Am J Physiol Cell Physiol 295: C378 –C387, 2008.
First published May 21, 2008; doi:10.1152/ajpcell.90624.2007.
Rho kinases regulate corneal epithelial wound healing
Jia Yin and Fu-Shin X. Yu
Departments of Ophthalmology, Anatomy and Cell Biology, Kresge Eye Institute, Wayne State University School of Medicine,
Detroit, Michigan
Submitted 13 December 2007; accepted in final form 15 May 2008
corneal epithelial cells; cell migration; cell proliferation; cell-matrix
adhesion; cell-cell junctions
is continuously subjected to physical,
chemical, and biological insults, often resulting in a wound and
the loss of barrier functions. The proper healing of corneal
wounds is vital to maintaining a clear, healthy cornea and to
preserving vision (24). The corneal epithelium responds rapidly to injury by migrating as a sheet to cover the defect and to
reestablish its barrier function (24). Successful wound healing
involves a number of processes including cell migration, proliferation, cell-matrix adhesion, and tissue remodeling, which
are often driven by growth factors and other factors released
coordinately into the injured area by epithelial cells (27, 65).
Prominent among these epithelium-derived factors are ligands
for epidermal growth factor (EGF) receptor (EGFR), such as
EGF and heparin-binding EGF-like growth factor (HB-EGF;
Ref. 24). In addition, other cellular components, such as ATP
and lipid mediator lysophosphatidic acid (1-acyl-2-hydroxysn-glycero-3-phosphate, LPA), have been shown to be released
from the injured cells to enhance epithelial migration and
wound healing in the cornea (54, 57, 60, 64).
The Rho family of small GTPases, including Rho, Rac, and
Cdc42, are small monomeric G proteins that cycle between an
inactive GDP-bound form and an active GTP-bound form and
THE CORNEAL EPITHELIUM
Address for reprint requests and other correspondence: F.-S X. Yu, Kresge
Eye Institute, Wayne State Univ. School of Medicine, 4717 St. Antoine Blvd,
Detroit, MI, 48201 (e-mail: [email protected]).
C378
regulate actin cytoskeleton, cell migration, and proliferation
(10, 15). Rho regulates actin polymerization, resulting in the
formation of stress fibers and the assembly of focal adhesion
complex (19, 32, 38). Rho has been implicated in cell migration, actin organization, focal adhesion formation, as well as
adherens and gap junction assembly in the corneal epithelium
(3, 31, 40). We recently showed that wounding, HB-EGF, and
LPA induced RhoA activation, and Rho activity is required for
modulating both cell migration and proliferation, through cytoskeleton reorganization and focal adhesion formation in response to wounding in corneal epithelial cells (63).
Rho kinases (ROCKs), the first identified and best characterized Rho downstream effectors, are protein serine/threonine
kinases with a molecular mass of ⬃160 kDa. Two isoforms
have been identified: ROCK 1 and ROCK 2 (14, 21, 22, 28).
They were initially characterized for their roles in mediating
the formation of RhoA-induced stress fibers and focal adhesions, as well as actomyosin contractility, through their effects
on the phosphorylation of myosin light chain (21, 39, 47). In
addition, they are involved in many other cell behaviors such
as cell-substrate and cell-cell adhesion, cell migration, differentiation, apoptosis, and proliferation (23, 29, 39, 44, 58).
ROCKs have been suggested to play roles in many pathological conditions such as neurological and cardiovascular disorders (23, 29). In the cornea, ROCKs have been suggested to be
involved in epithelial differentiation (49), cell cycle progression (4), cell-cell adhesion (3), endothelial barrier integrity (8,
42, 43), stromal cell phenotype conversion (2, 11), cytoskeleton reorganization, contractility (17), and cell-matrix interaction (16, 20, 34). However, the precise role of ROCKs during
corneal epithelial wound healing and the underlying mechanisms remain elusive.
In the present study, we demonstrated that ROCK 1 and
ROCK 2 are activated in response to wounding, LPA, and
HB-EGF stimulations and that inhibition of ROCK enhances
wound closure by modulating cell migration, proliferation,
cell-matrix adhesion, and cell-cell adhesion in HCECs.
MATERIALS AND METHODS
Materials. Defined keratinocyte serum-free medium (DK-SFM)
and TRIzol Reagent were purchased from Invitrogen (Grand Island,
NY). Keratinocyte basal medium (KBM) was from BioWhittaker
(Walkersville, MD). Human recombinant HB-EGF was obtained from
R&D Systems (Minneapolis, MN). The ROCK inhibitor Y-27632 was
from Calbiochem (La Jolla, CA). Antibodies against phosphoERK1/2 (p42/p44), ERK2 (p42 MAPK), ROCK 1, and ROCK 2 were
from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase conjugate secondary antibodies were from Bio-Rad (Hercules, CA). Antibodies against phospho-myosin phosphatase target
The costs of publication of this article were defrayed in part by the payment
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0363-6143/08 $8.00 Copyright © 2008 the American Physiological Society
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Yin J, Yu F-S. Rho kinases regulate corneal epithelial wound
healing. Am J Physiol Cell Physiol 295: C378 –C387, 2008. First
published May 21, 2008; doi:10.1152/ajpcell.90624.2007.—We have
previously shown that Rho small GTPase is required for modulating
both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the
present study, we investigated the role of Rho kinases (ROCKs),
major effectors of Rho GTPase, in mediating corneal epithelial wound
healing. Both ROCK 1 and 2 were expressed and activated in THCE
cells, an SV40-immortalized human corneal epithelial cell (HCEC)
line, in response to wounding, lysophosphatidic acid, and heparinbinding EGF-like growth factor (HB-EGF) stimulations. The ROCK
inhibitor Y-27632 efficiently antagonized ROCK activities without
affecting Rho activation in wounded HCECs. Y-27632 promoted
basal and HB-EGF-enhanced scratch wound healing and enhanced
cell migration and adhesion to matrices, while retarded HB-EGF
induced cell proliferation. E-cadherin- and ␤-catenin-mediated cellcell junction and actin cytoskeleton organization were disrupted by
Y-27632. Y-27632 impaired the formation and maintenance of tight
junction barriers indicated by decreased trans-epithelial resistance and
disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a
role in regulating corneal epithelial wound healing.
RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
AJP-Cell Physiol • VOL
proteins of ROCK 1, ROCK 2, MYPT, and ERK in cell lysates were
detected by Western blotting using corresponding antibodies.
Scratch wound healing studies. Growth factor-starved THCE cells
were wounded with a sterile 0.1–10 ␮l pipette tip (TipOne, USA
Scientific, Ocala, FL) to remove cells by two perpendicular linear
scrapes. After suspended cells were washed away, the cells were refed
with KBM containing 50 ng/ml HB-EGF and/or 10 ␮M Y-27632.
Wound closure was photographed both immediately and 24 h postwounding at the same spot with an inverted Zeiss microscope
equipped with a SPOT digital camera. The extent of healing was
defined as the ratio of the difference between the original and the
remaining wound areas vs. the original wound area.
Determination of cell proliferation. Cell proliferation was determined by two methods: BrdU incorporation and MTT cell proliferation assay. BrdU incorporation assay was carried out following the
manufacturer’s instructions. Briefly, THCE cells were grown on glass
chamber sides, growth factor starved, and pretreated with 10 ␮M
Y-27632 for 1 h. Confluent cells were wounded with a sterile 0.1–10
␮l pipet tip and refed with fresh KBM containing 50 ng/ml HB-EGF
and/or 10 ␮M Y-27632 and BrdU-labeling reagent for 24 h. Cells
were fixed with 3.7% formaldehyde, permeablized with 0.1% Triton
X-100, blocked with 5% horse serum and 1% BSA in PBS for 1 h at
room temperature, and then incubated with anti-BrdU antibody (1:10)
overnight at 4°C followed by incubation with anti-mouse IgG-fluorescent antibody (1:10). Cell proliferation was also measured using an
MTT cell proliferation assay kit, following the manufacturer’s instruction (American Type Culture Collection). Growth factor-starved
THCE cells were cultured in KBM containing 50 ng/ml HB-EGF
and/or 10 ␮M Y-27632 for 24 h. Cells were incubated in 100 ␮l KBM
containing 10 ␮l MTT reagent for 2 h. Detergent reagent (100 ␮l) was
added to each well and cells were further incubated for 2 h. Absorbance at 595nm was quantified on a GENios Fluorometer.
Boyden chamber analysis for cell migration. Growth factor-starved
THCE cells were pretreated with 10 ␮M Y-27632 for 30 min. Cells
were detached by 0.05% trypsin and 0.53 mM EDTA, washed with
10% donor calf serum in PBS to neutralize the trypsin, and adjusted
to an equal cell number of 6 ⫻ 104/ml; 170 ␮l of cell suspension were
added to top wells of the Boyden chambers, separated by a polycarbonate membrane (pore size: 14 ␮m) precoated with FNC (a 1:3 mix
of fibronectin and collagen 1) from the lower chambers loaded with
basal medium with or without 50 ng/ml HB-EGF. Cells were
allowed to migrate for 3 h. After incubation, cells on the top of
the filter membrane were removed by scraping. The filter membrane was then stained with a modified staining kit (Diff-Quik;
Dade Behring, Dudingen, Switzerland). Cell migration was quantified as the number of migrated cells on the lower surface of the filter
membrane in three random fields under ⫻400 magnification.
Cell-matrix adhesion assay. The adhesion of THCE cells to two
matrices, FNC (a 1:3 mix of fibronectin and collagen 1) and Matrigel
(BD Biosciences), were tested. The 96-well plates were coated with
FNC or Matrigel (1:5) diluted in PBS for 60 min before blocked with
10 mg/ml heat-denatured BSA for 30 min to prevent nonspecific
binding. Fifty microliters of basal medium containing 100 ng/ml
HB-EGF, and/or 20 ␮M Y-27632 or 20 ␮g/ml C3 were added to each
well. Growth factor-starved THCE cells were pretreated with 10 ␮M
Y-27632 for 30 min or 10 ␮g/ml C3 for 24 h. Cells were detached by
0.05% trypsin and 0.53 mM EDTA, washed with 10% donor calf
serum in PBS, and adjusted to an equal cell number of 1 ⫻ 106/ml. 50
␮l of cell suspension was added to each well (final concentration: 50
ng/ml HB-EGF, 10 ␮M Y-27632, and 10 ␮g/ml C3). Cells were
allowed to adhere to the matrices for 30 min; nonadherent cells were
then washed off with PBS. Cells were fixed with 5% glutaldehyde and
stained with 0.1% crystal violet solution for 60 min. Plates were
extensively washed with water to remove excessive staining, and the
dye was solubilized with 10% acetic acid. Absorbance at 570 nm was
quantified on a GENios Fluorometer.
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subunit 1 (MYPT1, Thr-696) and MYPT1, as well as recombinant
MYPT1 protein were from Upstate Signaling Technologies (Lake
Placid, NY). RhoA and occludin antibodies were from Cell Signaling
(Danvers, MA) and Zymed Laboratory (South San Francisco, CA),
respectively. 5-Bromo-2⬘-deoxy-uridine (BrdU) labeling and detection
kit were from Roche Applied Science (Indianapolis, IN). MTT cell
proliferation assay kit was purchased from American Type Culture
Collection (Manassa, VA). The 12-well Boyden chamber was from
Neuroprobe (Cabin John, MD). Polycarbonate membranes (14-␮m
pores) were from Osmonics (Livermore, CA). Fibronectin-collagen
coating mix (FNC, a 1:3 mix of fibronectin and collagen 1) was from
Athena Environmental Service (Baltimore, MD). Growth factor reduced Matrigel matrix was from BD Biosciences (San Jose, CA).
Transwell Polycarbonate Transwell plates (0.4-␮m pore size) were
from Costar (Cambridge, MA). Mouse E-cadherin and mouse ␤-catenin antibodies were from BD Biosciences. Rhodamine phalloidin was
from Invitrogen (Carlsbad, CA). FITC-conjugated antibody against
mouse or rabbit IgG was from Jackson ImmunoResearch Laboratories
(West Grove, PA). All other reagents and chemicals were purchased
from Sigma-Aldrich (St. Louis, MO).
Cell culture and extensive wounding study. THCE cells, an SV40immortalized human corneal epithelial cell (HCEC) line (5), were
grown in DK-SFM in a humidified 5% CO2 incubator at 37°C and
growth factor starved in KBM for 16 h before experiments. To create
extensive wounding for biochemistry studies, cells cultured on
100-mm dishes were wounded by multiple linear scratches using a cut
of 48-well shark’s tooth comb for DNA sequencing gel (Bio-Rad,
Hercules, CA) going from one side of the dish to the other. The dish
was then rotated, and scrapes were made similarly to the original
scrapes at 45, 90, and 135°. After wounding or other treatments, cells
were lysed with RIPA buffer (150 mM NaCl, 100 mM Tris 䡠 HCl pH
7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM NaF, 100
mM sodium pyrophosphate, 3.5 mM sodium orthovanadate, proteinase inhibitor cocktail, and 0.1 mM PMSF), and protein concentrations
were determined using a Micro BCA protein assay kit (Pierce,
Rockford, IL).
RNA isolation and semiquantitative RT-PCR. Total RNA was
isolated from THCE cells using the TRIzol solution according to the
manufacturer’s instructions (Invitrogen), and 2 ␮g of total RNA were
reverse transcribed with a first-strand synthesis system for RT-PCR
(SuperScript, Invitrogen). cDNA was amplified by PCR using primers
for human ROCK 1 (sense 5⬘-GAAGAAAGAGAAGCTCGAGAAGAAGG-3⬘; antisense 5⬘-ATCTTGTAGCTCCCGCATCTGT-3⬘)
and ROCK 2 (sense 5⬘-AATTCACTGTGTTTCCCTGAAGATA-3⬘;
antisense 5⬘-TTCATTTTTCCTTGATTGTATGGAA-3⬘; Ref. 9). The
PCR products were subjected to electrophoresis on 1% agarose gels
containing ethidium bromide. Stained gels were captured using a
digital camera.
Rho and ROCK activity assays and Western blotting. THCE cells
were pretreated with 10 ␮g/ml Rho inhibitor Clostridium botulinum
exoenzyme C3 (C3) for 24 h or 10 ␮M ROCK inhibitor Y-27632 for
1 h before being extensively wounded. Five hundred micrograms of
proteins were immunoprecipitated with 4 ␮g ROCK 1 or ROCK 2
antibody overnight at 4°C. Immunocomplexes were incubated with 20
␮l of protein A/G plus agarose for 3 h at 4°C, washed three times with
PBS, and resuspended in 50 ␮l of Tris-ATP buffer (50 mM Tris 䡠 HCl,
pH 7.5, 0.1 mM EGTA, 0.1% ␤-mercaptoethanol, 10 mM magnesium
acetate, and 100 ␮M ATP). The kinase assay was performed following Upstate’s protocol in the presence of 1 ␮g of recombinant MYPT1
at 30°C for 30 min. Phosphorylation and total levels of MYPT1
recombinant protein were detected using phospho-MYPT1 and
MYPT1 antibodies, respectively. The activation status of Rho was
assayed using fusion protein GST-21 as described previously (63).
Briefly, 1 mg cell lysate was incubated with GST-C21 fusion protein
and glutathione S-transferase beads. The beads were washed three
times with lysis buffer, and bound GTP-Rho was detected by immunoblot analysis with RhoA antibody. ERK phosphorylation and total
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RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
RESULTS
We first determined the expression of ROCK 1 and ROCK
2 in HCECs. As shown in Fig. 1, the mRNAs for both ROCK
1 and 2 were detected in THCE cells. To assess if ROCK was
involved in HCEC response to epithelial injury, the kinase
activities of ROCK 1 and 2 were examined at different time
points postwounding. As shown in Fig. 2, ROCK activities,
assessed by the phosphorylation of exogenously added ROCK
substrate MYPT1, were increased as early as 10 min (the
earliest time tested) and remained elevated 2 h postwounding,
while the levels of total ROCK 1 and ROCK 2 proteins in cell
lysates and exogenous MYPT1 protein were relatively unchanged. Since we have observed that both HB-EGF and LPA
enhanced corneal wound healing and induced RhoA activation
in vitro (59, 60, 63), we also assessed the effects of these
factors on ROCK activation. Both LPA and HB-EGF increased
ROCK kinase activities, suggesting that the RhoA/ROCK
pathway may act as a downstream effector of these extracellular stimuli. Phosphorylation of ERK1/2 confirmed cell activation in response to wounding, HB-EGF and LPA.
To determine whether ROCKs function as Rho downstream
effectors in HCECs, we first assessed the effects of ROCK
Fig. 1. Rho kinase (ROCK) 1 and ROCK 2 are expressed in human corneal
epithelial cells. RT-PCR was performed to determine the mRNA expression of
ROCK 1 and 2 in THCE cells. PCR products were separated and stained as
described in MATERIALS AND METHODS.
AJP-Cell Physiol • VOL
Fig. 2. ROCK 1 and 2 kinase activities are increased in response to wounding,
heparin-binding EGF-like growth factor (HB-EGF), and lysophosphatidic acid
(LPA). Growth factor-starved THCE cells were extensively wounded for
various time (10 –120 min) or stimulated with 50 ng/ml HB-EGF or 10 ␮M
LPA for 10 min and then lysed. Cell lysates were immunoprecipitated (IP) with
ROCK 1 (A) or ROCK 2 (B) antibodies, and ROCK kinase activities were
indicated by Thr-696 phosphorylation of recombinant myosin phosphatase
target subunit 1 protein (p-MYPT) by respective immunoprecipitants. Reprobing stripped membranes with MYPT antibody indicated equal addition of
recombinant MYPT protein (MYPT). Total ROCK 1 and 2, phosphorylated
ERK (p-ERK), and ERK proteins were also determined by Western blotting
(C). Figure represents 3 independent experiments.
inhibitor Y-27632 and Rho inhibitor exoenzyme C3 on Rho
and ROCK activities, respectively. Wounding-enhanced kinase
activities of both ROCK 1 and 2, indicated by increased
MYPT1 phosphorylation, were attenuated by both C3 and
Y-27632, suggesting that ROCKs act downstream to Rho in
wounded HCECs and Y-27632 was effective in inhibiting
ROCK activities (Fig. 3, A and B). Wounding-induced Rho
activation, however, was unaffected by Y-27632, confirming
Fig. 3. ROCK 1 and 2 act downstream to Rho in wounded HCECs. Growth
factor-starved THCE cells were pretreated with 10 ␮g/ml Rho inhibitor
exoenzyme C3 (C3) for 24 h or 10 ␮M ROCK inhibitor Y-27632 (Y) for 1 h
before being extensively wounded for 30 min. ROCK 1 (A) and ROCK 2 (B)
kinase activities were determined as described in Fig. 2. C: active RhoA
protein was pulled down by incubating cell lysates with GST-C21, separated
on SDS-PAGE, and detected by Western blot analysis using RhoA antibody.
Total RhoA protein in cell lysates was determined by Western blot analysis.
Figure represents 2 independent experiments.
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Immunostaining of E-cadherin, ␤-catenin, occludin, and actin.
THCE cells cultured on 4-well glass chamber slides (E-cadherin and
␤-catenin), or 12-mm Transwell inserts (occludin) were fixed with
3.7% formaldehyde, permeablized with 0.1% Triton X-100, blocked
with 1% BSA in PBS for 1 h at room temperature, and then incubated
with E-cadherin (1:250), ␤-catenin (1:50), occludin (1:100) antibodies, or mouse or rabbit IgG isotype controls overnight at 4°C, followed
by incubation with FITC-conjugated secondary antibody (1:100) and
rhodamine-phalloidin (1:50) to visualize actin filaments. The slides
were then washed in PBS and mounted with Vectashield mounting
medium with DAPI and examined under a Carl Zeiss fluorescence
microscope Axioplan 2 equipped with an ApoTome digital camera.
Negative controls (mouse or rabbit IgG isotypes) exhibited no fluorescence (data not shown).
Measurement of transepithelial electrical resistance. THCE cells
were plated on 12-mm Transwell filter with 0.4-␮m pore polyester
membrane insert (1⫻106 cells per insert) and cultured in DK-SFM
with or without 10 ␮M Y-27632. Transepithelial electrical resistance
(TER) was measured with STX-2 electrodes and an EVOM voltohmmeter (World Precision Instruments, Sarasota, FL) daily. The background TER of blank Transwell filters was subtracted from the TER
of cell monolayers.
Statistical analysis. Results are means ⫾ SE. Statistical parameters
were ascertained by software (SigmaStat) with Student’s t-test between two groups. P ⬍ 0.05 indicates a significant difference.
RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
Fig. 4. Y-27632 promotes basal and HB-EGF-enhanced scratch wound healing in THCE cells. Growth factor-starved THCE cells were wounded with a
0.1–10 ␮l pipet tip and allowed to heal in keratinocyte basal medium containing 50 ng/ml HB-EGF with or without 10 ␮M Y-27632. Wound closure was
photographed immediately after wounding (0 h) or 24 h postwounding (24 h).
Micrographs (A) represent 1 of 3 samples performed each time. B: statistical
analysis of healing extent. Values are means ⫾ SE (n ⫽ 3). *P ⬍ 0.05
(Student’s t-test).
AJP-Cell Physiol • VOL
of ROCK inhibition on these processes in cultured HCECs. To
elucidate the effects of Y-27632 on cell proliferation, BrdU
incorporation assay was performed (Fig. 5A). While unwounded cells exhibited minimum number of BrdU-positive
cells; in the wounded epithelial monolayer, there were some
proliferating cells near the leading edge, indicated by positive
BrdU staining. Inhibition of ROCK activities by Y-27632 had
a minimal effect on the basal cell proliferation, as there was no
apparent change in the number of BrdU-positive cells. HBEGF greatly increased the number of BrdU-positive cells,
indicating a mitogenic effect of the growth factor on HCECs.
This HB-EGF-mediated increase in cell proliferation was diminished in the presence of Y-27632. The effects of ROCK
inhibition on cell proliferation were also examined using MTT
cell proliferation assay (Fig. 5B). Exogenously added HB-EGF
significantly increased cell proliferation rate by 70%, compared with the control. While it exhibited no effect on basal
cell proliferation, Y-27632 attenuated the HB-EGF-enhanced
proliferation to a level similar to that of the control, indicating
that the enhancing effect of Y-27632 on wound healing observed in Fig. 4 may result from mechanisms other than cell
proliferation.
We next investigated the effects of Y-27632 on HCEC
migration by Boyden chamber assay (Fig. 6). Consistent with
a previous report (61), the presence of HB-EGF in the bottom
chamber increased the migration of HCECs. Y-27632 significantly enhanced HCEC migration toward FNC (a 1:3 mix of
fibronectin and collagen 1) and chemotaxis toward HB-EGF,
suggesting that ROCKs may negatively regulate cell motility.
HCEC adhesion to different matrices, FNC and diluted
Matrigel, was also investigated (Fig. 7). HB-EGF slightly
increased cell adhesion to FNC and Matrigel, while Y-27632
greatly enhanced cell adhesion both in the presence and absence of HB-EGF, suggesting the accelerated wound healing
by Y-27632 may result from enhanced cell-matrix adhesions.
Rho inhibitor C3, on the other hand, attenuated cell adhesion,
indicating that the differences between Rho and ROCKs in the
regulation of cell adhesion may account for their opposing
effects on wound closure.
To analyze whether ROCKs participate in the formation of
adherence junctions, the effects of Y-27632 on the cellular
distribution of E-cadherin were determined by immunostaining. In confluent control THCE cells, E-cadherin was localized
at cell-cell borders either as typical membrane protein or in a
diffuse pattern (Fig. 8A). Y-27632 treatment resulted in a
decrease in E-cadherin staining intensity and loss of continuity
at some cell-cell junctions (b). E-cadherin exhibited a more
defused staining pattern in the presence of HB-EGF (c). Remarkably, cells treated with Y-27632 and HB-EGF simultaneously appeared to be much larger in size and displayed a
more “spread and flattened” morphology with much reduced
E-cadherin staining at cell-cell junctions (d).
To understand the role of ROCKs in mediating adherence
junction in migratory cells, the effects of Y-27632 on actin
cytoskeleton and ␤-catenin staining in scratch-wounded THCE
cells were investigated (Fig. 8B). Migrating THCE cells displayed a strong continuous cortical actin network with apparent
actin bundle staining parallel to the wounding edge (a). The
presence of Y-27632 enhanced the actin bundle staining but
disrupted cortical actin staining at cell-cell junctions (arrowheads in e) at the leading edge. In control migratory cells,
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the linear relationship between Rho and ROCK in HCECs and
the specificity of Y-27632 (Fig. 3C).
To determine the role of ROCKs in epithelial wound healing, we assessed the effects of Y-27632 on epithelial wound
closure of THCE monolayer (Fig. 4). The presence of Y-27632
slightly increased the basal healing of THCE cells from 25% to
35.5% 24 h postwounding. Compared with the control, exogenously added HB-EGF significantly promoted epithelial
wound closure (43% healed), which was further enhanced by
Y-27632 to nearly 70%, suggesting that ROCKs may negatively regulate corneal epithelial wound healing. Since we
previously demonstrated that Rho inhibitor C3 delays wound
closure (63), Rho and ROCKs, albeit along the same pathway,
exert opposite effects on HCEC wound healing.
Since many cellular processes, such as proliferation, migration, and adhesion to extracellular matrix contribute to corneal
epithelial wound closure (24), we next investigated the effects
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RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
DISCUSSION
Fig. 5. Y-27632 attenuates HB-EGF-enhanced cell proliferation. A: growth
factor-starved THCE cells were pretreated with Y-27632 for 1 h then wounded
with a 1–10 ␮l pipette tip and allowed to reepithelialize in keratinocyte basal
medium containing 5-bromo-2⬘-deoxy-uridine (BrdU)-labeling reagent in the
presence of absence of 50 ng/ml HB-EGF with or without 10 ␮M Y-27632 for
24 h. BrdU incorporation was visualized using anti-BrdU antibody and an
FITC-conjugated second antibody. Corresponding nuclear staining was performed with DAPI (scale bar ⫽ 100 ␮M). Unwounded and untreated THCE
cells served as a negative control and showed minimal BrdU incorporation.
Figure 5A represents 2 independent experiments. B: growth factor-starved
THCE cells were incubated in the presence or absence of 50 ng/ml HB-EGF
with or without 10 ␮M Y-27632 for 24 h. Cell proliferation was determined
using an MTT cell proliferation kit as described in MATERIALS AND METHODS.
Values are means ⫾ SE (n ⫽ 4). **P ⬍ 0.001 (Student’s t-test).
␤-catenin staining was uninterrupted at cell-cell contact and
was also seen in some areas as “line of dots” structure (b).
Cells treated with Y-27632, however, displayed diminished
staining of ␤-catenin along cell-cell junctions and strong staining at the wounding edge (arrows in f), which colocalized with
thick actin bundles (h).
The role of ROCKs in the formation and maintenance of
tight junction-based corneal epithelial barrier was investigated
using TER measurement and occludin staining. To determine
the role of ROCKs in the maintenance of barrier function,
THCE cells were allowed to grow on porous Transwell membranes in growth medium and TER was measured daily. As
shown in Fig. 9A, the TER reading increased gradually from
⬃17 to ⬃220 ⍀ within 6 days, suggesting the formation of
tight junction barrier. These barrier-forming cells displayed
strong occludin immunoreactivity with a well-defined tight
junction like structure at cell-cell borders (Fig. 9C, a). From
day 6 to day 7, the TER reading continued to increase (control
4 – 6) in cells cultured in growth medium, whereas cells with
Y-27632 added to the culture medium had a decreased TER
reading (control 1–3). The net changes in the resistance after
drug treatment were significantly different from the controls
AJP-Cell Physiol • VOL
In the present study, we examined the role of ROCKs in
corneal epithelial wound healing and barrier formation. We
showed that both isoforms of ROCKs are expressed in HCECs
at mRNA and protein levels. Wounding, as well as LPA and
HB-EGF, rapidly and robustly increased the kinase activities of
ROCK 1 and ROCK 2. ROCK inhibitor Y-27632 was specific
and efficient in inhibiting ROCK kinase activities without
affecting Rho activity. Y-27632 accelerated basal and HBEGF-enhanced wound healing. Y-27632 attenuated cell proliferation induced by HB-EGF, promoted cell migration, and
enhanced cells adhesion to extracellular matrices; while Rho
inhibitor C3 attenuated wound closure (63) and cell adhesion.
ROCK activities were also required for cell-cell adhesion
mediated by E-cadherin and ␤-catenin, as well as the formation
and maintenance of barrier integrity. Our data indicate that
ROCKs play important roles in corneal epithelial homeostasis
and wound healing.
Fig. 6. Y-27632 promotes spontaneous haptotactic migration and HB-EGFinduced chemotaxis. Growth factor-starved THCE cells were pretreated with
10 ␮M Y-27632 and allowed to migrate towards fibronectin-collagen coating
mix (FNC)-coated polycarbonate membrane with or without 50 ng/ml HBEGF present in the bottom chamber for 3 h in Boyden Chamber assay.
Numbers of migrated cells per field were counted. Number of cells per field is
the average of wells counted. Values are means ⫾ SE (n ⫽ 3). **P ⬍ 0.001
(Student’s t-test).
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(Fig. 9A, inset). Interestingly, although the TER reading was
still relatively high, occludin staining was dramatically altered
in Y-27632-treated cells with more diffuse intracellular staining, suggesting that the disruption of cell-cell border localization of occludin preceded the dysfunction of tight junction
barrier in HCECs. To determine the role of ROCKs in barrier
formation, THCE cells were cultured in the presence of
Y-27632. Inhibition of ROCKs prevented the tight junction
barrier from forming, as indicated by low TER readings (⬍30
⍀; Fig. 9B, Y1–3) and by weak and defused occludin staining
(Fig. 9C, c). Withdrawal of Y-27632 from the culture medium
resulted in a rapid increase in TER readings (an increase of
⬃100 ⍀ within 24 h; Fig. 9B, Y4 – 6) and the reformation of
tight junction like structures (Fig. 9C, d) in THCE cells.
Western blot analysis revealed no detectable changes of occludin protein level in the control and Y-27632-treated cells
(data not shown). Taken together, these results suggest that
ROCKs play a role in the tight junction barrier formation
and that the inhibitory effect of Y-27632 on barrier formation was reversible.
RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
C383
Small GTPases of the Rho family, including Rho, Rac, and
Cdc42, are essential regulators of several cell functions, such
as actin cytoskeleton organization, cell migration, and proliferation (10, 15). Rho regulates actin polymerization, resulting
in the formation of stress fibers and the assembly of focal
adhesion complex. Rac and Cdc42 induce the formation of
filopodia and lamellipodia, respectively, which contribute to
the cytoskeletal rearrangements required for cell migration (19,
32, 38). ROCKs were identified as the downstream effectors of
Rho (14, 21, 22, 28) and were suggested to link Rho to stress
fiber formation through enhancing myosin light chain phos-
phorylation (18, 46, 51). Two isoforms have been identified:
ROCK 1 and ROCK 2 (14, 21, 22, 28), which share a 65%
overall identity at the amino-acid sequence level and a 92%
identity in their kinase domains (30). In the corneal epithelium,
Rho has been implicated in cell migration, actin organization,
focal adhesion formation, as well as adherens and gap junction
assembly (3, 31, 40), while ROCKs have been suggested to be
involved in corneal epithelial differentiation (49), cell cycle
progression (4), and cell-cell adhesion (3). Our recent study
revealed that Rho activity is required for cell migration, proliferation and focal adhesion formation during the healing
Fig. 8. Y-27632 impairs adherence junctions and
alters actin cytoskeleton. A: confluent THCE cells
were cultured in the presence (c and d) or absence
(a and b) of 50 ng/ml HB-EGF with (b and d) or
without (a and c) 10 ␮M Y-27632 for 24 h.
E-cadherin staining was visualized as described in
MATERIALS AND METHODS (scale bar ⫽ 50 ␮M).
Arrows and arrowheads indicate membrane and
intracellular localization of E-cadherin, respectively. B: confluent THCE cells were wounded
with a pipette tip and allowed to heal in the
presence (e–h) or absence (a–d) of 10 ␮M
Y-27632 for 48 h. ␤-Catenin and actin were visualized as described in MATERIALS AND METHODS.
Arrows in f indicated concentrated ␤-catenin staining at cell-cell contact among cells at the wounding
edge (scale bar ⫽ 20 ␮M). Figure represents of 2
independent experiments.
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Fig. 7. Spontaneous and HB-EGF-enhanced cell
adhesion were promoted by Y-27632 and attenuated by C3. Growth factor-starved THCE cells
were pretreated with 10 ␮M Y-27632 for 30 min
(A and B) or 10 ␮g/ml C3 for 24 h (C and D) and
then allowed to attach to FNC (A and C) or 1:5
diluted Matrigel (B and D) in the presence or
absence of 50 ng/ml HB-EGF for 30 min. Adherent cells were stained with crystal violet, which
was dissolved in acetic acid. Cell adhesion was
indicated by readings at 570 nm and are means ⫾
SE (n ⫽ 8). *P ⬍ 0.05 and **P ⬍ 0.001 (Student’s t-test).
C384
RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
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Fig. 9. Y-27632 disrupts barrier formation.
A and B: THCE cells, plated onto Transwell
membranes, were cultured in growth medium without (A; C1– 6) or with (B; Y1– 6)
10 ␮M Y-27632 for 6 days. At day 6,
Y-27632 was added to three samples in A
(C1–3) and removed from three samples in B
(Y4 – 6). Cells were further incubated for
indicated time and TER was measured daily.
Insert in A showed daily changes in TER
readings from day 4 to day 5, day 5 to day 6,
and day 6 to day 7. Significant difference in
the changes of TER readings was observed
when Y-27632 was added (a decrease in
TER reading) compared with that of the
control. **P ⬍ 0.001, Student’s t-test.
C: THCE cells, plated onto Transwell membranes, were allowed to growth in the absence (a and b) or the presence (c and d) of
10 ␮M Y-27632 for 5 days. Y-27632 was
then either added to (b, corresponding to
C1–3; A) or removed from (d, corresponding
to Y1–3; B) the culture media. Cells were
further cultured for 1 or 2 days before
stained for occludin (scale bar ⫽ 10 ␮M).
2 Transwell inserts for each condition.
process and contributes to the regulation of wound healing in
corneal epithelial cells (63). In the current study, we demonstrated that both ROCK 1 and ROCK 2 are expressed in
HCECs and activated in response to wounding. Although
ROCK 1 and ROCK 2 can be differentially regulated in certain
circumstances, there is no evidence that ROCK 1 and ROCK 2
have different functions (39). Hence, similar expression and
activation patterns of both ROCK 1 and 2 suggest they may
function as a unit in the regulation of HCEC wound healing.
The biological functions of ROCKs were effectively inhibited by Y-27632, a specific antagonist against ROCK 1 and
ROCK 2 with equal potency, and by Rho inhibitor exoenzyme
C3, indicating that Y-27632 indeed functions downstream to
Rho in wounded HCECs. Moreover, Y-27632 had a minimum
effect on wound-induced Rho activation, confirming the upstream-downstream linear relation between Rho and ROCKs
AJP-Cell Physiol • VOL
and suggesting that the effects of Y-27632 on cell behaviors
were specific to ROCKs but not Rho. While Rho inhibition
delayed wound closure (63), Y-27632 promoted healing of a
scratch wound in cultured HCECs, suggesting distinct roles of
Rho and ROCKs in regulating corneal epithelial wound healing. One explanation for the differential effects of Rho and
ROCK inhibitions is the multiple downstream effectors for
Rho (6). Other effector proteins for Rho include protein
kinase N (1), rhotekin (37), citron (25, 26), and noticeably
mDia (55, 56). A recent report suggested that mDia1 works
concurrently with ROCKs during Rho-induced actin reorganization and that various actin fiber patterns rely on the
balance between mDia and ROCK actions (55). Interestingly, both ROCK and Rho GTPase inhibitors have been
shown to increase aqueous humor drainage through the
trabecular meshwork, leading to a decrease in intraocular
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RHO KINASES IN CORNEAL EPITHELIAL WOUND HEALING
AJP-Cell Physiol • VOL
Interestingly, while ROCK inhibition resulted in changes in the
occludin staining pattern, the cells with disrupted occluding
staining still possessed a relatively high TER reading. A
similarly disturbed occluding staining was also observed in
cells cultured continuously in Y-27632, and removal of the
inhibitor induced redistribution of occludin to cell membrane,
resulting in a rapid increase in TER reading (100 ⍀/day).
These results suggest that alteration of occludin localization
precedes barrier breakdown or tight junction formation in
cultured HCECs. Alternatively, maintaining tight junction
function may not require the presence of occludin at cell-cell
borders. Indeed, the barrier function of the intestinal epithelium
was normal in occludin knockout mice (41), suggesting that
occludin may not be directly involved in barrier function in
corneal epithelial cells. Interestingly, inhibiting ROCK significantly reduced the smoke-induced lung epithelial tight junction permeability to both ions and macromolecules (33).
Hence, the function of ROCK in mediating tight junction
barrier formation and maintenance may be cell-type specific.
Pharmacological analyses revealed that ROCKs mediate formation of the vacuolar apical compartments and endocytosis of
tight junction proteins in a myosin light chain kinase-independent manner (52, 53). Our study provides further evidence
suggesting that ROCK is required for the assembly and maintain of tight junctions, probably through its effects on the
F-actin cytoskeleton organization during junctional formation.
It should be noted that while stimulating wound healing, the
ROCK inhibitor Y-27632 blocked tight junction barrier formation. Thus ROCK inhibition may have beneficial as well as
adverse effects on corneal epithelial cells. The contradicting
roles of Y-23623 may be related to the distinct functions of
ROCKs in preventing cell activation at an early stage of
wound healing, including converting epithelial cells from
stationary to migratory state and promoting cell differentiation, a late stage event occurring when a wound is closed.
Hence, ROCK inhibitors may be useful therapeutics to treat
delayed epithelial wound healing such as that observed in
diabetic corneas where accelerating wound closure is important for reducing the risk of sight-threatening complications such as bacterial infection (35).
In summary, ROCKs negatively regulate corneal epithelial
wound healing via modulating cell behaviors including cell
migration, proliferation, cell-matrix adhesion, and cell-cell
junctions. Since ROCK inhibitors such as fasudil and Y-27632
have been in clinical trials (12, 45) and side effects were
reported to be minimal, understanding the role and mechanism
of ROCK actions in corneal wound healing may lead to their
therapeutic application in treating corneal diseases. One potential use of these inhibitors is the treatment of diabetic keratopathy, which may lead to delayed wound healing, persistent
defects, and recurrent erosion of epithelium, all of which can
be attributed to defects in epithelium-basement membrane
interaction and adhesion (50).
GRANTS
This work was supported by NIH/NEI EY10869 (to F. X. Yu), a Midwest
Eye Banks Student Stipend (to J. Yin), and an unrestricted grant from the
Research to Prevent Blindness (Department of Ophthalmology, Wayne State
University School of Medicine).
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pressure and to enhance ocular blood flow, retinal ganglion
cell survival, and axon regeneration (36).
Using BrdU labeling and MTT cell proliferation assays, we
showed that Y-27632 did not affect basal cell proliferation near
a scratch wound. As Y-27632 accelerated epithelial wound
closure in cultured THCE cells, the minimal effect of Y-27632
on cell proliferation would indicate that cell migration primarily accounted for the observed spontaneous (unstimulated)
wound healing in these cells. The effects of Y-27632 on cell
proliferation in wound epithelial cells are in sharp contrast with
that of Rho inhibitor C3, which greatly attenuated cell proliferation (63). Hence, the function of ROCKs differs from its
activator Rho in the regulation of cell proliferation during the
healing of an epithelial wound in vitro. Intriguingly, although
Y-27632 accelerated HB-EGF-enhanced epithelial wound closure, it significantly inhibited the HB-EGF-induced increase in
the number of proliferating cells at the wound edge. How
ROCK kinase activity might influence growth factor-induced
cell proliferation remains elusive but may be related to its
ability to alter the levels of cell cycle regulatory proteins via
Ras and the MAPK pathway, a major mitogenic pathway of
receptor tyrosine kinases such as EGFR (7).
We previously demonstrated that Rho inhibition delayed
wound closure in the presence of hydroxyurea, a cell cycle
blocker, suggesting that Rho also participates in cell migration
during wound healing (63). In this study, we demonstrated that
inhibition of ROCK activity accelerated epithelial wound closure in a cell proliferation-independent manner. Consistent
with this observation, it was found that both haptotactic migration (towards matrix proteins) and chemotaxis induced by
HB-EGF are enhanced by Y-27632 in Boyden chamber assay.
The ability of ROCKs to negatively regulate cell migration
may be related to the fact that inhibition of ROCK activities
promotes cell adhesion to different matrixes as cell migration
requires a dynamic interaction between the cell and its substrate (13). Rho inhibition by C3, on the other hand, led to
enhanced cell adhesion, which may also account for the differential roles of Rho and ROCKs during wound healing. In
line with the enhanced cell-matrix adhesion, we observed that
Y-27632 in the presence of HB-EGF greatly increased the size
of culture cells, resulting in a more “spread and flattened”
morphology. It is likely that these “flattened” cells are strongly
attached to the substratum. It has been previously reported that
the Rho/ROCK signaling pathway influences the assembly of
E-cadherin adherens junctions in rabbit corneal epithelium (3).
It is worth noting that the third type of cell junction, gap
junction, also requires Rho/ROCK signaling (3). A recent
study (48) demonstrated that mice lacking the Rho-inhibitory
protein, p190-B RhoGAP, are reduced in size and such reduction can be rescued by Y-27632, suggesting that ROCKs
participate in the control of cell size. Similarly, migrating
epithelial cells near the wound edge exhibited a large size and
more apparent lamellipodia-type extension into the denuded
area with strong cortical actin staining.
Disruption of epithelial barriers has been associated with an
increase in microbial infection of the cornea (62). Using TER
as an indication of HCEC barrier function, we observed that
ROCK activities were required for the formation and maintenance of barrier properties. Staining of occludin in cells cultured on transwell inserts further confirmed that the inhibitory
effects of ROCK inhibition on tight junction are reversible.
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