Activation of Epidermal Growth Factor Receptor during Corneal Epithelial Migration

Abstract

purpose. Epidermal growth factor (EGF) and related growth factors: transforming growth factor (TGF)-α, heparin-binding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to a common EGF receptor (EGFR), tyrosine kinase. We have used in vivo and organ culture wound-healing models to examine the kinetics and extent of EGFR activation during corneal epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process.

methods. In the in vivo model, 3-mm débridement wounds were made in rat corneas and allowed to heal in situ. Activation of EGFR was analyzed by 1) indirect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF–fluorescein isothiocyanate (FITC). Relative levels of mRNA for EGF, TGF-α, HB-EGF, and AR were determined using reverse transcription–polymerase chain reaction. To determine whether inhibiting EGFR activation slows epithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0–50 μM), a specific inhibitor of EGFR kinase activity.

results. In unwounded corneas, EGFR was localized in basal cells and appeared to be membranous. Within 1 hour after wounding, EGFR was no longer immunolocalized in the membranes of cells migrating into the wound area. EGF-FITC–binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyrosine residues within 30 minutes after wounding and that phosphorylation levels increased after wounding. Levels of mRNA for TGF-α, HB-EGF, and AR all appeared to increase after wounding. In organ culture experiments, tyrphostin AG1478 inhibited migration rates in a dose-dependent manner.

conclusions. These data indicate that EGFR was activated during corneal epithelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signaling cascade significantly slowed migration rates.

Corneal epithelial wound repair, similar to that of other stratified squamous epithelia, occurs in three overlapping phases. ¹ In the first phase, the wound area is re-epithelialized as cells adjacent to the wound flatten and migrate. This migration phase is sufficient to resurface small wounds and does not require cell proliferation. In the second phase of wound repair, cell proliferation occurs to allow repopulation of the wound site. This proliferation generally is observed in a location distal to the edge of migrating epithelium. ^{2 3 4} In the final phase of wound repair, basement membrane components and underlying extracellular matrix are synthesized, assembled, and remodeled. A variety of growth factors, including epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor (TGF)-β, TGF-α, and heparin-binding (HB)-EGF have been shown to stimulate one or more of the phases of wound repair (see References 5–8 for review). 

Among the numerous growth factors that have been associated with wound repair, EGF and its related family members have been implicated in all the repair phases. They have been shown to stimulate cell migration, proliferation, and synthesis of basement membrane and extracellular matrix components and to accelerate healing rates in corneal and epidermal wounds. ^{5 6 7 8} EGF is a small polypeptide (6-kDa molecular mass) originally isolated from the submandibular glands of male mice. ⁹ Since the discovery of EGF, a family of structurally related growth factors has been identified that includes TGF-α, HB-EGF, amphiregulin (AR), and betacellulin. ^{10 11 12} All the members of the EGF family exert their effects by binding to a 170-kDa transmembrane tyrosine kinase receptor termed the EGF receptor (EGFR). ¹¹ After binding to one of the specific ligands, EGFR dimerizes and undergoes autophosphorylation of specific tyrosine residues. ^{13 14 15} The presence of these phosphotyrosine (PT) residues is considered the most definitive evidence that EGFR has been activated. ¹⁵ The phosphorylated tyrosine residues become binding sites for a group of cytoplasmic signaling proteins including phospholipase C, phosphatidylinositol 3-kinase, and the guanosine triphosphatase (GTPase)–activating protein Ras. These signaling proteins can then activate the cell to undergo migration, proliferation, and/or differentiation. ^{13 14 15}  

EGF has been known for many years to stimulate the proliferation of corneal epithelial cells in culture ¹⁶ and to stimulate the rate of epithelial migration. ¹⁷ More recently, TGF-α and HB-EGF have been shown to stimulate proliferation of corneal epithelial cells in culture. ¹⁸ However, there is still some question of whether the addition of exogenous growth factors demonstrates a beneficial effect on repair of the cornea. Several clinical studies on a variety of wound types have indicated that EGF has a beneficial effect, ^{19 20 21} but others have shown only little or no beneficial effect. ^{22 23 24 25} In the current investigation, we tested the hypothesis that EGFR is activated by endogenous growth factors during epithelial wound repair. 

Materials and Methods

Animal Model

Adult Sprague–Dawley rats of either sex were used in all experiments. Rats were anesthetized with an intramuscular injection of rodent anesthesia cocktail containing ketamine (43 mg/ml), xylazine (8.6 mg/ml), and acepromazine (1.4 mg/ml) followed by topical application of 0.5% proparacaine. An epithelial wound, 3 mm in diameter, was made by demarcating an area on the central cornea with a 3-mm trephine and removing the epithelium within the circle with a small scalpel, leaving an intact basement membrane. ²⁶ The corneas were allowed to heal from 5 minutes to 48 hours. Rats were killed with an intraperitoneal injection of pentobarbital sodium. All protocols in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Immunofluorescence Microscopy

Immunofluorescence, using cryostat sections, was performed as previously published. ²⁷ Polyclonal antibody against EGFR (Upstate Biotechnology, Lake Placid, NY) was placed on the sections and incubated for 1 hour at room temperature, followed by a 1-hour incubation of secondary antibody, FITC-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch, West Grove, PA). Coverslips were mounted with a medium consisting of phosphate-buffered saline (PBS), glycerol, and paraphenylene diamine. Negative control tissue sections (primary antibody omitted) were routinely run with every antibody-binding experiment. Control experiments were also performed with unrelated polyclonal antibodies to ensure specificity. The sections were viewed and photographed under a fluorescence microscope (Axiophot; Carl Zeiss, Thornwood, NY) equipped for epi-illumination. At least four eyes were examined for each time point. 

Detection of EGF Binding Sites

Three-millimeter wounds were made as described and allowed to heal 1 or 18 hours in vivo. At 5, 15, 30, and 60 minutes before the rats were killed, 40 μl EGF-FITC (4 μg/ml; Molecular Probes, Eugene, OR) was applied. Rats were reanesthetized 17 hours after wounding in the 18-hour experiment. Unwounded corneas were also examined. The rats were killed, and the corneas were fixed in situ for 10 minutes with 4% paraformaldehyde. The eyes were enucleated and then prepared as either wholemounts or sections. For wholemounts, the eyes were fixed for 45 minutes and washed three times, each time for 15 minutes in PBS, and the posterior portion of the eye, including the lens and iris, were removed. The corneas were cut into quarters and placed on gelatin-coated slides. These corneas were then viewed with a confocal microscope (TCS 4D; Leica, Heidelberg, Germany), and X–Y scans were performed through the full thickness of the epithelium. Depending on the area of interest, the upper half, lower half, or all the X–Y scans were merged together to view the superficial cells, basal and suprabasal cells, or the entire epithelium, respectively. For sections, eyes were fixed for 1 hour, and then corneas were dissected and fixed for an additional 3 hours. Corneas were then cryopreserved overnight in 30% sucrose and prepared as cryosections. Sections were also viewed using the confocal microscope. As a control, 40 μg/ml unconjugated EGF (R&D Systems, Minneapolis, MN) was mixed with the EGF-FITC before exposure to the eye. At least four eyes were examined per time point. 

Immunoprecipitation and Immunoblot Analysis

Three-millimeter débridement wounds were made as described, and either 15 μl human recombinant EGF (10 μg/ml; R&D Systems) or PBS was applied to the cornea and allowed to heal for 30 minutes. In addition, corneas without any application of EGF or PBS were allowed to heal for 0.5, 1, 2, 4, and 24 hours. Corneal epithelium from limbus-to-limbus was harvested by scraping with a small scalpel. Epithelium from unwounded corneas was used as a control. Ten eyes were used for each time point. Samples were homogenized in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mg/l aprotinin, 0.5 mg/l leupeptin, 0.7 mg/l pepstatin A, and 2 mM sodium orthovanadate and incubated for 30 minutes at 4°C. Tissue extracts were then centrifuged at 14,000g for 30 minutes, and protein amounts were determined using a commercial protein assay (Bio-Rad, Hercules, CA). The protein concentration of the supernatants was adjusted using RIPA buffer so that 500 μl of the supernatants contained 100 μg protein. The supernatants were then immunoprecipitated with anti-PT, or anti-EGFR. For anti-PT, the supernatants were incubated with 20 μl anti-PT IgG-conjugated agarose beads (500 μg IgG/0.25 ml agarose; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at 4°C. The beads were pelleted by centrifugation, washed, and analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis ²⁸ using rabbit anti-EGFR (1005; Santa Cruz Biotechnology) at a 1:30 dilution and horseradish peroxidase–conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories [KPL], Gaithersburg, MD) at a 1:4000 dilution. For anti-EGFR immunoprecipitation, the supernatants were incubated with 25μ l of anti-EGFR (1005) overnight, and protein A agarose beads (50μ l/500 μl of supernatant) were then added to each sample and incubated for 2 hours at 4°C. The beads were pelleted by centrifugation, washed, and analyzed by SDS-PAGE and Western blot analysis using either a 1:30 dilution of rabbit anti-EGFR (1005) or a 1:50 dilution of mouse anti-PT and either horseradish peroxidase–labeled goat anti-rabbit IgG at 1:4000 dilution or goat anti-mouse IgG (KPL) at a 1:1000 dilution. Antibody binding was detected by chemiluminescence with a substrate kit (Lumi GLO; KPL). Immunoprecipitation experiments were repeated four times. Relative levels of reactive proteins were determined using scanning laser densitometry (model 300 A; Molecular Dynamics, Sunnyvale, CA). The means of phosphorylated EGFR/total EGFR were analyzed statistically using a paired t-test. P < 0.05 was considered significant. 

Isolation of Total RNA

At the time points of 0.5, 1, 2, 4, 8, 16, and 24 hours after wounding, rats were killed, and whole corneal epithelium from limbus to limbus was removed with a small scalpel and immediately frozen in liquid nitrogen. Corneal epithelium from unwounded rats was used as a control. Epithelium from five corneas was used for each time point. Total RNA was isolated from samples by the acid guanidinium thiocyanate-phenol chloroform extraction method ²⁹ using an RNA isolation kit (Stratagene, La Jolla, CA). Before further use, the total RNA was treated with DNase I (amplification grade; 1 U DNase/1μ g total RNA; Life Technologies, Grand Island, NY). 

RT-PCR

Reverse transcription–polymerase chain reaction (RT-PCR) was performed as previously described ³⁰ using specific primers for EGF, HB-EGF, TGF-α, AR, and glyceraldehyde 3-phosphate dehydrogenase (G3PDH; Table 1 ). The primer sets and exon location for HB-EGF, TGF-α, EGF, and AR were derived from previously published sequences. ^{31 32 33 34 35 36 37} Primer sets were devised using primer analysis software (Oligo; National Biosciences, Plymouth, MN) that selects primers based on minimal hairpin formation, minimal duplex formation, and guanine cytosine composition. Primer sets for G3PDH were purchased from Clontech (Palo Alto, CA). Samples were denatured for 1.5 minutes at 94°C, followed by 25 PCR cycles of denaturation for 1.5 minutes at 94°C, annealing 1 minute at 50°C or 52°C (Table 1) , and extension for 1 minute at 72°C. The final elongation step was performed at 72°C for 7 minutes. Twenty microliters of the PCR fragment was then resolved on a 1.2% agarose gel containing 0.5 μg/ml ethidium bromide. Quality of cDNA was confirmed using primers for G3PDH. Samples with no cDNA were also amplified and served as negative controls. RT-PCR experiments were repeated three times. 

The specificity of the PCR products was checked by digestion of the products with restriction enzymes that would recognize known restriction sites in the cDNA sequence. In all cases, the restriction enzyme digestion produced fragments of the expected sizes, verifying that the PCR products corresponded to the appropriate cDNA. 

Organ Culture and Tyrphostin AG1478

Rats were killed with an overdose of pentobarbital sodium, and 3-mm débridement wounds were made as described. The anterior portion of the eyes were excised and filled with 40 μl 1% agar–1% collagen type I, as described by Foreman et al. ³⁸ The corneas were allowed to heal in organ culture in a serum-free, completely defined medium ²⁶ for 18 hours at 35°C in 5% CO₂. Six eyes were used for each concentration of tyrphostin AG1478 (0–50 μM; ICN, Costa Mesa, CA), a specific inhibitor of EGFR kinase activity. ^{39 40} After incubation, the corneas were treated with Richardson’s stain ⁴¹ to mark the remaining wound area. The corneas were photographed, and the wound area was quantified using image analysis (NIH Image, Version 1.61, National Institutes of Health, Bethesda, MD). As a toxicity control, an additional set of samples were incubated in 30 μM tyrphostin AG1478 for 18 hours, washed, and incubated for an additional 24 hours in medium without tyrphostin AG1478. The means of the remaining wound areas of the tyrphostin AG1478–treated and untreated corneas were analyzed statistically using a paired t-test. P < 0.01 was considered significant. 

Results

In an initial study to ascertain whether EGFR was involved in epithelial migration, immunofluorescence microscopy was used to determine whether the localization of EGFR was altered during epithelial migration. The characteristic immunofluorescence-staining pattern of anti-EGFR in unwounded corneal epithelium is seen in Figure 1A . EGFR was concentrated in the basal cell layer, with weak labeling extending into the suprabasal cell layers. The distribution of EGFR appeared to be primarily membranous, with highest levels along the apical–lateral borders. 

One hour after wounding, the cells making up the leading edge of migrating epithelium exhibited a greatly diminished membranous localization of EGFR (Fig. 1B) compared with unwounded corneal epithelium (Fig. 1A) . At 3 hours, the localization of EGFR was cytoplasmic at or near the leading edge (Fig. 1C) . In contrast, cells distal to the wound area still exhibited membranous labeling (Fig. 1D) . Binding of anti-EGFR to its normal membranous pattern was not restored in the wound area until after epithelial wound closure (Fig. 1E) . Wound closure occurs after approximately 24 hours in this model. 

To further characterize the potential role of EGFR, EGF-FITC was used to detect EGF binding to its receptor during corneal epithelial wound healing. EGF-FITC appeared to bind the surface of basal and suprabasal cells at the wound edge, with binding confined to the basal cells away from the wound edge (Fig. 2A ). Binding reached approximately one third of the way to the limbus after a 15-minute incubation with EGF-FITC and all the way to the limbus by 1 hour (Fig. 2A 2C-F ). Binding of EGF-FITC to the basal cells was not detected in tissues incubated with an excess of unconjugated EGF (Fig. 2B) . In addition, no binding of EGF-FITC was detected when the ligand was added to unwounded corneas (Fig. 2G) . Similar results were observed when corneas were allowed to heal in organ culture (data not shown). 

As seen in Figure 2 , EGF-FITC was localized in both superficial and basal cells. When EGF-FITC localization was examined using wholemount preparations, similar results were observed (Fig. 3A 3B 3C ). The localization pattern varied between the cell layers, with the superficial cells exhibiting cytoplasmic and nuclear localization (Fig. 3A) , whereas the binding in the basal cell layer appeared to be membranous (Fig. 3B) . During these experiments, we observed that EGF-FITC appeared to be aggregated. Therefore, we conducted a time course experiment. In these tissues, uniform membranous-appearing binding was seen 5 minutes after addition of EGF-FITC (Fig. 3D 3E) . At 15 minutes, aggregation of the receptors was apparent (Fig. 3F) , and by 30 minutes, EGF-FITC appeared to be internalized (Fig. 3G) . 

To determine whether EGFR was activated, as indicated by the presence of PT residues, epithelium removed from unwounded or wounded corneas was solubilized, immunoprecipitated with anti-PT, blotted and reacted with anti-EGFR, as described in the Materials and Methods section. As seen in Figure 4 , unwounded corneal epithelium contained very low levels of phosphorylated EGFR (at 170 kDa). In contrast, corneal epithelium harvested 30 minutes after wounding and treated with PBS only showed a distinct band (fourfold enhancement compared with control; Fig. 4 ). This band comigrated with the band activated by the addition of exogenous EGF (Fig. 4 ; 10-fold enhancement compared with control). These data indicate that EGFR is activated by endogenous growth factors. Activation of EGFR was further examined in a time course study (Fig. 5) . In this experiment, phosphorylated EGFR peaked 30 minutes to 2 hours after wounding (eight- to ninefold increase) and remained higher than control (1.7-fold) 24 hours after wounding. 

Because one potential source of growth factors that may regulate epithelial wound repair is the epithelium itself, we investigated whether the epithelium was expressing EGFR ligands that might be involved in an autoregulatory activation of the receptor. RT-PCR was used to examine alterations in levels of four members of the EGF family (Fig. 6) . mRNA for EGF itself was present at low levels and did not appear to increase after wounding (Fig. 6B) . However, mRNA levels for three other EGFR ligands appeared to be upregulated after wounding (Fig. 6A 6B) . 

Finally, tyrphostin AG1478 (a specific inhibitor of EGFR kinase activity) ^{39 40} was used to determine whether EGFR activation was required for epithelial migration. Corneas were wounded, excised, and allowed to heal in organ culture in varying concentrations of tyrphostin AG1478. When the remaining wound areas were quantified, tyrphostin AG1478 was seen to inhibit migration in a concentration-dependent manner (Fig. 7) . The maximum inhibition of migration (46%) was observed at 30 μM tyrphostin AG1478. The effect of the tyrphostin AG1478 was reversible. Corneas treated with the inhibitor for 18 hours and then washed with minimum essential medium healed completely. 

Discussion

Epithelial wound repair generally proceeds at a rapid rate to allow the reformation of a protective barrier to prevent entry of a variety of pathogens. It has been known since the early 1970s that the addition of exogenous EGF stimulates the rate of this healing response in a variety of epithelia. ^{16 17 42 43 44 45 46} Subsequently, the alterations in the localization and amounts of EGFR in these epithelia during wound repair has provided circumstantial evidence that EGFR is involved in the healing response. ^{47 48 49 50 51 52} However, several clinical studies have shown that EGF has little or no beneficial effects on corneal wound repair. ^{22 23 24 25} In addition, Wilson et al. ⁵³ found that mRNA for EGF and EGFR was not significantly altered during epithelial wound repair in mice. Thus, the physiological role of EGFR in corneal wound healing in vivo remains unclear. In the current investigation, we used an in vivo wound-healing model of the corneal epithelium to examine the dynamics and extent of EGFR activation to determine what physiological role EGFR plays in epithelial wound repair. 

Because activation of EGFR in vitro has been associated with both migration and proliferation, the question can be raised whether its physiological role in vivo involves the early re-epithelialization stage and/or the later proliferative stage. Initially, corneal epithelial cells adjacent to the wound flatten, elongate, and migrate to cover the wound area. At approximately the time of wound closure (24 hours for a 3-mm wound), cells distal to the original wound site undergo proliferation. ^{2 3 4} Our data suggest that the activation of EGFR could be involved in stimulating both proliferation and migration during wound repair. Perhaps the major finding of our study is that the level of phosphorylation of EGFR on tyrosine residues is increased within 15 to 30 minutes after wounding. These data suggest that the receptor is rapidly activated at an early stage of wound repair. This time of activation is consistent with the initiation of the re-epithelialization phase. Immunolocalization and ligand-binding experiments also indicate that EGFR activation may be involved in the stimulation of migration. Within 1 hour after wounding, EGFR loses its membranous localization and appears to be internalized. This is in agreement with previous findings in skin wounds ⁵¹ and is consistent with the mechanism in which EGFR is bound by one of its ligands, activated, and then internalized. Alternatively, it is possible that the epithelial wing cells, which express lower levels of EGFR than the basal cells, have migrated to form the leading edge. 

We further investigated the fate of EGFR and its ligands during corneal wound repair by using EGF conjugated to a fluorescein tag. These experiments clearly documented that EGF binds to the basal cells specifically, and that within minutes after binding EGFR, the complex appears to be aggregated and internalized. That EGFR activation is involved in migration is also supported by the finding that tyrphostin AG1478 inhibits the migration rate by almost 50%. Because inhibition of proliferation of corneal epithelium has been shown to have only minimal effects on the rate of corneal epithelial wound closure in débridement wounds, ^{54 55 56} it appears that activation of EGFR stimulates a pathway that plays a major role in the migration of cells in the re-epithelialization phase of wound repair. 

Our data are also consistent with the activation of EGFR’s playing a role in stimulating cell proliferation in the limbus and cells distal to the original wound. This is supported by the finding that EGFR phosphorylation levels remain elevated as long as 24 hours after wounding. One potentially confounding effect of the measurements performed at 24 hours is that at this time point some of the wounds are closed, whereas others are not. Thus, some of the observed changes may be associated with wound closure rather than migration. Further support is provided by the EGF-FITC experiments, which indicate that growth factors present in the tear film or released by disrupted cells can penetrate to the limbus. Thus, the growth factors could stimulate migration of cells at the leading edge and proliferation of cells distal to the wound area. It is not clear whether the EGF-FITC moved along the basement membrane zone or through the epithelium after disruption of tight junctions. Our data suggest both are possible, because it appears that the superficial cells became leaky after epithelial débridement (Figs. 2 3) . Our data regarding activation of EGFR are in agreement with the findings of Relan et al., ⁵² who observed that in gastric mucosa, EGFR was activated as early as 30 minutes ⁵² after injury. They also observed that EGFR was still activated 6 hours after wounding. These authors postulated that activation of EGFR was involved in both migration and proliferation of the gastric mucosa after wounding. ⁵⁷  

One of the major surprises of our study was the finding that the level of activated EGFR remained elevated for several hours after wounding. This is in contrast to cells in culture where addition of growth factors generally leads to a spike of receptor phosphorylation lasting seconds to minutes. ^{10 11 58} This led us to investigate whether the epithelium itself was producing growth factors capable of activating EGFR. Using RT-PCR, we determined that rat corneal epithelium is synthesizing mRNA for at least four EGFR ligands including EGF, TGF-α, HB-EGF, and AR. This is in agreement with previous reports that mouse and human corneal epithelia express EGF and TGF-α mRNA and protein. ^{59 60} Of note, mRNA levels for three of these growth factors, TGF-α, HB-EGF, and AR, appeared to increase after wounding. Although the RT-PCR technique used is only semiquantitative, our results that EGF mRNA levels did not change during the healing process are in agreement with those of Wilson et al., ⁵³ who used in situ hybridization to examine wound healing in mice corneas. These data support the concept ^{5 7 61 62} that the epithelium induces an autocrine loop of EGFR and its ligands during wound repair. This autocrine loop may provide a mechanism to propagate the original signal to initiate repair. Auto- and cross-induction between members of the EGF family have been observed in epidermal ⁶² and intestinal ⁶¹ epithelia. These data also suggest that an extremely redundant system is used during healing. This may be relevant to the findings that TGF-α knockout mice do not exhibit an impaired healing response, ^{63 64} in that other members of the EGF family can compensate for the absence of TGF-α. It is also interesting that two heparin-binding growth factors, HB-EGF and AR, were upregulated during the healing response. It could be speculated that these growth factors adhere to the denuded basement membrane and provide a signal to the actively migrating cells covering the wound area. This would allow a differential signal to be transmitted to these cells in contrast with the epithelium at a site distal to the original wound area. In a differential mechanism, TGF-α may be expected to filter through to the distal cells, potentially stimulating cell proliferation. 

In summary, we have used an in vivo corneal epithelial wound healing model to demonstrate a physiological role for the activation of EGFR in the healing of corneal epithelium. Our results demonstrate that EGFR is rapidly activated by endogenous growth factors and that EGFR is active throughout both the re-epithelialization and repopulation phases of repair. Potential sources of endogenous growth factors include the tear film, the keratocytes, and the epithelium itself. Our data suggest that the epithelium may produce several EGFR ligands that may enhance the healing process. Furthermore, wound-healing data using the organ culture model (Fig. 7) suggest that continuous exposure to growth factors in the tear film is not required for epithelial wound closure. Finally, we have demonstrated that inhibition of EGFR slowed healing by almost 50%, indicating that activation of EGFR was a major component of a signaling cascade for epithelial migration. These data also suggest, however, that other pathways are involved in the healing process. Wilson et al. ⁵³ have demonstrated that both keratinocyte growth factor receptor and hepatocyte growth factor receptor are upregulated during corneal repair in mice. Thus, it appears that corneal wound repair may involve interaction of several growth factor–stimulated pathways. 

 Supported by Grant R01 EY-O5665 from the National Eye Institute (JDZ).

 Submitted for publication May 14, 1999; revised November 17, 1999; accepted December 20, 1999.

 Commercial relationships policy: N.

 

Table 1.

 

Oligonucleotide Primer Sequences

Primer	Oligonucleotide Sequence	Annealing Temperature	Fragment Size	Exon Number
Amphiregulin	5′-AGCCCAAGAAAAACAAGACAG-3′	50°C	227 bp	3
	5′-TTTTTCTCCACACCGTTCACC-3′			4
EGF	5′-GACAACTCCCCTAAGGCTTA-3′	50°C	566 bp	16
	5′-CATGCACACGCCACCATTGA-3′			20
G3PDH	5′-ACCACAGTCCATGCCATCAC-3′	52°C	452 bp	8
	5′-TCCACCACCCTGTTGCTGTA-3′			9
HB-EGF	5′-GGAAAGTGAGGAGAAAGTGAA-3′	50°C	444 bp	5
	5′-TCTCTTCCAGTCCATAAACCA-3′			6
TGF-α	5′-TGGCTGTCCTCATTATCACCT-3′	50°C	532 bp	4
	5′-GTTTCCCCTTGTCCATTTTGT-3			6

Figure 1.

 

Immunolocalization of EGFR in (A) unwounded central corneal epithelium, (B) migratory epithelium at the leading edge 1 hour after wounding, (C) migratory epithelium at the leading edge 3 hours after wounding, (D) epithelium peripheral to the wound area 3 hours after wounding, and (E) central corneal epithelium 48 hours after wounding. Bars, 50 μm. (A, B, and E) and (C, and D) are at the same magnifications.

Figure 2.

 

Localization of EGF-FITC in corneas 1 hour after wounding (A through F) and in unwounded corneas (G). (A) Cornea after 15-minute incubation with EGF-FITC. (B) Cornea after 15-minute incubation with EGF-FITC plus a 10-fold excess of unconjugated EGF. Note that binding to basal cells was blocked by cold EGF. Binding to superficial cells appeared to be nonspecific. (C through F) Montage of cornea after 1-hour incubation with EGF-FITC. Binding extended from leading edge (F), to near leading edge (E), to periphery (D), to limbus (C). (G) Unwounded cornea after 1-hour incubation with EGF-FITC. EGF-FITC did not appear to penetrate intact epithelium. Bars, 50 μm. (A, B, and G) and (C through F) are at the same magnification.

Figure 3.

 

Localization of EGF-FITC to corneas 1 (A, B, and C) and 18 (D through F) hours after wounding in flatmount preparations. Corneas were exposed to EGF-FITC for 5 (D, E), 15 (A, B, C, F), or 30 (G) minutes. An X–Z scan is shown in (C). Time course experiment was performed at 18 hours, because little superficial binding was observed at this time point. Bars, 50 μm. (A, B, and D) and (E, F, and G) are at the same magnifications.

Figure 4.

 

Western blot of (C) unwounded corneal epithelium immunoprecipitated with anti-PT and (W) corneal epithelium harvested 30 minutes after wounding and immunoprecipitated with anti-PT. Corneas were treated with EGF (+) or with PBS only (−). The blot was reacted with anti-EGFR.

Figure 5.

 

(A) Representative Western blot analysis of unwounded corneal epithelium (Cont) and epithelium harvested 0.5, 1, 2, 4, and 24 hours after wounding and immunoprecipitated with anti-EGFR. Identical blots were reacted with anti-PT or anti-EGFR. Note, equal volumes of immunoprecipitated supernatant were loaded in all lanes except for the unwounded epithelium (Cont), which was overloaded to allow the visualization of PT. (B) Quantitation of the relative ratio of phosphorylated EGFR to total EGFR from four separate experiments. Values are means ± SEM. All time points were significantly enhanced (P < 0.05) compared with control. There was no significant difference between the 0.5-, 1-, and 2-hour time points.

Figure 6.

 

RT-PCR analysis of RNA isolated from unwounded corneal epithelium (Con) and epithelium (A) 0.5, 1, 2, and 4 hours after wounding and (B) 4, 8, 16, and 24 hours after wounding. The PCR products are visualized on agarose gels stained with ethidium bromide. Standard markers (Std) are shown.

Figure 7.

 

Quantitation of remaining wound areas after incubation for 18 hours with various concentrations of tyrphostin AG1478. Epithelial migration was significantly slowed (P ≤ 0.01) at concentrations of 5, 10, 20, 30, and 50 μM. It should be noted that corneal wounds in this organ culture model, which contains no added growth factors in the medium, heal in 22 hours. ²⁶ The original wound size was 7.07 mm².