|Molecular Vision 2006;
Received 21 July 2006 | Accepted 5 November 2006 | Published 16 November 2006
Transactivation of EGFR mediates insulin-stimulated ERK1/2 activation and enhanced cell migration in human corneal epithelial cells
Jungmook Lyu, Kyung-Suk Lee,
Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea
Correspondence to: Choun-Ki Joo, Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-ku, Seoul 137-701, Korea; Phone: 82-2-590-2613; FAX: 82-2-533-3801; email: firstname.lastname@example.org
Purpose: Insulin activates phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK)-1/2 in human corneal epithelial cells. These events have been shown to be involved in wound healing. However, the mechanism of insulin-induced ERK pathway is not clear during corneal wound healing. In this study, the effect of insulin associated with epidermal growth factor receptor (EGFR) on wound healing in transformed human corneal epithelial cells was investigated to determine the signaling mechanism involved.
Methods: SV40-immortalized human corneal epithelial (THCE) cells were cultured on a diluted Matrigel matrix that resembled the basement membrane of the corneal epithelium. A wound was introduced with a micropipette tip, and closure of the scratch wound was photographed 12 h after exposure to insulin. Activation of EGFR was analyzed by immunoprecipitation, and cytoskeletal rearrangements were visualized with rhodamine-conjugated phalloidin.
Results: Exposure of corneal epithelial cells to insulin induced phosphorylation of EGFR. Inhibition of EGFR activation by AG1478 or the MMP inhibitor, GM6001, reduced phosphorylation of insulin-induced ERK in the presence of insulin and delayed wound closure. In addition, cells exposed to insulin contained stress fibers and their submembranous cortical actin was depleted. These effects were inhibited by AG1478.
Conclusions: Inhibition of EGFR activity decreases cell migration involved in insulin-induced wound repair, an effect that mimics inhibition of MMP activation. Inhibition of MMP activity leads to decreased EGFR phosphorylation. Our data show that insulin stimulates wound healing in the corneal epithelium by activating EGFR, and point to a novel insulin signaling pathway that acts during corneal wound healing.
Renewal of the corneal epithelium is a complex process involving the migration, proliferation, and differentiation of epithelial cells. These events play an important role in maintaining barrier function and corneal transparency. Several growth factors are implicated in corneal reepithelialization, a process relying on cell migration and proliferation. These growth factors: namely epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), and insulin-like growth factor (IGF), all play roles in the migration and proliferation of corneal epithelial cells during wound repair [1-3].
Corneal wound healing, including reepithelialization, is delayed or impaired in insulin-dependent diabetes mellitus, a pathologic disorder resulting from insulin deficiency or resistance to insulin. Patients with this disorder can develop vision loss . Insulin therapy is known to be beneficial for the treatment of epidermal wounds in diabetes patients . In addition, a recent study showed that insulin therapy prevents the delay of corneal wound healing in the diabetic rat . Insulin, which is involved in wound repair, is found in human tear film, and its receptors are present in the human ocular surface . A recent study showed that insulin enhanced reepithelialization in an in vitro monolayer model of human corneal epithelial cells . The mechanisms of action of the insulin is not fully understood, although studies have suggested that corneal wound healing is, at least in part, mediated by insulin. Binding of insulin to the insulin receptors on cell membranes stimulates their tyrosine kinase activity and results in the phosphorylation of insulin receptor substrates (IRSs) [9,10].
Phosphorylated IRS-1 and IRS-2 activate diverse signal pathways, including the Ras/mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) pathways. PI3K is an important signaling intermediate that affects the behavior of several types of cells , while the MAP kinase pathway plays a central role in the mitogenic effect of insulin [10,12]. Furthermore, insulin activates PI3K/Akt and extracellular signal-regulated kinase (ERK)-1/2 in human corneal epithelial cells, and the cellular response to insulin is prevented by inhibitors of PI3K or ERK1/2 .
Here we focused on the EGF receptor (EGFR) as a potential target of insulin because its activation induces closure of corneal epithelial wounds as well as phosphorylation of ERK [3,13,14]. Transactivation of the EGFR, which controls a wide variety of biological responses such as proliferation, differentiation, and migration, can occur in response to diverse stimuli, including activation of G-protein-coupled and cytokine receptors, oxidative stress, UV light, and the autocrine release of soluble EGFR ligands, such as heparin-binding EGF . In addition, IGF-1 also stimulates ERK1/2 phosphorylation via transactivation of the EGFR in COS-7 cells . In this study, we show that exposure of corneal epithelial cells to insulin induces phosphorylation of the EGFR, and of ERK, and that inhibition of EGFR activation by AG1478 reduces the insulin-induced phosphorylation of ERK. In addition, wound closure in the presence of insulin was delayed by inhibiting the EGFR. Our results reveal a novel regulatory mechanism involving the EGFR-linked ERK signaling pathway in insulin-induced corneal reepithelialization.
The polyclonal antibody against Erk1/2 and the monoclonal antibody against EGFR were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) The polyclonal antibodies against Akt, phospho-Akt, and phospho-Erk1/2 were from Cell Signaling Technology, Inc. (Danvers MA), and the monoclonal antibody against phosphotyrosine (PY20H) was from Transduction Laboratories (San Jose, CA). The MEK inhibitor, U0126, the PI3K inhibitor, wortmannin, and the MMP inhibitor, GM 6001, were from Calbiochem (San Diego, CA), and the NADPH oxidase inhibitor, diphenyleneiodonium (DPI) and the EGFR-specific tyrosine kinase inhibitor, AG1478, were obtained from Sigma (St. Louis, MO).
SV40-immortalized human corneal epithelial (THCE) cells were kindly provided by Dr. Kaoru Araki-Sasaki (Osaka University, School of Medicine, Osaka, Japan). Cells were cultured for 24 h in DMEM/F-12 medium containing 5% FBS, 5 μg/ml insulin, 0.1 μg/ml cholera toxin, 10 ng/ml human EGF, and antibiotics and were then transferred to growth factor- and serum-free medium for the assays carried out in this study. The cells were plated on a diluted Matrigel matrix (Roche Applied Science, Mannheim, Germany), which is similar to the basement membrane of the corneal epithelium.
THCE cells were seeded in six-well culture dishes coated with diluted Matrigel matrix and grown to 80% confluence. Cells were then starved in DMEM/F-12 medium for 24 h, and a wound was introduced with a micropipette tip. The wounded cells were washed to remove any suspended cells and further incubated with insulin, AG1478, wortmannin, or UO126. An Axiovert model S100 microscope (Carl Zeiss, Oberkochen, Germany) and a digital camera (AxioCam; Carl Zeiss) were used to photograph healing cells 12 h after wounding. Each experiment was performed at least three times.
Immunoprecipitation and immunoblotting
After stimulation, THCE cells were placed on ice, washed with ice-cold PBS, and lysed in Nonidet P-40 solubilization buffer (250 mM NaCl, 50 mM HEPES, 0.5% Nonidet P-40, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 1.2 mM MgCl2, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). The lysate was centrifuged, and the supernatant incubated with 1 μg of anti-EGFR antibody at 4 °C for 2 h. Protein A-Sepharose beads were added, and the mixture was incubated at 4 °C for 8 h, and subsequently washed three times with Nonidet P-40 solubilization buffer. Immunocomplexes were resolved by 8% SDS-PAGE, blotted, and probed with antiphosphotyrosine (PY20H) antibody followed by horseradish peroxidase-conjugated antibody as secondary antibody, and visualized with an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). To measure ERK1/2 and Akt phosphorylation, cells were lysed in RIPA buffer containing 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. The lysates were fractionated by 10% SDS-PAGE, and blots were incubated with antiphospho-ERK1/2 antibody or antiphospho-Akt antibody. To confirm equal protein loading, the blots were stripped and reprobed with rabbit polyclonal anti-ERK1/2 antibody or rabbit polyclonal anti-Akt antibody.
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Trion X-100. After washing with PBS, cells were incubated with 2% bovine serum albumin for 30 min and then stained with rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR). Photographs were taken with a fluorescence microscope and digital camera.
Cells (2x102) were seeded in 96-well plates, incubated in growth factor- and serum-free medium for 24 h, and further incubated with AG1478, wortmannin, or UO126 in the presence of insulin for 24 h. Cell growth was determined with the cell proliferation reagent WST-1 (Roche Applied Science), according to the manufacturer's recommendations.
Insulin-induced wound closure involves PI3K and ERK activation
To determine whether the PI3K and ERK pathways are involved in wound repair in THCE cells, we examined the rate of wound repair in medium containing serum and growth factors, after wortmannin, a PI3K inhibitor, and U0126, an ERK inhibitor, were added. Wound repair was delayed in the presence of either inhibitor (Figure 1A). Insulin is reported to activate PI3K and ERK1/2 and to be involved in wound repair in corneal epithelial cells . When the cells were exposed to insulin for 10 min, phosphorylation of Akt (a major substrate of PI3K) and of ERK1/2 increased (Figure 1B). This result implies that PI3K and ERK are activated by insulin. We therefore examined the role of insulin-induced activation of PI3K and ERK in wound healing. THCE cells were scratched using a micropipette tip, exposed to DMSO, as a control, or to wortmannin or U0126, for 1 h and cultured with 5 μg/ml insulin or vehicle for 12 h. As shown in Figure 1C, insulin-induced wound closure was delayed by the presence of the inhibitors of PI3K and ERK1/2 activation.
Transactivation of EGFR by insulin
Phosphorylation of ERK points to activation of the MAPK pathway and is generally considered to be involved in EGFR signaling. The insulin-induced activation of ERK1/2 suggests that insulin promotes the activation of EGFR and leads to phosphorylation of ERK1/2. To test this idea, we assessed tyrosine phosphorylation of EGFR, an indication of its activation, by immunoprecipitation with anti-EGFR antibody. Figure 2A shows the time course of EGFR activation. Insulin-induced phosphorylation of EGFR was observed after as little as 3 min, peaked at 20 min, and decreased after 30 min. Similarly, treatment with insulin rapidly increased phosphorylation of ERK1/2 (Figure 2A, bottom). To determine whether activated EGFR contributes to the phosphorylation of ERK1/2 in response to insulin, we assayed the effect of AG1478, an EGFR-specific tyrosine kinase inhibitor. THCE cells were incubated with 1 μM AG1478 before exposing them to insulin. As shown in Figure 2B, insulin-induced phosphorylation of Akt was unaffected by AG1478. In contrast, insulin-stimulated ERK1/2 phosphorylation was markedly decreased in the presence of AG1478, suggesting that insulin-induced activation of EGFR accounts for ERK1/2 activation in response to insulin.
Several studies have shown that transactivation of EGFR is sensitive to matrix metalloproteinase (MMP) inhibitor and inhibitor of heparin-binding EGF (HB-EGF) activity. To identify the mechanism underlying insulin-induced transactivation of the EGFR, THCE cells were treated with GM6001 or CRM197. Cells pretreated with 10 μM GM6001, a hydroxamate MMP inhibitor, had no effect on EGFR phosphorylation (data not shown). However, inhibition of MMP activity with GM6001 attenuated EGFR phosphorylation in the presence of insulin, when cells were preincubated with highly concentrated reagent (50 μM; Figure 3A). Phosphorylation of ERK1/2 was similarly reduced in cells pretreated with GM6001 (Figure 3A). Since proteolysis of HB-EGF precursor contributes to transactivation of the EGFR and this process is mediated by members of the MMP family , we tested whether shedding of HB-EGF contributes to insulin-induced activation of the EGFR. CRM197, a catalytically inactive [Glu52] mutant of diptheria toxin, binds specifically to the extracellular domain of HB-EGF and inhibits HB-EGF activity . THCE cells were incubated with 10 μg/ml CRM197 for 1 h to inhibit HB-EGF shedding. Interestingly, this treatment did not affect EGFR and ERK1/2 phosphorylation (Figure 3B). Activation of NADPH oxidase can lead to Src-dependent EGFR transactivation , and insulin stimulates the activation of NADPH oxidase and increases intracellular superoxide anions via PI3K activation [20,21]. We therefore assessed the effect of NADPH oxidase on insulin-induced EGFR transactivation and found that addition of the NADPH oxidase inhibitor, DPI, had no effect on EGFR and ERK phosphorylation in the presence of insulin (Figure 3C). Taken together, these results show that insulin stimulates EGFR activation, leading to ERK phosphorylation, and insulin-stimulated EGFR transactivation is attenuated by inhibitors of MMP but not inhibitors of HB-EGF or NADPH oxidase. In turn, this points to the involvement of MMP-dependent shedding of other EGFR activators in insulin activation of ERK1/2.
Insulin induces migration of THCE cells via transactivation of EGFR
To examine the effect of the EGFR transactivation that mediates ERK1/2 activation on corneal epithelial wound healing, we examined the migration and proliferation of THCE cells. These cells were pretreated with aphidicolin to inhibit cell proliferation , exposed to DMSO, AG1478, wortmannin, or GM6001, and wounded. When the cells were incubated with insulin, wound closure was delayed in the cells treated with AG1478 or wortmannin. Addition of GM6001 also inhibited insulin-induced wound closure (Figure 4A). Insulin stimulated the growth of THCE cells, and, as shown in Figure 4B, this effect was slightly reduced by AG1478 and GM6001. The reduction in cell growth, was, however, not statistically significant. Thus, insulin-induced transactivation of EGFR stimulates the cell migration involved in wound healing, and MMP activity may be involved in this process.
Effect of EGFR transactivation on cytoskeletal organization
To test whether EGFR phosphorylation affects cytoskeletal organization, THCE cells pretreated with DMSO, wortmannin, GM6001, or AG1478, were incubated with or without insulin for 1 h and stained with rhodamine-phaloidin. In the absence of inhibitors, cells exposed to insulin displayed a network of fine fibrils along with depletion of submembranous cortical actin (Figure 5B). By contrast, in the presence of AG1478, cells exposed to insulin possessed submembranous cortical actin and few fine fibrils (Figure 5E). A similar pattern of actin staining was seen in cells exposed to vehicle, wartmannin, or GM6001. Together, these finding indicate that insulin-induced EGFR transactivation provokes cytoskeletal changes including the formation of actin stress fibers, which presumably enhance cell motility.
Diabetic corneal disorder is often associated with the form of nonhealing corneal epithelial defect. This abnormality can result from surgical and nonsurgical trauma [6,23]. Although insulin therapy may be beneficial for the treatment of wounds in patients with diabetes, the signaling mechanism in response to insulin during wound healing are poorly understood. Here, we have provided evidence that insulin stimulates the healing of wounds in the corneal epithelium by activating EGFR. Inhibition of EGFR activity reduced cell migration during insulin-induced wound repair, and this effect was mimicked by inhibiting MMP activity. Moreover inhibition of MMP activity led to decreased EGFR phosphorylation. These data define a new insulin signaling pathway involved in corneal wound healing.
Insulin activates both the PI3K and ERK signaling pathways in a number of cell types [24-26], and as shown here, has a similar action in corneal epithelial cells. Inhibition of PI3K or ERK activation impaired closure of in vitro scratch wounds, suggesting an important role of PI3K and ERK in the process of wound healing [13,27-29]. Several studies have reported that EGFR activates PI3K/Akt and ERK signaling in corneal epithelial cells. For example, inhibition of EGFR activity by AG1478 affected both Akt and ERK phosphorylation in corneal epithelial cells, and impaired their migration [3,27]. Interestingly, our immunoprecipitation experiments showed that tyrosine phosphorylation of EGFR increased in THCE cells incubated with insulin. Furthermore, blocking of EGFR decreased insulin-stimulated cell migration. Together with previous reports that the PI3K and ERK signal pathways are necessary for epithelial wound healing, these data have thus shown the EGFR signaling cascade that includes both the PI3K and ERK pathways. However, we found that inhibition of EGFR activity decreased insulin-stimulated ERK activation but not insulin stimulated PI3K activation. Although insulin induces cell migration via both PI3K and ERK signaling during wound healing, our results revealed that only insulin-stimulated ERK activation is dependent on the EGFR.
The effect of MMP inhibitors suggests that MMP-mediated release of some type of EGFR ligand is required for EGFR transactivation. The proteolytic release of HB-EGF, an EGFR ligand, generates an autocrine factor that activates EGFR and subsequently Erk during corneal epithelial healing [3,13,17]. To examine its possible involvement directly, we employed CRM197, which is known to neutralize HB-EGF . However, CRM197 did not inhibit the insulin-stimulated activation of EGFR, and had no affect on healing of wounded THCE cells (data not shown). This suggests that EGFR phosphorylation results from some other intracellular mechanism. Insulin promotes activation of NADPH oxidase that can lead to Src-dependent EGFR transactivation [19-21]. However, as with HB-EGF, we showed that NADPH oxidase is not involved in insulin-induced EGFR transactivation. The role of MMP activity in EGFR phosphorylation and healing of scratch wounds is consistent with the results of study showing that the MMP inhibitors, 1,10-phenanthrolin or GM6001, reduced epithelial cell migration and proliferation in cultured corneas and in THCE cells [3,13]. Therefore, our observations suggest that, during corneal epithelial wound healing, insulin induces the release of some other factor that is sensitive to MMP activity. EGFR ligands, such as EGF, HB-EGF, TGF-α, and amphiregulin, are expressed in the corneal epithelium and their expression, at least in part, mediates migration and proliferation during corneal epithelial wound healing [30,31]. Release of TGF-α and amphiregulin is stimulated by phorbol esters and sensitive to MMP inhibitors, and has been shown to be associated with wound healing [3,32]. We propose therefore that insulin-induced EGFR phosphorylation in epithelial wound healing is mediated by other EGFR ligands, such as TGF-α and amphiregulin rather than by HB-EGF, and that this event requires MMP activation.
Cell migration and proliferation have key roles in corneal wound healing. In this study, we showed that although cell proliferation is not influenced significantly via the PI3K and ERK pathways, insulin enhances cell migration. However, we found that it was not sufficient to fully restore the wound healing response of corneal epithelial cells, compared with repair in the presence of serum and growth factors. EGF, one of the growth factors involved in corneal wound healing, has been shown to be an important regulator of cell migration and proliferation [33,34], and the application of EGF together with insulin permitted complete repair of wounded corneal epithelial cells . Thus, it is likely that other factors involved in cell migration and proliferation are required for efficient repair of wounded corneal epithelial cells.
Rearrangement of the actin cytoskeleton is essential for cell migration during wound healing [35-37]. Block et al. have shown that in the cells at the wound edge, F-actin moves from a cortical localization to form stress fibers and lamellipodia. Furthermore, inhibition of EGFR activation suppressed the formation of stress fibers and lamellipodia. Consistent with these results, submembranous cortical actin decreased in the presence of insulin, and was replaced by stress fibers. Interestingly, stress fiber formation was reduced by inhibiting PI3K as well as by inhibiting activation of EGFR and MMP. In addition, the patterns of the lamellipodia were similar with those of the stress fibers even though the lamellipodia did not strongly stain for F-actin (Figure 5, arrow). PI3K controls actin cytoskeletal rearrangements via its effects on the Rho family of small GTPases [38,39]. Therefore, given that insulin-stimulated EGFR transactivation induces ERK activation but not PI3K activation, these results suggest that PI3K and the EGFR independently regulate actin cytoskeletal reorganization during insulin-enhanced cell migration. The mechanism of EGFR/ERK regulation of cytoskeletal rearrangement during the migration of corneal epithelial cells needs further investigation.
In conclusion, our results demonstrate that insulin stimulates the phosphorylation of EGFR in corneal epithelial cells and that this event requires the activation of MMPs. Furthermore, insulin-induced EGFR transactivation enhances the migration of corneal epithelial cells. Our findings will be useful for further studies of the mechanism by which insulin signaling is involved in the corneal wound healing process, and imply that EGFR could serve as a guide for developing therapeutic targets against the corneal wounds in insulin-dependent diabetes.
We thank Eek-hoon Jho for supporting this study with suggestions, and critical reading of the manuscript. We also thank Hyun-Jung Kim for technical assistance. This work was supported by grant (R01-2005-000-11271-0) from the Basic Research Program of the Korea Science & Engineering Foundation.
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