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Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma.
Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma ASHOK KUMAR,1 SAVITA LNU,1 RAHUL MALYA, DAVID BARRON, JOAN MOORE, DAVID B. CORRY, AND ALADIN M. BORIEK2 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA We investigated the effects of mechanical stretch and induced stimulation of lung parenchyma on the activation of proinflammatory transcription factors in normal mice and in a mouse model of asthma. Mechanical stretching of lung parenchyma led to increased activation of NF-␬B and AP-1 transcription factors. Incubation of lung parenchyma with methacholine increased the activation of NF-␬B, which was further augmented by stretch. Activation of NF-␬B in response to mechanical stretch was associated with the phosphorylation and degradation of I␬B␣ and the activation of I␬B kinase. Stretch-induced activation of NF-␬B involves activation of stretch-activated (SA) channels and the production of free radicals. Mechanical stretch and/or treatment with methacholine resulted in an increased activation of ERK1/2 and p38 MAP kinase, and the inhibition of the activity of these kinases partially blocked the stretch-induced NF-␬B and AP-1 activation. A greater level of NF-␬B and ERK1/2 activity was observed in the asthmatic mice, which was further increased by mechanical stretching. The level of cyclooxygenase-2, an NF-␬B-regulated enzyme, was also higher in lung parenchyma from asthmatic mice than in normal mice. Our data suggest that mechanical stretching of lung parenchyma activates NF-␬B and AP-1, at least in part, through the activation of MAP kinase signaling pathways.—Kumar, A., Lnu, S., Malya, R., Barron, D., Moore, J., Corry, D. B., Boriek, A. M. Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma. FASEB J. 17, 1800 –1811 (2003)

ABSTRACT

Key Words: 䡠 mechanotransduction 䡠 lung mechanics 䡠 nuclear factor-kappaB 䡠 activator protein-1 䡠 free radicals 䡠 stretch-activated channels 䡠 asthma 䡠 MAP kinases 䡠 cyclooxygenase-2 Although asthma is traditionally thought of as a disease of the pulmonary airways, it is becoming increasingly clear that abnormalities of lung parenchyma could play a significant role in this disease. Lung biopsies of patients with nocturnal asthma (1) as well as patients dying of status asthmatics (2) provide compelling pathological evidence that asthmatic inflammatory processes involve the lung parenchyma. Despite this 1800

evidence of peripheral inflammation, which could lead to possible altered mechanical function, the mechanisms involved in mechanical signal transduction in the lung parenchyma are unknown. Airway narrowing and acute hyperinflation are two forms of mechanical loading that asthmatics may experience during their lifetime (1– 4). Therefore, to understand the pathogenesis of asthma it is critical to investigate the mechanisms by which these mechanical loads trigger signal transduction in lung cells. The inflammatory process in asthma results in constriction of not only the bronchial tubes at the level of the primary airway, but also at the level of the peripheral airway and lung tissue. There is increasing evidence that nuclear factor-kappa B (NF-␬B) transcription factor plays a critical role in the activation and expression of inflammatory genes in asthma (5–7). The NF-␬B family consists of the members p50, p52, p65 (Rel A), c-Rel, and Rel B, which form various homo- and heterodimers (8). The NF-␬B dimers in resting cells reside in the cytoplasm in an inactive form bound to an inhibitory protein known as IkappaB (I␬B). Diverse signal transduction cascades mediate the stimulation of NF-␬B pathways. These signals activate the I␬B kinase (IKK) complex by phosphorylation of its IKK␣ and/or IKK␤ subunits by upstream kinases (9). Active IKK complex then phosphorylates I␬B proteins at serine residues to result in their ubiquitization and degradation by proteasome. This results in the activation of NF-␬B, which translocates to the nucleus and binds to the promoter or the enhancer regions of specific genes, initiating transcription (8 –11). The genes encoding many different cytokines and chemokines, receptors involved in immune recognition such as members of major histocompatibility complex (MHC), proteins involved in antigen presentation, and receptors required for neutrophil adhesion and migration contain multiple NF-␬B consensus binding sites, suggesting that 1

These authors contributed equally to this study. Correspondence: Pulmonary and Critical Care, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: [email protected] doi: 10.1096/fj.02-1148com 2

0892-6638/03/0017-1800 © FASEB

NF-␬B plays a major role in regulating host inflammatory and immune responses (5, 8, 12). Besides NF-␬B, activator protein-1 (AP-1) is another proinflammatory transcription factor that contributes to the initiation of inflammatory response in disease state, including asthma (13). AP-1 binding sites are found in the promoter region of many proinflammatory genes, including cytokines, adhesion molecules, and cell proliferation growth factors (14, 15). AP-1 is composed of two DNA binding subunits belonging to the Fos (c-Fos, FosB, Fra1, and Fra2) and Jun (c-Jun, JunB, and JunD) multigene family of transcription factors (14). The activity of AP-1 is regulated at least in part by the direct or indirect phosphorylation by mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinases (ERKs), c-Jun-N-terminal kinases (JNKs), and p38 kinase (16). We propose the hypotheses that 1) constant static mechanical stretch or constriction of the contractile cells in the normal lungs can trigger the activation of signaling pathways leading to the activation of NF-␬B and AP-1; 2) in asthma, mechanical loading of the lung leads to exaggerated activation of NF-␬B and its target genes, aggravating constriction of lung tissue and airways, which is a form of a mechanical loading, and this causes subsequent worsening of asthma. Thus, inflammation begets mechanical loading; the latter begets more inflammation, and a vicious cycle ensues. To test these hypotheses, we used a whole-lung lobe parenchyma preparation from either normal or an experimental mouse model of asthma and investigated the effects of constant mechanical stretch and/or the exogenous constrictor methacholine on the activation of NF-␬B, AP-1, and MAP kinase signaling pathways. Our results demonstrate that: 1) Constant mechanical stretch of lung parenchyma activates NF-␬B and AP-1 transcription factors; 2) the stretch-induced activation of NF-␬B involves the activation of stretch-activated (SA) channels and the production of free radicals; 3) the activity of NF-␬B and MAP kinases is higher in the mouse model of asthma, and such activity is further enhanced upon the application of mechanical stretch.

MATERIAL AND METHODS Materials Rabbit polyclonal antibodies to I␬B␣, IKK␣, p50, p52, p65, c-Rel, RelB, JNK1, Raf1, cyclooxygenase-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Normal as well as phospho-specific rabbit anti-p44/42 (Thr202/Tyr204), anti-p38 (Thr180/Tyr182), and anti-I␬B␣ (Ser32) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Gadolinium (III) chloride, curcumin, and N-acetyl cysteine (NAC) were from Sigma Chemical Co. (St. Louis, MO, USA). PD98059 and SB203580 were obtained from CalBiochem (San Diego, CA, USA). NF-␬B and AP-1 consensus oligonucleotide were obtained from Promega (Madison, WI, USA). Poly dI·dC was from Amersham Biosciences (Arlington Heights, IL, USA). ␥-32P-ATP [specific MECHANICAL STRETCH ACTIVATES NF-␬B IN LUNG PARENCHYMA

activity, 3000 (111 TBq) Ci/mmol] was obtained from PerkinElmer (Boston, MA, USA). Mice and tissue preparation The mice (strain C57BL6/ScSn) used in this study were purchased from Jackson Laboratory (Bar Harbor, ME, USA). These mice were housed and fed in stainless steel cages on a 12 h on and 12 h off lighting schedule. The animal facility is a virus-free facility. Experimental protocols were approved by the Animal Protocol Review Committee of the Baylor College of Medicine Animal Program (animal welfare assurance no. A-3823– 01) and are assigned protocol # AN-1727. All procedures were conducted in strict accordance with public health service animal welfare policy. Normal mice (3- to 4-wk-old) with a body weight ranging between 18 and 20 g and age-matched asthmatic mice (16 –18 g) were studied. The mice were anesthetized with an intravenous injection of pentobarbital (0.5– 0.7 mL/kg). Whole-lung lobe parenchyma was excised from each animal and immediately immersed into a tissue bath containing a modified KrebsRingers solution (in mM: 137 NaCl, 5 KCL, 1 NaH2PO4 , 24 NaHCO3 , 2 CaCl2 , 1 MgSO4 and pH 7.4) bubbled with 95% O2–5%CO2 (14). To study the effect of Gd3⫹, HEPES buffer (in mM: 10 HEPES, 137 NaCl, 5 KCL, 2 CaCl2 , 1 MgSO4 and pH 7.4) was used. The solution was maintained at a temperature of 25°C throughout the experimental phase of this study. To study the mechanical properties of lung parenchyma, four silk suture position markers (7-0 or 8-0 Surgilene) were sutured onto the surface of the lung parenchyma. All markers were placed in the central region of the lung parenchymal lobe to minimize the boundary effects as described by St. Venant’s principles of mechanics. The markers were placed in a square configuration ⬃1 mm apart with opposing sides aligned in orthogonal directions. Mechanical testing equipment The tissue testing apparatus used to apply mechanical stretch along the longer axis of the intact lung parenchymal lobe has been described (17). The apparatus consisted of a Cooper Instruments (Warrenton, VA, USA); LQB 630 force transducer (linearity of 0.1% full scale, sensitivity of 1 mV/volt/g, and hysteresis of 0.03% full scale). The force signals were amplified by a Validyne C019A system. A small metal alligator clip (10 mm width) is connected to the force transducer and the transducer is mounted on a linear ball carriage (Ball Slides, Inc., Medfield, MA, USA) with a right-hand threaded shaft (McMaster-CARR Supply Company, Atlanta, GA). Opposing the force transducer is an identical alligator clamp mounted on a ball carriage with a left-hand threaded shaft. The left- and right-hand threaded shafts are coupled together in the center of the apparatus. A stepper motor (Applied Motion Products, Inc., Aptos, CA, USA) with a 0.24° step is connected to one end of the right-hand shaft. The stepper motor is driven by a custom-built circuit using a Motorola driver (SAA 1042). As the shaft is rotated in the clockwise and counter-clockwise direction, the two opposing alligator clamps encroach and withdraw, respectively. A strain rate of ⬇0.4 or 1 N/cm2/s was maintained in applying mechanical stretch. The force data were collected at a sample rate of 10 Hz using a data acquisition board (model Lab-PC-1200/AI, National Instruments, Austin, TX, USA) and LabVIEW software (v 4.0). The force data were stored in an ASCII file for postanalysis. Displacements of the silk suture position markers were recorded (SONY SLV-620HF) on videocassette tapes using a CCTV type camera (HV-7200, Hitachi). Each loading 1801

cycle of the experiment began at the unstressed length (defined as intermarker length that produced no passive tension). Passive stress can be computed as the ratio of applied passive tension to the unstressed thickness (stress⫽tension/thickness), where stress is in N/cm2, tension is in N/cm (computed by the measured force in grams and divided by the average tissue width), and thickness is in centimeters. Thickness measurements were obtained from excised lung parenchyma. A digital image was created of the surface of the lung parenchyma and the surface area was determined using Image Tool (v 2.0). Lung tissue was blotted dry and weighed, and thickness was computed using the measured surface area, mass, and lung lobe density. In vitro mechanics protocol Constant mechanical stretch protocols were conducted on isolated lung parenchymal using the mechanical stretching apparatus as described above. Constant static stretch protocol was conducted by mechanically stretching the lung tissue by either 0.5 g or 1 g force, then stress relaxation was allowed to occur for 15 min. The applied forces resulted in mechanical stretches of ⬃12 or 20%, respectively. To examine the effect of exogenous constriction by methacholine at baseline, the mechanical data were collected continuously for 15 min at a sample rate of 10 Hz; 10 min after recording had begun, methacholine was added to the organ bath to yield a final concentration of 10⫺4 M. The same protocol of constant static stretch was applied to the lung parenchymal lobe in the presence of methacholine. Upon completion of the static stretch protocol, the whole-lung parenchymal lobes were removed from the organ bath and fixed immediately in lysis buffer for biochemical assays described below. Strain calculations Mechanical strains were calculated based on methods established in our previous work (17). Coordinates for any three markers on the surface of the lung parenchyma are denoted xi and yi (i⫽1, 2, 3), where three markers on the surface of the lung parenchyma define a triangle in a plane. Subsequently, the four-sutured markers would define four triangles. For example, triangle A would be defined by markers 1, 2, and 3 while triangle B would be defined by markers 1, 2, and 4. The displacements of the markers from an unstressed state to higher stressed states of loading are denoted ui for displacement in the principal direction of applied force and vi for the displacement in the orthogonal direction. For example, ui ⫽ a1 ⫹ a2xi ⫹ a3yi This equation, with known values of the displacements and the coordinates of three markers substituted for ui , xi , and yi , provides a set of three equations for the three coefficients a1, a2, and a3. Similarly, the coordinates of the markers provide information for the following equation: vi ⫽ a4 ⫹a5xi ⫹a6 yi The values of the coefficients a2, a3, etc., were used to find the partial derivatives, which were substituted into the following defined to calculate strains. εx ⫽ ␦u/␦x where εx denotes strain developed along the principal direction of applied force. Strains were computed relative to unstressed length. Unstressed length was defined as the length of the markers in the absence of applied forces. The 1802

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final strains computed as the average of the strains from the four triangles. Our mechanical data in Fig. 1 are plotted using extension ratios (1.0⫹strain values). Allergen challenge and analysis of asthma phenotype Complete Aspergillus allergen (CAA) prepared from a clinical isolate of Aspergillus fumigatus along with OVA was used to induce the asthma in mice as described (18). Mice were given a 3.5% isofluorane vapor/oxygen mixture until unconscious and breathing had stabilized at 2 respirations/s. Mice were then held upright and 50 ␮L of CAA was applied to the nares until completely aspirated. After resuming full consciousness (⬃1 min), mice were returned to their cages. Allergen challenge was repeated every 4 days for 5 total challenges. Mechanical and biochemical experiments were executed 24 h after the final challenge (18, 19). In one group of mice, curcumin (10 ␮g/kg of body weight) in 50 ␮L phosphatebuffered saline (PBS, 1⫻) was applied to the nares 1 h before the 4th and 5th CAA challenges. The asthmatic phenotype of these mice was confirmed by measuring the airway hyper-responsiveness as the provocative concentration of acetylcholine causing ⬃200% increase in lung resistance (PC200). Bronchoalveolar lavage cytology, OVA-specific and total IgE, lung histopathology, and measuring the lung cytokine profiles by ELISA were conducted exactly as described previously (18). Electrophoretic mobility shift assays To determine the activity of NF-␬B and AP-1 transcription factors, electrophoretic mobility shift assays (EMSA) were carried as described recently (20, 21) with some modification. After being subjected to mechanical stretch or exogenous constriction by methacholine, parenchyma tissues were weighed, frozen using dry ice, and grounded using a manual grinder in low-salt lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 ␮g/mL leupeptin, 2.0 ␮g/mL aprotinin, and 0.5 mg/mL benzamidine (18 ␮L buffer/mg of muscle tissue). The tissue extract in low-salt lysis buffer was allowed to swell on ice for 10 min, vortexed vigorously for 10 s, followed by two freeze/thaw cycles. The homogenate was centrifuged for 10 s at 4°C, the supernatant (cytoplasmic extract) was removed, and the nuclear pellet was resuspended (5 ␮L/mg of original tissue weight) in ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM MgCl2, 20% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 ␮g/mL leupeptin, 2.0 ␮g/mL aprotinin, 0.5 mg/mL benzamidine) and incubated on ice for 30 min with intermittent vortexing. Samples were centrifuged for 5 min at 4°C, and the nuclear extract was either used immediately or stored at –70°C. The protein content was measured by the method of BioRad Protein Assay Reagent. EMSAs were performed by incubating 10 ␮g of nuclear extract with 16 fmol of the 32P-end-labeled NF-␬B or 32P-endlabeled AP-1 consensus oligonucleotide for 15 min at 37°C. The incubation mixture included 2.5 ␮g of poly dI·dC in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl). The DNA–protein complex thus formed was separated from free oligonucleotide on 7.5% native polyacrylamide gel using buffer containing 50 mM Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA. The gel was then dried and the radioactive bands were visualized by exposing the gel to the X-ray film for a suitable amount of time. The bands obtained were quantitatively assessed with Personal Densitometer Scan version 1.30

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using ImageQuant software version 3.3 (Amersham Biosciences, Piscataway, NJ, USA). The specificity of the bands in EMSA was confirmed by using mutated probes, cold competition, and supershift assays. I␬B kinase (IKK) assay The IKK assay was performed as described in our previous work (21). The IKK complex was precipitated by treating 600 ␮g cytoplasmic extracts with 1 ␮g anti-IKK␣ antibody (Santa Cruz, San Diego, CA, USA), overnight at 4°C followed by treatment with 40 ␮L protein A/G-Sepharose (Amersham Biosciences, Arlingon Heights, IL, USA). After 2 h, the beads were washed three times with lysis buffer and three times with the kinase assay buffer, then resuspended in 20 ␮L of kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl2, 2 mM DTT, 20 ␮Ci ␥-32P-ATP, 10 ␮M unlabeled ATP, and 2 ␮g of substrate GST-I␬B␣ (amino acid residues 1–54). After incubation at 30°C for 15 min, reaction was terminated by boiling with 20 ␮L of 2⫻ SDS sample buffer for 3 min. Finally, the protein was resolved on 10% polyacrylamide gel, the gel was dried, and the radioactive bands were visualized and quantified by PhosphorImager. Total amounts of IKK␣ in each sample were determined by Western blot using antiIKK␣. Western blot The lung lobe parenchymal tissues obtained after the application of mechanical stretch were washed with PBS and homogenized in lysis buffer A (20 mM HEPES (pH 7.4), 2 mM EDTA, 200 mM NaCl, 0.3% NP-40, 2 ␮g/mL leupeptin, 2 ␮g/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 ␮g/mL benzamidine, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 50 mM NaF, and 10 mM ␤-glycerophosphate). The protein concentration of the samples was measured using BioRad protein assay reagent and 50 –70 ␮g of protein was resolved on 10% SDS-PAGE gel. The proteins were then electrotransferred to a nitrocellulose membrane blocked with 3% nonfat milk and probed with suitable antibodies (1:2000) for 4 –5 h. The blot was washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h, and detected by chemiluminescence (ECL, Amersham Biosciences, Arlington Heights, IL, USA). The bands obtained were quantitated with Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Amersham Biosciences, Piscataway, NJ, USA).

RESULTS Quasi-static cyclic force-length relationship of wholelung parenchyma from normal mice A representative set of force-length relationships of the normal lung parenchyma is shown in Fig. 1. The data were collected during five loading and unloading cycles. The following features of this example are representative of the data for all samples examined. Applied force increased continuously over the range of strains that were imposed. The force-length data are mildly nonlinear over a sizable range of strains that our data cover. There is very little hysteresis, as evident by the superposition of the data during passive loading and unloading. We assumed that linear dimensions vary MECHANICAL STRETCH ACTIVATES NF-␬B IN LUNG PARENCHYMA

Figure 1. Force-length relationships of excised normal mouse whole-lung parenchyma. Representative data of five slowcycling force stretch shortening relationships during uniaxial loading and unloading of whole-lung parenchyma is shown. There is a nonlinear relationship between force and stretch ratio over the applied strains. The data for stretching and shortening are indistinguishable over the imposed strain. We assumed that linear dimensions vary with the cube root of the total lung volume. We also assume that unstressed length is equivalent to residual lung volume and total lung capacity is ⬃fourfold that at residual lung volume. Therefore, a total lung capacity is equivalent to ⬃60% linear stretch of lung tissue. Thus, a 12% linear stretch (achieved at ⬃0.5 g force) is equivalent to a lung volume that is ⬃20% of the total lung capacity.

with the cube root of the total lung volume. We also assume that unstressed length is equivalent to residual lung volume and total lung capacity is ⬃fourfold that at residual lung volume. Therefore, a total lung capacity is equivalent to ⬃60% linear strains. Thus, a 20% linear stretch is equivalent to a lung volume that is ⬃1/3 of the total lung capacity. Mechanical stretch of whole-lung parenchyma activates NF-␬B We investigated the effects of mechanical stretch of whole-lung parenchymal lobe on the activation of NF␬B. The time course analysis of EMSA revealed that NF-␬B/DNA binding activity was increased as early as 5 min after the application of mechanical stretch (Fig. 2A). In the dose-dependent studies as depicted in Fig. 2B, NF-␬B/DNA binding activity reached maximum with 0.5 g force (or 12% stretch) and was marginally decreased in response to greater forces. The quantitative estimation of the NF-␬B/DNA complex after 30 min of stretch from multiple experiments revealed that mechanical stretching of lung parenchyma causes a ⬃threefold increase in NF-␬B activity (Fig. 2C). These data strongly suggest that mechanical stretching of lung parenchyma activates NF-␬B transcription factor. 1803

Figure 2. Activation of NF-␬B by mechanical stretch of lung parenchyma. A) Lung parenchyma isolated from normal mice was subjected to mechanical force of 0.5 g (12% linear stretch) for different periods; the nuclear extracts were made and analyzed with EMSA. A representative blot presented here shows that mechanical stretch activates the NF-␬B as early as 5 min. B) Lung parenchyma was stretched with different magnitudes of force ranging from 0.5 to 2 g (12– 40% linear stretch). It appears there is a higher activation of NF-␬B in response to a 0.5 g force (⬃12% stretch) and that the activation was decreased in response to greater force. C) The quantitative estimation of NF-␬B activity after 30 min of stretch from 3 independent experiments shows that mechanical stretch significantly increases the NF-␬B activation (*P⬍0.05).

Mechanical stretch-induced NF-␬B/DNA complex contains p50, p65, and c-Rel proteins Different combinations of Rel/NF-␬B proteins can constitute an active NF-␬B heterodimer that binds to specific sequences in DNA (23). To confirm that the retarded band seen in EMSA is indeed NF-␬B and to investigate which subunits of NF-␬B are activated in response to mechanical stretch, we performed a supershift assay. Preincubation of nuclear extracts from 30 min mechanically stretched parenchyma with antibodies against p50, p65, or c-Rel proteins of NF-␬B family shifted the band to higher levels of molecular weight indicating that NF-␬B/DNA complex analyzed by EMSA constitutes these proteins. The multiple supershifted bands observed in p50 and p65 indicate that these proteins make heterodimers with other NF-␬B family proteins. Antibodies against other members of NF-␬B family such as Rel B and p52 and an irrelevant antibodies against Raf-1 did not affect the mobility of the NF-␬B/DNA complex (Fig. 3). Effect of mechanical stretch on the cellular level of I␬B␣ The activation of NF-␬B by inflammatory cytokines such as TNF-␣ is achieved through the phosphorylation of 1804

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Figure 3. Analysis of mechanical stretch-induced NF-␬B/DNA complex. Nuclear extracts (12 ␮g protein) of 30 min mechanically stretched lung parenchyma were preincubated with 1 ␮g antibodies against p50, p52, p65, RelB, or c-Rel proteins of NF-␬B family or with Raf-1 (as a control) antibody for 15 min followed by EMSA using NF-␬B probe. The data show that NF-␬B/DNA complex seen in EMSA contains p50, p65, and c-Rel proteins. NSB, nonspecific band.

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I␬B␣ at Ser-32 and Ser-36 residue, followed by its polyubiquitination and degradation (8). The degradation of I␬B␣ leads to the nuclear translocation of NF-␬B. We next investigated whether mechanical stretch-induced activation of NF-␬B is associated with the phosphorylation and degradation of I␬B␣ protein. After mechanical stretching of the lung parenchyma, the resultant cytoplasmic extracts were subjected to Western blot using normal I␬B␣ antibodies or with phosphospecific I␬B␣ (Ser-32) antibodies. As shown in Fig. 4 (upper panel), the level of I␬B␣ protein is significantly decreased in response to 15 min mechanical stretch of lung parenchyma. The level of I␬B␣ protein again reached basal level after 60 min of stretch (Fig. 4, upper panel). Furthermore, the cytoplasmic level of phosphorylated I␬B␣ protein was significantly increased after 15 min of mechanical stretching (Fig. 4, middle panel). The level of an unrelated protein ␤-actin was not affected by mechanical stretching (Fig. 4, lower panel). These results clearly suggest that mechanical stretching of lung parenchyma leads to the phosphorylation and degradation of I␬B␣.

Figure 5. Time kinetics of activation of I␬B kinase (IKK) in mechanically stretched lung parenchyma. The cytosolic extracts made from mechanically stretched lung parenchyma were immunoprecipitated with IKK␣ antibody and the kinase activity was measured using GST-I␬B␣ (1–54) as substrate. A significant increase in the activity of IKK was observed as early as 15 min of the onset of mechanical stretch (upper panel). The total level of IKK␣ protein as determined by Western blot did not change during mechanical stretch (lower panel). The data are representative of 2 independent experiments.

suggesting that mechanical stretching of lung parenchyma increases the activity of IKK.

Effect of mechanical stretch on the activation of I␬B kinase

Effect of tissue constriction and mechanical stretch on the activation of NF-␬B in lung parenchyma

We studied the effect of mechanical stretch of lung parenchyma on the activation of IKK, a kinase that phosphorylates I␬B␣ (9). Whole-lung parenchymal lobe were mechanically stretched by 0.5 g force (or 12% stretch) and held at the stretched state for different intervals. The activity of IKK was measured using GST-I␬B␣(1-54) proteins. A significant increase in the IKK activity was observed after the application of the mechanical stretch (Fig. 5, upper panel) whereas the level of IKK␣ was unaffected (Fig. 5, lower panel),

Methacholine is a synthetic chemical related to the natural neurotransmitter acetylcholine (24). Methacholine challenge causes airway narrowing and constriction of the lung tissue similar to that which occurs in asthmatic patients (24, 25). We investigated the effects of methacholine on the activation of NF-␬B in lung parenchyma. Excised mouse whole-lung parenchymal lobes were incubated in 100 ␮M methacholine for different intervals, and in some samples lung parenchyma was subjected to mechanical force of 0.5 g force (12% stretch) for 5 min. The activity of NF-␬B in nuclear extracts was then measured using EMSA. Treatment of parenchyma with methacholine alone caused a significant increase in the NF-␬B/DNA binding activity (Fig. 6). The level of NF-␬B activation in methacholinetreated parenchyma was further increased after 5 min of constant static mechanical stretch. These data strongly suggest that constriction of lung parenchyma causes an up-regulation of NF-␬B activity and that additional applied external mechanical stretch further exaggerates the NF-␬B activation. Role of stretch-activated (SA) channels and free radicals in the mechanical stretch-induced activation of NF-␬B

Figure 4. Time course analysis of phosphorylation and degradation of I␬B␣ in the cytosolic extracts of lung parenchyma. A representative immunoblot of I␬B␣ proteins shows that mechanical stretching of lung parenchyma causes a significant decrease in the amount of total I␬B␣ protein (upper panel) with a concomitant increase in the phosphorylated form of I␬B␣ protein (middle panel). The level of an unrelated protein ␤-actin was not affected by mechanical stretching (lower panel). The data are representative of 4 independent experiments. MECHANICAL STRETCH ACTIVATES NF-␬B IN LUNG PARENCHYMA

Because mechanical forces are applied directly to the cell membrane, it is possible that permeability of the cellular membrane to various ions is affected in response to mechanical stress. Indeed, the activities of SA channels have been reported in lung cells (26, 27). We investigated whether SA channels are involved in the mechanical stretch or methacholine-induced activation of NF-␬B in lung parenchyma. Pretreatment of lung 1805

Figure 6. Effects of tissue constriction and mechanical stretch on the activation of NF-␬B in lung parenchyma. Excised mouse whole-lung parenchyma were preincubated in 100 ␮M methacholine alone or followed by application of 0.5 g force (equivalent to ⬃12% strain relative to the excised length) for 5 min. The activity of NF-␬B in nuclear extracts was then measured using EMSA. The data show that treatment of parenchyma with methacholine alone caused a significant increase in the NF-␬B/DNA binding activity, which was further enhanced by application of mechanical stretch.

parenchyma with 100 ␮M GdCl3 (an inhibitor of SA channels) for 1 h inhibited the mechanical stretchinduced activation of NF-␬B, suggesting that SA channels play a role in the process of activation of NF-␬B in response to mechanical stretch (Fig. 7A). On the other hand, similar pretreatment of lung parenchyma with GdCl3 did not alter the activation of NF-␬B in response to methacholine (Fig. 7B). The role of reactive oxygen intermediates (ROI) in the activation of NF-␬B in response to stimuli such as TNF-␣ and phorbol 12-myristae 13-acetate (PMA) has been well documented in the literature (28, 29). Whether free radicals have any role in mechanical stretch-induced activation of NF-␬B in lung paren-

chyma is unknown. Whole-lung parenchymal lobes were preincubated with 30 mM N-acetyl cysteine (NAC), a free radical scavenger, for 1 h followed by the application of 0.5 g force (12% stretch) or treatment with 100 ␮M methacholine for 15 or 30 min. A complete inhibition in the stretch-induced NF-␬B activation was observed in NAC-treated lung tissue (Fig. 7A). The activation of NF-␬B in response to methacholine was also decreased in presence of NAC (Fig. 7B). These data indicate that ROI plays an essential role in the process of NF-␬B activation in response to mechanical stretch and tissue constriction by methacholine in lung parenchyma. Mechanical stretching of lung parenchyma activates ERK1/2, p38 and AP-1 transcription factor

Figure 7. Role of stretch-activated (SA) channels and reactive oxygen species in the activation of NF-␬B. Lung parenchyma isolated from normal mice were preincubated with either 100 ␮M GdCl3 or with 30 mM N-acetyl cysteine (NAC) for 1 h, followed by the application of either ⬃12% mechanical stretch (0.5 g force) or 100 ␮M methacholine treatment for 15 and 30 min. The NF-␬B DNA binding activity in the nuclear extracts was measured by EMSA. A representative EMSA gel of 2 independent experiments shows that both Gd3⫹ ions completely inhibited the activation of NF-␬B in lung parenchyma in response to only mechanical stretch (A) but not in response to tissue constriction by methacholine (B). Pretreatment of lung parenchyma with NAC completely inhibited the activation of NF-␬B in response to mechanical stretch whereas similar treatment resulted in partial inhibition of NF-␬B in response to methacholine. 1806

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Since MAP kinase signaling pathways are involved in regulating the activity of many transcription factors, including AP-1 and NF-␬B (30), we studied the effect of mechanical stretch of lung parenchyma on the activation of ERK1/2 and p38 kinases. As shown in Fig. 8A, constant mechanical stretch of lung parenchyma increased the phosphorylation of ERK1/2 and p38 MAP kinase in a time-dependent manner. The increased phosphorylation was not due to alteration of total cellular level of these kinases, as the total amount of ERK1/2 and p38 was essentially the same in both the normal and stretched lung tissues (data not shown). We also studied the effects of methacholine on the activation of ERK1/2 and p38 kinase. As shown in Fig. 8B, constriction of lung parenchyma with methacholine alone increased the phosphorylation of ERK1/2 and p38 MAP kinase, and such activation was not affected by the application of mechanical stretch. Activation of MAP kinase signaling pathways usually leads to the activation of various subunits of AP-1 transcription factor, which in its turn initiates the transcription of AP-regulated genes (14, 15). To understand whether mechanical stretching of lung parenchyma activates the AP-1, the lung parenchyma were mechanically stretched by 0.5 g force (⬃12% stretch) and held at stretched state for different intervals, and the activity of AP-1 was measured by EMSA. As shown in Fig. 8C, a rapid time-dependent activation of AP-1 was observed after the application of mechanical stretch.

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transcription factors (Fig. 9). On the other hand, inhibition of p38 MAP kinase decreased the mechanical stretch-induced activation of AP-1 and NF-␬B (Fig. 9). Activation of NF-␬B and MAP kinases in the mouse model of asthma in response to mechanical stretch

Figure 8. Effect of mechanical stretch on the activation of ERK1/2, p38, and AP-1. A) Lung parenchyma were stretched for indicated time intervals and the activation of ERK1/2 (upper panel) and p38 kinase (lower panel) was measured using phosphospecific antibodies. The data show that mechanical stretch leads to significant up-regulation in the activity of ERK1/2 and p38 MAP kinase. B) Lung parenchyma were preincubated for 10 min with methacholine (100 ␮M) followed by the application of mechanical stretch for 15 min. The activity of ERK1/2 and p38 kinase was then measured. Data clearly show that constriction of parenchyma with methacholine alone activates ERK1/2 and p38 kinase. C) Time course analysis of DNA binding activity of AP-1 using EMSA shows that stretch activates the AP-1 transcription factor.

We also evaluated the effects of mechanical stretch on NF-␬B, AP-1, and MAP kinase activities in lung parenchyma of an experimental mouse model of asthma (18, 19). This mouse model has been proven to be a robust experimental model of allergic lung inflammation that mimics many salient features of human asthma (18). We investigated whether mechanical stretch has any effect on the activation of either NF-␬B or MAP kinases in these asthmatic mice. The basal level of NF-␬B/DNA binding activity was significantly greater than that in normal mice. Compared with normal mice, the application of mechanical stretch further increased NF-␬B activity to a significantly higher level, suggesting that asthmatic parenchyma are more responsive to mechanical stretch-induced activation of NF-␬B (Fig. 10A). The supershift analysis of NF-␬B/DNA complex of asthmatic mice revealed that it contains p50 and c-Rel proteins (Fig. 10B). The basal level of ERK1/2 activity was also higher in lung parenchyma from asthmatic mice than that in normal mice, which was further augmented in response to mechanical stretching (Fig. 10C). Although the basal level of the activities of AP-1 was not different in normal and asthmatic mice, the activation level of AP-1 in response to mechanical

Involvement of ERK1/2 and p38 MAP kinase in the mechanical stretch-induced activation of NF-␬B and AP-1 Since MAP kinases are involved in regulating the activity of various transcription factors and these kinases are activated in response to mechanical stretching of lung parenchyma, we next investigated the role of ERK1/2 and p38 MAP kinase in mechanical stretch-induced activation of NF-␬B and AP-1. Lung parenchyma from normal mice were preincubated with either 50 ␮M PD98059 (MEK1/2 inhibitor) or 20 ␮M SB203580 (p38 kinase inhibitor) for 30 min prior to the application of mechanical stretch for 30 min. The inhibition of ERK1/2 using PD98059 significantly inhibited the activation of AP-1 without affecting the activity of NF-␬B MECHANICAL STRETCH ACTIVATES NF-␬B IN LUNG PARENCHYMA

Figure 9. Effect of inhibition of ERK1/2 and p38 MAP kinase on the activation of NF-␬B and AP-1 in response to mechanical stretching of lung parenchyma. Lung parenchyma from normal mice was pretreated for 30 min with A) 50 ␮M PD98059 (a MEK1/2 inhibitor) or B) 20 ␮M SB203580 (a p38 kinase inhibitor) followed by application of ⬃12% mechanical stretch (0.5 g force) for 30 min. The data presented here show that inhibition of both ERK1/2 and p38 MAP kinase inhibits the activation of AP-1 whereas inhibition of p38 kinase only affected the activation of NF-␬B transcription factor. 1807

Figure 10. Increased activation of NF-␬B, ERK1/2 and expression of cyclooxygenase-2 (COX-2) in asthmatic mice. A) Lung parenchyma from control or experimentally induced asthma (see materials and methods) was stretched for 15 or 30 min with 0.5 g force (12% stretch). NF-␬B activity as measured by EMSA shows that basal level of NF-␬B activity is significantly higher in asthmatic mice. Furthermore, the level of NF-␬B activity in response to applied mechanical stretch was significantly higher in asthmatic mice compared with the control mice. B) Supershift analysis of nuclear extracts (20 ␮g protein/lane) from asthmatic mice in absence of mechanical stretch shows that NF-␬B/ DNA complex contains p50 and c-Rel proteins. C) The level of ERK1/2 activity in normal and asthmatic mice. A representative blot shows a significant higher level of ERK1/2 activation in asthmatic mice that was further increased by mechanical stretch. D) A higher level of COX-2 protein was observed in asthmatic mice, which was inhibited by administration of NF-␬B inhibitor curcumin before antigen challenge to the mice.

stretch was marginally higher in asthmatic mice (data not shown). We also evaluated the level of cyclooxygenase-2 (COX-2), an enzyme that plays a major role in inflammation and contains NF-␬B binding sites within its promoter/enhancer region (31–33). The level of COX-2 was significantly higher in allergen-challenged asthmatic mice than the corresponding controls (Fig. 10D). The increased expression of COX-2 was blocked by curcumin (an inhibitor of NF-␬B), suggesting that higher activation of NF-␬B in these mice is associated with the increased expression of COX-2 (Fig. 10D).

DISCUSSION Data from the last few decades have suggested that distal lung tissue and lung parenchyma contribute to asthma pathogenesis. More recently peripheral airways, including lung tissue, have been recognized as a major site of airflow obstruction in asthmatic patients (3, 4, 34, 35). Wagner et al. showed that individual peripheral airway resistance increased in mild asthmatics up to sevenfold compared with controls, and these measurements correlated with responsiveness to methacholine (36). Quantitative analyses based on histology (37–39) have shown that the majority of airway hyper-responsiveness among asthmatic persons occurs within the peripheral airways. Research in the past few years has established that physical forces influence the structure and function of lung cells whereas excessive mechanical forces may lead to many pathophysiologic conditions (26, 40). However, the role of either mechanical stretch or induced stimulation of lung parenchyma on 1808

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the activation of transcription factors such as NF-␬B and AP-1 and MAP kinase signaling pathways remains to be elucidated. In this study we have demonstrated that either the application of mechanical stretch or the induced stimulation of lung parenchyma activates MAP kinase, AP-1, and NF-␬B signaling pathways in normal mice. NF-␬B activation is a key early event in a variety of cell and animal model systems developed to elucidate the pathobiology of lung diseases (41– 44). Although the exact role NF-␬B plays in asthma is not known, the following line of evidence suggests it may play a pivotal role in the initiation and perpetuation of inflammation that occurs in asthmatic patients. 1) Activated NF-␬B has been identified in the key locations in the airways of the asthmatic patients. 2) Agents such as allergens, ozone, and viral infections, which are associated with exacerbation of asthma, stimulate activation of NF-␬B. 3) The major effective treatment of asthma, corticosteroids, is a potent blocker of NF-␬B activation. 4) Most of the immune and inflammatory genes that are overexpressed in asthma, such as those encoding proinflammatory cytokines, chemokines, adhesion molecules, and inflammatory enzymes contain ␬B sites for NF-␬B within their promoter. 5) Finally, activation of NF-␬B has been shown to induce the proliferation of smooth muscle cells that causes further airway narrowing and hyperresponsiveness (7, 8, 12, 45–52). Although the role of NF-␬B in the pulmonary diseases has been investigated recently, the stimulus that causes the activation of this pathway remains poorly understood. Asthmatic patients experience a wide variety of mechanical loading. We postulate that mechanical loading of the lung could trigger the activation of

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NF-␬B. There are isolated reports that suggest that in vitro mechanical stretching of fibroblast (53) or smooth muscle cells (54) that constitute the lung causes the activation of NF-␬B pathways in these cells. How mechanical stretching of these cells in complex tissue environment alters the activation of NF-␬B signaling pathways remains unknown. The complexity of cell– cell and cell cell–matrix interactions and the complicated intracellular communications between different cell types in lung tissue cannot be completely simulated in cell culture (26). We used an ex vivo system to investigate the effect of mechanical stretching on the activation of NF-␬B in lung parenchyma. Our technique using isolated whole-lung parenchymal lobe therefore offers a useful approach to bridge cell culture studies to those conducted at the whole animal level. The normal lung parenchyma preparation used in this study contained only a minimal amount of trapped air. The stretch applied to the lung parenchyma was similar to that experienced in vivo (Fig. 1). Our data clearly demonstrated that mechanical stretching of the lung parenchyma rapidly activates the NF-␬B signaling pathways as evident by gel retardation assays and IKK assays (Figs. 2 and 5). The prototypic NF-␬B heterodimer consists of p50 and p65 (Rel A) subunits, but other complexes have recently been described with different members of Rel family modulating DNA-binding and transactivation (8 –12). The activation of different subunits of NF-␬B is governed by an upstream kinase IKK that consists two catalytic subunits IKK␣ and IKK␤ and a regulatory subunit IKK-␥. The targeted disruption of IKK␣ and IKK␤ in mice revealed that their functions are not interchangeable. IKK␣ plays a crucial role in the lymphoid organ development and adaptive immunity, but it is not required for the cytokine-induced activation of NF-␬B. In contrast, the IKK␤ is essential for activation of canonical NF-␬B pathways, which is based on I␬B degradation and is activated in response to many proinflammatory stimuli, including TNF-␣ and IL-1␤ (10). While p50 and p65 subunits constitute the NF-␬B complex in canonical pathway, other proteins of NF-␬B family such as c-Rel, Rel B, and p52 are predominately present in a NF-␬B complex activated by an alternative pathway, which involves the activation of IKK␣ (10). Some reports suggest that mechanical stretch activates NF-␬B in a variety of cell types (20, 53, 54), but it is not yet clear whether the activation of NF-␬B involves IKK␣ or IKK␤ pathways. Our data in the current study showed that the NF-␬B/DNA complex constitutes p50 and p65 proteins as well as the c-Rel protein (Fig. 3), suggesting that both canonical and alternative pathways might contribute to the activation of NF-␬B in response to mechanical stretching of lung parenchyma. Although we observed the activation of IKK␣ kinase in response to mechanical stretch, it does not rule out the possible activation of IKK␤ catalytic subunit in the IKK complex. This is further supported by the fact that mechanical stretching of lung parenchyma reduced the cytosolic level of NF-␬B inhibitory MECHANICAL STRETCH ACTIVATES NF-␬B IN LUNG PARENCHYMA

protein I␬B␣ (Fig. 4), which is a physiological substrate of IKK␤. We also observed a significant increase in the NF-␬B/ DNA binding activity in response to methacholine (Fig. 6), an exogenous constrictor of the contractile elements in lung parenchyma (24, 25). The activation of NF-␬B in response to methacholine was transient, which reached basal level after 30 – 45 min of treatment. However, mechanical stretching of methacholine-constricted parenchymal tissues led to a sustained and higher level of activation of NF-␬B, suggesting that mechanical stretch further exaggerates the activation of NF-␬B in already constricted lung tissues. The mechanism by which NF-␬B is activated in response to methacholine is unclear. Tissue constriction and mechanical stretch are two distinct forms of mechanical loads. However, it is possible that a common mechanism of activation of NF-␬B in lung parenchyma exists in response to either of these mechanical loads. To understand the upstream events that lead to the activation of NF-␬B, we investigated the role of mechanosensitive ion channels and the oxygen free radicals. The activation of stretch-activated (SA) channels allows the influx of Ca2⫹, K⫹, Na⫹ ions in the cells and has been proposed as the transduction mechanisms between mechanical stress and various cellular responses (40, 55, 56). Recently, Inoh et al. showed that the direct Ca2⫹ influx through the SA channels is required for the cyclic stretch-activated NF-␬B in cultured human fibroblast cells (53). Our present data using Gd3⫹ (SA channel inhibitor) ions clearly suggest that the activation of SA channels is essential for the mechanical stretch-induced activation of NF-␬B in lung parenchyma (Fig. 7). Furthermore, the activation of NF-␬B in response to mechanical stretch and tissue constriction by methacholine seems to involve the generation of reactive oxygen intermediates, as the pretreatment of lung parenchyma with NAC, a free radical scavenger (29), inhibited the activation of NF-␬B (Fig. 7). The precise role of free radicals in the activation of NF-␬B in lung parenchyma in response to mechanical stress and tissue constriction is unknown. However, many other studies suggest that free radicals act as a second messenger in the activation of NF-␬B in response to NF-␬Bactivating stimuli such as phorbol myristate acetate (PMA) and TNF-␣ (28, 29). An increased amount of oxidized glutathione has been reported in the airways of asthmatics, which is indicative of increased oxidative stress (57–59). It is therefore possible that mechanical stretching leads to the development of an oxidant/ antioxidant imbalance in the lungs that may lead to the activation of redox-sensitive transcription factors such as NF-␬B and AP-1. Our data also provides evidence that constant static stretch as well as induced stimulation of lung parenchyma by methacholine activates ERK1/2 and p38 MAP kinase (Fig. 8) and that the activation of these kinases contributes to the activation of NF-␬B and AP-1 transcription factors (Fig. 9). MAP kinase signaling pathways have been shown to be activated by mechanical 1809

stress in a variety of lung cells. For example, cyclic stretch activates ERK1/2 in epithelial cells (60), endothelial cells (61, 62), and vascular smooth muscle cells (63, 64). Since MAP kinases regulate the activity of many nuclear transcription factors, including AP-1 and NF-␬B (65, 66), the activation of MAP kinases in lung parenchyma may link the effects of mechanical stretch to biological responses and gene expression. To explore the role of stretch-induced activation of NF-␬B in asthmatic subjects, we used an experimental mouse model of asthma that was developed to investigate immune response to allergic inflammation (18). A higher basal level of NF-␬B activity was observed in lung parenchyma of asthmatic mice (Fig. 10A). The exact stimuli that increased the activity of NF-␬B in those parenchyma is not clear, however, a more pronounced constriction in this lung tissue could possibly contribute to the higher level of activation of NF-␬B. Our data are consistent with published reports documenting higher level of NF-␬B activation in the mouse and horse model of asthma (41– 44). Elevated protein level of COX-2, an NF-␬B-regulated enzyme involved in inflammatory response (31–33), in lung parenchyma of our asthmatic mice indicates that the activation of NF-␬B is associated with the increased expression of target proteins. This is further confirmed by our data, which show that treatment of mice with NF-␬B inhibitor curcumin (67– 69) before allergen challenge completely inhibited the increased COX-2 level in asthmatic lungs (Fig. 10). The results presented in this study support the hypothesis that mechanical stretch activates proinflammatory transcription factor NF-␬B and AP-1 in lung parenchyma, which might initiate the expression of several NF-␬B- or AP-1-regulated inflammatory molecules. These molecules in turn further augment the activity of these transcription factors in an autocrine fashion. The increased expression of these proinflammatory molecules might contribute to the pathogenesis of asthma. This work was supported in part by National Institutes of Health grant 63134 to A.B.

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