Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell-Cell Communication in Corneal Endothelium

Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 06/2011; 52(8):5174-82. DOI: 10.1167/iovs.11-7272
Source: PubMed
To determine how corneal endothelial (CE) cells respond to acute genotoxic stress through changes in connexin-43 (Cx43) and gap junction intercellular communication (GJIC).
Cultured bovine CE cells were exposed to mitomycin C or other DNA-damaging agents. Changes in the levels, stability, binding partners, and trafficking of Cx43 were assessed by Western blot analysis and immunostaining. Live-cell imaging of a Cx43-green fluorescent protein (GFP) fusion protein was used to evaluate internalization of cell surface Cx43. Dye transfer and fluorescent recovery after photobleaching (FRAP) assessed GJIC.
After genotoxic stress, Cx43 accumulated in large gap junction plaques, had reduced zonula occludens-1 binding, and displayed increased stability. Live-cell imaging of Cx43-GFP plaques in stressed CE cells revealed reduced gap junction internalization and degradation compared to control cells. Mitomycin C enhanced transport of Cx43 from the endoplasmic reticulum to the cell surface and formation of gap junction plaques. Mitomycin C treatment also protected GJIC from disruption after cytokine treatment.
These results show a novel CE cell response to genotoxic stress mediated by marked and rapid changes in Cx43 and GJIC. This stabilization of cell-cell communication may be an important early adaptation to acute stressors encountered by CE.


Available from: James Funderburgh, Feb 03, 2014
Rapid Changes in Connexin-43 in Response to
Genotoxic Stress Stabilize Cell–Cell Communication in
Corneal Endothelium
Danny S. Roh and James L. Funderburgh
PURPOSE. To determine how corneal endothelial (CE) cells
respond to acute genotoxic stress through changes in con-
nexin-43 (Cx43) and gap junction intercellular communication
ETHODS. Cultured bovine CE cells were exposed to mitomy-
cin C or other DNA-damaging agents. Changes in the levels,
stability, binding partners, and trafficking of Cx43 were as-
sessed by Western blot analysis and immunostaining. Live-cell
imaging of a Cx43–green fluorescent protein (GFP) fusion
protein was used to evaluate internalization of cell surface
Cx43. Dye transfer and fluorescent recovery after photobleach-
ing (FRAP) assessed GJIC.
ESULTS. After genotoxic stress, Cx43 accumulated in large gap
junction plaques, had reduced zonula occludens-1 binding, and
displayed increased stability. Live-cell imaging of Cx43–GFP
plaques in stressed CE cells revealed reduced gap junction
internalization and degradation compared to control cells. Mi-
tomycin C enhanced transport of Cx43 from the endoplasmic
reticulum to the cell surface and formation of gap junction
plaques. Mitomycin C treatment also protected GJIC from
disruption after cytokine treatment.
ISCUSSION. These results show a novel CE cell response to
genotoxic stress mediated by marked and rapid changes in
Cx43 and GJIC. This stabilization of cell–cell communication
may be an important early adaptation to acute stressors en-
countered by CE. (Invest Ophthalmol Vis Sci. 2011;52:
5174–5182) DOI:10.1167/iovs.11-7272
he corneal endothelium (CE) is a monolayer of neural
crest–derived cells that is essential for corneal transpar-
ency. Located at the posterior surface of the cornea, the CE
separates the cornea from the aqueous humor, relying on
cell–cell junctions to control corneal hydration. The CE is a
fragile cell layer that is vulnerable to the effects of intraocular
surgery, systemic and ocular disease, and topical drugs.
addition, endogenous oxidative stress appears to play a signif-
icant role in the degeneration of CE with age
and in Fuchs’
Human CE does not regenerate in vivo, so exist-
ing cells must compensate for cell loss to maintain the pump
and barrier functions required for corneal homeostasis. It is
clear that surviving cells respond to CE cell loss by spreading to
cover the posterior corneal surface. This spreading is associ-
ated with thinning of the cell layer,
but the full range of
physiological responses of the CE to cell loss has not been
characterized. CE function is dependent on the cell–cell junc-
tions, which maintain the integrity of this monolayer. The goal
of the present study was to examine how CE cells mediate
these junctions in response to genotoxic stressors.
Gap junctions are intercellular channels composed primar-
ily of the connexin family of proteins. These channels provide
rapid intercellular transfer of small signaling molecules, such as
nucleotides, inositol trisphosphate (IP
), glutathione, and Ca
between connected cells.
Such gap junction intercellular
communications (GJIC) function in the maintenance of tissue
homeostasis and influence cellular survival and death in re-
sponse to oxidative stress,
metabolic stress,
reperfusion injury,
and genotoxic stress.
may also mediate cell survival by GJIC-independent mecha-
nisms in addition to functions associated with gap junctions.
With a half-life of 1.5 to 5 hours, connexin proteins respond
rapidly to physiologic changes by altering gap junction cou-
pling between cells. The Cx43 C terminus contains 14 docu-
mented phosphorylation sites,
and different phosphorylated
species of Cx43 can be distinguished experimentally by SDS-
PAGE. Aggregation of Cx43 into functional gap junction
plaques, opening of the junctional pores, and Cx43 degrada-
tion have all been linked to site-specific phosphorylation of the
Cx43 protein.
Given the pervasive role of connexins in the
maintenance of cell and tissue homeostasis, we hypothesized
that exogenous genotoxic stress would alter homeostasis-reg-
ulating proteins such as Cx43 in the CE. Previously, we re-
ported DNA damage in goat CE after brief doses of the DNA
interstrand cross-linking agent mitomycin C (MMC) during
procedures emulating photorefractive keratectomy.
In this
study, we identified specific changes that occur in Cx43 and in
GJIC in CE as a result of genotoxic stress induced by exposures
such as MMC. Determining how CE cells respond to various
stressors may provide opportunities for CE protection and
Cell Culture and Reagents
Primary bovine CE cells were isolated as previously described
cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM;
Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum
and antibiotics/antimycotics in a humidified 5% CO
, 37°C environ-
ment. Passage 1 to 4 cells, split 1:4, were used and grown 2 to 3 days
past confluency for every experiment unless otherwise indicated. At
least 24 hours before treatment with MMC, cycloheximide, forskolin,
and epidermal growth factor (all from Sigma, Saint Louis, MO), the
medium was changed to serum-free DMEM containing no antibiotics/
antimycotics. MMC was added to cells by changing medium with
From the Department of Ophthalmology, University of Pittsburgh,
Pittsburgh, Pennsylvania.
Supported by National Institutes of Health Grants EY016415 (JLF),
EY009368 (JLF), P30-EY008098, F30-AG035443 (DSR), Research to
Prevent Blindness, and the Eye and Ear Foundation of Pittsburgh.
Submitted for publication January 24, 2011; revised May 10, 2011;
accepted May 17, 2011.
Disclosure: D. Roh, None; J. Funderburgh, None.
Corresponding author: James L. Funderburgh, Department of
Ophthalmology, University of Pittsburgh, 1009 Eye and Ear Institute,
203 Lothrop Street, Pittsburgh, PA 15213; jlfunder@pitt.edu.
Investigative Ophthalmology & Visual Science, July 2011, Vol. 52, No. 8
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
Page 1
prewarmed media containing 5
M freshly diluted MMC. Primary
bovine corneal fibroblasts were isolated and grown as previously de-
Cells were fixed in 3.2% paraformaldehyde (PFA) solution (Electron
Microscopy Science, Hatfield, PA) in PBS for 20 minutes at room
temperature. Permeabilization with 0.1% Triton X-100 (Thermo Fisher,
Pittsburgh, PA) in PBS for 1 minute was followed by blocking with 10%
heat-inactivated goat serum in PBS for 1 hour at room temperature.
Cells were incubated with a polyclonal rabbit anti-Cx43 antibody to the
C terminus (Invitrogen), monoclonal mouse anti-Cx43 to the C termi-
nus (Millipore, Billerica, MA), or monoclonal mouse anti-zonula oc-
cludens-1 (ZO-1; Invitrogen) diluted 1:200 in 1% bovine serum albumin
(BSA) in PBS solution overnight at 4°C. After multiple washes in PBS,
cells were incubated for 1 hour at room temperature with species-
appropriate Alexa-conjugated secondary antibodies (Invitrogen) di-
luted to 0.8
g/mL in 1% BSA in PBS containing 0.5
g/mL 4,6-
diamidino-2-phenylindole (DAPI; Invitrogen). Cells were observed on a
laser scanning confocal microscope (Olympus Fluoview FV1000,
Olympus, Center Valley, PA) and images captured and processed using
Olympus Fluoview software. To quantify plaque size and intensity,
ImageJ (developed by Wayne Rasband, National Institutes of Health,
Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) was
used to manually trace Cx43 plaques in three separate high magnifi-
cation confocal images. Area and intensity values were calculated and
compared with the Mann–Whitney U test.
Cells were lysed directly in 1X SDS sample buffer [1.6% sodium dode-
cyl sulfate, 0.06M Tris, 5.5% glycerol, and 0.002% bromophenol blue],
scraped into tubes, heated at 95°C for 5 minutes, then sonicated until
solubilized. For alkaline phosphatase treatment of cell lysates, cells
were lysed in RIPA buffer (Thermo Fisher) [25 mM Tris-HCl pH 7.6,
150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS] contain-
ing protease inhibitor cocktail (Sigma), sonicated, and incubated with
0.1 U/
L cell lysate of calf alkaline phosphatase (Sigma) for 1 hour at
37°C. Protein concentration was determined by Bio-Rad DC Protein
Assay (Bio-Rad, Hercules, CA) and then 2-mercaptoethanol was added
to a final concentration of 1% to the lysates and heated at 70°C for 20
minutes. An equal amount of protein was added to precast 4 –20%
gradient or 10% polyacrylamide gels (Bio-Rad) and electrophoresis was
performed for 1 hour at 200 V. Protein was transferred to PVDF
membrane (Millipore) and blocked for 1 hour at room temperature in
0.2 M Tris, 0.15 M NaCl, 0.01% thimeresol, 0.2% Tween-20, pH 7.4
(TTTBS) for ECL detection or with fluorescent blocker (Millipore) for
Odyssey infrared imaging (LI-COR, Lincoln, NE). Membranes were
incubated with the following primary antibodies as indicated: mouse
anti-Cx43NT1, which recognizes the N-terminal region of Cx43 (Fred
Hutchinson Cancer Center, Seattle, WA); rabbit anti-Cx43, which rec-
ognizes the C-terminal region of Cx43 (Invitrogen); mouse anti-ZO-1
(Invitrogen); and mouse anti-
-tubulin (Sigma), all diluted in 1% BSA in
TTTBS or fluorescent blocker. Secondary antibodies included horse-
radish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for enhanced
chemiluminescence (ECL) detection and IRDye800 and IRDye680 la-
beled secondary antibodies (LI-COR) for infrared imaging. Where indi-
cated, blots were stripped for 15 minutes at room temperature (Re-
store Western Blot Stripping Buffer, Thermo Fisher Scientific,
Rockford, IL). Chemiluminescent signal was visualized with ECL sub-
strate (Millipore) followed by detection and capture of 16-bit images
with a Bio-Rad FX imager (Bio-Rad), and fluorescent signal was de-
tected with Odyssey infrared imaging system (LI-COR). Densitometry
was performed using digital analysis software for ECL (Bio-Rad Quan-
tity One) and fluorescent detection (LI-COR) followed by appropriate
statistical analysis for comparisons with GraphPad (GraphPad Software
Inc., La Jolla, CA). All forms of Cx43 (phosphorylated and nonphos-
phorylated) were quantified in densitometry.
Cells were lysed directly in cold coimmunoprecipitation buffer [0.1%
Nonidet P40, 25 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA] contain-
ing HALT protease and phosphatase inhibitors (Thermo Fisher) and
briefly sonicated until solubilized. Lysates were precleared with mag-
netic protein G beads (Millipore) for 1 hour at room temperature to
remove any nonspecific binding of cell proteins to beads. The beads
were discarded and then cleared lysates were incubated with 1
antibody per 500
g of lysate using a rabbit polyclonal anti-Cx43
antibody (Invitrogen) overnight at 4°C. Beads were then washed mul-
tiple times with lysis buffer and heated at 95°C for 5 minutes in 1X SDS
sample buffer with 2-mercaptoethanol. A magnetic rack was used to
separate the beads from lysate before loading for SDS-PAGE separation.
Mouse anti-ZO-1 (Invitrogen) and rabbit anti-Cx43 (Invitrogen) anti-
bodies were used for immunoblotting.
Triton X-100 Insolubility
Cells were lysed in cell lysis buffer containing 1% Triton X-100 and 1
mM EDTA with protease inhibitors (Sigma) and then chilled on ice for
30 minutes with mixing. Cell lysates were spun at 10,000 g for 30
minutes at 4°C. The soluble fraction was removed and the Triton X-100
insoluble pellet was further dissolved in 1X SDS buffer followed by
sonication. Both the insoluble and soluble fractions were used for
Live-Cell Imaging
CE cells grown on Bioptech dishes (Bioptechs Inc., Butler, PA) to 70%
to 80% confluency were transfected with 1
g of Cx43–GFP plasmid
(gift of Professor Matthias Falk, Lehigh University) using Lipofectamine
2000 (Invitrogen). This construct was previously described to have
GFP at the most C-terminal end of Cx43,
and 24 hours after trans
fection, cells expressing the Cx43–GFP fusion protein were imaged
with the Nikon Eclipse TE200-E microscope (Nikon Instruments Inc.,
Melville, NY) on a heated stage with 5% CO
. Images were captured
using software (Metamorph, Molecular Devices, Sunnyvale, CA) every
2 minutes over a 4- to 6-hour period after treatment with MMC or
medium alone. To determine internalization and degradation fre-
quency, Cx43–GFP assembled into cell surface plaques were identified
and followed frame-by-frame exactly 24 hours after transfection.
Plaques were counted if they were internalized and/or degraded by
reduced fluorescent signal over a 3-hour timeframe. Comparison of the
frequency of internalization/degradation events between medium and
MMC-treated cells was performed using the Fisher exact test. A 2 2
contingency table was analyzed with GraphPad, with P 0.05 being
considered statistically significant.
GJIC Assays
Lucifer yellow (LY) dye (Sigma) scrape-load assays were performed by
scraping confluent monolayers of CE cells with a sterile pipette tip in
the presence of 1 mg/mL LY in warm PBS. After 2 minutes, cells were
rinsed to remove LY dye solution and cells were immediately fixed in
3.2% PFA without permeabilization. Images were captured using a
Nikon epifluorescent microscope. Dye transfer images were analyzed
using ImageJ software by measuring the distance of dye transfer from
the scrape wound at 10 different sites per image. Average distances
were then analyzed with ANOVA and a post-hoc Tukey’s test for
multiple comparisons. P 0.05 was considered statistically significant.
For fluorescent recovery after photobleaching (FRAP), cells grown on
35-mm coverslip-bottom dishes were loaded with 1
M of Calcein-AM
(Invitrogen) for 30 minutes at 37°C. Cells were allowed to recover for
1 to 2 hours then treated with 5
M MMC for 15 minutes. Individual
cells were photobleached using a laser scanning confocal microscope
(Olympus) and images of dye recovery were captured. ImageJ software
IOVS, July 2011, Vol. 52, No. 8 CE Response to Genotoxic Stress Involves Cx43 5175
Page 2
was used to measure dye intensity in image stacks. Photobleached cells
were normalized to control unbleached cells. Data were exported into
GraphPad for exponential curve fitting and calculation of rate con-
stants (k). Half-life of dye recovery (t
) was calculated based on the
formula: t
ln(2)/k. The Student’s t test was used for statistical
comparison of t
Genotoxic Stress to CE Generates Rapid Changes
in the Cx43 Abundance and Localization
We previously reported the rapid induction of DNA lesions
after brief exposure to low levels of MMC in CE cells.
a similar timeframe, low dose MMC exposure induced an in-
crease in total cellular Cx43 levels as detected by immunoblot-
ting (Fig. 1A). Increases occurred in each of the three character-
istic protein bands representative of differentially phosphorylated
Cx43 species present in most eukaryotic cells. The phenomenon
was highly repeatable, with total cellular Cx43 increasing
somewhat more than twofold 60 to 90 minutes after treatment.
These results were confirmed using two Cx43 antibodies tar-
geting different regions of Cx43, the C-terminal (Fig. 1A, top)
and the N-terminal (Fig. 1A, bottom). The use of antibodies
targeting different regions of the protein reduces the possibil-
ity that the observed quantitative changes result from epitope
masking. Alkaline phosphatase (AP) treatment of whole cell
lysates before electrophoresis dephosphorylates Cx43 collaps-
ing the characteristic multiple bands into a single faster moving
band (Fig. 1B). The rapid and transient increase in Cx43 was
maintained after AP treatment, indicating that total levels of
Cx43, independent of phosphorylation state, occurred. A sim-
ilar transient increase in cellular Cx43 was observed after
exposure to other genotoxic agents, including ultraviolet-C
radiation, etoposide, and hydrogen peroxide (data not shown).
Immunostaining of CE cells (Fig. 2A) revealed increased
Cx43 at cell–cell interfaces, with an increased size of the
punctate staining in this locale suggestive of increased accu-
mulation of Cx43 in gap junction plaques. Staining also in-
creased in perinuclear regions of the treated cells, suggesting
recently synthesized Cx43 in transit to the cell surface to be
increased as well. Cell fractionation of the treated cells found
increased Cx43 largely in the Triton X-100 detergent-insoluble
fraction (Fig. 2C). Association with gap junction plaques con-
fers detergent insolubility on connexin
; therefore, increases
in insoluble Cx43 in response to MMC is consistent with
immunostaining results, suggesting that MMC induces trans-
port of the increased Cx43 into cell– cell junctional plaques.
Similar changes were not observed in other cell– cell junction
proteins, such as ZO-1, cadherin, and
-catenin, based on
immunoblotting (data not shown).
Quantitative image analysis of cells treated with MMC con-
firmed a significant increase in both the gap junction plaque
area and Cx43 signal intensity compared to medium alone (Fig.
2B) after 60 minutes of treatment with MMC. Previous studies
have shown that interactions between Cx43 and the tight
junction protein ZO-1 can regulate the size of Cx43 gap junc-
To explore the potential involvement of ZO-1, we
used coimmunoprecipitation (Co-IP) and colocalization to ex-
amine changes in Cx43:ZO-1 association after MMC treatment.
Co-IP revealed a relative decrease in the abundance of ZO-1
immunoprecipitating with Cx43 after 60 minutes of MMC
treatment (Fig. 3A). This alteration was also evident in images
staining for ZO-1 and Cx43 (Fig. 3B). Large Cx43 gap junction
plaques displaying reduced ZO-1 colocalization occasionally
were evident in untreated cells (Fig. 3B); however, these were
present with significantly higher frequency in MMC-treated
MMC Treatment Increases Cx43 Stability
Connexin protein half-life ranges from 1.5 to 5 hours in many
cell types and organ systems.
Treatment of cultured CE
with 10
g/mL cycloheximide (CHX) to inhibit protein synthe-
sis was used to examine decay rates of Cx43. In the untreated
control, the half-life of decay was in the expected range of 1 to
FIGURE 1. Rapid accumulation of Cx43 protein in response to genotoxic stress with mitomycin C. Cells were treated with prewarmed media
containing 5
M MMC for indicated times in minutes. (A) Western blot for total Cx43 using C-terminal antibody (top) and N-terminal antibody
(bottom) from independent experiments. Blots were stripped and reprobed for
-tubulin as a loading control. Quantification below is ratio of
-tubulin normalized to values at the “0” time point. Asterisks indicate statistically significant differences from time “0” (ANOVA, P 0.05;
Dunnett’s multiple comparison test, P 0.05). Blots are representative of eight independent experiments and the mean fold change SEM is
shown in the graph. (B) Alkaline phosphatase (AP) treatments of cell lysates were followed by immunoblotting. Quantification in the graph is the ratio
of Cx43/
-tubulin normalized to “0” for either AP
or AP
. Differences between “–AP” and AP” at each time point were not statistically significant
(2-way ANOVA, P 0.05). The blot is representative of three independent experiments and the mean fold change SEM is shown in the graph.
5176 Roh and Funderburgh IOVS, July 2011, Vol. 52, No. 8
Page 3
2 hours (Fig. 4, left). Treatment with MMC, however, stabilized
the Cx43, delaying degradation for up to 120 minutes (Fig. 4,
right). After this period of stability, however, the decay rate
returned to one similar to that of untreated cells.
Gap Junction Internalization is Decreased
after MMC Treatment
Degradation of Cx43 present in cell surface plaques occurs via
processes involving the internalization of small and/or large
endocytic double-membrane vesicles.
These processes
can be followed directly by micrography using fluorescently
tagged Cx43.
We examined this internalization and deg
radation of Cx43 after MMC treatment by live-cell imaging of
CE that had been transiently transfected with a Cx43–GFP
fusion protein.
Figure 5
shows a series of images from
live-cell video micrography after Cx43–GFP in MMC-treated
cells or medium-only controls. Sixty-three individual plaques
were identified and followed in the videos starting 24 hours
after transfection at the time of MMC treatment. The pres-
ence or absence of each of these was scored 3 hours later to
assess the relative stability of the plaque-associated Cx43
under the two conditions (Fig. 5A). In medium-treated con-
trol transfected cells, 40 of 46 (89.6%) disappeared after 3
hours, whereas in the MMC-treated samples, only 8 of 17
(52.9%) of the plaques had been internalized and vanished
(Fig. 5B). This significant (P 0.01) difference in plaque
stability suggests that the reduced degradation of Cx43 iden-
tified in Figure 4 relates to alterations in the mechanism by
which cells endocytose and degrade the gap junction
Genotoxic Stress Increases Forward Trafficking
of Cx43 into Gap Junctions
To distinguish MMC effects in the formation of new mem-
brane-associated Cx43 from its effects on Cx43 internaliza-
tion/degradation, we examined transfer of Cx43 from the
endoplasmic reticulum (ER) compartment to the cell surface
after an 8-hour block with brefeldin A (BFA). BFA has been
used in previous studies to investigate gap junction assem-
bly because of its reversible inhibition of vesicle transport
from the ER to the Golgi and subsequently to the cell
When BFA is removed, the transport of proteins
is restored and the accumulation of proteins at the cell
surface becomes briefly representative of the rate at which
this forward trafficking process is occurring. Therefore, gap
junction formation can be assessed after BFA removal. As
shown in Figure 6, BFA treatment significantly reduced
Cx43 at the cell surface (Fig. 6A, BFA). After BFA washout
and a 2-hour recovery, Cx43 appeared at the cell surface in
gap junction plaques and in the perinuclear compartment in
FIGURE 2. Changes in Cx43 gap junction plaque size and intensity after genotoxic stress. (A) Cells were treated with prewarmed media containing
M MMC for the indicated time in minutes then stained for Cx43 (green). DAPI (nuclei). Orange arrows indicate apparent gap junction plaques.
(B) Gap junction plaques were imaged by confocal microscopy and analyzed for area (
) and Cx43 fluorescence intensity (arbitrary units) after
60 minutes of 5
M MMC (n 25 plaques) or medium alone (n 25 plaques). Individual data points are shown along with the median. Area and
intensity distributions were compared for statistically significant differences using the Mann–Whitney U test (area, P 0.01; intensity, P 0.0001).
Similar results were obtained in three independent experiments. (C) Western blot for 1% Triton X-100 soluble and insoluble Cx43. Quantification
below is insoluble Cx43 normalized to “0.” Asterisks indicate statistically significant differences from time “0” (ANOVA, P 0.05; Dunnett’s
multiple comparison test, P 0.05). Results are representative of three independent experiments and the mean fold change SEM is shown in
the graph. Scale bar: (A)20
IOVS, July 2011, Vol. 52, No. 8 CE Response to Genotoxic Stress Involves Cx43 5177
Page 4
both control and MMC-treated cells (Fig 6A, BFA 2hr, BFA
MMC 2hr). However, the presence of MMC led to a clear
increase of Cx43 in plaques at the cell– cell interfaces (Fig 6A,
BFA MMC 2hr). These plaques were significantly larger
than those formed without MMC (Fig. 6B).
Gap Junction Communication is Stabilized by
MMC Treatment
LY dye, which transfers from cell to cell via gap junction
channels, was used to assess the temporal rate of GJIC
during a 2-minute incubation after scrape loading. As shown
in Figure 7A, no significant differences were observed in the
amount of dye transferred or the distance the dye moved
comparing control and MMC-treated cells (Fig. 7A). FRAP
using Calcein AM, a similarly gap junction permeable dye,
also revealed identical transfer kinetics between control and
MMC-treated CE cells (Fig. 7C). Forskolin is an agent known
to maximize GJIC and markedly increases scrape-loaded dye
transfer in many cell types.
In CE cells, however, fors
kolin produced no significant increase on the dye transfer
FIGURE 3. MMC induces alteration in Cx43 association with ZO-1. Cells were treated with prewarmed
media containing 5
M MMC for 60 minutes or media alone (CTRL). (A) Coimmunoprecipitation with a
rabbit polyclonal anti-Cx43 was performed followed by immunoblotting with mouse monoclonal anti-
ZO-1. The blot was stripped and reprobed for Cx43. Quantification below is the ratio of ZO-1:Cx43, which
is normalized to “CTRL.” Denatured heavy chain immunoglobulin G (IgG) is indicated as HC IgG. Asterisks
indicate a statistically significant difference (Student’s t-test, P 0.001). Results are representative of three
independent experiments and mean fold change to “CTRL” SEM is shown below. (B) Representative
confocal images of a large gap junction plaque after MMC treatment. In the higher magnification inset
(below), the areas of Cx43:ZO-1 colocalization are highlighted in white. Cx43 (green), ZO-1 (red), DAPI
(blue). Scale bar, 10
FIGURE 4. The stability of preexist-
ing Cx43 is altered after MMC treat-
ment. Cells were treated with pre-
warmed media containing 10
CHX with or without 5
M MMC for
indicated times in minutes. Blot shows
total Cx43 using a C-terminal–specific
antibody. Blots were stripped and re-
probed for
-tubulin as a loading con-
trol. Quantification below is ratio of
-tubulin normalized to the zero
time (0) for “CHX” or “CHX MMC.”
Asterisks indicate statistically signifi-
cant differences between “CHX” and
“CHX MMC” (2-way ANOVA, P
0.01; Bonferroni multiple compari-
sons, P 0.05). Results are represen-
tative of three independent experi-
ments and mean fold change SEM is
shown in the graph.
5178 Roh and Funderburgh IOVS, July 2011, Vol. 52, No. 8
Page 5
rate (Fig. 7A, far right panel). Both forskolin and MMC,
however, increased dye transfer rates in primary bovine
corneal fibroblasts (Fig. 7B), suggesting that CE cells may be
communicating via gap junctions at near maximal levels,
making further increases in GJIC undetectable.
To determine whether the increased Cx43 at the cell
surface induced any alteration in gap junction function of CE
cells, we inhibited GJIC using epidermal growth factor
(EGF). EGF treatment has been shown to decrease GJIC in
cultured cells through activation of the mitogen-activated
kinase pathway.
This response was clearly detected in CE
cells using the LY dye transfer assay (Fig. 8). MMC-treated
cultures partially rescued the EGF-induced GJIC inhibition,
suggesting that increases in cell surface Cx43 induced by
MMC are indeed functional in promoting intercellular com-
The CE is a nonregenerating tissue in which the total number
of cells declines throughout life. As a result of damage or
various acute stressors, this process can occur more rapidly.
With cell loss, CE cells spread and flatten, covering a greater
area. Apart from this process, little of the physiologic response
to stressors in these cells is known, particularly regarding how
cell–cell junctions are maintained. In the present study, we
document several novel observations regarding how CE cells
respond to an acute stress: Cx43 rapidly accumulates in deter-
gent-insoluble plaques at cell– cell junctions; the plaques are
stabilized, showing reduced endocytosis and degradation;
transport of Cx43 from the ER to the cell surface is increased;
and gap junction communication is stabilized against down-
regulation by EGF. These observations describe a previously
unidentified response of CE cells to stress, rapidly increasing
the abundance of functional gap junctions in the tissue.
The mechanism mediating this response is not straightfor-
ward. In initial experiments, we found no statistically signifi-
cant increase in Cx43 mRNA (data not shown); therefore, the
rapid change in Cx43 abundance in response to MMC seemed
likely to involve decreased Cx43 degradation. A temporary halt
to Cx43 degradation after MMC treatment was indeed ob-
served (Fig. 4), as was a significant reduction in endocytosis
and dispersion of plaque-associated Cx43 (Fig, 5), providing a
mechanism that could be responsible for the observed increase
in total cellular Cx43. Cx43 stabilization has been described in
other cell types in response to stress. Long-lived gap junctions
after arsenite and heat-shock exposure were observed in in
CHO and S180 cell lines.
In the case of CHO cells, cell surface
Cx43 was stabilized by a mechanism dependent on functional
proteasomal action.
The CE response to MMC observed in
FIGURE 6. MMC induces increased assembly of gap junction plaques.
Confluent monolayers were treated with prewarmed media containing
g/mL BFA for 8 hours followed by washout with warm medium and
a 2-hour recovery. Five
M MMC was added as indicated during the
2-hour recovery period. (A) Representative confocal images of Cx43
(green) before treatment (NOTX), after 8 hour BFA (BFA), after recov-
ery (BFA 2hr), and after recovery in the presence of MMC (BFA
MMC 2hr). DAPI (nuclei). (B) Gap junction plaques were imaged by
confocal microscopy and analyzed for area (
) and Cx43 fluores
cence intensity (arbitrary units) similar to Figure 2B. BFA 2hr (n
25 plaques) or BFA MMC 2hr (n 25 plaques). Individual data
points are shown along with median. Area and intensity distributions
were compared using the Mann–Whitney U test (area, P 0.0001;
intensity, P 0.0001). Similar results were obtained in three indepen-
dent experiments.
FIGURE 5. Genotoxic stress results in reduced internalization of Cx43
plaques. Seventy percent to 80% confluent cells were transfected with
a Cx43–GFP plasmid. (A) Twenty-four hours after transfection, cells
were treated with either medium (“CTRL,” upper figures) or medium
with 5
M MMC (“MMC,” lower figures) and live-cell imaging was
performed. Images were captured every 2 minutes over a 4- to 6-hour
period after treatment. Images are stills of Cx43–GFP plaques over time
in minutes. “CTRL” images illustrate an example of internalized and
degraded plaques. MMC images illustrate a long-lived plaque. (B)To
determine internalization and degradation frequency, Cx43–GFP as-
sembled into cell surface plaques were identified and followed frame-
by-frame exactly 24 hours after transfection. Plaques were counted if
they were internalized and/or degraded by reduced fluorescent signal
over a 3-hour timeframe. Statistical comparison of the frequency of
internalization/degradation events between medium “CTRL” and MMC-
treated cells was performed using Fisher’s exact test, P 0.01. Results
are from two independent live-cell experiments tracking a combined
63 plaques (46 CTRLs, 17 MMCs). Scale bar: (A)20
IOVS, July 2011, Vol. 52, No. 8 CE Response to Genotoxic Stress Involves Cx43 5179
Page 6
this study could, therefore, be typical of a generalized cellular
mechanism by which cells stabilize GJIC in response to stress.
Another potential effector of GJIC stabilization is ZO-1.
ZO-1 is present in gap junctions, binding the C-terminal intra-
cellular region of Cx43.
We observed Cx43 but not ZO-1 to
increase in response to MMC, and subsequently found a de-
creased association of ZO-1 with Cx43 by coimmunoprecipi-
tation (Fig. 3). There is reason to believe that this reduced
ZO-1:Cx43 ratio could contribute to the stress-induced Cx43
plaque stabilization. Association of Cx43 with ZO-1 has been
linked to an increased endocytosis of Cx43
; therefore,
reduced association of Cx43 with Z0 –1 in gap junction plaques
(such as we observed) could delay or prevent Cx43 endocyto-
sis and internalization. ZO-1 may also have an effect on the gap
junction plaque size (Fig. 2). ZO-1 has been reported to regu-
late gap junction size by limiting access of ZO-1-associated
Cx43 into preexisting gap junctions.
Reduced ZO-1 asso
ciation with cell surface Cx43 is therefore consistent with the
increased gap junction plaques we observed.
The in vitro CE layers used in this study did not appear to
restrict the intercellular transmission of dyes via gap junctions
(Fig. 7). However, in the presence of EGF—an agent that
disrupts GJIC—it was clear that GJIC in MMC-treated cells was
more resistant to this inhibition than control cells (Fig. 8).
Therefore, the rapid alterations in Cx43 protein in response to
genotoxic stress produce a cell– cell communication array that
is more robust than that in nonstressed cells. This response
gains relevance in light of the documentation that exposure to
genotoxic stressors releases cytokines and growth factors
that could compromise GJIC in the CE.
The tight junctions in CE cells serve to control movement of
water and nutrients into the cornea,
but there is not yet a
clearly demonstrated role for gap junctions in corneal physiol-
FIGURE 8. MMC treatment generates GJIC resistant to EGF-mediated
downregulation. (A) Confluent monolayers were pretreated for 60
minutes with either prewarmed media containing 5
M MMC or
medium (“CTRL”) alone. Medium was replaced with 100 ng/mL EGF
for an additional 30 minutes and then GJIC was assessed with a
scrape-load dye transfer assay as described in the Methods section. Ten
individual sites were measured along different regions of the scrape
wound for distance of LY dye transfer to determine cell– cell commu-
nication as shown in the graph. Data points are mean distance SD of
the dye transfer. ANOVA was used for statistical comparison among
treatment groups (P 0.0001). Tukey’s multiple comparison test
reported untreated versus MED EGF (P 0.001), untreated versus
MMC EGF (P 0.05), MED EGF versus MMC EGF (P 0.001).
Results are representative of two independent experiments.
FIGURE 7. MMC and forskolin do not significantly increase GJIC. (A)
Confluent monolayers were treated with prewarmed media containing
M MMC, 10
M forskolin, or medium alone (“CTRL”) for 30 minutes
Medium was replaced with 1 mg/mL Lucifer yellow (LY) dye in PBS
and scrape-load dye transfer assay was performed as described in the
Methods section. Ten individual sites were measured along different
regions of the scrape wound for distance of LY dye transfer to deter-
mine cell– cell communication as shown in the graph below. Data
points are mean distance SD. There was no statistically significant
difference between treatments (ANOVA [P 0.3]). (B) Confluent
monolayers of bovine corneal fibroblasts were treated similar to (A).
Asterisks indicate statistically significant differences (ANOVA [P
0.001]; Dunnett’s test: medium versus Forksolin [P 0.01], medium
versus MMC [P 0.01]). (C) FRAP was performed after loading
confluent CE monolayers with 1
M calcein AM and observing dye
recovery. Half-life of recovery in seconds (t
) shown on the y-axis was
compared between untreated (n 4 cells) and MMC-treated (n 4)
cells. Mean SD t
shown. No statistically significant difference was
found between treatments (Student’s t-test [P 0.8]).
5180 Roh and Funderburgh IOVS, July 2011, Vol. 52, No. 8
Page 7
ogy. Our results suggest that alterations in Cx43 may serve as
survival factor for CE cells. In other biologic systems in which
cells are subjected to physical stress or injury, gap junction
coupling activation results in the mediation of injury or in-
creased survival.
In the CE, upregulation of Cx43 mRNA
and protein, via release of ciliary neurotrophic factor and its
receptor, promote CE cell survival during oxidative stress and
ex vivo corneal storage.
Also, point mechanical stimula
tion of CE cell monolayers results in a Cx43-dependent in-
crease in ATP release and calcium-wave propagation between
Therefore, Cx43 and GJIC provide a response in CE to
changes in cellular environment that is likely to represent a
survival response.
The rapid rate at which CE responds to MMC suggests a
mechanism not reliant on changes in gene expression but
instead on posttranslation modifications of Cx43. Cx43 C-ter-
minal phosphorylation controls multiple aspects of this pro-
tein, including channel gating, subcellular localization, second-
ary structure, and stability.
Increased phosphorylation of
Cx43 C-terminal, for example is responsible for decreased
ZO-1 binding.
Site-specific Cx43 phosphorylation has also
been linked to internalization and degradation of this pro-
Casein kinase 1 is a likely candidate for an active par
ticipant in the response to MMC. Phosphorylation of Cx43 by
casein kinase 1 has been proposed to stimulate incorporation
of Cx43 into gap junction plaques,
and it appears to exert a
general role in regulating the stability of its numerous sub-
In addition, casein kinase 1 acutely responds to geno
toxic stress resulting in increased kinase activity, changes in
subcellular location, and increased mRNA and protein lev-
In conclusion, we have documented rapid changes in Cx43
in response to genotoxic stress and identified the changes in
binding partners and reduced degradation of Cx43. Mitomycin
C is widely used in ocular surgery, and we recently showed
that such treatment generates DNA cross-linking in CE.
however, may not be the only genotoxic agent to which these
cells are exposed. The high metabolic rate of the CE generates
reactive oxygen species, which are capable of DNA damage,
and as a possible consequence, CE exhibits an accumulation of
DNA modifications with increasing age.
The implications of
the stabilization of Cx43 and GJIC on CE cell homeostasis/
survival are yet to be fully elucidated but may serve to protect
against detrimental effects of natural DNA damage and acute
stress generated by agents such as MMC or ultraviolet light.
Future areas of interest include determining the functional
consequences of stabilized cell surface Cx43 and GJIC on CE
cell viability. One potential function influencing cell survival
may be to increase the intercellular transfer of cytoprotective
molecules that increase survival and function of intercon-
nected cells and/or disperse cytotoxic molecules and prevent
their accumulation in any one cell.
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  • Source
    • "In contrast, cultured bovine corneal endothelial cells exposed to DNA-damaging agents (e.g. mitomycin C), exhibited Cx43 accumulation in large GJ plaques, with reduction of ZO-1 binding, and displayed increased plaque stability [91]. Applied to acute skin lesions, the mimetic peptide corresponding to the last 9 amino acids of the Cx43 tail was reported [92] to accelerate wound healing while reducing scar formation in a mouse model of cutaneous injury, but the peptide effects on wound healing were in some respects not similar to those reported for Cx43 antisense treatment [93], making unclear whether the Cx43 antisense or the peptide designed to target ZO-1 acted via related mechanisms. "
    [Show abstract] [Hide abstract] ABSTRACT: Zonula Occludens (ZOs) proteins are ubiquitous scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of the plasma membrane and linking transmembrane proteins to the filamentous cytoskeleton. They belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. ZO proteins were originally described to localize specifically to tight junctions, or Zonulae Occludentes, but this notion was rapidly reconsidered since ZO proteins were found to associate with adherens junctions as well with gap junctions, particularly with connexin-made intercellular channels, and also with a few other membrane channels. Accumulating evidences reveal that in addition to having passive scaffolding functions in organizing gap junction complexes, including connexins and cytoskeletals, ZO proteins (particularly ZO-1) also actively take part in the dynamic function as well as in the remodeling of junctional complexes in a number of cellular systems. This article is part of a Special Issue entitled "Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters".
    Full-text · Article · Jul 2013 · Biochimica et Biophysica Acta
  • Source
    • "Digested and undigested samples were separated by electrophoresis on 4%–20% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with streptavidin-IR700 dye and imaged on a LiCor Odyssey Imaging System [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.
    Full-text · Article · Feb 2013 · PLoS ONE
  • [Show abstract] [Hide abstract] ABSTRACT: HYS-32 [4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone] is a new analogue of the anti-tumor compound combretastatin A-4 containing a cis-stilbene moiety. In this study, we investigated its effects on Cx43 gap junction intercellular communication (GJIC) and the signaling pathway involved in rat primary astrocytes. Western blot analyses showed that HYS-32 dose- and time-dependently upregulated Cx43 expression. A confocal microscopic study and scrape-loading/dye transfer analyses demonstrated that HYS-32 (5 μM) induced microtubule coiling, accumulation of Cx43 in gap junction plaques, and increased GJIC in astrocytes. The HYS-32-induced microtubule coiling and Cx43 accumulation in gap junction plaques was reversed when HYS-32 was removed. Treatment of astrocytes with cycloheximide resulted in time-dependent degradation of Cx43, which was delayed by co-treatment with HYS-32 by increasing the half-life of Cx43. Co-treatment with HYS-32 also prevented the LPS-induced downregulation of Cx43 and inhibition of GJIC in astrocytes. HYS-32 induced activation of PKC, ERK, and JNK, and co-treatment with the PKC inhibitor Go6976 or the ERK inhibitor PD98059, but not the JNK inhibitor SP600125, prevented the HYS-32-induced increase in Cx43 expression and GJIC. Go6976 suppressed the HYS-32-induced PKC phosphorylation and increase in phospho-ERK levels, while PD98059 did not prevent the HYS-32-induced increase in phospho-PKC levels, suggesting that PKC is an upstream effector of ERK. In conclusion, our results show that HYS-32 increases the half-life of Cx43 and enhances Cx43 expression and GJIC in astrocytes via a PKC-ERK signaling cascade. These novel biological effects of HYS-32 on astrocyte gap junctions support its potential for therapeutic use as a protective agent for the central nervous system.
    No preview · Article · Mar 2013 · Neurochemistry International
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