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CALL FOR PAPERS Translational Research in Acute Lung Injury and Pulmonary

Fibrosis

c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-inducedendothelial dysfunction and lung injury

Panfeng Fu,1 Peter V. Usatyuk,1 Abhishek Lele,1 Anantha Harijith,2 Carol C. Gregorio,4

Joe G. N. Garcia,5 Ravi Salgia,6 and Viswanathan Natarajan1,3

1Department of Pharmacology, University of Illinois, Chicago, Illinois; 2Department of Pediatrics, University of Illinois,Chicago, Illinois; 3Department of Medicine, College of Medicine, University of Illinois, Chicago, Illinois; 4Department ofCellular and Molecular Medicine, The University of Arizona College of Medicine, Tucson, Arizona; 5Department of Medicine,The University of Arizona College of Medicine, Tucson, Arizona; and 6Department of Medicine, University of Chicago,Chicago, Illinois

Submitted 22 October 2014; accepted in final form 19 March 2015

Fu P, Usatyuk PV, Lele A, Harijith A, Gregorio CC, GarciaJG, Salgia R, Natarajan V. c-Abl mediated tyrosine phosphorylationof paxillin regulates LPS-induced endothelial dysfunction and lunginjury. Am J Physiol Lung Cell Mol Physiol 308: L1025–L1038, 2015.First published March 20, 2015; doi:10.1152/ajplung.00306.2014.—Paxillin is phosphorylated at multiple residues; however, the role oftyrosine phosphorylation of paxillin in endothelial barrier dysfunctionand acute lung injury (ALI) remains unclear. We used siRNA andsite-specific nonphosphorylable mutants of paxillin to abrogate thefunction of paxillin to determine its role in lung endothelial permea-bility and ALI. In vitro, lipopolysaccharide (LPS) challenge of humanlung microvascular endothelial cells (HLMVECs) resulted in en-hanced tyrosine phosphorylation of paxillin at Y31 and Y118 with nosignificant change in Y181 and significant barrier dysfunction. Knock-down of paxillin with siRNA attenuated LPS-induced endothelialbarrier dysfunction and destabilization of VE-cadherin. LPS-inducedpaxillin phosphorylation at Y31 and Y118 was mediated by c-Abltyrosine kinase, but not by Src and focal adhesion kinase. c-AblsiRNA significantly reduced LPS-induced endothelial barrier dys-function. Transfection of HLMVECs with paxillin Y31F, Y118F, andY31/118F double mutants mitigated LPS-induced barrier dysfunctionand VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957attenuated LPS-induced pulmonary permeability in mice. Together,these results suggest that c-Abl mediated tyrosine phosphorylation ofpaxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascularpermeability and injury.

paxillin; c-Abl; lipopolysaccharide; tyrosine kinase; permeability;endothelial dysfunction; cytoskeleton

GRAM-NEGATIVE BACTERIA-INDUCED sepsis affects more than750,000 patients annually in the United States (23). Acuterespiratory distress syndrome (ARDS), the most severe form ofacute lung injury (ALI), is characterized by endothelial hyper-permeability leading to lung inflammation and injury. Lipo-polysaccharide (LPS), the bacterial endotoxin and an outermembrane component, induces proinflammatory cytokines, ad-hesion molecules, endothelial permeability changes, and apo-ptosis regulated by TLR-4 signaling via activation of Rho

GTPase-dependent signaling cascade, NF-�B activation, andrearrangement of cytoskeletal proteins (15, 31). Despite recentadvances in understanding the pathogenesis of ALI/ARDS,effective pharmacological therapies are not currently availableand the molecular mechanisms regulating endothelial barrierdysfunction in ALI/ARDS are not completely defined.

Interactions between focal adhesions, extracellular matrix,and cytoskeletal proteins have been implicated in endothelialcell (EC) remodeling and barrier dysfunction associated withLPS- and ventilator-induced lung injury (15, 31, 48). Specificposttranslational modifications such as phosphorylation of fo-cal adhesion proteins are also known to play a role in endo-thelial barrier regulation to agonists and endotoxin (8, 31).Paxillin, a focal adhesion adaptor protein of �68 kDa, servesas an important scaffolding protein at focal adhesions byrecruiting and binding to structural and signaling molecules (6,40). Paxillin is tyrosine phosphorylated by focal adhesionkinase (FAK) and Src family kinases (5) at Y31 and Y118,which are important for paxillin redistribution and interactionwith other proteins and disassembly of focal adhesion at theleading edge of migrating cells (47). Tyrosine phosphorylationof paxillin at Y31 and Y118 also activates Rho family ofGTPases leading to the formation of filopodia, lamellipodia,and stress fibers, which are actin-based structures primarilylocalized at the leading edge of migrating cells (3, 33, 44).Paxillin has been shown to differentially regulate endothelialbarrier function by growth factors via modulation of Rac-Rhosignaling (4). Recent studies showed that LPS treatment isknown to induce phosphorylation of paxillin in macrophages(19) and bovine pulmonary arterial ECs (7); however, little isknown about the potential role of tyrosine phosphorylation ofpaxillin in regulating LPS-induced endothelial barrier dysfunc-tion.

We hypothesized that tyrosine phosphorylation of paxillin atY31 and Y118 is essential for LPS-mediated endothelial per-meability changes. In the present study, we provide evidencethat LPS challenge of human lung microvascular endothelialcells (HLMVECs) significantly enhanced paxillin tyrosinephosphorylation at Y31 and Y118, but not Y181, which wasmediated by c-Abl but not c-Src and FAK tyrosine kinase.

Address for reprint requests and other correspondence: P. Fu, COMRBBldg., Rm. #3140, 909 S. Wolcott Ave., Chicago, IL 60612 (e-mail: [email protected]).

Am J Physiol Lung Cell Mol Physiol 308: L1025–L1038, 2015.First published March 20, 2015; doi:10.1152/ajplung.00306.2014.

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Furthermore, phosphorylation of paxillin at Y31 and Y118 wascritical for LPS-induced endothelial hyperpermeability be-cause nonphosphorylable mutants of Y31F and/or Y118F sup-pressed LPS-induced EC permeability. We next investigatedthe in vivo role of c-Abl and paxillin in LPS-induced lunginjury. Inhibition of c-Abl in mouse by AG957 attenuatedpaxillin tyrosine phosphorylation and lung permeability, andknockdown of paxillin in mouse lung by use of paxillin siRNAsignificantly attenuated LPS-induced lung inflammation andinjury. These results provide further insight into the role oftyrosine phosphorylation of paxillin in LPS-induced endothe-lial permeability.

MATERIALS AND METHODS

Reagents and cell culture. QuikChange II Site-Directed Mutagen-esis Kit was purchased from Agilent Technologies (La Jolla, CA).Paxillin antibody (BD Transduction Laboratories, San Diego, CA);phospho Y31, Y118 paxillin, and c-Abl antibodies (Cell Signaling,Beverly, MA); phospho Y181 paxillin antibody, Src antibody, andscrambled and siRNA for paxillin and c-Abl were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA). Phospho-Src Y418 wasprocured from Sigma (St. Louis, MO). Phospho-VE-cadherin Y658antibody, phospho-tyrosine antibody, and c-Abl inhibitor AG957were purchased from EMD Millipore (Millipore, Temecula, CA).RNA transfection reagent Gene Silencer was from Genlantis (SanDiego, CA). PolyJet DNA in vitro transfection reagent was obtainedfrom SignaGen Laboratories (Rockville, MD). In vivo RNA transfec-tion reagent jetPEI was from Polyplus transfection (Illkirch, France).HLMVECs, essential basal medium (EBM-2), and growth factor kitwere obtained from Lonza (Walkersville, MD).

Plasmid constructs. Enhanced green fluorescent protein (EGFP)-tagged wild-type paxillin and control plasmids were prepared asdescribed previously (28). Nonphosphorylable mutants Y31F, Y118F,Y31/118F were engineered by using a point mutation kit from AgilentTechnologies (Santa Clara, CA). Briefly, a pair of mutagenic primerscontaining the mutated tyrosine/coding sequence (tac/ttc) for each orboth sites was designed according to manufacturer’s instruction.Mutant strands were synthesized by PCR method, and PCR productswere digested by 10 U/�l of DpnI restriction enzyme and 1 �l of thedigested DNA were used for bacterial transformation. Generation ofmutants was confirmed by DNA sequencing at Research ResourceCenter of University of Illinois at Chicago.

Transfection of small interfering RNA. Depletion of endogenouspaxillin and c-Abl in HLMVECs was carried out by using gene-specific siRNA as described previously (12). In brief, predesignedhuman paxillin, c-Abl siRNA, or nonspecific/nontargeting siRNAwere used to transfect HLMVECs (passage of 5–7). Before transfec-tion, cells were starved in 2% fetal bovine serum (FBS) EBM-2medium overnight. The next day, 50 nM scrambled, paxillin, or c-AblsiRNA complexes were prepared in Gene Silencer transfection re-agent according to the manufacturer’s recommendation, cells weretransfected in serum-free medium for 4 h, and the medium wasreplaced with fresh complete EBM-2 medium supplemented with10% FBS and growth factors. Cells were used 72 h posttransfection.

Immunoblotting and immunoprecipitation. Immunoblotting andimmunoprecipitation (IP) were performed as described previously(42). In brief, after appropriate treatments, cells were pelleted inice-cold PBS, lysed in standard lysis buffer (Cell Signaling, Beverly,MA), and sonicated. Lysates were then centrifuged at 1,000 g for 10min at 4°C, supernatants were collected, and protein was assayed witha BCA protein assay kit. For IP experiments, equal amounts of protein(1 mg) from each sample were precleared with control IgG conjugatedto A/G agarose beads at 4°C for 1 h; supernatants were collected andincubated overnight with primary antibody conjugated to A/G agarosebeads at 4°C. The next day, the samples were centrifuged at 1,000 g

for 1 min in a microfuge centrifuge, and the pellet containing theagarose beads was washed three times with lysis buffer at roomtemperature (RT). After centrifugation at 1,000 g for 1 min, the beadswere collected by removing supernatant buffer, and 40 �l of SDSsample buffer [100 mM Tris·HCl (pH 6.8), 4% SDS, 0.1% bromophe-nol blue, 20% glycerol, 200 mM DTT] were added to the beads andboiled. Lysates were then subjected to 10% SDS-PAGE followed byWestern blotting. Proteins were detected by immunoblotting withappropriate primary antibodies and horseradish peroxidase-conju-gated anti-rabbit or anti-mouse secondary antibodies. Band intensitieswere quantified by densitometry using Image J software.

Measurement of endothelial permeability by transendothelial elec-trical resistance. The endothelial permeability changes were mea-sured by the highly sensitive biophysical assay with an electricalcell-substrate impedance sensing system (Applied Biophysics, Troy,NY) as described previously (12). Briefly, cells were grown on goldmicroelectrodes (10�4 cm2) in complete culture medium containing10% FBS and growth factor supplements as described (43). Twenty-four hours prior to transendothelial electrical resistance (TER) mea-surements, the culture medium was replaced with EBM-2 mediumcontaining 1% FBS. The TER was measured dynamically across themonolayer, and the effect of LPS challenge was monitored over aperiod of 16 h. Decrease in TER resulted from cell retraction,paracellular gap formation, rounding, or loss of adhesion. Resistancewas normalized to the initial voltage and expressed as a fraction of thenormalized resistance value.

Endothelial permeability measurement by FITC-dextran Transwellassay. Transwell tracer experiments with fluorescent dextran wereperformed with the PET track-etched membrane 24-well Transwellsystem (1.0 �m pore; BD). HLMVECs were transfected with scram-bled RNA or siRNA for paxillin as described above. After 48 h oftransfection, cells (2 � 104) were reseeded on a Transwell insertmembrane, and monolayers were allowed to grow to �95% conflu-ence. Cells were challenged with LPS (100 ng/ml) for 6 h prior toaddition of FITC-dextran (3 mg/ml, 40 kDa, Sigma) to the luminalchambers, and incubation was continued for an additional 2 h. Theinserts were removed and fluorescence of the medium in the lowerchamber was measured at an excitation wavelength of 485 nm andemission wavelength of 530 nm by use of the Aminco Bowman Series2 luminescence spectrophotometer (12).

Immunofluorescence staining and image analysis. HLMVECsgrown on chamber slides (Millipore) were stimulated with vehicle orLPS (100 ng/ml) for 1–6 h. Cells were fixed and subjected toimmunofluorescence staining for paxillin, VE-cadherin and F-actin.Image analysis was performed as described (43). Briefly, HLMVECsafter the challenge were fixed with 3.7% formaldehyde solution inPBS for 10 min at RT, washed three times with PBS, permeabilizedwith 0.1% Triton X-100 in PBS for 5 min at RT, and blocked with 2%bovine serum albumin in PBS for 1 h. Incubation with antibodies ofinterest was performed in blocking solution at RT for 1 h followed bystaining with Alexa Fluor 488-conjugated secondary antibodies (Mo-lecular Probes, Eugene, OR). Actin filaments were stained at RT withTexas red phalloidin (Molecular Probes) for 1 h. After immunostain-ing, slides were examined with a Nikon TE 2000-S fluorescencemicroscope and Hamamatsu digital camera via a �60 oil-immersionobjective lens. Paxillin distribution at focal adhesions was quantifiedby using a segmentation algorithm in MetaVue software (UniversalImaging, West Chester, PA), and a minimum of 20 typical cells fromeach experimental condition were analyzed for paxillin distribution.

In vivo transfection of mouse lung with paxillin siRNA. The micewere housed in a pathogen-free barrier facility maintained by theUniversity of Illinois at Chicago, Biologic Resources Laboratory. Allexperiments involving mice were conducted with approved protocolsby the Institutional Animal Care and Use Committee of the Universityof Illinois at Chicago. In vivo paxillin knockdown in mouse lung wascarried out by intratracheal paxillin siRNA transfection using jetPEIas a transfecting agent (1 mg/kg body wt of the mouse). Before

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transfection, siRNA was diluted with 5% glucose to make a 10–15 �lsolution per mouse. In parallel, jetPEI polymer at N/P ratio (nuclear/polymer ratio) of 7 was diluted with 5% glucose in the same volumeas siRNA solution. The diluted polymer was added to the dilutedsiRNA, vortexed gently, and incubated for 15 min at RT. Adult maleC57BL/6J mice, 8–10 wk old, with average weight 20–25 g wereanesthetized and intubated intratracheally with a 20-G catheter (Penn-Century, Philadelphia, PA) and were administered 20–30 �l ofsiRNA working solution. Seventy-two hours after transfection, micewere challenged with vehicle or LPS for 24 h.

Intratracheal administration of LPS or c-Abl inhibitor AG957 tomice. Mice were anesthetized by intraperitoneal injection of ketamine(75 mg/kg) and acepromazine (1.5 mg/kg). LPS (2 mg/kg body wt,Escherichia coli O55:B5, dissolved in sterile water) or vehicle control(sterile water) was instilled intratracheally in a small volume (20–30�l) using a 20-gauge catheter to scramble siRNA control, scramblesiRNA LPS, siRNA paxillin control, and siRNA paxillin LPS groupsof mice. In another experiment, mice were pretreated with AG957 (10mg/kg) intratracheally 1 h before LPS treatment as described above.After 24 h of LPS treatment, mice were killed, and bronchoalveolarlavage (BAL) fluids were collected by intratracheal injection of 1 mlof sterile Hanks’ balanced salt solution followed by gentle aspiration.The lavage was used for the measurement of cell count, and proteinconcentration. For histopathology, lungs were flushed with 1 ml ofice-cold PBS containing 5 mM EDTA, excised, and dipped in saline;the right lobes were blotted dry, quickly snap-frozen in liquid nitro-gen, and stored at �80°C for further analysis and the left lobes werefixed in 4% formalin and sectioned for hematoxylin and eosin stain-ing.

Assessment of pulmonary vascular leakage by Evans blue. Evansblue dye (EBD, 30 ml/kg) was injected into the external jugular vein2 h before termination of experiment to assess vascular leak. In brief,at the end of the experiment, thoracotomy was performed, and thelungs were perfused free of blood with PBS containing 5 mM EDTA.Left lung and right lungs were excised and blotted dry, weighed, andhomogenized in PBS (1 ml/100 mg tissue) and used for quantitativeanalysis of EBD tissue accumulation. Briefly, homogenized tissue wasincubated with two volumes of formamide (18 h, 60°C) and centri-fuged at 12,000 g for 20 min. Optical density of the supernatant wasdetermined by spectrophotometry at 620 and 740 nm. EBD concen-tration in the lung tissue homogenates (�g EBD/g tissue) was calcu-lated against a standard curve.

Myeloperoxidase assay. Myeloperoxidase (MPO) activity wasmeasured in lung tissue homogenates to reflect neutrophil and mac-rophage parenchymal infiltration. Lung tissue was homogenized in 5mM potassium phosphate buffer (pH 6.0) and homogenates werecentrifuged (30,000 g, 30 min at 4°C); pellets were suspended inextraction buffer [50 mM potassium phosphate buffer (pH 6.0) con-taining 0.5% hexadecyl trimethylammonium bromide] and subjectedto three cycles of freezing and thawing. The supernatants generated(13,000 g, 15 min at 4°C) were assayed for MPO activity by kineticreadings over 3 min (200 �l sample plus 800 �l reaction buffercontaining 50 mM potassium phosphate buffer, 0.167 mg/ml ofO-dianisidine dihydrochloride, and 0.0006% H2O2). Absorbance wasmeasured at 460 nm. The results are presented as MPO units per 100milligram protein.

Statistical analysis. Results are expressed as means � SE of threeto five independent experiments. Statistical significance was assessedby ANOVA, followed by Bonferroni multiple-comparison post hoctests. A value of P � 0.05 was considered statistically significant.

RESULTS

LPS induces paxillin accumulation at focal adhesions. LPSis known to induce cytoskeletal rearrangement; therefore, theeffect of LPS on paxillin rearrangement was investigated inHLMVECs. Cells were treated with LPS (100 ng/ml) for 1, 3,

and 6 h, and paxillin distribution was visualized by immuno-fluorescence staining. LPS challenge induced significant accu-mulation of paxillin at focal adhesions and stress fibers after 3h compared with vehicle control. In LPS-treated cells, paxillinredistribution showed a pattern of enlarged focal adhesions(0.39 � 0.18 �m2) compared with the relatively small-sizefocal adhesion observed in untreated control (0.11 � 0.04�m2, P � 0.05) (Fig. 1A). Furthermore, the redistributedpaxillin mainly flanked the two ends of stress fibers, suggestingthat it serves as anchoring sites for actomyosin filaments.

Knockdown of paxillin with siRNA attenuates LPS-inducedendothelial barrier dysfunction and VE-cadherin dissociationfrom adherens junctions. Having demonstrated paxillin accu-mulation at focal adhesions after LPS challenge, we nextinvestigated whether paxillin plays a role in LPS-inducedendothelial permeability. HLMVECs were transfected withscrambled or paxillin-specific siRNA to knock down endoge-nous paxillin expression, and EC permeability was assessed bymeasuring TER and leakage of FITC-dextran. LPS at a con-centration of 100 ng/ml induced a gradual decrease in TER,suggesting increased permeability in scrambled siRNA-trans-fected HLMVECs (Fig. 1B), which was significant after 10 h ofLPS challenge (Fig. 1E). Paxillin depletion with siRNA (Fig.1C) significantly attenuated LPS-induced increase in EC per-meability as determined by TER (Fig. 1, B and E). Similarly,downregulation of paxillin with siRNA reduced LPS-inducedHLMVEC permeability as measured by FITC-dextran leakage(Fig. 1F).

The effect of paxillin on LPS-induced EC permeability wasfurther characterized by determining disruption of VE-cad-herin, a major adherens junction protein in ECs. As shown inFig. 1G, in untreated control cells, the expression of VE-cadherin was concentrated in interendothelial junctions; how-ever, LPS challenge induced significant dissociation of VE-cadherin from adherens junctions and downregulation of pax-illin by siRNA restored VE-cadherin distribution within theadherens junctions. Taken together, these results demonstratean essential role of paxillin in LPS-induced VE-cadherin dis-tribution in adherens junctions and endothelial barrier dysfunc-tion.

LPS stimulates paxillin tyrosine phosphorylation inHLMVECs. Earlier studies have demonstrated that LPS en-hanced paxillin tyrosine phosphorylation in macrophages andbovine pulmonary arterial endothelial cells (7, 19); however,the role of tyrosine phosphorylation of paxillin on LPS-inducedendothelial barrier function is not well defined. We determinedwhether LPS modulates paxillin tyrosine phosphorylation inHLMVECs. By using site-specific phosphor antibodies againstY31, Y118, and Y181, we found LPS treatment of HLMVECsresulted in enhanced tyrosine Y31 and Y118 phosphorylationwith maximal phosphorylation seen at 1 h compared withvehicle control (1.9- and 3.2-fold, respectively, Fig. 2, A andB). In contrast to Y31 and Y118, LPS did not cause Y181phosphorylation in HLMVECs (Fig. 2, A and B). These resultssuggest site-specific tyrosine phosphorylation of paxillin atY31 and Y118 by LPS in HLMVECs.

Role of paxillin phosphorylation at Y31 and Y118 in LPS-induced endothelial barrier dysfunction. To further determinewhether LPS-induced specific tyrosine phosphorylation at Y31and Y118 participates in pulmonary EC barrier disruption,HLMVECs were transfected with each of the following: empty

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vector, paxillin wild-type, and nonphosphorylable single Y31Fand Y118F and double-mutant Y31F and Y118F. As shown inFig. 2, C and D, LPS-induced profound barrier disruption incells transfected with control vector and wild-type paxillinplasmids; however, ectopic expression of Y31 and Y118 pax-illin single mutants in HLMVECs partially attenuated LPS-induced barrier dysfunction, whereas transfection with thedouble mutant of paxillin conferred maximum protectionagainst LPS-induced barrier dysfunction. These results furthersupport a role for tyrosine phosphorylation of paxillin at Y31and Y118 in LPS-mediated barrier disruption in HLMVECs.

LPS-induced paxillin tyrosine phosphorylation is not medi-ated by Src or FAK in HLMVECs. Having demonstrated thatLPS stimulates paxillin tyrosine phosphorylation at Y31 and

Y118, but not Y181, we next sought to identify the kinase(s)that mediates LPS-induced paxillin tyrosine phosphoryla-tion. Src and FAK are two potential kinases that are knownto mediate paxillin phosphorylation in different cell types(5). LPS treatment of HLMVECs resulted in increasedphosphorylation of Src at Y418 (Fig. 3, A and B); however,inhibition of Src by PP2 (10 �M) showed no significanteffect on paxillin Y31 and Y118 phosphorylation in re-sponse to LPS (Fig. 3, C and D). In contrast to paxillintyrosine phosphorylation, PP2 attenuated LPS-induced FAKphosphorylation compared with cells treated with vehicle(Fig. 3, C and D). Furthermore, LPS treatment of HLM-VECs did not result in increased interaction of paxillin withSrc or FAK (Fig. 4A). In contrast, there was a significant

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Fig. 1. Paxillin mediates LPS-induced human lung microvascular endothelial cell (HLMVEC) monolayer permeability. A: confluent HLMVEC monolayer wasexposed to LPS (100 ng/ml) for varying time points as indicated followed by immunofluorescent staining of paxillin (Pxn) and F-actin. Scale bar 10 �m. B–D:HLMVECs were transfected with scrambled RNA (scRNA) or paxillin siRNA (siPxn). After 24 h, transfected cells were reseeded onto gold electrode plates orTranswell inserts and cultured for an additional 48 h to form confluent monolayer. Western blotting was performed to confirm knockdown of paxillin (C andD). Statistical analysis of the Western blot showing paxillin expression was normalized to total actin and presented as ratio of paxillin to actin. *P � 0.01, n 5. B, E, and F: cells were then treated with LPS (100 ng/ml), and monolayer permeability was monitored by measurement of transendothelial electrical resistance(TER) (B and E) or leaked FITC-dextran from inner to outer compartment (F). *Significantly different compared with cells transfected with scrambled siRNA(P � 0.01); #significantly different compared with scrambled siRNA transfected cells challenged with LPS (P � 0.05). Knockdown of paxillin was confirmedby Western blot (C). G: immunofluorescent staining of VE-cadherin and actin was performed after LPS treatment (100 ng/ml, 6 h) in scRNA or paxillinsiRNA-transfected HLMVECs. Scale bar 10 �m. Images were captured by fluorescence microscope (�60); shown are representative images of 3 independentexperiments.



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interaction between paxillin and c-Abl after LPS treatmentcompared with untreated cells (Fig. 4A). Similar to Srcinhibition, transfection of HLMVECs with the FAK domi-nant-negative plasmid, FAK-related nonkinase (FRNK), hadno effect on paxillin tyrosine phosphorylation by LPS (Fig.4, C and D). Also, PP2 had no effect on LPS-inducedpaxillin accumulation at focal adhesion as indicated byimmunofluorescent staining (Fig. 4E). These results showthat although Src and FAK were activated by LPS, they didnot mediate paxillin tyrosine phosphorylation at Y31 andY118 in HLMVECs.

c-Abl tyrosine kinase mediates LPS-induced paxillin ty-rosine phosphorylation and barrier dysfunction in HLMVECs.An earlier study has demonstrated that integrins regulate phos-phorylation of paxillin by c-Abl (22), and we also showed thatc-Abl mediates NADPH oxidase activation by hyperoxia inhuman lung ECs (34). Therefore, we investigated whetherLPS-induced tyrosine phosphorylation of paxillin is dependenton c-Abl tyrosine kinase in HLMVECs. LPS stimulated phos-phorylation of c-Abl at Y245 in HLMVECs (Fig. 5, A and B),indicating activation of c-Abl kinase. Downregulation of c-Ablwith siRNA (Fig. 5C) resulted in �85% loss of c-Abl expres-

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Fig. 2. LPS-induced paxillin tyrosine phosphorylation at Y31 and Y118 residues mediates endothelial cell (EC) permeability. A: HLMVECs (�95% confluence)were exposed to LPS (100 ng/ml) for varying time points. Total cell lysates (20 �g protein) were subjected to SDS-PAGE and Western blotting with Y31-, Y118-,and Y181-specific phospho-paxillin antibodies. B: Western blots were scanned and analyzed by image analyzer and expressions were normalized to total paxillinand presented as ratio p-Pxn/Pxn. *P � 0.01 compared with vehicle control, #P 0.05 compared with vehicle control (n 3). HLMVECs were transfectedwith control plasmid, paxillin mutant plasmids (PxnY31F, PxnY118F, or PxnY31/118F double site mutant plasmids). LPS-induced permeability was monitoredby measurement of TER (C) or by FITC-dextran method (D). *P � 0.01, compared with vehicle control, #P � 0.05 compared with control plasmid � LPS.E: Western blotting was performed with anti-paxillin antibody to confirm expression of paxillin-GFP or GFP-tagged paxillin mutants.



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sion and LPS-induced paxillin phosphorylation at Y118 but notY31 compared with scramble siRNA-transfected cells (Fig. 5,C and D). Also pretreatment of HLMVECs with c-Abl specificinhibitor AG957 (20 �M) for 1 h blocked LPS-induced Y31and Y118 phosphorylation of paxillin (Fig. 5E) compared withcells without the inhibitor. These results suggest a role forc-Abl in LPS-mediated tyrosine phosphorylation of paxillin atY31 and Y118 in HLMVECs.

c-Abl siRNA attenuates LPS-induced EC barrier dysfunction.Having established a role for c-Abl in paxillin tyrosine phos-phorylation, we next determined the functional involvement ofc-Abl in LPS-mediated loss in EC barrier integrity. Downregu-lation of c-Abl by using specific siRNA significantly reducedLPS-induced barrier disruption as indicated by restoration ofTER (Fig. 5G) and inhibition of FITC-dextran leakage (Fig.5H). These results demonstrate a key role for c-Abl in endo-thelial barrier dysfunction mediated by LPS.

Paxillin Y31 and Y118 mutants mitigate LPS-induced VE-cadherin destabilization in HLMVECs. VE-cadherin is a majorconstituent of adherens junctions and forms homophilic dim-mers by binding through its extracellular region to anotherdimmer of VE-cadherin expressed in adjacent cells (13). Ty-rosine phosphorylation of VE-cadherin has often been pro-posed as a mechanism that controls the integrity of the adhe-rens junctions and endothelial barrier function (9). To furtherstudy the mechanism(s) by which paxillin regulates LPS-induced barrier dysfunction, we determined the effect of LPSon VE-cadherin phosphorylation. As shown in Fig. 6, A and B,LPS stimulated VE-cadherin phosphorylation at Y658 in atime-dependent manner. To determine whether LPS-inducedVE-cadherin phosphorylation is regulated by paxillin, paxillinexpression was downregulated by siRNA or paxillin functionwas blocked by expression of paxillin mutant in ECs. Asshown in Fig. 6, C and D, paxillin siRNA significantly inhib-ited VE-cadherin Y658 phosphorylation. Similarly, transfec-tion of paxillin mutants Y31 and Y118 or the double mutantY31/Y118 also blocked LPS-mediated VE-cadherin Y658phosphorylation (Fig. 6, E and F). These results suggest thatLPS-induced VE-cadherin phosphorylation is dependent onpaxillin and paxillin tyrosine phosphorylation at Y31 and Y118in HLMVECs. We next determined the role of paxillin phos-phorylation on LPS-induced VE-cadherin remodeling. Takingadvantage of VE-cadherin monoclonal antibody (BV6), whichrecognizes the extracellular domain of human VE-cadherin, weobserved that LPS challenge disrupted VE-cadherin disruptionat adherens junctions with increased intracellular staining incells, which was prevented by overexpression of paxillin mu-tants at Y31 and Y118 (Fig. 6G). The expression of paxillindouble mutant at Y31 and Y118 showed the most significantinhibitory effect on VE-cadherin disruption at adherens junc-tions and endocytosis. These results suggest a role for paxillinY31 and Y118 phosphorylation by LPS in VE-cadherin dis-ruption at adherens junctions of HLMVECs.

Inhibition of c-Abl attenuates LPS-induced paxillin phos-phorylation and lung injury in mice. To further investigate therole of c-Abl in LPS-induced paxillin phosphorylation andpulmonary permeability in vivo, mice were treated intratrache-ally with DMSO (1:2,000 in sterile PBS, 20 �l/mouse) orAG957 (10 mg/kg) to block c-Abl activity in the lung. LPS wasadministered 1 h after AG957 treatment. At the end of theexperiment, lung injury was evaluated by measurements ofBAL cell count, protein concentration, histological analysis oflung sections, and measurements of EBD accumulation in thelung tissue. In vivo, LPS induced significant phosphorylationof paxillin at Y31 and Y118 (Fig. 7A). Inhibition of c-Abl byAG957 attenuated LPS-induced phosphorylation of paxillin atboth sites (Fig. 7A). Also, LPS induced significant phosphor-ylation of VE-cadherin in DMSO-treated mice (Fig. 7A), whichwas attenuated in AG957-treated mice (Fig. 7A). In DMSO

LPS (min) 0 5 10 30

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Fig. 3. Src and focal adhesion kinase (FAK) activation are not required forpaxillin tyrosine phosphorylation by LPS. A: HLMVECs (�95% confluence)in 35-mm dishes were challenged with LPS (100 ng/ml) for multiple timepoints and total cell lysates (20 �g protein) were subjected to SDS-PAGE andWestern blotting with anti-phospho-Src Y418 antibody. Shown is a represen-tative blot from 3 independent experiments. B: Western blots were scanned andquantified by image analyzer. *P � 0.01 compared with vehicle control. C:HLMVECs (95% confluence) were pretreated with PP2 (10 �M) prior tostimulation with LPS (100 ng/ml) for 2 h. Total cell lysates (20 �g protein)were subjected to SDS-PAGE and Western blotting with anti-phospho-FAKY576, phospho-paxillin Y31, phospho-paxillin Y118, phospho-SrcY418, andrespective non-phospho antibodies. Shown is a representative blot from 3experiments. D: Western blots were scanned and quantified by image analyzer.Values are means � SE from 3 independent experiments. *P � 0.001; #P �0.05.



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control mice, LPS instillation caused pronounced lung inflam-mation, as reflected by elevation of protein content, total cellsin BAL fluid, and MPO activity in the lung (Fig. 7, B, C, E, andF). Compared with DMSO controls, AG957-treated mice ex-hibited strong protective effects. The protective effects ofAG957 against LPS-induced lung vascular leak were furtherassessed by measurement of EBD leakage into the lung tissue.LPS induced noticeable EBD accumulation in the lung paren-chyma, which was significantly decreased by AG957 (Veh �LPS: 20.08 � 3.78 �g/g wet lung tissue vs. AG957 � LPS:12.81 � 1.51 �g/g wet lung tissue, P � 0.05) (Fig. 7D). Theseresults demonstrate a role for c-Abl in the mediation of LPS-induced paxillin phosphorylation and pulmonary permeability.

Knockdown of paxillin with siRNA in mouse lungs inhibitsLPS-induced lung inflammation. Our in vitro results suggest arole for paxillin and its tyrosine phosphorylation in LPS-

mediated endothelial barrier dysfunction in HLMVECs; there-fore, we next examined whether paxillin mediates LPS-in-duced lung injury and pulmonary permeability in vivo. Paxillinin mouse lungs was downregulated by intratracheally instillingpaxillin siRNA for 72 h prior to LPS challenge. As shown inFig. 8, A–D, LPS challenge induced significant leakage ofprotein in scrambled RNA (scRNA)-treated mice [vehicle(Veh): 0.16 � 0.02 mg/ml vs. LPS: 0.53 � 0.11 mg/ml, P �0.01] and infiltration of inflammatory cells into the alveolarspace (Veh: 1.28 � 0.21 � 105/ml vs. LPS: 25.02 � 3.65 �105/ml, P � 0.001); EBD (Veh: 5.81 � 1.91 �g/g vs. LPS:18.63 � 3.96 �g/g wet lung tissue, P � 0.01; MPO activity inthe lung tissue: 25.3 � 6.2 U/mg protein vs. 138.6 � 31.2U/mg protein, P � 0.01). The above indicators of pulmonaryleak and inflammation were significantly attenuated in paxillinknockdown lungs [nsRNA � LPS: 25.02 � 3.65 � 105/ml vs.

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Fig. 4. LPS does not induce association betweenpaxillin, Src and FAK. A: HLMVECs (95% con-fluence) were treated with LPS (100 ng/ml) for 2h. Cell lysates (1 mg protein) were subjected toimmunoprecipitation (IP) with anti-paxillin anti-body conjugated to A/G agarose beads, and im-munoprecipitates were subjected to SDS-PAGEand Western blotting with anti-FAK, -Src, -c-Abland -paxillin antibodies. Immunoprecipitationwith IgG antibody served as a control. No sig-nificant increase in the interaction between pax-illin with FAK or Src was observed after LPStreatment. Shown is a representative blot from 3experiments. In contrast, LPS induced associa-tion between paxillin and c-Abl. B: immunoblotswere scanned and quantified by image analyzerand normalized to total paxillin. *P � 0.05. C:HLMVECs (�60% confluence) were transfectedwith control plasmid or FAK dominant-negativeplasmid FRNK (50 nM) for 72 h prior to LPS(100 ng/ml) challenge for 2 h. Cell lysates (20 �gprotein) were subjected to SDS-PAGE and West-ern blotting with anti-phospho-paxillin Y31,Y118, anti-FAK and anti-actin antibodies.Shown is a representative blot from 3 experi-ments. D: Western blots were scanned and quan-tified by image analyzer. *P � 0.05, comparedwith control plasmid; #P � 0.05, compared withFRNK plasmid control. E: HLMVECs grown onglass slides (�95% confluence) were pretreatedwith EBM-2 medium plus 2% serum containingDMSO (1 �l/ml) or PP2 (10 �M) in DMSO (1�l/ml) for 1 h prior to LPS treatment. After 2 hof LPS treatment, paxillin rearrangement wasstudied by immunofluorescent staining of paxillin.Scale bars 10 �m. Images were captured by fluo-rescent microscope (�60); shown are representa-tive images from 3 independent experiments.



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paxillin siRNA (siPxn) � LPS: 16.01 � 3.13 � 105/ml, P �0.05; protein content: nsRNA � LPS: 0.53 � 0.11 mg/ml vs.siPxn � LPS: 0.31 � 0.07 mg/ml, P � 0.05; EBD: nsRNA �LPS: 18.63 � 3.96 �g/g vs. siPxn � LPS: 10.23 � 3,21 �g/gwet lung tissue, P � 0.05; MPO activity in the lung tissue:138.6 � 31.2 U/mg protein vs. 63.1 � 12.3 U/mg protein, P �0.05]. Furthermore, histochemical analysis of paraffin-embed-ded mouse lungs revealed inflammatory cell recruitment and

areas of alveolar hemorrhage indicative of vascular disruption inscrambled siRNA-treated mice. In contrast, there was a markedattenuation of lung injury in paxillin siRNA-treated mice (Fig.8E). Knockdown of paxillin in mouse lungs was confirmed byWestern blotting of lung tissue lysates obtained from scrambledand paxillin siRNA-administered mice (Fig. 8F). These resultssuggest that paxillin is an important regulator of LPS-mediatedpulmonary vascular permeability in vivo.

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DISCUSSION

The integrity of the pulmonary endothelium is an essentialrequirement for lung function preservation as movement offluid, macromolecules, and leukocytes into the interstitium andalveolar space during ALI severely compromises oxygenationof the blood (46). This is potentially life threatening whenincreased hypoxemia leads to pulmonary leak, tissue edema,and multiorgan failure. Several mechanistic studies have re-vealed that paracellular gap formation is regulated by thebalance between competing contractile forces and adhesivecell-cell and cell-matrix tethering forces, which together reg-ulate cell shape changes (11). In addition, focal adhesionslinking pulmonary ECs to underlying matrix may play animportant role in defining EC remodeling and barrier integrityin healthy lungs and its failure in ALI (16, 32, 36). Althoughfocal adhesion-associated proteins such as FAK and paxillinplay a key role in focal adhesion assembly via interaction withthe cytoskeleton, the mechanism(s) of endothelial barrier reg-ulation by focal adhesion proteins remains poorly defined. Theeffects of LPS on pulmonary EC barrier function have beenextensively studied (2), whereas the role of paxillin in LPS-induced barrier function has not been characterized. We haveshown that LPS stimulates actin reorganization and paxillinaccumulation at focal adhesions in HLMVECs. This studypresents several novel observations that paxillin, a multido-main adapter focal adhesion protein, regulates LPS-mediatedlung injury both in human lung endothelium and in mouselungs. Furthermore, our data show tyrosine phosphorylation ofpaxillin at Y31 and 118, but not Y181, mediated by c-Ablnonreceptor tyrosine kinase activation, is important in LPS-induced EC barrier dysfunction. Additionally, tyrosine phos-phorylation of paxillin-mediated destabilization of VE-cad-herin at adherens junctions contributed to LPS-induced barrierdysfunction in the endothelium. The physiological relevance ofthe in vitro studies on the role of paxillin and tyrosine phos-phorylation in barrier function were verified in an in vivomurine model of LPS-induced ALI wherein paxillin in mouselung was knocked down by instillation of paxillin siRNA, orc-Abl was inhibited by AG957.

An interesting observation is that knockdown of paxillinreduced LPS-mediated endothelial barrier dysfunction invivo and in vitro. The mechanism by which paxillin medi-ates this effect on endothelial barrier function is unclear.Paxillin can interact with integrins at focal adhesions, whichmediate adhesion of cells to extracellular matrix proteins.

Integrins themselves have no enzymatic activity but theybind to actin through paxillin. Furthermore, blockade ofintegrin �v 5 prevented development of lung vascular per-meability in two different models of ALI: ischemia-reper-fusion in rats and ventilation-induced lung injury in mice(35). Similar to their findings, in this study, blockade ofpaxillin prevented lung vascular permeability in a LPS-induced ALI murine model as well. Since both integrins andpaxillin are focal adhesion components, both the present andearlier study (35) indicate that blockade of focal adhesionsis beneficial to vascular integrity. This can be interpreted bythe dynamic control between cell-matrix and cell-cell inter-action. We hypothesize that weakening of cell-matrix inter-action goes along with a concurrent strengthening of cell-cell contact and the overall outcome of enhanced barrierfunction. Since focal adhesion is a docking site for stressfibers, small focal adhesion generates less centripetal ten-sion. This hypothesis needs further testing in other modelsand with a broad array of focal adhesion proteins. Of note,in fibroblasts integrin induces paxillin phosphorylation in ac-Abl-dependent manner (22), and our results revealed thatLPS-induced paxillin phosphorylation was c-Abl dependentand independent of Src and FAK. In this study, paxillinsiRNA was delivered intratracheally to the lung, whichassures reasonable siRNA delivery to the lung. However,intratracheal administration of paxillin siRNA does notensure that knockdown is limited to the lung ECs and it islikely that other cells in the lung may be affected.

Another novel observation is the role of c-Abl in mediatingLPS-induced endothelial permeability via phosphorylation ofpaxillin at Y31 and Y118 in human lung ECs. Paxillin wasoriginally identified as a major substrate for the v-Src tyrosinekinase (14), and c-Src and FAK have been identified as keykinases responsible for paxillin tyrosine phosphorylation inresponse to a variety of stimuli (1, 5, 24, 27, 37, 47). AlthoughLPS stimulated c-Src and FAK phosphorylation, knock-down of Src with siRNA or overexpression FRANK, adominant-negative form of FAK, did not block LPS-inducedtyrosine phosphorylation of paxillin in HLMVECs. How-ever, inhibition or downregulation of c-Abl attenuated LPS-induced paxillin phosphorylation at Y118 in HLMVECs.Interestingly, in c-Abl siRNA-transfected cells, the basallevel of paxillin phosphorylation at Y31 increased comparedwith nsRNA-transfected cells. But after LPS treatment,there was no further increase in Y31 phosphorylation, which

Fig. 5. c-Abl interacts with paxillin and mediates paxillin tyrosine phosphorylation and EC permeability by LPS. A: HLMVECs (�95% confluence) were treatedwith LPS (100 ng/ml) for indicated time periods. Phosphorylation of c-Abl tyrosine 245 was detected by Western blotting with anti-phospho-c-Abl Y245antibody. Shown is a representative blot from several independent experiments. B: Western blots were scanned, analyzed by image analyzer and normalized tototal c-Abl and presented as p-c-Abl/c-Abl. *P � 0.01 compared with vehicle control. C: HLMVECs (�60% confluence) were transfected with scrambled orc-Abl siRNA (50 nM) for 72 h before LPS (100 ng/ml) challenge for 2 h. Cell lysates (20 �g protein) were subjected to SDS-PAGE and Western blotting withanti-phospho-paxillin Y31 and Y118, anti-c-Abl, and anti-paxillin antibodies. Shown is a representative blot from 3 independent experiments. D: Western blotswere scanned and quantified by image analyzer. *P � 0.01, compared with scRNA control; **P 0.05 compared with scRNA � LPS; #P � 0.01 comparedwith scRNA � LPS. E: HLMVECs (90% confluence) in 35-mm dishes were pretreated with c-Abl inhibitor AG957 (20 �M) for 1 h prior to LPS (100 ng/ml)challenge for 2 h. Total cell lysates (20 �g protein) were subjected to SDS-PAGE and Western blotting with anti-phospho-paxillin Y31 and Y118 andanti-paxillin antibodies. Shown is a representative blot from 3 independent experiments. F: Western blots from (E) were scanned, quantified by image analyzer,and normalized to total paxillin. *P � 0.001 compared with vehicle control; #P 0.05 compared with AG957 control. G: HLMVECs grown on gold electrodes(60% confluence) were transfected with scrambled or c-Abl siRNA (100 nM) for 48 h prior to LPS (100 ng/ml) challenge. Permeability changes were monitoredby TER. H: HLMVECs grown on a 24-Transwell system were transfected with scrambled or c-Abl siRNA (100 nM) for 48 h and changes in permeability afterLPS challenge were measured by the FITC-dextran method as described in MATERIALS AND METHODS. Shown is a representative tracing from 3 independentexperiments. Values are means � SE from 3 independent experiments. *P � 0.01, compared with vehicle control, #P � 0.05, compared with scRNA � LPS.



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may be attributed to a compensatory mechanism. Down-regulation of c-Abl expression may result in the upregula-tion other tyrosine kinase(s), which can increase basalpaxillin phosphorylation at Y31. Intratracheal administra-tion of AG957, an inhibitor of c-Abl, attenuates LPS-induced paxillin tyrosine phosphorylation and lung injury inmice. Similar to paxillin siRNA delivery, intratracheal ad-ministration of AG957 may be inhibiting c-Abl on bothendothelial and nonendothelial cells. There is evidence forthe involvement of c-Abl in mediating LPS-induced lym-phoid B cells differentiation and macrophage activation (10,21); however, the mechanism of LPS-induced activation ofc-Abl is unknown. c-Abl can be activated by reactiveoxygen species (ROS) and LPS has been shown to stimulate

ROS production in human lung endothelium (20). Further-more, in our study LPS stimulated the association betweenpaxillin and c-Abl, but not with Src or FAK. It is very likelythat the SH3 and SH2 domains in c-Abl may dock withspecific proline-rich domain or phosphorylated tyrosine-based motifs in paxillin. Furthermore, the proline-rich re-gion in paxillin can provide a potential binding site for SH3containing proteins such as c-Abl that could facilitate ty-rosine phosphorylation of paxillin.

Our finding that paxillin Y31F and Y118F mutants pre-vented LPS-induced VE-cadherin redistribution from cell pe-riphery, VE-cadherin phosphorylation, and barrier dysfunctionin HLMVECs is compelling. Paxillin tyrosine residues Y31,118, and 181 are embedded in the SH2 domain binding site,

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Fig. 6. Paxillin mediates LPS-induced VE-cadherin (VE-cad) tyrosine phosphorylation and remodeling. A: HLMVECs (�90% confluence) were challenged withLPS (100 ng/ml) for 0–12 h. Total cell lysates (20 �g protein) were subjected to SDS-PAGE and Western blotting with anti-phospho-VE-cadherin Y658,anti-VE-cadherin and anti-actin antibodies. Shown are representative blots from 3 experiments. B: blots were scanned and quantified by image analyzer, andnormalized to VE-cadherin. *P � 0.001, compared with vehicle control. C: HLMVECs (�60% confluence) were transfected with scRNA or paxillin siRNA (100nM), for 72 h prior to LPS (100 ng/ml) challenge for 2 h. Total cell lysates (20 �g protein) were subjected to SDS-PAGE and Western blotting withanti-phospho-VE-cadherin Y658, anti-VE-cadherin, and anti-actin antibodies. Shown are representative blots from 3 independent experiments. D: blots in C werescanned and quantified by image analyzer. *P � 0.01, compared with scRNA control; #P � 0.05, compared with scRNA � LPS. E: HLMVECs (�60%confluence) were transfected with control or paxillin mutant plasmids, PxnY31F, PxnY118F, and PxnY31/118F (100 nM) for 72 h prior to LPS (100 ng/ml)challenge for 2 h. Total cell lysates (20 �g protein) were subjected to SDS-PAGE and Western blotting with anti-phospho-VE-cadherin Y658 andanti-VE-cadherin antibodies. Shown are representative blots from 3 separate experiments. F: blots from E were scanned and quantified by image analyzer. *P �0.01, compared with plasmid control; #P � 0.01, compared with control plasmid � LPS; ##P � 0.05, compared with PxnY31F � LPS or PxnY118F � LPS.G: HLMVECs (�60% confluence) grown on glass slides were transfected with control plasmid or paxillin mutant Y31F, Y118F, or Y31F/Y118F plasmids (1�g/ml) for 72 h. Transfected cells were challenged with vehicle or LPS (100 ng/ml) for 6 h, and images were visualized by immunofluorescence microscope(�60) after staining for VE-cadherin with VE-cadherin monoclonal antibody (BV6). Scales bar 10 �m. Shown are representative micrograph from 3 independentexperiments.



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Fig. 7. Inhibition of c-Abl attenuates LPS-induced lung permeability and injury. C57BL/6J mice were pretreated with DMSO (1:2,000 in sterile PBS) or AG957(10 mg/kg) intratracheally 1 h before intratracheal LPS challenge. A: lung tissues were homogenized and subjected to SDS-PAGE and Western blotting forphosphorylation of VE-cadherin, paxillin, and c-Abl. B–E: lung permeability and injury were assessed by measurement of cell counts, and protein concentrationin bronchoalveolar lavage (BAL), Evans blue accumulation in the lung tissue; MPO activity was assayed in lung tissue homogenates as described in MATERIALS

AND METHODS. Values are means � SE from 5 animals. *P � 0.01, compared with DMSO control; #P � 0.05, compared with DMSO � LPS. F:hematoxylin-eosin staining of lung sections from mice at 24 h after intratracheal instillation of 2 mg/kg of LPS. The sections are representative lung sectionsfrom 5 mice per group. Magnification �20, scale bar, 100 �m.



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which upon phosphorylation allows docking of binding part-ners that include FAK, GIT-2, PIX, p190RhoGAP (29, 38,41). Agonist stimulated paxillin phosphorylation at Y31 andY118 is known to enhance cell motility that can be neutralizedby the expression of Y31F/Y118F mutants (39). Thus Y31 andY118 are two key tyrosine phosphorylation sites in paxillinwhose phosphorylation is induced by a variety of stimuli thatinduce cell motility (17, 18, 25, 26, 30, 45) and regulate barrierfunction. Paxillin is also known to indirectly activate Rac1 andinhibit RhoA in an Y31 and Y118 phosphorylation-dependentmanner (39). Since the balance between Rac1 and RhoA playsa critical role in determining endothelial barrier function, theY31/Y118 tyrosine phosphorylation of paxillin-dependent reg-ulation of Rac1/RhoA activity may serve as an alternativemechanism underlying paxillin-mediated barrier regulation byLPS, which needs further investigation.

In summary, our findings presented here demonstrate anessential role for tyrosine-phosphorylated paxillin at Y31 andY118 by c-Abl nonreceptor tyrosine kinase in LPS-induced

endothelial barrier dysfunction. LPS/endotoxin mediates lunginjury in animals and humans and therapeutic targeting ofc-Abl and its target paxillin may provide new avenues in themanagement and amelioration of ALI in patients.

ACKNOWLEDGMENTS

The authors thank Dr. Prasad Kanteti for valuable discussions.

GRANTS

This work was supported by National Heart, Lung, and Blood InstituteGrants P01HL58064 and P01HL98050 to V. Natarajan.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

P.F. and V.N. conception and design of research; P.F., P.V.U., A.L., andA.H. performed experiments; P.F., P.V.U., A.L., and A.H. analyzed data; P.F.,J.G.G., R.S., and V.N. interpreted results of experiments; P.F. prepared figures;

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Fig. 8. Knockdown of paxillin in mouse lung attenuates LPS-induced lung permeability and injury. C57BL/6J mice were instilled with scrambled or paxillinsiRNA (5 mg/kg body wt) for 48 h prior to intratracheal LPS challenge. A–D: lung permeability and injury was assessed by measurement of BAL fluid cell count,BAL protein concentration, Evans blue accumulation in the lung tissue; MPO activity was assayed in lung tissue homogenates as described in MATERIALS AND

METHODS. Values are means � SE from 5 animals. *P � 0.01, compared with scRNA control; #P � 0.05, compared with scRNA � LPS. E: hematoxylin-eosinstaining of lung sections from scRNA or paxillin siRNA-transfected mice at 24 h after intratracheal instillation of 2 mg/kg of LPS. The sections are representativelung sections from 5 mice per group. Magnification �20, scale bar, 100 �m. F: lung tissue lysates (30 �g protein) from scramble and paxillin siRNA-transfectedmice subjected to SDS-PAGE and Western blotting with anti-paxillin and anti-actin antibodies. Immunoblotting confirmed downregulation of paxillin by siRNAtransfection in lung tissue. G: statistical analysis of Western blot (F), and paxillin expression was normalized to total actin and presented as ratio of paxillin toactin. *P � 0.05, n 5.



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P.F. drafted manuscript; P.F., P.V.U., C.C.G., J.G.G., and V.N. edited andrevised manuscript; P.F. and V.N. approved final version of manuscript.

REFERENCES

1. Aponte M, Jiang W, Lakkis M, Li MJ, Edwards D, Albitar L, VitonisA, Mok SC, Cramer DW, Ye B. Activation of platelet-activating factorreceptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer. Cancer Res 68: 5839–5848, 2008.

2. Bannerman DD, Goldblum SE. Direct effects of endotoxin on theendothelium: barrier function and injury. Lab Invest 79: 1181–1199, 1999.

3. Birukova AA, Alekseeva E, Cokic I, Turner CE, Birukov KG. Crosstalk between paxillin and Rac is critical for mediation of barrier-protectiveeffects by oxidized phospholipids. Am J Physiol Lung Cell Mol Physiol295: L593–L602, 2008.

4. Birukova AA, Cokic I, Moldobaeva N, Birukov KG. Paxillin is in-volved in the differential regulation of endothelial barrier by HGF andVEGF. Am J Respir Cell Mol Biol 40: 99–107, 2009.

5. Brown MC, Cary LA, Jamieson JS, Cooper JA, Turner CE. Src andFAK kinases cooperate to phosphorylate paxillin kinase linker, stimulateits focal adhesion localization, and regulate cell spreading and protrusive-ness. Mol Biol Cell 16: 4316–4328, 2005.

6. Brown MC, Turner CE. Paxillin: adapting to change. Physiol Rev 84:1315–1339, 2004.

7. Chatterjee A, Snead C, Yetik-Anacak G, Antonova G, Zeng J, Catra-vas JD. Heat shock protein 90 inhibitors attenuate LPS-induced endothe-lial hyperpermeability. Am J Physiol Lung Cell Mol Physiol 294: L755–L763, 2008.

8. Chen XL, Nam JO, Jean C, Lawson C, Walsh CT, Goka E, Lim ST,Tomar A, Tancioni I, Uryu S, Guan JL, Acevedo LM, Weis SM,Cheresh DA, Schlaepfer DD. VEGF-induced vascular permeability ismediated by FAK. Dev Cell 22: 146–157, 2012.

9. Daniel JM, Reynolds AB. Tyrosine phosphorylation and cadherin/cateninfunction. Bioessays 19: 883–891, 1997.

10. Daniel R, Chung SW, Eisenstein TK, Sultzer BM, Wong PM. Specificassociation of Type I c-Abl with Ran GTPase in lipopolysaccharide-mediated differentiation. Oncogene 20: 2618–2625, 2001.

11. Dudek SM, Garcia JG. Cytoskeletal regulation of pulmonary vascularpermeability. J Appl Physiol 91: 1487–1500, 2001.

12. Fu P, Mohan V, Mansoor S, Tiruppathi C, Sadikot RT, Natarajan V.Role of nicotinamide adenine dinucleotide phosphate-reduced oxidaseproteins in Pseudomonas aeruginosa-induced lung inflammation and per-meability. Am J Respir Cell Mol Biol 48: 477–488, 2013.

13. Giannotta M, Trani M, Dejana E. VE-cadherin and endothelial adherensjunctions: active guardians of vascular integrity. Dev Cell 26: 441–454,2013.

14. Glenney JR Jr, Zokas L. Novel tyrosine kinase substrates from Roussarcoma virus-transformed cells are present in the membrane skeleton. JCell Biol 108: 2401–2408, 1989.

15. Gonzalez-Lopez A, Astudillo A, Garcia-Prieto E, Fernandez-GarciaMS, Lopez-Vazquez A, Batalla-Solis E, Taboada F, Fueyo A, Al-baiceta GM. Inflammation and matrix remodeling during repair of ven-tilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 301:L500–L509, 2011.

16. Holinstat M, Knezevic N, Broman M, Samarel AM, Malik AB, MehtaD. Suppression of RhoA activity by focal adhesion kinase-induced acti-vation of p190RhoGAP: role in regulation of endothelial permeability. JBiol Chem 281: 2296–2305, 2006.

17. Huang Z, Yan DP, Ge BX. JNK regulates cell migration throughpromotion of tyrosine phosphorylation of paxillin. Cell Signal 20: 2002–2012, 2008.

18. Iwasaki T, Nakata A, Mukai M, Shinkai K, Yano H, Sabe H, SchaeferE, Tatsuta M, Tsujimura T, Terada N, Kakishita E, Akedo H.Involvement of phosphorylation of Tyr-31 and Tyr-118 of paxillin inMM1 cancer cell migration. Int J Cancer 97: 330–335, 2002.

19. Kleveta G, Borzecka K, Zdioruk M, Czerkies M, Kuberczyk H,Sybirna N, Sobota A, Kwiatkowska K. LPS induces phosphorylation ofactin-regulatory proteins leading to actin reassembly and macrophagemotility. J Cell Biochem 113: 80–92, 2012.

20. Kratzer E, Tian Y, Sarich N, Wu T, Meliton A, Leff A, Birukova AA.Oxidative stress contributes to lung injury and barrier dysfunction viamicrotubule destabilization. Am J Respir Cell Mol Biol 47: 688–697,2012.

21. Le Q, Daniel R, Chung SW, Kang AD, Eisenstein TK, Sultzer BM,Simpkins H, Wong PM. Involvement of C-Abl tyrosine kinase in lipopo-

lysaccharide-induced macrophage activation. J Immunol 160: 3330–3336,1998.

22. Lewis JM, Schwartz MA. Integrins regulate the association and phos-phorylation of paxillin by c-Abl. J Biol Chem 273: 14225–14230, 1998.

23. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology ofsepsis in the United States from 1979 through 2000. N Engl J Med 348:1546–1554, 2003.

24. Pasapera AM, Schneider IC, Rericha E, Schlaepfer DD, WatermanCM. Myosin II activity regulates vinculin recruitment to focal adhesionsthrough FAK-mediated paxillin phosphorylation. J Cell Biol 188: 877–890, 2010.

25. Petit V, Boyer B, Lentz D, Turner CE, Thiery JP, Valles AM.Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cellmigration through an association with CRK in NBT-II cells. J Cell Biol148: 957–970, 2000.

26. Romanova LY, Hashimoto S, Chay KO, Blagosklonny MV, Sabe H,Mushinski JF. Phosphorylation of paxillin tyrosines 31 and 118 controlspolarization and motility of lymphoid cells and is PMA-sensitive. J CellSci 117: 3759–3768, 2004.

27. Roy S, Ruest PJ, Hanks SK. FAK regulates tyrosine phosphorylation ofCAS, paxillin, and PYK2 in cells expressing v-Src, but is not a criticaldeterminant of v-Src transformation. J Cell Biochem 84: 377–388, 2002.

28. Salgia R, Li JL, Ewaniuk DS, Wang YB, Sattler M, Chen WC,Richards W, Pisick E, Shapiro GI, Rollins BJ, Chen LB, Griffin JD,Sugarbaker DJ. Expression of the focal adhesion protein paxillin in lungcancer and its relation to cell motility. Oncogene 18: 67–77, 1999.

29. Scheswohl DM, Harrell JR, Rajfur Z, Gao G, Campbell SL, SchallerMD. Multiple paxillin binding sites regulate FAK function. J Mol Signal3: 1, 2008.

30. Shephard V. Comments on “The effects of a 12-wk group exerciseprogramme on physiological and psychological variables and function inoverweight women” by Grant et al. (Public Health 2004; 118: 31–42).Public Health 118: 310, 2004.

31. Shikata Y, Birukov KG, Birukova AA, Verin A, Garcia JG. Involve-ment of site-specific FAK phosphorylation in sphingosine-1 phosphate-and thrombin-induced focal adhesion remodeling: role of Src and GIT.FASEB J 17: 2240–2249, 2003.

32. Shikata Y, Birukov KG, Garcia JG. S1P induces FA remodeling inhuman pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin.J Appl Physiol 94: 1193–1203, 2003.

33. Shikata Y, Rios A, Kawkitinarong K, DePaola N, Garcia JG, BirukovKG. Differential effects of shear stress and cyclic stretch on focaladhesion remodeling, site-specific FAK phosphorylation, and small GT-Pases in human lung endothelial cells. Exp Cell Res 304: 40–49, 2005.

34. Singleton PA, Pendyala S, Gorshkova IA, Mambetsariev N, Moitra J,Garcia JG, Natarajan V. Dynamin 2 and c-Abl are novel regulators ofhyperoxia-mediated NADPH oxidase activation and reactive oxygen spe-cies production in caveolin-enriched microdomains of the endothelium. JBiol Chem 284: 34964–34975, 2009.

35. Su G, Hodnett M, Wu N, Atakilit A, Kosinski C, Godzich M, HuangXZ, Kim JK, Frank JA, Matthay MA, Sheppard D, Pittet JF. Integrinalphavbeta5 regulates lung vascular permeability and pulmonary endothe-lial barrier function. Am J Respir Cell Mol Biol 36: 377–386, 2007.

36. Sun X, Shikata Y, Wang L, Ohmori K, Watanabe N, Wada J, ShikataK, Birukov KG, Makino H, Jacobson JR, Dudek SM, Garcia JG.Enhanced interaction between focal adhesion and adherens junction pro-teins: involvement in sphingosine 1-phosphate-induced endothelial barrierenhancement. Microvasc Res 77: 304–313, 2009.

37. Teranishi S, Kimura K, Nishida T. Role of formation of an ERK-FAK-paxillin complex in migration of human corneal epithelial cells duringwound closure in vitro. Invest Ophthalmol Vis Sci 50: 5646–5652, 2009.

38. Tomar A, Lim ST, Lim Y, Schlaepfer DD. A FAK-p120RasGAP-p190RhoGAP complex regulates polarity in migrating cells. J Cell Sci122: 1852–1862, 2009.

39. Tsubouchi A, Sakakura J, Yagi R, Mazaki Y, Schaefer E, Yano H,Sabe H. Localized suppression of RhoA activity by Tyr31/118-phosphor-ylated paxillin in cell adhesion and migration. J Cell Biol 159: 673–683,2002.

40. Turner CE. Paxillin interactions. J Cell Sci 113: 4139–4140, 2000.41. Turner CE, Brown MC, Perrotta JA, Riedy MC, Nikolopoulos SN,

McDonald AR, Bagrodia S, Thomas S, Leventhal PS. Paxillin LD4motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: a role in cytoskeletal remodeling. J Cell Biol 145: 851–863,1999.



by 10.220.33.5 on October 29, 2017


ownloaded from


42. Usatyuk PV, Burns M, Mohan V, Pendyala S, He D, Ebenezer DL,Harijith A, Fu P, Huang LS, Bear JE, Garcia JG, Natarajan V.Coronin 1B regulates S1P-induced human lung endothelial cell che-motaxis: role of PLD2, protein kinase C and Rac1 signal transduction.PLoS One 8: e63007, 2013.

43. Usatyuk PV, Natarajan V. Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesionkinase and adherens junction proteins. Am J Physiol Lung Cell MolPhysiol 289: L999–L1010, 2005.

44. Valles AM, Beuvin M, Boyer B. Activation of Rac1 by paxillin-Crk-DOCK180 signaling complex is antagonized by Rap1 in migrating NBT-IIcells. J Biol Chem 279: 44490–44496, 2004.

45. Vindis C, Teli T, Cerretti DP, Turner CE, Huynh-Do U. EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118. J Biol Chem 279: 27965–27970, 2004.

46. Ware LB, Matthay MA. The acute respiratory distress syndrome. N EnglJ Med 342: 1334–1349, 2000.

47. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE,Parsons JT, Horwitz AF. FAK-Src signalling through paxillin, ERKand MLCK regulates adhesion disassembly. Nat Cell Biol 6: 154 –161,2004.

48. Yuan D, He P. Vascular remodeling alters adhesion protein and cytoskel-eton reactions to inflammatory stimuli resulting in enhanced permeabilityincreases in rat venules. J Appl Physiol 113: 1110–1120, 2012.



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