FAK activation was critical in intercellular adhesion and barrier disruption. In FRNK-overexpressing ECs, the H2O2-induced drop in the mathematical parameter Rb that reflects modulation of intercellular adhesion and gap formation was almost completely abolished, suggesting cell-cell gap formation as a major contributor of TER changes (Fig. 4C). In contrast to Rb, challenge of cells with H2O2 had no influence on the parameter {alpha} that reflects cell-matrix interaction; however, overexpression of FRNK decreased {alpha} in response to H2O2 treatment. Interestingly, treatment of ECs with a lower dose of H2O2 (50 µM) resulted in Rb decrease followed by recovery to near basal level (Fig. 1C), suggesting possible involvement of endothelial antioxidant defense mechanism. However, in FRNK-transfected cells, a higher dose of H2O2 (100 µM) and prolonged treatment, up to 3–4 h, resulted in a decrease of Rb (Fig. 4C), suggesting possible involvement of ROS-mediated FAK activation. The change in {alpha} is consistent with a decrease in the number of focal contacts and phosphorylation of FAK and paxillin seen in cells expressing FRNK. As decreases in TER due to H2O2 challenge were mainly regulated by intercellular adhesion, the protection offered by FRNK overexpression suggests that FAK-dependent modulation of intercellular adhesion but not cell-matrix interactions play a key role in barrier dysfunction. Our current data on the effect of FRNK on H2O2-induced changes in TER are consistent with a recent study on attenuation of neutrophil-mediated increase in venular permeability and tyrosine phosphorylation of FAK by FRNK (19). Furthermore, as observed in the present study, overexpression of FRNK had no effect on basal venular permeability (19). Interestingly, exposure of cells to either H2O2 or neutrophils (19) resulted in membrane ruffling and punctuated localization of FAK and VE-cadherin in membrane ruffles as evidenced by immunocytochemistry.

Recent studies indicate at least five possible mechanisms underlying the role of FAK in EC barrier regulation: 1) agonist-dependent activation of FAK leads to disassembly and redistribution of focal adhesions, leading to EC rounding and gap formation; 2) activation of FAK tightens the intercellular gaps, resulting in assembly rather than disassembly; 3) activation of FAK regulates myosin light chain phosphorylation, development of contractile force, and gap formation; 4) inactivation of FAK by FRNK or FAK dominant negative reduces tyrosine phosphorylation of adherens junction proteins and tightening of the intercellular junctions; and 5) activation of FAK modulates intracellular signaling molecules such as Src family of nonreceptor kinases, paxillin, and Rho that alter barrier function. In this study, we have demonstrated that H2O2 not only upregulates tyrosine phosphorylation of FAK and paxillin but also the adherens junction proteins, namely {beta}-catenin and VE-cadherin, in ECs (Fig. 6). Furthermore, overexpression of FRNK attenuated tyrosine phosphorylation of FAK, paxillin, {beta}-catenin, and VE-cadherin as determined in coimmunoprecipitation studies with anti-phosphotyrosine (Fig. 5). The paracellular pathway is essentially regulated by strengthening or weakening of intercellular junctions, and tyrosine phosphorylation of adherens junction proteins is associated with disruption of the intercellular adhesive properties of these proteins and enhanced cell motility and invasiveness (3). Also, in subconfluent and sparsely confluent cells, both {beta}-catenin and VE-cadherin are highly tyrosine phosphorylated compared with confluent cells where the tyrosine phosphorylation status is much lower (4). Our results clearly show that H2O2-induced tyrosine phosphorylation of both {beta}-catenin and VE-cadherin is decreased in cells overexpressing FRNK but not in vector control or FAK wild-type transfected cells. These data suggest that FRNK-dependent attenuation of tyrosine phosphorylation of adherens junction proteins could be related to the decrease in TER and barrier function induced by H2O2. In a related study with human umbilical vein ECs, the VEGF-elicited hyperpermeability was attenuated by FRNK, suggesting a role for FAK phosphorylation in vascular barrier response to VEGF (57). The present study does not address the direct role of FAK in altering EC barrier function via Src and Rho family of GTPases (14, 22, 32, 34, 38). Earlier studies have shown that oxidant-mediated permeability changes are regulated by protein kinase C (23, 40, 60), mitogen-activated protein kinases (24, 33, 50), Src family of nonreceptor kinases (14, 49), calcium/calmodulin-dependent protein kinase (33), and nitrite oxide synthase (48). Although FAK is a well-defined substrate of Src, there is evidence that FAK can also phosphorylate Src and activate Rho, which in turn induces cell contraction and adherens junction disruption (22, 41, 55, 56). Additionally, activated FAK functions as a scaffolding protein and recruits a variety of kinases and focal adhesion proteins (53) that cause changes in distribution of cytoskeleton and/or intercellular adhesion proteins, resulting in altered barrier function (15). Also, H2O2 inhibits protein tyrosine phosphatases, and, in particular, those associated with adherens junction and influences their phosphorylation status (9, 25, 44). In addition to paracellular pathway, there are reports on the transcellular pathway for transfer of plasma components across endothelial barrier (54); however, this study has not addressed the relative contribution of the transcellular pathway in H2O2-induced permeability changes in human lung ECs. Further studies are necessary to characterize the role of transcellular pathway and a balance between protein tyrosine kinases and phosphatases in modulating agonist-induced phosphorylation of FAK and adherens junction proteins in regulating EC barrier function.

In summary, we have demonstrated that H2O2-induced activation of FAK regulates TER changes in the vascular endothelium. Furthermore, inhibition of FAK with FRNK attenuated H2O2-dependent tyrosine phosphorylation of FAK, paxillin, {beta}-catenin, VE-cadherin, and TER, suggesting a role for FAK and adherens junction proteins in regulating EC barrier function (Fig. 8). Future studies will focus on signaling pathways that modulate agonist-induced cross talk and interaction between focal adhesion and adherens junction proteins and protein platforms that recruit protein kinases, phosphatases, and adaptor proteins to better understand the pathophysiology of vascular leak and lung injury.



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  		Fig. 8. Schematic depicts modulation of cytoskeleton, focal adhesion, and adherens junction proteins by reactive oxygen species (ROS) resulting in changes in EC barrier function.

 

   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institutes Grants RO1-HL-69909 and P01-HL-58064 (to V. Natarajan).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Dr. J. L. Guan, Cornell University, Ithaca, NY, for providing HA-tagged FAK cDNA plasmids in mammalian expression vectors and Dr. C. R. Keese, Applied Biophysics, New York, NY, for helpful discussions. The authors gratefully acknowledge Dr. V. P. Fomin, Indiana University School of Medicine, Indianapolis, IN, for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Natarajan, Dept. of Medicine, Section of Pulmonary and Critical Care Medicine, Univ. of Chicago, C/S Bldg., Rm. 408, 929 E. 57th St., Chicago, IL 60637 (e-mail: vnataraj{at}medicine.bsd.uchicago.edu)

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


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