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The Journal of Immunology, 2007, 179, 7001 -7011
Copyright © 2007 by The American Association of Immunologists, Inc.
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Src Tyrosine Kinases Mediate Activations of NF-{kappa}B and Integrin Signal during Lipopolysaccharide-Induced Acute Lung Injury1

Hui S. Lee*, Changsuk Moon*, Hye W. Lee*, Eun-Mi Park{dagger}, Min-Sun Cho{ddagger} and Jihee L. Kang2,*

* Department of Physiology, {dagger} Department of Pharmacology, and {ddagger} Department of Pathology, Division of Cell Biology, Ewha Medical Research Center, School of Medicine, Ewha Womans University, Seoul, South Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-{kappa}B activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-{alpha} and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-{kappa}B activation, and phosphorylation and degradation of I{kappa}B-{alpha}. The inhibition of NF-{kappa}B activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin β3, and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-{alpha}v, anti-β3, or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-{kappa}B activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-{kappa}B and integrin ({alpha}vβ3) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nuclear factor {kappa}B (NF-{kappa}B) is transcriptional regulatory proteins that modulate the expressions of immunoregulatory genes related to critical organ injury. Recent evidence indicates that LPS-induced NF-{kappa}B activation in lung tissue is associated with cytokine and chemokine production, lung neutrophilia, epithelial permeability, and lipid peroxidation (1, 2). Furthermore, the blocking of NF-{kappa}B activation by inhibition of protein tyrosine kinases or mitogen-activated protein (MAP) kinases, such as JNK and p38 MAP kinase, was found to attenuate LPS-induced acute lung injury in rodent (3, 4). Thus, these findings suggest that the inhibition of the signaling pathway upstream of NF-{kappa}B activation has therapeutic potential in terms of providing a means of preventing inflammatory cascade-associated lung injury.

Src tyrosine kinases (TKs)3 are signaling proteins that participate in several cell signaling pathways toward cytoskeletal, membrane and nuclear targets. Consequently, Src TKs mediate a wide spectrum of cellular activities, e.g., proliferation, differentiation, survival, adhesion, and migration. Src TKs have also been shown to participate in cytokine signaling and inflammatory response (5, 6). Moreover, accumulating evidence indicate that activation of c-Src, an ubiquitous Src TK, is required for NF-{kappa}B activation in macrophages and epithelial cells stimulated with TNF-{alpha}, pervanadate, hypoxia/reoxygenation, silica, or LPS (7, 8, 9, 10). Src activation in animal models of acute lung injury has been shown and the inhibition of the kinase activity attenuated LPS-induced acute lung injury (11). However, the cellular sources of c-Src during lung injury have not been identified. Src-mediated downstream signaling effectors, such as NF-{kappa}B pathway and integrin signaling, have not previously been linked to pulmonary inflammatory response.

The integrins mediate the cellular adhesions and motilities on extracellular matrix (ECM) molecules. Moreover, the Arg-Gly-Asp (RGD) motif of ECM entities that associate with integrin {alpha}vβ3 signaling appears to be involved in NF-{kappa}B activation (12). Furthermore, in macrophages, integrins play a complementary role in LPS signaling, i.e., in the activations of MAP kinases (such as ERK and JNK), and in TNF-{alpha} production (13, 14). These relations suggest that integrin signaling in macrophages mediates or amplifies inflammation. Likely, integrin signaling is involved in neutrophil activation and transmigration in vivo (15).

Src TKs are important transducers during integrin-mediated signaling (16). For example, c-Src has been shown to directly phosphorylate proteins downstream of integrin signaling (e.g., focal adhesion kinases (FAK), Cas, and paxillin) following integrin ligation (17). Moreover, recently, Src TKs were demonstrated to participate in both "inside-out" signaling leading to integrin activation and to "outside-in" signaling as a result of interactions between activated integrins and the ECM (18).

Lower respiratory tract exposure to LPS by intratracheal (i.t.) instillation is a well-recognized model of acute lung injury, and mimics, in many important pathological aspects, the clinical development of acute lung injury induced by Gram-negative pulmonary infection (19, 20, 21). Although i.p. injections of LPS also evoke acute lung injury, the pulmonary responses of animal (e.g., BAL fluid cytokine/chemokine profiles and BAL fluid cellular constituents) are different due to different LPS approaches (22, 23). We cannot find reports on the use of a selective Src TK inhibitor, PP1, in i.t. LPS-induced lung injury model. Here, we i.p. administered mice with PP1 before or after i.t. LPS treatment, and then examined the key pulmonary inflammatory responses, including neutrophil recruitment, damage to the blood-gas barrier of the lung, and inflammatory mediator production. Furthermore, we focused on the roles of Src TKs in NF-{kappa}B pathway and in ({alpha}vβ3) signaling during acute lung injury induced by i.t. LPS treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

LPS (Escherichia coli LPS, 055:B5), Arg-Gly-Asp-Ser (RGDS) and Arg-Gly-Glu-Ser (RGES) were purchased from the Sigma-Aldrich. PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), was purchased from BIOMOL. The Abs used in this study were: anti-I{kappa}B-{alpha} rabbit polyclonal, antiphospho-I{kappa}B-{alpha} (Serine 32) (New England Biolabs), rabbit anti-phospho-c-Src (pY418), anti-c-Src, anti-phospho-integrin β3 (pY773) rabbit polyclonal (BioSource), anti-integrin {alpha}v (RMV-7) mAb (24), anti-integrin β3 (HMβ) mAb (BioLegend) (25), anti-phospho-FAK (pY397), anti-FAK, anti-phospho-Pyk2 (pY402), anti- Pyk2 (Santa Cruz Biotechnology). DNA polymerase and dNTP were purchased from Invitrogen Life Technologies, and fibrinogen conjugated to Alexa Fluor-488 was purchased from Molecular Probes.

Animal protocols

Specific, pathogen-free male BALB/C mice (Daehan Biolink) weighing 19–21 g were used in all experiments. The Animal Care Committee of the Ewha Medical Research Institute approved the experimental protocol. Mice were cared for and handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Mouse pharyngeal aspiration was performed as described by Rao et al. (26). Animals were anesthetized with a mixture of ketamine and xylazine (45 and 8 mg/kg, i.p., respectively). Following anesthesia, animals were placed individually on a board in a near vertical position and tongues were withdrawn with a lined forceps. 30 µl of the test solution containing LPS (1.5 mg/kg) was then placed posterior in the throat and aspirated into lungs. Control mice were administrated sterile saline (0.9% NaCl). Mice revived unassisted after 10–20 min. Animals were administered once with PP1 (a selective Src TK inhibitor; i.p., 2 mg/kg body weight) one hour before LPS treatment and sacrificed 4 h post-LPS (27). In addition, animals administered with multiple doses of PP1 (at 2, 8, and 14 h after LPS treatment, i.p., 2 mg/kg body weight) were sacrificed at 24 h post-LPS (28).

Isolation of bronchoalveolar lavage (BAL) cells, lung tissue, and cell counts

Mice were sacrificed 4 or 24 h post-LPS, and BAL was performed through a tracheal cannula using 0.8 ml aliquots of ice-cold Ca2+/Mg2+-free phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 9.35 mM Na2HPO4, and 5.5 mM dextrose; at pH 7.4) to a total of 2.4 ml for each mouse. BAL samples so obtained were centrifuged at 500 x g for 5 min at 4°C, and cell pellets were washed and resuspended in phosphate-buffered medium. Cell counts were determined using an electronic Coulter counter fitted with a cell sizing analyzer (Coulter model ZBI with a channelizer 256; Coulter Electronics), as described by Lane and Mehta (29). Neutrophils and alveolar macrophages were identified by their characteristic of cell diameter (30). Recovered cells were 98% viable, as determined by trypan blue dye exclusion. After BAL, lungs were removed, immediately frozen in liquid nitrogen, and stored at –70°C.

Measurement of total protein

BAL sample protein concentration was used as an indicator of blood-pulmonary epithelial cell barrier integrity. Total protein was measured according to the method of Hartree (31), using BSA as a standard.

Measurement of TNF-{alpha}

First BAL fluid samples were assayed using TNF-{alpha} (BioSource International) enzyme-linked immunosorbent assay (ELISA) kits (R &D Systems) according to the manufacturer’s instructions.

Zymographic analysis of matrix metalloproteinase-9 (MMP-9)

The gelatinolytic activities of BAL fluid samples or lung tissue homogenate samples were determined by zymography using gelatin copolymerized with acrylamide in gel, as described previously (32). Aliquots, normalized for equal amounts of protein (5 or 10 µg) were electrophoresed in 10% SDS-PAGE gels using 0.1% gelatin as substrate without boiling under nonreducing conditions. After removing SDS with 2.5% Triton X-100 for 2 h, gels were incubated for 20 h at 37°C in 50 mM Tris-Cl (pH 7.4) containing 10 mM CaCl2 and 0.02% NaN3. The gels were then stained for 1 h in 7.5% acetic acid/10% propanol-2 containing 0.5% Coomassie Brilliant Blue G250 and destained in the same solution without dye. Positions of gelatinolytic activity were unstained on a heavily stained background, and clear zymogram bands were photographed using negative Polaroid 665 film and MMP-9 activities were quantified by densitometry using an UltroScan XL laser densitometer (LKB, model 2222-020). To confirm MMP-9 activity, aliquots of BAL fluid were analyzed by Western blotting using anti-human MMP-9 mAb.

Western blot analysis

Lung tissue homogenate samples (50 µg protein/lane) or aliquots of acellular BAL fluid (5 µg protein/lane for MMP-9) were separated on 10% SDS-polyacrylamide gels. Separated proteins were electrophoretically transferred onto nitrocellulose paper and blocked for 1 h at room temperature with Tris-buffered SAL containing 3% BSA. Membranes were then incubated with the indicated Ab and visualized by chemiluminescence (ECL).

p38 MAP kinase immunoprecipitation assays

Kinase activity of p38 MAP kinase in lung tissue was assayed from immunoprecipitated samples by the ability to phosphorylate ATF-2 as previously described (33). Equal amounts of protein were used for the immunoprecipitation procedures. Anti-phospho-p38 mAb (Cell Signaling Technologies) immobilized by cross-linkage to agarose hydrazide beads was added to each lysate and incubated overnight at 4°C. For immune complex kinase assays, immunoprecipitates were resuspended in 25 µl of kinase buffer (25 mM Tris, 5 mM β-glycerolphosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM MgCl2) supplemented with 200 µM ATP and 2 µg ATF-2 fusion protein (Cell Signaling Technologies) and incubated for 1 h at 30°C. After incubation, the reaction was terminated using Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-ME). The samples were loaded onto a 10% SDS-polyacrylamide gel with boiling. Phospho-ATF-2 rabbit polyclonal Ab (Cell Signaling technologies) was used for detection at a dilution of 1:1000.

Nuclear extracts

Nuclear extracts were prepared using a modification of the method described by Kang et al. (32). Typically, BAL samples from 10 mice in each experimental group were pooled to obtain a sufficient cell number to conduct EMSA. Pooled BAL cells were resuspended in DMEM (Mediatech), supplemented with 5% FBS (HyClone), 2 mM glutamine, and 1,000 U/ml penicillin-streptomycin. DMEM (5 ml) containing 5 x 106 alveolar macrophages, was added to 6-well plates and incubated at 37°C in a humidified 5% CO2 atmosphere for 1 h. Nonadherent cells were then removed with two 1 ml aliquots of DMEM. Adherent cells (>95% alveolar macrophages) were harvested and resuspended in hypotonic buffer A (100 mM HEPES, pH 7.9, 10 mM KCl, 0.1 M EDTA, 0.5 mM DTT, 0.6% Nonidet P-40, and 0.5 mM PMSF) for 10 min on ice, vortexed for 10s, and nuclei were pelleted by centrifugation at 12,000 rpm for 30 s. Nuclear extracts were also prepared from lung tissue samples. Aliquots of frozen tissue were mixed with liquid nitrogen and ground to a powder using a mortar and pestle. Ground tissues were placed in a Dounce tissue homogenizer (Kontes) containing 4 ml of buffer A to lyse cells. Supernatants containing intact nuclei, were incubated on ice for 5 min, and centrifuged for 10 min at 5000 rpm. Nuclear pellets were resuspended in buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.42 M NaCl, 1 mM EDTA, and 0.5 mM PMSF) for 30 min on ice. The supernatants, which contained nuclear proteins, were collected by centrifuging at 10,000 rpm for 2 min and stored at –70°C.

EMSA

Binding reaction mixtures (10 µl), containing 5 µg (4 µl) of nuclear extract protein, 2 µg of poly(dI-dC)·poly(dI-dC) (Sigma-Aldrich), and 40,000 cpm 32P-labeled probe in binding buffer (4 mM HEPES, pH 7.9, 1 mM MgCl2, 0.5 mM DTT, 2% glycerol, and 20 mM NaCl) were incubated for 30 min at room temperature. Protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gels in 1x TBE buffer, and autoradiographed. Autoradiograph signals of activated NF-{kappa}B were quantitated by densitometry using an UltroScan XL laser densitometer (LKB, model 2222-020) to determine band intensities.

The oligonucleotide used as a probe for EMSA was a dsDNA fragment, containing the NF-{kappa}B consensus sequence (5'-CCTGTGCTCCGGGAATTTCCCTGGCC-3'), and labeled with [{alpha}-32P]dATP (Amersham Biosciences) using DNA polymerase Klenow fragment (Invitrogen Life Technologies). Cold competition was performed by adding 100 ng of unlabeled double-stranded probe to reaction mixtures.

Fibrinogen binding assay

BAL cells were resuspended in DMEM, supplemented with 5% FBS, 2 mM glutamine, and 1,000 U/ml penicillin-streptomycin. DMEM (5 ml) containing 1 x 105 alveolar macrophages was then added to 24-well plates and incubated at 37°C in a humidified 5% CO2 atmosphere for 1 h. Nonadherent cells were then removed using two 1-ml aliquots of DMEM, and adherent cells (>95% alveolar macrophages) were incubated in 100 µl of DMEM containing 80 µg/ml fibrinogen conjugated to Alexafluor-488, 2 mM CaCl2, and 4 mg/ml BSA for 20 min at room temperature in the absence or presence of MnCl2 (1 µM) as a positive control. Cells were then washed once with PBS containing 2 mM CaCl2, and 4 mg/ml BSA, resuspended in a fixation solution, and after a 10-min incubation on ice rewashed five times. Nuclei were stained with Hoechst no. 33258 (5 µg/ml; Calbiochem) for 10 min at room temperature and slides were then mounted. Images were collected using an LSM Image Examiner on a confocal laser scanning microscope (LSM-5 Pascal Exciter; Carl Zeiss).

Lung histology

Lungs were fixed with 10% buffered formalin at room temperature for 48 h, dehydrated, and embedded in paraffin. Sections (4 µm) were stained with H&E.

Immunohistochemical localization of phospho-c-Src

Sections (4 µm) were taken from formalin-fixed, paraffin-embedded tissue blocks and immunostained. Briefly, slides were deparaffinized twice in xylene for 5 min and rehydrated through graded ethanol solutions to distilled water. Ag retrieval was performed by heating sections in citrate buffer (pH 6.0). Sections were then immunostained using an automated unit (Bond Automated Immunohistochemistry, Vision Biosystems) and the Bond polymer detection system with counterstain (Vision Biosystems). The process used included endogenous peroxidase blocking by 3% hydrogen peroxide for 10 min, incubation with primary Abs for phospho-c-src (pY418, 1:400) for 15 min at room temperature, and the use of polymeric HRP-linker Ab conjugate as a secondary Ab. DAB (3,3-diaminobenzidine) substrate was used as the chromogen to yield a brown reaction product.

Statistical analysis

Values are expressed as means ± SEs. Intergroup comparisons were made using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was accepted for p values <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phosphorylation of c-Src in lung

c-Src is known to be a key mediator of LPS-induced NF-{kappa}B activation in vitro, and as a component of focal-adhesion complexes, to be a selective regulator of integrin-cytoskeleton interactions. Therefore, we first investigated whether c-Src is activated in the lungs of mice i.t. treated with LPS by analyzing lung lysates for c-Src phosphorylation using an anti-specific c-Src-pY418. The phosphorylation of c-Src in lung tissue was found to be enhanced by 2.1-fold 4 h after LPS treatment (post-LPS) (Fig. 1A, left). The level of phosphorylation of this kinase was slightly decreased 24 h post-LPS (Fig. 1A, right). The administration of PP1, a selective Src TK inhibitor, at 1 h pre-LPS, decreased the LPS-induced phosphorylation of c-Src in lung tissue at 4 h post-LPS. Posttreatment with PP1 (three times at 2, 8, and 14 h post-LPS) also inhibited c-Src phosphorylation in lung tissue at 24 h post-LPS. c-Src activation was barely detectable in mice treated with saline or saline-PP1 at 4 and 24 h post-LPS.


Figure 1
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  		FIGURE 1. Phosphorylations of c-Src (A and C) and activations of p38 MAP kinase (B) in lung tissue at 4 and 24 h post-LPS. Where indicated, mice were i.p. administered PP1 once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 three times at 2, 8, and 14 h post-LPS, were sacrificed at 24 h post-LPS. A, Western blots with anti-c-Src-pY418/c-Src were employed to monitor c-Src. Relative values for phosphorylated c-src vs nonphosphorylated c-src are indicated below the gel. B, p38 MAP kinase was immunoprecipitated from lung tissue homogenates and immunoprecipitates were combined with ATF-2 in the presence of ATP for immune complex kinase assays. Relative values for p38 activities are expressed as comparison with saline controls. Values represent means ± SEM of results from 5 mice per group. *, Significantly different vs saline, p < 0.05, and +, significantly different vs the LPS only treated animals, p < 0.05. C, Immunohistochemical staining of lungs with anti-c-Src-pY418 Ab at 4 and 24 h post-LPS. Lung sections were immunostained using anti-c-Src-pY418 Ab, as described in Methods. Representative results from 6 mice per group are shown. Long arrows represent neutrophils and short arrows represent alveolar macrophages. Original magnification, x400.

 
Liu et al. (34) have shown that non-Src TKs, including c-Abl and p38 MAP kinase, are moderately (IC50 ~ 1 µM) inhibited by PP1. Previously, we found that inhibition of p38 MAP kinase by SB203580 attenuated LPS-induced pulmonary inflammation (4). Therefore, we examined whether PP1 at the dose used affects p38 MAP kinase activation in the lungs after LPS treatment in mice. To determine p38 MAP kinase activation in the lung tissue from LPS-treated animals, immunoprecipitation studies were employed, where the ability of p38 to phosphorylate the substrate ATF-2 was determined directly. Pre or posttreatment with PP1 had little effect on LPS-induced p38 MAP kinase activation at 4 or 24 h post-LPS, respectively (Fig. 1B). We observed the same result as the Western blot analysis with a phospho-specific p38 MAP kinase Ab (data not shown). These data suggest that PP1 selectively inhibits Src TK activity in vivo and some of the kinase-mediated effects during LPS-induced acute lung injury without blocking p38 MAP kinase.

Localization of phospho-c-Src in lung

To identify the cells expressing phospho-c-Src during the development of acute lung injury post-LPS, serial sections of lung tissue were immunostained with anti-c-Src-pY418. Weakly positive c-Src-pY418 cells were observed within a few neutrophils in vascular spaces in saline-treated controls (Fig. 1C). However, at 4 h post-LPS, substantial increases in c-Src phosphorylation were observed in vascular margination in concert with alveolar infiltration of neutrophils (Fig. 1C). Furthermore, c-Src phosphorylation was also found to be higher in alveolar macrophages in LPS treated animals at 4 h post-LPS than saline controls (Fig. 1C). At 24 h post-LPS, c-Src phosphorylation was observed in many recruited inflammatory cells (mainly neutrophils) and in alveolar macrophages (Fig. 1C). These findings suggest that LPS induces substantial c-Src activation in both recruited neutrophils and alveolar macrophages in lung during the initiation and progression of lung injury. PP1 treatment at 2, 8, and 14 h post-LPS resulted in decreases in c-Src phosphorylation in alveolar cells, including alveolar macrophages and neutrophils, and in numbers of recruited neutrophils at 24 h post-LPS (Fig. 1C). Taken together, the Western blot data show that PP1 is effective in blocking LPS-induced c-Src activation in these cell types and thus, their mediation of pulmonary inflammation.

Total protein in BAL fluid

BAL protein levels in LPS-treated mice were substantially increased at 4 h post-LPS and were further increased at 24 h post-LPS at 1.8- and 2.2-fold that of saline controls, respectively (Fig. 2A). Treatment with PP1 before or after LPS inhibited LPS-induced BAL protein increases by 95 and 80% at 4 or 24 h post-LPS, respectively.


Figure 2
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  		FIGURE 2. Reduction in LPS-induced lung inflammation by PP1. Where indicated, mice were i.p. administered PP1 once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 three times at 2, 8, and 14 h after LPS were sacrificed at 24 h post-LPS. A, Total levels of protein in bronchoalveolar lavage (BAL) fluid. Values represent means±SEM of results from 10 mice per group. *, Significantly different vs saline, p < 0.05, and +, significantly different vs the LPS only treated animals, p < 0.05. B, Lung sections were H&E stained, as described in Materials and Methods. Representative results from 6 mice per group are shown.

 
BAL inflammatory cell yields

BAL inflammatory cells were differentially analyzed to evaluate the effect of PP1 on BAL inflammatory cell yields induced by LPS treatment. The number of neutrophils recovered in BAL fluid samples from LPS-treated mice increased by 3.0- and 23-fold vs unstimulated controls at 4 and 24 h post-LPS, respectively (Tables I and II). At these times, recruited cells were predominantly neutrophils. Pretreatment with PP1 (1 h pre-LPS) significantly inhibited neutrophil number increases at 4 h post-LPS by 84% (p < 0.05). Similarly, at 24 h post-LPS, treatment with PP1 after LPS significantly inhibited neutrophil numbers by 50% (vs LPS animals, p < 0.05), whereas neutrophil numbers in mice treated with saline-followed by PP1 were similar to those of saline treated controls (p < 0.05).


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  		Table I. Effects of PP1 on neutrophil and alveolar macrophage numbers in bronchoalveolar lavage fluid at 4 h post-LPSa

 

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  		Table II. Effects of PP1 on neutrophil and alveolar macrophage numbers in bronchoalveolar lavage fluid at 24 h post-LPSa

 
Alveolar macrophage numbers were not significantly altered at 4 h post-LPS, but were elevated by 2.0-fold at 24 h post-LPS (Tables I and II, p < 0.05). PP1 treatment after LPS significantly inhibited this increase by 57% (p < 0.05).

Histological lung sections obtained at 24 h post-LPS confirmed BAL findings. H&E sections of lungs fixed with paraformaldehyde revealed significant reductions in peribroncheal and intraalveolar infiltration of neutrophils in lungs treated with PP1 compared with those treated with LPS at 24 h (Fig. 2B).

TNF-{alpha} production and MMP-9 activity

TNF-{alpha} and MMP-9 are representative proinflammatory mediators, which play major roles in neutrophil influx and lung damage, and their gene regulations are known to be NF-{kappa}B dependent. Levels of TNF-{alpha} in BAL fluid were measured by ELISA. As shown in Fig. 3A, at 4 or 24 h post-LPS, TNF-{alpha} levels increased 71- and 7.6-fold that of the saline treated controls, respectively. Moreover, these increases were significantly reduced by the pre or posttreatment with PP1 at 4 or 24 h post-LPS (by 71 and 81% vs LPS, respectively, p < 0.05).


Figure 3
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  		FIGURE 3. Inhibition of LPS-induced TNF-{alpha} production and MMP-9 activity by PP1 at 4 and 24 h post-LPS. Where indicated, mice were i.p. administered PP1once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 at 2, 8, and 14 h post-LPS, were sacrificed at 24 h post-LPS. TNF-{alpha} levels in bronchoalveolar lavage fluid were quantified using enzyme-linked immunosorbent assay (A). Gelatinolytic activity in bronchoalveolar lavage fluid (B) and lung tissue homogenates (C). BAL fluid and lung tissue lysates were analyzed by zymography followed by scanning densitometry. The 92-kDa genolytic bands correspond to MMP-9. Densities are expressed as percentages of saline controls. Western blot of BAL fluid samples from each group with an anti-human-MMP-9 mAb (D). Values represent means ± SEM of results from 5 or 10 mice per group. *, Significantly different from saline controls, p < 0.05, and +, significantly different from LPS treated animals, p < 0.05.

 
BAL fluid and lung tissue homogenates were analyzed for evidence of MMP-9 activity by gelatin zymography. LPS induced a distinct increase in gelatinolytic activity and the most prominent band was observed at 92-kDa (corresponding to MMP-9 by Western blot analysis with anti-MMP-9 mAb) in BAL fluid (Fig. 3B, left) and lung homogenate samples (Fig. 3C, left) at 4 h post-LPS. At 24 h post-LPS, unlike TNF-{alpha}, MMP-9 activity in BAL fluid (Fig. 3B, right) and lung homogenates (Fig. 3C, right) further increased. Pre or posttreatment with PP1 inhibited LPS-induced MMP-9 activity in BAL fluid at 4 and 24 h (by 82 and 96% vs LPS, respectively) (Fig. 3B). Similarly, PP1 inhibited this activity in the lung homogenates by 55 and 43% at 4 and 24 h, respectively (Fig. 3C). MMP-9 activity was undetectable in samples obtained from saline control and saline-PP1 treated animals. PP1 was also observed to have an inhibitory effect on MMP-9 expression in BAL fluid at 4 and 24 h (Fig. 3D, upper and lower).

NF-{kappa}B activation in lung tissue and alveolar macrophages

The DNA-binding activities of NF-{kappa}B in lung tissue were elevated 4.6-fold at 4 h post-LPS vs saline controls, and reduced to 3.7-fold at 24 h (Fig. 4, A and B). PP1 treatment before or after LPS blocked these increases by 60 and 67% in lung tissue at 4 and 24 h post-LPS, respectively (Fig. 4, A and B, lane 3). In addition, 5.2-fold increases in the DNA-binding activity of NF-{kappa}B were observed in alveolar macrophages at 4 h post-LPS vs saline treated controls (Fig. 4C). The inhibitory effect of PP1 (58%) was also observed in alveolar macrophages at 4 h post-LPS (Fig. 4C, lane 3).


Figure 4
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  		FIGURE 4. Inhibition of LPS-induced NF-{kappa}B activation by PP1 in lung tissue at 4 (A) and 24 h (B) post-LPS and in alveolar macrophages at 4 h post-LPS (C). Where indicated, mice were i.p. administered PP1 once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 at 2, 8, and 14 h post-LPS, were sacrificed at 24 h post-LPS. Nuclear extracts were prepared from lung tissues or alveolar macrophages. Densities of NF-{kappa}B bands on EMSA are expressed as percentages of those of saline controls. Results are means ± SEM of results from 5 mice per group. *, Significantly different vs saline, p < 0.05, and +, significantly different vs the LPS only treated animals, p < 0.05.

 
The addition of the cold competitor eliminated the specific bands in the samples from LPS-treated mice, indicating that the band on the autoradiogram was specific for NF-{kappa}B binding (data not shown).

Phosphorylation and degradation of I{kappa}B-{alpha} in lung tissue

To examine the mechanism underlying the actions of PP1 on the LPS induction of pathways leading to NF-{kappa}B activation, we analyzed serine phosphorylation and degradation of I{kappa}B-{alpha} in lung tissue from LPS and LPS-PP1 treated mice by Western blotting with anti-phospho-I{kappa}B-{alpha} (serine-32), and anti-I{kappa}B-{alpha} Ab. LPS treatment resulted in increases in serine phosphorylation and degradation of I{kappa}B-{alpha} at 4 h post-LPS (Fig. 5, A and B left). In-line with the change of NF-{kappa}B activation at different time points, LPS-induced serine phosphorylation and degradation of I{kappa}B-{alpha} decreased slightly at 24 h vs at 4 h post-LPS, but these were still greater than those of the saline controls (Fig. 5, A and B right). PP1 treatment before or after LPS blocked LPS-induced phosphorylation and degradation of I{kappa}B-{alpha} at 4 h and 24 h post-LPS. However, PP1 alone had little effect on these events.


Figure 5
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  		FIGURE 5. Inhibition of LPS-induced phosphorylation (A) and degradation of I{kappa}B-{alpha} (B) in lung tissue by PP1 at 4 and 24 h after LPS treatment. Where indicated, mice were i.p. administered PP1 once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 at 2, 8, and 14 h post-LPS, were sacrificed at 24 h post-LPS. Western blots with anti-serine phospho-I{kappa}B{alpha} (Ser32)/I{kappa}B{alpha} Ab were employed to monitor phosphorylated I{kappa}B-{alpha} or I{kappa}B-{alpha} in lung tissue lysates. Densities are expressed as percentages of saline controls. The results are presented as means±SEM of results from 5 mice per group. *, Significantly different vs saline, p < 0.05, and +, significantly different vs the LPS only treated animals, p < 0.05.

 
Activation of integrin ({alpha}vβ3) signaling

Integrin ({alpha}vβ3) signaling following ligation has been shown to activate NF-{kappa}B in concert with c-Src in vitro (35). The present study confirmed the involvement of Src TKs, like c-Src, in LPS-induced NF-{kappa}B activation during the initiation and progression of pulmonary inflammation and injury in our mouse model. To determine whether Src TKs regulate integrin ({alpha}vβ3) signaling during the development of acute lung injury, we first evaluated whether LPS activates of integrin ({alpha}vβ3) signaling, e.g., {alpha}v or β3 expression, and phosphorylations of integrin β3, and FAK and Pyk2 (FAK family members), in lung tissue, and if so, whether the inhibition of Src TKs by PP1 blocks LPS-induced integrin signaling. No difference was found in the expressions of integrin {alpha}v (data not shown) or β3 between LPS-treated mice and saline controls at 4 or 24 h (Fig. 6A, middle bands). However, the phosphorylations of integrin β3 (Fig. 6A, upper bands), FAK (Fig. 6B), and Pyk2 (Fig. 6C) in lung tissue were enhanced at both times post-LPS. Pre or posttreatment with PP1 suppressed LPS-induced phosphorylations of these integrin signaling proteins at 4 or 24 h, respectively.


Figure 6
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  		FIGURE 6. Inhibition of the LPS-induced activation of integrin signaling in lung tissue by PP1: phosphorylations of integrin β3 (A), FAK (B), and Pyk2 (C) at 4 and 24 h post-LPS. Where indicated, mice were i.p. administered PP1 once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 at 2, 8, and 14 h post-LPS, were sacrificed at 24 h post-LPS. Western blots with anti-specific (phospho) Ab were employed in lung tissue homogenates. Relative values of phosphorylated integrin β3, FAK, or Pyk2 vs nonphosphorylated integrin β3, FAK, or Pyk2, respectively, are indicated below the gel. Values represent means ± SEM of results from 5 mice per group. *, Significantly different vs saline, p < 0.05, and +, significantly different vs the LPS only treated animals, p < 0.05.

 
In addition, we examined the fibrinogen binding activity of alveolar macrophages, because fibrinogen is known to be a specific and physiologic ligand for activated integrin complexes, such as, of {alpha}vβ3 (36). As was expected, direct addition of MnCl2, an agonist that increases fibrinogen binding to cells by activating integrin {alpha}vβ3 (37, 38), increased fibrinogen binding to alveolar macrophages obtained from saline-treated controls (Fig. 7, A and B). Increased fibrinogen binding activity of alveolar macrophages obtained from mice treated with LPS was found in many confocal microscope fields, whereas pre or posttreatment with PP1 suppressed this activity at 4 or 24 h post-LPS, respectively (Fig. 7, A and B). These findings suggest that Src TKs up-regulate LPS-induced integrin signaling during the development of acute lung injury.


Figure 7
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  		FIGURE 7. Inhibition of LPS-induced increases in fibrinogen binding activity of alveolar macrophages by PP1 at 4 (A) and 24 h (B) post-LPS. Where indicated, mice were i.p. administered PP1 once at 1 h before LPS, and sacrificed at 4 h post-LPS. Mice i.p. administered PP1 at 2, 8, and 14 h post-LPS, were sacrificed at 24 h post-LPS. Alveolar macrophages were incubated with fibrinogen conjugated to Alexafluor-488 for 20 min in the absence or presence of MnCl2. After exposure to fibrinogen for 20 min, cells were fixed and analyzed by confocal fluorescence microscopy. The results are representative results obtained from 10 mice per group.

 
Involvement of integrin ({alpha}vβ3) signaling in NF-{kappa}B activation

To further investigate whether integrin ({alpha}vβ3) signaling associated with Src TKs is involved in NF-{kappa}B activation during LPS-induced acute lung injury, we assessed the functional link between integrin signaling and LPS-induced NF-{kappa}B activation. To block integrin {alpha}v or β3 binding, mice received anti-{alpha}v, anti-β3,or the tetrapeptide RGDS (5 mg/kg body weight of each blockade, i.p.) at 1h before LPS treatment. Animals were sacrificed at 4 h post-LPS. Fig. 8, A and B show that pretreatment with anti-{alpha}v, anti-β3 or RGDS markedly blocked LPS-induced NF-{kappa}B activation in lung tissue in the order anti-β3 ≥RGDS > anti-{alpha}v. However, IgG Ab or RGES (a negative control), had little effect on LPS-induced NF-{kappa}B activation in lung tissue at 4 h post-LPS. NF-{kappa}B activity in lung tissue was not detectable at any time in animals treated with saline-RGDS.


Figure 8
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  		FIGURE 8. Inhibition of LPS-induced NF-{kappa}B activation and integrin ({alpha}vβ3) signaling in lung tissue by anti-integrin {alpha}v, β3, or RGDS at 4 h post-LPS. Where indicated, mice were i.p. administered anti-{alpha}v, -β3, IgG, RGDS, or RGES (5 mg/kg body weight) once at 1 h before LPS, and sacrificed at 4 h post-LPS. Nuclear extracts were then prepared from lung tissues. Densities of NF-{kappa}B bands on EMSA are expressed as percentages of saline controls (A and B). Western blots with anti-specific (phospho) Ab were employed in lung tissue homogenates. Relative values of phosphorylated c-Src, FAK, or Pyk2 vs nonphosphorylated c-Src, FAK or Pyk2, respectively, are indicated below the gel (C–E). Results are means±SEM of results from 5 mice per group. *, Significantly different vs saline, p < 0.05, and +, significantly different vs the LPS only treated animals, p < 0.05.

 
In addition, we examined whether c-Src and other tyrosine kinases, such as FAK and Pyk2, participate in "outside-in" integrin ({alpha}vβ3) signaling toward NF-{kappa}B activation during LPS-induced acute lung injury. As shown Fig. 8C, blocking Abs to {alpha}v or β3 integrin subunit inhibited LPS-induced c-Src phosphorylation in lung tissue at 4 h post-LPS in the order anti-β3 > anti-{alpha}v. Furthermore, these Abs markedly inhibited LPS-induced phosphorylations of FAK and Pyk2 (Fig. 8, D and E). Collectively, these data suggest that c-Src along with FAK and Pyk2 participating in "outside-in" integrin ({alpha}vβ3) signaling partly mediates NF-{kappa}B activation during LPS-induced acute lung injury.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our previous study indicates that Src TKs, like c-Src, mediate LPS-induced NF-{kappa}B activation through phosphorylation and degradation of I{kappa}B-{alpha} in RAW 264.7 macrophages (10). Here, we report a functional link between Src TKs, like c-Src,-mediated NF-{kappa}B activation and inflammatory lung injury in response to LPS. Initially, we confirmed that c-Src activation was significantly increased in lung at 4 h after the intratracheal instillation of LPS and that this level was only slightly reduced at 24 h post-LPS (1.5 mg/kg). Others have also found that this Src TK activity is increased in the lungs of mice treated with LPS i.p. at 2 h post-LPS (11), but, at 24 h post i.p. LPS at the higher concentration of 6 mg/kg, c-Src phosphorylation in lung was not observed. This disparity between the timings of c-Src activation may be related to different routes of LPS administration. Our immunohistochemical findings are the first evidence that active, phosphorylated, c-Src was mainly localized to alveolar macrophages and neutrophils recruited to the lung during the initiation and progression of acute lung injury. Kang et al. (10) and others (39) have reported that LPS or TNF-{alpha} activates c-Src in RAW 264.7 macrophages and HL60 cells (a neutrophil cell line) in vitro, respectively. These findings suggest that c-Src plays important roles in alveolar macrophage- or neutrophil-mediated inflammatory responses, and that these include the productions of proinflammatory cytokines and chemokines, and the migration of inflammatory cells during acute lung injury. Western blot and histological findings, show that pre or posttreatment with PP1 (a selective Src TK inhibitor), inhibits LPS-induced c-Src activation at 4 or 24 h post-LPS, respectively, which indicates that this Src TK inhibitor is effective at blocking c-Src activation in lung inflammatory cells.

In the present study, posttreatment with PP1 resulted in significant inhibitions of LPS-induced increases in total protein in BAL fluid, which serves as a marker of damage to the lung blood-gas barrier, and of LPS-induced neutrophil recruitment to lung at 24 h post-LPS, although these inhibitory effects of PP1 in the posttreatment model are less effective as compared with the pretreatment model at 4 h post-LPS. In addition, the lung histologic findings indicate that posttreatment with PP1 effectively reduced LPS-induced lung parenchymal infiltration of neutrophils at 24 h post-LPS. The inhibitory effects of PP1 on TNF-{alpha} levels and MMP-9 activity in BAL fluid in the posttreatment model at 24 h were slightly greater than the pretreatment model at 4 h. There were no significant differences in the LPS-induced changes (total protein, neutrophil recruitment, and TNF-{alpha} levels in BAL fluid) at 24 h between groups from the posttreatment with PP1(at 2, 8, and 16 h after LPS) and the pretreatment with PP1 (at 1 h before as well as 8 and 14 h after LPS) (data not shown). The fact that the protective effect of PP1 at 24 h post-LPS was evident when it was administered after LPS treatment, suggests that selective Src TK inhibitors like PP1 have therapeutic potential for the treatment of acute lung injury. The protective effect of another Src TK inhibitor, TyrA1 or SU-6656, administered at 6 h after i.p. LPS injection, has been demonstrated in mice with acute lung injury (28). In addition to lung, PP2 treatment 30 min after middle cerebral artery occlusion also reduced the infarct size of focal ischemic brain injuries (40). Moreover, the results of Akiyama et al. (41) indicate that PP1 treatment 10 min after compression reduces secondary damage after spinal cord injury by significantly reducing contusion-induced lesion sizes, macrophage infiltration and the expressions of TNF-{alpha} and IL-1β mRNA. Moreover, following myocardial infarction, genetic or pharmacological blockade (with PP1) of Src TKs preserved endothelial cell barrier function and suppressed vascular permeability and infarct volume at 24 h, and improved long-term cardiac function, fibrosis, and survival (27). The protective effects of Src inhibitors observed suggest that Src TKs could be a potential therapeutic target in the pathogenesis of different organ injury.

Consistent with our previous kinetic results in rats (32), the DNA binding activity of NF-{kappa}B was enhanced at 4 h after the intratracheal instillation of LPS in mice and this was reduced slightly at 24 h post-LPS. Treatment with PP1 before or after LPS blocked NF-{kappa}B activation in lung tissue via down-regulations of the phosphorylation and degradation of I{kappa}B-{alpha} at 4 or 24 h post-LPS, respectively. In addition, the inhibitory effect of PP1 on LPS-induced NF-{kappa}B activation was evident in alveolar macrophages at 4 h post-LPS. These findings indicate that Src TKs-mediated NF-{kappa}B activation in alveolar macrophages plays a key role in the initiation of lung injury in vivo. The present study is the first to demonstrate the role of Src TKs, like c-Src, in NF-{kappa}B activation in an animal model of LPS-induced acute lung injury.

Several previous studies have suggested that integrins participate in LPS signaling in macrophages (13, 42, 43). Furthermore, the binding of ligands to integrin {alpha}vβ3 was found to induce in NF-{kappa}B activation (35), thus suggesting that integrin {alpha}vβ3 engagement influences an NF-{kappa}B-dependent program of gene expression. Integrin activation involves both a conformation change in {alpha}β heterodimer, and the clustering of laterally diffusing integrins in the plasma membrane (44, 45). Ligand-induced phosphorylation on β3 results in integrin clustering and subsequent direct and indirect recruitment of a signaling and adaptor protein complex to the cytoplasm tail, including Src, Syk, FAK, phosphatide kinase, vinculin, talin and {alpha}-actin proteins (46). Our in vivo experiments demonstrate increases in the fibrinogen binding activity of alveolar macrophages of mice treated with LPS at 4 or 24 h post-LPS, suggesting that LPS induces integrin activation, leading to integrin engagement with a soluble adhesion ligand fibrinogen. Furthermore, we found that LPS induces integrin signaling, e.g., the phosphorylations of integrin β3, FAK, and Pyk2 in lung tissue at these time points. These findings suggest that LPS activates integrins, increasing integrin ligation and clustering, and thus triggering an "outside-in" signaling that amplifies the action of LPS. Consistent with our in vivo data, Monick et al. (14) proposed the hypothesis that in vitro treatment of macrophages with LPS results in "inside-out" integrin activation, and increased cellular adherence, providing integrin "outside-in" signaling to MAP kinases. Indeed, signals from integrin receptors seem to be required for optimal LPS signaling in macrophages.

Bouaouina et al. (18) demonstrated that constitutively active Src TKs in neutrophils stimulated with TNF-{alpha} are involved in the integrin signaling that leads to β2 integrin activation in neutrophils. In contrast, the findings of the present study suggest that the inducible active Src TKs are involved in the integrin signaling activation induced by LPS. Furthermore, our immunostaining findings clearly indicate increases in c-Src activation by LPS in neutrophils recruited to the lung. Thus, we consider that disparities in the nature of active Src TKs (constitutive vs inducible) in induction of integrin signaling is probably due to different stimuli, i.e., TNF-{alpha} or LPS, and the in vitro and in vivo experimental models used.

Recent evidence indicates the direct involvement of c-Src in the phosphorylations of down-stream integrin signaling proteins, such as integrin β3, FAK, CAS, and paxillin, following integrin ligation (17). Therefore, the LPS-induced activation of c-Src may affect integrin signaling via two ways, i.e., via activating "inside-out" signal leading to integrin activation and by directly affecting integrin signaling proteins during acute lung injury.

The cell types that express high levels of integrin {alpha}vβ3 include bone resorbing osteoclasts, activated macrophages, a small number of neutrophils, angiogenic endothelial cells, and migrating smooth muscle cells (47). Recently, integrin {alpha}vβ3 was suggested to play a key role in the initiation or progression of several diseases, including rheumatoid arthritis, osteoporosis, atherosclerosis and cancer, and to play a role in the pathogenesis of HIV type 1 infection (48, 49, 50, 51). However, integrin {alpha}vβ3 has received little attention in terms of its potential role in acute lung injury. In the present study, we found that LPS-induced NF-{kappa}B activation in lung at 4 h post-LPS was inhibited by blocking {alpha}v or β3 integrin binding. These findings suggest the possible involvement of integrin ({alpha}vβ3) signal associated with Src TKs in LPS-induced NF-{kappa}B activation as an upstream pathway. Furthermore, the data from previous studies suggest the possible effects of integrin {alpha}vβ3 on proinflammatory mediator productions by monocytes and neutrophils stimulated with pathogens (52, 53). Whether integrin {alpha}vβ3 signal contributes LPS-induced inflammatory lung injury is the focus of our current investigation.

However, the manner in which integrin {alpha}vβ3 is linked to NF-{kappa}B activation is unclear. The present in vivo study and a previous in vitro study (35, 54) suggest the possibility of a downstream pathway initiated by the activations of c-Src along with FAK and Pyk2. Future studies are required to determine the nature of the downstream pathway that links the LPS induced activation of integrin {alpha}vβ3 and NF-{kappa}B during acute lung injury. Data from our previous study showed that c-Src is physically associated with I{kappa}B-{alpha}, and that it is involved in the phosphorylation and degradation of I{kappa}B-{alpha} in LPS-stimulated macrophages (10). Collectively, c-Src, directly, or via cross-talk with integrin {alpha}vβ3 mayenhance the NF-{kappa}B activating signal in the context of LPS stimulation.

Interestingly, in addition to the action of Src TKs on NF-{kappa}B activation, the present study also suggests that Src TKs directly contribute in LPS-induced inflammatory cell responses, including cell adhesion and transmigration into the lung, via its interaction with integrins. To understand which integrin types are involved in this complex signaling associated with the cell functions, and how Src TKs and the integrin receptors are linked, further study is required.

The present study suggests that Src TK activation is required for the NF-{kappa}B activation and integrin ({alpha}vβ3) signaling during acute lung injury induced by i.t. LPS treatment. We found LPS-induced c-Src activation in neutrophils recruited and alveolar macrophages. Specifically, posttreatment with PP1 attenuated LPS-induced pulmonary inflammatory responses, the NF-{kappa}B activation and integrin ({alpha}vβ3) signaling. Therefore, the inhibition of Src TK, like c-Src, activity appears to be important therapeutic target in terms of preventing NF-{kappa}B-mediated uncontrolled inflammation and consequent lung injury.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by Korea Research Foundation Grant KRF-2005- 041-E00034. Back

2 Address correspondence and reprint requests to Dr. Jihee Lee Kang, Department of Physiology, School of Medicine, Ewha Womans University, 911-1 Mok-6-dong, Yangcheon-ku, Seoul, Korea. E-mail: jihee{at}ewha.ac.kr Back

3 Abbreviations used in this paper: TK, tyrosine kinase; FAK, focal adhesion kinase; RGDS, Arg-Gly-Asp-Ser; RGES, Arg-Gly-Glu-Ser; ECM, extracellular matrix; BAL, bronchoalveolar lavage; MMP-9, matrix metalloproteinase-9. Back

Received for publication April 2, 2007. Accepted for publication August 23, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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