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Published ahead of print on July 15, 2004, doi:10.1165/rcmb.2003-0433OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 528-537, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0433OC

Epidermal Growth Factor and Trefoil Factor Family 2 Synergistically Trigger Chemotaxis on BEAS-2B Cells via Different Signaling Cascades

Caroline E. Chwieralski, Ingo Schnurra, Lars Thim and Werner Hoffmann

Institut für Molekularbiologie und Medizinische Chemie, Otto-von-Guericke-Universität, Magdeburg, Germany; and Department of Protein Chemistry, Novo Nordisk A/S, Bagsvaerd, Denmark

Address correspondence to: Prof. Werner Hoffmann, Institut für Molekularbiologie und Medizinische Chemie, Universitätsklinikum, Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail: Werner.Hoffmann{at}Medizin.Uni-Magdeburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal–regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor {kappa}B. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.

Abbreviations: bisindolylmaleimide I hydrochloride, BIS • Dulbecco's modified Eagles medium, DMEM • extracellular matrix, ECM • ethylenediamine-N,N,N',N'-tetraacetic acid, EDTA • ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, EGTA • epidermal growth factor, EGF • extracellular signal–regulated kinase, ERK • inhibitory protein {kappa}B, I{kappa}B • interleukin, IL • c-Jun-N-terminal kinase, JNK • mitogen-activated protein kinase, MAPK • nuclear factor {kappa}B, NF-{kappa}B • polyacrylamide gel electrophoresis, PAGE • phosphate-buffered saline, PBS • phosphoinositide 3-kinase, PI3K • phosphatidylinositol-3,4,5-triphosphate, PIP3 • protein kinase C, PKC • phospholipase C, PLC • sodium dodecyl sulfate, SDS • trefoil factor family, TFF • transforming growth factor, TGF • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Damage to the respiratory epithelium is a frequent process, e.g., after inhaling toxic agents or infection with microorganisms or during inflammatory diseases, such as asthma. The initial rapid repair of such damage occurs by a process termed "restitution." This immediate response involves the migration of epithelial cells adjacent to the injured area (1–2). Rapid re-epithelization is promoted by adhesive interactions between the migrating cells and extracellular matrix (ECM) proteins, as well as several motogenic peptides, including insulin, calcitonin gene–related peptide, epidermal growth factor (EGF), and trefoil factor family (TFF) peptides.

ECM proteins have been shown to be attachment substrates for bronchial epithelial cells (3). Fibronectin plays a particularly important role, being exclusively expressed by the migratory cells in the wounded area, but not in epithelial cells of normal airway mucosa (4). Furthermore, the normal pattern of integrin expression is dramatically modified during wound repair; for example, the {alpha}5-integrin subunit, which is part of the cellular fibronectin receptor {alpha}5ß1-integrin, is exclusively observed in repairing cells (4).

Members of the EGF family, such as EGF, transforming growth factor (TGF)–{alpha}, heparin-binding EGF-like growth factor, amphiregulin, betacellulin, epiregulin, and heregulin, are likely to be important regulators of epithelial restitution. For example, EGF induces restitution in various in vitro wound models and EGF receptor immunoreactivity increases dramatically in patients with severe asthma (5–9). These ligands are secreted from the apical surface, whereas the corresponding receptors are localized at the basolateral surface. Thus, these ligands act as luminal surveillance peptides (10) by reaching their receptors only when surface integrity is lost (i.e., when tight junctions between adjacent cells are opened) after mucosal damage. Such a discrete localization ensures that members of the EGF family launch their repair program only when needed. Such a simple but effective control mechanism has been demonstrated in the gut and airway epithelia where EGF or heregulin act as luminal surveillance factors (10, 11).

TFF peptides, such as TFF1, TFF2, and TFF3, are also typical motogens for a variety of epithelial as well as immune cells (see reviews in Refs. 12–14). They play a particularly important role for the airway epithelium. TFF3 is the predominant TFF peptide of normal human airways, where it is secreted mainly from submucosal glands and, to a lesser extent, from goblet cells (15). TFF peptides enhance migration of BEAS-2B cells in an in vitro wound assay (9). They also promote the migration of BEAS-2B cells as well as normal human bronchial epithelial cells in modified Boyden chambers, and this effect is dependent upon protein kinase C (PKC) and extracellular signal–regulated kinase (ERK) 1/2 activation (9, 16). TFF peptides also augment tumor necrosis factor (TNF)-{alpha}–induced interleukin (IL)-6 and IL-8 secretion in BEAS-2B cells (16). Furthermore, TFF2 was recognized recently as an allergen-induced gene in the murine lung (17).

Interestingly, EGF and TFF peptides show synergistic effects. For example, the presence of EGF at a concentration that, on its own, had no motogenic effect, enhanced TFF2-triggered migration of BEAS-2B cells after in vitro wounding (9). A similar effect has been reported for TFF1- or TFF3-triggered migration of HT-29 cells (18, 19). Furthermore, EGF and TFF3 cooperate in regulating epithelial chloride transport in Col-29 cells (20).

The data presented here were obtained using modified Boyden chamber assays and demonstrate the influence of different ECM proteins on the EGF-triggered haptotactic migration of BEAS-2B cells. The signaling cascades responsible for the motogenic activity of EGF were also investigated and compared with data from TFF2-triggered migration. This revealed surprising differences between both motogenic peptides. The TFF experiments presented here were performed with TFF2, making the results fully comparable with all of the cell migration data on BEAS-2B cells published recently (9, 16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of BEAS-2B Cells
The BEAS-2B cell line is from normal human bronchial epithelial cells immortalized using an SV40/adenovirus-12 hybrid virus (21). Cells were maintained in 75-cm2 cell culture flasks with filter caps or in culture dishes (Greiner, Frickenhausen, Germany) with Dulbecco's modified Eagles medium (DMEM)/Ham's F-12 (PAA Laboratories GmbH, Cölbe, Germany) supplemented with 1% 100x nonessential amino acids, 1 mM sodium pyruvate (both from Biochrom, Berlin, Germany), and 2 mM glutamine (Gibco Invitrogen Corp., Karlsruhe, Germany), and 5% AC2 (Cell Concept, Umkirch, Germany) and without supplement of antibiotics, as described previously (9, 16). The cells were starved for 18–24 h with DMEM/Ham's F-12 without any supplements before beginning the experimental procedures.

TFF Peptides, EGF
The following TFF peptides were used: recombinant glycosylated human TFF2 (TFF2/glyc; average relative molecular mass [Mr]: 14,465 [22]), recombinant nonglycosylated human TFF2 (TFF2; Mr: 11,961.5 [22]), and recombinant human TFF3/dimer (TFF3/di; Mr: 13,147 [23]). Recombinant human EGF (Mr: 6222; stock solution at 50 µg/ml in 10 mM acetic acid and 0.1% bovine serum albumin) was obtained from Sigma (Taufkirchen, Germany).

Antibodies and Reagents
Polyclonal antisera against the mitogen activated protein kinases (MAPKs) ERK1/2 and goat anti-rabbit antibodies coupled with horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specifically recognizing activated ERK1/2 (phospho-T202/Y204), c-Jun-N-terminal kinase (JNK; phospho-T183/Y185), p38 MAPK (phospho-T180/Y182), Akt/protein kinase B (phospho-S473), p70S6K (phospho-T389; phospho-T421/S424), or inhibitory protein {kappa}B (I{kappa}B; phospho-S32) were from Cell Signaling Technology Inc. (Beverly, MA). The polyclonal antiserum recognizing activated PKC{alpha} (phospho-S657) as well as the monoclonal antibody against nonphosphorylated PKC{alpha} (clone M4) were from Upstate Biotechnology (Lake Placid, NY). Different ECM molecules were used as substrates for coating the underside of the Boyden chamber membranes: collagen type I from rat tail (Upstate Biotechnology), fibronectin, laminin, collagen type IV (all from Sigma), and vitronectin (Promega, Mannheim, Germany). The inhibitors AG1478, UO126, PD98059, BAY11–7082, curcumin, wortmannin, SB203580, Gö6976, bisindolylmaleimide I hydrochloride (BIS), SP600125, and U-73122 were from Calbiochem-Novobiochem (Schwalbach, Germany). The protease inhibitor mixture "Complete" was purchased from Roche Diagnostic GmbH (Mannheim, Germany). Poly-L-lysine, sodium orthovanadate, dithiothreitol, and Triton X-100 were from Sigma. Bovine serum albumin was purchased from PAA Laboratories GmbH (for immunoblotting) or from Sigma (for cell migration assays), and milk powder was from Roth (Karlsruhe, Germany).

Immunoblotting of Proteins
Immunoblotting of proteins was performed as described previously (16). BEAS-2B cells were grown to confluence, starved overnight, and stimulated with EGF at given concentrations for indicated times, washed with ice-cold phosphate-buffered saline (PBS) and lysed at 4°C in lysis buffer (20 mM Hepes/pH 7.5, 10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 40 mM ß-glycerophosphate, 1% Triton X-100, 25 mM MgCl2, 2 mM sodium orthovanadate, 1 mM dithiothreitol, and the protease inhibitor mixture "Complete" according to the manufacturer's recommendations). The lysates were clarified by centrifugation at 15,000 x g for 10 min at 4°C, and the protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL). Samples were boiled with sodium dodecyl sulfate (SDS) sample buffer. 30 µg of protein were loaded per lane, separated by SDS–polyacrylamide gel electrophoresis (PAGE; 10% gel) and analyzed by Western blotting using different polyclonal antibodies against activated ERK1/2, p38, JNK, Akt, p70S6K, I{kappa}B or PKC{alpha} as well as the non-phosphorylated forms of ERK1/2 or PKC{alpha}. Immunoreactivity was detected with the ECL Western blotting analysis system (Amersham Pharmacia Biotech, Freiburg, Germany) using a Biomax ML film (Kodak, Rochester, NY).

Cellular Fractionation and PKC{alpha} Immunoreactivity
BEAS-2B cells were cultivated in 100-mm dishes, starved for 20 h, and stimulated with EGF (1.5 x 10–10 M) for indicated times (see Figure 5). The following cellular fractionation was similar to that described previously (24). Cells were washed with ice-cold PBS and lysed with PKC buffer (15 mM Tris/pH7.5, 2 mM ethylenediamine-N,N,N',N'-tetraacetic acid [EDTA], 10% glycerol, 50 mM ß-mercaptoethanol, and the protease inhibitor mixture "Complete" according to the manufacturer's recommendations) for 10 min at 4°C. Cells were harvested and disrupted with a glass–Teflon homogenizer. The resulting suspension was centrifuged at 24,000 x g for 30 min at 7°C. The clear supernatant was taken as the cytosolic fraction, whereas the pellet was resuspended in PKC buffer plus 0.1% NP-40 and taken as the membrane fraction. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany), and the samples were subjected to Western blot analysis for PKC{alpha} immunoreactivity.

Cell Migration Assays Using Boyden Chambers
Cell migration assays were performed with modified Boyden chambers (6.5 mm diameter, 8 µm pores, Transwell; Costar Corp., Cambridge, MA) as described previously in detail (9, 25) with minor modifications. Briefly, starved cells were removed from culture dishes with trypsin/EDTA (0.05% trypsin and 0.53 mM EDTA), washed once with migration buffer (DMEM/Ham's F-12 supplemented with 0.25% bovine serum albumin), and then resuspended in migration buffer (106 cells/ml). A total of 105 cells was then added to the top of each migration chamber and allowed to migrate to the underside of the top chamber membrane, which was coated with various ECM substrates at given concentrations in Dulbecco–PBS (PAA Laboratories GmbH) at 37°C for 2 h. The lower chambers contained 500 µl migration buffer with different peptides, inhibitors, or vehicle solutions as indicated. Nonmigratory cells on the upper membrane surface were removed with a cotton swab. The migratory cells attached to the bottom surface of the membrane were stained with freshly prepared 0.025% crystal violet in 0.1 M borate buffer (pH 9.0) and 2% ethanol for 30 min at room temperature. The stained cells were eluted with 10% acetic acid and the absorbance was determined at 590 nm.

Experiments with different inhibitors were performed by preincubation of starved cells for 30 or 60 min with the inhibitor prior to trypsin treatment. The corresponding inhibitors at the given concentrations were also present in the lower chamber during cell migration. Each determination represents the average of three or four individual wells.

Statistical Analysis
Error bars in the figures represent ± SEM). Significance by Student's t test is indicated in the figures by one asterisk (P <= 0.05), two asterisks (P <= 0.01), and three asterisks (P <= 0.001).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF Enhances Haptotactic Migration of BEAS-2B Cells Cultured in Boyden Chambers: Chemotactic Activity of EGF
Migration of BEAS-2B cells was routinely measured using modified Boyden chambers, where the underside of the membrane was coated with 10 µg/ml collagen type I as a haptotactic substrate (9, 16). The lower chamber contained various concentrations of EGF as a chemoattractant and the migratory cells attached to the bottom surface of the membrane were quantified by crystal violet staining. A typical dose–response curve is shown in Figure 1, which clearly exhibits a biphasic motogenic response with a maximum migration rate at 1.5 x 10–10 M EGF.



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  		Figure 1. Motogenic response of BEAS-2B cells to EGF as a function of concentration. Starved BEAS-2B cells were allowed to migrate for 6 h in the presence of various EGF concentrations in the lower chamber using modified Boyden chambers coated on the underside with 10 µg/ml collagen type I. Cell migration was quantified as described in MATERIALS AND METHODS.

 
A simplified checkerboard analysis was performed to distinguish between chemotactic and chemokinetic activity of EGF. Here, EGF was applied in either the lower or upper chamber or in both chambers. The results presented in Figure 2 demonstrate that the majority of EGF-stimulated migration result from chemotaxis rather than chemokinesis.



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  		Figure 2. EGF promotes mainly chemotactic migration of BEAS-2B cells in modified Boyden chambers. Starved BEAS-2B cells were allowed to migrate for 6 h in the absence (control) or presence of 1.5 x 10–10 M EGF in the different compartments of the Boyden chamber. The undersides of the membranes were coated with 10 µg/ml collagen type I. Significance between control cells and EGF-treated cells is indicated: ***extremely high significance (P <= 0.001).

 
EGF-Induced Haptotactic Migration of BEAS-2B Cells: Influence of Different ECM Proteins
The following extracellular matrix components as well as poly-L-lysine were tested as haptotactic substrates: collagen type I, collagen type IV, laminin, vitronectin, and fibronectin (Figure 3). There was only low basic migratory activity of BEAS-2B cells in the absence of the chemoattractant EGF regardless of the haptotactic substrate tested (white bars in Figure 3). However, when EGF was present, all ECM components drastically enhanced haptotactic migration in a dose-dependent manner, whereas only low basic migratory activity was observed when a haptotactic substrate was missing. Thus, the motogenic response to EGF was clearly dependent on a haptotactic substrate, such as collagen type I, collagen type IV, laminin, vitronectin, or fibronectin. Only poly-L-lysine did not serve as a positive haptotactic substrate in this assay.



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  		Figure 3. Migration of BEAS-2B cells toward different haptotactic substrates in Boyden chambers. Starved BEAS-2B cells were allowed to migrate for 6 h in the absence (white bars, control) or presence of 1.5 x 10–10 M EGF (black bars) using membranes coated on the underside with the various ECM substrates (collagen type I, collagen type IV, laminin, vitronectin, fibronectin, or poly-L-lysine) at the concentrations indicated.

 
EGF-Induced Phosphorylation of Signaling Proteins in BEAS-2B Cells
ERK, JNK, and p38 are three well-characterized MAPKs, whereas both Akt and p70S6K are members of the phosphoinositide 3-kinase (PI3K) signaling cascade. Activation of these enzymes, as well as I{kappa}B by EGF, was tested in BEAS-2B cells at three different time points (8, 15, and 40 min; Figure 4). Phosphorylation of ERK1/2, JNK, p38, Akt, and p70S6K was transient with maxima between 8 and 15 min. Only for ERK1/2 was low-level activation observed, even at 40 min after stimulation. In contrast, significant phosphorylation of I{kappa}B was not detectable under these conditions.



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  		Figure 4. EGF induces phosphorylation of ERK1/2, JNK, p38, Akt, and p70S6K in BEAS-2B cells. Starved BEAS-2B cells were exposed to 1.5 x 10–10 M EGF for 8, 15, or 40 min. As negative controls, cells were incubated with an equal volume of 10 mM acetic acid, 0.1% bovine serum albumin, which was used as the solvent for EGF (cEGF); additionally, untreated cells were analyzed after 8 min (c). Cells were lysed, subjected to SDS-PAGE, and blotted onto nitrocellulose membranes. Immunoblots were obtained using polyclonal antisera against activated ERK1/2, JNK, p38, Akt, p70S6K, or I{kappa}B. Immunostaining of nonphosphorylated ERK1/2 served as an internal control for the amount of protein analyzed. The molecular size standard is shown on the left.

 
Furthermore, activation of PKC{alpha} by EGF was tested after cell fractionation at three different time points (2, 6, and 15 min). There was a transient phosphorylation of PKC{alpha} detectable in the membrane fraction after 6 min, but not in the cytosolic fraction (Figure 5).



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  		Figure 5. EGF induces phosphorylation of PKC{alpha} in BEAS-2B cells. Starved BEAS-2B cells were exposed to 1.5 x 10–10 M EGF for 2, 6, or 15 min. As negative controls, cells were incubated with an equal volume of 10 mM acetic acid, 0.1% bovine serum albumin, which was used as the solvent for EGF (cEGF); additionally, untreated cells were analyzed after 2 min (c). Cells were lysed, separated into a cytosolic and a membrane fraction (see MATERIALS AND METHODS), subjected to SDS-PAGE (15 µg or 20 µg, respectively), and blotted onto nitrocellulose membranes. Immunoblots were obtained using a polyclonal antiserum against activated PKC{alpha}. Immunostaining of nonphosphorylated PKC{alpha} (monoclonal antibody) served as an internal control for the amount of protein analyzed. The molecular size standard is shown on the left.

 
In addition, activation of ERK1/2, JNK, p38, or Akt by EGF was tested in the presence of specific signal transduction inhibitors (Figure 6). These experiments were designed to gain more insight into the possible interrelationships between the different signaling cascades. The PKC{alpha}-specific inhibitor Gö6976 (26) blocked activation of both p38 and Akt. Furthermore, the ERK kinase inhibitor PD98059 blocked activation of JNK, and the PI3K inhibitor worthmannin blocked the activation of p38.



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  		Figure 6. Influence of various inhibitors on the EGF-induced phosphorylation of ERK1/2, JNK, p38, and Akt. Starved BEAS-2B cells were preincubated for 1 h with various inhibitors or dimethylsulfoxide (solvent for the inhibitors) as a control and then additionally exposed to 1.5 x 10–10 M EGF for 8 min. As negative controls (c), the preincubated cells were exposed for 8 min to an equal volume of 10 mM acetic acid and 0.1% bovine serum albumin, which was used as the solvent for EGF. Cells were lysed, subjected to SDS-PAGE, and blotted onto nitrocellulose membranes. Immunoblots were obtained using polyclonal antisera against activated ERK1/2, JNK, p38, or Akt. Immunostaining of nonphosphorylated ERK1/2 served as an internal control for the amount of protein analyzed. The following inhibitors were used: Gö6976 (GÖ; 5 µM), BAY11–7082 (BAY; 25 µM), PD98059 (PD; 50 µM), and wortmannin (Wo; 100 nM). The molecular size standard is shown on the left.

 
Influence of Various Inhibitors on the EGF-Induced Haptotactic Migration of BEAS-2B Cells
Motogenic effects were tested in a standardized haptotaxis assay using modified Boyden chambers coated with collagen type I and EGF in the lower chamber. A number of specific inhibitors were tested to investigate the signaling cascades responsible for cell migration processes triggered by EGF. Figure 7 represents the results obtained with the ERK kinase inhibitors PD98059 and UO126, the p38 MAPK inhibitor SB203580, the PKC inhibitors BIS and Gö6976 (26), the inhibitor BAY11–7082 (27), which prevents degradation of I{kappa}B, the inhibitor curcumin (28), which suppresses nuclear factor {kappa}B (NF-{kappa}B) activation, the PI3K inhibitor wortmannin, and the specific EGF receptor kinase inhibitor AG1478. The motogenic effect of EGF could be significantly inhibited by all inhibitors tested with the exception of the two ERK kinase inhibitors, which even enhanced the migratory response of BEAS-2B cells to EGF.



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  		Figure 7. Influence of various inhibitors on the EGF-induced migration of BEAS-2B cells in Boyden chambers. Starved BEAS-2B cells were allowed to migrate for 6 h in the absence (control) or presence of 1.5 x 10–10 M EGF as well as in the absence or presence of various inhibitors. Membranes were coated on the underside with 10 µg/ml collagen type I. Cell migration was quantified as described in MATERIALS AND METHODS. The following inhibitors were used: PD98059 (PD; 50 µM), UO126 (UO; 5 µM), SB203580 (SB; 20 µM), BIS (5 µM), Gö6976 (GÖ; 5 µM), BAY11–7082 (BAY; 25 µM), curcumin (Cur; 100 µM), wortmannin (Wo; 100 nM), and AG1478 (AG; 1 µM). *Significant difference between cells treated with (hatched bars) or without (solid bars) inhibitor (P <= 0.05); **high significance (P <= 0.01); ***extremely high significance (P <= 0.001).

 
Chemotactic Activity of TFF2 and TFF3 on Haptotactic Migration of BEAS-2B Cells: Influence of Different ECM Proteins
A simplified checkerboard analysis revealed that both TFF2 and TFF3/dimer display mainly chemotactic activity (Figure 8). Only the TFF3/dimer stimulated low but significant level of chemokinesis.



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  		Figure 8. TFF2 and TFF3/dimer promote mainly chemotactic migration of BEAS-2B cells in modified Boyden chambers. Starved BEAS-2B cells were allowed to migrate for 7.5 or 17 h in the absence (control) or presence of 1.6 x 10–6 M TFF2 or TFF3/dimer, respectively. The TFF peptides were added in the different compartments of the Boyden chamber. The undersides of the membranes were coated with 10 µg/ml collagen type I. Cell migration was quantified as described in MATERIALS AND METHODS. *Significant difference between control cells and TFF-treated cells (P <= 0.05); **high significance (P <= 0.01); ***extremely high significance (P <= 0.001).

 
In a further experimental series, we tested whether the motogenic activity of TFF2 requires haptotactic substrates (Figure 9). In the absence of any substrate, BEAS-2B cells showed a low basic migratory activity, which could not be enhanced by TFF2. The motogenic effect of TFF2 was only detectable when a haptotactic substrate was offered. Collagen type I and fibronectin were the preferred substrates, whereas collagen type IV was not a suitable haptotactic substrate for TFF2-triggered migration.



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  		Figure 9. Relative migration of BEAS-2B cells toward different haptotactic substrates in Boyden chambers. Starved BEAS-2B cells were allowed to migrate for 6 h in the presence of 1.5 x 10–10 M EGF (gray bars) or 1.2 x 10–6 M TFF2 (black bars) using membranes coated on the underside with the various ECM substrates (10 µg/ml collagen type I, 10 µg/ml collagen type IV, 10 µg/ml laminin, 10 µg/ml fibronectin or 2 µg/ml vitronectin). Shown is the fold increase when compared with migration in the absence of EGF or TFF2.

 
Synergistic Motogenic Effect of EGF and TFF2 on Haptotactic Migration of BEAS-2B Cells
The motogenic effects of EGF and TFF-peptides were shown in the past to be synergistic in an in vitro wound assay of BEAS-2B cells (9). Thus, we tested whether such an effect could also be observed in the standardized Boyden chamber assay. Figure 10 demonstrates that the motogenic effects of EGF and TFF2 are synergistic. The contribution by EGF can be blocked by the specific EGF receptor kinase inhibitor AG1478, whereas the motogenic activity of TFF2 is not sensitive to this inhibitor.



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  		Figure 10. Synergistic motogenic effect of EGF and TFF2 on migration of BEAS-2B cells in modified Boyden chambers. Starved BEAS-2B cells were allowed to migrate for 6 h in the presence or absence of 3 x 10–11 M EGF and/or 1.6 x 10–6 M TFF2 and/or the specific EGF receptor inhibitor AG1478 (1 µM; 1 h preincubation). The undersides of the membranes were coated with 10 µg/ml collagen type I. Significance between control cells (no EGF, no TFF2, no AG1478) and the cells treated with EGF and/or TFF2 and/or AG1478 is indicated: ***extremely high significance (P <= 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EGF-Induced Chemotactic Migration of BEAS-2B Cells
EGF is a typical motogen for a variety of cells (5–7, 29–31), including BEAS-2B cells (9). In the present study, the motogenic activity of EGF on BEAS-2B cells was investigated systematically by the use of modified Boyden chambers for the first time. The characteristic biphasic dose–response observed (maximum migration rate at 1.5 x 10–10 M EGF; Figure 1) is typical of chemoattractants (7, 31–33). The simplified checkerboard analysis (Figure 2) clearly demonstrated that the motogenic activity of EGF was indeed mainly due to chemotaxis (i.e., directed movement along an EGF gradient) and not to chemokinesis (i.e., nondirected movement). Generally, the chemotactic effect of EGF on BEAS-2B cells was comparable with that observed on intestinal epithelial cells (5).

Blocking ERK1/2 activation did not inhibit EGF-triggered migration, but rather appears to have enhanced the migratory response (Figure 7). This is surprising because the integrin-dependent migration of many cells was shown to be dependent upon phosphorylation of ERK1/2 and, thereby, enhancing myosin light-chain kinase activity (25). However, a lack of involvement of ERK1/2 in the EGF-triggered chemotactic response has been documented in the past (34, 35). Nevertheless, EGF certainly triggered transient ERK1/2 phosphorylation in BEAS-2B cells (Figure 4), which might be responsible for enhancing the proliferative pathway. Such competition between the proliferative and the migratory responses has been observed previously (36, 37).

The experiments using specific inhibitors (see Figure 7) indicate that the motogenic activity of EGF is dependent upon several signaling cascades (i.e., activation of PKC, PI3K, NF-{kappa}B, and p38). The implication of PKC would favor a "classical" mechanism, in which EGF triggers activation of phospholipase C{gamma} (PLC{gamma}), which hydrolyzes phosphatidylinositol-4, 5-biphosphate (PIP2) to produce inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG), the latter being the natural activator of PKC (38). In the past, such activation of PLC{gamma} has repeatedly been shown to be required for EGF-induced cell motility (30, 36). However, all attempts to block EGF-stimulated migration of BEAS-2B cells by treatment with the PLC inhibitor U-73122 failed (data not illustrated), suggesting a lack of involvement of the PLC{gamma} pathway.

Thus, as an alternative, the PI3K pathway (38) might be responsible for the motogenic effect of EGF (see Figure 7). Selective activation of PI3K is sufficient to initiate chemotaxis; PI3K is also required for membrane ruffling due to cortical actin rearrangement (39). Indeed, phosphatidylinositol-3,4,5-triphosphate (PIP3) is one of the first molecules to become polarized in response to a chemotactic agent, and localized activation of PI3K at the cell front produces rapid accumulation of PIP3 there (40). This lipid is known to recruit other molecules with pleckstrin homology domains, such as Akt, to the plasma membrane (41). Activation of the PI3K/Akt pathway via EGF has been reported for various cell lines (42), and this pathway has also been shown to regulate cell migration (35, 43). Recently, the roles of PI3K, Akt, and p70S6K1 for actin filament remodeling and cell migration were described (44). Furthermore, Akt was shown to phosphorylate Pak, which stimulated cell migration (45). Thus, the transient activation of Akt and p70S6K observed after EGF treatment of BEAS-2B cells (Figure 4), together with the inhibitory effect by wortmannin on cell migration (Figure 7) as well as on Akt phosphorylation (Figure 6), are in agreement with this PI3K/Akt/p70S6K cell migration model (44). Additionally, a PI3K-dependent activation of the GTPase Rac could account for alterations in the actin cytoskeleton (46).

PKC has been found to be another downstream target of PIP3 increasing cell motility (47). The results obtained with the isotype-specific inhibitors BIS and Gö6976 (26) suggest that PKC{alpha} is involved in the EGF-induced migration of BEAS-2B cells (see Figure 7). This is in line with results indicating that EGF treatment of BEAS-2B cells leads to a transient phosphorylation of PKC{alpha} (Figure 5). Taken together, PKC{alpha} is expected to play a central role for EGF-induced signaling in BEAS-2B cells, because even the activation of p38, as well as Akt, depends on PKC{alpha} (Figure 6). However, the underlying complex mechanism has not been elucidated thus far; it may be that integrins are involved.

The strong inhibitory effects of BAY11–7082 and curcumin on EGF-induced migration of BEAS-2B cells in Boyden chambers (Figure 7) are an indication that activation of the transcription factor NF-{kappa}B is involved in the motogenic activity. Activation of NF-{kappa}B could occur, for example, via mitogen-activated protein kinase kinase kinase (MEKK1) in the course of activating the JNK pathway, or from being a downstream target of PKC. The latter can be triggered by EGF via PI3K (48). Activation of NF-{kappa}B via PKC was detected even in BEAS-2B cells (49) and is a known signaling cascade, regulating cell migration and epithelial restitution (50, 51) by influencing gene expression, such as that of integrins. However, it has been reported that EGF does not induce NF-{kappa}B activation in BEAS-2B cells (52), a finding that is in agreement with our own observations (Figure 4). This is not in direct contrast to our data presented in Figure 7, as the culture conditions for BEAS-2B cells differ drastically (i.e., by the presence of collagen I as a haptotactic substrate). Furthermore, NF-{kappa}B can also be activated in BEAS-2B cells via metal-induced EGF receptor signaling (e.g., by V5+) (52).

The inhibition of EGF-induced migration of BEAS-2B cells by SB203580 (Figure 7) and the demonstration of EGF-triggered p38 activation (Figure 4) are in full agreement with previous reports on the importance of p38 activation for epithelial wound healing and chemotactic responses (35, 37, 53, 54). Aside from regulating gene expression, p38 is known to activate MAPK–activated protein kinase-2 (MAPKAPK2), which phosphorylates HSP25/27, inducing actin fiber polymerization (55). p38 activation represents a second route essential for chemotaxis other than the PI3K/Akt pathway. Furthermore, in neutrophils there is a report on a cross-talk between p38 MAPK and PI3K/Akt pathways establishing an intracellular signaling hierarchy (53). Here we show that EGF-induced activation of p38 in BEAS-2B cells depends on PI3K (Figure 6). Rac would be suited to connect these two pathways (35, 46). Generally, many details of the motogenic signaling of EGF in BEAS-2B cells are reminiscent of the report by Klekotka and colleagues (35), in which the {alpha}2-integrin cytoplasmic domain supported EGF-stimulated cell migration by activation of the p38 pathway. There is also cross-talk between p38 and ERK1/2 coordinating cellular migration and proliferation (37). ERK1/2 activation induced proliferation, whereas ERK1/2 inhibition (e.g., by PD98059) increased migration. The latter effect was also observed with BEAS-2B cells (Figure 7) together with augmented basal phosphorylation of p38 after treatment with PD98059 (Figure 6).

EGF also induced transient phosphorylation of JNK (Figure 4). Well known substrates of JNK are nuclear factor c-Jun and cytoplasmic actin-associated paxillin, the latter being important for cell migration (56). However, experiments with the specific JNK inhibitor SP600125 (56) suggest that activation of JNK does not play a major role for the motogenic effect of EGF in BEAS-2B cells (data not shown). This situation again is similar to the report by Klekotka and colleagues (35).

The motogenic activity of EGF was strictly dependent upon the presence of a haptotactic substrate (e.g., collagen I, collagen IV, laminin, fibronectin, or vitronectin) (Figures 3 and 9). Normal components of the basement membrane, such as collagen IV and laminin, were the most effective substrates. This clearly indicates that integrins are an essential prerequisite for the motogenic effect of EGF. The distribution of integrins on airway epithelial cells was studied in detail previously (57) and the following integrins were reported to occur on BEAS-2B cells: {alpha}1–{alpha}6, {alpha}V, {alpha}IIb, ß1, ß3, and ß5 (58, 59). This allows formation of functional integrins known to bind collagens, laminin, fibronectin, and vitronectin. The requirement of integrin ligation for EGF-triggered migration is in line with previous reports (29, 31, 35, 36, 60). Furthermore, EGF modulates integrin expression (58, 61). However, it has been reported that integrin expression on BEAS-2B cells is not affected by EGF (58). Thus, cooperative signals generated from both growth factors and integrins are required for cell locomotion (62, 63); CAS/Crk coupling provides the adhesion-dependent component of this signaling cascade and phosphorylation of CAS changes with the integrin ligand (63). Furthermore, the transmembrane integrin and EGF receptors are clustered by an integrin ligand, leading to an intensive cross-talk. Recent analysis of such a complex, interactive system (64) clearly revealed mechanisms that could also account for the sensitivity of the EGF-triggered motogenic response of BEAS-2B cells to inhibition of PKC or NF-{kappa}B (Figure 7).

Motogenic Activity of TFF2 for BEAS-2B Cells
It has been shown previously that the motogenic activity of TFF2 on BEAS-2B cells depends upon the activation of ERK1/2 as well as PKC (16). In this study, it is demonstrated that both TFF2 and TFF3/dimer have clear chemotactic activity in a haptotactic migration assay (Figure 8), whereas there is either little or no chemokinetic activity of TFF3/dimer or TFF2, respectively. This is in line with results obtained previously with TFF1 and human breast cancer cells (65).

Furthermore, the motogenic activity of TFF2 was dependent upon the haptotactic substrate offered. (Figure 9). Collagen I and fibronectin were the preferred substrates, whereas collagen IV was not a suitable substrate. Remarkably, the substrate specificities differed markedly when compared with EGF, the other motogen used in this study. This is an indication that the pattern of integrin ligation necessary for the chemotactic activity of EGF or TFF2, respectively, differs considerably.

Motogenic Activities of EGF and TFF2 are Synergistic but Depend on Different Mechanisms
A synergistic motogenic effect of TFF peptides with EGF was demonstrated after in vitro wounding of different epithelial cell lines in the past (9, 18, 19). It is also known that TFF2 does not trigger phosphorylation of the EGF receptor in BEAS-2B cells (9). Here it is shown for the first time that the motogenic effects of TFF2 and EGF synergize also in a modified Boyden chamber assay (Figure 10). Generally, the motogenic effect of EGF is more pronounced than that of TFF2 in BEAS-2B cells.

Both peptides show mainly chemotactic activity, and the motogenic responses depend upon the activation of PKC. However, the most surprising result was certainly that the major signaling cascades involved in the motogenic responses of TFF2 or EGF differ drastically (compare Ref. 16 and Figure 7). Major differences are as follows: (i) The TFF2-triggered motogenic response is inhibited by PD98059 and UO126, whereas the EGF-triggered motogenic effect is enhanced by these ERK kinase inhibitors; (ii) the TFF2-triggered motogenic response is insensitive to wortmannin, whereas the EGF-triggered motogenic effect is partially blocked by this PI3K inhibitor; (iii) the TFF2-triggered motogenic response is completely insensitive to BAY11–7082, whereas the EGF-triggered motogenic effect is totally blocked, preventing phosphorylation of I{kappa}B-{alpha}; (iv) the TFF2-triggered motogenic response is insensitive to SB203580, whereas the EGF-triggered motogenic effect is partially blocked by this p38 MAPK inhibitor. Thus, the TFF2-triggered response requires activation of ERK1/2, whereas the EGF-triggered response depends upon the activation of PI3K, p38, and, possibly, NF-{kappa}B. The latter is also supported by the inhibitory effect of curcumin (Figure 7).

Taken together, there are still open questions concerning the signaling cascades responsible for the motogenic activity of EGF. Compared with other epithelial cells, there were some surprises: for example, the absence of ERK1/2 activation as a prerequisite for migration, and the dependence on PI3K and p38. Also the point(s) of convergence between the TFF2- and the EGF-triggered signals are currently not known. However, the results presented clearly support the hypothesis that TFF peptides and EGF act synergistically to enhance restitution of the bronchial epithelium.


   Acknowledgments
 
The authors thank I. Schmidl-Kunz, J. Reising, and B. Glöckner for excellent technical assistance, Dr. A. Graness for helpful discussions, and Dr. J. Lindquist for comments on the manuscript. This work was generously supported by the Bundesministerium für Bildung, Wissenschaft, Forschung, und Technologie (BMBF; NBL3/01ZZ0107/PP20 to W.H.), the Land Sachsen-Anhalt (1918A/0025H, 1918A/2587B to W. H.), and the Fonds der Chemischen Industrie (0163615 and 0500058 to W.H.).

Received in original form December 3, 2003

Received in final form June 22, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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