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Published ahead of print on June 26, 2003, doi:10.1165/rcmb.2003-0121OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 76-83, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0121OC

Mechanical Stretch Induces Fetal Type II Cell Differentiation Via an Epidermal Growth Factor Receptor–Extracellular-Regulated Protein Kinase Signaling Pathway

Juan Sanchez-Esteban, Yulian Wang, Philip A. Gruppuso and Lewis P. Rubin

Department of Pediatrics, Division of Neonatology, and Program in Fetal Medicine, Women & Infants' Hospital of Rhode Island; Division of Pediatric Endocrinology and Metabolism, Hasbro Children's Hospital; and Brown Medical School, Providence, Rhode Island

Address correspondence to: Juan Sanchez-Esteban, M.D., Department of Pediatrics, Women & Infants' Hospital, 101 Dudley Street, Providence, RI 02905. E-mail: jsesteba{at}wihri.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mechanical forces are important for fetal alveolar epithelial cell differentiation. However, the signal transduction pathways regulating this process remain largely unknown. Based on the importance of the extracellular-regulated protein kinase (ERK) pathway in cell differentiation, we hypothesized that this cascade mediates stretch-induced fetal type II cell differentiation. We demonstrate that ERK1/2 was maximally activated (> 3-fold) after 15 min of cyclic stretch. Blockage of the ERK pathway with U0126 (a selective MEK1/2 inhibitor) significantly decreased stretch-inducible surfactant protein-C (SP-C) mRNA expression. We examined upstream activators of ERK1/2 and found that stretch induced phosphorylation of Raf-1 and activation of Ras. Moreover, GW5074, a selective c-Raf-1 inhibitor, decreased stretch-inducible SP-C mRNA accumulation. Mechanical stretch also stimulated epidermal growth factor receptor (EGFR) phosphorylation. Finally, blockage of the EGFR, either with tyrphostin AG1478 or neutralizing antibody, decreased stretch-inducible SP-C mRNA expression. We conclude that stretch, at least in part, induces differentiation of fetal epithelial cells via EGFR activation of the ERK pathway. These results suggest that EGFR may be a mechanosensor during fetal lung development. These findings may have significant implications for the design of strategies to accelerate lung maturation.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • dithiothreitol, DTT • epidermal growth factor, EGF • EGF receptor, EGFR • extracellular-regulated protein kinase, ERK • G-protein coupled receptors, GPCRs • horseradish peroxidase, HRP • myelin basic protein, MBP • receptor tyrosine kinase, RTK • surfactant protein, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The importance of mechanical forces in fetal lung development has been well established (1, 2). During intrauterine life, the fetus makes episodic breathing movements generating ~ 5% changes of the alveolar surface area (3, 4). The fetal lung also actively secretes fluid into the tissue lumen, creating a constant transpulmonary pressure (~ 2.5 mm Hg) in the potential airways and airspaces (5). In experimental animals, abolition of fetal breathing movements by section of the phrenic nerve or cervical spinal cord causes lung hypoplasia (6, 7). These studies demonstrate that fetal breathing movements provide physical stimuli necessary for normal lung development.

During the transition from the canalicular to saccular stage of lung development, mechanical forces contribute to remodeling of the distal lung cytoarchitecture. Along with reductions in the volume and cellularity of interstitial tissue, distal epithelial cells begin to differentiate into type II cells and secrete surfactant, the phospholipid–protein complex that lines the air–liquid alveolar interface and prevents alveolar collapse during expiration.

Hormonal factors are important regulators of lung differentiation (8). Recently, several lines of evidence have suggested that mechanical forces also play a major role in modulating lung differentiation. Chronic pulmonary overdistension produced by tracheal occlusion in fetal lambs causes an increase in lung growth but a decrease in the content of pulmonary surfactant components (saturated phosphatidylcholine, surfactant protein [SP]-A and SP-B) and in the abundance of type II cells (9). In contrast, tracheal occlusion performed later in ovine pregnancy preserves the type II cell population (10). Gutierrez and coworkers (11) showed that rat adult lung explant cultures subjected to a continuous 21% increase in cellular surface area for 16 h decreased mRNA expression of SP-B and SP-C and increased expression of a type I cell marker. When lower pressures are applied in similar in vitro systems, mechanical stretch results in increased SP-B and SP-C mRNA expression (1, 12, 13). These studies suggest that the developing lung epithelium differentially responds to the magnitude of stretch.

To date, no studies have addressed the contribution of different signaling pathways in stretch-inducible type II cell differentiation. The extracellular-regulated protein kinase (ERK) pathway is activated by mechanical stresses in endothelial cells (14), cardiomyocytes (15), and vascular smooth muscle cells (16). Mechanical forces also stimulate ERK in human lung adenocarcinoma clara cell-like H441 cells (17) and in adult alveolar type II cells (18).

Epidermal growth factor (EGF) is a critical regulator of fetal lung development (19, 20). Targeted disruption of the EGF receptor (EGFR) gene in mice by homologous recombination results in altered branching morphogenesis, deficient alveolarization and septation, and lack of type II cell maturation (21, 22, 23). Klein and colleagues (24) showed that inhibition of EGFR with antisense oligonucleotides decreased SP-A expression in human fetal lung explants. These studies demonstrate the important role of EGF in type II cell differentiation.

Based on our previous experiments on mechanical stretch in fetal type II cell differentiation (1, 12) and the importance of the ERK pathway in cell differentiation in other systems, we undertook the current studies. We show that mechanical stretch activates the ERK pathway in fetal type II cells and contributes to stretch-inducible pulmonary epithelial cell differentiation. We also demonstrate that stretch-inducible EGFR activation is a proximal signal for this intracellular differentiation pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Isolation and Mechanical Distention
Fetal rat lungs were obtained from timed-pregnant Sprague-Dawley rats (Charles Rivers, Wilmington, MA) on E19 of gestation (term = 21 d). The time of breeding was designated as Day 0. After hysterotomy, fetal lungs were dissected under sterile conditions and cleared of major airways. The tissues were finely minced and digested with 0.5 mg/ml collagenase type I and 0.5 mg/ml collagenase type IA (Sigma Chemical Co., St. Louis, MO) using vigorous pipetting for 15 min at 37°C as previously described (25). The suspension was centrifuged at 400 x g for 5 min, and the pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% (vol/vol) fetal bovine serum and sequentially filtered through 100-, 30-, and 20-µm nylon meshes. The filtrate was plated onto 75-cm2 flasks and incubated for 30–60 min at 37°C in an atmosphere of 95% air/5% CO2 to allow fibroblasts to adhere. Nonadherent cells were collected and cultured overnight in 75-cm2 flasks containing DMEM with 10% fetal bovine serum for attachment of epithelial cells (26). Purity (90 ± 5%) was assessed by microscopy for epithelial cell morphology and immunocytochemistry using cytokeratin and vimentin as markers of epithelial cells and fibroblasts, respectively. After overnight culture, epithelial cells were harvested with 0.25% (wt/vol) trypsin in 0.4 mM EDTA and plated at a density of 5–8 x 105 cells/well in Bioflex silicone elastomer multiwell plates precoated with collagen I (Flexcell Corporation, Hillsborough, NC). Cultures were maintained for an additional 24 h in serum-free DMEM. Under these conditions, monolayers express type II cell markers including SP-B and SP-C and synthesize saturated phosphatidylcholine (12). Plates were mounted in a Flexercell FX-3000 Strain Unit (Flexcell Corp.) atop flat-head Delrin cylinders. Application of vacuum stretches each membrane over the central cylinder post, creating uniform radial and circumferential strain across the membrane surface. Equibiaxial elongation of 5% was applied at intervals of 60 cycles/min for different lengths of time. For Northern blot experiments, cells were subjected to 5% cyclic stretch at 60 cycles/min for 15 min and 2.5% static distention for the remaining 45 min of each hour, for a total of 16 h. This regimen was chosen to simulate mechanical forces during fetal lung development and is based on previous observations in human (27) and ovine (3, 28) fetuses. Cells grown in nonstrained collagen-coated plates were otherwise treated in an identical manner and served as controls.

Assessment of ERK Activation
Monolayers were lysed with ice-cold RIPA buffer (150 mM NaCl, 100 mM Tris-Base pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, 3.5 mM Na3VO4, 2 mM phenylmethylsulphonylfluoride [PMSF], 50 mM NaF, 100 mM sodium pyrophosphate) with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 143.5 µM aminoethyl benzenesulfonyl fluoride). Lysates were centrifuged at 15,000 x g and the supernatants were stored at -80°C until processing. Total protein contents were determined by the bicinchoninic acid method. Protein samples (20 µg/lane) were separated by one-dimensional SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). Membranes were incubated for 1 h at room temperature in blocking buffer (TBST with 5% nonfat dry milk) to reduce nonspecific binding. To detect phosphorylated (activated) ERK, blots were incubated with anti-phospho-ERK1/2 antibody (Cell Signaling, Beverly, MA) for 1 h at RT. After washing, secondary antibody (donkey anti-rabbit–horseradish peroxidase [HRP], diluted 1:2,000 in blocking buffer) was added for 1 h at room temperature. Immunoreactive phospho-ERK1/2 was detected by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL). To control for protein loading, membranes were stripped and reprobed with antibody to total ERK1/2 (Cell Signaling) and processed as described before. The intensity of the bands was analyzed by densitometry.

Endogenous ERK activity was assayed by immunoprecipitation. Two-hundred-microgram samples of protein lysate phospho-ERK1/2 (Thr202/Tyr204) monoclonal antibody (Cell Signaling) (immobilized by cross-linking to agarose hydrazide beads) were mixed and incubated with gentle rocking overnight at 4°C. After centrifugation, pellets were washed twice with 0.5 ml of 1x lysis buffer and 0.5 ml of 1x kinase buffer (25 mM Tris, 5 mM ß-glycerophosphate, 2 mM dithiothreitol [DTT], 0.1 mM sodium orthovanadate, 10 mM MgCl2). ERK activity was measured by suspending the pellet in 50 µl of 1x kinase buffer plus 200 µM ATP and 2 µg of Elk1 fusion protein for 30 min at 30°C. Reactions were terminated by addition of 25 µM 3x SDS sample buffer (187.5 mM Tris-HCl, pH 6.8, 6% SDS [wt/vol], 30% glycerol, 150 mM DTT, 0.3% bromphenol blue [wt/vol]), boiled for 5 min, vortexed, and then centrifuged for 2 min. Thirty-microliter samples were fractionated by SDS-PAGE, blotted to nitrocellulose membranes and incubated for 3 h at room temperature in 25 ml of blocking buffer (1x TBS, 0.1% Tween 20, 5% nonfat dry milk [wt/vol]) and then overnight at 4°C with phospho-specific anti-Elk1 (Ser383) antibody (1:1,000) in 10 ml of antibody dilution buffer. Membranes were washed three times with 1x TBS, 0.05% Tween 20, and incubated with HRP-conjugated anti-rabbit secondary antibody (1:2,000) for 1 h at room temperature. After three washes in TBS, immunoreactive bands were detected by ECL.

Phosphorylation of EGFR
E19 cells were lysed in RIPA buffer and subjected to Western blot analysis using a polyclonal anti-phospho-EGFR (Tyr845) antibody (Cell Signaling). Anti-rabbit IgG conjugated to HRP was used as the secondary antibody. Phosphorylation of EGFR was visualized using ECL.

Raf-1 Kinase Assay
Raf-1 kinase activity was determined using a commercially available kit (Upstate Biotechnology, Lake Placid, NY) according to manufacturer's recommendations. Subconfluent E19 type II cells grown in Bioflex plates were subjected to stretch (5% elongation, 60 cycles/min) for 15 min. Monolayers were lysed in ice-cold RIPA buffer as described above. Cell lysates were centrifuged and the supernatants (1 mg of protein) were incubated with 2 µg of anti-Raf-1 antibody and 50 µl of 50% protein G sepharose slurry (Amersham Pharmacia Biotech, Piscataway, NJ) with gentle agitation for 2 h at 4°C. Protein G sepharose with immunoprecipitated Raf-1 was washed and incubated with inactive MEK1 and ERK2 in the presence of assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT) and magnesium/ATP cocktail. Activated ERK was then used to phosphorylate myelin basic protein (MBP) in the presence of [{gamma}-32P]ATP. The radiolabeled substrate was allowed to bind to P81 phosphocellulose paper, and radioactivity was measured in a scintillation counter. Nonspecific binding was assessed using sheep IgG.

Ras Activation Assay
Activated Ras was measured using an affinity precipitation assay (Upstate Biotechnology). Briefly, E19 type II cells were subjected (or not) to the stretching protocol for 15 min. Monolayers were washed with cold PBS and lysed in 125 µl of Mg2+ lysis/wash buffer (MLB) containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 1 mM NaF, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF. Lysates diluted to ~ 1 µg/ml total cell protein in MLB were precleared with glutathione agarose. Cell protein lysate (1 mg) was incubated with 15 µl Raf-1 Ras binding domain agarose conjugate and gently agitated at 4°C for 30 min. After centrifugation, beads were washed three times with MLB, resuspended in Laemmli sample buffer and boiled for 5 min. Samples were fractionated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with 1 µg/ml of anti-Ras antibody (clone RAS10; Upstate Biotechnology) overnight at 4°C. HRP-conjugated anti-mouse secondary antibody was added for detection by ECL.

Northern Blot Analysis
Total cellular RNA was isolated using a single-step method as previously described (1). RNA (10 µg) was denatured at 65°C for 5 min and fractionated by 1.4% agarose, 2.2 M formaldehyde gel electrophoresis. RNA was blotted, transferred to GeneScreen (NEN, Boston, MA) nylon membranes in 10x SSC (1x SSC = 150 mM NaCl, 15 mM sodium citrate) and immobilized by ultraviolet cross-linking. SP-C and GAPDH antisense complementary RNA (cRNA) probes were synthesized from linearized recombinant phagemid template using in vitro transcription (Promega, Madison, WI), T7 RNA polymerase and [{alpha}-32P]UTP (Amersham). Unincorporated nucleotides were separated from the RNA probes by affinity chromatography on Elutip columns (Schleicher and Schuell, Keene, NH). Blots were hybridized with 106–107 cpm of probe for 18–22 h at 65°C and washed in 0.5x SSC/1% sodium dodecyl sulfate (SDS) at the same temperature. The intensity of mRNA bands of interest in each lane was normalized to the constitutively expressed GAPDH mRNA and to 18S rRNA fluorescence to control for differences in samples loading. Blots were exposed to X-ray films with intensifying screens at -80°C. Autoradiographs were measured by densitometry.

Cytotoxicity Assay
The cytotoxic effects of U0126, AG1478, and GW5074 on E19 type II cells were measured using a CytoTox nonradioactive cytotoxicity assay (Promega) to detect the release of cellular lactate dehydrogenase (LDH) into the culture medium. Under the experimental conditions, none of these chemicals caused any significantly increased cytotoxicity when compared with samples without inhibitor.

Statistical Analysis
Results are expressed as means ± SEM from at least three experiments, using different litters for each experiment. Control and stretched samples were compared by unpaired Student's t test. For multiple comparisons, data were analyzed with ANOVA followed by post hoc tests. P < 0.05 was considered statistically significant. Instat 3.0 (GraphPad Software, San Diego, CA) was used for statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of the ERK Pathway by Mechanical Stretch in Fetal Type II Cells
To analyze the role of the ERK cascade on stretch-induced type II cell differentiation we first determined whether mechanical stretch activates ERK signaling in E19 type II cells. In previous experiments (12) we determined that stretch-inducible alveolar differentiation is maximal on E19. Monolayers were subjected to cyclic stretch for different periods of time, and ERK activation was assayed using ERK1/2 phospho-specific antibodies. Cyclic stretch-activated ERK (pERK-1, pERK-2) by 2 min. ERK activation increased to > 3-fold by 15 min and decreased thereafter (Figure 1A).



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  		Figure 1. Effects of mechanical stretch on ERK activation. (A) E19 type II cells were subjected to cyclic stretch for the indicated periods of time. Unstretched (static) samples served as controls. Proteins were extracted and the levels of ERK1/2 phosphorylation were analyzed by Western blot using specific anti–phospho-ERK1/2 (pERK) antibodies. Blots were then stripped and re-hybridized with total ERK (tERK) antibody to control for protein loading. The upper panel shows a representative E19 blot. Data shown in the lower panel are representative of three independent experiments (*P < 0.01 versus control, Dunnett's post hoc test). (B) E19 monolayers were subjected or not to stretch (5% cyclic distention, 60 cycles/min) for 15 min. ERK activity was assessed by an in vitro kinase assay using ELK-1 as substrate. The upper panel shows a representative kinase blot. The data shown in the lower panel are representative of three independent experiments (P < 0.03).

 
Mechanical stretch also phosphorylated ERK-1/2 in E18 and E20 fetal epithelial cells in a similar pattern, although the magnitude of activation was less than that on E19 (data not shown). ERK activation by mechanical stretch could also be demonstrated using an in vitro kinase assay with phospho-ELK-1 (pELK-1) as substrate (Figure 1B).

Effects of ERK Inhibition on SP-C Gene Expression
Next, we determined whether the ERK pathway is involved in stretch-inducible type II cell differentiation. We tested two MEK1 inhibitors, PD98059 and U0126; U0126 was a more potent inhibitor of ERK phosphorylation (Figure 2), consistent with previously published findings (29). Mechanical stretch significantly increased SP-C mRNA expression over unstretched controls (n = 4, P < 0.001) (Figure 3). Preincubation with U0126 effectively prevented stretch-inducible SP-C mRNA accumulation in a concentration-dependent fashion (n = 4, P < 0.001). These data indicate that ERK-mediated signaling is involved in stretch-inducible SP-C mRNA expression.



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  		Figure 2. Effects of MEK inhibitors on ERK phosphorylation. E19 alveolar type II cells were preincubated with the MEK inhibitors PD98059 or U0126 at different concentrations and subjected to stretch (as described in MATERIALS AND METHODS) for 15 min. ERK activation was measured using anti–phospho-ERK1/2 antibody. This blot is representative of two separate experiments.

 


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  		Figure 3. U0126 inhibits stretch-inducible surfactant protein-C (SP-C) mRNA expression. E19 type II cells were plated on silastic membranes and subjected to mechanical stretch (as described in MATERIALS AND METHODS) for 16 h in the presence of varying concentrations of the ERK pathway inhibitor U0126. Samples were analyzed by Northern blot to determine the abundance of SP-C mRNA. The upper panels show a representative blot. Data shown in the lower panel are representative of four independent experiments, U0126 (40 µM) (*P < 0.001, control, vehicle versus stretch, vehicle; #P < 0.001 stretch, vehicle versus stretch, U0126; Tukey's multiple comparison test).

 
Role of Raf-1 in Stretch-Inducible ERK Activation and SP-C mRNA Expression
We examined potential proximal effectors linking mechanical stretch to ERK1/2 activation. We found that Raf-1 was activated by cyclic stretch in E19 cells measured by the phosphorylation of MBP via the Raf-1-MEK1-ERK2 kinase cascade. Raf-1 kinase activity doubled after 15 min of cyclic stretch when compared with static control samples (n = 3, P = 0.02) (Figure 4A). We also analyzed the role of Raf-1 on stretch-inducible type II cell differentiation by selectively blocking Raf-1 with GW5074 (30). Preincubation of E19 cells with this specific Raf-1 inhibitor significantly attenuated stretch-inducible SP-C mRNA accumulation (Figure 4B). Taken together, these data further demonstrate a role for the ERK signaling cascade in stretch-inducible type II cell differentiation.



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  		Figure 4. (A) Mechanical stretch activates Raf-1 in fetal alveolar epithelial cells. E19 monolayers were subjected to cyclic stretch for 15 min. Raf-1 kinase activity was assayed by the phosphorylation of MBP through the Raf-1-MEK1-ERK2 kinase cascade in the presence of [{gamma}-32P]ATP. Radioactivity was measured by scintillography. Data represent three separate experiments, P = 0.02. (B) Effects of Raf-1 inhibitor on SP-C mRNA accumulation. E19 type II cells were subjected to stretch for 16 h in the presence or absence of the selective Raf-1 inhibitor GW5074 (10 µM). RNA was extracted and samples were assayed by Northern blot to detect the abundance of SP-C mRNA (*P < 0.05 control, negative versus control, GW5074; #P < 0.001 control, negative versus stretch, negative; **P < 0.001 stretch, negative versus stretch, GW5074; Tukey's multiple comparison test).

 
Activation of Ras by Mechanical Stretch
An important step in Raf-1 activation involves interaction with Ras. Therefore, we determined whether cyclic stretch also activates Ras. Ras activation was measured using an affinity precipitation assay based on the specific interaction between Ras-GTP and the Ras binding domain of Raf-1. Results (Figure 5) showed that after 30 s of cyclic stretch, Ras was activated by > 2-fold (n = 3, P < 0.05). These data indicate Ras–Raf-1 is a proximal step in stretch-induced ERK activation in fetal type II cells.



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  		Figure 5. Cyclic stretch induces Ras activation in fetal type II cells. E19 monolayers were subjected to cyclic stretch for 30 s. Unstretched samples served as controls. Ras activation was analyzed using an affinity precipitation assay based on the specific interaction between Ras-GTP and the Ras binding domain of Raf-1 (n = 3, *P < 0.05).

 
The Role of EGFR in Stretch-Induced ERK Activation
The best-described activators of Ras-Raf-1-ERK signaling are members of the family of receptor tyrosine kinases (RTKs), including the EGFR. To explore whether mechanical stretch uses a similar cell surface activation mechanism, we pharmacologically blocked EGFR activation with the selective inhibitor tyrphostin AG1478. Preincubation with AG1478 had no independent effect on ERK phosphorylation in static type II cells. In contrast, AG1478 decreased stretch-inducible ERK activation by 40% after 15 min of cyclic stretch (n = 4, P < 0.01) (Figure 6). This effect is not generalized among type II cell RTKs, since similar treatment with AG1295, a selective platelet-derived growth factor RTK inhibitor, had no effect on ERK1/2 activation (data not shown).



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  		Figure 6. Effects of EGFR inhibition on stretch-induced ERK activation. E19 cells were subjected to cyclic stretch for 15 min in the presence or absence of AG1478, a selective EGFR tyrosine kinase inhibitor. Equal amounts of protein were loaded and immunoblotted to detect ERK activation using an anti-phospho-ERK1/2 antibody. The data are from four separate experiments (*P < 0.01 control, vehicle versus stretch, vehicle; #P < 0.05 stretch, vehicle versus stretch, AG1478; Tukey's multiple comparison test).

 
To directly demonstrate the activation of EGFR by mechanical stretch, E19 cells were subjected to cyclic stretch for up to 15 min. Samples were subjected to Western blot analysis using an anti-phospho-EGFR (Tyr845) antibody. We found that EGFR was maximally phosphorylated (by 2-fold) after 30 s of cyclic stretch, returning to the baseline levels by around 15 min (Figure 7). Taken together, these data suggest that the EGFR may function as a mechanosensor in fetal type II cells.



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  		Figure 7. Mechanical stretch activates EGFR. E19 cells were subjected to cyclic stretch for the indicated periods of time. Unstretched samples served as controls. Samples were immunobloted with an anti–phospho-EGFR (Tyr845) antibody to detect the active form (upper panel, two separate experiments). The lower panel shows equal protein loading (Coomassie Blue stain).

 
Effect of EGFR Inhibitors on SP-C mRNA Expression
To determine whether EGFR stimulation is necessary for stretch-inducible fetal type II cell SP-C expression, E19 monolayers were subjected to mechanical stretch for 16 h (as described in MATERIALS AND METHODS) in the presence or absence of EGFR inhibition by AG1478 or neutralizing antibody 151-IgG. Preincubation of monolayers with AG1478 had no effect on SP-C mRNA accumulation in static type II cells, but significantly attenuated stretch-inducible SP-C mRNA expression compared with samples without inhibitor (n = 3, P < 0.01) (Figure 8A). Similarly, blockage of the extracellular ligand-binding domain of the EGFR with the monoclonal antibody 151-IgG (15 µg/ml; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) significantly decreased stretch-inducible SP-C mRNA accumulation (n = 3, P < 0.001) (Figure 8B). Furthermore, under static conditions, the addition of EGF (10 µg/ml) to the culture media for 16 h doubled the expression levels of SP-C when compared with samples without EGF. Under conditions of mechanical stretch, EGF did not additionally increase SP-C mRNA levels compared with samples without EGF (data not shown).



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  		Figure 8. (A) AG1478 inhibits stretch-induced SP-C mRNA accumulation. E19 type II cells were subjected to stretch protocol (as described in MATERIALS AND METHODS) for 16 h in the presence or absence of AG1478 (1 µM), a selective EGFR tyrosine kinase inhibitor. Samples were processed to detect the abundance of SP-C mRNA expression by Northern blot. The upper panel depicts a representative blot. Data in the lower panel are representative of three separate experiments (*P < 0.01 control vehicle versus stretch vehicle; #P < 0.01 stretch vehicle versus stretch AG1478). (B) 151-IgG inhibits stretch-induced SP-C mRNA accumulation. E19 type II cells were subjected to cyclic stretch for 16 h in the presence or absence of 151-IgG (15 µg/ml), an EGFR neutralizing antibody. Samples were processed to detect the abundance of SP-C mRNA expression by Northern blot. The upper panel depicts a representative blot. Data in the lower panel are representative of three separate experiments (*P < 0.001 control, negative versus stretch negative; #P < 0.001 stretch, negative versus stretch, 151-IgG).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous in vitro experiments have shown the importance of mechanical forces as a regulator of lung growth (31), cytoarchitectural remodeling (32), and type II cell differentiation (12, 13). Although mechanical forces modify epithelial development, the specific mechanosensors and signaling pathways involved in this process are largely unknown.

Mechanical forces activate the MAP kinase ERK pathway in a variety of cells and tissues (14, 15, 16). Chess and colleagues (17) showed that stretch activates ERK in Clara cell–like H441 cells, suggesting a possible role in lung epithelial cell proliferation. Edwards and coworkers (33) demonstrated that osmotic stress in adult type II cells activates ERK. Recently, Correa-Meyer and associates (18) observed ERK activation in adult type II cells subjected to mechanical stretch. Our data show that mechanical stretch also activates this pathway in fetal rat type II cells. Using this model, we now demonstrate that mechanotransduction at the cell surface signals through ERK to promote alveolar epithelial development. SP-C, which is selectively expressed in epithelial cells committed to the type II cell lineage (34), is a useful marker of type II cell differentiation. By blocking the ERK pathway with the specific MEK1/2 inhibitor U0126, we observed a significant decrease in stretch-inducible SP-C mRNA expression.

The present investigation identifies proximal mediators of stretch-activated ERK1/2, specifically Raf-1 and Ras. Raf-1 plays a key role in the ERK cascade through its action as a MEK-activating kinase (35). Among the different Raf isoforms (A-Raf, B-Raf, and C-Raf-1), Raf-1 may be a critical regulator of fetal lung development. Mice in which the Raf-1 gene has been disrupted display absence of lung maturation (36). Consistent with these observations, we found that Raf-1 is activated by mechanical stretch in fetal type II cells and appears to be a critical proximal regulator of stretch-inducible fetal alveolar epithelial cell differentiation.

The monomeric G protein Ras occupies a central position in cell signaling by mediating the intracellular responses to a variety of extracellular stimuli including growth factors, cytokines, and hormones (37). One of the best-defined signaling pathways that leads from the cell membrane to ERK activation involves receptor tyrosine kinases. Stimulation of these receptors by their cognate ligands results in an increase in receptor catalytic activity, autophosphorylation of receptor tyrosine residues, and the subsequent formation of multiprotein complexes and downstream signaling events. RTK activation of Ras is achieved by the recruitment of adaptor proteins, such as Shc and Grb2. The guanine nucleotide exchange factor Sos then becomes engaged with the complex and induces Ras to exchange GDP for GTP. Ras-GTP directly interacts with a number of effectors, including Raf-1 (38).

Our data show that mechanical stretch maximally activates Ras after 30 s of cyclic stretch. These findings are consistent with its role in early recruitment to the membrane upon complex activation. Correa-Meyer and colleagues (18), using adult type II cells, found that stretch activated ERK and inhibition of the EGFR tyrosine kinase with AG1478 decreased ERK phosphorylation. However, they observed neither Ras nor Raf-1 activation. They suggested that EGFR might be transactivated through G-protein coupled receptors (GPCRs), acting as a scaffold for growth factor–independent downstream signaling. Our studies demonstrate the presence of a stretch-inducible EGFR-Ras-Raf-1-ERK1/2 signaling cascade. These different results may emphasize the developmentally dependent diversity and complexity of cell signaling. Prenatal stretch-induced ERK activation may play a different biological role (e.g., type II cell differentiation) and use different signaling pathways compared with the mature lung, where the type II cell phenotype is fully developed.

EGF is critical for pulmonary branching morphogenesis and type II cell maturation (19, 20). In various animal models, EGF increases alveolarization (39), stimulates surfactant phospholipid synthesis (40), and induces alveolar type II cell differentiation (41). EGFR may serve as mechanosensor in cardiomyocytes (42), bladder and vascular smooth muscle cells (43, 44), and bronchial epithelial cells (45) subjected to mechanical forces. These studies have linked stretch-induced EGFR activation to several pathologic conditions such as cardiac and bladder hypertrophy and asthma. Our in vitro experiments also demonstrate that EGFR is maximally phosphorylated after 30 s of cyclic stretch, suggesting its role as a mechanosensor in fetal alveolar type II cells. The EGFR tyrosine kinase inhibitor, tyrphostin AG1479, and neutralizing antibody to the EGFR ectodomain both decreased stretch-inducible SP-C mRNA expression, indicating that this receptor may be important for mechanotransduction in fetal type II cell differentiation.

The mechanisms by which mechanical forces might activate the EGFR remain unknown. Physical deformation of the cell membrane may induce conformational changes in the receptor leading to its activation. In addition, various other signals, aside from EGF, also can directly or indirectly activate the EGFR. These diverse stimuli include GPCR agonists, cytokine receptors, integrins, ion channels, other RTKs, and environmental stress factors (46). Activation of the EGFR by seemingly unrelated stimuli was mechanistically difficult to understand until Prenzel and coworkers (47) demonstrated transactivation of the EGFR by GPCR agonists. This group showed that treatment with various GPCR agonists increased conversion of the transmembrane heparin-binding EGF precursor to the soluble heparin-binding EGF ligand for the EGFR. This proteolytic cleavage is mediated by metalloproteinases. Since then, numerous studies have shown the importance of this mechanism to activate EGFR (48). Our data suggest that EGFR activation may be, at least in part, mediated through a similar mechanism because blockage of the extracellular ligand–binding domain of the EGFR with neutralizing antibodies decreased stretch-induced SP-C mRNA expression. Whether mechanical activation of the EGFR requires ligand binding is currently under investigation.

In summary, our data show that mechanical stretch activates ERK in fetal rat type II cells and plays an important role in stretch-inducible type II cell maturation. We propose a signaling model in which mechanical stretch increases expression of SP-C via activation of the EGFR and the ERK signaling pathway. These results may be relevant to the design of therapeutic strategies to accelerate lung maturation in clinical conditions where lung development is impaired.


   Acknowledgments
 
The authors thank E. Filardo, Ph.D., for helpful discussions, Virginia Hovanesian for technical assistance with image analysis, and Brian P. Johnston for manuscript preparation. This work was funded by the Rhode Island Foundation and a Richard B. Salomon Faculty Award (Brown University) to J.S.E.

Received in original form April 1, 2003

Received in final form June 12, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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