Matrix metalloproteinase inhibitors suppress transforming growth factor-beta-induced subcapsular cataract formation

Article (PDF Available)inAmerican Journal Of Pathology 168(1):69-79 · February 2006with35 Reads
DOI: 10.2353/ajpath.2006.041089 · Source: PubMed
Abstract
The pleotropic morphogen transforming growth factor-beta (TGFbeta) plays an important role in the development of fibrotic pathologies, including anterior subcapsular cataracts (ASCs). ASC formation involves increased proliferation and transition of lens epithelial cells into myofibroblasts, through epithelial-mesenchymal transformation that results in opaque plaques beneath the lens capsule. In this study, we used a previously established TGFbeta-induced rat cataract model to explore the role of matrix metalloproteinases (MMPs) in ASC formation. Treatment of excised rat lenses with TGFbeta resulted in enhanced secretion of MMP-2 and MMP-9. Importantly, co-treatment with two different MMP inhibitors (MMPIs), the broad spectrum inhibitor GM6001 and an MMP-2/9-specific inhibitor, suppressed TGFbeta-induced ASC changes, including the epithelial-mesenchymal transformation of lens epithelial cells. Using an anti-E-cadherin antibody, we revealed that conditioned media from lenses treated with TGFbeta contained a 72-kd E-cadherin fragment, indicative of E-cadherin shedding. This was accompanied by attenuated levels of E-cadherin mRNA. Conditioned media from lenses co-treated with TGFbeta and MMPIs exhibited attenuated levels of the E-cadherin fragment compared with those from TGFbeta-treated lenses. Together, these findings demonstrate that TGFbeta-induced E-cadherin shedding in the lens is mediated by MMPs and that suppression of this phenomenon might explain the mechanism by which MMPIs inhibit ASC plaque formation.
Epithelial and Mesenchymal Cell Biology
Matrix Metalloproteinase Inhibitors Suppress
Transforming Growth Factor-
-Induced Subcapsular
Cataract Formation
Dhruva J. Dwivedi,* Giuseppe Pino,* Alice Banh,
Zahra Nathu,* Derek Howchin,* Peter Margetts,
Jacob G. Sivak,
and Judith A. West-Mays*
From the Department of Pathology and Molecular Medicine * and
the Division of Nephrology,
McMaster University, Hamilton; and
the School of Optometry,
University of Waterloo, Waterloo,
Ontario, Canada
The pleotropic morphogen transforming growth fac-
tor-
(TGF
) plays an important role in the develop-
ment of fibrotic pathologies, including anterior sub-
capsular cataracts (ASCs). ASC formation involves
increased proliferation and transition of lens epithe-
lial cells into myofibroblasts, through epithelial-mes-
enchymal transformation that results in opaque
plaques beneath the lens capsule. In this study, we
used a previously established TGF
-induced rat cata-
ract model to explore the role of matrix metallopro-
teinases (MMPs) in ASC formation. Treatment of ex-
cised rat lenses with TGF
resulted in enhanced
secretion of MMP-2 and MMP-9. Importantly, co-treat-
ment with two different MMP inhibitors (MMPIs), the
broad spectrum inhibitor GM6001 and an MMP-2/9-
specific inhibitor, suppressed TGF
-induced ASC
changes, including the epithelial-mesenchymal trans-
formation of lens epithelial cells. Using an anti-E-
cadherin antibody, we revealed that conditioned me-
dia from lenses treated with TGF
contained a 72-kd
E-cadherin fragment, indicative of E-cadherin shed-
ding. This was accompanied by attenuated levels of
E-cadherin mRNA. Conditioned media from lenses
co-treated with TGF
and MMPIs exhibited attenu-
ated levels of the E-cadherin fragment compared
with those from TGF
-treated lenses. Together,
these findings demonstrate that TGF
-induced E-
cadherin shedding in the lens is mediated by MMPs
and that suppression of this phenomenon might
explain the mechanism by which MMPIs inhibit
ASC plaque formation.
(Am J Pathol 2006, 168:69 –79;
DOI: 10.2353/ajpath.2006.041089)
Loss of transparency of the lens, or cataract, is the lead-
ing cause of blindness worldwide, despite the availability
of effective surgery in developed countries.
1
Currently,
there are no pharmacological agents to prevent the onset
or to inhibit the progression of cataract formation. Thus,
an understanding of the cellular and molecular mecha-
nisms regulating the normal and pathological differentia-
tion of the lens may lead to the development of therapeu-
tic strategies for the treatment and/or prevention of
cataracts.
The lens is a relatively simple tissue composed of two
cell types: epithelial cells and fiber cells. In the embryo,
the lens consists of a highly proliferative monolayer of
lens epithelial cells (LECs) that cover the anterior half of
the lens.
2
At the lens equator, these cells are stimulated
to terminally differentiate into fiber cells by a gradient
concentration of growth factors within the ocular media.
3
In adults, lens proliferation and differentiation occurs near
the lens equator, albeit at a slower rate than in the em-
bryo. However, in a pathological situation such as occurs
after ocular trauma, surgery, or systemic diseases like
atopic dermatitis and retinitis pigmentosa, the anterior
LECs can be triggered to proliferate and multilayer be-
neath the lens capsule.
3,4
A proportion of these cells
transform into plaques of large “spindle shaped” cells, or
myofibroblasts, by a phenomenon known as epithelial-to-
mesenchymal transition (EMT).
5–7
The cytokine transforming growth factor-
(TGF
) has
been shown to play a role in lens disease and to induce
these aberrant changes in LECs, including their conver-
sion to myofibroblasts.
3
The resultant myofibroblasts ex
-
press contractile elements, like
-smooth muscle actin
Supported by research grants from the National Institutes of Health
project grants (EY013845-01 and EY015006-01 to J.A.W.-M.), Canada
Foundation for Innovation (to J.A.W.-M.), and Natural Sciences and En-
gineering Research Council of Canada (to J.G.S.). J.A.W.-M. was a Re-
search to Prevent Blindness Career Development Awardee (2000–2003).
D.J.D. and G.P. contributed equally to this study.
Accepted for publication August 30, 2005.
Address reprint requests to Judith A. West-Mays, Ph.D., Department of
Pathology and Molecular Medicine, McMaster University, HSC 1R10,
Hamilton, ON, Canada L8N3Z5. E-mail: westmayj@mcmaster.ca.
American Journal of Pathology, Vol. 168, No. 1, January 2006
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2006.041089
69
(
-SMA), and begin to secrete an abnormal accumulation
of type I and III collagen.
8
Additional extracellular matrix
proteins are deposited, including tenascin and fibronec-
tin, and as a result, fibrous anterior subcapsular cataract
(ASC) plaques form that develop into distinct opacities in
the lens. Similar to ASC, in secondary cataract, also
known as posterior capsular opacification, LECs that
remain within the capsule after cataract surgery are
triggered to proliferate and migrate to the posterior
lens capsule, where they frequently transform into
myofibroblasts.
9,10
Matrix metalloproteinases (MMPs) are a family of zinc-
dependent endopeptidases that act as key regulators of
tissue remodeling
11
and have been shown to participate
in a number of ocular diseases, including retinal disease,
glaucoma, and corneal disorders.
12
MMPs and tissue
inhibitors of matrix metalloproteinases have also been
examined in the normal and cataractous lens. Although
some MMPs, such as MMP-9 and membrane-type
1-MMP, have been found to be constitutively expressed
in the lens,
13–15
other MMPs, such as MMP-2, are typi
-
cally expressed after treatment with growth factors or
during cataract formation. For example, Seomun et al
16
reported that TGF
triggers MMP-2 mRNA expression in
a human lens epithelial cell line and induces immunore-
activity for both MMP-2 and
-SMA in the subcapsular
plaques of rat lenses. TGF
has also been shown to
stimulate secretion of MMP-2 and MMP-9 in cultured
annular pad cells of the chick lens
17
and in human cap
-
sular bags.
18
Induction of the proforms of both MMP-2
and MMP-9 has also been reported during hydrogen
peroxide-induced cataract formation and after sham cat-
aract surgery.
19
Multiple MMPs and tissue inhibitors of
matrix metalloproteinases have also been detected in the
extracellular matrix and LECs from human capsules de-
rived from post-cataract/intraocular lens surgery tissue,
whereas normal anterior lens capsules did not.
20
Evidence that MMP-2 may have an active role in me-
diating the EMT that occurs in ASC has been provided by
findings in which overexpression of MMP-2 in the human
HLE B-3 lens cell line caused a conversion of the cells
into a myofibroblastic phenotype.
16
The ability of other
MMPs to induce LEC conversion, however, has not been
investigated. In addition, the requirement for MMPs in
mediating the formation of the TGF
-induced ASC
plaques in the ex vivo lens and the mechanism(s) by
which MMPs participate in ASC formation are not known.
Investigations in other systems, including cancer, have
shown that MMPs promote EMT by altering the E-cad-
herin/
-catenin pathway through a phenomenon known
as E-cadherin shedding.
21–24
Specifically, the associa
-
tion between E-cadherin and
-catenin is vulnerable to
enzymatic attack by multiple MMPs, including MMP-9
and MMP-2, resulting in the formation of E-cadherin ex-
tracellular domain fragments with reported sizes ranging
from 50 to 84 kd.
24 –26
Induction of MMPs by TGF
in the
lens may lead to the EMT of LECs through a specific
disruption in E-cadherin.
In the current study we directly tested the requirement
of MMP activity in TGF
-induced ASC formation using a
well-established ex vivo rat lens model
27
in conjunction
with the two different MMP inhibitors (MMPIs), the broad
spectrum inhibitor GM6001 and a MMP-2/9-specific in-
hibitor (MMPI2/9). Additional experiments examined the
capacity of these MMPIs to prevent decreases in optical
quality of the lens induced by TGF
and to affect the cell
adhesion molecule E-cadherin. Together, the findings of
this study demonstrate that co-culture of lenses with two
different MMPIs effectively suppressed TGF
-induced
ASC formation and suggest attenuation of E-cadherin as
a possible mechanism.
Materials and Methods
Ex Vivo Rat Lens Cataract Model
The previously established TGF
-induced rat cataract
model was used for these studies.
27
Briefly, lenses were
obtained from adult male Wistar rats and cultured over-
night in 3.5 ml of serum-free M199 medium supple-
mented with 50 IU/ml penicillin, 50
g/ml streptomycin,
and 2.5
g/ml fungizone (Invitrogen, Burlington, ON,
Canada). The following day, lenses were either left un-
treated or treated with TGF
-2 (R&D Systems, Minneap-
olis, MN) at a final concentration of 1 or 2 ng/ml. Some
lenses were co-treated with TGF
-2 and the broad spec-
trum MMP inhibitor GM6001 (Ilomastat) (Chemicon Inter-
national, Temecula, CA) at concentrations ranging from
10 to 25
mol/L or the MMP-2/9 inhibitor ((2R)-[(4-biphe-
nylylsulfonyl)amino]-N-hydroxyl-3-phenylpropionamide)
(Chemicon) at concentrations of 10 or 25
mol/L. The
GM6001 negative control (N-t-butoxycarbonyl-
L-leucyl-L-
tryptophan methylamide) (25
mol/L) (Calbiochem, San
Diego, CA) was also used. Lenses were subjected to
optical analysis at subsequent time points of 2, 4, or 6
days and photographed using a digital camera mounted
to a dissecting scope. The lenses were then fixed for
histology and immunofluorescence or subjected to laser
capture microdissection (LCM). The conditioned media
were also collected from each treatment group for zy-
mography and Western blot analysis.
Optical Analysis
Lens optical qualities (the average back vertex distance
[BVD]) and sharpness of focus (BVD error) were as-
sessed using the automated laser scanning system that
was developed at the University of Waterloo.
28 –30
This
system consists of a single collimated scanning helium-
neon laser source that projects a thin (0.05 mm) laser
beam onto a plain mirror mounted at 45° on a carriage
assembly. A digital camera captures the actual position
and slope of the laser beam at each step. When all steps
have been made (20 beam positions across each lens),
the captured data for each step position are used to
calculate the back vertex distance. BVD (in millimeters) is
defined as the measurement of the distance from the
surface of the lens to the focal point where the laser beam
crosses the optical axis of the lens being scanned. When
portrayed graphically, the average BVD for the lenses is
70 Dwivedi et al
AJP January 2006, Vol. 168, No. 1
plotted for each eccentric position (Figure 1). With less
spherical aberration, the data points line up as a straight
line. The poorer the quality of the lens, the greater the
variation in BVD (BVD error) is with eccentricity, as shown
for lenses treated with TGF
(Figure 1). Because BVD
error is a more sensitive measure of lens damage, the
results are expressed in terms of BVD error.
28 –30
Repeat
-
ed-measures analysis of variance was determined using
SPSS 11.0 statistical software and was used to assess
treatment, concentration of TGF
, and temporal effects
on the back vertex variability for the first experiment in
Figure 2. One-way analysis of variance was used for
analyzing data in the second experiment in Figure 3,
which examines the dosage effect of GM6001 on TGF
-
induced BVD error at a single time point. A probability
value (P) 0.05, indicating a 95% confidence interval,
was considered significant.
Histology and Immunohistochemistry
Lenses were collected from different treatment groups
and fixed overnight in an acetic acid:ethanol solution
(1:99), dehydrated, embedded in paraffin, and pro-
cessed for routine histology. Immunofluorescence was
performed on 5-
m-thick paraffin-embedded sections.
Sections were incubated with primary antibody specific
for
-SMA (1:100; Sigma, Oakville, ON, Canada) for 1
hour at room temperature, and bound primary antibodies
were visualized with a fluorescein-isothiocyanate anti-
mouse secondary antibody (1:50; Jackson ImmunoRe-
search Laboratories, West Grove, PA). All sections were
mounted in Vectashield mounting medium with 4,6-dia-
minodino-2-phenylindol (Vector Laboratories, Burlington,
ON, Canada) to visualize the nuclei.
Zymography
Conditioned media from all treatment groups were con-
centrated using 3.5-ml 10K Microsep concentrating de-
vices (Viva Sciences, Hanover, Germany). Before con-
centration, refrigerated media were warmed to 37°C. The
media were centrifuged at 1000 g (at room tempera-
ture) for 5 minutes to pellet any debris before loading.
Each device was loaded with an equal volume of super-
natant, and the concentration was performed by centrif-
ugation at 25°C for 30 minutes at 3000 g. An equal
Figure 1. Optical scans of rat lenses. The scatter plots represent the back
vertex measurements (focal length) of a control lens (left) and a TGF
-
treated lens (right). The y axis indicates the eccentricity of the laser beam
from the optical axis and the x axis demonstrates BVD measurements. Each
point on the scatter plot represents the back vertex distance from each beam
location. In a control (noncataractous) lens, there is little difference in back
vertex distance, demonstrating sharpness in focus. In a TGF
-treated lens,
there is substantial variation in the back vertex distances.
Figure 2. Back vertex variability for lenses left untreated, treated with TGF
, and treated with TGF
and GM6001. The graph shows the changes in back vertex
variability (BVD error; mm SEM) from day 0 (initial measurements before treatment) and 2, 4, and 6 days after treatment. Two concentrations of TGF
(1 and
2 ng/ml) were used in this experiment. Repeated-measures analysis of variance demonstrated that there are both treatment and temporal effects (P 0.05). At
day 6 after treatment, both concentrations of TGF
(
1 and *2 ng/ml) showed significantly higher BVD error measurements than other treatment groups, including
the control and the TGF
(1 ng/ml), GM6001 and TGF
(2 ng/ml), and GM6001 groups. Apparent differences between the TGF
(1 ng/ml) and TGF
(2 ng/ml)
groups did not reach statistical significance.
MMPIs Suppress TGF
-Induced Subcapsular Cataract Formation 71
AJP January 2006, Vol. 168, No. 1
volume of each concentrate was electrophoresed on
10% SDS-polyacrylamide gels containing 0.1% gelatin as
the substrate. After electrophoresis, the gels were devel-
oped as described previously
31
and stained in 0.5%
Coomassie brilliant blue for 1 hour followed by destaining
with 10% isopropanol. Sites of gelatinase activity were
detected as clear bands against a background of uniform
staining, which was digitally photographed.
Western Blot Analysis
Concentrated samples derived from the conditioned me-
dia were also examined by Western blot analysis. Equal
volumes of sample were electrophoresed on a 10% SDS-
polyacrylamide gel. The resolved bands were electro-
transferred onto a nitrocellulose membrane (Pall Corpo-
ration, Pensacola, FL). Membranes were blocked with 5%
skimmed milk powder in Tris-buffered saline (50 mmol/L
Tris base and NaCl [pH 8.5]) and 0.1% Tween-20 and
then incubated overnight at 4°C with primary antibodies
generated against MMP-9 (1:500; Chemicon Interna-
tional), MMP-2 (1:500; Chemicon), or E-cadherin (1:1500;
BD Transduction Laboratories). After this incubation,
membranes were probed with an horseradish peroxi-
dase-conjugated secondary antibody (1:7500; Amer-
sham Biosciences, Little Chalfont, Buckinghamshire, UK)
and ECL detection reagents (Amersham Biosciences).
The Western blots were scanned and analyzed by image
quantification software (ImageJ; National Institutes of
Health, Bethesda, MD). The relative density versus con-
trol ratio was estimated using GraphPad Prism Software
(GraphPad). Quantitative data were analyzed statistically
using a Student’s t-test and expressed as mean SEM.
A value of P 0.05 was considered significant.
Laser Capture Microdissection, RNA Extraction,
and cDNA Synthesis
After treatment, lenses were placed in a cryostat mold
containing Tissue-Tek OCT (Sakura Finetek, Torrance,
CA), frozen on dry ice, and then stored at 70°C. The
frozen tissue was then sectioned at 5 to 8
mina
cryostat, mounted on noncoated clean glass slides, and
stored again at 70°C. Immediately before LCM, the
frozen sections were thawed for 10 seconds and then
stained with HistoGene LCM Frozen Section Staining kit
(Arcturus, Mountain View, CA) using the protocol pro-
vided, with strict adherence to RNase-free conditions.
LCM was then performed using the PixCell II (Arcturus),
as described by others.
32,33
The HistoGene stain allowed
for the identification of the general morphology of the
epithelium. Cells from the epithelial region of the lens
were then captured on CapSure Macro LCM Caps (Arc-
turus) using the PixCell II LCM Microscope (Arcturus)
with a minimal beam diameter of 7.5
m. Total cellular
RNA was then extracted from lifted cells using the Pico-
Pure RNA Isolation kit (Arcturus). Purified RNA was ana-
lyzed both qualitatively and quantitatively using an Agi-
lent 2100 Bioanalyzer (Agilent Technologies, Foster City,
CA) and subjected to standard reverse transcription re-
actions (SuperScript II; Life Technologies).
Quantification of Gene Expression Using Real
Time Quantitative Polymerase Chain Reaction
(RT-QPCR)
E-cadherin and
-SMA gene expression from recov-
ered cDNA was analyzed with RT-PCR using a 96-well
Figure 3. Dosage effect of GM6001 on TGF
-induced BVD variability. This bar graph represents the back vertex variability (BVD error, mm) SEM of lenses
left untreated or treated with TGF
(2 ng/ml) or TGF
(2 ng/ml) plus four different concentrations of GM6001 (I) as indicated for 6 days. These measurements
show a decrease in BVD error as the GM6001 concentration increases to 25
mol/L. One-way analysis of variance was used to determine the dosage effect of
GM6001 on TGF
-treated lenses (P 0.05). BVD errors from TGF
-treated lenses co-treated with 20 or 25
mol/L GM6001 were not significantly different from
the control lenses. In contrast, lenses treated with TGF
(2 ng/ml) alone or with TGF
(2 ng/ml) plus GM6001 at 10 and 15
mol/L had BVD errors that were
significantly different from control lenses, as indicated by asterisks. Lenses treated with TGF
and the negative control for GM6001 (25
mol/L) also had BVD
errors that were significantly different from the controls.
72 Dwivedi et al
AJP January 2006, Vol. 168, No. 1
TaqMan optical reaction plate format on an ABI Prism
7700 sequence detection system (Applied Biosystems,
Foster City, CA). RNA was normalized to 18S and
GAPDH for each reaction. Each 25-
l PCR reaction
(including controls) contained TaqMan Universal Mas-
ter Mix (Applied Biosystems), gene-specific forward
and reverse primers (Mobix, Hamilton, ON, Canada),
and probes for target and endogenous control genes
(Applied Biosystems). Serial dilutions (one- to fivefold)
of standard samples were prepared in separate wells
in duplicate for endogenous control genes (18S and
GAPDH), E-cadherin, and
-SMA gene targets. Stan-
dard and unknown samples were added in a volume of
5
l. Thermal cycling parameters consisted of the fol-
lowing: 2 minutes at 50°C, 10 minutes at 95°C followed
by 40 cycles of 15 seconds at 95°C, and 1 minute at
60°C. The number of target gene copies was calcu-
lated from a standard curve generated in parallel with
each batch of samples. A linear relationship was de-
tected over at least 5 orders of magnitude. In all ex-
periments, the correlation coefficient was between
0.997 and 0.986. The normalization of samples was
performed by dividing the number of copies of both
E-cadherin and
-SMA by the number of copies of 18S
and GAPDH. PCR reactions for E-cadherin and
-SMA
cDNA quantification were performed using standard
cDNA dilution curves. Quantitative data were analyzed
statistically using a Student’s t-test and expressed as
means SEM. A value of P 0.05 was considered
significant.
Results
MMPIs Suppress Anterior Subcapsular Cataract
Formation
In the following experiments, the broad spectrum MMP
inhibitor GM6001 was used in the rat subcapsular cata-
ract model to determine whether it could effectively sup-
press TGF
-induced subcapsular cataract formation. To
perform these experiments, excised rat lenses were
treated with exogenous TGF
2 for a period of 6 days. The
TGF
-treated lenses exhibited multiple, distinct opacities
on the anterior surface of the lens, as previously de-
scribed
27,34
(Figure 4
B), whereas the untreated control
lenses remained transparent and devoid of opacities
(Figure 4A). In comparison with lenses treated with TGF
alone, lenses co-cultured with TGF
and GM6001 (25
mol/L) for the 6-day period did not exhibit visible sub-
capsular cataracts (Figure 4C) but resembled the control
lenses. Histological cross-sections of the lenses treated
with TGF
revealed the presence of numerous plaques
consisting of a multilayering of cells beneath the lens
capsule (Figure 4F) in contrast to the simple cuboidal
monolayer of epithelial cells observed in the control lens
(Figure 4E). Strong immunoreactivity of
-SMA was ob-
served in a proportion of the cells of the subcapsular
plaques in the TGF
-treated lenses (Figure 4F). In com-
parison, the lenses co-cultured with TGF
and GM6001
(25
mol/L) did not exhibit multilayering of the lens epi-
Figure 4. TGF
-induced subcapsular formation in the rat lens is inhibited by co-culture with GM6001 and specific MMP-2/9 inhibitor. An untreated control lens
(A), a lens treated with TGF
(2 ng/ml) (B), a lens co-cultured with TGF
(2 ng/ml) and GM6001 (25
mol/L) (C), and a lens co-cultured with TGF
(2 ng/ml)
and MMPI2/9 (25
mol/L) (D) are shown after 6 days of culture. The TGF
(2 ng/ml)-treated lens (B) exhibited distinct subcapsular plaques unlike the untreated
lens (A) or those co-cultured with GM6001 (25
mol/L) (C) and MMPI2/9 (25
mol/L) (D), which remained devoid of opacities. Immunolocalization of
-SMA
in cross-sections of lenses revealed strong immunoreactivity of
-SMA (green) in sections of lenses treated with TGF
(2 ng/ml) (F), confirming the presence of
subcapsular plaques. Control lenses (E), lenses co-cultured with TGF
(2 ng/ml) and GM6001 (25
mol/L) (G), and lenses co-cultured with TGF
(2 ng/ml) and
MMPI2/9 (25
mol/L) (H) showed no observable immunoreactivity to
-SMA. All sections were mounted in a medium with 4,6-diaminodino-2-phenylindol to
co-localize the nuclei (blue). Scale bars 100
m.
MMPIs Suppress TGF
-Induced Subcapsular Cataract Formation 73
AJP January 2006, Vol. 168, No. 1
thelium and no
-SMA immunoreactivity was observed
under the same immunolocalization conditions as out-
lined above (Figure 4G).
In parallel with the lenses above, lenses were co-
treated with TGF
and the specific MMP2/9 inhibitor at
concentrations of 10 or 25
mol/L for 6 days. Similar to
the findings for GM6001, these studies revealed that
co-treatment with the MMP2/9 inhibitor suppressed the
appearance of ASC plaques and
-SMA expression.
Lenses co-treated with TGF
and the MMP-2/9-specific
inhibitor (10
mol/L) exhibited slight multilayering of the
epithelium (two layers observed) in some regions of the
lens with very faint
-SMA immunoreactivity (data not
shown). However, lenses co-treated with TGF
and the
MMP-2/9 inhibitor (25
mol/L) resembled control lenses
and did not exhibit
-SMA immunoreactivity (Figure 4, D
and H). Note that lenses treated with each of the MMPIs,
in the absence of TGF
, resembled the control lenses
(not shown).
A laser scanning system (ScanTox) was next used to
determine quantitative differences in the optical quality of
the lenses from three different treatment groups (control,
TGF
, and TGF
plus 25
mol/L GM6001) at three time
points: days 2, 4, and 6. Analyses of BVD errors showed
that there was both a treatment and temporal effect of
TGF
on the cultured rat lens (Figure 2). Although the
BVD errors for the control lenses did not change signifi-
cantly from day 0 to 6, treatment with either 1 or 2 ng/ml
of TGF
caused a significant increase in BVD error by
day 6 of the time course. In contrast, the groups of lenses
co-treated with TGF
(at 1 and 2 ng/ml) and GM6001 (25
mol/L) did not exhibit a significant change in BVD error
over the 6-day period. Statistical differences between the
treatment groups at each time point were also deter-
mined. At days 0, 2, and 4, no significant difference in
BVD error was observed between all groups (Figure 2).
However, at day 6, both of the TGF
-treated groups of
lenses (1 and 2 ng/ml) exhibited a significantly larger
BVD error versus the control group and the GM6001
co-treated lenses (Figure 2). Lenses cultured with the
inhibitor alone did not exhibit changes in BVD error rela-
tive to untreated lenses (not shown).
A dose-dependent effect of the broad spectrum inhib-
itor GM6001 in preventing the TGF
-induced cataracts
was also observed. Similar to earlier experiments, treat-
ment with TGF
for 6 days resulted in BVD errors that
were statistically greater than control lenses. Similarly,
those lenses co-treated with TGF
and GM6001 (10 or 15
mol/L) significantly differed from controls and exhibited
cataracts. In contrast, lenses co-treated with higher con-
centrations of GM6001, such as 20 and 25
mol/L, ex-
hibited BVD errors that were very similar to the controls,
further demonstrating the dramatic suppression in cata-
ract formation (Figure 3). Treatment with an analog of
GM6001, with no MMP inhibitory activity (negative con-
trol), exhibited a similar BVD error to the TGF
-treated
lens, indicating that the effect of GM6001 on maintaining
lens optical quality was related to its MMP inhibitory
activity.
TGF
-Induced ASC Formation Is Accompanied
by Enhanced Secretion of MMP-2 and MMP-9
To examine the timing and level of induction of MMPs
in the rat lens after TGF
treatment and subcapsular
cataract formation, zymography was performed on
conditioned media of lenses taken at the 6-day time
point from the following treatment groups: control,
TGF
(2 ng/ml), TGF
(2 ng/ml) plus GM6001 (25
mol/L), and TGF
(2 ng/ml) plus the MMP-2/9-specific
inhibitor (25
mol/L). Conditioned media from all treat-
ment groups exhibited distinct bands on gelatin gels,
indicating the presence of MMPs with gelatinolytic
and/or collagenolytic activity (Figure 5A). Conditioned
media from control lenses exhibited expression of a
92-kd band, corresponding to the proform of MMP-9.
11
In comparison with control lens media, media from
lenses treated with TGF
exhibited additional bands of
62, 65, and 72 kd, corresponding to the active and
proforms of MMP-2;
11
MMP-9 levels were also in
-
creased. Media obtained from lenses co-treated with
TGF
and either GM6001 or MMP-2/9 inhibitor for 6
days exhibited reduced levels of all gelatinolytic bands
relative to that of TGF
-treated lenses (Figure 5A).
Confirmation and quantification of MMP-2 and
MMP-9 from all treatment groups at each time period
was performed using Western blot analysis. Blots de-
veloped with an MMP-2-specific antibody revealed the
presence of latent and active species of MMP-2 in
conditioned media from lenses treated with TGF
,
whereas the control lenses at all time points did not
exhibit detectable levels of MMP-2 protein (Figure 5B).
In comparison, conditioned media from the lenses co-
cultured with TGF
plus GM6001 or TGF
plus the
MMP-2/9 inhibitor showed undetectable levels of
MMP-2 similar to control lenses (Figure 5B). Blots
probed with the MMP-9-specific antibody revealed a
band at 92 kd, corresponding to the proform of MMP-9
(Figure 5B). Similar to the zymography results, consti-
tutive MMP-9 protein expression was evident in the
conditioned media of control lenses. In comparison,
media from lenses treated with TGF
exhibited signif-
icantly higher levels of MMP-9 after 2, 4, and 6 days of
treatment (Figure 5, B and C). Co-treatment with TGF
and GM6001 revealed significant attenuation of MMP-9
in conditioned media relative to TGF
treatment alone
at all three time points, whereas conditioned media
from co-treatment of TGF
and the specific MMP-2/9
inhibitor showed a significant attenuation of MMP-9 at
days 2 and 4 only (Figure 5, B and C). Note that lenses
treated with either MMPI alone exhibited levels of
MMP-9 and MMP-2 similar to that of control lenses
(data not shown).
MMPIs Attenuate TGF
-Induced E-Cadherin
Shedding in the Rat Lens
Because previous studies had shown the ability of
MMPs to induce E-cadherin shedding,
22–24
the follow
-
ing experiments were designed to determine whether
74 Dwivedi et al
AJP January 2006, Vol. 168, No. 1
TGF
treatment of the rat lens results in an induction of
E-cadherin shedding and whether this can be modu-
lated by the MMPIs. To accomplish this, previously
concentrated conditioned media were obtained from
the 2-, 4-, and 6-day treatment groups outlined earlier
and subjected to Western blot analysis using an anti-
body specific for the extracellular domain of E-cad-
herin, which has been used in previous studies to
detect the presence of soluble E-cadherin fragments.
An E-cadherin fragment of approximately 72 kd was
detected in the conditioned media from lenses treated
with TGF
for 6 days and was not observed in media
from untreated lenses (Figure 6A). Media from lenses
treated with TGF
for shorter time periods (days 2 and
4) did not exhibit detectable levels of the fragment (not
shown). Importantly, a significant twofold reduction in
the levels of this fragment was detected in media from
lenses co-treated with TGF
and the broad spectrum
inhibitor GM6001, whereas levels were undetectable in
media from lenses co-treated with TGF
and the MMP-
2/9 inhibitor (Figure 6A). Lenses treated with either of
the MMPIs alone also exhibited undetectable levels of
the E-cadherin fragment (data not shown). Thus, co-
culture of lenses with either of the MMPIs tested re-
sulted in attenuated levels of TGF
-induced E-cad-
herin fragment.
MMP inhibitors have been shown to augment cell-
cell adhesion and specifically increase expression
of cadherins.
23
We therefore examined the levels of
E-cadherin mRNA using RT-QPCR. After 6 days of cul-
ture, we examined epithelial cells or plaque cells of
lenses from the following treatment groups: control
lenses, lenses treated with TGF
(2 ng/ml), TGF
(2
ng/ml) plus GM6001 (25
mol/L), or GM6001 (25
mol/L) alone. For these experiments, cryostat sec-
tions of lenses were subjected to LCM to specifically
isolate the cells in the plaques. RT-QPCR findings re-
vealed that whereas E-cadherin mRNA was detected in
the normal lens epithelium (Figure 6B), its expression,
relative to that of GAPDH, was suppressed nearly four-
fold in the plaque tissue of TGF
-treated lenses. In
comparison, E-cadherin levels in cells from the epithe-
lial region of lenses co-treated with TGF
and GM6001
(25
mol/L) were significantly higher (6.9-fold) than
those treated with TGF
alone and significantly greater
than that of controls. Treatment with 25
mol/L
GM6001 alone also produced a significant increase in
E-cadherin when compared with control lenses ( P
0.01) (2.5-fold). When these experiments were per-
formed using the 18S housekeeping gene, data exhib-
ited the same trends seen with GAPDH (not shown).
Thus, treatment of lenses with GM6001 prevented the
attenuation of E-cadherin mRNA induced by TGF
and
Figure 5. Effect of TGF
and MMPIs on MMP protein levels. A: Gelatin
zymography of conditioned media from cultured lenses after 6 days of
treatment. MMP-9 (92 kd) was detected in media from all of the untreated
( C) lenses at day 6 of the culture period. After treatment with TGF
(2
ng/ml ) (T), up-regulated levels of MMP-9 were observed relative to
controls. Conditioned media obtained from lenses treated with TGF
(2
ng/ml ) and GM6001 ( 25
mol/L ) (TI ) exhibited attenuated bands and
resembled levels secreted by control lenses. However, conditioned media
obtained from lenses co-cultured with TGF
( 2 ng/ml) and MMPI2/9 ( 25
mol/L ) ( T2/9) exhibited up-regulated levels of MMP-9, relative to
controls. Gelatinolytic bands for proMMP-2 ( 72 kd) and active MMP-2 (65
kd ) were observed in conditioned media from the TGF
-treated ( 2 ng/ml)
( T) lenses but not in control ( C) lenses. Media from TGF
( 2 ng/ml ) and
GM6001 ( 25
mol/L ) (TI) and lenses co-cultured with TGF
( 2 ng/ml)
and MMPI2/9 ( 25
mol/L ) (T2/9) exhibited negligible activity for
MMP-2. B: Representative Western blot. Conditioned media obtained
from control lenses (C) and lenses treated with TGF
( 2 ng/ml) (T), TGF
( 2 ng/ml) and GM6001 (25
mol/L ) (TI), and TGF
( 2 ng/ml) and
MMPI2/9 (25
mol/L ) (T2/9 ) at 2, 4, and 6 days were examined by
Western blot analysis to confirm the identity of MMP-2 and MMP-9. A
representative blot demonstrates that MMP-9 was constitutively expressed
in all of the control lenses (C ) over 2, 4, and 6 days. An up-regulation in
MMP-9 protein was observed in media from lenses treated with TGF
(2
ng/ml ) (T) at day-2, -4, and -6 time points compared with control lenses
( C), whereas the lenses co-cultured with TGF
and GM6001 ( 25
mol/L )
(TI) exhibited levels similar to the controls at all three time points.
Lenses co-cultured with TGF
( 2 ng/ml) and MMPI2/9 (25
mol/L )
(T2/9) exhibited an up-regulation in MMP-9 protein at day 6 compared
with control lenses (C), whereas at day-2 and -4 time points, the lenses
exhibited levels similar to the controls. At all three time points, MMP-2
protein was only detected in media from lenses treated with TGF
(2
ng/ml ) ( T ), whereas the other treatment groups did not exhibit detectable
levels. C: Densitometric analysis of MMP-9 protein levels in conditioned
media. The Western blot data for MMP-9 from three separate experiments
were analyzed by densitometry. Values are expressed as the relative
density versus control ratio (RD/C) SEM of three blots. Note a signifi-
cant up-regulation ( *P 0.05) of MMP-9 in the conditioned media of
TGF
( 2 ng/ml ) (T)-treated lenses compared with control. A significant
reduction ( *P 0.05 ) was observed in the expression of MMP-9 in
conditioned media of lenses co-treated with TGF
( 2 ng/ml ) and GM6001
(25
mol/L ) (TI ) compared with TGF
( 2 ng/ml) ( T). In the condi-
tioned media of lenses co-cultured with TGF
( 2 ng/ml) and MMPI2/9 (25
mol/L ) (T2/9), a significant reduction (*P 0.05 ) was observed in the
expression of MMP-9 at day-2 and -4 time points compared with TGF
(2
ng/ml ) ( T ), whereas there was no significant reduction at day 6 compared
with TGF
( 2 ng/ml) ( T).
MMPIs Suppress TGF
-Induced Subcapsular Cataract Formation 75
AJP January 2006, Vol. 168, No. 1
further increased the constitutive levels of E-cadherin
mRNA.
For comparison, we also examined the levels of
-SMA
mRNA in the same tissues used to perform the E-cadherin
studies (Figure 6C). These experiments revealed little to
no expression of
-SMA mRNA in the cells obtained from
the epithelial region of control lenses whereas its expres-
sion, relative to that of GAPDH, was significantly induced
in the plaque tissue of TGF
-treated lenses. This corre-
sponds with our earlier experiments, which showed in-
creased
-SMA protein immunoreactivity in the subcap-
sular plaques (Figure 4F).
-SMA mRNA levels in the
LECs co-treated with the MMPI were substantially re-
duced compared with the plaque cells of TGF
-treated
lenses, demonstrating that co-culture with the MMPI re-
sulted in suppression in induction of
-SMA by TGF
.
Addition of GM6001 (25
mol/L) alone to lenses did not
alter
-SMA mRNA levels when compared with those of
control lenses (Figure 6C).
Discussion
Increasing evidence regarding the importance of
MMPs in development and pathology has accumulated
over the last 10 years. In the eye, MMPs have been
shown to contribute to a number of ocular diseases
and disorders including retinal disease, glaucoma,
corneal ulcers and corneal postoperative haze.
12
More
recently, induction of MMP expression has been cor-
related with the formation of cataracts.
16,18,19
However,
the role of MMPs and the mechanism by which they
contribute to cataractogenesis are not well under-
stood. In the current study, we used the previously
established ex vivo rat lens subcapsular cataract
model to directly test the involvement of MMPs in the
initiation and progression of TGF
-induced ASC. We
demonstrated that co-treatment with TGF
and two
different MMPIs, the broad spectrum inhibitor GM6001
and the MMP-2/9-specific inhibitor, resulted in sup-
pression of TGF
-induced ASC formation, including
the appearance of
-SMA-expressing cells indicative
Figure 6. Effect of TGF
and GM6001 on
-SMA and E-cadherin mRNA and
shedding. A: Representative Western blot and densitometric analysis of
E-cadherin protein released into conditioned media. The Western blot re-
vealed the presence of a 72-kd E-cadherin fragment in the conditioned media
from lenses treated with TGF
(2 ng/ml) (T) that was not detected in media
from control lenses (C) and lenses co-treated with TGF
(2 ng/ml) and
MMPI2/9 (25
mol/L) (T2/9). Levels of the E-cadherin fragment were
suppressed in media from lenses co-cultured with TGF
and GM6001 (25
mol/L) (TI), relative to that from TGF
-treated lenses. Values are ex-
pressed as optical density units (ODU) SEM of three blots. Note the
presence of E-cadherin fragment in conditioned media of TGF
-treated (2
ng/ml) (T) lenses compared with control (C). A significant (*P 0.05)
reduction in the levels of the E-cadherin fragment was observed in media
from lenses co-treated with TGF
(2 ng/ml) and GM6001 (25
mol/L)
compared with TGF
(T). B: E-cadherin mRNA expression using RT-QPCR.
E-cadherin mRNA levels, relative to GAPDH, were also measured for the
lenses treated in A using RT-QPCR, and the values are given as SEM of
three separate experiments. All comparisons were two tailed. The RT-QPCR
results revealed that cataractous plaques derived from lenses treated with
TGF
(2 ng/ml) (T) for 6 days exhibited a significant suppression (*P 0.01)
in E-cadherin mRNA levels compared with epithelial cells from control
lenses (C). Lenses co-cultured with TGF
and GM6001 (25
mol/L) (TI)
exhibited significantly higher levels (**P 0.001) of E-cadherin compared
with those treated with TGF
(2 ng/ml) (T) (nearly sevenfold). The GM6001
(25
mol/L) alone group (I) exhibited significantly elevated levels of E-
cadherin relative to control (C) lenses (*P 0.01). C:
-SMA mRNA expres-
sion using RT-QPCR. The
-SMA mRNA levels were measured relative to
GAPDH, and the values are given as SEM. All comparisons were two tailed.
The RT-QPCR results revealed that the control lens epithelium (C) expressed
minimal levels of
-SMA mRNA. The cells isolated from the cataractous
plaques of the TGF
-treated (2 ng/ml) (T) lenses expressed significantly
higher levels (*P 0.001) of
-SMA mRNA compared with epithelial cells
from control lenses (C). Lens epithelium from lenses co-cultured with TGF
(2 ng/ml) and GM6001 (25
mol/L) (TI) exhibited significantly less (*P
0.001)
-SMA mRNA compared with those treated with TGF
(2 ng/ml) (T).
Lenses treated with GM6001 alone (25
mol/L) did not exhibit statistically
significant elevated levels of
-SMA when compared with control lenses (C).
76 Dwivedi et al
AJP January 2006, Vol. 168, No. 1
of the EMT of LECs. Further evidence is provided to
show that treatment of the rat lens with TGF
resulted
in appearance of proteolytic fragments of the cell-cell
adhesion molecule E-cadherin, an event that was at-
tenuated by co-treatment with either of the two MMPIs
tested. Together, these data suggest that the suppres-
sion of TGF
-mediated E-cadherin shedding and deg-
radation is a possible mechanism by which MMPIs
reduce the appearance of ASC plaques.
Earlier work has shown that MMP-2 expression is in-
duced in the rat lens after treatment with TGF
.
16
Fur
-
thermore, overexpression of MMP-2 via stable transfec-
tion results in conversion of cultured human LECs into
-SMA-expressing cells.
16
Similar to these findings, we
also report induced secretion of MMP-2 in the rat lens
after treatment with TGF
, accompanied by enhanced
secretion of MMP-9. A recent study revealed that MMP-9
and MMP-2 mRNA are expressed in the normal rat lens
epithelium.
35
This suggests that constitutive mRNA ex
-
pression of these MMPs does not result in the EMT of
LECs and cataract formation. However, induced levels of
MMP-2 and/or MMP-9, above those of constitutive ex-
pression, and accompanying secretion of MMPs could
result in EMT in the lens. Induced levels of MMP-2 and
membrane-type 1-MMP have been correlated with EMT
in the embryonic heart, and overexpression of MMP-3
has been shown to cause conversion of mammary epi-
thelial cells into mesenchymal cells.
21,36
The fact that
co-treatment with GM6001 and the specific MMP2/9 in-
hibitor resulted in suppression of TGF
-induced
-SMA
expression in the rat lens, further corroborates these
earlier findings and suggests that MMPs are important for
mediating the EMT of LECs. We further show that treat-
ment with MMPIs led to a substantial reduction in subse-
quent plaque formation in the ex vivo lens and mainte-
nance of lens optical quality. Because MMP-2 and
MMP-9 were induced after TGF
treatment and the
MMPIs used have inhibitory activity against both, it can-
not be discerned whether one or both of these MMPs
participate in ASC formation. Future studies that target
the individual expression of MMP-2 or MMP-9, such as
through gene knockdown experiments or the use of
specific MMP-2 and MMP-9 knockout mouse models, will
help to further determine whether one or both of these
MMPs is critical for ASC formation.
MMPs are principally known for their role in extracel-
lular matrix (ECM) remodeling.
11
However, additional
roles for MMPs have emerged, including their ability to
regulate cell migration, invasion, and EMT.
21,36
It has
been suggested that MMPs may contribute to EMT by
participating in the separation of epithelial cells from
their basement membrane,
37,38
thereby promoting
their migration during cellular transformation.
7,37,38
However, more recent findings suggest that MMPs par-
ticipate in the initial activation stages of EMT through
dissociation of the E-cadherin/
-catenin complex.
22–24
Proteolytic cleavage of the N-terminal extracellular do-
main of E-cadherin by MMPs, referred to as “E-cad-
herin shedding,” results in the formation of an E-cad-
herin extracellular domain fragment with reported sizes
ranging from 50 to 84 kd, compared with the intact
120-kd protein.
39
Here, we report the appearance of a
72-kd E-cadherin fragment in the conditioned media of
lenses treated with TGF
that was not detected in the
media from untreated lenses. To the best of our knowl-
edge, this is the first report of E-cadherin shedding in
a cataract model. The appearance of the E-cadherin
fragment in the TGF
-treated rat lenses was also
shown to coincide with enhanced levels of secreted
MMP-2 and MMP-9, MMPs that have been implicated
in E-cadherin shedding and degradation in other sys-
tems.
24,40
We further demonstrated that the TGF
-in
-
duced levels of the E-cadherin fragment were attenu-
ated by co-treatment with either MMPI, broad spectrum
GM6001, or MMP-2/9 inhibitor, suggesting that TGF
-
induced fragmentation of E-cadherin is mediated by
MMP activity. Interestingly, the ability of GM6001 to
suppress E-cadherin shedding has been shown in nitric
oxide-treated murine colonic epithelial cells, and this led to
further stabilization of E-cadherin junctions.
22,24
Along with E-cadherin shedding, we also reported a
significant decrease in the E-cadherin mRNA levels in
the subcapsular plaque tissue of TGF
-treated lenses.
Decreased E-cadherin expression has also been re-
ported after dexamethasone-induced cataract forma-
tion in the cultured rat lens; however, evidence of
E-cadherin shedding in this cataract model was not
investigated.
41
Cadherins are known to act as cell
signaling receptors by controlling the localization of
-catenin. Proteolytic shedding of E-cadherin causes
dissociation of
-catenin from membrane-bound
E-cadherin, resulting in increases in the levels of free
-catenin in the cytoplasm.
24
-Catenin can then local
-
ize to the nucleus where it associates with the T-cell
factor/lymphoid enhancer factor(s) to activate or re-
press target gene expression.
22
Thus, the changes in
E-cadherin and
-SMA mRNA levels that we observed
may be associated with changes in
-catenin localiza-
tion. The levels of E-cadherin mRNA in lenses co-
treated with TGF
and GM6001 were found to be well
above that of control lenses, and treatment of lenses
with GM6001 alone also resulted in a substantial ele-
vation in the constitutive amount of E-cadherin mRNA.
These findings suggest that E-cadherin mRNA expres-
sion was either stimulated or stabilized by GM6001.
The findings of the current study also revealed that
treatment with the broad spectrum inhibitor GM6001 sup-
pressed TGF
-induced levels of both MMP-2- and MMP-
9-secreted protein, whereas the MMP-2/9 inhibitor spe-
cifically blocked MMP-2 induced levels. The primary
function of both MMPIs is to inhibit MMP enzymatic ac-
tivity. However, MMPIs such as GM6001 have been
shown to have an inhibitory effect on MMP expression in
other systems, yet this has remained unexplained.
42,43
Previous work has shown that the myofibroblast-like cells
in the subcapuslar plaques of rat lenses treated with
TGF
exhibited immunoreactivity for both
-SMA and
MMP-2.
16
Thus, the absence of MMP-2-secreted protein
in the media from lenses co-treated with either of the two
MMPIs may simply be due to the fact that both MMPIs
suppress the appearance of myofibroblasts, the cell type
that expresses MMP-2.
MMPIs Suppress TGF
-Induced Subcapsular Cataract Formation 77
AJP January 2006, Vol. 168, No. 1
In summary, the findings of the current study have
shown that treatment with the broad spectrum MMP
inhibitor GM6001 and the specific MMP2/9 inhibitor
significantly suppressed the formation of TGF
-in-
duced ASC plaques in the rat lens. This suppression
was associated with decreased
-SMA expression and
a significant decrease in the 72-kd soluble E-cadherin
fragment, indicative of E-cadherin shedding. Together,
these data suggest the novel finding that MMPIs sup-
press the EMT of LECs in ASC formation through inhi-
bition in MMP-mediated disruption of E-cadherin.
Based on these findings, two possible therapeutic
strategies for the prevention of ASC formation can be
proposed: inhibition of MMP activity and/or attenuation
of aberrant E-cadherin shedding.
Acknowledgments
We thank Giuseppe Pontoriero and Paula Deschamps for
their technical assistance.
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MMPIs Suppress TGF
-Induced Subcapsular Cataract Formation 79
AJP January 2006, Vol. 168, No. 1
    • "A number of signaling pathways, including TGFβ, are involved in promoting EMT during both development and disease (Kalluri and Weinberg, 2009; Thiery et al., 2009). The phenotypic changes found in EMT are associated with molecular changes including reduced expression of epithelial proteins such as the adherens junction protein, E-cadherin, and the tight junction protein, zonula occludens 1 (ZO-1), and upregulation in the expression of mesenchymal proteins (Kalluri and Weinberg, 2009; Thiery et al., 2009), and proteins involved in remodeling of the extracellular matrix such as certain matrix metalloproteinases (Dwivedi et al., 2006). While it is known that factors such as E-cadherin and ZO-1 are essential components of cell adhesion and polarity, and their loss promotes EMT, much less is known about the upstream factors that are required to establish and maintain epithelial structure in vertebrates. "
    [Show abstract] [Hide abstract] ABSTRACT: The integrity and function of epithelial tissues depends on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.
    Full-text · Article · Oct 2013
    • "Previous studies from our laboratory have shown that MMP-2 and -9 are involved in TGFβ-induced EMT in the lens. For example, we have shown that in whole rat lenses, the MMP-2/9 inhibitor (MMPi) can suppress TGFβ-induced EMT in the lens (as determined by αSMA expression) and subsequent subcapsular cataract formation [38]. To determine the involvement of MMP-2 and -9 in TGFβ-induced MRTF-A translocation, rat lens explant cultures were treated with TGFβ or cotreated with TGFβ, and 25 µM of the MMP-2/9 inhibitor (MMPi) for 48 h and subsequently stained for MRTF-A and αSMA. "
    [Show abstract] [Hide abstract] ABSTRACT: Transforming growth factor beta (TGFβ) is a known inducer of epithelial to mesenchymal transition (EMT), and studies in other systems have shown that nuclear localization of the myocardin-related transcription factor (MRTF) is downstream of TGFβ. In the following study, we investigated whether nuclear translocation of MRTF-A or MRTF-B is involved in TGFβ-induced EMT of lens epithelial cells (LECs). We further investigated the relationship between matrix metalloproteinase-2 and -9 (MMP-2/9) and MRTF in the EMT of LECs. Rat lens explant cultures were used as the model system. Explants were treated with TGFβ, an MMP-2/9 inhibitor, or actin binding drugs and immunostained for alpha smooth muscle actin (αSMA), MRTF-A, and MRTF-B. Cytoplasmic and nuclear intensities of cells were measured using ImageJ. Production of αSMA was measured using western blot analysis and ImageJ. Untreated explant cells exhibited little αSMA expression, and MRTF-A and B were found to reside primarily in the cytosol. However, when stimulated with TGFβ, a significantly greater number of cells exhibited nuclear expression of MRTF-A, accompanied by an increase in αSMA expression. However, MRTF-B remained in the cytoplasm following TGFβ treatment. Cotreatment with an MMP-2/9 inhibitor and TGFβ resulted in reduced MRTF-A nuclear localization and αSMA expression compared to cells treated with TGFβ alone. Our results are the first to demonstrate the expression of MRTF-A in LECs and that its nuclear translocation can be stimulated by TGFβ. Our data further suggest that MMP-2 and -9 are involved in the translocation of MRTF-A in LECs during TGFβ-induced EMT.
    Full-text · Article · May 2013
    • "Our laboratory has shown that MMP inhibition can suppress TGFβ-stimulated fibrosis in the lens. For example, using a TGFβ-induced model of anterior subcapsular cataracts in rats, we demonstrated that cotreatment with the MMP-2/9 specific inhibitor effectively prevented anterior subcapsular cataract formation and the associated deposition of matrix [43] . In addition , MMP inhibition has also been shown to prevent matrix deposition in injury-induced tissue remodeling [44,45]. "
    [Show abstract] [Hide abstract] ABSTRACT: Extracellular matrix remodeling is thought to have profound effects on tissue architecture and associated function. We have shown previously that overexpression of transforming growth factor beta (TGFβ), which stimulates matrix accumulation, results in altered morphology, cataract, and ocular hypertension in rodents. We have further shown that TGFβ-induced cataracts can be mitigated through inhibition of the matrix metalloproteinases (MMP) MMP-2 and MMP-9. We therefore sought to determine whether loss of MMP expression also altered TGFβ-induced changes in intraocular pressure (IOP). To carry out this study, TGFβ1 transgenic mice were bred onto a MMP-9 null background. IOP measurements were made at 1- to 2-, 2- to 3-, and 3- to 4-month time points using a TonoLab rebound tonometer. Histological and immunofluorescence findings were obtained at the same time points. Our results demonstrate that lens-specific expression of TGFβ1 in mice results in altered morphology of the anterior segment and an accompanying significant increase in IOP. TGFβ1 transgenic mice bred onto the MMP-9 null background exhibited a further increase in IOP. Interestingly, the MMP-9-deficient animals (without the TGFβ transgene), which exhibited normal angle morphology, had increased IOP levels compared to their wild-type littermates. These results indicate that TGFβ and MMP-9 likely act independently in regulating IOP. Additionally, MMP-9 plays an important role in maintaining IOP, and further investigation into the mechanisms of MMP-9 activity in the anterior angle may give clues to how extracellular matrix remodeling participates in ocular hypertension and glaucoma.
    Full-text · Article · Mar 2013
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