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Originally published In Press as doi:10.1074/jbc.M606533200 on November 29, 2006

J. Biol. Chem., Vol. 282, Issue 5, 3213-3220, February 2, 2007
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Airway Epithelial Cell Migration and Wound Repair by ATP-mediated Activation of Dual Oxidase 1*

Umadevi V. Wesley{ddagger}1, Peter F. Bove§1, Milena Hristova§, Sean McCarthy§, and Albert van der Vliet§2

From the Departments of §Pathology and {ddagger}Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405

Received for publication, July 10, 2006 , and in revised form, November 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signal-regulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the primary functions of the airway epithelium is to provide a protective barrier against inhaled environmental toxins and airborne pathogens, and airway epithelial cells express a number of intrinsic factors that serve to minimize invasion by infectious agents and to promote repair processes following injury (1, 2). A newly discovered NADPH oxidase homolog, dual oxidase (Duox),3 has been recently identified within the tracheobronchial epithelium (3-6) and is thought to contribute to innate epithelial host defense based on structural similarities with the phagocyte NADPH oxidase system (7, 8). Of the two known Duox isoforms, Duox1 is primarily expressed within the ciliated epithelium and believed to be responsible for epithelial H2O2 production after cell stimulation (5), whereas Duox2 has been localized to salivary or submucosal glands, thus constituting a functional host defense system with co-localized lactoperoxidase (4, 5). Other than recent observations in Drosophila (9), a direct role for Duox1/2 in host defense has not yet been directly demonstrated, and other functions of airway epithelial Duox1 have been postulated (6, 10).

Both Duox isozymes contain two EF-hand Ca2+-binding domains and are activated by Ca2+-mobilizing stimuli (11, 12). A critical mechanism of Ca2+-mediated signaling within epithelial cells after bacterial infection or mechanical or oxidative injury involves the activation of purinergic receptors at the cell surface by release of ATP (13-15). ATP-mediated autocrine or paracrine signaling is known to regulate diverse processes involved in host defense, including anion transport, ciliary function, and mucin expression (16-19), and some of these events have recently been associated with Duox1 activation (5, 6, 10).

The airway epithelium is known to undergo continuous denudation and regeneration, especially in inflammatory airway diseases such as asthma, and rapid re-epithelialization by epithelial cell de-differentiation and migration are important features of epithelial repair in vivo (20). Several recent studies have implicated ATP in in vitro cell migration and/or proliferation in various cell types through purinergic signaling (14, 21). Moreover, NADPH oxidases have been linked to endothelial or smooth muscle cell migration (22). We therefore explored whether ATP release mediates airway epithelial cell migration and wound repair following injury and investigated the potential involvement of Duox1 activation in this process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human primary tracheobronchial epithelium (NHBE) cells were obtained from Clonetics (Cambrex Biosciences, Walkersville, MD) and used at passages 3-5. For experiments, NHBE cells were cultured to confluence in 24-well plates or 8-well chamber slides (Nunc International, Naperville, IL). Immortalized human bronchial epithelial cells (HBE1), kindly provided by Drs. R. Wu and J. Yankaskas, were cultured in Ham's F-12 nutrient medium supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 µM), cholera toxin (10 ng/ml), and bovine hypothalamus extract (15 µg/ml), as described previously (23), and grown to confluence in 24-well plates or chamber slides for experimentation, unless otherwise indicated.

In Vitro Wound Assay—In vitro injury was induced in NHBE or HBE1 monolayers by creating 1-2 linear scratches of ±0.2 mm wide, after which detached cells were removed by washing with phosphate-buffered saline and fresh medium was added. Wound closure was followed over 24 h, using a phase contrast microscope interfaced with a digital camera, and initial and remaining wound areas were determined using NIH Image J software for calculation of the percentage of wound closure. Unless otherwise indicated, all reagents used in these studies were from Sigma.

Transwell Cell Migration Assay—HBE1 cells (1 x 105) were placed in Boyden-like chambers containing 10-mm polycarbonate membrane inserts (8 µm pore size) (Nunc International), which were coated with 10 µg/ml fibronectin (Invitrogen) for 60 min. Following incubation for 4 h at 37 °C to allow cell adhesion, test reagents were added or cells were transfected with Duox1-specific small interfering RNA (siRNA) or negative siRNA control. Untreated and siRNA-treated cells were incubated for 24 h at 37 °C, after which non-migrated cells were removed from the membranes with a cotton swab. The remaining migrated cells were stained with crystal violet and extracted in 200 µl of 0.2 M sodium acetate buffer (pH 4.5) for analysis of absorbance (A) at 540 nm.

Total RNA Isolation and Semiquantitative Reverse Transcription (RT)-PCR—Total RNA was isolated using the RNAeasy extraction kit according to the manufacturer's protocol (Qiagen, Inc., Valencia, CA). The cDNAs synthesized from total RNA (2 µg) using superscript reverse transcriptase (Invitrogen) were used as templates for RT-PCR reaction. The primer set used for Duox1 amplification was 5'-GCA GGA CAT CAA CCC TGC ACT CTC-3' and 5'-CTG CCA TCT ACC ACA CGG ATC TGC-3' and for Duox2 amplification was 5'-GAT GGT GAC CGC TAC TGG TT-3' and 5'-GCC ACC ACT CCA GAG AGA AG-3'. GAPDH amplification using the primer set 5'-ATC TTC CAG GAG CGA GAT CC-3' and 5'-ACC ACT GAC ACG TTG GCA GT-3' was used as the control. PCR amplifications were carried out in 30 cycles of denaturation (94 °C, 1 min), annealing (58 °C, 30 s), and extension (72 °C, 1 min), and the products were analyzed on 1% agarose gels.

Silencing of Duox1 Expression by RNA Interference Assay—Expression of Duox1 was silenced using predesigned siRNA (catalog number 16708) from Ambion RNA Company (Austin, TX). Negative control siRNAs that have no significant sequence similarity to mouse, rat, or human gene sequences and functionally proven to have minimal effects on cell proliferation and viability (silencer negative control number 1 siRNA, catalog number 4611, Ambion) were used as controls. HBE1 cells were transfected with 100 nM Duox1 and negative control siRNA using Lipofectamine 2000 reagent (Invitrogen) 72 h prior to experimentation. Effects on Duox1 expression were verified by RT-PCR and immunofluorescence analysis of Duox protein, which was detected using polyclonal antibodies against Duox (kindly provided by Drs. J. David Lambeth and Francoise Miot) and visualized using a Cy3-conjugated goat anti-rabbit secondary antibody (Jackson Laboratories, Bar Harbor, ME).

Measurement of ATP Release—ATP release into the medium was monitored using a luciferase/luciferin bioluminescence ATP determination kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Confluent HBE1 cell monolayers in 24-well plates were wounded and immediately placed in 1 ml of fresh medium. At various time points, aliquots (100 µl) were analyzed with luciferase/luciferin in a Lumat LB 9507 luminometer (Oak Ridge, TN). The amount of ATP released was calculated using external ATP standards (1-1000 nM).

Analysis of Epithelial H2O2 Production—Confluent HBE1 cell monolayers were incubated in the presence of L-tyrosine (1 mM) and lactoperoxidase (10 µg/ml) in Hanks' balanced salt solution and production of H2O2 was measured via lactoperoxidase-catalyzed oxidation of tyrosine to o,o'-dityrosine, which was measured by high pressure liquid chromatography with fluorescence detection (24). This assay is sensitive and allows detection of as little as 1 nM H2O2. For analysis of cellular H2O2 production in situ, confluent HBE1 cells in chamber slides were preincubated with 10 µM 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) for 15 min, after which the cells were washed with phosphate-buffered saline and wounded. DCF fluorescence was monitored 30 min after wounding using an inverted fluorescence microscope (Nikon Eclipse TE2000-U) connected to a RT Slider Spot digital camera (Diagnostic Instruments). Images were generated using the software SPOT, version 3.2.

Analysis of ERK1/2 Activation—Confluent HBE1 cell monolayers in 24-well plates were wounded by creating several scratches in a cross pattern. Cell lysates were collected 10 min after wounding, and phosphorylated and total ERK1/2 were analyzed by SDS-PAGE and Western blotting using polyclonal antibodies against phosphorylated ERK (ppERK) and total ERK1/2 (Cell Signaling Technology, Beverly, MA), which were visualized using horseradish-conjugated secondary antibody (Cell Signaling Technology) and enhanced chemiluminescence (Pierce). Alternatively, HBE1 cell monolayers in chamber slides were wounded and fixed after 10 min of incubation with 4% paraformaldehyde (10 min at room temperature), after which slides were blocked for 45 min with phosphate-buffered saline containing 2% milk and 0.1% Triton X-100 and stained with a polyclonal antibody against phospho-ERK1/2 (Cell Signaling Technololgy; 1:100 dilution for 45 min) and a Cy3-conjugated goat anti-rabbit (red) secondary antibody (Jackson Laboratories). Phospho-ERK1/2 immunostaining was visualized using an inverted fluorescence microscope (Nikon Eclipse TE2000-U).

Analysis of MMP-9 Expression and Activity—MMP-9 expression in HBE1 cells was analyzed by semiquantitative RT-PCR, as described previously (23). Gelatinase activity in injured HBE1 cells was determined using an in situ zymography procedure based on proteolytic cleavage of a fluorescently labeled gelatin substrate, DQ-gelatin (Invitrogen). Confluent HBE1 cells in multichamber slides were wounded and incubated for 24 h, after which cells were incubated with 100 µg/ml DQ-gelatin for 2 h at 37 °C, washed, and fixed with 4% paraformaldehyde and analyzed by fluorescence microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ATP Release Promotes Epithelial Cell Migration by Purinergic Signaling—To investigate the signaling mechanisms in epithelial cell migration and repair, we used an in vitro model of epithelial injury, which involves generating a linear scratch of ±0.2 mm width in confluent monolayers of either NHBE cells or immortalized human tracheobronchial epithelial cells (HBE1). Because chemical or mechanical epithelial cell injury is known to promote release of ATP by as yet incompletely defined mechanisms (19, 25), we first determined ATP release in response to linear scratch injury in confluent HBE1 cell monolayers. Indeed, extracellular ATP levels rapidly increased following epithelial wounding and reached a maximum after 15-20 min, thereafter declining rapidly to near normal levels after 40 min (Fig. 1A). The gradual rise in extracellular ATP levels is indicative of an active mechanism of ATP release from remaining cells after linear wounding. The rapid decline in extracellular ATP is most likely due to its hydrolysis by cell surface ecto-ATPases (19). Accordingly, extracellular ATP persisted longer in the presence of the ectonucleotide pyrophosphatase inhibitor beta,{gamma}-methylene ATP (300 µM; data not shown), consistent with previous findings (26).


Figure 1
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  		FIGURE 1.
ATP-mediated P2 purinergic receptor activation promotes bronchial epithelial cell migration and wound repair. Confluent HBE1 cell monolayers in 24-well plates were wounded by introducing a linear scratch of ~0.2 mm width using a sterile pipette tip, and the extent of wound closure was monitored and quantitated by the analysis of the remaining wound areas using ImageJ software. A, measurement of ATP release after linear scratch injury in confluent HBE1 cell monolayers. Cells were washed after wounding to remove cellular debris, and 1 ml of fresh medium was added. ATP levels in the medium were determined at the indicated times after wounding using a luciferase/luciferin bioluminescence assay (mean ± S.D.; n = 3). B, bright field images of the wound areas taken immediately (a) or 24 h after linear scratch injury in confluent HBE1 cell monolayers, incubated in the absence (b) or presence of 100 µM reactive blue 2 (c) or 10 units/ml apyrase (d), which were added directly after wounding. C, quantitative analysis of the percentage of wound closure 24 h after linear wound injury in HBE1 cell monolayers in the absence or presence of the P2 purinergic receptor inhibitors suramin (100 µM) or reactive blue 2 (100 µM) or the ATPases hexokinase (10 units/ml) or apyrase (10 units/ml) (mean ± S.D.; n = 3-5; *, p < 0.05 compared with untreated controls using unpaired Student's t test). D, effect of exogenous ATP or its stable analog ATP{gamma}S on the extent of wound closure 12 h after linear scratch injury (mean ± S.D.; n = 2-3; *, p < 0.05 compared with control). E, effect of inhibitors of purinergic signaling on HBE1 cell migration in a Transwell migration assay (mean ± S.D.; n = 3-5; *, p < 0.05 compared with control). F, effect of exogenous ATP (10 µM) in the absence or presence of suramin (100 µM) on HBE1 cell migration in a Transwell migration assay (mean ± S.D.; n = 3-4; *, p < 0.05).

 
We next determined the contribution of ATP to the rate or extent of wound closure after linear injury to NHBE or HBE1 monolayers. Although these cells normally rapidly migrate into the wound area to cause almost complete wound closure within 24 h (Fig. 1B, panels a and b), the extent of wound closure was markedly inhibited in the presence of two different P2 purinergic receptor antagonists, suramin and Cibracon blue (reactive blue 2) (Fig. 1, B (panel c) and C). Epithelial wound closure was also markedly attenuated in the presence of the ATPase/ADPase apyrase, indicating the involvement of extracellular ATP (Fig. 1, B (panel d) and C). In contrast, the addition of hexokinase, which converts ATP to ADP, did not affect wound closure, presumably because ADP is also an agonist for P2 receptors and capable of promoting epithelial migration. Consistent with a stimulatory role of released ATP on epithelial wound closure, the addition of exogenous ATP (1-10 µM) or its non-hydrolyzable analog ATP{gamma}S (1 µM) also enhanced wound closure in HBE1 cells (Fig. 1D). Interestingly, higher concentrations of ATP or ATP{gamma}S inhibited wound closure, consistent with earlier findings in other cell types (14), presumably because of nonspecific effects due to the persistent presence of higher extracellular concentrations of these purines. Because wound closure in this in vitro injury model is an integrated process of cell migration and proliferation, we used a Transwell cell migration assay to more directly determine the effects of ATP-dependent signaling on epithelial cell migration (27). Indeed, migration of HBE1 cells across the Transwell membrane was markedly inhibited by treatment with the P2 receptor antagonists suramin and reactive blue 2 and in the presence of apyrase (Fig. 1E). Moreover, HBE1 cell migration could be stimulated by the addition of exogenous ATP, an effect that could be reversed by suramin (Fig. 1F).

Extracellular ATP Generates Epithelial H2O2 Production by Activation of Duox1—Recent studies have shown that P2 receptor stimulation by extracellular ATP enhances cellular H2O2 production in various cell types, including bronchial epithelial cells (5, 28). To determine the contribution of P2 receptor activation to H2O2 generation in bronchial epithelial cells, we monitored cellular H2O2 production using a high pressure liquid chromatography-based assay. Consistent with earlier findings (5), the addition of exogenous ATP (10-100 µM) resulted in a dose-dependent increase in H2O2 production by both NHBE cells or HBE1 cells (Fig. 2A). Similarly, the addition of exogenous ATP (10-100 µM) also enhanced fluorescence in HBE1 cells that were preloaded with the oxidant-sensitive fluorescence indicator 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), indicating increased H2O2 production (not shown). Moreover, epithelial production of extracellular H2O2 was enhanced after linear scratch injury in confluent HBE1 cell monolayers, and this was attenuated in the presence of the P2 receptor antagonist suramin or the ATPase/ADPase apyrase (Fig. 2B), consistent with involvement of ATP release and P2 receptor stimulation in cellular H2O2 production. Because only a small fraction of cells was expected to be stimulated in this scratch wound model, we determined localized H2O2 production in injured cell monolayers using fluorescence microscopy after cell preloading with H2DCF-DA prior to wounding. Indeed, we observed localized DCF fluorescence after linear wounding of H2DCF-DA-loaded cells primarily in cells at the wound edge (Fig. 2C, panels a-d). Again, this was largely suppressed in the presence of either suramin or apyrase (Fig. 2C, panels e-h), indicating the involvement of ATP/ADP-mediated P2 receptor activation.


Figure 2
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  		FIGURE 2.
ATP-mediated H2O2 production in bronchial epithelial cells. A, confluent NHBE (black bars) or HBE1 cells (white bars) in 24-well plates were placed in Hanks' balanced salt solution supplemented with 1 mM L-tyrosine and 10 µg/ml lactoperoxidase. After 30 min, the cells were stimulated with the indicated concentration of ATP for an additional 30 min, and dityrosine in the medium was analyzed by high pressure liquid chromatography as a measure of H2O2 production (24). Data (mean ± S.E.; n = 3) are presented relative to H2O2 production by unstimulated cells typically ranging from 0.1-0.5 nmol/30 min/106 cells (*, p < 0.05 compared with corresponding controls). B, confluent HBE1 monolayers in 24-well plates were wounded by two linear scratches, and H2O2 production was monitored 30 min after wounding in the absence or presence of suramin (100 µM) or apyrase (10 units/ml) (mean ± S.E.; n = 3; *, p < 0.05 compared with control). C, fluorescence analysis of cellular H2O2 production by HBE1 cells preloaded with the oxidant-sensitive probe H2DCF-DA (Molecular Probes; 10 µM for 30 min). HBE1 cell monolayers were wounded, and after removal of detached cells, the remaining cells were incubated for an additional 30 min in the absence (c and d) or presence of suramin (100 µM) (e and f) or apyrase (10 units/ml) (g and h). Representative bright field (a, c, e, and g) and fluorescence (b, d, f, and h) images of at least duplicate experiments are shown.

 


Figure 3
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  		FIGURE 3.
Duox1 is primarily responsible for ATP-mediated H2O2 production in bronchial epithelial cells. A, analysis of Duox expression in NHBE and HBE1 cells and silencing of Duox1 expression in HBE1 cells by siRNA. HBE1 cells were transfected with Duox1-targeted siRNA (Ambion catalog number 16708) or control siRNA (Ambion catalog number 4611) for 72 h, and Duox1 and Duox2 mRNA expression was analyzed by RT-PCR and expressed quantitatively relative to GAPDH expression (mean ± S.E.; n = 3; *, p < 0.05 compared with control). B, immunohistochemical analysis of Duox protein in untreated HBE1 cells or cells transfected with either control or Duox1 siRNA using a polyclonal antibody against Duox1 (kindly provided by Dr. J. David Lambeth; used at 1:50 dilution for 45 min). C, HBE1 cells were preincubated with DPI (1 µM) for 15 min or transfected with Duox1-targeted siRNA 48 h prior to cell stimulation with 100 µM ATP in the presence of Tyr and lactoper-oxidase, and H2O2 production was measured by high pressure liquid chromatography (mean ± S.E.; n = 3-4; *, p < 0.05 compared with corresponding control). D, fluorescence analysis of cellular H2O2 production by HBE1 cells transfected for 72 h with either control siRNA (a and b) or Duox1 siRNA (c and d) and preloaded with the oxidant-sensitive probe H2DCF-DA (Molecular Probes; 10 µM for 30 min) after linear scratch injury. Alternatively, HBE1 cells were preincubated with DPI (10 µM) (e and f) 30 min prior to wounding. DCF fluorescence was monitored 30 min after linear scratch injury, and representative results of two separate experiments are shown in comparison with corresponding bright field images.

 
According to several recent studies, airway epithelial production of H2O2 originates primarily from activation of the recently identified NADPH oxidase homologs, Duox1 and Duox2 (3-5). As illustrated in Fig. 3A, NHBE or HBE1 cells express primarily Duox1 and to a lesser extent Duox2, consistent with previous findings suggesting that Duox1 is the major enzyme responsible for airway epithelial H2O2 production (5, 6). To directly demonstrate the contribution of Duox1 NADPH oxidase activation in ATP-mediated H2O2 production, we used the pharmacological NADPH oxidase inhibitor diphenylene iodonium (DPI) or silenced Duox1 expression in HBE1 cells using siRNA. Transfection of HBE1 cells with Duox1 siRNA was found to suppress Duox1 expression by 60-70% without significantly affecting Duox2 expression (Fig. 3A) and resulted in markedly decreased Duox immunoreactivity (Fig. 3B). Both basal and ATP-stimulated H2O2 production by HBE1 cells was markedly reduced by DPI and after transfection with Duox1 siRNA (Fig. 3C), indicating that Duox1 is primarily responsible for epithelial H2O2 production. The extent of inhibition of H2O2 production by Duox1 siRNA was 50-70%, equivalent to the relative extent by which Duox1 expression was suppressed (Fig. 3A). In addition, DPI and Duox1 siRNA also markedly reduced localized H2O2 production after epithelial wounding, as measured by DCF fluorescence (Fig. 3D). Collectively, these findings demonstrate that epithelial injury results in localized ATP-mediated production of H2O2 due to the activation of Duox1.


Figure 4
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  		FIGURE 4.
Duox1 contributes to bronchial epithelial cell migration and wound repair. A, confluent NHBE or HBE1 cell monolayers were wounded as described, and wound closure was determined in the absence or presence of 1 µM DPI (mean ± S.D.; n = 3-5; *, p < 0.05 compared with corresponding control). B, HBE1 cells were transfected with Duox1 siRNA or control siRNA 72 h prior to wounding, and effects on wound closure were determined after 24 h (mean ± S.D.; n = 3-5; *, p < 0.05 compared with untreated cells). C, HBE1 cells transfected with Duox1-targeted siRNA or control siRNA were plated on Boyden-like polycarbonate filters (8 µm pore size) for analysis of cell migration, which was determined after 24 h and expressed relative to untreated cells (mean ± S.D.; n = 3-4; *, p < 0.05 compared with untreated cells). D, confluent HBE1 cell monolayers in 24-well plates were stimulated with IL-4 or IL-13 (R & D Systems; 100 ng/ml) for 48 h prior to wounding. Duox1 expression was determined by RT-PCR in relation to the housekeeping gene GAPDH (top), and effects on wound closure were determined after 24 h (bottom) (mean ± S.D.; n = 2-3).

 
Duox1 Activation Contributes to Airway Epithelial Cell Migration and Repair—Based on the observed stimulatory effects of ATP on epithelial cell migration and wound repair, the localized ATP-mediated activation of Duox1 at the wound margin of injury in the epithelial monolayers, and the suggested involvement of NADPH oxidases in cell migration in other cell types (22), we postulated that Duox1 activation might actively contribute to airway epithelial cell migration and wound repair. Consistent with this notion, wound closure in injured NHBE or HBE1 cell monolayers was markedly suppressed in the presence of DPI (Fig. 4A). More specifically, HBE1 transfection with Duox1-targeted siRNA (but not control siRNA) markedly suppressed wound closure to an extent that was comparable with the degree of Duox1 inhibition (Fig. 4B). The contribution of Duox1 to epithelial cell migration could also be demonstrated more directly in a quantitative Transwell migration assay showing marked inhibition of HBE1 cell migration after transfection with Duox1 siRNA (Fig. 4C). Finally, because epithelial cell stimulation with the Th2 cytokines interleukin (IL)-4 and IL-13 was recently found to induce the expression of Duox1 (3), we tested whether prestimulation of HBE1 cells with IL-4 or IL-13 would enhance their ability to migrate and promote wound closure. As illustrated in Fig. 4D, HBE1 preincubation with either cytokine was found to enhance the extent of wound closure, in association with the up-regulation of Duox1 expression, consistent with a role of Duox1 in cell migration.

Involvement of ATP-mediated Duox1 Activation in ERK1/2 Activation—Epithelial repair following injury is a complex process involving many diverse cellular and extracellular processes. Among these, activation of extracellular signal-regulated kinases (ERK1/2) is known to represent a critical signaling mechanism in epithelial cell migration, and extracellular ATP has been shown to be capable of activating ERK1/2 through P2 receptor stimulation (29-32). We confirmed the activation of ERK1/2 in linearly wounded HBE1 cells (Fig. 5) and NHBE cells (not shown) by using Western blot analysis of cell lysates as well as immunohistochemical analysis of wounded HBE1 cell monolayers. Both approaches revealed rapid ERK1/2 activation immediately after wounding, specifically in cells near the wound margin (Fig. 5A and B (panels a and b)). Moreover, pharmacological inhibition of ERK signaling with U0126 (10 µM) markedly inhibited HBE1 wound closure (results not shown), consistent with earlier reports (29, 32). ERK activation in response to epithelial injury was markedly reduced in the presence of the P2 receptor antagonist suramin or the ATPase apyrase (Fig. 5B (panels c and d)), indicating the critical involvement of ATP-dependent P2 receptor activation. Moreover, inhibition of Duox1 activation with DPI or after siRNA-mediated specific Duox1 silencing significantly inhibited localized ERK activation in wounded HBE1 monolayers (Fig. 4, C and D), demonstrating the importance of this NADPH oxidase in ATP-mediated ERK1/2 activation in response to airway epithelial injury.

MMP-9 Expression and Activation by ATP-mediated Duox1 Stimulation—Among the effector proteins that mediate epithelial cell migration and repair are various matrix metalloproteases (MMPs), including gelatinase B (MMP-9). Because oxidative mechanisms, including those mediated by Duox1 activation, have been suggested to contribute to activation or up-regulation of MMPs or related proteases that contribute to epithelial cell migration and repair (10, 33, 34), we determined the involvement of ATP-mediated signaling and Duox1 activation in expression and activation of MMP-9. Consistent with previous observations (35, 36), we revealed localized activation of gelatinase activity at the wound edge of injured HBE1 monolayers, using an in situ zymography assay (Fig. 6A, panels a and b). Although HBE1 cells express both gelatinase A (MMP-2) and gelatinase B (MMP-9), our previous studies indicate that gelatinase activity in wounded HBE1 cells originates primarily from MMP-9 (36). The appearance of gelatinase activity was dramatically reduced after P2 receptor blockade with suramin (Fig. 6A, panels c and d). Similarly, gelatinase activation was also dramatically inhibited in the presence of 1 µM DPI (not shown) and after siRNA knockdown of Duox1 (Fig. 6A, panels e-h), suggesting the importance of ATP-mediated Duox1 activation in this process. The involvement of MMP-9 (gelatinase B) in airway epithelial migration was demonstrated more directly in studies using MMP-9-targeted siRNA, which confirmed the active contribution of MMP-9 in HBE1 cell wound closure (Fig. 6B).


Figure 5
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  		FIGURE 5.
ATP-mediated signaling and Duox1 contribute to ERK activation in response to epithelial injury. A, HBE1 cells in 24-well plates were untreated (a) or wounded (b), and cell lysates were collected after 10 min for Western blot analysis of phosphorylated and total ERK. A representative blot and quantitative analysis by densitometry are shown (mean ± S.E.; n = 3; *, p < 0.05). B, immunofluorescence analysis of ppERK, unwounded HBE1 cells (a), or HBE1 cell monolayers 10 min after wounding in the absence (b) or presence of 100 µM suramin (c) or 10 units/ml apyrase (d). A representative experiment of three is shown. C, Western blot analysis of wound-induced ERK activation after transfection with control siRNA (b) or Duox1 siRNA (c) or after inhibition of Duox activity by DPI (1 µM) (d). A representative blot and quantitative analysis by densitometry are shown (mean ± S.E.; n = 3; *, p < 0.05). D, analysis of ppERK immunostaining in wounded HBE1 cells under similar conditions as in C. Representative images of at least two experiments are shown.

 


Figure 6
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  		FIGURE 6.
Duox1 contributes to ATP-mediated expression and activation of MMP-9. A, in situ analysis of gelatinase activity in response to HBE1 cell wounding. Untransfected or Duox1 siRNA-transfected HBE1 cells were plated in chamber slides, wounded, and subsequently incubated for 24 h. Gelatinase activity was assessed by incubation with gelatin-DQ and visualized by fluorescence microscopy (green). Fluorescence images were overlaid with bright field images for orientation. Arrows indicate the wound margin and the direction of cell migration. B, inhibition of MMP-9 expression in HBE1 by siRNA inhibits wound closure. HBE1 cells were transfected with MMP-9 siRNA as described previously (36), and effects of MMP-9 expression (top) or wound closure (bottom) were analyzed (mean ± S.E.; n = 3-4; *, p < 0.05 compared with untreated cells). C, semiquantitative RT-PCR analysis of MMP-9 expression in HBE1 cells 24 h after stimulation with the indicated concentrations of ATP (in µM) or 24 h after wounding in the absence or presence of sumarin (100 µM) or apyrase (10 units/ml). Representative data and densitometry analysis (mean ± S.E.; n = 3; *, p < 0.05) are shown. D, effect of DPI (1 µM) or Duox1 siRNA knockdown on MMP-9 expression analyzed 24 h after wounding. Representative data and densitometry analysis (mean ± S.E.; n = 3; *, p < 0.05) are shown.

 
Consistent with earlier studies (36), epithelial injury resulted in increased expression of MMP-9 (Fig. 6C), which was found to depend on ATP-mediated purinergic signaling, as illustrated by the inhibitory effects of either suramin or apyrase. Accordingly, MMP-9 expression was also enhanced after cell stimulation with exogenous ATP (Fig. 6C). Both basal and ATP-mediated MMP-9 expression were found to depend on ERK signaling and were markedly inhibited by U0126 (10 µM; data not shown). Finally, MMP-9 induction after epithelial injury was markedly inhibited by DPI, or after transfection, with Duox1-targeted siRNA (Fig. 6D), illustrating the involvement of Duox1-mediated NADPH oxidase activity in MMP-9 expression in response to epithelial injury.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results illustrate a previously unidentified role for Duox1 as an important component of airway epithelial repair in response to injury. Activation of Duox1 appears to be critical in the activation of ERK1/2-dependent signaling pathways and in the induction and activation of critical metalloproteases, such as MMP-9. Moreover, activation of Duox1 is mediated by purinergic P2 receptor activation at the epithelial surface in response to extracellular ATP released from epithelial cells following stimulation or injury. In this regard, Duox1 may be one of the participants in epithelial defense mechanisms against bacterial infection that induces epithelial ATP release and promotes purinergic signaling (13).

Although our studies clearly link ATP-mediated P2 receptor activation to cell migration and epithelial repair, measured concentrations of extracellular ATP following linear scratch injury in HBE1 monolayers were much lower (up to 50 nM) than those required to fully activate purinergic receptors (typically in the µM range) (19). Indeed, in studies using exogenous ATP, concentrations of 1-100 µM were required to stimulate H2O2 production, MMP-9 expression, or cell migration (Figs. 1, 2, and 6). This apparent discrepancy has been recognized in several studies of ATP-mediated signaling in response to mechanical or chemical cellular stress and was recently addressed by Okada et al. (37). Using chimeric protein A-luciferase constructs that bind to endogenous antigens on the epithelial surface, these investigators demonstrated that extracellular ATP can reach µM concentrations at the apical surface in response to hypotonic cell stress, thus confirming that ATP release can occur to an extent sufficient to activate P2 receptors (37). These observations also strengthen the notion that measurements of extracellular ATP in bulk media in epithelial cell culture will most likely underestimate the extracellular ATP concentrations that may occur in vivo within the thin epithelial surface layer.

The same consideration also applies to the interpretation of measured H2O2 concentrations upon ATP stimulation or after epithelial injury. Although our measurements indicate extracellular H2O2 concentrations in the nanomolar range in response to cell injury or after stimulation with ATP, epithelial H2O2 release into the thin epithelial surface fluid in vivo will most likely yield substantially higher concentrations. Therefore, attempts to mimic these conditions by adding similar concentrations of these mediators to bulk media may sometimes yield confusing or contradictory findings. For example, consistent with previously reported findings (14), the administration of exogenous ATP at elevated concentrations (100 µM) was found to inhibit epithelial wound closure. This may be due to prolonged activation of P2 receptors or nonspecific stimulation of other purinergic receptors under these conditions, with adverse effects. Moreover, excessive ATP stimulation may result in overproduction of H2O2 and thereby inhibit cell migration (38).

With regard to the downstream mechanisms by which Duox1 activation mediates ERK activation and MMP expression and activation, many questions still remain to be answered. Analogous to other NADPH oxidases, Duox1 activation could promote ERK1/2 activation because of H2O2-mediated inhibition of redox-sensitive protein phosphatases (39). However, questions remain regarding the localization of H2O2 production, which may be restricted to subcellular compartments (40, 41). In this case, the extent of extracellular H2O2 production may not necessarily relate to downstream cell signaling events. Consistent with this scenario, our observations of Duox1-dependent increased DCF fluorescence in response to epithelial injury are also suggestive of a potential intra- or subcellular source of H2O2 that may be involved in cell signaling.

Another possibility that will need to be addressed in future studies is the potential involvement of H2O2-independent mechanisms in Duox1-mediated cellular events. In this regard, it is becoming increasingly appreciated that activation of NADPH oxidases, including Duox1, is associated with intracellular pH changes and H+ transport, which includes the activation of Na-H exchangers (6, 42). Indeed, Na-H exchangers have recently been linked to ERK-mediated signaling and are critically involved in polarized cell organization and migration in several cell types (42, 43). Finally, Duox1 may also control ERK activation and epithelial repair by more indirect mechanisms through activation of growth factor/chemokine receptors that control cell migration or proliferation (10).

The involvement of Duox1 activation in MMP-9 activation, as demonstrated in our studies, appears consistent with the reported ability of oxidants to activate latent MMPs by direct actions on the cysteine switch (34). Analogously, Duox1-derived H2O2 was recently suggested to directly activate a related metalloproteinase, tumor necrosis factor-{alpha}-converting enzyme (10). However, our results also revealed a role for Duox1 in ATP-mediated induction of MMP-9 expression following epithelial injury in association with ERK1/2 activation, a finding that is consistent with several reports showing oxidant-mediated MMP-9 induction in other cell systems (33, 44). Moreover, studies with recombinant pro-MMP-9 failed to show significant gelatinase activation by H2O2 (1-100 µM; data not shown). Collectively, these findings suggest that Duox1-mediated epithelial MMP-9 activation most likely occurs by mostly indirect mechanisms regulating MMP-9 expression and activation, although the possible contribution of Duox1-derived H2O2 or secondary oxidants in extracellular MMP activation cannot be ruled out.

In summary, the present findings illustrate a previously unrecognized function of airway epithelial Duox1 in cell migration and repair, which is of critical importance in rapid reepithelialization after epithelial shedding to maintain airway mucosal barrier properties. Much is still to be learned regarding airway epithelial Duox1 regulation and activation, illustrated by the recent discovery of Duox-associated factors that control Duox maturation and cellular localization (45). At present, the biological significance of such maturation factors and their potential role in the airway epithelium are unknown. Moreover, recent observations of the regulation of airway epithelial Duox expression by pro-inflammatory cytokines (3) may point to the potential importance of Duox1 as an integral component of intrinsic epithelial repair mechanisms under pathological conditions associated with airway infection or inflammation.


   FOOTNOTES
 
* This work was supported in part by grants from the NHLBI, National Institutes of Health (HL068865 and HL074295), a training grant from the National Institute for Environmental Health Sciences (T32ES07122), and an intramural grant from the University of Vermont College of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Pathology, D205 Given Bldg., University of Vermont, 89 Beaumont Ave., Burlington, VT 05405. Tel.: 802-656-8638; Fax: 802-656-8892; E-mail: Albert.van-der-Vliet{at}uvm.edu.

3 The abbreviations and trivial names used are: Duox, dual oxidase; DCF, dichlorofluorescein; DPI, diphenylene iodonium; ERK, extracellular signal-regulated kinase; IL, interleukin; MMP, matrix metalloproteinase; NHBE, normal human bronchial epithelium; siRNA, small interfering RNA; RT, reverse transcription; ATP{gamma}S, adenosine 5'-3-O-(thio)triphosphate. Back


   ACKNOWLEDGMENTS
 
We thank Karen Lounsbury for use of the luminometer, Cedric Wesley for assistance with fluorescence microscopy, J. David Lambeth and Francoise Miot for kindly providing Duox antibodies, and Takaaki Akaike for providing recombinant proMMP-9.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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