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From the Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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-, 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-, 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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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-, 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 culture16
and to stimulate the
rate of epithelial migration.17
More recently, TGF- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
analysis28
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 25
µl of anti-EGFR (1005) overnight, and protein A agarose beads (50
µl/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 method29
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/1
µg total RNA; Life Technologies, Grand Island, NY).
RT-PCR
Reverse transcription–polymerase chain reaction (RT-PCR) was
performed as previously described30
using specific primers
for EGF, HB-EGF, TGF-, AR, and glyceraldehyde 3-phosphate
dehydrogenase (G3PDH; Table 1
). The primer sets and exon location for HB-EGF, TGF-, 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.
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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 medium26
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 stain41
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 wounds51 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 minutes52 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-, HB-EGF, and AR. This is in agreement with
previous reports that mouse and human corneal epithelia express EGF and
TGF- mRNA and protein.59
60
Of note, mRNA levels for
three of these growth factors, TGF-, 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
concept5
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 epidermal62
and
intestinal61
epithelia. These data also suggest that an
extremely redundant system is used during healing. This may be relevant
to the findings that TGF- 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-. 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- 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.
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Footnotes |
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Submitted for publication May 14, 1999; revised November 17, 1999; accepted December 20, 1999.
Commercial relationships policy: N.
Corresponding author: James D. Zieske, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. zieske{at}vision.eri.harvard.edu
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J. D. Zieske, A. E. K. Hutcheon, X. Guo, E.-H. Chung, and N. C. Joyce TGF-{beta} Receptor Types I and II Are Differentially Expressed during Corneal Epithelial Wound Repair Invest. Ophthalmol. Vis. Sci., June 1, 2001; 42(7): 1465 - 1471. [Abstract] [Full Text]
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V. E. Klepeis, A. Cornell-Bell, and V. Trinkaus-Randall Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells J. Cell Sci., January 12, 2001; 114(23): 4185 - 4195. [Abstract] [Full Text] [PDF]
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R. Mohan, S. K. Chintala, J. C. Jung, W. V. L. Villar, F. McCabe, L. A. Russo, Y. Lee, B. E. McCarthy, K. R. Wollenberg, J. V. Jester, et al. Matrix Metalloproteinase Gelatinase B (MMP-9) Coordinates and Effects Epithelial Regeneration J. Biol. Chem., January 11, 2002; 277(3): 2065 - 2072. [Abstract] [Full Text] [PDF]
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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.