Departments of 1Physiology & Biophysics, 2Pediatrics, and 3Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, and 4Arkansas Children's Hospital Research Institute, Little Rock, Arkansas; and 5Department of Pediatrics, Shands Children's Hospital, University of Florida, Gainesville, Florida
Submitted 15 January 2003 ; accepted in final form 1 September 2004
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ABSTRACT |
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wound healing; cell motility
Peptide growth factors provide signals that can enhance wound-healing responses. Epidermal growth factor (EGF), a prototypical growth factor, binds a plasma membrane receptor tyrosine kinase, the EGF receptor (9, 10, 46), and enhances cell proliferation and motility in both fibroblasts and epithelial cells. For example, EGF promotes healing of superficial tongue wounds in mice. The removal of submandibular salivary glands (the major source of salivary EGF) reduced the rate at which wounds healed, and the inclusion of EGF in drinking water restored the rate of wound healing to normal levels (35). Thus EGF appears to play a physiologically relevant role in wound healing in oral epithelium. Another EGF receptor ligand, TGF-, enhances the repair of scrape-wounded alveolar epithelial cells (21). Similarly, in cultured guinea pig airway epithelial cells, EGF promotes the repair of scrape-wounded monolayers by stimulating cell migration with no effect on cell proliferation in the wound margin (23).
EGF also accelerates the repair of scrape wounds in human bronchial epithelial cell monolayers (16HBE14o_) (39) and enhances migration in the human bronchial epithelial cell line BEAS-2B (36). However, in the latter example, the wound-healing response is marginal. This contrast in migratory behavior between two different bronchial epithelial cell lines is not unique. In keratinocyte cell lines, EGF induces the dispersion of colonies at low cell density and the closure of scrape wounds in monolayer cultures (30). These contrasting behaviors prompt a question: How is the enhanced motility harnessed to generate the vectorial migration of cells necessary to close a wound? Conversely, how do differences between cell lines or in culture conditions such as cell density contribute to distinct cell behaviors? Although it is generally recognized that epithelial motility is coordinated, it is not well established that fibroblast motility is also coordinated (16, 19). Indeed, uncoordinated fibroblast motility is considered an advantage in studies of cell motility, because the analysis is not complicated by cell-to-cell adhesion (49).
One possible explanation for differences in the behavior of different cell types is that the level of expression of EGF receptors regulates the rate of wound healing by controlling motility. Indeed, in cultured keratinocytes transfected with EGF receptors, a positive correlation between EGF receptor levels and ligand-induced cell motility was demonstrated (30). Furthermore, the delivery of expression vectors encoding human EGF receptors into porcine skin with the use of a gene gun delivery system enhanced wound healing (32). Similarly, in asthmatic bronchiolar epithelium, where epithelial damage is a feature of the disease, EGF receptor expression is elevated compared with normal epithelium (12). Other mechanisms exist whereby erb24 (members of the EGF receptor family) are segregated on basolateral surfaces and are inaccessible to a ligand restricted to the apical surface (47). In contrast to the highly organized migration of epithelial sheets, fibroblast migration in wound repair is less coordinated because there are fewer of the cell-to-cell junctions that define epithelial cells (49). Nevertheless, EGF still plays an important role in wound repair by fibroblasts. For example, in senescent fetal lung fibroblasts (3) or in fibroblasts derived from older donors (40), reductions in EGF receptor levels and activation are correlated with reductions in cell motility.
Because EGF receptors are efficiently downregulated within an hour of EGF treatment (24) and wound closure may take many hours to complete, we reasoned that EGF must persistently activate some pathways to promote the motility essential to efficient wound closure. To begin to define these persistent pathways, we performed a quantitative analysis of wound healing in vitro using scrape-wounded African Green monkey kidney cell monolayers expressing endogenous EGF receptors. We used EGF alone or in combination with serum and monitored wound closure. EGF treatment in the absence of calf serum led to an uncoordinated increase in the motility of individual cells with fibroblastic morphology but did not lead to efficient wound closure. By contrast, we found that EGF treatment in the presence of calf serum accelerated the rate of CV-1 cell monolayer wound healing by promoting a coordinated or syncytial migratory behavior more reminiscent of epithelial cells. The promotion of wound healing by EGF persisted even in the absence of exogenous EGF, provided that serum was present. This response was not simply due to the presence of low levels of EGF in serum and was dependent on transcription and translation. Thus EGF promotes the stable expression of a set of proteins mediating enhanced motility, and serum factors promote cell-to-cell adhesion resulting in the coordinated migration of cells necessary for efficient wound closure. Together, our results also define a fibroblast-to-epithelium differentiation pathway complementary to the epithelium-to-mesenchyme transition.
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MATERIALS AND METHODS |
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Video microscopy and image analysis.
Cells were cultured in Bioptechs (Butler, PA) dishes with 0.5-mm-thick coverglass bottoms fitted to Zeiss Axiovert microscopes (Carl Zeiss, Thornwood, NY). The dishes were maintained at 37°C with a Bioptechs TC controller under a 1 liter/min stream of 5% CO2 and 95% air. To prevent evaporation, the medium was overlaid with Serva silicone DC200 fluid (Crescent Chemical, Hauppauge, NY). Images were collected at 6-min intervals for 24 h using a chilled charge-coupled device camera (C5985; Hamamatsu Photonic Systems, Bridgewater, NJ) and a CG-7 frame grabber (Scion, Frederick, MD) installed in a personal computer. The monolayers were "wounded" by being scratched with a sterile, disposable 200-µl plastic pipette tip (Fisher, St. Louis, MO). A macro was written in Scion Image to control image acquisition and storage from two or three microscopes operated in parallel. The macro operated Uniblitz shutters (Vincent Associates, Rochester, NY) so that cultures were only illuminated for image acquisition. Once collected, the series of TIFF files were converted into AVI files using Adobe Premier (San Jose, CA), and plates were generated using Adobe Photoshop (San Jose, CA). Quantitative analysis of wound healing was also performed using a custom Scion Image macro. The denuded area in every file in a series was measured by applying an edge filter and by selecting a range of densities (density slice) corresponding to the denuding area. This processed image was converted into a binary image, and the number of pixels (black) corresponding to the denuded area were counted. The resulting pixel counts were exported to Microsoft Excel (Redmond, WA), converted to areas, and plotted as a function of time. This analysis yields an "offset" attributed to the presence of areas (including spaces between cells) with a density identical to that of the wound. The offset is about 10% of the starting wound area and is noted in the figures. To control for the quality of the automated analysis, the binary images were saved and examined to ensure that the denuded areas were actually being measured. Scion Image is available free of charge at http://www.scioncorp.com/.
To quantify wound closure, we measured the rate of migration of the monolayer. To ensure that the size of the wound, or the size of the microscope field, did not influence the measurements, all experiments were performed in parallel with one control culture and one experimental culture. All wounds were made with the same tool and were similar in width. To permit comparison between different sets of experiments, the instantaneous rate of closure (µm2/h) was calculated using differentials and was normalized to the length of the wound sampled. Depending on the objective used, the length was either the width of the camera frame (310 µm for a x20 objective) for wound closure from only one side of the field or twice the width of the camera frame (620 µm for a x10 objective) if the wounds closed from both the top and the bottom of the field. This calculated parameter corresponded to the rate of movement of the monolayer across the substrate. To reduce the variability in this parameter, it was calculated as a rolling average over nine intervals of 6 min each, and the mean and standard deviation were calculated. A maximal rate of monolayer migration was similarly calculated using linear regression where the rates were constant. Both calculations gave equivalent results over intervals, provided the rate of migration was constant. In EGF-pretreated cells displaying a robust wound-healing response, the linear migration rate was 20 µm/h. By contrast, in cells cultured in 10% serum, the linear migration rate peaked at
10 µm/h. Despite the utility of the time-lapse assay, there are limitations in the cost and availability of the instrumentation and, thus, in the number of assays that can be performed simultaneously. Therefore, static assays were also performed in which the wound areas were simply measured after intervals of 624 h.
Statistical comparisons were made using SigmaStat for Windows (version 3.0; SPSS, Rochester, MN). Significance was assessed using one-way analysis of variance followed by the Holm-Sidak method of evaluating all pairwise multiple comparisons, using P < 0.05 to define significant differences.
Western blot analysis.
CV-1 cells were grown to confluence and serum-starved for at least 24 h. Cells were treated with 10 nM EGF and harvested by scraping in boiling lysis buffer (1% SDS, 10 mM Tris, pH 7.4, and 1 mM sodium orthovanadate, supplemented with protease inhibitors). After the viscosity was reduced via several passages through a tuberculin syringe, lysates were clarified by centrifugation at 13,000 g and the protein concentrations were determined with the BCA assay (Pierce, Rockford, IL), using bovine serum albumin as the standard. The total mass of EGF receptors, Shc, E-cadherin, N-cadherin, -catenin,
-catenin,
-catenin, integrin
1, integrin
2, integrin
3, integrin
4, integrin
5, and fibronectin in the lysates was measured with the use of monoclonal antibodies from BD Transduction Laboratories (San Diego, CA) by performing Western blotting as previously described (20). An ERK1/2 antibody was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA), and the phosphospecific ERK1/2 antibody was obtained from Cell Signaling Technology (Beverly, MA).
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RESULTS |
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We considered that our results might be compromised by the presence of EGF in serum. Using an estimate for the serum concentration of EGF of 1 nM (45), we calculated that the final concentration of endogenous EGF in medium supplemented with 10% serum is
0.1 nM. Although this is low compared with the 10 nM EGF we used in our experiments, it is high enough to stimulate half-maximal thymidine incorporation in a fibroblast growth assay and is at the low end of the 110 nM range used for EGF as a growth supplement (www.roche-applied-science.com/pack-insert/1376454a.pdf). Nevertheless, we observed a fourfold enhancement in the rate of wound healing by adding 10 nM EGF to serum for a final concentration of
10.1 nM. We typically use 10 nM EGF in our experiments, but we have observed similar effects with concentrations as high as 100 nM EGF. To determine the potential effects of
0.1 nM serum-derived EGF, we tested IgG1 MAb-225, a neutralizing monoclonal antibody specific for the EGF receptor (44), and found that it attenuated both EGF- and serum-induced wound closure (Fig. 6A). When assays were performed in the presence of the EGF receptor tyrosine kinase inhibitor AG-494, the stimulation of wound closure by both serum alone and EGF+serum was blocked (Fig. 6B). A structurally related benzylidenecyanothioacetamide inhibitor specific for the NGF receptor tyrosine kinase (AG-879) was without effect when used at the same concentration. Thus serum-derived EGF does stimulate wound closure, and this is mediated via activation of the EGF receptor kinase. However, wound closure stimulated by serum-derived EGF is not maximal, because it is enhanced by the addition of exogenous EGF. Our experimental observations are most consistent with a role for EGF in stimulating cellular motility and with a role for another serum-derived factor in coordinating EGF-induced motility.
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Potential mechanisms for persistent effects of EGF + serum.
Video microscopy indicates that cell migration is coordinated by an enhancement of cell-to-cell contacts in serum-treated cultures. Given the morphological conversion of CV-1 cells from a fibroblastic to an epithelial behavior in EGF+serum-treated cultures compared with cultures treated with EGF alone, the abundance of a variety of proteins involved in cell adhesion and signaling was determined by Western blot analysis of whole cell extracts (Fig. 7A). Compared with cultures maintained in serum-free medium, no detectable differences in the total mass of E- or N-cadherin, -,
-, or
-catenin, or integrin
1,
3, or
5 were observed in cultures treated with EGF, serum, or EGF+serum. There was a modest increase in the abundance of integrin
5 and an apparent stabilization of fibronectin and
-catenin to proteolysis by EGF, but this does not explain the coordination of cell motility by serum.
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To more generally test the hypothesis that EGF promotes the expression of proteins facilitating wound healing, we performed experiments using the protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D to block gene expression. Cells were cultured in plastic dishes and scrape wounded, and the size of the wounds was measured after 24 h in the presence of varying concentrations of cycloheximide and actinomycin D. Transient treatment (6 h) and washout of the protein synthesis inhibitor cycloheximide (10 µg/ml) had little effect on the basal rate of closure of scrape wounds in the presence of calf serum (Fig. 8A). By contrast, sustained treatment with cycloheximide (24 h) blocked wound closure, demonstrating the efficacy of the inhibitor. Similarly, transient treatment and washout of the transcription inhibitor actinomycin D (5 µg/ml) also had little effect on the basal rate of closure of scrape wounds in the presence of calf serum, whereas sustained treatment with actinomycin D (24 h) blocked wound closure. These experiments demonstrate that these two compounds can be used to reversibly inhibit wound healing, presumably by blocking gene transcription (actinomycin D) and mRNA translation (cycloheximide). Time-lapse experiments were performed to determine whether gene transcription and mRNA translation are required for the persistent effects of EGF on wound healing. Cultured CV-1 cells were scrape wounded and cultured in calf serum-containing medium supplemented with 10 nM EGF in the absence or presence of actinomycin D or cycloheximide. Wound healing was monitored for 24 h and was blocked by both inhibitors, as expected (data not shown). The medium was then exchanged for calf serum without EGF or inhibitors, a new scrape wound was made, and its closure was monitored during the second 24 h of the experiment. Treatment with either inhibitor for 024 h blocked the EGF-enhanced increase in the rate of wound closure from 24 to 48 h without effect on the rate of basal wound closure. The rate of closure in cultures treated with EGF was 2.6-fold higher than in cultures treated with EGF and cycloheximide during the first 24 h and was 3.0-fold higher than in cultures treated with EGF and actinomycin D during the first 24 h (Fig. 8B and Video 4). Treatment intervals of from 6 to 24 h on day 1 were used with similar, albeit slightly lower, inhibitory effects on wound healing on day 2 (data not shown). Thus both gene transcription and de novo protein synthesis are required for the persistent stimulation of scrape wound closure by EGF in the presence of serum. Together, the results support the hypothesis that EGF induces the expression of a stable set of proteins responsible for enhanced cell motility.
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DISCUSSION |
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The observation that serum coordinates EGF-induced motility has important implications. Confluent monolayers of CV-1 cells displayed a robust wound closure response in the presence of serum, and removal of serum for at least 24 h before scrape wounding largely abolished this response. EGF enhanced this response greater than twofold when combined with serum. Under the most optimal conditions we have defined, this reduced the time it took to close a scrape wound from 24 h to
8 h. Although low levels of EGF present in serum appear to be responsible for basal motility that results in scrape wound closure within 24 h, EGF alone does not promote efficient wound closure. The use of serum in combination with EGF in the present study contrasts many studies with growth factors in which the experiments were performed in serum-free medium. We used serum-free medium in our initial studies and encountered variability in the assays that was traced to differences in the length of culture in serum-free medium before the initiation of experiments. Thus, although basal signaling activity can be suppressed by overnight serum starvation, suppression of functional responses important for wound closure requires serum starvation for 2448 h.
The present experiments are most relevant to the problem of reepithelialization of partial thickness wounds. In many ways, the behavior of CV-1 cells is similar to that of the basal keratinocyte in skin epithelia and MDCK cells. In the absence of serum, EGF induces CV-1 cell scattering, whereas in its presence, EGF promotes coordinated wound closure. In keratinocytes (30), EGF induces scattering in isolated colonies and closure of scrape wounds in confluent monolayers. In MDCK cells, hepatocyte growth factor (HGF) promotes scattering of isolated colonies and coordinated migration of wounded, polarized monolayers cultured on permeable substrates (1). The present findings prompt two questions for further study: 1) What is the factor (or factors) in serum that promotes a coordinated motile response to EGF?, and 2) what molecules are activated or induced by serum that mediate the coordinated motile response to EGF?
Serum factors that regulate wound healing include insulin-like growth factors, PDGF, and TGF-, among many others (51). In addition, LPA has been shown to enhance migration in an intestinal epithelial restitution model (43) and in scrape-wounded endothelial cell monolayers (25). Furthemore, EGF+LPA synergistically stimulate mitogenesis in airway smooth muscle cells (5) and LPA+PDGF-BB synergistically enhance human gingival fibroblast migration in scrape wound assays (4). Although LPA did stimulate wound closure in combination with EGF+serum, it failed to substitute for serum when combined with EGF alone. TGF-
is a logical candidate to test in future experiments, given reported synergistic enhancements of EGF-stimulated hepatocyte motility (42). However, TGF-
is also reported to promote an epithelium-to-mesenchyme transition in renal proximal tubular epithelial cells (37), which is essentially the opposite of the coordination of motility we observe by combining serum and EGF.
The abundance of a variety of molecules involved in cell adhesion and signaling was examined by immunoblotting in an attempt to identify serum-induced proteins. Among the candidate proteins tested, only -catenin, integrin
5, and fibronectin varied with treatment. However, these responses were small and were related to EGF, and not to serum treatment, and thus do not help to explain how serum coordinates cellular motility. Disassembly of cell-cell adhesive complexes may be important for cell migration into the denuded area of the culture, but this is expected to be a local property of the wound margin and not one of the bulk culture. Reductions in tight and adherens junction complexes are typical of an epithelial-to-mesenchymal cell transition associated with tumor invasion and metastasis (48) and have been linked to tyrosine phosphorylation of E-cadherin and
-catenin (2), so changes in phosphorylation status may be more relevant than the changes in mass measured in the present experiments. In any event, the morphological responses strongly indicate that one or more cell adhesion molecules are necessary for the epithelial behavior adopted by these cells.
In serum-free medium, EGF stimulated CV-1 cell motility but had only a modest effect on wound closure. This uncoupling of EGF activity and wound closure occurred because the EGF-stimulated CV-1 cell motility was not well coordinated. Although cells did move into the wound area, the cells adopted the typical morphology of migrating fibroblasts and did not cover the substrate effectively. Cells moved freely around and past one another, indicating that there was very little adhesion of cells to one another. However, by contrast to IMR-90 fibroblasts, CV-1 cells exhibit contact inhibition and do not grow on top of one another at high cell densities the way fibroblasts do. Human bronchiolar epithelial cells (16HBE cells) adhered to one another, and the motility of individual cells was constrained. Treatment of CV-1 cells with EGF+serum modified their behavior such that the monolayers were more reminiscent of epithelial cell sheets, although the cell-to-cell adhesions were not as extensive as they were for 16HBE cells.
Upon binding the EGF receptor, EGF activates a variety of processes leading to enhanced growth and motility. The pathways that mediate these responses diverge and involve both short-term (enzymatic) and long-term (transcriptional) mechanisms (6, 7, 50). Enzymes whose activation is necessary for EGF-induced cell motility in fibroblasts include phospholipase C for mediating cytoskeletal reorganization (7), MEK for reducing adhesion to substrates (53), and calpain, necessary for detachment of the rear of the cells from the substrate (17, 38). It is postulated that these pathways should be negatively regulated to limit or terminate the wound-healing response and thereby minimize scarring. Indeed, the chemokine IP-10 has been shown to suppress the activation of calpain by EGF and thereby inhibit fibroblast motility (41). Negative regulation of these pathways is likely mediated by the same mechanisms that lead to contact inhibition of growth and to growth arrest at high cell density.
The present observation that wound closure rates accelerate 68 h after the initiation of EGF treatment is consistent with earlier observations that maximal fibroblast motility occurs 68 h after EGF stimulation (27). We reasoned that this period of time was sufficient for the induction of new protein expression. Indeed, transient treatment with either an inhibitor of transcription or translation blocked the ability of EGF to acutely enhance wound closure. Inhibitor concentrations were selected such that serum-stimulated wound closure was not blocked by transient treatment. Both inhibitors also blocked the sustained stimulation of wound healing by EGF. On the basis of these results, we conclude that EGF treatment promotes the expression of one or more new proteins that mediate enhanced motility. These proteins appear stable because their effects persist after the downregulation of receptors and removal of EGF. Thus a simple activation of enzymes by EGF receptors is not adequate for an enhanced wound closure response by CV-1 cells. However, enhancement of cell motility alone is not adequate to promote efficient wound healing.
To make the best use of the ability of EGF to promote wound healing, it is necessary to understand 1) the mechanisms by which EGF promotes motility of the individual cells, 2) how enhanced motility is channeled to promote the appropriate behavior of a sheet of cells (vectorial movement of cells essential for rapid wound closure), and 3) how the motility process is terminated once the wound is closed. The present study is most relevant to the second question and defines a clear and robust synergy between EGF and serum factors in enhancing the rate of wound healing in vitro. This interaction between EGF and an unidentified serum factor(s) is functionally relevant to reepithelialization of wounds. Identification of the factor and the molecular basis for its activity could prove important for the combinatorial optimization of therapeutic approaches for the treatment of impaired wound healing. Our findings also indicate that chronic administration of EGF (and possibly other growth factors) is not necessary in wound- healing therapies and may actually be counterproductive because of receptor downregulation.
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GRANTS |
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FOOTNOTES |
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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.
1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00024.2003/DC1.
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