December 2009
Volume 50, Issue 12
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Cornea  |   December 2009
Role of Formation of an ERK-FAK-Paxillin Complex in Migration of Human Corneal Epithelial Cells during Wound Closure In Vitro
Author Affiliations & Notes
  • Shinichiro Teranishi
    From the Departments of Ophthalmology and
  • Kazuhiro Kimura
    Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
  • Teruo Nishida
    From the Departments of Ophthalmology and
    Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
  • Corresponding author: Kazuhiro Kimura, Department of Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan; k.kimura@yamaguchi-u.ac.jp
Investigative Ophthalmology & Visual Science December 2009, Vol.50, 5646-5652. doi:10.1167/iovs.08-2534
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      Shinichiro Teranishi, Kazuhiro Kimura, Teruo Nishida; Role of Formation of an ERK-FAK-Paxillin Complex in Migration of Human Corneal Epithelial Cells during Wound Closure In Vitro. Invest. Ophthalmol. Vis. Sci. 2009;50(12):5646-5652. doi: 10.1167/iovs.08-2534.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Migration of corneal epithelial cells is an important step in the corneal wound healing. The role of extracellular signal regulated kinase (ERK) for the regulation of cell migration during wound closure was examined.

Methods.: Scratch wounds were introduced into human corneal epithelial cells in the absence or presence of PD98059, an ERK signaling inhibitor. The phosphorylation and localization of ERK during wound closure were examined by immunoblot and immunofluorescence analyses. The tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as the association of FAK with paxillin and ERK, were evaluated by immunoprecipitation and immunoblot analysis. The effect of a mutant form of MEK1 on cell migration and proliferation was determined by transfection.

Results.: PD98059 inhibited cell migration in a concentration- and time-dependent manner. Wounding increased the phosphorylation of ERK as well as the tyrosine phosphorylation of FAK and paxillin in a manner sensitive to PD98059. Furthermore, wounding induced the formation of an ERK-FAK-paxillin complex and this effect as well as the wounding-induced formation of focal adhesions, membrane ruffles, and bundles of F-actin, were inhibited by PD98059. Phosphorylated ERK localized at the wound margin, and such localization was not observed in the presence of PD98059. Expression of dominant negative mutant form of MEK1 inhibited cell migration during wound closure without the effect of cell proliferation.

Conclusions.: ERK regulates cell migration during wound healing in vitro by modulating the phosphorylation of FAK and paxillin and the consequent formation of focal adhesions. An ERK-FAK-paxillin signaling pathway may play an important role in corneal epithelial wound healing in vivo.

The healing of corneal wounds is essential for maintenance of corneal structure and function. 1 Injury of the corneal epithelium induces migration of the remaining epithelial cells surrounding the wound to cover the wounded area. 2 The corneal epithelial cells attach to and migrate over a fibronectin matrix that is deposited at the wound site. 3,4 Attachment of corneal epithelial cells to fibronectin induces reorganization of the actin cytoskeleton, membrane ruffling, and the formation of focal adhesions containing focal adhesion kinase (FAK) and paxillin. 5,6 We previously showed that inhibition of the formation of focal adhesions results in impaired adhesion and migration of corneal epithelial cells. 6  
Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine kinases that contribute to the regulation of various cellular processes, including cell proliferation, differentiation, and apoptosis. Moreover, the MAPKs extracellular signal–regulated kinase (ERK), 7 p38 MAPK, 8,9 and c-Jun NH2-terminal kinase (JNK) 10,11 have been implicated in the regulation of cell migration, including that of corneal epithelial cells. We have now investigated how ERK signaling might contribute to the regulation of corneal epithelial cell migration during wound closure. 
Materials and Methods
A mixture of Dulbecco's modified Eagle's medium (DMEM) and nutrient mixture F-12 (DMEM/F-12) as well as general purpose cell medium (OPTI-MEM), fetal bovine serum, trypsin-EDTA, gentamicin, and a lipophilic transfection reagent (Lipofectamine 2000) were obtained from Invitrogen-Gibco (Carlsbad, CA). Bovine insulin, cholera toxin, recombinant human epidermal growth factor, Nonidet P-40, and a protease inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO). Plastic 24-well culture plates and 60 mm culture dishes were obtained from Corning (Corning, NY), and PD98059 was obtained from Merck (Whitehouse Station, NJ). Rabbit polyclonal antibodies to ERK1 or ERK2 (ERK1/2) and to phosphorylated ERK1/2 were obtained from Cell Signaling (Danvers, MA). Mouse monoclonal antibodies to phosphotyrosine were obtained from UBI (Temecula, CA), and those to paxillin and to FAK were obtained from BD Biosciences (Franklin Lakes, NJ). A nuclear counterstain (TOTO-3), AlexaFluor 488–labeled goat antibodies to rabbit or mouse immunoglobulin G, and rhodamine-phalloidin were obtained from Invitrogen (Carlsbad, CA). Protein G–sepharose, horseradish peroxidase–conjugated goat secondary antibodies, and ECL plus detection reagents were obtained from Amersham Biosciences (Little Chalfont, UK). An enzyme-linked immunosorbent assay (ELISA) kit for detection of bromodeoxyuridine (BrdU) was obtained from Roche (Indianapolis, IN). 
Cells and Cell Culture
Simian virus 40–immortalized human corneal epithelial (HCE) cells 12 were obtained from RIKEN Biosource Center (Tsukuba, Japan). They were passaged in supplemented hormonal epithelial medium (SHEM), which consists of DMEM/F-12 supplemented with 15% heat-inactivated fetal bovine serum, bovine insulin (5 μg/mL), cholera toxin (0.1 μg/mL), recombinant human epidermal growth factor (10 ng/mL), and gentamicin (40 μg/mL). For experiments, HCE cells were plated at a density of 5 × 105 cells per 60 mm dish or 1 × 105 cells per well in 24 well plates and were cultured for 48 hours before serum deprivation by incubation in unsupplemented DMEM/F-12 for 24 hours. 
Plasmids and Transfection
The plasmids pCS2=MEK1DA and pCS2=MEK1DN, which encode constitutively active and dominant negative mutants of human MEK1, respectively, were constructed as described previously. 13 HCE cells in 60 mm dishes were transfected for 3 hours with 2 μg of either plasmid mixed with reagent (Lipofectamine 2000; Invitrogen-Gibco) in 2 mL of OPTI-MEM medium. The cells were then incubated for an additional 6 hours in SHEM, replated in 60 mm dishes, and cultured for 48 hours before experiments. 
Wound Closure Assay
HCE cells cultured in 24-well plates or 60 mm dishes and deprived of serum for 24 hours were incubated for 1 hour with PD98059 or vehicle (0.1% dimethyl sulfoxide, or DMSO) in unsupplemented DMEM/F-12. The cell monolayer was then scraped with the narrow end of a micropipette tip to generate a wound ∼0.1 cm in width. Phase-contrast images were acquired at various times thereafter with an inverted microscope (Zeiss Axiovert; Carl Zeiss, Hallbergmoos, Germany) equipped with a charge-coupled device camera (Carl Zeiss). The wound area in each image was determined by computerized planimetry with NIH Image version 1.62f software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). . 
Immunofluorescence Microscopy
HCE cells cultured on 12 mm cover glasses in 24-well plates were treated and wounded as described above. At various times thereafter, the cells were fixed for 15 minutes at 37°C with 3.7% formalin, washed with Ca2+- and Mg2+-free phosphate-buffered saline [PBS(–)], permeabilized with 0.1% Triton X-100 in PBS(–) for 5 minutes at room temperature, and incubated for 1 hour at room temperature with 1% bovine serum albumin (BSA) in PBS(–). They were then incubated for 1 hour with antibodies to phosphotyrosine or to phosphorylated ERK1/2 [each at a dilution of 1:100 in PBS(–) containing 1% BSA], washed with PBS(–), and incubated for 1 hour with corresponding AlexaFluor 488–conjugated secondary antibodies (1:1000 dilution), rhodamine-phalloidin (1:200 dilution), and TOTO-3 (1:1000 dilution) in PBS(–) containing 1% BSA. The cells were finally examined with a laser confocal microscope (LSM5, Zeiss). 
Immunoblot Analysis
HCE cells cultured in 60 mm dishes and treated and wounded as described above were lysed on ice in 0.5 mL of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 15 minutes at 4°C, and the resulting supernatants (20 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to blocking buffer [20 mM Tris-HCl (pH 7.4), 5% skim milk, 0.1% Tween 20] for 1 hour at room temperature before incubation for 1 hour with antibodies to ERK1/2 or to phosphorylated ERK1/2 at a dilution of 1:1000 in blocking buffer. The membrane was washed with washing buffer [20 mM Tris-HCl (pH 7.4), 0.1% Tween 20], incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies (1:1000 dilution in washing buffer), washed again, incubated with ECL Plus detection reagents for 5 minutes, and then exposed to film. 
Immunoprecipitation Analysis
HCE cells cultured in 60 mm dishes were treated, wounded, and lysed as described for immunoblot analysis. The cell lysates were centrifuged at 15,000g for 10 minutes at 4°C, and portions of each supernatant (200 μg of protein) were incubated for 16 hours at 4°C in a final volume of 100 μL with antibodies to FAK or to paxillin (1:100 dilution) and 20 μL of protein G–sepharose beads. The beads were isolated by centrifugation and washed twice with cell lysis solution, and the bound proteins were fractionated by SDS-polyacrylamide gel electrophoresis on a 10% gel and then transferred to a nitrocellulose membrane. After incubation for 16 hours at 4°C with blocking buffer, the membrane was exposed for 16 hours at 4°C to antibodies specific for FAK, paxillin, phosphotyrosine, or phosphorylated ERK1/2 (each at a dilution of 1:1000 in blocking buffer), washed with washing buffer, and incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies (1:1000 dilution in washing buffer). The membrane was washed again, and immune complexes were then detected with ECL Plus reagents. 
Assay of Cell Proliferation
HCE cells cultured in 60 mm dishes were treated and wounded as described above. After incubation of the cells for 12 or 24 hours, cell proliferation was measured with the use of an assay kit. In brief, BrdU solution was added to the culture medium and the cells were incubated for an additional 2 hours. The medium was then removed from each dish, and the cells were processed for colorimetric detection of incorporated BrdU with ELISA reagents and measurement of absorbance at 370 nm with a fluorescence plate reader. 
Statistical Analysis
Quantitative data are presented as mean ± SE and were analyzed with Dunnett's test. A P value of <0.05 was considered statistically significant. 
Results
We examined the possible role of ERK signaling in corneal epithelial cell migration during wound closure in vitro with the use of PD98059, an inhibitor of the activity of MEK1, a protein kinase that phosphorylates and thereby activates ERK. HCE cells in a confluent monolayer were deprived of serum for 24 hours and then exposed to PD98059 (10 μM) or vehicle (0.1% DMSO) for 1 hour before wounding. The cells were fixed 24 hours after wounding, and the wounded area was examined by phase-contrast microcopy. Whereas the cells incubated with vehicle had migrated into and largely covered the original wound area, those incubated with PD98059 failed to cover a substantial portion of the wound (Fig. 1A). Quantitation of the remaining wound area revealed that PD98059 inhibited HCE cell migration in a time- and concentration-dependent manner (Fig. 1B). Whereas the cells incubated with vehicle had covered ∼75% and ∼100% of the original wound area after 12 and 24 hours, respectively, those incubated with 30 μM PD98059 had covered only ∼2% and ∼37% of the wound area at these times. 
Figure 1.
 
Inhibition of HCE cell migration by PD98059 in an in vitro model of corneal epithelial wound healing. (A) Confluent monolayers of HCE cells were deprived of serum for 24 hours, incubated for 1 hour with 10 μM PD98059 or vehicle (0.1% DMSO), and subjected to scratch-wounding. The cells were fixed immediately or 24 hours after wounding, and the wounded area of the cell monolayer was examined by phase-contrast microcopy. (B) HCE cells were treated with various concentrations of PD98059 and wounded as in (A), and the remaining area of the wound was determined at the indicated times thereafter. Data are mean ± SE of triplicates from an experiment that was repeated three times with similar results. *P < 0.05 versus corresponding value for cells treated with 0.1% DMSO.
Figure 1.
 
Inhibition of HCE cell migration by PD98059 in an in vitro model of corneal epithelial wound healing. (A) Confluent monolayers of HCE cells were deprived of serum for 24 hours, incubated for 1 hour with 10 μM PD98059 or vehicle (0.1% DMSO), and subjected to scratch-wounding. The cells were fixed immediately or 24 hours after wounding, and the wounded area of the cell monolayer was examined by phase-contrast microcopy. (B) HCE cells were treated with various concentrations of PD98059 and wounded as in (A), and the remaining area of the wound was determined at the indicated times thereafter. Data are mean ± SE of triplicates from an experiment that was repeated three times with similar results. *P < 0.05 versus corresponding value for cells treated with 0.1% DMSO.
We investigated whether ERK is indeed activated in response to wounding of HCE cell monolayers. Immunoblot analysis of control cells treated with vehicle revealed a marked increase in the amount of phosphorylated (activated) ERK1/2 that was maximal at 5 minutes after wounding but remained apparent at 60 minutes (Fig. 2A). In contrast, this response was greatly attenuated in cells treated with PD98059 (10 μM). The total amount of ERK1/2 was not affected by wounding in cells treated with vehicle or PD98059. Wounding also had no effect on HCE cell proliferation in the absence or presence of PD98059 (Fig. 2B). 
Figure 2.
 
Phosphorylation of ERK1/2 induced by wounding in HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for the indicated times (time 0 corresponds to non-wounded cells), the cells were lysed and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total ERK1/2 (upper panels). The intensity of the phosphorylated ERK1/2 bands was determined by densitometry and normalized by that of the corresponding total ERK1/2 bands (lower panel); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO. (B) Cells incubated with 10 μM PD98059 or vehicle (0.1% DMSO) for 1 hour and wounded (or not) as in (A) were assayed for cell proliferation by measurement of BrdU incorporation after 12 or 24 hours. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
Figure 2.
 
Phosphorylation of ERK1/2 induced by wounding in HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for the indicated times (time 0 corresponds to non-wounded cells), the cells were lysed and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total ERK1/2 (upper panels). The intensity of the phosphorylated ERK1/2 bands was determined by densitometry and normalized by that of the corresponding total ERK1/2 bands (lower panel); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO. (B) Cells incubated with 10 μM PD98059 or vehicle (0.1% DMSO) for 1 hour and wounded (or not) as in (A) were assayed for cell proliferation by measurement of BrdU incorporation after 12 or 24 hours. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
We examined changes in the morphology of HCE cells at the wound margin 12 hours after wounding. Immunofluorescence analysis with antibodies to phosphotyrosine revealed numerous small dotlike structures, presumably corresponding to focal adhesions, that were associated with lamellipodia containing bundles of F-actin in control cells exposed to vehicle (Fig. 3). In contrast, cells treated with PD98059 (10 μM) exhibited only a thin rim of F-actin staining and only a few small dotlike structures containing phosphotyrosine immunoreactivity at the cell periphery. 
Figure 3.
 
Effects of PD98059 on the morphology of HCE cells at the wound edge. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1 and were then cultured for 12 hours. They were then fixed, permeabilized, and subjected to staining for F-actin (red) with rhodamine-phalloidin, for phosphotyrosine with specific antibodies (green), and for nuclei with TOTO-3 (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Figure 3.
 
Effects of PD98059 on the morphology of HCE cells at the wound edge. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1 and were then cultured for 12 hours. They were then fixed, permeabilized, and subjected to staining for F-actin (red) with rhodamine-phalloidin, for phosphotyrosine with specific antibodies (green), and for nuclei with TOTO-3 (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
To examine further the role of ERK in wound closure, we transfected HCE cells with an expression vector either for MEK1DN, a dominant negative mutant of MEK1, or for MEK1DA, a constitutively active mutant of MEK1. The rate of wound closure was significantly reduced in cells expressing MEK1DN compared with that apparent for cells transfected with the empty vector (Fig. 4A). Conversely, expression of MEK1DA resulted in acceleration of wound closure (Fig. 4A). Expression of neither MEK1 mutant had an effect on HCE cell proliferation before or after wounding (Fig. 4B). 
Figure 4.
 
Effects of dominant negative and constitutively active forms of MEK1 on HCE cell migration during wound closure. (A) Monolayers of cells transfected with expression vectors for dominant negative (DN) or constitutively active (DA) forms of MEK1, or with the corresponding empty vector (control), were deprived of serum for 24 hours and then wounded. The extent of wound closure was determined at the indicated times thereafter as in Figure 1. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05, **P < 0.01 versus the corresponding value for cells transfected with the empty vector (pCS-2=). (B) Cells transfected and wounded as in (A) were assayed for cell proliferation by measurement of BrdU incorporation at the indicated times thereafter (time 0 corresponds to non-wounded cells). Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
Figure 4.
 
Effects of dominant negative and constitutively active forms of MEK1 on HCE cell migration during wound closure. (A) Monolayers of cells transfected with expression vectors for dominant negative (DN) or constitutively active (DA) forms of MEK1, or with the corresponding empty vector (control), were deprived of serum for 24 hours and then wounded. The extent of wound closure was determined at the indicated times thereafter as in Figure 1. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05, **P < 0.01 versus the corresponding value for cells transfected with the empty vector (pCS-2=). (B) Cells transfected and wounded as in (A) were assayed for cell proliferation by measurement of BrdU incorporation at the indicated times thereafter (time 0 corresponds to non-wounded cells). Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
We next examined whether the focal adhesion proteins FAK and paxillin are indeed activated in response to wounding of HCE cell monolayers. We incubated HCE cells with PD98059 (10 μM) or vehicle for 1 hour before wounding and then subjected cell lysates to immunoprecipitation with antibodies to FAK or to paxillin at various times thereafter. Immunoblot analysis of the resulting precipitates from vehicle-treated cells with antibodies to phosphotyrosine revealed marked and transient increases in the amounts of phosphorylated (activated) FAK and paxillin that were maximal at 15 minutes after wounding, with the level of FAK and paxillin phosphorylation returning to baseline values by 60 minutes (Fig. 5). In contrast, the wounding-induced increases in the levels of FAK and paxillin phosphorylation were greatly attenuated in cells treated with PD98059. The total amounts of precipitated FAK and paxillin did not differ between cells treated with vehicle or PD98059. 
Figure 5.
 
Tyrosine phosphorylation of FAK and paxillin induced by wounding in HCE cells and its inhibition by PD98059. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 2. The cells were lysed at the indicated times after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK (A) or to paxillin (B). The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (p-Tyr) and with the antibodies used for immunoprecipitation (upper panels). The intensity of the tyrosine-phosphorylated FAK or paxillin bands was determined by densitometry and normalized by that of the total FAK or paxillin bands, respectively (lower panels); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO.
Figure 5.
 
Tyrosine phosphorylation of FAK and paxillin induced by wounding in HCE cells and its inhibition by PD98059. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 2. The cells were lysed at the indicated times after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK (A) or to paxillin (B). The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (p-Tyr) and with the antibodies used for immunoprecipitation (upper panels). The intensity of the tyrosine-phosphorylated FAK or paxillin bands was determined by densitometry and normalized by that of the total FAK or paxillin bands, respectively (lower panels); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO.
We determined the localization of phosphorylated ERK1/2 in HCE cells by immunofluorescence analysis 15 minutes after wounding (Fig. 6). Staining corresponding to phosphorylated ERK1/2 was markedly increased in cells at the wound margin of monolayers treated with vehicle but not in the corresponding cells of monolayers treated with PD98059 (10 μM). 
Figure 6.
 
Localization of phosphorylated ERK1/2 to HCE cells at the wound margin. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for 15 minutes, they were fixed, permeabilized, and stained with antibodies to phosphorylated ERK1/2 (green) as well as with rhodamine-phalloidin for F-actin (red) and with TOTO-3 for nuclei (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Figure 6.
 
Localization of phosphorylated ERK1/2 to HCE cells at the wound margin. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for 15 minutes, they were fixed, permeabilized, and stained with antibodies to phosphorylated ERK1/2 (green) as well as with rhodamine-phalloidin for F-actin (red) and with TOTO-3 for nuclei (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Finally, we tested whether association among FAK, paxillin, and ERK1/2 might be detected by immunoprecipitation analysis. Lysates of cells treated with PD98059 (10 μM) or vehicle were prepared 5 minutes after wounding and subjected to immunoprecipitation with antibodies to FAK. Immunoblot analysis of the resulting precipitates revealed that the associated amounts of paxillin and phosphorylated ERK1/2 were markedly greater for cells treated with vehicle than for those treated with PD98059 (Fig. 7). The association of FAK with paxillin or phosphorylated ERK1/2 was not detected in HCE cell monolayers not subjected to wounding (data not shown). 
Figure 7.
 
Association of FAK with paxillin and phosphorylated ERK1/2 in wounded HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. Cell lysates were prepared 5 minutes after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK. The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphorylated ERK1/2, to paxillin, or to FAK. (B) The intensity of phosphorylated ERK1/2 or paxillin bands in immunoblots similar to that in (A) was determined by densitometry and normalized by that of the corresponding FAK band. Data are mean ± SE of values from three independent experiments. **P < 0.01.
Figure 7.
 
Association of FAK with paxillin and phosphorylated ERK1/2 in wounded HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. Cell lysates were prepared 5 minutes after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK. The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphorylated ERK1/2, to paxillin, or to FAK. (B) The intensity of phosphorylated ERK1/2 or paxillin bands in immunoblots similar to that in (A) was determined by densitometry and normalized by that of the corresponding FAK band. Data are mean ± SE of values from three independent experiments. **P < 0.01.
Discussion
We have shown that ERK1/2 was activated in HCE cells during wound closure in vitro, and that the MEK1 inhibitor PD98059 markedly inhibited HCE cell migration during wound closure in a time- and concentration-dependent manner. Furthermore, the MEK1 inhibitor blocked membrane ruffling associated with the formation of focal adhesions and inhibited the phosphorylation of FAK and paxillin induced by wounding. Phosphorylated ERK1/2 was detected in HCE cells at the wound margin and interacted with FAK and paxillin during wound healing. These results thus implicate ERK-FAK-paxillin signaling in the formation of focal adhesions and consequent regulation of HCE cell migration associated with wound closure. 
Our results suggest that ERK activated in response to wounding promoted the formation of focal adhesions in HCE cells by affecting the tyrosine phosphorylation of both paxillin and FAK. Regulation of the formation of focal adhesions by ERK has been described in other epithelial cell types. 13,14 The MAPKs JNK and p38 are also activated in response to cell stress induced by injury, ultraviolet radiation, heat shock, or pro-inflammatory cytokines. 1519 In addition to roles in cell survival, proliferation, and differentiation, p38 MAPK has been shown to modulate cell migration in several cell types including corneal epithelial cells. 811 JNK is also thought to play a role in regulation of corneal epithelial cell migration by mediating the phosphorylation of c-Jun. 20 We previously showed that activation of the small GTPase Rho resulted in the phosphorylation of paxillin, but not in that of FAK, in HCE cells. 6 These results suggest that the activation of ERK in HCE cells during wound healing may induce the phosphorylation of paxillin and FAK through a Rho-independent signaling pathway. We found that regulation of FAK and paxillin phosphorylation by ERK1/2 in HCE cells during wound closure was associated with the formation of an ERK-FAK-paxillin complex, consistent with previous observations in other epithelial cell types. 1416  
The tyrosine phosphorylation of FAK and paxillin is upregulated during migration of corneal epithelial cells and other cell types induced by various stimuli. 1719 Furthermore, the phosphorylation of ERK was previously shown to be induced during corneal epithelial cell migration, and cell migration was inhibited by PD98059. 20,21 However, the relation between the tyrosine phosphorylation of FAK and paxillin and the activity of ERK during corneal epithelial cell migration has remained unclear. We have now shown that the phosphorylation of FAK and paxillin induced by the wounding of HCE cell monolayers was dependent on ERK activity, and that ERK formed a complex with FAK and paxillin in a manner dependent on ERK activity during wound closure. Moreover, phosphorylated ERK1/2 was found to be localized to HCE cells at the wound margin and to regulate the formation of lamellipodia and focal adhesions by these cells. These results suggest that ERK localizes to focal adhesions through interaction with FAK and paxillin during wound closure, thereby regulating corneal epithelial migration. 
ERK has also been shown to contribute to the regulation of cell proliferation. 2224 Various growth factors, including platelet-derived growth factor and fibroblast growth factor 2, induce cell proliferation as well as promote cell migration in experimental models of wound healing. 25,26 Both the cell migration and proliferation stimulated by these growth factors were inhibited by PD98059 or by expression of a dominant negative mutant of MEK1. However, in the model of wound healing used in the present study, ERK activity was found to be required for cell migration but not for cell proliferation during wound closure. Cell proliferation was also found not to be associated with cell migration during wound healing in a scratch-wound assay similar to that used in the present study. 27  
We previously showed that hepatocyte growth factor and keratinocyte growth factor each promote the migration of rabbit corneal epithelial cells in primary culture through the activation of p38 MAPK, but not through that of JNK or ERK. 28 Our present study investigated the role of ERK1/2 in the migration of HCE cells as a model system for wound healing in vitro. In this system, wound closure was studied in the absence of exogenous growth factors or extracellular matrix proteins, so that the response of the cells to wounding is mediated entirely by endogenous components. Our results thus suggest that regulation of corneal epithelial cell migration by members of the MAPK family of proteins may be context dependent. 
Activated Rho induces the assembly of actin stress fibers and promotes the formation of focal adhesions. 29 Rac and Cdc42, which are members of the Rho family of proteins, also induce the assembly of small focal adhesions termed focal contacts. 30,31 We previously showed that fibronectin activates Rac1 and induces the formation of lamellipodia and focal adhesions in HCE cells, resulting in increased cell adhesion and motility. 6 These previous and our present observations thus suggest that ERK1/2 may function upstream or downstream of Rho family proteins in the regulation of HCE cell migration during wound healing. The functional relation between ERK and Rho family proteins in regulation of HCE cell migration requires further investigation. 
In conclusion, our present results implicate an ERK-FAK-paxillin signaling pathway in the regulation of human corneal epithelial cell migration during wound closure. Further characterization of the mechanism of this regulation may provide a better understanding of corneal epithelial wound healing as well as a basis for the development of new treatments for corneal epithelial wounds. 
Footnotes
 Supported by Grant 19791271 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Footnotes
 Disclosure: S . Teranishi, None; K. Kimura, None; T. Nishida, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Yasumiko Akamatsu and the staff of Yamaguchi University Center for Gene Research for technical assistance. 
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Figure 1.
 
Inhibition of HCE cell migration by PD98059 in an in vitro model of corneal epithelial wound healing. (A) Confluent monolayers of HCE cells were deprived of serum for 24 hours, incubated for 1 hour with 10 μM PD98059 or vehicle (0.1% DMSO), and subjected to scratch-wounding. The cells were fixed immediately or 24 hours after wounding, and the wounded area of the cell monolayer was examined by phase-contrast microcopy. (B) HCE cells were treated with various concentrations of PD98059 and wounded as in (A), and the remaining area of the wound was determined at the indicated times thereafter. Data are mean ± SE of triplicates from an experiment that was repeated three times with similar results. *P < 0.05 versus corresponding value for cells treated with 0.1% DMSO.
Figure 1.
 
Inhibition of HCE cell migration by PD98059 in an in vitro model of corneal epithelial wound healing. (A) Confluent monolayers of HCE cells were deprived of serum for 24 hours, incubated for 1 hour with 10 μM PD98059 or vehicle (0.1% DMSO), and subjected to scratch-wounding. The cells were fixed immediately or 24 hours after wounding, and the wounded area of the cell monolayer was examined by phase-contrast microcopy. (B) HCE cells were treated with various concentrations of PD98059 and wounded as in (A), and the remaining area of the wound was determined at the indicated times thereafter. Data are mean ± SE of triplicates from an experiment that was repeated three times with similar results. *P < 0.05 versus corresponding value for cells treated with 0.1% DMSO.
Figure 2.
 
Phosphorylation of ERK1/2 induced by wounding in HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for the indicated times (time 0 corresponds to non-wounded cells), the cells were lysed and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total ERK1/2 (upper panels). The intensity of the phosphorylated ERK1/2 bands was determined by densitometry and normalized by that of the corresponding total ERK1/2 bands (lower panel); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO. (B) Cells incubated with 10 μM PD98059 or vehicle (0.1% DMSO) for 1 hour and wounded (or not) as in (A) were assayed for cell proliferation by measurement of BrdU incorporation after 12 or 24 hours. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
Figure 2.
 
Phosphorylation of ERK1/2 induced by wounding in HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for the indicated times (time 0 corresponds to non-wounded cells), the cells were lysed and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total ERK1/2 (upper panels). The intensity of the phosphorylated ERK1/2 bands was determined by densitometry and normalized by that of the corresponding total ERK1/2 bands (lower panel); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO. (B) Cells incubated with 10 μM PD98059 or vehicle (0.1% DMSO) for 1 hour and wounded (or not) as in (A) were assayed for cell proliferation by measurement of BrdU incorporation after 12 or 24 hours. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
Figure 3.
 
Effects of PD98059 on the morphology of HCE cells at the wound edge. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1 and were then cultured for 12 hours. They were then fixed, permeabilized, and subjected to staining for F-actin (red) with rhodamine-phalloidin, for phosphotyrosine with specific antibodies (green), and for nuclei with TOTO-3 (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Figure 3.
 
Effects of PD98059 on the morphology of HCE cells at the wound edge. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1 and were then cultured for 12 hours. They were then fixed, permeabilized, and subjected to staining for F-actin (red) with rhodamine-phalloidin, for phosphotyrosine with specific antibodies (green), and for nuclei with TOTO-3 (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Figure 4.
 
Effects of dominant negative and constitutively active forms of MEK1 on HCE cell migration during wound closure. (A) Monolayers of cells transfected with expression vectors for dominant negative (DN) or constitutively active (DA) forms of MEK1, or with the corresponding empty vector (control), were deprived of serum for 24 hours and then wounded. The extent of wound closure was determined at the indicated times thereafter as in Figure 1. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05, **P < 0.01 versus the corresponding value for cells transfected with the empty vector (pCS-2=). (B) Cells transfected and wounded as in (A) were assayed for cell proliferation by measurement of BrdU incorporation at the indicated times thereafter (time 0 corresponds to non-wounded cells). Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
Figure 4.
 
Effects of dominant negative and constitutively active forms of MEK1 on HCE cell migration during wound closure. (A) Monolayers of cells transfected with expression vectors for dominant negative (DN) or constitutively active (DA) forms of MEK1, or with the corresponding empty vector (control), were deprived of serum for 24 hours and then wounded. The extent of wound closure was determined at the indicated times thereafter as in Figure 1. Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05, **P < 0.01 versus the corresponding value for cells transfected with the empty vector (pCS-2=). (B) Cells transfected and wounded as in (A) were assayed for cell proliferation by measurement of BrdU incorporation at the indicated times thereafter (time 0 corresponds to non-wounded cells). Data are mean ± SE of triplicates from an experiment that was repeated a total of three times with similar results.
Figure 5.
 
Tyrosine phosphorylation of FAK and paxillin induced by wounding in HCE cells and its inhibition by PD98059. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 2. The cells were lysed at the indicated times after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK (A) or to paxillin (B). The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (p-Tyr) and with the antibodies used for immunoprecipitation (upper panels). The intensity of the tyrosine-phosphorylated FAK or paxillin bands was determined by densitometry and normalized by that of the total FAK or paxillin bands, respectively (lower panels); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO.
Figure 5.
 
Tyrosine phosphorylation of FAK and paxillin induced by wounding in HCE cells and its inhibition by PD98059. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 2. The cells were lysed at the indicated times after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK (A) or to paxillin (B). The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphotyrosine (p-Tyr) and with the antibodies used for immunoprecipitation (upper panels). The intensity of the tyrosine-phosphorylated FAK or paxillin bands was determined by densitometry and normalized by that of the total FAK or paxillin bands, respectively (lower panels); data are mean ± SE of values from three independent experiments. *P < 0.05, **P < 0.01 versus the corresponding value for cells cultured with 0.1% DMSO.
Figure 6.
 
Localization of phosphorylated ERK1/2 to HCE cells at the wound margin. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for 15 minutes, they were fixed, permeabilized, and stained with antibodies to phosphorylated ERK1/2 (green) as well as with rhodamine-phalloidin for F-actin (red) and with TOTO-3 for nuclei (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Figure 6.
 
Localization of phosphorylated ERK1/2 to HCE cells at the wound margin. Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. After culture for 15 minutes, they were fixed, permeabilized, and stained with antibodies to phosphorylated ERK1/2 (green) as well as with rhodamine-phalloidin for F-actin (red) and with TOTO-3 for nuclei (blue). Scale bar, 10 μm. Data are representative of three independent experiments.
Figure 7.
 
Association of FAK with paxillin and phosphorylated ERK1/2 in wounded HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. Cell lysates were prepared 5 minutes after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK. The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphorylated ERK1/2, to paxillin, or to FAK. (B) The intensity of phosphorylated ERK1/2 or paxillin bands in immunoblots similar to that in (A) was determined by densitometry and normalized by that of the corresponding FAK band. Data are mean ± SE of values from three independent experiments. **P < 0.01.
Figure 7.
 
Association of FAK with paxillin and phosphorylated ERK1/2 in wounded HCE cells and its inhibition by PD98059. (A) Cells were treated with 10 μM PD98059 or 0.1% DMSO and wounded as in Figure 1. Cell lysates were prepared 5 minutes after wounding and subjected to immunoprecipitation (IP) with antibodies to FAK. The resulting precipitates were then subjected to immunoblot analysis (IB) with antibodies to phosphorylated ERK1/2, to paxillin, or to FAK. (B) The intensity of phosphorylated ERK1/2 or paxillin bands in immunoblots similar to that in (A) was determined by densitometry and normalized by that of the corresponding FAK band. Data are mean ± SE of values from three independent experiments. **P < 0.01.
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