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1Centre de Recherche, Centre Hospitalier de l'Université de Montréal-Hôtel-Dieu, 2Département de Médecine and 3Groupe d’Étude des Protéines Membranaires, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada
Submitted 14 September 2006 ; accepted in final form 12 July 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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lung; adenosine 5'-triphosphate-stimulated potassium channel; KvLQT1 K+ channels; epidermal growth factor; alveolar cell repair; migration; proliferation
Role of Growth Factors in Lung Epithelia Repair
After injury, surviving epithelial cells and fibroblasts potentially release proinflammatory cytokines and growth factors. Among them, hepatocyte growth factor (HGF) is synthesized predominantly by fibroblasts and macrophages, whereas epidermal growth factor (EGF) is expressed and secreted by fibroblasts and epithelial cells (2, 5, 37, 39, 49). Afterward, HGF and EGF act, respectively, via paracrine and autocrine pathways through HGF receptors (HGF-R or c-Met) and EGF receptors (EGF-R). EGF ligands (EGF, transforming growth factor-
, heparin-binding EGF-like growth factor, etc...) bind several subtypes of EGF-R, erbB1 (or HER1), erbB2, erbB3, and/or erbB4, which are expressed at the cell surface of pulmonary epithelial cells (2, 18, 37–39, 52, 61). After EGF ligand binding and autophosphorylation at its multiple COOH-terminal tyrosine residues, activated EGF-R transmit intracellular signals through docking sites for downstream signaling molecules (20). Activated EGF-R induce mitogenic, motogenic, and morphogenic cellular responses, which are crucial in the airway and alveoli epithelial repair process (5, 15, 28, 29, 38, 49, 50, 53). Unfortunately, in cystic fibrosis patients, for example, elevated endogenous levels of growth factors are not sufficient to protect lung epithelia from injury (49). Thus it will be important to develop other strategies to potentiate their effects.
Role of K+ Channels in Repair Processes
K+ channels have been shown to play a major role in Cl– and Na+ transepithelial transport through lung epithelia (10, 26, 27, 31, 33, 34). In alveolar monolayers, we demonstrated recently that ATP-dependent K+ (KATP) and KvLQT1 channel activity regulated epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator activity and expression as well as fluid absorption (27). In addition to this role in epithelial transport, K+ channels could also be important in the repair process of lung epithelia by regulating cell proliferation and migration. Indeed, several studies have demonstrated that KATP channels, Ca2+-activated intermediate IKCa channels, and channels from the voltage-dependent Kv family modulate the proliferation of various cell types (6, 21, 24, 25, 32, 35, 36, 51, 54, 56, 57, 60). It has been postulated that K+ channel activity could affect cell proliferation by altering membrane potential, intracellular Ca2+ ([Ca2+]i), cell volume and/or growth factor-mediated mitogenic signaling (17, 35, 55, 60). KATP, KCa, and Kv K+ channels also regulate the migration process in several cell types (11, 23, 30, 40, 43, 46, 47). The role of IKCa channels in cell migration has been studied considerably. It has been proposed (44, 45, 47) that a rise in [Ca2+]i and massive K+ efflux through IKCa channels causes shrinkage of the cell pole and induces retraction of the rear part of the migrating cell, promoting cell movement. Thus K+ channels not only play a role in ion transport in lung epithelia but could also participate in repair processes. Up to now, the cell proliferation, migration, and repair functions of K+ channel activity have never been explored in lung epithelia.
A coupling mechanism could link K+ channel activity to growth factor signaling. It has been reported recently that EGF hyperpolarizes membrane potential through the modulation of KCa channels in PC-12 cells (48). Such activation of K+ channel activity and/or expression by EGF has also been demonstrated in mucous airway cells (22), corneal epithelial cells (41), myeloblastic cells (54), and vascular smooth muscle cells (VSMC; see Refs 21 and 25). Moreover, it has been shown that EGF-induced proliferation of corneal epithelial cells (41), VSMC (21, 25), myeloblastic cells (54, 60), and HGF-stimulated migration of Madin-Darby canine kidney (MDCK)-F cells (23) are dependent on K+ channel activation. The relationship between K+ channel activity and growth factor-stimulated cell proliferation and migration has, however, never been explored in pulmonary epithelia.
A better understanding of the cellular and molecular mechanisms, involved in repair processes of lung epithelia from in vitro models, may be crucial to elaborate strategies favoring the resolution of lung injuries that will be tested in the future on in vivo models. The aim of our in vitro study was to better characterize the role of EGF in the repair of alveolar monolayers after mechanical injury and in alveolar migration and proliferation. We also investigated, for the first time, the effects of K+ channel modulators on these repair processes in basic conditions and after stimulation with EGF. Finally, the impact of EGF on K+ currents and K+ channel expression was explored.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Alveolar epithelial type II (ATII) cells were isolated from adult male Sprague-Dawley rats (6–7 wk), according to a well-established protocol (14, 26). In brief, the lungs were washed to remove blood cells and alveolar macrophages before treatment with elastase. They were then minced, and the resulting suspensions were filtered. Alveolar cells were collected and purified by a differential adherence technique (12), which enhances the purity of the ATII cell pool to 86% (7, 26). This freshly isolated cell suspension was cultured on petri dishes or Costar Transwell permeant filters (1 x 106 cells/cm2; Costar Transwell, Toronto, ON) in minimal essential medium (MEM; Gibco, Invitrogen, Burlington, Toronto) containing 10% FBS (Gibco, Invitrogen), 0.08 mg/l gentamicin, septra (3 µg/ml trimethoprime + 17 µg/ml sulfamethoxazole), 0.2% NaHCO3, 10 mM HEPES, and 2 mM L-glutamine, as described previously (26). This MEM-FBS-septra medium was replaced after 3 days by the same MEM-FBS without septra.
Wound Healing
Alveolar epithelial cells, cultured for 3 days in MEM-FBS medium on petri dishes, were injured mechanically with a P10 Gilson pipette tip. After injury, the monolayers were washed with MEM-FBS to remove detached, injured cells. The monolayers were then photographed with a NIKON camera under light microscopy after varied repair times (0, 24, and 48 h) in control conditions and in the presence of the K+ channel modulators 25 ng/ml EGF (Sigma-Aldrich, Oakville, ON) and 5 µg/ml anti-EGF antibody (EGF-Ab; Santa Cruz Biotechnology, Santa Cruz, CA) and selective inhibitors of erbB1 (tyrphostin AG-1478, 10 µM; Sigma-Aldrich) and/or erbB2 (tyrphostin AG-825, 10 µM; Sigma-Aldrich) receptors.
Wound width was measured by ImageJ software and represented as percentage of the initial wound width. At the end of the experiment, the monolayers were fixed and processed for alkaline phosphatase assay to identify epithelial ATII cells as described previously (7). Briefly, the cells were fixed for 30 s at room temperature in cold paraformaldehyde/acetone solution [4% paraformaldehyde (wt/vol), 0.02% Na2HPO4 (wt/vol), 0.1% KH2PO4 (wt/vol), 45% acetone (vol/vol), pH 6.6], rinsed for 1 min in distilled water, and air-dried. Fixed cells were incubated for 90 min at 37°C in a fresh staining solution containing 0.05% Fast Blue BB salt in stock staining solution (30 mg naphtol-AS-phosphate in 0.5 ml N,N-dimethylformamide filled up to 100 ml with 0.2 M Tris base). The samples were then rinsed with distilled water for 1 min and air-dried. The cells were counterstained with 0.1% neutral red solution for 3 min at room temperature, rinsed for 1 min, air-dried, and photographed.
Conditioned media were also collected for migration assay. Briefly, alveolar monolayers were cultured for 3 days in MEM-FBS medium on petri dishes. A first group of petri dishes was washed three times with FBS-free MEM; one-half of them was injured mechanically, whereas the other half was not. All petri dishes were then washed with FBS-free MEM to remove detached or injured cells. The "wounded" or "unwounded", FBS-free conditioned media were collected after 1 h. The same protocol was repeated with a second group of petri dishes in the presence of MEM-FBS to obtain wounded or unwounded complete MEM-FBS conditioned media.
Time-Lapse Experiments
Time-lapse images were taken with a x10 objective and an Olympus microscope at 5-min intervals with a digital camera connected to a microscope over a 12-h time period. Small wounds (half-size compared with standard wound-healing experiments) were performed to reduce the duration of these experiments. Complete closure was observed after 
12 h.
Cell Migration in Boyden-Type Chamber
Alveolar cells were cultured on plastic supports for 2 days in MEM-FBS medium, washed in PBS, and separated with trypsin-EDTA (Gibco, Invitrogen). Cell suspensions were collected by centrifugation, and the cells were suspended in FBS-free MEM and counted. Cell viability after trypsinization was verified with Trypan blue. In addition, an alkaline phosphatase test was employed to verify the ATII phenotype of these cells. Briefly, an aliquot of cell suspension was seeded on plastic supports. After adherence, the cells were fixed and subjected to alkaline phosphatase assay, as described above. About 90% of the cells were positive, which indicated that the trypsinization of ATII cells did not affect their phenotype. For the migration assay, the cell suspension obtained after trypsinization was placed (150,000 and 75,000 cells for 6 and 24 h migration, respectively) in the upper compartment of 8-µm pore filters (0.33 cm2, Thin Certs-TC inserts, Greiner Bio-one; MJS Biolynx, Brockville, ON). The lower compartment was filled with FBS-free MEM in the presence or absence of the K+ channel modulators EGF or EGF-Ab. After a 6- or 24-h migration period, the filters were washed with PBS, and migrating cells that passed through the lower face of the filters were fixed with a paraformaldehyde/acetone solution and stained overnight with crystal violet (0.1% in 20% methanol). After some washings, nonmigrating cells of the upper compartment were scraped off with Q-Tips (Fischer, Napean, ON). The filters were examined by microscopy at x40 enlargement, and cells that had migrated were counted in five different fields.
In migration assays with conditioned medium, alveolar cell suspensions were resuspended in FBS-free MEM or MEM-FBS media and placed in the upper compartment of the filters. The lower compartment was filled with the relevant wounded or unwounded FBS-free MEM or MEM-FBS conditioned medium (see Wound Healing) in the presence or absence of 5 µg/ml EGF-Ab.
Cell Proliferation
Freshly isolated alveolar cells were seeded at low density (150,000 cells/well) in 12-well cell culture clusters (Costar; Corning, Corning, NY) and cultured for 3 days in MEM-FBS-septra medium (until 50% confluence). On day 3, the culture medium was replaced by MEM-FBS medium containing 0.5 µCi/ml [3H]thymidine (MP Biomedical, Irvine, CA) in the presence of the K+ channel modulators EGF or EGF-Ab. After a 24-h period, the [3H]thymidine medium was removed by inversion and replaced by ice-cold trichloroacetic acid (TCA 5%, 5–10 min). TCA was then discarded, and the plates were washed in three successive ice-cold water baths. The cells were lysed with 0.1 N NaOH, transferred to scintillation tubes, and counted (Bq/cm2) in a beta counter (Tri-carb, 1600TR liquid scintillation analyzer; Canberra Packard). Protein contents were estimated by Coomassie blue assay (Bradford, Pierce, Rockford, IL) of an aliquot of each sample. [3H]thymidine incorporation was corrected for protein content and presented as a percentage of control values (without modulators).
Immunoblotting
Total proteins were extracted from alveolar cells (obtained from at least 4 different rats), cultured on plastic dishes for 3 days, and treated or not with EGF or EGF-Ab. In some experiments, the monolayers were wounded 30 min before protein extraction.
For the detection of phosphorylated forms of EGF-R (p-EGF-R or p-erbB1) and erbB2, the monolayers were washed two times with ice-cold PBS, and proteins were extracted after cell scraping in the presence of 3x hot (95°C) Laemmli buffer (15% 
-mercaptoethanol, 7.5% SDS, 30% glycerol, 300 mM Tris·HCl, pH 6.8, bromophenol blue) for immediate inhibition of cellular proteases and phosphatases. Protein contents were estimated in an equivalent cell preparation after extraction with 1 N NaOH. Proteins (200–400 µg) were separated by SDS-PAGE (7.5%) and transferred to Hybond C extra nitrocellulose membranes (Amersham/GE, Baie D'Urfé, QC). The nitrocellulose membranes were first incubated with 4% fraction V BSA in TBS containing Tween 20 (TBST, 500 mM NaCl, 20 mM Tris HCl, and 0.1% Tween 20, pH 7.4) for 1 h at room temperature. The erbB1 and erbB1 phosphorylated on tyrosine-1148 or -845 as well as erbB2 and erbB2 phosphorylated on tyrosine-877 were then blotted with a 1:1,000 dilution of commercially available specific antibodies in TBST plus 2% fraction V BSA [erbB1 (no. 2232; Cell Signaling Technologies, Danvers, MA), phosphorylated (p)-EGF-R1148 (no. 4404; Cell Signaling Technologies), p-EGF-R845 (no. 2231; Cell Signaling Technologies), and erbB2 (no. E3451-27; USBiological)] for at least 2 h at room temperature and detected with goat horseradish peroxidase-labeled rabbit immunoglobulins (Jackson Immunoresearch). Ponceau staining was performed, and -actin protein was revealed with the monoclonal AC15 antibody at a 1:50,000 dilution (Sigma-Aldrich) to ensure equivalent loading.
For KvLQT1 and Kir6.1 K+ channel detection (27), the cells were detached by gentle scraping, and the cell suspension was centrifuged at 2,800 g at 4°C for 15 min. The cell pellet was solubilized in lysis buffer [150 mM NaCl, 50 mM Tris·HCl, pH 7.6, 1% Triton X-100, 0.1% SDS, protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail; Roche, Mannheim, Germany)] for 1 h on ice and centrifuged at 12,000 g for 15 min. The supernatants were collected, and protein contents were measured. The proteins were then solubilized at 95°C for 5 min in 2x sample buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 0.2% bromophenol blue, and 4% -mercaptoethanol). Proteins (40 µg) were separated by SDS-PAGE (7.5%) and transferred onto polyvinyl difluoride membranes. The membranes were first blocked with 10% dried milk in TBST for 1 h at room temperature and then stained with commercial polyclonal anti-Kir6.1 (dilution 1:200, sc-11224; Santa Cruz Biotechnology,) or anti-KvLQT1 (dilution 1:500, sc-10645; Santa Cruz Biotechnology) in TBST plus 10% milk for 16 h at 4°C. After being washed with TBST, the membranes were incubated with a donkey anti-goat IgG (Santa Cruz Biotechnology) linked to horseradish peroxidase for 1 h. Proteins were revealed by chemiluminescence reagent (ECL Plus; Amersham) and exposed to Kodak Biomax light film. The anti-Kir6.1 and anti-KvLQT1 antibodies, respectively, recognized an 50- and 75-kDa protein (27). The specificity of anti-Kir6.1 and anti-KvLQT1 antibodies was verified with their respective blocking peptide (sc-11224P and sc10645P; Santa Cruz Biotechnology; see Ref. 27). Ponceau staining was also performed, and -actin protein was revealed with a purified mouse anti--actin monoclonal Ab (dilution 1:2,500, CLT9001; Cerdarlane Laboratory Limited) to ensure equivalent loading. The intensity of each specific band corresponding to K+ channel protein was quantified with ImageJ software and normalized to the -actin signal.
Semiquantitative PCR
RNA purification. Total RNA from alveolar epithelial cells cultured on filters for 4 days was purified with TRIzol reagent according to the manufacturer's (Invitrogen) instructions.
PCR amplification of ionic channels.
Total RNA (5 µg) purified from cultured alveolar epithelial cells (on permeant filters) was reverse transcribed to cDNA with Moloney murine leukemia virus RT (Invitrogen) in the presence of oligo(dT) primers. cDNAs were amplified with Taq polymerase (Invitrogen), employing specific primers designed from sequences of the following cloned K+ channels: rat KvLQT1 (sense: 5'-cccatctcagaaaagagcaa-3', antisense: 5'-tcttggtgagctccagattc-3', 1 µM final concentration of each, 462-bp PCR product) and rat Kir6.1 (sense: 5'-cgcccacggggacatctatgc-3', antisense: 5'-agggggctacgcttatcaat-3', 1 µM final concentration of each, 544-bp PCR product). EGF expression was also evaluated using the rat EGF primer pair (sense: 5'-gaaggagtagacacgccaga-3', antisense: 5'-tgggtgacctacatcgttct-3', 1 µM, 408-bp PCR product). Finally, the -actin primer pair (sense: 5'-ctaaggccaaccgtgaaaag-3' and antisense: 5'-gccatctcttgctcgaagtc-3', 0.25 µM) amplified a 311-bp product (7, 27). Primer pairs were designed in distinct exons to avoid genomic DNA amplification. Semiquantitative RT-PCR amplification was undertaken according to a well-established laboratory protocol (7, 26, 27). Briefly, cDNA amplification curves as a function of the number of amplification cycles were first charted for each primer pair to define the optimum number of cycles in the linear phase of amplification. The KvLQT1 product was amplified for 24 cycles and Kir6.1 for 30 cycles, whereas -actin amplification was stopped after 17 cycles. Because -actin amplification remains stable under all test conditions, even in the linear phase of amplification (26), PCR products were normalized with the -actin signal for each cDNA sample. The RT-PCR products were finally separated on agarose gels, stained with ethidium bromide, and analyzed with the Typhoon Gel Imager (7, 26, 27).
Electrophysiology.
The electrophysiological characteristics of alveolar monolayers were studied by short-circuit current (Isc) measurements in an Ussing chamber (26, 27). First, alveolar epithelial cells were cultured on filters (4 cm2; Costar Transwell) for 3 days until they formed a tight epithelium (>1,200 
·cm2). The monolayers were then treated or not for 24 h with EGF. After being washed, the alveolar monolayers were mounted in a heated (37°C) Ussing chamber and perfused on the apical and basolateral sides with warm physiological solution. To quantify the amount of active K+ channels at the basolateral membrane after treatment with EGF, Isc was measured after establishment of a K+ gradient and permeabilization of the apical membrane with 7.5 µM amphotericin B. The apical-to-basolateral K+ gradient was created by bathing the apical side with a high-K+ physiological solution [containing (in mM): 81 NaCl, 65.4 KCl, 0.78 NaH2PO4, 0.8 MgCl2, 1.8 CaCl2, 5 glucose, and 15 HEPES, pH 7.4], whereas, for the basolateral side, 60 mM of KCl was replaced by an equivalent amount of N-methyl-D-glucamine chloride. Transepithelial potential difference was clamped to zero by an external voltage-clamp amplifier (VCCMC2; Physiological Instruments) with KCl agar-calomel half-cells and Ag-AgCl electrodes, and the resulting Isc was recorded continuously on a computer with a PowerLab system (ADInstrument, Toronto, ON; see Ref. 26). Membrane resistance was verified with 1-mV pulses every minute.
Statistics The data are represented as means ± SE collected from at least four different animals (n > 4). Comparison between groups was made by paired t-test or one-group t-test with StatView software (SAS Institute, Cary, NC). A probability of P < 0.05 was considered to be significant (P values were reported for each experiment).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To study the regulatory mechanisms of injury responses and repair processes of alveolar epithelia, we adapted a model of wound injury to primary cultured alveolar cell monolayers. A mechanical injury protocol was chosen because it gave more standardized and reproducible results than chemical injury in our cell model. The wound healing of alveolar monolayers, cultured in an undefined MEM-FBS medium (MEM supplemented with FBS) on plastic supports, was followed over a 48-h period (Fig. 1A). We found 54 ± 3% repair at 24 h after injury and 94.1 ± 2.4% at 48 h (Fig. 1A). We decided in future experiments to study the regulation of alveolar repair at 24-h postinjury to be able to observe a decrease and increase in cell repair. The presence of FBS in culture medium was crucial since an FBS starvation for 24 h affected monolayer integrity and dramatically reduced wound healing (15.5 ± 4.2% repair after 24 h, n = 4). Future wound-healing experiments were then performed in the presence of MEM-FBS medium.
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Because other ligands, such as TGF- or HB-EGF, could be involved in EGF-R signaling, we tested the impact on wound healing of erbB1 and erbB2 receptor inhibition by using tyrphostin AG-1478 and AG-825, respectively. AG-1478 (10 µM) significantly reduced wound healing; however, the observed inhibition was smaller (38.2 ± 4.4% inhibition, n = 5) than that measured in the presence of EGF-Ab (P = 0.03, Fig. 1D). Inhibition of both erbB1 and erbB2 receptors, in the presence of AG-1478 and AG-825, reduced alveolar repair by 48.2 ± 6.1% (n = 5), an effect comparable to that of EGF-Ab (difference: 5.9 ± 5.3%, P = 0.3, not significant). These results suggested that, in our experimental conditions, alveolar repair regulation by erbB1 signaling (and to a lesser extent, erbB2) is mostly the result of their activation through EGF. For this reason, we focused our study on EGF ligand in future experiments.
Activation of EGF-R Upon Injury Through an Autocrine EGF Loop
Because EGF-Ab reduced wound healing probably through binding to secreted EGF, we first verified the EGF expression in alveolar epithelial cells. As shown in Fig. 2A, the rat EGF primer pair amplified, as expected, a 408-bp product from the cDNA of primary cultured epithelial alveolar cells.
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Effect of K+ Channel Modulators on Alveolar Wound Healing
We recently identified the presence of functional KATP, KvLQT1, and IKCa channels in alveolar cells (26, 27). The impact of KATP, KvLQT1, and IKCa channel modulators was explored on wound healing of alveolar monolayers under basic and EGF-stimulated conditions. We observed that the presence of pinacidil (100 µM, an efficient dose to stimulate KATP when added in acute or chronic protocols; see Ref. 27), applied just after injury, did not significantly modify wound repair measured at 24 h (Fig. 3A) under basic conditions (without EGF) and after exogenous EGF addition (with EGF). On the other hand, KATP and KvLQT1 inhibitors [glibenclamide (15 µM) and clofilium (5 µM), respectively] slightly reduced basic (Fig. 3A, left) and EGF-stimulated (Fig. 3A, right) wound repair. Applied in combination, these inhibitors severely inhibited alveolar repair in basic (65.5 ± 6.2% in untreated monolayers vs. 31.1 ± 7% in glibenclamide + clofilium-treated monolayers, n = 8, P = 0.0003; Fig. 3A, left) and EGF-stimulated (74.4 ± 5.6% in untreated monolayers vs. 40.4 ± 6.8% in glibenclamide + clofilium-treated monolayers, n = 8, P = 0.004; Fig. 3A, right) conditions. Inhibitors of IKCa channels (charybdotoxin or Tram-34) were without effect (data not shown).
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The inhibitory effect of glibenclamide was also evaluated in time-lapse experiments under videomicroscopy. In these experiments, time-lapse images of wound healing in alveolar monlayers treated or not for 24 h with glibenclamide were taken at 5-min intervals over a 12-h period. Observation of video 1 (control condition, time 0–7 h after injury) and video 2 (15 µM glibenclamide, time 0–7 h after injury) confirmed that inhibition of KATP channels severely reduced alveolar cell motility and the rate of repair (Supplemental material for this article can be found at the American Journal of Physiology: Lung, Cellular and Molecular Physiology website). Figure 3C shows images taken 3, 6, 9, and 12 h after injury.
Effect of EGF and K+ Channel Modulators on Alveolar Cell Migration and Proliferation
In a previous paper (7), we have reported that ATII cells progressively changed their phenotype in primary culture (decrease of surfactant protein expression and alkaline phosphatase activity, a marker of ATII cells). However, as shown in Fig. 3D, enlargement of the injured monolayer labeled for alkaline phosphatase activity revealed that intact cells still developed alkaline phosphatase activity (cells stained in blue). Conversely, alveolar cells in the wound changed their phenotype with a more widespread appearance and absence (red cells) of alkaline phosphatase activity. Similarly, it has been established in vivo that alveolar repair involves transdifferentiation from ATII cells onto ATI cells as well as cell migration and proliferation (3, 15). In our in vitro model, we observed 24 h after injury (Fig. 3D) that lamellipodia of some of the dedifferentiated cells stretched out to connect the wound edges, indicating a migratory process. Cell mitosis could also be seen by the presence of two nuclei in some cells.
Because wound healing was regulated by EGF and K+ channel activity, we decided to evaluate their effect on migration and proliferation processes involved in epithelial repair. Migration of ATII cells was first measured over a 6-h period in a Boyden-type migration chamber, in the presence of FBS-free and EGF-free MEM in both the upper and lower compartments. We first observed that FBS addition, in the lower compartment or in both the lower and upper compartments, enhanced the number of migrating cells by sixfold (n = 10, P = 0.006) or ninefold (n = 10, P = 0.012), respectively. The addition of EGF-Ab (5 µg/ml) decreased by 50% the number of migrating cells measured in the presence of FBS, suggesting that part of the FBS stimulatory effect was because of the presence of EGF in FBS medium. For better control of medium composition, further migration experiments were performed in the absence of FBS.
The stimulatory effect of exogenous EGF was then evaluated. As illustrated in Fig. 4A, addition of EGF in FBS-free medium induced fourfold (n = 15, P = 0.006, Fig. 4A) increases in cell migration. The hypothesis that EGF release by injured cells stimulates cell migration was then explored. In this experiment, conditioned FBS-free MEM, collected from alveolar monolayers 1 h after injury, was applied in the lower compartment of the Boyden-type chamber. The conditioned medium increased the number of migrating cells by threefold (Fig. 4B, compare bars 1 and 2, n = 6, P = 0.026); the stimulatory effect was completely abolished by the presence of EGF-Ab in the lower compartment (Fig. 4B, bar 3, n = 6, P = 0.035). This observation favors an autocrine EGF loop modulating cell migration and wound healing through activation of EGF-R following EGF release after injury. The presence of conditioned medium from noninjured monolayers did not significantly augment cell migration, even if a tendency toward an increase was found (Fig. 4B, bars 4 and 5).
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The impact of EGF on the presence of active K+ channels at the basolateral membrane was then evaluated in an Ussing chamber after permeabilization of the apical membrane of alveolar monolayers (Fig. 7). In these experiments, an apical-to-basolateral K+ gradient was first created. The apical membrane was then permeabilized with amphotericin B. After stabilization of the K+ currents, clofilium (Fig. 7A) or glibenclamide (Fig. 7B) was applied at the basolateral membrane to specifically inhibit KvLQT1 or KATP currents. We observed that the clofilium-sensitive current (Iclofi; 4 ± 1 µA/cm2) was higher than the glibenclamide-sensitive current (2.2 ± 0.5 µA/cm2). The effect of acute (10 min) or chronic (24 h) exposure to EGF on KvLQT1 and KATP currents was then evaluated. EGF was applied at the basolateral side, since EGF-R have been found to be segregated at this membrane (52). Iclofi increased by 1.7-fold (n = 4, P = 0.022) after a 10-min application of EGF (Fig. 7A, right). Similarly, 2.2 ± 0.5 µA/cm2 of glibenclamide-sensitive current (Iglib) rose to 3.4 ± 0.3 µA/cm2 after 10 min in the presence of EGF (1.55-fold increment, n = 6, P = 0.0107; Fig. 7B, right). Longer EGF stimulation was also tested. In these experiments, the monolayers were pretreated with EGF for 24 h, and the cells were then washed just before Ussing experimentation to evaluate changes in the active number of K+ channels at the basolateral membrane in the absence of immediate EGF stimulation. We found that this pretreatment induced a large increase in Iclofi that reached 9.4 ± 0.6 µA/cm2 (n = 4, P = 0.007, Fig. 7A, right panel). Similarly, Iglib was elevated by 2.3-fold (n = 4, P = 0.008, Fig. 7B, right) after long-term exposure to EGF.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Role of EGF in Alveolar Repair Processes
We first explored the role of the EGF/EGF-R pathway in alveolar repair processes. EGF expression in primary cultured epithelial alveolar cells was first confirmed by PCR, as demonstrated previously by Raaberg et al. (39). In addition, we performed Western blotting experiments to evaluate the nature of EGF-R in these cells. We detected erbB1 and traces of erbB2 expression in alveolar ATII cell extracts. Studies on the identity of EGF-R subtypes along the respiratory tract have shown that their expression varies with cell type. The erbB1 is expressed in the majority of lung epithelial cells (2, 5, 37), including alveolar cells (2, 18, 39). However, erbB2 and erbB3 expression has also been localized in the airways (37, 52). On the other hand, the level of EGF-R could be regulated after injury. Higher EGF-R expression has been reported, for example, in asthma (5, 38).
Our results have shown that exogenous addition of EGF enhanced alveolar wound healing as well as alveolar cell migration and proliferation. The contribution of EGF signaling in alveolar and airway repair (38, 61) and/or remodeling (5) has already been observed. More precisely, EGF ligand is thought to activate EGF-R at the wound edge, thus stimulating the migration and proliferation of epithelial cells and favoring the restoration of epithelial integrity. Indeed, our results showing an increase of cell migration (Fig. 4) and proliferation (Fig. 6) as well as erbB1 phosphorylation (Fig. 2) after EGF treatment or early after wounding are consistent with those obtained in other studies (1, 13, 29). In addition, we noted (Fig. 1) that EGF titration (by EGF-Ab) or erbB1/erbB2 inhibition (by AG-1478 and AG-825) similarly reduced wound healing, indicating that EGF-R activation was mostly the result of EGF binding.
We found that the effect of exogenous EGF on cell migration was higher (4-fold) than that occurring on wound healing (15%). However, it must first be noted that the migration assays were performed on noninjured alveolar cells for 6 h in FBS-free MEM, whereas the wound-healing experiments were undertaken in the presence of FBS, which contains traces of EGF. These 24- to 48-h wound-healing experiments could not be conducted without FBS, since the integrity of the alveolar monolayers was altered in the absence of serum. Second, the injury of lung epithelial cells most probably induces EGF release, which could basically control its repair. Elevated EGF levels have been reported in cystic fibrosis patients, for example (49). In our rat alveolar cells, mature EGF protein released from injured monolayers could not be measured with commercially available ELISA kits, since the EGF antibodies used are directed toward human EGF, which does not cross-react with rat EGF. However, we observed that conditioned FBS-free medium collected 1 h after injury of alveolar monolayers stimulated cell migration, whereas conditioned medium from noninjured cells had no significant effect. Interestingly, the stimulatory effect of injured conditioned medium was completely abolished by EGF-Ab (Fig. 4). In addition, this EGF-Ab severely inhibited wound healing. Our results showing an increase of EGF-R phosphorylation levels in wounded alveolar monolayers (Fig. 2) also confirmed EGF release by injured cells. Similarly, EGF-R activation (phosphorylation), in the absence of exogenous EGF, has been seen at the wound edge of 16HBE14o– cells (38). All together, our results suggested injury-induced autocrine EGF-R activation, following EGF release, during cell repair. This autocrine hypothesis could nicely explain the low additional increase of wound repair in the presence of exogenous EGF compared with the high stimulatory effect of EGF or wounded conditioned medium on the migration of intact alveolar cells. Such an autocrine EGF pathway is consistent with the results reported by Geiser et al. (16).
EGF-R activation induces a cascade of cellular events (20), including the phosphorylation of COOH-terminal tyrosine residues, as mentioned above. This activation of EGF-R then allows the docking of numerous downstream signaling molecules inducing the Ras/Raf-mitogen-activated protein kinase (59) and phosphatidylinositol-3-phosphate/AKT (63) transduction pathways, both involved in migration and proliferation processes (59, 63), as well as phospholipase C/protein kinase C (9) that are implicated in EGF stimulation of cell motility. As demonstrated in the present study, K+ channel activity could also be a crucial step in EGF-mediated EGF-R signaling.
Role of K+ Channel Activity in Alveolar Repair Processes
Our results have shown that modulation of KATP and KvLQT1 channel activities regulated alveolar wound healing in basic conditions. In the absence of pretreatment, the KATP and KvLQT1 inhibitors (glibenclamide and clofilium) slightly reduced (by 25%) alveolar repair. The effect of K+ inhibitors was additive (50% inhibition with combined treatment; Fig. 3A). Pretreatment with glibenclamide or clofilium, 24 h before injury, reduced wound healing by >50% (Fig. 3B), inhibition similar to that observed following the combined action of these agents in the absence of pretreatment (Fig. 3A). This observation suggested that inhibition of one type of K+ channel (KATP or KvLQT1) could be compensated by activity of the other in a short period.
In the absence of pretreatment, KATP activation with pinacidil did not increase wound healing. Similarly to experiments with K+ channel inhibitors, it might be interesting to test the effect of combined activation of KATP and KvLQT1 channels. Unfortunately, no KvLQT1 activator channel was available commercially. In conditions where pinacidil was pretreated for 24 h before injury, a slight increment of wound repair was observed. It should be noted that the presence of EGF (from FBS and released by injured cells) already stimulated wound healing in basic conditions. A common stimulatory pathway of EGF and pinacidil could explain the absence or low level of stimulation in the presence of pinacidil. Accordingly, the small stimulatory effect of pinacidil (Fig. 3B, left) was not observed in the presence of EGF (Fig. 3B, right). Moreover, in conditions where the EGF/EGFR pathway was not basically activated, i.e., during cell migration experiments of intact cells in the absence of FBS, pinacidil was highly effective (2.2-fold increment of cell migration).
Because alveolar repair mainly depends on migration and proliferation processes, we evaluated the impact of K+ channel modulators on these processes. We observed that KATP modulation regulated alveolar migration but had no significant effect on cell proliferation in basic conditions. This result suggested that KATP-dependent alveolar wound healing was mostly controlled through the impact of KATP activity on cell migration. Conversely, KvLQT1 inhibition affected basic cell migration and proliferation. Regulation of alveolar cell repair by KvLQT1 activity could be because of a cumulative action on alveolar cell migration and proliferation. An impact of K+ channel activity on cell proliferation (6, 21, 24, 25, 32, 35, 36, 51, 54, 56, 57) and migration (11, 23, 30, 40, 43, 46, 47) has already been reported in several cell types, but we have demonstrated it for the first time in lung epithelial cells. Our results thus give a better understanding of the control of migration and proliferation processes, which are crucial in alveolar cell regeneration.
We also studied the effect of K+ channel activity on EGF-stimulated alveolar cell repair processes to evaluate the direct involvement of K+ channels in EGF-modulated wound-healing. We saw, for the first time, that K+ channel inhibition severely reduced EGF-stimulated alveolar wound healing as well as cell migration and proliferation in the presence of EGF. These results are consistent with K+-dependent growth factors stimulation demonstrated in several other cell types. Indeed, it has been shown that HGF-stimulated migration of MDCK cells (23) as well as EGF-induced proliferation of corneal epithelial cells (41), VSMC (25), and myeloblastic cells (54, 60) are dependent on K+ channel activation by these growth factors. Conversely, pinacidil slightly potentiated the EGF stimulation of alveolar cell migration and proliferation. However, the stimulation of cell migration in the presence of EGF (33%) was lower than the 2.2-fold increase measured in the absence of EGF. Again, this result supports the hypothesis that pinacidil and EGF stimulatory mechanisms are, for the most part, the result of a common pathway.
K+ Channel Activation by EGF
We observed that acute application of EGF increased KATP and KvLQT1 currents, measured in an Ussing chamber, on apical permeabilized alveolar monolayers by 1.5- and 1.8-fold, respectively. It has already been shown that growth factors enhance intermediate (23, 25) and large-conductance (21) KCa channels in VSMC and MDCK cells. In addition, the open probability of 4-aminopyridine-sensitive Kv channels from myeloblastic cells was increased with EGF through a cAMP (protein kinase A)-dependent pathway (54). In the lungs, EGF was found to augment acetylcholine-induced K+ currents of the tracheal acinar gland (22). In alveolar cells, our results demonstrated, for the first time, a stimulation of KATP and KvLQT1 currents in lung epithelial cells. These short-term effects of EGF could be because of the modulation of K+ channel gating through a second messenger (step 3 of the recapitulative scheme presented in Fig. 9). However, in the absence of patch-clamp experiments, we cannot prove that single KATP and KvLQT1 channel activities were increased with EGF. Rapid membrane insertion of new K+ channels could not be excluded.
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Coupling of K+ Channels, EGF, and Repair Processes
Based on the results obtained from in vitro experiments, we propose an integrated model (Fig. 9) where EGF, secreted by injured alveolar cells, stimulates the activity and expression of KATP and KvLQT1 channels through erbB1 and, to a lower extent, erbB2 activation. According to our results, EGF-stimulated alveolar cell migration, proliferation, and wound healing are dependent on K+ channel stimulation, since their inhibition prevented the major part of EGF stimulation. A change in K+ channel activity could affect migration and proliferation processes through several pathways, which remain to be determined. Subsequent change in cell volume, after the modification of K+ flux, could be a possible candidate. It has been proposed, for example, that cell shrinkage of the cell pole, after K+ efflux through IKCa channels, could facilitate migration of the rear part of MDCK cells (47). An increase in [Ca2+]i and a change in cell volume have also been proposed to be responsible for the K+ stimulation of cell proliferation (58). On the other hand, K+ channel activity could affect EGF signaling. Indeed, Xu et al. (60) have suggested that K+ channel activity is required for initiation of growth factor-mediated mitogenic signal transduction through mitogen-activated protein kinase pathways in myeloblastic cells.
In conclusion, our study constitutes the first demonstration of such an involvement of K+ channel activity in in vitro EGF-mediated alveolar epithelia repair.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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