Am J Physiol Cell Physiol 306: C1154 –C1166, 2014. First published April 9, 2014; doi:10.1152/ajpcell.00415.2012.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling Najmunnisa Nasreen,1,2 Nazli Khodayari,1,2 Peruvemba S. Sriram,1,2 Jawaharlal Patel,1,2 and Kamal A. Mohammed1,2 1

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Florida, Gainesville, Florida; and 2North Florida/South Georgia Veterans Health Care System, Malcom Randall Veterans Affairs Medical Center, University of Florida, Gainesville, Florida Submitted 26 December 2012; accepted in final form 4 April 2014

Nasreen N, Khodayari N, Sriram PS, Patel J, Mohammed KA. Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling. Am J Physiol Cell Physiol 306: C1154 –C1166, 2014. First published April 9, 2014; doi:10.1152/ajpcell.00415.2012.—Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury. EphA2 receptor; tobacco smoke; permeability; paxillin; E-cadherin

is the major etiological factor in the development of chronic obstructive pulmonary disease (COPD) and is currently the fourth leading cause of morbidity and mortality (5). In the United States alone, about 250,000 people die with COPD each year. COPD is characterized by the destruction of lung tissue and loss of alveolar cells secondary to inflammation (41). Bronchial airway epithelial cells (BAEpCs) that line the airways are the first cells to come in contact with tobacco smoke (TS), and airway epithelial injury is the important pathological event in the development of COPD (38, 42). BAEpCs from COPD patients were susceptible to TS-mediated

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Address for reprint requests and other correspondence: K. A. Mohammed, Div. of Pulmonary, Critical Care and Sleep Medicine, Dept. of Medicine, Univ. of Florida, Gainesville, FL (e-mail: [email protected]). C1154

hyperpermeability (40). However, the mechanisms underlying TS-induced BAEpC barrier dysfunction are not well established. The airway epithelium is a selectively permeable barrier that separates the airways from submucosal tissue and the vasculature. The integrity of the epithelium is critical to prevent exposure of the subepithelial compartment to environmental irritants, such as TS. The apical junctional complexes consisting of apical tight junctions and underlying adherens junctions maintain cell-cell adhesions in the epithelium (35, 39). Under normal conditions bronchial airway epithelial barrier function is dependent on both cell-cell and cell-extracellular matrix interactions. The adherens junction protein E-cadherin maintains the integrity of the airway epithelium, and inflammatory stimuli are believed to increase transepithelial permeability by inducing disassembly in cell-cell adhesions in the airway epithelium (21). Cellular adhesion to the extracellular matrix (ECM) occurs via specialized plasma membrane regions called focal adhesions (14, 17). Focal adhesion protein paxillin activation is associated with a decrease in cellular adherence to ECM. Phosphorylation on serine and threonine sites near the carboxyl terminus promotes paxillin accumulation at focal adhesion plaques (44), and inhibition of paxillin phosphorylation decreases cell-ECM interactions (10). Moreover, deregulation in focal adhesion proteins may compromise the barrier function of epithelium. Besides, decreased expression of the adherens junction protein E-cadherin leads to gap formation and increases permeability to macromolecules (1). TS exposure decreased E-cadherin expression in mice bronchial airway epithelium in vivo (24). However, the mechanisms whereby TS mediates bronchial airway epithelial barrier dysfunction are not clear. Cellular response to external stimuli, such as TS, is controlled by a complex array of mitogen-activated protein kinase (MAPK) signaling pathways (8, 27, 32). The MAPK are an important group of serine/threonine signaling kinases that play a key role in converting stress stimuli into nuclear responses. Three major groups of MAPK have been identified in mammalian cells: the extracellular signal-regulated kinases (ERK1/ ERK2), p38, and JNK. Activation of these signaling cascades leads to translocation of nuclear factors, such as NF-kB, to the nucleus, which in turn affects the expression of target gene or phosphorylation of transcription factors in the nucleus (47). In a recent study, we reported that TS exposure activates MAPK in BAEpCs (32). However, whether these activated MAPK in TS-exposed BAEpCs play a role in the modulation of airway epithelial E-cadherin expression and the permeability have not been investigated. http://www.ajpcell.org

TOBACCO SMOKE AND RECEPTOR EphA2 SIGNALING

The erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ligands the Ephrins represent the largest family of receptor tyrosine kinases that play a key role during embryonic neuronal development, vascular assembly, and oncogenesis (4, 11). EphA2 receptor and its cognate ligand EphrinA1 are known to be associated with inflammation, epithelial development, and homeostasis (26, 36). In addition, EphA2 and EphrinA1 contribute to migration and angiogenesis in endothelial cells (3, 16). Furthermore, injury to the lung by viral infection and hypoxia induced the expression of EphA2 and its ligand EphrinA1 in a mouse model (9). EphA2 receptor signaling leads to activation of MAPK. Pratt and Kinch (37) demonstrated that, upon crosstalk with its ligand EphrinA1, EphA2 transmits signals to the nucleus via MAPK. Since EphA2 is associated with several important cellular functions in lung injury, it is possible that EphA2 signaling may play a key role in TS-induced airway epithelial permeability. However, whether TS modulates EphA2 expression and affects the cell-cell or cell-ECM adhesion proteins in BAEpCs and compromises epithelial permeability, are not known. Therefore, we hypothesized that TS may induce permeability in BAEpC monolayer by modulating receptor EphA2 expression, adherens junction, and focal adhesion proteins. In the present study we investigated TS-mediated structural and functional changes in BAEpCs and the role of EphA2 signaling in modulation of adherens junctions. We also studied TSinduced EphA2 signaling on BAEpC monolayer barrier functions in vitro. MATERIALS AND METHODS

Cell culture and TS exposure. Primary human BAEpCs were cultured in bronchial epithelial cell growth medium (BEGM) as per manufacturer’s instructions (Cell Application, San Diego, CA) in serum-free media as reported earlier (30). In brief, BAEpCs were cultured submerged in 60-mm cell culture dishes, and when they reached near confluence, BAEpC cultures were exposed to either room air or TS. Some cultures were transfected with siRNA-EphA2 or control-siRNA prior to TS exposure. BAEpCs were exposed to varying concentrations of TS, i.e., 1 exposure unit (EU), 2EU, 3EU, or 4EU, as reported earlier (32). Briefly, the 1EU of TS consists of 15 min of TS exposure followed by 45 min of incubation in 5% CO2 and 95% ambient air. BAEpC cultures were harvested 24 h post-TS exposure and subjected to Western blot or quantitative PCR analysis. Transient transfection of BAEpCs. BAEpCs were transfected with vector containing EphA2 construct to delineate the EphA2-mediated signaling where necessary. The gene transfer vector pcDNA3.2/V5DEST was used as an expression vector for receptor EphA2 expression, and pcDNA3.2/V5/CAT was used as a control vector (Invitrogen, Carlsbad, CA) as reported earlier (22). In brief, the pENTR TOPO vector containing EphA2 insert expanded in One-shot Top10 cells, and cloned into the destination vector pcDNA3.2/V5-DEST according to the manufacturer’s instructions (Invitrogen). The BAEpCs were transfected with pcDNA-EphA2 or empty vector using lipofectamine-2000 reagent (Invitrogen). Some transfected cells were treated with recombinant EphrinA1 (R and D systems, Minneapolis, MN) and used for further experiments. The overexpression of EphA2 was analyzed by Western blot and by real-time PCR analysis. Transfection of BAEpCs with siRNA-EphA2. BAEpCs were transfected with siRNA-EphA2 or scrambled-siRNA (Sc-siRNA). The sequences used in this study were as follows: siRNA-EphA2, sense CGCAAGAAGGGAGACUCCAACAGCU and antisense AGCUGUUGGAGUCUCCCUUCUUGCG; and Sc-siRNA, sense CGAGAGAGUCG GCGACUAUUGGUCA and antisense UGAC-

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CAAUAGUCGCCGACUCUCUCG, as reported earlier (31). The transfection medium was changed with BEGM culture medium, and the experiments were carried out 36 h posttransfection. BAEpC monolayer permeability. TS-induced permeability changes in BAEpC monolayer was evaluated by measuring electrical resistance in real-time using an electrical cell-substrate impedance sensing (ECIS) system as described earlier (1). Briefly, BAEpCs were cultured on ECIS electrode arrays that consist of eight 1-cm2 chambers. Each chamber contains 10 gold electrodes of 10⫺4 cm2 each, and the culture medium functions as the electrolyte. Confluent cultures of BAEpCs were exposed to increasing concentrations (increasing EU) of TS. Some of the wells were exposed to room air. Some of the cultures were pretreated with EphA2 anti-sense siRNA to evaluate the effect of EphA2 function. Some cultures were pretreated with MAPK pharmacological inhibitors to evaluate the role of MAPK in TSmediated epithelial barrier function. The total electrical resistance measured dynamically across the BAEpC monolayer, determined as the combined resistance between the basal surface of the cell and the electrode, reflects alterations in cell matrix adhesion and/or cell-cell adhesion. The differences between cell-cell and cell-matrix component total transepithelial resistance was resolved into values reflecting resistance to current flow beneath the monolayer (␣) and resistance to current flow between adjacent cells (Rb), by following the method of Keese and Giaever (19). Evans blue dye albumin protein permeability. Bovine serum albumin (BSA) was labeled with Evans blue dye (10 mg of dye/g of BSA) as reported by English et al. (12). BAEpCs were cultured to confluence on polycarbonate membranes of Transwell chambers as reported earlier (28). BAEpC confluence was confirmed by microscopic analysis. The Transwell plates have an inner and an outer chamber separated by a polycarbonate membrane. This membrane contained 3-␮m pores. To evaluate the role of EphA2 on TS-induced protein permeability in BAEpCs, confluent BAEpC monolayers in Transwell chambers were transfected with either EphA2-siRNA or scrambledsiRNA and exposed to TS. After 8 h of incubation, 0.1 ml of Evans blue dye BSA complex was added into the inner chambers of Transwell and incubated for 2 h at 37°C and 5% CO2 atmosphere. Some of the chambers were left untreated (controls). The lower chambers of Transwells were filled with 1.5 ml of HBSS. Protein leak across the membrane was determined by measuring optical density of Evans blue dye transferred into the lower chamber at 620 nm using a spectrophotometer. Permeability across BAEpC monolayers was expressed as mean permeability coefficient (percent of control). Immunofluorescence staining. TS-induced modulation of junctional proteins paxillin and E-cadherin in the primary BAEpCs was determined by confocal microscopy as reported earlier (29). In brief, BAEpCs were grown to confluence on gelatinized glass cover slips and maintained in BEGM culture media. After TS exposure BAEpCs were fixed with 5% paraformaldehyde (with 50 mM phosphate buffer) in 50% Tris wash buffer (TWB) for 5 min. TWB contained 150 mM NaCl and 50 mM Tris, pH ⫽ 7.6, with 0.1% NaN3. The glass cover slips were rinsed three times and permeabilized with 1.2% Triton X-100 for 5 min, rinsed three times, incubated with 1% BSA in 100% TWB for 1 h, and then paxillin and E-cadherin expression was detected using rabbit anti-human paxillin and rabbit anti-human Ecadherin antibodies (BD Transduction Labs, Franklin Lakes, NJ), respectively. The cells were stained with secondary antibodies conjugated with FITC (Life technologies, Grand Island, NY). The slides were visualized using confocal laser scanning microscope [Zeiss LSM 510 (Axiovert 100M), Zeiss, Thornwood, NY], as reported earlier (30). Western blot analysis. BAEpCs cultured in 60-mm culture dishes (Corning, Tewksbury, MA) were exposed to varying concentration of TS, and the expression of EphA2 and EphrinA1 was detected using rabbit anti-human EphA2 and rabbit anti-human EphrinA1, respectively. Some BAEpC cultures were exposed to TS either in the presence of MAPK inhibitors or siRNA-EphA2, and focal adhesion

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proteins paxillin and E-cadherin were detected using rabbit antihuman paxillin, and rabbit anti-human E-cadherin (BD Transduction Labs, Franklin Lakes, NJ), respectively, as reported earlier (32, 33). In brief, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate. Protein concentration was measured as reported earlier (34) using Pierce’s BCA Protein Assay Kit (Pierce, Rockford, IL). Typically, 20 ␮g of total protein was resolved on 4%-20% Tris glycine SDS-PAGE gels (Bio-Rad, Hercules, CA). Proteins were transferred to PVDF membranes, blocked, and incubated with respective antibodies at 1:200 dilutions at 4°C overnight. The blots were washed three times and incubated with goat anti-rabbit secondary antibodies at 1:2,000 dilutions for 1 h at room temperature. Proteins were detected by enhanced Immuno-Star HRP Chemiluminescent Kit (Bio-Rad). Quantitative real-time PCR. Total cellular RNA was isolated from BAEpCs after TS exposure using RNeasy kit (QIAGEN, Valencia CA) according to manufacturer’s recommendations as reported earlier (6). RNA (100 ng/␮l) was reverse transcribed into cDNA using M-MLV Reverse Transcriptase and oligo(dT). The SYBR Green JumpStart Taq Ready Mix was used to perform PCR as reported earlier (33). After reverse transcription, 10 ␮l of diluted cDNA product was mixed with 25 ␮l of SYBR Green JumpStart Taq ReadyMix, 0.5 ␮l of Internal Reference Dye, and 14.5 ␮l of specific oligonucleotide primers (80 nM final concentration) to a total of 50-␮l volume for quantitative real-time PCR. The primer sequences used for EphrinA1 were 5=-CTTTACCCGGGAGCTTGAT-3= (forward) and 5=-ATATGCGGGTGGCTAAACTG-3= (reverse) with product size of 214 bp; and the sequence for EphA2 was 5=-TTCAGCCACCACAACATCAT-3= (forward) and 5=-TCAGACACCTTGCAGACCAG-3= (reverse) with product size of 263 bp. PCR amplification consisted of 40 cycles (95°C for 15 s, 60°C for 1 min, and 72°C for 45 s) after the initial denaturation step (95°C for 2 min). The gene expression level was based on the amount of the target message relative to the h18S RNA. Statistical analysis. The statistical analyses were performed by using SigmaStat 3.5 (SYSTAT Software, San Jose, CA). Results were expressed as means ⫾ SE. To assess the overall significance for the experiments with more than one treatment group, we used the Kruskal-Wallis and the Mann-Whitney U-tests. Differences were considered significant if P values were ⬍0.05. RESULTS

TS upregulated EphA2 receptor and its ligand EphrinA1 expression in BAEpCs in a dose-dependent manner. BAEpC culture was exposed to varying concentrations (1EU, 2EU, 3EU, and 4EU) of TS or left alone in medium as control and total RNA was isolated. The expression of EphA2 and EphrinA1 was determined by real-time quantitative PCR (Fig. 1, A and C). TS induced EphA2 and EphrinA1 mRNA expression in a dose-dependent manner in BAEpCs. Peak expression of EphA2 was noticed with 2EU of TS whereas with 4EU of TS the expression declined significantly; however, the magnitude of expression was much higher compared with control. The EphrinA1 mRNA expression steadily increased with increasing dose of TS (1EU– 4EU) in BAEpCs (Fig. 1C). BAEpCs were exposed to TS for 24 h, total cell lysates were prepared and subjected to Western blot analysis, and ␤-actin was probed to demonstrate equal sample loading. TS significantly increased the expression of EphA2 receptor and EphrinA1 protein in BAEpCs compared with control cells (Fig. 1, B and D). Both EphA2 and EphrinA1 expression was dependent on the number of units of TS exposure in BAEpCs. The EphA2 expression increased up to 2EU, whereas with 3EU and 4EU the EphA2

expression started to decline. Although a decrease in the receptor expression was noted at 3EU and 4EU, it was significantly higher compared with control. The EphrinA1 protein expression steadily increased with the increasing dose of TS. To determine time response some cultures were exposed with 2EU of TS, harvested over time (3–24 h) and subjected to Western blot analysis. TS induced both EphA2 and EphrinA1 expression in a time-dependent manner (Fig. 1E). The EphA2 expression peaked at 3 h whereas the EphrinA1 expression peaked at 24 h. Thus TS caused both an early increase in EphA2 and delayed increase in EphrinA1 mRNA expression in BAEpCs. In addition, we noticed pcDNA-EphA2 transfected BAEpCs expressing high levels of EphA2, and transfection with empty vector did not induce EphA2, suggesting absence of nonspecific expression by the vector transfection. Besides, when BAEpCs were treated with siRNA-EphA2, TS-induced EphA2 expression was significantly blocked (Fig. 1F). Taken together these data suggest that TS induces both EphA2 and EphrinA1 expression in BAEpCs in a dose- and time-dependent manner that may play a critical role in airway epithelial function and remodeling. TS downregulated adherens junction protein E-cadherin expression in BAEpCs. E-cadherin expression was determined in BAEpCs exposed to various doses of TS by Western blot analysis. The E-cadherin expression was significantly decreased in BAEpCs exposed to TS in a dose-dependent manner compared with control cells (Fig. 2A). Exposure to 4EU of TS showed a complete downregulation of E-cadherin in BAEpCs. We also determined TS-mediated E-cadherin expression in BAEpCs by exposing the cells to 2EU of TS over time. In BAEpCs TS downregulated the E-cadherin expression in a time-dependent manner (Fig. 2B). TS inhibited E-cadherin expression in BAEpCs as early as 3 h, and it steadily declined further at 8 and 24 h post-TS exposure. We also confirmed TS mediated E-cadherin expression in BAEpCs by immunofluorescence analysis. In the control BAEpCs, E-cadherin expression was strong and located toward the peripheral region of cells, indicating the cell-cell contacts are maintained by the expression of E-cadherin (Fig. 2C). When BAEpCs were exposed to TS, the E-cadherin expression was significantly downregulated, indicating that TS affects the adherens junction protein. At 4EU, the expression of E-cadherin was significantly decreased. To understand the role of EphA2 in modulation of E-cadherin, the BAEpCs were transfected with vector containing EphA2 or empty vector and treated with recombinant EphrinA1. The overexpression of EphA2 significantly inhibited the expression of E-cadherin (Fig. 2D), whereas silencing EphA2 with siRNA before TS (2EU) exposure blocked TSmediated downregulation of E-cadherin expression in BAEpCs. These data together demonstrate that TS induces EphA2 expression, regulates the expression of E-cadherin, and thereby affects bronchial airway epithelial cell-cell adhesions that lead to epithelial hyperpermeability. The alteration in adherens junction leads to gap formation and compromises the permeability of airway epithelial monolayer. TS did not affect the expression of the focal adhesion protein paxillin in BAEpCs. Focal adhesion protein paxillin expression was determined in BAEpCs exposed to various doses of TS. The expression of paxillin was not affected in TS-exposed BAEpCs compared with control cells (Fig. 3A). The expression of paxillin in BAEpCs was also confirmed by immunofluores-

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Fig. 1. Tobacco smoke (TS) upregulates erythropoietin-producing human hepatocellular carcinoma A2 (EphA2) and EphrinA1 expression in bronchial airway epithelial cells (BAEpCs). BAEpCs were exposed to 1– 4 exposure units (EU) of TS. TS showed significant upregulation in the expression of EphA2 and EprhinA1 compared with control. A and C represent mRNA expression of EphA2 and EphrinA1, respectively, as detected by real-time quantitative PCR analysis. Data presented are means of 3 experiments performed at different times. *P ⬍ 0.001 vs. control. B and D represent EphA2 and EphrinA1 expression, respectively, as detected by Western blot analysis. Lane C represents BAEpCs incubated in the absence of TS as control. The experiments were performed 3 times. Mean values of optical density were normalized to ␤-actin. *P ⬍ 0.001 vs. control. E: Western blot analysis for EphA2 and EphrinA1 expression in control and TS-exposed BAEpCs over time. F: Western blot analysis showing EphA2 expression in TS-exposed, pcDNA-EphA2transfected, and siRNA-EphA2-transfected BAEpCs. The experiments were performed 3 times. Mean values of relative optical density were normalized to ␤-actin. Statistical significance: #P ⬍ 0.001 vs. empty (Emp) vector-transfected cells; $P ⬍ 0.001 vs. scrambled siRNA (Sc-siRNA) ⫹ TS-exposed and TS-exposed cells.

cence staining (Fig. 3B). A strong green signal was noticed in the cytoplasm of the cells. In addition, the transient transfection of BAEpCs by EphA2 vector (pcDNA-EphA2) and subsequent treatment with its ligand recombinant EphrinA1 did not affect the paxillin expression compared with empty vector-transfected plus recombinant EphrinA1-treated BAEpCs (Fig. 3C). These data demonstrate that in BAEpCs acute TS exposure shows little or no effect on focal adhesion protein paxillin expression. TS downregulated E-cadherin expression via Erk1/Erk2, p38, and JNK MAPK activation in BAEpCs. E-cadherin expression was determined in BAEpCs by treating with

PD98059, an inhibitor of Erk1/Erk2; SB203580, an inhibitor of p38; and an inhibitor of JNK MAP kinases. The pretreatment of BAEpCs with inhibitors of Erk1/Erk2, p38, and JNK MAP kinases and subsequent TS (2EU) exposure significantly blocked the downregulation of E-cadherin expression compared with BAEpCs exposed with TS alone (Fig. 4A). These data suggest that TS-induced downregulation of E-cadherin was mediated through Erk1/Erk2, p38, and JNK MAPK signaling in BAEpCs. However, the expression of paxillin remained unaffected, suggesting its nonparticipation in the injury mediated by acute TS exposure of BAEpCs (data not shown). In BAEpCs, TS induced the activation of downstream MAPK

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Fig. 2. TS downregulates E-cadherin expression in BAEpCs. BAEpCs were exposed to 1EU, 2EU, and 4EU of TS, and E-cadherin expression was measured by Western blot and immunofluorescence analysis. TS significantly downregulated the expression of E-cadherin in BAEpCs. A: Western blot analysis of E-cadherin expression in control and 1EU, 2EU, and 4EU of TS-exposed BAEpCs. The experiments were performed 3 times, and mean optical density values of respective bands were normalized to ␤-actin. Statistical significance: *P ⬍ 0.001 vs. control. B: E-cadherin expression over time in control and in 2 EU TS-exposed BAEpCs as determined by Western blot analysis. Statistical significance: *P ⬍ 0.001 compared with respective control. C: E-cadherin expression in BAEpCs as determined by immunofluorescence staining. The cells were labeled with anti-E-cadherin antibody and fluorescein isothiocyanate (FITC)conjugated secondary antibody. DAPI was used as nuclear stain. Magnification is 400⫻. D: Western blot showing E-cadherin expression in BAEpCs transfected either with siRNA-EphA2 or Sc-siRNA prior to exposure of TS (2EU) or pcDNA-EphA2 vector and treated with EphrinA1. This is a single representative of three independent experiments performed at different times. Statistical significance: *P ⬍ 0.001, pcDNA-EphA2 ⫹ rec-EphrinA1 vs. empty vector ⫹ rec-EphrinA1-treated cells; **P ⬍ 0.001, siRNA-EphA2 ⫹ TS vs. Sc-siRNA ⫹ TS and TS alone.

signaling pathway, and it was dose dependent (Fig. 4B). TS exposure at 2EU and 4EU enhanced phosphorylation of Erk1/ Erk2, p38, and JNK in BAEpCs compared with control, i.e., cells treated with DMSO alone (Fig. 4B). In addition, inclusion of inhibitors before exposure to TS downregulated the activation of Erk1/Erk2, p38, and JNK compared with BAEpCs exposed to TS alone (Fig. 4B). Taken together, these data indicate that TS-induced regulation of the adherens junction protein E-cadherin is dependent on Erk1/Erk2, p38, and JNK signaling pathways in BAEpCs. TS induced hyperpermeability across BAEpC monolayer, and silencing EphA2 blunted this response. We measured the permeability changes across the BAEpC monolayer in real

time using Electric Cell Substrate Impedance-Sensing (ECIS) in vitro. Figure 5A demonstrates that TS induces hyperpermeability across BAEpC monolayer in a dose-dependent manner. TS at 1EU did not induce any permeability changes, and the resistance remained comparable to control levels. However, TS at 2EU significantly decreased the resistance across the BAEpC monolayer, and at 4EU TS further decreased the resistance across the monolayer. To evaluate if TS-induced permeability changes across the monolayer are due to overexpression of EphA2, we transfected the BAEpCs with siRNA-EphA2 to silence EphA2 and measured the electrical resistance over time. We noticed a significant protection in barrier function that increased resistance across the siRNA-EphA2 transfected

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Fig. 3. TS fails to affect paxillin expression in BAEpCs. BAEpCs were exposed to 1EU, 2EU, and 4EU of TS, and paxillin expression was measured by Western blot and immunofluorescence analysis. A: Western blot analysis of paxillin expression in control and 1EU, 2EU, and 4EU of TS-exposed BAEpCs. The experiments were performed 3 times, and mean optical density values of respective bands were normalized to ␤-actin. B: paxillin expression in control and TS-exposed BAEpCs as determined by immunofluorescence microscopy. Green fluorescence indicates paxillin expression. DAPI was used as nuclear stain. Magnification is 400⫻. C: Western blot showing paxillin expression in BAEpCs transfected either with siRNA-EphA2 or Sc-siRNA or pcDNA-EphA2 vector or empty vector prior to exposure of TS. This is a single representative of 3 independent experiments each performed at different times, and mean values were considered statistically significant if P ⬍ 0.05. NS ⫽ not significant, control vs. pcDNA-EphA2 and siRNA EphA2 vs. TSexposed BAEpCs.

BAEpC monolayer when compared with Sc-siRNA-transfected, TS-exposed BAEpCs (Fig. 5B). In contrast, transient overexpression of EphA2 and subsequent treatment with its ligand EphrinA1 increased the permeability to the levels comparable to TS exposure in BAEpCs. EphrinA1 treatment in the empty vector-transfected BAEpCs did not induce any significant change in BAEpC permeability (Fig. 5C). The insignificant increases in resistance may be due to the low levels of EphA2 expression in control cells. Furthermore, we also evaluated protein permeability across BAEpC monolayer with Evans blue dye albumin complex. We noticed significantly high protein permeability across TS-exposed BAEpC monolayer (Fig. 5D). We noticed a significant reduction in TSinduced protein permeability across the siRNA-EphA2-transfected BAEpC monolayer compared with Sc-siRNA-transfected BAEpCs. In addition, the protein permeability was also significantly high in pcDNA-EphA2-transfected, EphrinA1treated BAEpC cultures compared with empty vector-transfected, EphrinA1-treated BAEpC cultures. Taken together these data demonstrate that TS-induced permeability changes are modulated by upregulation of EphA2 receptor-mediated signaling in BAEpCs, suggesting that EphA2 plays a key role in the barrier function of BAEpCs during acute TS exposure. TS-induced hyperpermeability across BAEpCs monolayer was mediated through Erk1/Erk2, p38, and JNK MAP kinases. We noticed a remarkable decrease in resistance across the

BAEpC monolayers after 5 h of TS exposure. After 10, 15, and 20 h of TS exposure, significant decreases in resistance were observed compared with control (room air exposed) BAEpCs. In a recent study we reported that TS (1EU– 4EU) exposure induced the phosphorylation of Erk1/Erk2 and p38 MAPK in BAEpCs in a concentration-dependent manner (32). To determine the potential role of MAPKs in TS-induced decrease in cellular resistance, we pretreated the BAEpC monolayers with pharmacological inhibitors to Erk1/2, p38, and JNK and exposed them to TS or room air and measured the resistance over time. Pretreatment of BAEpC monolayers with Erk1/Erk2, p38, and JNK inhibitors (PD98059, SB203580, and JNK inhibitor II, respectively) significantly attenuated the TS-induced increases in transepithelial resistance (Fig. 6). These data indicate that MAPKs play a critical role in regulating permeability across BAEpC monolayers during acute TS exposure. TS-induced hyperpermeability in BAEpCs was due to modulation of cell-cell adhesions but not due to cell-matrix adhesions. The total electrical resistance measured across the BAEpC monolayer was determined as the combined resistance between basal surface of the cell and the electrode, which reflects alterations in cell matrix adhesions and/or cell-cell adhesions. The differences between cell-cell and cell-matrix components, the total transepithelial resistance, are resolved into values reflecting resistance to current flow beneath the monolayer (␣) and resistance to current flow between adjacent

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Fig. 4. TS-mediated downregulation of E-cadherin is dependent on Erk1/Erk2, p38, and JNK in BAEpCs. A: TS-mediated E-cadherin expression in BAEpC in presence and absence of Erk1/Erk2, p38, and JNK MAP kinase inhibitors. BAEpC pretreatment with MAPK inhibitors [10 ␮g/ml of PD98059 (PD), 10 ␮g/ml of SB203580 (SB), and 5 ␮g/ml of JNK inhibitor (JNK-inh)] prior to TS exposure restored E-cadherin expression. Data presented are means ⫾ SE of 3 experiments, each performed at different time, and mean optical density values of respective bands were normalized to ␤-actin. Statistical significance: *P ⬍ 0.001, BAEpCs exposed to 2EU of TS vs. BAEpCs exposed to 2EU of TS plus either PD98059 or SB203580 or JNK inhibitor-treated cells. B: Western blot showing TS-induced phosphorylation of MAPK in presence and absence of pharmacological inhibitors to MAPK. TS exposure induced phosphorylation of Erk1/Erk2, p38, and JNK in BAEpCs, and inclusion of PD98059, SB203580, and JNK inhibitors blocked TS-mediated phosphorylation of Erk1/Erk2, p38, and JNK, respectively.

cells (Rb) by following the method of Keese and Giaever (19). Figure 7A illustrates the measurement of permeability across BAEpC monolayers due to cell-cell adhesions and cell-matrix adhesions. We noticed a significant decrease in Rb values in TS (2EU)-exposed BAEpCs, suggesting a breach in paracellular permeability due to loss of cell-cell adhesions in BAEpCs, whereas the cell-matrix adhesion ␣ was not affected (Fig. 7B) compared with control. Exposure of TS induced ⬃65% downregulation of the electrical resistance due to loss of cell-cell adhesions, and the decline in resistance started as early as 8 h of incubation, which continued to decline up to 20 h. Transfection of BAEpCs with siRNA-EphA2 prior to TS exposure prevented the integrity of monolayer compared with Sc-siRNA (Fig. 7C). These data suggest that TS-induced BAEpC monolayer barrier dysfunction is mediated through EphA2 signaling. Moreover, TS-mediated electrical resistance decrease between the cells indicated that the paracellular permeability (Rb) across the BAEpC monolayer may be due to decreases in E-cadherin expression. TS modulated the actin filaments in BAEpCs. Actin cytoskeleton disruption is a key event in TS-induced injury in BAEpCs. We investigated whether TS (2EU) exposure induces any changes in the organization of actin cytoskeleton in BAEpCs. We used rhodamine phalloidin staining to determine TS-induced cytoskeletal changes in BAEpCs. We noticed a drastic change in the actin filament organization in TS-exposed BAEpCs compared with control. An increase in the stress fiber formation was noticed in TS-exposed cells. The actin filaments formed thick bundles and were mostly located toward the

periphery in TS-exposed BAEpCs (Fig. 8). To investigate if EphA2 is involved in TS-mediated actin cytoskeletal changes, we transfected BAEpCs with siRNA-EphA2 or Sc-siRNA before TS exposure. Silencing the EphA2 expression attenuated the stress fiber formation and restored the actin filaments comparable to that of control cells. In addition, BAEpCs were transfected with plasmid-containing EphA2 construct and treated with EphrinA1, and we examined the actin cytoskeleton changes. In EphA2-overexpressed BAEpCs, EphrinA1 treatment induced actin stress fiber formation, suggesting that disruption of actin cytoskeleton noticed in the TS-exposed cells was due to the TS-mediated upregulation of EphA2 (Fig. 8). To investigate if TS-mediated actin cytoskeleton changes are dependent on MAPK signaling pathways, inhibitors for Erk1/ Erk2 (10 ␮M of PD98059), p38 (10 ␮M of SB203580), and JNK (5 ␮M of JNK inhibitor-II) were introduced before exposure of BAEpCs to TS. As demonstrated in Fig. 8, the stress fiber formation attenuated in the presence of pharmacological inhibitors to Erk1/Erk2, p38, and JNK MAPK. These data suggest that TS-mediated disruption of actin cytoskeleton was dependent on activation of MAPK signaling pathway in BAEpCs. DISCUSSION

One of the most critical events in the pathogenesis of COPD at the cellular level in the airway epithelium is the disruption of the actin cytoskeleton which is associated with loss of polarity and weakening of both cell-cell junctions and cell-matrix

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Fig. 5. TS decreases resistance across the BAEpC monolayer. A: TS mediated decreases in electrical resistance across BAEpC monolayer over time. TS at 2EU and 4EU significantly decreased transepithelial resistance in BAEpC monolayer compared with control. B: TS mediated transepithelial electrical resistance in BAEpCs transfected with siRNA-EphA2 or Sc-siRNA. C: EphrinA1 mediated transepithelial electrical resistance in BAEpCs transfected with pcDNA-EphA2 vector or empty vector. Data presented are means of 3 separate experiments. Statistical significance: *P ⬍ 0.001 vs. control; NS ⫽ not significant vs. control. D: protein permeability coefficient in BAEpCs transfected either with siRNA-EphA2 or Sc-siRNA prior to exposure of TS (4EU) or pcDNA-EphA2 vector transfected, plus EphrinA1 treated. Data presented are means ⫾ SE of 3 separate experiments performed at different times. Statistical significance: *P ⬍ 0.001, pcDNA-EphA2 ⫹ EphrinA1 vs. empty vector ⫹ EphrinA1-treated BAEpCs; $P ⬍ 0.001, TS ⫹ siRNA EphA2 vs. TS-exposed BAEpCs, as well as Sc-siRNA ⫹ TS-treated cells.

adhesions. These disturbances result in loss of epithelial barrier function as well as shedding of cells. In the present study, we have demonstrated that EphA2 and EphrinA1 are overexpressed in TS-exposed BAEpCs compared with control cells and that EphA2 signaling contributes to epithelial barrier dysfunction via downregulation of the adherens junction protein E-cadherin. In addition the relationship between the overexpression of EphA2 and airway epithelial barrier dysfunction due to varying levels of TS (1EU– 4EU) exposure have been determined in vitro. We found that silencing EphA2 expression with siRNA blocked the TS-mediated downregulation of Ecadherin expression and significantly restored TS-induced epithelial permeability. Furthermore, TS-induced EphA2 signaling as well as epithelial permeability in BAEpCs were dependent on the activation of Erk1/Erk2 and p38 MAPK signaling. Our findings from the present study indicate that EphA2 may

be an important mediator of epithelial barrier function during TS exposure in BAEpCs. EphA2 was found to be highly expressed in neuronal and vascular tissue during early stages of development, but minimal levels of EphA2 mRNA expression have been reported in adult normal tissues, including lung (3, 11, 15). EphA2 signaling has been widely studied in angiogenesis, endothelial migration, and vascular assembly (3, 18, 23, 48); however, little is known about its effects on epithelial barrier function. Recent reports indicate that EphA2 signaling was associated with inflammation (18), and during hypoxia EphA2 overexpression was noticed to mediate increased vascular permeability (7). We noticed acute TS exposure enhanced EphA2 and its ligand EphrinA1 expression at transcriptional and translational levels in BAEpCs in a dose (EU)-dependent manner. Our observations are consistent with those of Brannan et al. who noticed

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Fig. 6. TS-mediated transepithelial resistance decrease in BAEpCs monolayer is dependent on Erk1/Erk2, p38, and JNK. BAEpCs were treated with Erk1/ Erk2, p38, or JNK inhibitors (PD98059, 10 ␮g/ml; SB203580, 10 ␮g/ml; JNK inhibitor-II, 5 ␮g/ml) prior to TS (4EU) exposure, and electrical resistance was evaluated in real time. Data represent means ⫹ SE of 3 separate experiments. DMSO included as vehicle showed no significant effect. *P ⬍ 0.001, TS vs. control; #P ⬍ 0.001, TS ⫹ SB vs. TS alone; $P ⬍ 0.001, TS ⫹ PD vs. TS alone; ¥P ⬍ 0.001, TS ⫹ JNK Inhibitor-II vs. TS alone.

elevated levels of EphA2 in tobacco smokers compared with nonsmokers (2, 20), and patients with increased EphA2 levels had poor survival in lung cancer. Under homeostasis E-cadherin, an adherens junction protein expressed in epithelial cells, helps maintain cell-cell adhesions, and cell matrix interactions are maintained by the focal adhesion protein paxillin. In earlier studies TS exposure has been observed to decrease E-cadherin expression in mice bronchial epithelium in vivo (24). In addition, TS extract was also found to downregulate E-cadherin expression in BAEpCs in vitro (46); however, in both these studies the mechanism of Ecadherin downregulation was not known. We noticed TS exposure significantly decreased E-cadherin expression in BAEpCs. In addition, silencing EphA2 with siRNA significantly blunted TS-mediated E-cadherin downregulation in BAEpCs, suggesting that E-cadherin downregulation in BAEpCs during TS exposure was in part due to EphA2 signaling. Furthermore, when pcDNA-EphA2-transfected BAEpCs were treated with recombinant EphrinA1, E-cadherin expression was downmodulated; however, paxillin expression was not affected. These data indicate that during acute TS exposure EphA2 signaling modulates the expression of the cell-cell adhesion protein E-cadherin in BAEpCs. Besides, in response to external stimuli, the tight junction proteins are known to play an important role in paracellular permeability in airway epithelium (35, 39), and EphA2 signaling is known to modulate the tight junction protein claudin-4 in H29 carcinoma cells (43). However, it remains to be investigated whether TS-mediated EphA2 signaling also modulates the tight junction proteins in airway epithelial cells. TS-induced permeability increases across BAEpC monolayer were associated with downregulation of E-cadherin. However, the signaling mechanisms underlying the TS-mediated downregulation of E-cadherin remain unknown. Earlier reports indicate that bronchoalveolar lavage (BAL) cells obtained from smokers exhibited remarkable activation of all three classes of MAP kinase, including Erk1/2 (27). In a recent study we noticed phosphorylation of Erk1/Erk2 and p38 MAPK in TS-exposed BAEpCs (32). Here we report that inhibition of Erk1/Erk2, p38, and JNK MAPK signaling by

inhibitors PD98059, SB203580, and JNK inhibitor, respectively, significantly blunted TS-induced downregulation of E-cadherin expression in BAEpCs, indicating TS-induced downmodulation of E-cadherin was mediated via an MAPK signaling pathway. Since TS exposure was found to activate all three MAPK, consequently Erk1/Erk2, p38, and JNK MAPK are seen to regulate TS-mediated BAEpC permeability, and thus inhibition of these MAPK restored TS-mediated epithelial barrier dysfunction. Astoundingly, these MAPK are seen to be associated with TS-mediated downregulation of E-cadherin expression in BAEpCs. It is tempting to speculate that this may be a TS- specific response in BAEpCs. Interestingly, inhibition of EphA2 expression by siRNA also blunted the TS-induced epithelial hyperpermeability, indicating that EphA2 signaling was associated with TS-induced airway epithelial permeability. In addition, crosstalk between EphA2 and its ligand EphrinA1 transmits signals to the nucleus via MAP kinases (37). Taken together these data indicate that TS-mediated MAPK activation in BAEpCs is downstream to the EphA2. BAEpCs adhere to ECM through the focal adhesion protein paxillin (14). Activation of focal adhesion protein decreases cell adherence to ECM. Paxillin expression is associated with cell-ECM adhesions and maintains epithelial monolayer integrity. In addition, paxillin expression is also found to facilitate cell survival (25). We noticed no significant change in paxillin expression in TS-exposed BAEpCs. Our data indicate that TS-induced permeability changes in BAEpCs were not associated with cell-matrix adhesions, and hence the epithelial injury noticed in tobacco smokers may not be due to loss of epithelial paxillin expression. However, the present work focused on acute TS (1EU– 4EU) exposure of BAEpCs, and further investigations are required to understand the role of paxillin in chronic TS exposure-induced epithelial injury. In endothelial cells hypoxia and infection induced EphA2 expression and vascular leak, which indicates that EphA2 signaling could play a role in lung injury (7, 45). However, the expression of EphA2 and the associated mechanisms in BAEpCs remain poorly defined. In epithelial cells EphA2 signaling modulated the localization and function of claudin-4, a tight junction protein, and loss of tight junction protein promoted paracellular permeability (43). We have noticed TS to decrease resistance in BAEpCs in a dose-dependent manner compared with control cells. At lower dose TS failed to induce significant change in BAEpC resistance. In addition, TS decreased transepithelial resistance and increased the protein permeability in BAEpCs. TS-induced hyperpermeability was blocked when EphA2 expression was silenced using siRNAEphA2. Furthermore, transient overexpression of EphA2 and subsequent treatment with its ligand EphrinA1 significantly decreased electrical resistance and the protein permeability across BAEpC monolayer, suggesting EphA2 receptor signaling modulates barrier function in BAEpCs. However, we noticed EphrinA1 treatment failed to decrease transepithelial electrical resistance or to increase protein permeability in control BAEpCs. This poor response to EphrinA1 may be due to the low levels of EphA2 expressed in the control cells. This indicates that EphrinA1 may not be effective in inducing permeability changes in BAEpCs when its receptor is expressed in low levels. In mammary epithelial cells Fang et al. (13) reported that overexpression of EphA2 receptor destabilizes adherens junctions, and it was dependent on RhoA kinase

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Fig. 7. TS decreases paracellular permeability across BAEpC monolayer. A: schematic presentation of permeability changes due to loss of cell-cell adhesions Rb (paracellular) and/or due to cell-matrix adhesions ␣ (subcellular) in BAEpCs. To measure the differences between cell-cell and cell-matrix components, total transepithelial resistance was resolved into values reflecting resistance to current flow beneath the monolayer (␣) and resistance to current flow between adjacent cells (Rb). B: subcellular transepithelial permeability changes in control and TS (4EU)-exposed BAEpCs in presence and absence of MAPK inhibitors or siRNAEphA2 over time. C: paracellular transepithelial permeability changes in control and TS (4EU)-exposed BAEpCs in presence and absence of MAPK inhibitors or siRNA-EphA2 over time. BAEpCs transfected with siRNA-EphA2 and exposed to TS. Data presented are means ⫾ SE of 3 independent experiments performed at different times that gave similar results. Statistical significance: $P ⬍ 0.001, TS ⫹ PD, TS ⫹ SB, and TS ⫹ JNK-Inh vs. TS; #P ⬍ 0.001, Sc-siRNA ⫹ TS vs. siRNA-EphA2 ⫹ TS; *P ⬍ 0.001, TS vs. control.

(13). In addition, Carpenter et al. (6) reported that EphA2 receptor knockout mice were protected from bleomycininduced lung injury. Since silencing EphA2 attenuated TSinduced permeability in BAEpC, it can be speculated that EphA2 knockout mice may be protected from TS-induced lung injury. However, this remains to be investigated. The permeability increases in epithelium occur either due to loss of cell-cell adhesions or due to loss of cell-matrix interactions, which can be measured as Rb and ␣, respectively (19). In this study, we have demonstrated that TS-induced permeability changes in BAEpCs are due to the loss of cell-cell adhesions (Rb), and TS did not affect cell-matrix adhesions, the subcellular permeability (␣). Interestingly silencing

EphA2 expression significantly blunted TS-induced paracellular permeability (Rb) in BAEpCs, whereas the scrambled siRNA failed to protect TS-induced paracellular permeability. Our findings suggest that TS-induced EphA2 expression in BAEpCs modulates epithelial barrier function and thus may play a key role in the pathogenesis of lung injury in COPD. In summary, we have demonstrated that TS exposure induces EphA2 expression and downregulates the expression of the adherens junction protein E-cadherin in BAEpCs. The inhibition of EphA2 expression by siRNA blocked TSmediated hyperpermeability across BAEpC monolayers. The present data suggest that the barrier function of the BAEpCs

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Fig. 8. Immunofluorescence analysis showing actin cytoskeletal changes in TS-exposed BAEpCs. BAEpCs were either exposed to room air (control) or to TS (2EU) in presence and absence of MAPK inhibitors, or treated with rec-EphrinA1, or transfected with pcDNA-EphA2 or siRNA-EphA2. The immunofluorescence data presented are a single representation of 3 similar observations. Magnification, 400⫻.

could be associated with the levels of expression of EphA2, and higher levels of EphA2 receptor and its ligand may promote dysregulation of epithelial integrity. Moreover, TSmediated hyperpermeability across BAEpC monolayer was dependent on MAPK signaling. We conclude that during acute TS exposure EphA2 may play a significant role in airway epithelial barrier function, and thus it may represent a potential

therapeutic target to protect epithelial injury in patients with COPD. GRANTS This work was supported by New Investigator Research (NIR) Grant 09KN-09 from the Florida Department of Health to N. Najmunnisa, and Veterans Affairs Merit Grant and Univ. of Florida-Gatorade Grants to K. A. Mohammed.

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TOBACCO SMOKE AND RECEPTOR EphA2 SIGNALING DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: N.N. and K.A.M. conception and design of research; N.N., N.K., and K.A.M. performed experiments; N.N. and K.A.M. analyzed data; N.N., J.P., and K.A.M. interpreted results of experiments; N.N. and K.A.M. prepared figures; N.N. and K.A.M. drafted manuscript; N.N., P.S.S., J.P., and K.A.M. edited and revised manuscript; N.N., N.K., P.S.S., J.P., and K.A.M. approved final version of manuscript. REFERENCES 1. Antony AB, Tepper RS, Mohammed KA. Cockroach extract antigen increases bronchial airway epithelial permeability. J Allergy Clin Immunol 110: 589 –595, 2002. 2. Brannan JM, Dong W, Prudkin L, Behrens C, Lotan R, Bekele BN, Wistuba I, Johnson FM. Expression of the receptor tyrosine kinase EphA2 is increased in smokers and predicts poor survival in non-small cell lung cancer. Clin Cancer Res 15: 4423–4430, 2009. 3. Brantley-Sieders DM, Chen J. Eph receptor tyrosine kinases in angiogenesis: from development to disease. Angiogenesis 7: 17–28, 2004. 4. Brennan C, Monschau B, Lindberg R, Guthrie B, Drescher U, Bonhoeffer F, Holder N. Two Eph receptor tyrosine kinase ligands control axon growth and may be involved in the creation of the retinotectal map in the zebrafish. Development (Cambridge, England) 124: 655–664, 1997. 5. Calverley PM, Walker P. Chronic obstructive pulmonary disease. Lancet 362: 1053–1061, 2003. 6. Carpenter TC, Schroeder W, Stenmark KR, Schmidt EP. Eph-A2 promotes permeability and inflammatory responses to bleomycin-induced lung injury. Am J Respir Cell Mol Biol 46: 40 –47, 2012. 7. Cercone MA, Schroeder W, Schomberg S, Carpenter TC. EphA2 receptor mediates increased vascular permeability in lung injury due to viral infection and hypoxia. Am J Physiol Lung Cell Mol Physiol 297: L856 –L863, 2009. 8. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 410: 37–40, 2001. 9. Chauhan SD, Seggara G, Vo PA, Macallister RJ, Hobbs AJ, Ahluwalia A. Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice. FASEB J 17: 773–775, 2003. 10. Cohen AW, Carbajal JM, Schaeffer RC Jr. VEGF stimulates tyrosine phosphorylation of beta-catenin and small-pore endothelial barrier dysfunction. Am J Physiol Heart Circ Physiol 277: H2038 –H2049, 1999. 11. Dodelet VC, Pasquale EB. Eph receptors and ephrin ligands: embryogenesis to tumorigenesis. Oncogene 19: 5614 –5619, 2000. 12. English D, Cui Y, Siddiqui R, Patterson C, Natarajan V, Brindley DN, Garcia JG. Induction of endothelial monolayer permeability by phosphatidate. J Cell Biochem 75: 105–117, 1999. 13. Fang WB, Ireton RC, Zhuang G, Takahashi T, Reynolds A, Chen J. Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism. J Cell Sci 121: 358 –368, 2008. 14. Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124: 619 –626, 1994. 15. Hafner C, Becker B, Landthaler M, Vogt T. Expression profile of Eph receptors and ephrin ligands in human skin and downregulation of EphA1 in nonmelanoma skin cancer. Mod Pathol 19: 1369 –1377, 2006. 16. Hunter SG, Zhuang G, Brantley-Sieders D, Swat W, Cowan CW, Chen J. Essential role of Vav family guanine nucleotide exchange factors in EphA receptor-mediated angiogenesis. Mol Cell Biol 26: 4830 –4842, 2006. 17. Ivanov AI, Parkos CA, Nusrat A. Cytoskeletal regulation of epithelial barrier function during inflammation. Am J Pathol 177: 512–524, 2010. 18. Ivanov AI, Romanovsky AA. Putative dual role of ephrin-Eph receptor interactions in inflammation. IUBMB Life 58: 389 –394, 2006. 19. Keese CR, Giaever I. Substrate mechanics and cell spreading. Exp Cell Res 195: 528 –532, 1991. 20. Kinch MS, Moore MB, Harpole DH Jr. Predictive value of the EphA2 receptor tyrosine kinase in lung cancer recurrence and survival. Clin Cancer Res 9: 613–618, 2003. 21. Koch S, Nusrat A. Dynamic regulation of epithelial cell fate and barrier function by intercellular junctions. Ann NY Acad Sci 1165: 220 –227, 2009.

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22. Lai YM, Mohammed KA, Nasreen N, Baumuratov A, Bellew BF, Antony VB. Induction of cell cycle arrest and apoptosis by BCG infection in cultured human bronchial airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 293: L393–L401, 2007. 23. Lin YG, Han LY, Kamat AA, Merritt WM, Landen CN, Deavers MT, Fletcher MS, Urbauer DL, Kinch MS, Sood AK. EphA2 overexpression is associated with angiogenesis in ovarian cancer. Cancer 109: 332–340, 2007. 24. Liu L, Yuan Y, Li F, Liu H. Relationship between apoptosis and E-cadherin expression in bronchial epithelium of smoking mouse. J Huazhong Univ Sci Technolog Med Sci 23: 216 –218, 2003. 25. Mackinnon AC, Tretiakova M, Henderson L, Mehta RG, Yan BC, Joseph L, Krausz T, Husain AN, Reid ME, Salgia R. Paxillin expression and amplification in early lung lesions of high-risk patients, lung adenocarcinoma and metastatic disease. J Clin Pathol 64: 16 –24, 2011. 26. Miao H, Wei BR, Peehl DM, Li Q, Alexandrou T, Schelling JR, Rhim JS, Sedor JR, Burnett E, Wang B. Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway. Nat Cell Biol 3: 527–530, 2001. 27. Mochida-Nishimura K, Surewicz K, Cross JV, Hejal R, Templeton D, Rich EA, Toossi Z. Differential activation of MAP kinase signaling pathways and nuclear factor-kappaB in bronchoalveolar cells of smokers and nonsmokers. Mol Med 7: 177–185, 2001. 28. Mohammed KA, Nasreen N, Hardwick J, Logie CS, Patterson CE, Antony VB. Bacterial induction of pleural mesothelial monolayer barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 281: L119 –L125, 2001. 29. Mohammed KA, Nasreen N, Hardwick J, Van Horn RD, Sanders KL, Antony VB. Mycobacteria induces pleural mesothelial permeability by downregulating beta-catenin expression. Lung 181: 57–66, 2003. 30. Mohammed KA, Nasreen N, Tepper RS, Antony VB. Cyclic stretch induces PlGF expression in bronchial airway epithelial cells via nitric oxide release. Am J Physiol Lung Cell Mol Physiol 292: L559 –L566, 2007. 31. Mohammed KA, Wang X, Goldberg EP, Antony VB, Nasreen N. Silencing receptor EphA2 induces apoptosis and attenuates tumor growth in malignant mesothelioma. Am J Cancer Res 1: 419 –431, 2011. 32. Nasreen N, Khodayari N, Sukka-Ganesh B, Peruvemba S, Mohammed KA. Fluticasone propionate and Salmeterol combination induces SOCS-3 expression in airway epithelial cells. Int Immunopharmacol 12: 217–225, 2012. 33. Nasreen N, Mohammed KA, Antony VB. Silencing the receptor EphA2 suppresses the growth and haptotaxis of malignant mesothelioma cells. Cancer 107: 2425–2435, 2006. 34. Nasreen N, Mohammed KA, Mubarak KK, Baz MA, Akindipe OA, Fernandez-Bussy S, Antony VB. Pleural mesothelial cell transformation into myofibroblasts and haptotactic migration in response to TGF-beta1 in vitro. Am J Physiol Lung Cell Mol Physiol 297: L115–L124, 2009. 35. Niessen CM. Tight junctions/adherens junctions: basic structure and function. J Invest Dermatol 127: 2525–2532, 2007. 36. Pasquale EB. Eph receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol 6: 462–475, 2005. 37. Pratt RL, Kinch MS. Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade. Oncogene 21: 7690 –7699, 2002. 38. Puchelle E, Zahm JM, Tournier JM, Coraux C. Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease. Proc Am Thorac Soc 3: 726 –733, 2006. 39. Rezaee F, Georas SN. Breaking barriers: new insights into airway epithelial barrier function in health and disease. Am J Respir Cell Mol Biol 2014 Jan 27. [Epub ahead of print] 40. Rusznak C, Mills PR, Devalia JL, Sapsford RJ, Davies RJ, Lozewicz S. Effect of cigarette smoke on the permeability and IL-1beta and sICAM-1 release from cultured human bronchial epithelial cells of neversmokers, smokers, and patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 23: 530 –536, 2000. 41. Sutherland ER, Martin RJ. Airway inflammation in chronic obstructive pulmonary disease: comparisons with asthma. J Allergy Clin Immunol 112: 819 –827; quiz 28, 2003. 42. Tam A, Wadsworth S, Dorscheid D, Man SF, Sin DD. The airway epithelium: more than just a structural barrier. Ther Adv Respir Dis 5: 255–273, 2011. 43. Tanaka M, Kamata R, Sakai R. EphA2 phosphorylates the cytoplasmic tail of Claudin-4 and mediates paracellular permeability. J Biol Chem 280: 42375–42382, 2005. 44. Turner CE. Paxillin and focal adhesion signalling. Nat Cell Biol 2: E231–E236, 2000.

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45. Vihanto MM, Plock J, Erni D, Frey BM, Frey FJ, Huynh-Do U. Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin. FASEB J 19: 1689 –1691, 2005. 46. Wang X, Wu R, Hao T, Chen F. Effects of cigarette smoke extract on E-cadherin expression in cultured airway epithelial cells. J Tongji Med Univ 20: 32–35, 2000.

47. Zhao J, Harper R, Barchowsky A, Di YP. Identification of multiple MAPKmediated transcription factors regulated by tobacco smoke in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 293: L480–L490, 2007. 48. Zhou N, Zhao WD, Liu DX, Liang Y, Fang WG, Li B, Chen YH. Inactivation of EphA2 promotes tight junction formation and impairs angiogenesis in brain endothelial cells. Microvasc Res 82: 113–21, 2011.

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Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling.

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