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Published ahead of print on January 24, 2005, doi:10.1165/rcmb.2004-0118OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 301-310, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0118OC

Stress-Activated Protein Kinases Mediate Cell Migration in Human Airway Epithelial Cells

Steven R. White, Roberta Tse and Bertha A. Marroquin

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

Correspondence and requests for reprints should be addressed to Steven R. White, M.D., University of Chicago, Section of Pulmonary and Critical Care Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637. E-mail: swhite{at}medicine.bsd.uchicago.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway epithelial cell (AEC) repair immediately after injury requires coordinated cell spreading and migration at the site of injury. Stress-activated protein kinases such as p38 MAPK and c-Jun N-terminal Protein Kinase (JNK) modulate several responses to cell stress and injury, but their role in AEC migration is not clear. We examined migration in confluent 16HBE14o– human AEC lines and in primary AEC grown on collagen-IV. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Inhibitors of either p38 extracellular signal–regulated kinase (ERK)1/2 (PD98059), mitogen-activated protein kinase (MAPK) (SB203580), or JNK (SP600125) could block cell migration substantially. Inhibiting JNK but not p38 MAPK or ERK1/2 blocked extension of cells into the wound region from the original line of injury. Initial migration was associated with phosphorylation of ERK, p38 MAPK, and JNK within 5–15 min. The downstream effector of p38, heat shock protein 27, also was phosphorylated rapidly after injury; phosphorylation could be blocked by prior treatment with SB203580 but not SP600125. The downstream effector of JNK, c-Jun, likewise was phosphorylated rapidly after injury and could be blocked by inhibiting JNK. Our data demonstrate that p38 MAPK, JNK, and ERK1/2 participate in the early stages of AEC migration.

Key Words: airway epithelium • migration • mitogen-activated protein kinase • c-Jun N-terminal protein kinase


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The airway epithelium is a target of inflammatory and physical insults in asthma. Injury to the epithelium is a common finding in pathologic studies of patients with asthma, even when the clinical state of disease is mild (1, 2). Repair of the airway mucosa after injury begins quickly with establishment of a provisional basement membrane made up of plasma proteins (3). Epithelial cells near the wound edge then shift their phenotype and migrate into the wound region (3, 4). Once the wound region is covered by new cells, these cells phenotype shift into required, more differentiated cells (5, 6). This repair process is regulated by both constitutive and inflammatory cells within the airway mucosa and submucosa.

Migration of epithelial cells in airway injury frequently will close small wounds quickly without the need for cell proliferation (3). Proliferation requires > 24 h given the cell cycle time of airway epithelium and may be more useful in repairing large areas. Although areas of mucosal damage may be extensive in severe or fatal asthma (7), epithelial injury as defined by bronchoscopic biopsies is more focal in mild to moderate asthma (8, 9). These sites are of a scale that would be predicted to heal primarily by cell spreading and migration. That these focal injury sites persist in asthma suggests that defects in repair processes such as cell migration and spreading may exist.

Many cytokines, chemokines, and growth factors transduce their signals via activation of defined kinase pathways. Among these are the extracellular signal–regulated protein kinases (ERKs) and the stress-activated protein kinases (SAPK), including c-Jun NH2-terminal kinases (JNK) and the p38 mitogen-activated protein kinases (MAPK). The ERKs have a role in signaling cell proliferation, whereas the SAPKs have been implicated in cell differentiation (10, 11), migration (12), and transduction of both inflammatory and anti-inflammatory signals (13). In cultured airway smooth muscle, p38 phosphorylation and cell migration is increased after treatment with transforming growth factor-ß, platelet-derived growth factor, or the cytokine interleukin-1ß; the effect of each was blocked by the p38 inhibitor SB203580, which inhibits both {alpha} and ß isoforms of p38 (14). JNK, like p38 MAPK, also may have a role in mediating cell migration by phosphorylating c-Jun, which then may dimerize with itself or c-Fos to form the transcription factor activating protein–1 (AP-1) (15), involved in several processes related to migration such as membrane ruffling and lamellipodia formation (16, 17). Re-epithelialization of dermal wounds results in elevated c-fos but not c-jun, the latter being more constitutively expressed (reviewed in Ref. 18). Both c-fos and c-jun expression are increased after corneal wounding (15), suggesting different, tissue-specific patterns of expression.

Repair and remodeling of the airway mucosa after injury requires the integration of multiple external signals from growth factors, cytokines and chemokines, and physical stimuli. MAPKs are ideally suited to integrate these signals and then initiate proliferation, migration, and differentiation as required to reconstitute the airway mucosa. Disordered or disrupted MAPK function may lead to aberrant signaling that may, in collaboration with changes in fibroblast (19) and smooth muscle (20, 21) function, lead to and perpetuate airway remodeling. Little is known about the role of MAPKs in airway epithelial cell (AEC) migration. As a first step to understanding this process, we examined the role of ERK1/2, p38, and JNK in cultured AECs. Our data demonstrate that both kinases are required for epithelial cell migration to occur after injury, and that both coordinate separate processes in migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The following antibodies were purchased from Cell Signaling, Inc. (Beverly, MA): mouse anti-human p38 MAPK, mouse monoclonal anti-human phospho p38 MAPK (clone 28B10) that recognizes the Thr180/Thr182 residues in p38 MAPK, rabbit polyclonal anti-human JNK, rabbit polyclonal anti-human phospho-JNK that recognizes the Thr183/Thr185 residues in JNK, rabbit polyclonal anti-human ERK (p42/44 MAPK), mouse monoclonal anti-human phospho-ERK (clone E10) that recognizes the Thr202/Tyr204 residues in ERK, mouse monoclonal anti-human heat shock protein 27 (HSP27) (clone G31), rabbit polyclonal anti-human phospho-HSP27 that recognizes the Ser82 residue in HSP27, and rabbit polyclonal anti-human phospho-c-Jun that recognizes the Ser63 residue in c-Jun. A rabbit polyclonal anti-human c-Jun antibody was purchased from Santa Cruz, Inc. (Santa Cruz, CA). PD98059, an MEK inhibitor, SB203580, a p38 MAPK inhibitor, and SP600125, a c-Jun NH2-terminal kinase (JNK) II inhibitor, and SB202474, an inactive control compound for SB203580, were purchased from CalBiochem, Inc. (San Diego, CA). U0126, an inhibitor of MEK kinase activity, was purchased from Promega, Inc. (Madison, WI). Inhibitors were diluted in DMSO immediately before use. Super Signal West Fempto Maximum Sensitivity Substrate was purchased from Pierce (Rockford, IL). BEGM medium was purchased from Clonetics, Inc. (Walkersville, MD). All other reagents were obtained from Sigma, Inc. (St. Louis, MO) and were of the highest quality available.

Cell Culture
The use of primary human AECs was approved by the University of Chicago Institutional Review Board. Human subjects with no history of lung disease, a normal physical examination, normal spirometry, a negative methacholine challenge, and a negative skin allergen test to 14 common allergens underwent fiberoptic bronchoscopy and endobronchial brushing to collect primary epithelial cells from the left lower lobe. These cells were collected in DMEM medium at 4°C, washed x 2, and then grown in BEGM medium containing 5 µg/ml insulin, 0.5 ng/ml hEGF, 10 µg/ml transferrin, 6.5 ng/ml triiodothyrinine, 0.5 µg/ml hydrocortisone, 0.5 µg/ml epinephrine, 30 µg/ml gentamicin, 15 µg/ml amphotericin, and 52 µg/ml bovine pituitary extract in 5% CO2 atmosphere at 37°C. Cells were used when 100% confluent at passage 1. Primary cells were maintained in defined medium for the 24-h imaging period after wound generation to prevent any confounding effects from withdrawal of growth factors.

The cell line 16HBE14o– was obtained from Dieter Gruenert (University of Vermont, Burlington, VT), and are SV40-transformed human central AECs (22) that have cell surface markers similar to primary airway basal epithelial cells (23). Cell lines were grown on collagen-IV–coated chamber slides, 6-well plates, or Petri dishes as required in DMEM containing 10% fetal calf serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin G. All cells were used when 100% confluent, and all cell lines were used before passage 30.

Wound Repair Assay
We have previously published details of this method (24, 25). Briefly, confluent monolayers were washed twice and placed in serum-free or defined medium appropriate for the cells being studied. Mediators or control diluent (DMSO) were added as appropriate, and 15 min later a small wound was made in the confluent monolayer with a rubber stylet. In all experiments, the concentration of DMSO was < 0.1% in culture wells. All wounds were viewed immediately after creation by phase-contrast microscopy to look for signs of matrix removal within the wound; wounds with evidence of matrix removal were discarded. Wound closure was measured serially for 24 h starting immediately after wound creation. Microscope images were photographed using a Nikon digital camera attached to a Nikon Diaphot inverted-stage microscope (Nikon Inc., Morton Grove, IL); image resolution was 0.04 µm at x40 total magnification. Images were assembled using Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA). Analysis of perimeter length and area of the remaining wound in each image was performed using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Values were normalized to time 0 values. Intra-operator variance was < 1% for wounds of 1.5 mm2, and inter-operator variance was < 3%.

Wound Edge Staining
Linear wounds of ~ 1–2 mm width were generated in 16HBE14o– cell monolayers, which were fixed using 10% neutral buffered formalin 5 min to 4 h later. After washing three times in TBS-T, monolayers were treated with 5% normal goat serum in TBS-T for 1 h, and then labeled using 1:500 of the mouse anti-human phospho-p38 MAPK monoclonal antibody or 1:500 of the mouse anti-human phospho-p38 JNK monoclonal antibody for 1 h at 37°C. Monolayers were washed twice in TBS-T, then labeled using goat anti-mouse antibody conjugated to biotin at 1:1 dilution in TBS-T for 10 min. Monolayers then were washed once and treated with streptavidin for 5 min followed by diaminobenzidine for 20 min. Monolayers were washed once and counterstained with hematoxylin. Slides were mounted and examined for evidence of specific labeling. Controls done at the same time omitted the primary antibody.

Western Blot Analysis
Confluent monolayers were wounded using a cell rake, adapted from a metal lice comb. Rake tines were 0.45–0.50 mm in width at the tips, and the gaps between tines were 0.30–0.40 in width. Monolayers were pre-treated with inhibitor or sham 15 min before wounding and afterwards incubated at 37°C for 5 min to 6 h. Cell lysates then were collected in buffer (1% NP-40, 0.25% Na-DOC, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM Na3VO4, and 1 mM NaF) at 4°C and then centrifuged at 14,000 rpm for 10 min at 4°C. Supernatants were frozen at –70°C until use. Proteins were separated on a SDS-PAGE mini-gel and transferred onto nitrocellulose membranes. Immunodetection was performed using an ECL protocol (26), and detected using a Fuji LAS-3000 ImageReader (Stamford, CT). Membranes were re-probed with an antibody for actin (Sigma, Inc.) when appropriate to control for differences in protein loading.

Statistical Analysis
Wound repair data are referenced to Time 0 wound area for each wound and are expressed as the mean ± SEM. To avoid confounding problems with multiple analyses along the time–response curve, differences were analyzed at 24 h. Differences were examined by ANOVA; when significant differences were found, post hoc analysis was done using Fisher's protected least significant difference test. Differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of SAPKs in AEC Migration
Wounds were created in 16HBE14o– cell line monolayers without difficulty. Starting area in all experiments was ~ 0.6–1.5 mm2, and the coefficient of variation was always < 25% within each series of experiments. In preliminary experiments, addition of DMSO control vehicle, up to 0.1% concentration, did not affect wound closure compared with untreated controls (n = 8, data not shown).

Addition of the p38 MAPK inhibitor, SB203580 (27, 28), 15 min before wound generation attenuated wound closure significantly in a concentration-dependent manner. After 24 h, wound area in control monolayers was 27 ± 2% of starting area, whereas wound area in monolayers treated with 20 µM SB203580 at the time of wound generation was 59 ± 3% of starting area (P < 0.0001, n = 8–16) (Figure 1A). Attenuated closure was not associated, however, with decreased movement of individual cells from neighboring cells into the wound region normally seen within 6–12 h (Figure 1B). Concurrent addition of 50 ng/ml EGF, an accelerant for wound closure (29), did not overcome the inhibitory effect of SB203580 (Figure 1C). Likewise, concurrent addition of 30 ng/ml hepatocyte growth factor/scatter factor, another chemotactic factor for AECs (30, 31), did not overcome the inhibitory effect of SB203580 (data not shown). In additional experiments, pretreatment of cells with the inactive control compound, SB202474 (20 µM) (32), did not significantly change wound closure (n = 8, data not shown).





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  		Figure 1. Response to p38 MAPK inhibition in 16HBE14o– AEC migration. (A) Concentration response using the inhibitor SB203580. Inhibitor was added 15 min before wound generation, and remaining wound area was followed for 24 h. *P < 0.0001 versus control (n = 8 at each data point except for control, where n = 16). (B) Wound edge immediately and 12 h after wound generation in control monolayers and monolayers treated with 20 µM SB203580. The number of cells migrating ahead of the main line of cells is similar in each treatment group. Bar, 100 µm. (C) Effect of epidermal growth factor on wound closure. Cells were treated with 50 ng/ml EGF ± 2 or 20 µM SB203580 15 min before wound generation, and remaining wound area was followed for 24 h. *P < 0.0001 versus control (n = 8 at each data point).

 
Addition of the JNK inhibitor, SP600125 (33, 34), 15 min before wound generation likewise attenuated wound closure substantially and in a concentration-dependent manner. After 24 h, wound area in control monolayers was 38 ± 4% of starting area, whereas wound area in monolayers treated with 20 µM SP600125 at the time of wound generation was 68 ± 5% of starting area (P < 0.001, n = 8) (Figure 2A). As in the experiments with inhibiting p38, co-incubation of SP600125 with EGF led to less slowing than that seen with SP600125 alone, but far less closure than seen with EGF alone (Figure 2B). In contrast to the results seen with the p38 inhibitor, attenuated closure after treatment with the JNK inhibitor was associated with substantially decreased movement of individual cells from neighboring cells into the wound region normally seen within 6–12 h (Figure 2C).





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  		Figure 2. Response to JNK inhibition in 16HBE14o– AEC migration. (A) Concentration response using the inhibitor SP600125. Inhibitor was added 15 min before wound generation, and remaining wound area was followed for 24 h. *P = 0.007, and {dagger}P < 0.0001 versus control (n = 8 at each data point). (B) Effect of epidermal growth factor on wound closure. Cells were treated with 50 ng/ml EGF ± 20 µM SP600125 15 min before wound generation, and remaining wound area was followed for 24 h. *P = 0.004 versus control; {dagger}P = 0.003 versus EGF alone (n = 8 at each data point). (C) Wound edge immediately and 12 h after wound generation in control monolayers and monolayers treated with 20 µM SP600125. The number of cells migrating ahead of the main line of cells is substantially decreased in cells treated with the JNK inhibitor. Bar, 100 µm.

 
Addition of the MEK inhibitor, PD98059 (35), 15 min before wound generation attenuated wound closure modestly. After 24 h, wound area in control monolayers was 25 ± 2% of starting area, whereas wound area in monolayers treated with 20 µM PD98059 at the time of wound generation was 36 ± 2% of starting area (P < 0.05, n = 4) (Figure 3A). Lower concentrations of the inhibitor had no apparent effect. There was no apparent effect of this MEK inhibitor on cell movement into the wound region (Figure 3B). In additional experiments, addition of the MEK activity inhibitor U0126 (36) also attenuated wound closure significantly. After 24 h, wound area in control monolayers was 19 ± 3% of starting area, whereas wound area in monolayers treated with 20 µM PD98059 at the time of wound generation was 42 ± 2% of starting area (P < 0.0001, n = 8) (Figure 3C). As in experiments using PD98059, addition of U0126 slowed cell migration without altering cell movement into the wound region (data not shown).





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  		Figure 3. Response to MEK inhibition in 16HBE14o– AEC migration. (A) Concentration response using the inhibitor PD98059. Inhibitor was added 15 min before wound generation, and remaining wound area was followed for 24 h. *P = 0.002 versus control (n = 8 at each data point). (B) Wound edge immediately and 12 h after wound generation in control monolayers and monolayers treated with 20 µM PD98059. The number of cells migrating ahead of the main line of cells is similar in each treatment group. Bar, 100 µm. (C) Concentration response using the inhibitor U0126. Inhibitor was added 15 min before wound generation, and remaining wound area was followed for 24 h. *P = 0.01; {dagger}P < 0.0001 versus control (n = 4–8 at each data point).

 
AECs collected from normal human volunteers responded in a similar manner as the 16HBE14o– cell line to inhibition of either p38 MAPK or JNK. Cells from two normal subjects were grown to confluence at first passage. After washing, cells were treated with either sham, 20 µM SB203580, or 20 µM SP600125, and wounds were generated 15 min later. Inhibition of either p38 MAPK or JNK attenuated wound closure substantially compared with no treatment: after 24 h, remaining wound area in control monolayers was 38 ± 6% of starting area, whereas wound area in monolayers treated with 20 µM SB203580 immediately before wound generation was 80 ± 4% of starting area (P < 0.001 versus control), and was 86 ± 2% in monolayers treated with 20 µM SP600125 (P < 0.001 versus control, n = 8) (Figure 4A).






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  		Figure 4. (A) Response to either p38 MAPK or JNK inhibition in the migration of primary human AECs. A quantity of 20 µM of either SB203580 or SP600125 was added 15 min before wound generation, and remaining wound area was followed for 24 h. *P < 0.001 versus control. Data points represent four wounds each in monolayers derived from cells collected from two normal subjects. (B) Response to inhibition of p38 MAPK, JNK, or MEK in the migration of primary human AECs. A quantity of 2 or 20 µM of each inhibitor was added 15 min before wound generation, and remaining wound area was followed for 24 h. *P < 0.05, {dagger}P < 0.003, §P < 0.001, and {ddagger}P < 0.0001 versus control. Data points represent four wounds each in monolayers derived from cells collected from 1 normal subject different than the subjects used in A.

 
Concentration–response relationships were examined using cells collected from another normal subject, grown to confluence at first passage. After washing, cells were treated with either sham, 2 or 20 µM SB203580, 2 or 20 µM SP600125, or 2 or 20 µM PD98059, and wounds were generated 15 min later. Inhibition of each kinase attenuated wound closure significantly compared with no treatment (Figure 4B). Wound repair was attenuated by inhibition of MEK to a greater degree in primary cells compared with the 16HBE14o– cell line; with the other two inhibitors, the degree of attenuated repair was similar between primary cells and the cell line.

Treatment with either the p38 or JNK inhibitor could slow migration and wound closure even when the inhibitor was added after wound generation. In these experiments, 2 or 20 µM of either inhibitor was added to 16HBE14o– cell monolayers 6 h after wound generation. At this point wound closure was similar in each group of monolayers (Figure 5). By 24 h after wound generation (18 h after addition of inhibitors), migration in monolayers treated with 20 µM of either SB203580 (45 ± 2% of original area) or SP600125 (44 ± 1% of original area) had slowed substantially compared with control monolayers (19 ± 3% of original area) (P < 0.001 versus either inhibitor, n = 8). These experiments suggested that the effect of p38 MAPK and JNK on epithelial cell migration was long-lived.




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  		Figure 5. Response to delayed inhibition of either p38 MAPK or JNK in 16HBE14o– AEC migration. A quantity of 20 µM of either SB203580 (A) or SP600125 (B) was added 6 h after wound generation (arrow), and remaining wound area was followed for an additional 18 h. *P = 0.005 versus control in each panel (n = 8 at each data point).

 
Identification of Activated SAPKs in Migrating Cells
If either p38 MAPK or JNK are important in cell migration, activity in cells at or near the wound edge should be evident. As a first attempt to assess this, we generated linear wounds in 16HBE14o– cell monolayers. Labeling to detect the presence of either phospho-p38 MAPK or phospho-JNK was done in monolayers fixed 5 min to 4 h after wound generation. As shown in Figure 6A, there was little evidence for phospho-p38 immediately after wound generation. By 30 min after generation phospho-p38 was abundant, and this persisted for 4 h after wound generation. Increased labeling was confined to cells within three to five cells of the wound edge in each case. Abundance of phospho-JNK was more variable but could be demonstrated within 30 min (Figure 6B). These experiments suggested that both p38 and JNK were activated in edge cells after wound generation.




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  		Figure 6. Localization of phospho-p38 MAPK (A) and phospho-JNK (B) in 16HBE14o– AEC monolayers after wound generation. Linear wounds ~ 1–2 mm in width were created, and monolayers were fixed at indicated time points and labeled for the presence of phospho-p38 MAPK or phospho-JNK using an immunoperoxidase protocol. Primary antibody (Ab) was omitted in controls. Labeling was apparent at time points >= 30 min. Representative of three experiments for each. Bar, 100 µm.

 
We then assessed activation of both p38 and JNK after wound generation using a protocol that created multiple wound edges in a single 16HBE14o– cell monolayer (Figure 7A). Protein lysates were collected 5 min to 6 h after wound generation, and the presence of total and phosphorylated p38 and JNK were assessed by Western blot. Total p38 did not change appreciably over 6 h, but a significant increase in phospho-p38 was seen within 15 min (Figure 7B). This was blocked by the addition of 20 µM SB203580 15 min before wound generation (Figure 7B), but not by addition of either the ERK1/2 inhibitor or the JNK II inhibitor (data not shown). Similarly, abundance of total JNK did not change over 6 h, but a significant increase in phospho-JNK was seen within 5 min (Figure 7C). This increase was inhibited by addition of 20 µM SP600125 15 min before wound generation (Figure 7C), but not by addition of the p38 inhibitor (data not shown).







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  		Figure 7. (A) Representative photomicrograph of a 16HBE14o– cell monolayer 30 min after wound generation using the multiple wound edge protocol. A significant proportion of cells in the plate are within five cells of a wound edge. Bar, 100 µm. (B––D) Abundance of p38 and phospho-p38 MAPK (B), JNK and phospho-JNK (C), and ERK1/2 and phospho-ERK1/2 (D) in 16HBE14o– cell monolayers after wound generation using the multiple wound edge protocol. Proteins were collected 5 min to 6 h after wound generation and resolved using the antibodies indicated in the text. Representative of two to three experiments for each kinase. For inhibition experiments, monolayers were treated with 20 µM SB203580 (B), 20 µM SP600125 (C), or 20 µM PD98059 15 min before wound generation. Bar graphs represent densitometry of three experiments for phospho-p38 MAPK done without addition of inhibitors.

 
Phosphorylation of ERK1/2 was also seen after wound generation. Abundance of total ERK1/2 did not change over 6 h, but a significant increase in phospho-ERK was seen within 5 min (Figure 7D). This increase was inhibited by addition of 20 µM PD98059 (Figure 7D), but not by addition of the p38 inhibitor (data not shown).

Activation of Downstream Targets of p38 MAPK and JNK
One of the downstream effectors of p38 MAPK via the signaling intermediate MAPK-activated protein kinase 2/3 (MAPKAP K 2/3) is HSP27 (37). Phosphorylation of HSP27 in response to heat shock leads to its localization to the nucleus and protection from the effects of heat shock (38). HSP27 mediates cytoskeletal reorganization by inhibiting actin polymerization (38) and stabilizing actin filament organization (39, 40), and is associated with actin filaments in motile cell protrusions such as filopodia and membrane ruffles (40, 41). Ectopic expression of HSP27 stimulates, and expression of a dominant-negative mutant of HSP27 inhibits, migration of endothelial cells (42), and phosphorylation of HSP27 confers motility to human breast carcinoma cells (43). We examined whether HSP27 was phosphorylated in the initial stages of AEC migration using the multiple-wound edge model over 5 min to 6 h. Total HSP27 did not change appreciably in this time interval, but increased abundance of phospho-HSP27 was noted within 15 min (Figure 8A). Pretreatment of cells with 20 µM SB203580 15 min prior to wound generation abolished the phosphorylation of HSP27 (Figure 8A).




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  		Figure 8. Abundance of HSP27 and phospho-HSP27 (A) and c-jun and phospho-c-jun (B) in 16HBE14o– cell monolayers after wound generation using the multiple wound edge protocol. Proteins were collected 5 min to 6 h after wound generation and resolved using the indicated antibodies. Representative of three experiments. For inhibition experiments, monolayers were treated with 20 µM SB203580 or 20 µM SP600125 15 min before wound generation.

 
A principal downstream effector of JNK is c-jun, a component of the transcription factor AP-1 (15). We examined whether c-jun was phosphorylated in the initial stages of AEC migration using the multiple-wound edge model over 5 min to 6 h. Total c-jun did not change appreciably in this time interval, but increased abundance of phospho-c-jun was noted within 15 min (Figure 8B). Pretreatment of cells with 20 µM SP600125 15 min before wound generation abolished the phosphorylation of c-jun (Figure 8B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Repair of the airway mucosa after injury requires the integration of multiple external signals from growth factors, cytokines and chemokines, and physical stimuli. These integrated signals prompt spreading and migration of cells into the sites of injury, repair of defects in and changes in the composition of the underlying basement membrane, and phenotype shifting of cells into required cell subtypes. Several growth factors and cytokines signal via SAPK pathways, and diverse inflammatory mediators and physical stimuli (e.g., stretch, compression) also activate upstream kinases that lead to SAPK activation. Some recent evidence to suggest that the SAPKs may modulate migration of other cell types (10–17), and thus can be postulated to have a similar role in airway epithelium. We demonstrate that p38 MAPK, ERK1/2, and JNK modulate the early steps in AEC migration after mechanical injury, such that inhibiting either substantially attenuates migration. SAPK activation is seen in wound edge cells and occurs within 5–15 min of injury, and is associated with subsequent activation of downstream effectors. These data represent the first demonstration of the role of SAPKs in AEC repair.

While ERK1/2, p38 MAPK, and JNK each have been implicated in regulating cell migration, the exact role of each may differ depending on cell type and context. Activation of ERK is important in chemotactic responses of some cell types such as eosinophils (44) and vascular smooth muscle cells (45), though the response in smooth muscle may be heterogeneous with substantial variability in the response of subtypes (46). Inhibiting JNK but not p38 nor ERK1/2 blocks migration in keratocytes (47). Infection with dominant-negative adenoviral constructs to inhibit upstream activation of either JNK or c-Jun reduced neural crest cell migration, whereas a dominant-negative inhibitor of the p38 pathway had no effect (48). In endothelial cells, activation of p38 but not ERK1/2 is essential for thrombin-activated migration (49). In contrast, endothelial cell activation by vascular endothelial growth factor elicited migration via p38 but not via either ERK or JNK (50). In our study, both the p38 and JNK inhibitors could block repair almost completely. However, inhibition of ERK1/2 using the MEK inhibitor PD98059 attenuated wound repair modestly in the 16HBE14o– cell line, even though ERK1/2 was phosphorylated after wound generation. The MEK inhibitor U0126 attenuated cell migration more substantially in this cell line, suggesting that ERK1/2 has a role in migration. Inhibition of ERK1/2 in primary AECs did attenuate wound repair significantly, suggesting some variance between the two cell types. Our data thus suggest some differences from migration seen in other cell types, and further reinforce the recognition that regulation of migration by different MAPKs is cell type– and context-specific.

Immunohistochemical labeling of wounded monolayers indicated that both p38 and JNK are activated in cells near the wound edge almost immediately after injury. This correlated with an increase in phosphorylation of both kinases seen in the cell rake experiments that created a large number of wound edges. These results suggest that both kinases are activated in migrating cells so that wound closure may begin quickly. Even when added 6 h after injury, either SP600125 to inhibit JNK or SB203580 to inhibit p38 attenuated repair. This suggests that both p38 and JNK activation drive migration for the entire time of wound closure.

Wound closure in our experiments proceeded without addition of growth factors or serum, suggesting that endogenous activation of migration, with attendant release of epithelial-derived growth factors or cytokines, was sufficient to initiate repair. Addition of EGF accelerated wound closure but could not overcome the inhibition of either p38 or JNK.

Activated p38 MAPK can phosphorylate transcription factors such as ATF-2 (51) and Max (52). It can also phosphorylate the MAPK-associated protein kinase-2 (MAPKAP K-2) (53), which in turn phosphorylates HSP27 (50, 54). Activation of HSP27 confers resistance to cell stress and modulates actin filament dynamics by stabilizing F-actin (50, 54). In our study, wounding phosphorylated HSP27 within 5–15 min, suggesting that this kinase is activated rapidly in response to injury. We did not further define the role of HSP27 in the regulation of AEC migration after injury; in other cell systems blocking HSP27 phosphorylation may block migration (43, 55).

Members of the Jun and Fos transcription factor families cooperate to form hetero- or homodimeric complexes known as AP-1 (56), which activates a number of genes encoding growth factors that may have a role in regulating cell migration and proliferation after injury (57, 58). ERK, p38, and JNK phosphorylate and activate Elk-1, ATF-2, or c-Jun, which can activate c-jun and c-fos expression (59, 60). Thus MAPK/SAPK signaling can influence AP-1 activity both by stimulating formation and by increasing abundance of AP-1 components. Our study demonstrates phosphorylation of c-Jun and strongly suggests that these signaling pathways transduce a signal to the nucleus. It is important to note that we did not directly evaluate phosphorylation of c-Jun in our studies.

Cell migration requires the integration of multiple processes including attachment and detachment of cells from the underlying matrix, actin reorganization, membrane ruffling, and lamellipodia extension. Assays measuring the passage of cells through a semipermeable membrane can assess the chemotactic potential of an agent but do not permit evaluation of signaling at different points. The two-dimensional migration assay used in our study permits repeated measurements of migration and evaluation of cell extensions, as well as assessment of kinase phosphorylation. As with all in vitro migration assays, this assay has the major limitation that cells are studied under conditions not found in vivo. Caution should therefore be taken in extending our observations to repair in native airways.

There are some significant potential limitations with our study. One potential concern is the use of pharmacologic inhibitors to block MAPK function. Inhibiting kinases generally is fraught with complications, particularly in inhibiting kinases other than the intended target. However, transfection to inhibit kinase function also introduces a number of potential problems. Among these are the ability to express a stable, dominant-negative kinase in sufficient abundance to block native kinase function without impairing other vital processes required survival. The use of regulated (e.g., tetracycline on or off) vectors may be complicated by breakthrough expression or, at the other end, insufficient expression to block native kinase pathways. The multiple isoforms for each of the MAPKs also presents a problem, as blocking one isoform may be insufficient to block a kinase pathway. The inhibitors used in the present study (PD98059, U0126, SB203580, and SP600125) have demonstrated utility and relative specificity (27, 28, 33–36) in blocking respective kinase function. However, data derived from the use of pharmacologic inhibitors should be interpreted with caution.

In summary, we demonstrate that activation of p38 MAPK, ERK1/2, and JNK modulate the early steps in AEC migration after mechanical injury. Activation occurs rapidly after injury and modulates migration and repair for the entire period of wound closure. Our data suggest that both SAPKs have important roles in the initial phase of airway epithelial repair after injury.


   Acknowledgments
 
The authors thank Stacy Raviv, M.D., for her technical assistance.


   Footnotes
 
This work was presented in part at the 2003 International Conference of the American Thoracic Society in Seattle, Washington, May 18, 2003.

This work was supported by AI-56352 and HL-63300, and by a grant to the General Clinical Research Center of the University of Chicago, from the National Institutes of Health.

Received in original form April 9, 2004

Received in final form December 28, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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