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Published ahead of print on March 30, 2006, doi:10.1165/rcmb.2006-0013OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 252-259, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2006-0013OC

Activation of Transforming Growth Factor-beta by the Integrin {alpha}vbeta8 Delays Epithelial Wound Closure

Claus Neurohr, Stephen L. Nishimura and Dean Sheppard

Department of Medicine, and Department of Pathology, Lung Biology Center, University of California, San Francisco, California

Correspondence and requests for reprints should be addressed to Dean Sheppard, Lung Biology Center, University of California, San Francisco, Box 2922, San Francisco, CA 94143-2922. E-mail: dean.sheppard{at}ucsf.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Transforming growth factor (TGF)-beta family members regulate multiple aspects of wound repair through effects on cell proliferation, matrix production, and tissue inflammation, but the effects of TGF-beta on wound closure itself have been controversial. We found that blocking antibodies to TGF-beta enhanced the degree of closure of scratch wounds in primary airway epithelial monolayers, while addition of exogenous TGF-beta1 inhibited the degree of closure, suggesting that endogenous activation of TGF-beta normally serves as a brake on the degree of wound closure. Although these cells secreted large amounts of TGF-beta2 and small amounts of TGF-beta1, blockade of TGF-beta1 enhanced the degree of wound closure, whereas blockade of TGF-beta2 had no effect. TGF-beta1 (but not TGF-beta2) can be activated by two members of the integrin family, {alpha}vbeta6 and {alpha}vbeta8, which are both expressed on airway epithelial cells. Wounding induced activation of TGF-beta through effects of both integrins, but antibodies against {alpha}vbeta8 enhanced the degree of wound closure, whereas antibodies against {alpha}vbeta6 did not.

Key Words: airway epithelium • integrin {alpha}vbeta6 • integrin {alpha}vbeta8 • TGF-beta • wound closure


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In response to injury to surface epithelium, transforming growth factor (TGF)-beta isoforms are secreted and activated. TGF-beta isoforms are thought to play important roles in inducing fibroblasts to produce extracellular matrix proteins to cover the wound and in limiting the local inflammatory response. However, the effects of TGF-beta on the behavior of the cells involved in primary closure of epithelial wounds have not been completely elucidated.

Injury to the airway epithelium has been shown to be an early, prominent feature of several respiratory diseases. For example, chronic asthma has been suggested to involve impaired ability of the epithelium to restitute itself after initial injury, which could contribute to the airway remodeling that characterizes this disease (1). A growing body of evidence suggests that idiopathic pulmonary fibrosis (IPF) also involves abnormal epithelial wound healing (2). Furthermore, in obliterative bronchiolitis (OB), the leading cause of limited long-term survival after lung transplantation, airway epithelial injury may be an early, crucial event (3). In each of these diseases, TGF-beta has been shown to contribute to disease pathology, suggesting that TGF-beta is likely to be activated in the presence of airway epithelial injury. TGF-beta family members play important roles at multiple steps in wound healing (4–6). These proteins regulate a broad array of biologic functions, including cellular proliferation, migration, apoptosis, differentiation, developmental specification, adhesion, immunoregulation, and extracellular matrix production (7, 8). Each of the TGF-beta isoforms is made as a large precursor that is cleaved in the endoplasmic reticulum to form a short carboxy-terminal fragment (the active cytokine) and a longer amino-terminal fragment (termed the latency-associated peptide [LAP]). These two fragments each form disulfide linked homodimers that noncovalently associate with one another to form what is called the small latent complex, since in this form the associated LAP prevents the mature cytokine from binding to its receptors and inducing TGF-beta's known effects. In virtually all cells, this complex is further linked to members of another protein family, the latent TGF-beta–binding proteins (LTBP), forming what is called the large latent complex. A number of mechanisms have been described by which these latent complexes can be activated, including direct interaction with members of the integrin family of cell surface adhesion receptors. A subset of integrins can bind to TGF-beta1 LAP and TGF-beta3 LAP, through an arginine-gylcine-aspartic acid (RGD) recognition sequence. This RGD site is not present in TGF-beta2 LAP, and TGF-beta2 has thus far not been shown to be activated by interaction with integrins (9).

Integrins are heterodimeric transmembrane receptors that themselves contribute to a variety of cellular responses, including survival, proliferation, and migration (10). At least eight members of the integrin family have been shown to be capable of binding to RGD sites (e.g., {alpha}vbeta1, {alpha}vbeta3, {alpha}vbeta5, {alpha}vbeta6, {alpha}vbeta8, {alpha}5beta1, {alpha}8beta1, {alpha}IIbbeta3) (11). At least three of these, {alpha}vbeta6, {alpha}vbeta8, and {alpha}vbeta5, have been reported to be capable of activating TGF-beta (12–14). Whereas previous data published by our laboratory suggested that the integrin {alpha}vbeta6 might not be expressed in normal subjects, we have been able to detect {alpha}vbeta6 on airway epithelial cells of normal human subjects in vivo by using newer, more sensitive antibodies (15; D. Sheppard and coworkers, unpublished observation). Moreover, the integrins {alpha}vbeta5 and {alpha}vbeta8 have been demonstrated to be expressed in airway epithelial cells in normal subjects in vivo (16, 17). However, the significance of each of these integrins in modulating effects of endogenous TGF-beta in injured conducting airways has not been evaluated.

Several previous studies have examined the effects of exogenous TGF-beta on migration of epithelial cells and have suggested that TGF-beta can enhance epithelial cell migration (18, 19). However, most of these have been done with either epithelial cell lines derived from carcinomas or cells immortalized by expression of transforming oncogenes. In contrast, mice that exhibit a defect in TGF-beta signaling (as a result of a null mutation of the TGF-beta signaling protein, SMAD3) have been reported to have acceleration of the rate of cutaneous wound closure, suggesting that in vivo endogenous activation of TGF-beta might actually suppress the rate of wound closure (20). Furthermore, at least one study of the effects of exogenous TGF-beta on the rate of closure of primary airway epithelial cell wounds found that TGF-beta slowed the rate of wound closure (21).

In the current study, we sought to determine the effects of epithelial wounding on the activation of endogenously produced TGF-beta and to determine how such activation might affect the degree of wound closure. We also sought to determine what role, if any, specific epithelial integrins play in this process.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Airway Epithelial Cell Culture and Reagents
Primary normal human bronchial epithelial cells (NHBE), bronchial epithelial basal medium, and cell culture supplements were purchased from Cambrex Bioscience (Walkersville, MD). Cells were expanded in 100-mm dishes (Corning Inc., Corning, NY) using complete bronchial epithelial cell growth medium (BEGM) according to the protocol provided by Cambrex (East Rutherford, NJ). At confluence, passage 2 NHBE cells were seeded onto Transwell culture inserts (6.5 mm diameter; Costar 3470; Costar, Cambridge, MA) coated with collagen I at a density of 300,000 cells/well. Cells were further cultivated using Dulbecco's minimal essential medium (DMEM)/BEGM (1:1) containing 10% FCS. Cells were maintained in culture for 10–14 d, and medium was replaced every other day. Differentiated cultures showed a typical "cobblestone" appearance and were selected for experiments by measuring the transepithelial resistance (Rt) using an ohmmeter (EVOM; World Precision Instruments, Sarasota, FL). Cultures were considered confluent and differentiated if the Rt was stable and > 500 {Omega}cm2. Cells were grown in supplement- and serum-free DMEM/BEGM (1:1) medium for 24 h before and throughout the experiments.

Scratch Wound Assay and Determination of Degree of Wound Closure
The cell monolayer was wounded with a sterile 0.1- to 10-µl pipet tip (TipOne; USA Scientific, Ocala, FL) by one perpendicular linear scratch, creating a wound of ~ 500 µm width across the diameter of the well. The wells were washed twice with PBS to remove detached cells or cell debris followed by incubation with the different stimuli. Human recombinant active TGF-beta1, TGF-beta2, and TGF-beta3, monoclonal anti-pan–TGF-beta (1D11), and affinity-purified anti–TGF-beta1, -beta2, and -beta3 antibodies were purchased from R&D Systems (Minneapolis, MN). Polyclonal TGF-beta2 antibody was purchased from BioVision (Mountain View, CA). The concentrations used were based on the neutralization doses and ED50 provided by the manufacturers. Mouse monoclonal antibody against human {alpha}vbeta6 (6.3G9, 10D5) and {alpha}vbeta8 (37E1) were generated as previously described (13, 22). A mouse monoclonal antibody against {alpha}vbeta5 (ALULA) was generated in our laboratory by immunizing beta5 knockout mice with murine L cells, which express {alpha}vbeta5, and screening by flow cytometry, immunoprecipation, and inhibition of cell adhesion to vitronectin. As negative control, wounded cultures were exposed to an isotype mouse monoclonal IgG1 control antibody with no observed reaction with human cell surface (Chemicon, Temecula, CA) in the same concentrations as the neutralizing antibodies, or to PBS.

Wound closure was monitored immediately after initial wounding with time-lapse video phase contrast microscopy (x50 magnification) at 37°C and 5% CO2 in a humidified and climatized chamber using a Leica DMI 6000 microscope (Wetzlar, Germany) over 24 h. Images of the wounds were taken with a digital camera (Leica DFC350) at various time points. Wound areas were calculated using the publicly available ImageJ (1.34 s) software from the NIH (http://rsb.info.nih.gov/ij/; Bethesda, MD). The degree of wound closure was quantified by calculating the difference between the initial wound area and the remaining wound area expressed as percentage of their respective controls, 10 h after wounding. For each condition, three wounded areas were analyzed in parallel, and all experiments were repeated at least three times.

TGF-beta Bioassay
To determine TGF activation, TMLC (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter) were co-cultured with NHBE as previously described (12). TMLC were suspended at 5 x 105 cells/ml in DMEM/BEGM with or without additions (e.g., antibodies). NHBE cell medium in the upper compartment was removed and immediately after wounding replaced with 100 µl of TMLC suspension. NHBE and TMLC were co-cultured for 16–20 h. Lysates were assayed for luciferase activity as described (23). Relative luciferase units were defined as activity minus the background activity of the TMLC reporter cells.

TGF-beta ELISA
The secretion of TGF-beta1, TGF-beta2, and TGF-beta3 was measured by determining their concentration in the culture medium in the apical and basal compartments of the filter system using commercial, sandwich ELISA systems (R&D Systems). The supernatants were removed, acid activated, and directly assayed using visualization with tetramethylbenzidine on an ELISA plate reader at 450 nm according to the manufacturer's instructions. All values were corrected using a seven-point standard curve with 2-fold serial dilutions and a high standard of 2,000 pg/ml.

Flow Cytometry
Cells were blocked with normal goat serum (10 mg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) for 10 min, washed with PBS, and incubated with primary antibody (10 µg/ml) for 20 min and then with phycoerythrin-conjugated goat anti-mouse secondary antibody (Boehringer Mannheim, Mannheim, Germany) for 20 min at 4°C. Cells were re-suspended with PBS and analyzed by FACScan (Becton Dickinson, Rutherford, NJ).

Immunocytochemistry
For immunohistochemistry studies, cells were grown on filters and wounded as described above. Primary antibodies used were mouse monoclonal anti-{alpha}vbeta6 antibody, 10D5, and rabbit polyclonal antibodies against TGF-beta1 (Promega, Madison, WI) or TGF-beta2 (Santa Cruz Biotech, Santa Cruz, CA). The medium was removed and cells were washed with PBS. Cells were then fixed for 10 min with PBS containing 2% paraformaldehyde. Cells were washed with staining buffer PBS (supplemented with 4 mM MgCl2 and 0.5% Tween 20 [Bio Rad, Hercules, CA] for 10D5 staining only), blocked for 1 h with staining buffer containing 10% Normal Goat Serum (NGS), and incubated for 1 h with primary antibody in staining buffer + 10% NGS at 37°C. Samples were washed and incubated for 45 min with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) or goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted to 3 µg/ml in staining buffer + 10% NGS. Cells were then washed, and Transwell culture insert membranes were mounted with coverslips using VECTASHIELD mounting medium (Vector Laboratories).

5-Bromo-2'-Doxyuridine Staining and Mitomycin C Treatment
Mitomycin C (10 µg/ml) was dissolved in the experimental medium and added to the lower and upper compartments of the inserts at the time of wounding. 5-Bromo-2'-doxyuridine (BrdU) labeling was performed to measure new DNA synthesis as an indication of cell proliferation. For BrdU studies, cells were grown on filters and wounded as described above. Nine hours after wounding, BrdU (10 µM; Amersham, Piscataway, NJ) was added for 60 min. The cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized with Triton-X-100 0.5% for 10 min. The denaturation step with 4 N HCl at 37°C lasted for 30 min followed by three washing steps with PBS. The cells were subsequently incubated with monoclonal antibody against BrdU diluted in PBS (1:1,000, Clone Bu20a; DakoCytomation, Glostrup, Denmark) at 4°C overnight. The reaction was visualized by fluorescein-conjugated anti-mouse antibody (1:200; Jackson Immunoresearch Laboratories) with a Leica DM 5000B microscope. The number of stained cells was determined by counting at least four high-power fields (x200 magnification) per culture insert.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism Software (GraphPad Software Inc., San Diego, CA). Comparison between groups was made using one-way ANOVA followed by the Tukey test. Data are reported as mean value ± SE of at least three independent experiments done in triplicate. A P value of < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of TGF-beta on Epithelial Wound Closure
Epithelial cell scratch wounds usually close as a consequence of collective cell (sheet) migration (24). Wound closure in our assay was the result of a continuous, multicellular sheet movement over 15–20 h (Figure 1A). To evaluate the role of endogenously secreted and activated TGF-beta in this process, wounded monolayers were incubated with either a control antibody (100 µg/ml) or an antibody that blocks all TGF-beta isoforms (1D11, 100 µg/ml) or with recombinant active TGFbeta1 (Figures 1A and 1B). Addition of pan–TGF-beta–blocking antibody induced a significant increase in the degree of wound closure, whereas treatment of scratch wounds with recombinant, active TGF-beta1 (10 ng/ml) significantly reduced the degree of wound closure.


Figure 1
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  		Figure 1. Effects of TGF-beta1 and anti–TGF-beta antibody on wound closure. (A) Phase contrast microscopy (x50 magnification) of NHBE cell monolayers on transwell culture inserts immediately after wounding (upper panels, 0 h) and after 10 h (lower panels). Incubation with either control antibody (100 µg/ml), anti-pan–TGF-beta (1D11, 100 µg/ml) or TGF-beta1 (10 ng/ml). Scale bar: 100 µm. (B). Degree of wound closure expressed as percent of control, 10 h after wounding. The data are shown as means ± SE. **P < 0.001 versus control.

 
The effect of TGF-beta–blocking antibody suggested that one or more TGF-beta isoforms was present in our culture system and was being endogenously activated by airway epithelial cells. To determine which TGF-beta isoforms were being secreted by these cells, we measured the concentration of each of the three mammalian TGF-beta isoforms (TGF-beta1, -beta2, and -beta3) in the culture medium in the apical and basal compartments of our filter culture system. Because of the large volume of basal medium, we could not detect any TGF-beta in the basal compartments of wounded or unwounded cultures. Both TGF-beta1 and TGF-beta2 were detectable in the apical compartment, but, as previously reported by others, these cells made ~ 20 times more TGF-beta2 than TGF-beta1 (Figure 2A). TGF-beta3, if present, was at a concentration below the limit of detection of our assay (30 pg/ml). Over the time course studied (18 h), wounding had no effect on the amount of either TGF-beta isoform secreted by these cells.


Figure 2
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  		Figure 2. Role of specific TGF-beta iosforms in slowing epithelial wound closure. (A) Culture supernatants (apical compartment) were analyzed by ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 (logarithmic scale). TGF-beta3 was not detectable (n.d.). (B) Degree of wound closure expressed as percent of control, 10 h after wounding in the absence of antibodies (control) or in the presence of blocking antibodies against all TGF-beta isoforms or against TGF-beta1 or -beta2. **P < 0.001 versus control. (C) Unwounded NHBE (test cells) and TMLC (reporter) cells were co-cultured for 16–20 h and lysed for measurement of luciferase activity. Measurements for unwounded cells in the absence of blocking antibody were adjusted to 100%. (D) Unwounded and wounded NHBE (test cells) and TMLC (reporter) cells were co-cultured for 16–20 h in the presence or absence of blocking antibodies against all TGF-beta isoforms or against TGF-beta1 or -beta2 and lysed for measurement of luciferase activity. Measurements for unwounded cells in the absence of blocking antibody were adjusted to 100%. The data are shown as means ± SE. *P < 0.01 versus control.

 
Since the airway epithelial cells we studied made both TGF-beta1 and -beta2, we next evaluated the contributions of each isoform to inhibition of the degree of wound closure. Blocking antibodies against TGF-beta1 substantially enhanced the degree of closure, whereas two different blocking antibodies to TGF-beta2 had no effect at concentrations up to 100 µg/ml (Figure 2B and data not shown).

Taken together, the above results suggested that airway epithelial wounding, at least over the time course we studied, does not alter the protein concentration of any TGF-beta isoforms, but does induce an effect on wound closure that is preferentially mediated by TGF-beta1. One possible explanation for these findings would be that wounding specifically activates TGF-beta1, but not TGF-beta2. To address this possibility we performed a co-culture bioassay in which we incubated either wounded or unwounded airway epithelial cells together with mink lung epithelial cells stably transfected with a TGF-beta–inducible luciferase reporter. As shown in Figure 2C, reporter cell activity in unwounded monolayers was not inhibited by TGF-beta–blocking antibody, meaning that there was no detectable TGF-beta activity in the absence of wounding. However, monolayer wounding induced a substantial increase in reporter cell activity that could be inhibited down to the levels seen for unwounded monolayer by anti-pan–TGF-beta. To determine which TGF-beta isoform was being activated, we also performed co-culture assays with wounded monolayers in the presence of isoform-specific blocking antibodies to TGF-beta1 or -beta2. TGF-beta1 antibody significantly inhibited reporter cell activity in this assay, whereas TGF-beta2 antibody had no effect (Figure 2D) suggesting that wounding induced activation of TGF-beta1 but not TGF-beta2.

Roles of {alpha}v Integrins in Wound-Induced Activation of TGF-beta
Having found that endogenous active TGF-beta plays a critical role in our wound repair model, we sought to determine whether endogenous activity could be explained by TGF-beta activation by epithelial integrins (25). Thus far, three integrins have been suggested to be capable of activating latent TGF-beta, {alpha}vbeta5 (14), {alpha}vbeta6 (12), and {alpha}vbeta8 (13). First, expression of integrins {alpha}vbeta5, {alpha}vbeta6, and {alpha}vbeta8 was assessed by flow cytometry. As shown in Figure 3A, NHBE cells in our culture model expressed all three integrins on their cell surface. Creation of the wound did not induce a significant shift in the expression pattern of these integrins over the 15-h time period used for our assays (Figure 3A). Moreover, as assessed by immunocytochemistry, wounding did not affect the spatial distribution of {alpha}vbeta6, TGF-beta1, or TGF-beta2 throughout the monolayer (data not shown). We could not assess the spatial distribution of {alpha}vbeta8 because no antibodies are available that are suitable for immunocytochemistry.


Figure 3
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  		Figure 3. Role of specific integrins in TGF-beta activation after epithelial wounding. (A) Flow cytometry of NHBE cells with and without wounding. Cells were stained with anti-{alpha}vbeta5, anti-{alpha}vbeta6, and anti-{alpha}vbeta8 antibodies. Open peaks represent control (i.e., secondary antibody alone), shaded peaks represent NHBE cells stained with primary antibodies indicated. (B) Unwounded NHBE (test cells) and TMLC (reporter) cells were co-cultured for 16–20 h in the presence or absence of antibodies to {alpha}vbeta5 (ALULA, 10 µg/ml), {alpha}vbeta6 (6.3G9, 10 µg/ml), or {alpha}vbeta8 (37E1, 100 µg/ml) integrins and lysed for measurement of luciferase activity. Measurements for unwounded cells in the absence of blocking antibody were adjusted to 100%. (C) Unwounded and wounded NHBE (test cells) and TMLC (reporter) cells were co-cultured for 16–20 h in the presence or absence of anti-integrin antibodies and lysed for measurement of luciferase activity. Measurements for unwounded cells in the absence of blocking antibody were adjusted to 100%. The data are shown as means ± SE. *P < 0.01 versus control.

 
To evaluate the potential contribution of these integrins to TGF-beta activation, co-culture bioassays were performed for wounded or unwounded monolayers incubated with monoclonal antibodies against human {alpha}vbeta5 (ALULA, 10 µg/ml), {alpha}vbeta6 (6.3G9, 10 µg/ml), or {alpha}vbeta8 (37E1, 100 µg/ml). None of these antibodies had any effect on the basal reporter cell activity in unwounded monolayers (Figure 3B). In wounded monolayers, both anti-{alpha}{nu}beta6 and anti-{alpha}vbeta8 significantly inhibited reporter cell activity, suggesting that both integrins contribute to the activation of TGF-beta1 in response to wounding (Figure 3C). Antibody to {alpha}vbeta5 had no effect.

Roles of {alpha}v Integrins in Epithelial Wound Repair
We next sought to determine whether TGF-beta activation by {alpha}vbeta6, {alpha}vbeta8, or both was responsible for the delay in wound closure caused by endogenously activated TGF-beta. As expected, antibody against {alpha}vbeta5 had no effect on the degree of wound closure. {alpha}vbeta8-blocking antibody significantly increased the degree of wound closure, but, surprisingly, antibody against {alpha}vbeta6 had no significant effect (Figure 4A).


Figure 4
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  		Figure 4. Role of specific integrins in regulating the degree of epithelial wound closure. (A) Degree of wound closure in the presence or absence of anti-pan–TGF-beta (1D11, 100 µg/ml) and antibodies to {alpha}vbeta5 (ALULA, 10 µg/ml), {alpha}vbeta6 (6.3G9, 10 µg/ml), or {alpha}vbeta8 (37E1, 100 µg/ml) integrins, 10 h after wounding. *P < 0.01 versus control. (B) Effects of anti-{alpha}vbeta6 antibody on the degree of wound closure in the presence or absence of combined treatment with antibodies against TGF-beta or {alpha}vbeta8, 10 h after wounding. The data are shown as means ± SE. *P < 0.01 versus treatment with either anti–TGF-beta or anti-{alpha}vbeta8 alone.

 
Since both {alpha}vbeta6 and {alpha}vbeta8 apparently contribute to activation of TGF-beta after airway epithelial wounding, we were initially puzzled by the failure of blocking antibody against {alpha}vbeta6 to accelerate the degree of wound closure. However, {alpha}vbeta6 has been previously shown to accelerate epithelial cell migration. We therefore tested the possibility that {alpha}vbeta6 might exert competing effects on wound closure—inhibition mediated by activation of TGF-beta and simultaneous acceleration through TGF-beta–independent effects on cell migration. To test this possibility, we evaluated the effects of blocking {alpha}vbeta6 on closure of wounds in which TGF-beta or {alpha}vbeta8 had already been inhibited. Although anti-{alpha}vbeta6 antibody again had no effect on its own, addition of anti-{alpha}vbeta6 antibody to either anti-{alpha}vbeta8 or anti–TGF-beta antibody significantly inhibited wound closure compared with treatment with anti-{alpha}vbeta8 or anti–TGF-beta alone (Figure 4B).

Contribution of Proliferation to Epithelial Wound Healing
TGF-beta is a well-established inhibitor of epithelial cell proliferation, an effect that could contribute to the degree of wound closure over the time course of our assay. To evaluate the contribution of proliferation in our scratch wound assay, we used the DNA-crosslinking inhibitor Mitomycin C. We verified that Mitomycin blocked DNA synthesis by demonstrating negligible BrdU incorporation in Mitomycin-treated cells. Inhibition of cell proliferation had little if any effect on the degree of wound closure and did not abrogate the effects of pan–TGF-beta– or {alpha}vbeta8-blocking antibodies on the degree of wound closure (Figure 5).


Figure 5
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  		Figure 5. Effects of mitomycin C on the degree of wound closure. Degree of wound closure expressed as percent of control, 10 h after wounding and incubation with the indicated stimuli in the absence or presence of mitomycin C (MitoC, 10 µg/ml). The data are shown as means ± SE. *P < 0.01.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The results of the present study show that confluent airway epithelial cells, despite secreting two TGF-beta isoforms and expressing two members of the integrin family that can activate TGF-beta, do not activate either isoform under resting conditions. In response to mechanical wounding, however, there is rapid induction of integrin-dependent activation of TGF-beta, which slows the degree of wound closure. TGF-beta activation following wounding appears to be mediated by both the {alpha}vbeta6 and {alpha}vbeta8 integrins, but {alpha}vbeta8-mediated activation is principally responsible for the reduction in the degree of wound closure, since blocking antibodies to {alpha}vbeta8 substantially accelerated wound closure, whereas antibodies to {alpha}vbeta6 had no effect on their own. This difference is explained in part by a TGF-beta–independent contribution of {alpha}vbeta6 to the degree of wound closure, as demonstrated by inhibition of the degree of closure by antibodies to {alpha}vbeta6 after TGF-beta activity was inhibited by antibodies to either TGF-beta or {alpha}vbeta8. Airway epithelial cells make substantial amounts of TGF-beta2 and smaller amounts of TGF-beta1, but the concentrations of each TGF-beta isoform are not affected by wounding, at least during the time course of our experiments. The {alpha}vbeta8-mediated decrease in the degree of wound closure is mediated by activation of TGF-beta1, but TGF-beta2 does not appear to contribute, consistent with the identification of the arginine-glycine–aspartic acid site in TGF-beta1 LAP as the site of binding of this integrin. The RGD site is present in TGF-beta1 and -beta3 LAP, but absent from TGF-beta2 LAP (25).

Numerous previous reports have examined effects of TGF-beta on individual migration of a variety of cell types. Based on these studies, it is clear that TGF-beta can exert dramatically different effects on different cell types and under different circumstances. TGF-beta isoforms have been shown to be chemotactic for macrophages, mast cells, and fibroblasts, enhancing the migration of each cell type toward gradients of diffusible TGF-beta (26–28). TGF-beta has also been reported to enhance the migration of epithelial tumor cells and immortalized epithelial cells (18, 29, 30). Finally, TGF-beta is a potent inducer of epithelial-to-mesenchymal transformation (EMT), inducing primary epithelial cells to take on mesenchymal cell characteristics, including enhanced detachment from epithelial sheets and enhanced speed of migration (31, 32). However, EMT occurs over a time course of several days, much longer than the time course of primary epithelial wound closure (33, 34).

Primary epithelial wounds, like the scratch wounds studied here, close by a process of continuous sheet migration, which appears to be distinct from migration of isolated individual cells. The one previous study that looked at the effects of exogenous TGF-beta on the rate of closure of airway epithelial wounds found significant slowing of the rate of closure, just as we did in the current study (21). Furthermore, this effect is consistent with results reported for the rate of cutaneous wound closure in vivo in mice with impaired responsiveness to TGF-beta as a consequence of a null mutation of the TGF-beta signaling molecule, SMAD3. These mice have dramatically accelerated wound closure, as expected if endogenously activated TGF-beta normally slows the rate of wound closure (20).

Many of the previous studies that examined the potential roles of TGF-beta on migration of epithelial cells focused primarily on exogenously administered TGF-beta. In the current study, we demonstrated that TGF-beta is activated by airway epithelial cells themselves in response to mechanical wounding, and that locally activated TGF-beta significantly slows the degree of wound closure. Based on studies with blocking antibodies, it is clear that TGF-beta1 is endogenously activated and contributes to this response. In response to mechanical wounding, TGF-beta1 is activated by the actions of at least two members of the integrin family, {alpha}vbeta6 and {alpha}vbeta8. Airway epithelial cells express one other integrin, {alpha}vbeta5, that has also recently been reported to be capable of activating TGF-beta on the surface of fibroblasts from patients with sceleroderma (14, 35). Blockade of {alpha}vbeta5 had no effect on either TGF-beta activity or on the degree of wound closure in our assay. These results are consistent with our own inability to demonstrate {alpha}vbeta5-mediated TGF-beta activation by any primary cells or cell lines we have examined (data not shown). We are uncertain why our results in this regard are different from those reported by Asano and coworkers (14, 35), but perhaps this function of {alpha}vbeta5 uniquely occurs on fibroblasts from patients with scleroderma. An unanswered question from our experiments is how wounding stimulates {alpha}vbeta6- and {alpha}vbeta8-mediated TGF-beta activation. This effect does not appear to be due to either a change in the level of expression of either integrin or a change in expression of any TGF-beta isoforms, since the levels of each of these proteins was unaffected by wounding over the time course of our experiments.

Our finding that airway epithelial cells make large amounts of TGF-beta2, but do not appear to activate it in response to injury raises the questions of how TGF-beta2 becomes activated and whether or not it might contribute to the slowing of wound closure or other TGF-beta-dependent effects in wounded airways in vivo. In addition to integrins, TGF-beta isoforms can be activated by a variety of proteases and by interaction with the secreted protein, thrombospondin and it is certainly possible that other cells that are present in or recruited to injured airways could secrete these other activators and lead to an important contribution of TGF-beta2 to in vivo responses (36, 37, 38).

Interestingly, although we found that both {alpha}vbeta6 and {alpha}vbeta8 contributed to TGF-beta activation after wounding, and the contribution of {alpha}vbeta6 was at least as large as that of {alpha}vbeta8, we could not demonstrate any contribution of {alpha}vbeta6 to the decrease in the degree of wound closure we observed. As noted above, one possible explanation for this confusing result might be that, in addition to activating TGF-beta (an effect that would be expected to slow the degree of wound closure), {alpha}vbeta6 itself enhances the degree of wound closure through a TGF-beta–independent mechanism. This possibility is supported by our finding that blockade of {alpha}vbeta6 actually slowed the degree of wound closure under conditions in which the effects of endogenously activated TGF-beta were prevented by addition of TGF-beta–blocking antibodies. However, there may be additional explanations for these results. Although {alpha}vbeta6 and {alpha}vbeta8 both bind to the same RGD sites in TGF-beta1 and -beta3, the mechanisms of TGF-beta activation by these two integrins are actually quite different. {alpha}vbeta6 induces activation of latent complexes through a mechanism that depends on actin polymerization and appears to induce a conformational change in latent TGF-beta but does not release any free TGF-beta from the latent complex. Rather, this pathway is completely dependent of direct contact between the cell expressing the activated integrin and an adjacent target cell expressing TGF-beta receptors (12). In contrast, {alpha}vbeta8-mediated TGF-beta activation involves presentation of the latent complex to transmembrane proteases, with resultant proteolytic cleavage of the LAP and release of freely diffusible active TGF-beta from the cell surface (13). It is thus conceivable that TGF-beta locally activated by wounding of the epithelial cells at the wound edge might not be sufficient to globally affect the migration of a continuous sheet of epithelial cells, whereas the diffusible TGF-beta released by {alpha}vbeta8-mediated activation could have more long-range effects on the entire epithelial sheet. However, we have not been able to demonstrate any difference in TGF-beta activity between the wound edge and the rest of the wounded epithelial sheet, so we have no data to support this possibility.

In summary, we have found that airway epithelial cells respond to injuring by inducing TGF-beta activation mediated by two different integrins, {alpha}vbeta6 and {alpha}vbeta8. Although these cells make large amounts of TGF-beta2 and small amounts of TGF-beta1, only TGF-beta1 is activated by this pathway. At least in the case of {alpha}vbeta8, activation of endogenous TGF-beta1 slows the degree of wound closure, an effect that can be prevented by blocking either TGF-beta or the {alpha}vbeta8 integrin. These observations could have important implications for understanding the mechanisms underlying a variety of lung and airway diseases that are characterized by abnormal airway repair.


   Acknowledgments
 
The authors thank Mark Travis and Greg deHart for valuable scientific discussion and technical assistance.


   Footnotes
 
This work was funded by Deutsche Forschungsgemeinschaft (DFG) grant NE 1168/1–1 (to C.N.) and by National Heart, Lung and Blood Institute grants HL56385, HL64353, HL66600, and HL53949 (to D.S.)

Originally Published in Press as DOI: 10.1165/rcmb.2006-0013OC on March 30, 2006

Conflict of Interest Statement: C.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.L.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.S. is PI on a sponsored research agreement with Biogen Idec ($99,000/year since 2003) and is a co-owner of a patent that covers antibody 3G9.

Received in original form January 10, 2006

Accepted in final form March 22, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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