Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells

Abstract

Purpose: Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells.

Methods: Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP.

Results: In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor-sensitive manner.

Conclusions: HB-EGF released on wounding acts as an autocrine-paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.

Figure 1

Time course of tyrosine phosphorylation of EGFR and ERK in wounded THCE cells. THCE cells were cultured in 100-mm dishes and starved in KBM overnight. Cells were extensively injured by sequential comb scratching and incubated for different time points in KBM. (A) Wounded THCE cells were lysed and 600 μg protein of each reaction was immunoprecipitated (IP) with 10 μg agarose-conjugated rabbit anti-EGFR, subjected to SDS-PAGE, and immuno-blotted with mouse anti-PY99 antibody (top). After stripping, the membrane was reprobed with EGFR antibody (bottom) to assess the amount of protein precipitated. (B) To assess ERK phosphorylation, 10 μg cell lysates of the same samples were subjected to Western blot analysis with either anti-ERK2 (ERK2) or anti-phospho-ERK1 and 2 (pERK). The figure is a representative of three independent experiments. Left: molecular mass (kDa).

Figure 2

Corneal epithelial wound healing in cultured porcine corneas. (A) Representative epithelial wound closure in cultured porcine corneas treated with different reagents. Porcine corneas were injured, and one cornea was stained immediately with Richardson staining solution to show the area of initial wound (T0) (a). Injured corneas were cultured for 48 hours in MEM (control, b), MEM containing 50 ng/mL HB-EGF (c), 1 μM AG1478 (d), 100 μM GM6001 (e), 100 μM GM6001 + 50 ng/mL HB-EGF (f), 10 μg/mL CRM197 (g), or 10 μg/mL CRM197 + 50 ng/mL HB-EGF (h). Bar, 4 mm. (B) Changes in the extent of healing in cultured porcine corneas treated with different reagents. (□) Treatment with inhibitors alone; () treatments with in the presence of HB-EGF. The coverage of the wound (0% for no migration and 100% for a complete covering of the wound bed) was then calculated. Statistically, there are significant decreases of corneal epithelial healing rate in AG1478-, GM6001-, and CRM197-treated groups compared with MEM-treated (cont, **P < 0.01). Data are the mean ± SE of at least six corneas from two or more independent experiments.

Figure 3

Healing of scratch wound in cultured human corneal epithelial cells. Growth factor-starved THCE cell monolayers cultured in 12-well plates were injured with a sterile 0.1- to 10-μL pipet tip (KBM d0). Cells were incubated in KBM-plus for 24 hours (KBM d1), in KBM containing 0.5 μM AG1478 (AG d1), 0.5 μM AG1478 with 50 ng/mL HB-EGF (AG+HB d1), 50 μM GM6001 (GM d1), 50 μM GM6001 with 50 ng/mL HB-EGF (GM+HB d1), 10 μg/mL CRM197 (CRM d1), or 50 ng/mL HB-EGF (HB d1). Photomicrographs represent one of four samples. Bar, 100 μm.

Figure 4

Effects of HB-EGF and EGFR inhibitors on wound-induced EGFR phosphorylation. Growth factor-starved THCE cells were pretreated with 10 μg/mL HB-EGF neutralizing antibody, 0.5 μM AG1478, 50 μM GM6001, 10 μg/mL CRM197 (A) or 0 to 50 μM GM6001 (B) for 20 minutes and then wounded by comb-scratching, with unwounded (W −) serving as the control. Cells were lysed 15 minutes after wounding. For each reaction, 600 μg of protein was immunoprecipitated (IP) by 10 μg EGFR antibody and immunoblotted by mouse anti-PY99 (top), followed by reprobing with EGFR antibody (bottom). Data are representatives of three independent experiments. Left: molecular mass (kDa).

Figure 5

Wound-induced release of soluble HB-EGF. CM was collected at indicated times after extensive wounding of THCE cells and combined with 2 NaCl extract of cell surface proteins. The mixture was then applied to a heparin HP affinity column to purify heparin-binding proteins. Bound proteins were eluted. The eluents were used to stimulate growth-factor–starved THCE cells and cell lysates were subjected to immunoprecipitation (IP: EGFR) and/or immunoblot analysis with pY99 antibody (WB: pY99) for EGFR phosphorylation, anti-phospho-ERK1/2 (WB: pERK1/2) for ERK phosphorylation and anti-ERK for normalization of protein loading. Lane 1: KBM; lane 2: eluent from nonwounded cells; lanes 3 to 5: the eluents from wounded, 15, 30, and 60 minutes, respectively, THCE cells; lane 6: KBM plus 50 ng/mL HB-EGF.

Figure 6

Wound-induced HB-EGF-AP release. THCE cells were stably transfected with plasmid pHB-EGF-AP. HB-EGF-AP–expressing cells were cultured on six-well plates and one group was pretreated with GM6001 (50 μM) for 1 hour. Cells, GM6001 treated (◆) and non-treated (■), were then scratched sequentially with a comb, washed, and cultured in KBM for up to 90 minutes, with the nonwounded cells serving as the control (▲). Fifteen microliters were taken from each group at indicated times and the AP activity in the media were measured. The results are expressed in relative light units. Data represents the mean ± SD of results in triplicate wells from a representative experiment. Similar results were obtained in two independent experiments.