Role of small GTPase Rho in regulating corneal epithelial wound healing

Abstract

Purpose: To determine the role of small GTPase Rho and its relation with epidermal growth factor receptor (EGFR) in mediating corneal epithelial wound healing.

Methods: Rho activity in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, and primary HCECs was assessed by pull-down assay followed by Western blotting. Rho functions were inhibited with specific inhibitor exoenzyme C3 (C3) and confirmed by knockdown with small interference RNA (siRNA) transfection. Effects of Rho inhibition on wound healing were determined in porcine corneal organ culture and HCEC scratch wound models. Effects of C3 on cell proliferation and focal adhesion formation were determined by BrdU incorporation assay and immunocytochemistry, respectively.

Results: Wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) induced rapid and strong RhoA activation. HB-EGF-, but not wounding-, enhanced RhoA activity was sensitive to EGFR inhibition. In corneal organ and cell culture models, C3 attenuated spontaneous and HB-EGF-induced wound closures, confirmed by delayed wound healing in cells transfected with RhoA siRNA. C3 also retarded spontaneous wound healing in the presence of hydroxyurea, a cell cycle blocker. C3 significantly reduced the number of BrdU-positive cells near the leading edge. Treatment with C3 resulted in the disruption of the cortical actin cytoskeleton and in the disappearance of paxillin-containing focal adhesion and lamellipodia.

Conclusions: Wounding induces RhoA activation through an EGFR-independent pathway. Rho activity is required for modulating cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding.

Figure 1

RhoA is activated by wounding, LPA, and HB-EGF in THCE cells. (A) Growth factor-starved THCE cells were extensively wounded and incubated for the indicated time. Active RhoA protein was pulled down by incubating cell lysates with GST-fusion protein (GST-C21), separated on SDS-PAGE, and detected by Western blot analysis using mouse anti–RhoA antibody. Aliquots of respective cell lysate were also used to analyze total RhoA protein by Western blot. (B) Growth factor-starved THCE cells were stimulated with 1 μM LPA, 50 ng/mL HB-EGF, or extensive wounding for 10 minutes before they were lysed. Active and total RhoA proteins were determined as in (A). Results are representative of four independent experiments.

Figure 2

Wound-induced RhoA activation requires Ca²⁺ and Src, but not EGFR, activities. (A) Growth factor-starved THCE cells were pre-treated with 1 μM EGFR inhibitor AG1478 for 1 hour and then stimulated with 50 ng/mL HB-EGF or extensive wounding for 10 minutes before they were lysed. Active RhoA was pulled down by incubating cell lysates with GST-fusion protein (GST-C21), separated on SDS-PAGE, and detected by Western blot analysis using rabbit anti–RhoA antibody. Cell lysates were also subjected to Western blot with phospho-EGFR (Tyr1068) antibody. (B) Growth factor-starved THCE cells were pretreated with 50 μM Ca²⁺ blocker BAPTA/AM (BA), 40 nM PKC inhibitor staurosporine (Stau), or 25 μM Src inhibitor PP2 (PP2) before they were extensively wounded and lysed. Active and total RhoA proteins were determined as in Figure 1. Results are representative of three independent experiments.

Figure 3

Effects of exoenzyme C3 on RhoA activation and Rho GTPase expression. (A) Growth factor-starved THCE cells were pre-treated with 10 μg/mL exoenzyme C3 for 3, 8, or 24 hours before they were extensively wounded for 10 minutes. RhoA activation was determined as described in Figure 1. (B) Growth factor-starved THCE cells were pretreated with 10 μg/mL exoenzyme C3 for 24 hours, and cytotoxicity was assayed by measuring LDH release using a cytotoxicity kit. Values are expressed as mean ± SEM in fold increase compared with the negative control; 2% Triton X-100 treatment served as a positive control, as recommended by the manufacturer’s instruction. (N = 3) (C) Growth factor-starved THCE cells were pretreated with 10 μg/mL exoenzyme C3 for 24 hours and mRNA expressions of RhoA, RhoB, and RhoC were determined by RT-PCR with GAPDH as the internal control.

Figure 4

Exoenzyme C3 attenuates spontaneous and HB-EGF-enhanced wound healing in cultured porcine corneas. A 4-mm diameter epithelial wound was made and allowed to heal in MEM containing 50 ng/mL HB-EGF, with or without 10 μg/mL exoenzyme C3, for 48 hours. Wounded corneas were stained with Richardson staining solution to show the initial (0 hr) and the remaining (48 hr after wounding [p.w.]) wounds. Micrographs (A) represent one of four samples performed each time (scale bar, 1 mm). (B) Statistical analysis of the extent of healing. Values are expressed as mean ± SEM (N = 4). **P < 0.001 (Student’s t-test).

Figure 5

Inhibition of RhoA activity attenuates scratch wound healing in THCE cells. (A, B) Growth factor-starved THCE cells were pretreated with 10 μg/mL exoenzyme C3 for 24 hours and then wounded with a 0.1- to 10-μL pipette tip. Cells were allowed to heal in KBM containing 50 ng/mL HB-EGF, with or without 10 μg/mL C3. Wound closure was photographed immediately (0 hr) or 24 hours (24 hr) after wounding. Micrographs (A) represent one of three samples performed each time. Original magnification, × 100. Scale bar, 200 μM. (B) Statistical analysis of extent of healing. (C, D) THCE cells were transfected with RhoA siRNA or nonsilencing siRNA. Forty-eight hours after transfection, cells were wounded and allowed to heal in defined keratinocyte SFM for 30 hours. After wound closure was photographed, cells were lysed, and RhoA protein expression was examined by Western blotting. Micrographs (C) represent one of three samples. Original magnification, × 100. Scale bar, 200 μm. (D) Statistical analysis of extent of healing with RhoA expression (inset). Values are expressed as mean ± SEM (N = 3). *P < 0.05; **P < 0.001 (Student’s t-test).

Figure 6

Exoenzyme C3 inhibits THCE cell proliferation. Growth factor-starved THCE cells were pre-treated with 10 μg/mL C3 for 16 hours, then wounded with a 1- to 10-μL pipette tip and allowed to reepithelialize in KBM containing BrdU, with or without 10 μg/mL C3, for 24 hours. BrdU incorporation was visualized using anti–BrdU antibody and an FITC-conjugated secondary antibody. Corresponding nuclear staining was performed with DAPI. Micrographs (A) represent one of five random fields visualized, and positively staining cells (green) are proliferating cells. Original magnification, ×100. Scale bar, 200 μm. (B) Statistical analysis of the numbers of BrdU-positive cells per field. Values are expressed as mean ± SEM (N = 5). **P < 0.001 (Student’s t-test).

Figure 7

Exoenzyme C3 attenuates scratch wound healing in the presence of hydroxyurea. Growth factor-starved THCE cells were pre-treated with 10 μg/mL exoenzyme C3 for 16 hours and then wounded with a 0.1- to 10-μL pipette tip. Cells were allowed to heal in KBM containing 10 μg/mL C3, with or without 0.5 mM hydroxyurea (HU). Wound closure was photographed immediately (0 hr) or 24 hours (24 hr) after wounding. Micrographs (A) represent one of three samples performed each time Original magnification, ×100. Scale bar, 200 μm. (B) Statistical analysis of the extent of healing. Values are expressed as mean ± SEM (N = 3). *P < 0.05; **P < 0.001 (Student’s t-test).

Figure 8

Exoenzyme C3 disrupts actin organization and focal adhesion in THCE cells. Growth factor-starved THCE cells were pretreated with 10 μg/mL C3 for 24 hours. Confluent cells (B) were wounded with a 0.1- to 10-μL pipette tip and further incubated in the presence or absence of C3 for 1 hour. Subconfluent (A) and wounded (B) cells were then fixed and incubated with mouse IgG1 isotype (negative control) or paxillin antibody, followed by visualization with FITC-conjugated secondary antibody (green). Actin organization was visualized with rhodamine-phalloidin (red). (asterisks) Wound edge. Arrows: intercellular gaps. Arrowheads: Paxillin-positive cell protrusions. Details are displayed in two right panels in (B). Results are representative of two independent experiments. Original magnification ×400. Scale bar, 50 μm.