Abstract
purpose. To determine the levels of mRNAs encoding markers of fibrosis in lens
epithelial cells (LECs) from patients with anterior polar cataracts and
to test whether transforming growth factor (TGF)-β enhances the
expression of mRNAs for mesenchymal markers in LECs.
methods. LECs attached to the anterior capsules of patients with nuclear or
anterior polar cataracts were analyzed by reverse
transcription–polymerase chain reaction (RT-PCR) for the expression of
mRNAs encoding pathologic extracellular matrix proteins, a marker of
myofibroblast transformation, growth factors, and growth factor
receptors, and by western blot analysis for the proteins encoded by
these mRNAs. Bovine lens epithelial explants and intact rabbit lenses
cultured with or without TGF-β1 were also subjected to RT-PCR and
western blot analysis.
results. The levels of fibronectin, type I collagen, and α-smooth muscle actin
(SMA) mRNAs were higher in LECs from patients with anterior polar
cataracts than in those from patients with nuclear cataracts.
Expression of mRNAs for TGF-β1, TGF-β2, TGF-β receptor type II,
and connective tissue growth factor (CTGF) was significantly greater in
anterior polar type than in nuclear type cataracts. In contrast,
expression of mRNAs for epidermal growth factor (EGF), epidermal growth
factor receptor (EGFR), fibroblast growth factor (FGF)-2, and FGF
receptor-1 was similar in LECs from the two types of cataracts.
TGF-β1 markedly increased the levels of fibronectin, type I collagen,
and α-SMA mRNA in bovine lens epithelial explants and intact rabbit
lenses.
conclusions. This is the first finding showing altered mRNA expression in LECs from
anterior polar cataracts. Enhanced expression of TGF-β and the
TGF-β receptor suggests that TGF-β derived from LECs may function
in an autocrine fashion as the prime mediator of transdifferentiation
and pathogenesis in human LECs. Elevated levels of CTGF mRNA suggest
that this growth factor may play a role in the increased deposition of
extracellular matrix in metaplastic LECs.
Epithelial–mesenchymal transformation is a dynamic process in
which cells change from the epithelial state of differentiation into a
mesenchymal phenotype. This transition is known to occur normally
during embryogenesis. Epithelial–mesenchymal transformation is,
however, thought to be a critical pathologic event in certain diseases,
such as metastasis of cancer cells and pathologic fibrosis in some
abnormal conditions.
1 2 Transforming growth factor-β
(TGF-β), a multifunctional cytokine, has been implicated in
epithelial–mesenchymal transformation of many cell types, such as
cardiac endothelium and mammary epithelial cells.
3 4 TGF-β also promotes the production and deposition of extracellular
matrix, which is essential to normal tissue repair after
injury.
5 However, overexpression of TGF-β can cause
tissue fibrosis in many pathologic conditions such as scleroderma,
idiopathic pulmonary fibrosis, and liver fibrosis.
6 7 8 9 TGF-β stimulates the expression of other growth factors, including
platelet-derived growth factor and connective tissue growth factor
(CTGF) that may act locally in fibrotic lesions. CTGF is implicated in
connective tissue formation and appears to mediate some of the actions
of TGF-β in fibrosis.
10 It is transiently expressed
during normal repair processes but may be permanently overexpressed in
various fibrotic conditions. TGF-β and CTGF are coordinately
expressed at sites of wound repair and in fibrotic disorders such as
atherosclerosis, pulmonary fibrosis, and renal
fibrosis.
11 12 13
Lens epithelial cells (LECs) can transdifferentiate into
mesenchyme-like cells during the formation of anterior subcapsular
cataracts and after-cataracts.
14 15 Abnormal extracellular
materials, including type I and type III collagen, accumulates around
the transdifferentiated cells in these cataracts.
16 17 18 Embryonic LECs also undergo transdifferentiation into fibroblasts when
suspended within native type I collagen gels in
vitro.
19 20 These mesenchyme-like cells stop producing
type IV collagen, laminin, and lens crystallins and, instead,
synthesize type I collagen. In cultured rat lenses and lens epithelial
explants, TGF-β added to the culture medium induces cataractous
changes resembling anterior subcapsular cataract.
21 22 23 In
addition, overexpression of active TGF-β by lens fiber cells causes
anterior subcapsular cataract in mice.
24 However, there
have been few studies attempting to correlate expression level of
TGF-β or other growth factors with pathogenic changes in LECs
obtained from human cataracts. Moreover, precise molecular mechanisms
whereby TGF-β mediates transdifferentiation and overproduction of
extracellular matrix materials of LECs are not clearly understood.
Therefore, the objective of this study was to characterize
transdifferentiated LECs in anterior subcapsular cataracts by analyzing
the levels of mRNAs encoding proteins characteristic of myofibroblasts.
We also assayed growth factors and growth factor receptors that may be
involved in the transdifferentiation of LECs. These studies were
supplemented by experiments designed to test whether TGF-β can induce
mRNAs and proteins characteristic of fibroblasts in LECs in vitro.
Lens capsules with attached LECs were obtained during cataract
surgery from 40 patients with the clinical diagnosis of nuclear or
anterior polar cataracts. The ages of the patients ranged from 38 to 91
years. Lens capsules of noncataractous lenses were obtained during
clear lens extraction for correction of high myopia. Briefly, after
injecting viscoelastic material into the anterior chamber, continuous
curvilinear capsulorrhexis was performed, and the anterior capsule was
removed carefully with forceps. Lens capsules were immediately placed
in TRIzol reagent (Gibco, Gaithersburg, MD) for RNA preparation or
frozen in liquid nitrogen and stored at −70°C for protein
extraction.
Young bovine eyes were obtained from a local abattoir and
transported to the laboratory in a 4°C cold chamber. After the cornea
was excised, the iris and ciliary body were removed, and the lens
capsule was torn with a bent syringe needle. The lens capsule was
grasped with fine forceps and removed from the remainder of the lens
with the assistance of fine scissors. The separated layer containing
LECs was transferred to a six-well plate coated with type IV collagen
(Sigma, St. Louis, MO) in serum-free minimum essential medium (MEM;
Gibco) containing 0.2% (wt/vol) bovine serum albumin (Sigma). The
fresh explants were incubated with the cell layer downward on cell
culture dishes in the absence or presence of 10 ng/ml human recombinant
TGF-β1 (Sigma). After the indicated periods, total cellular RNA was
prepared and subjected to RT-PCR. For western blot analysis, cell
lysate was prepared as will be described.
All procedures in this study adhered to the ARVO Statement for the
Use of Animals in Ophthalmic and Vision Research. The eyes were removed
from New Zealand albino rabbits weighing approximately 2 kg immediately
after euthanasia, and the lenses were carefully dissected by a
posterior approach. Each of the dissected lenses was placed in a well
of a 12-well culture plate containing 2 ml MEM for 1 to 2 hours.
Transparent eyes without surgical damage were selected for
experimentation. For treatment, each sample containing two transparent
lenses was incubated in 2 ml MEM containing bovine serum albumin with
TGF-β1 (20 ng/ml), fibroblast growth factor (FGF)-2 (20 ng/ml;
Sigma), or medium only. After the indicated periods, total RNA or
protein extract from LECs was used for RT-PCR or western blot analysis.
Total cellular RNA was isolated from LECs attached to the anterior
capsules of human lenses or animal lenses by using TRIzol reagent. One
microgram RNA was reverse transcribed in a 20-μl reaction mixture by
using a kit (1st Strand cDNA Synthesis; Boehringer Mannheim,
Indianapolis, IN). The cDNA (0.2–1 μl) was amplified in a 20-μl
reaction mixture. Conditions for PCR were as follows: 0.4 μM each
primer, 0.2 mM deoxynucleoside triphosphate mixture (Perkin Elmer,
Foster City, CA), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM
MgCl
2, and 1.0 U
Taq DNA polymerase
(Perkin Elmer). Reaction mixtures were incubated in a thermal
controller (model PTC-100; MJ Research, Watertown, MA) for 25 to 35
cycles (denaturation at 94°C for 45 seconds, annealing at 60°C for
30 seconds, extension at 72°C for 1 minute). The amounts of amplified
products were analyzed using an image documentation system (ImageMaster
VDS; Pharmacia Biotech, Uppsala, Sweden). DNA size markers were run in
parallel to validate the predicted sizes of the amplified bands (D-15
DNA marker; Novex, San Diego, CA). The primer sequences specific for
the genes examined and predicted product sizes are shown in
Table 1 . All primer sequences were designed by using primer selection
software offered through a Web site (Primer 3; Center for
Genome Research, the Whitehead Institute for Biomedical Research,
Cambridge, MA, www-genome.wi.mit.edu), except those indicated in
Table 1 .
Lens epithelial cells were lysed on ice in lysis buffer (20 mM
Tris-HCl [pH 7.5], 120 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM
EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 20 μM leupeptin, and 1 μM aprotinin; Sigma). For sodium
dodecyl sulfate (SDS)- and urea-soluble fractions, cells were lysed in
buffer containing 1% SDS and 8 M urea, respectively. After
centrifugation for 10 minutes at 10,000g, the supernatant
was stored at −20°C. Protein concentrations were determined by using
a protein assay kit (DC; Bio-Rad Laboratories, Hercules, CA). The
lysates containing 20 μg protein were boiled for 5 minutes in SDS
sample buffer, fractionated by SDS−10% polyacrylamide gel
electrophoresis, and transferred to a nitrocellulose membrane (Hybond;
Amersham Life Science, Cleveland, OH) using an electroblot apparatus
(Bio-Rad). Membranes were blocked for 1 hour in
Tween–phosphate-buffered saline (PBS-Tween) containing 5% nonfat milk
powder. The membranes were then incubated with primary antibody at
1:1000 dilution in PBS-Tween containing 5% nonfat milk powder at room
temperature for 45 minutes and washed in PBS-Tween. The primary
antibodies used in this study were rabbit anti-human fibronectin
antibody (Sigma) and mouse anti-human smooth muscle actin (SMA)
antibody (Sigma). Blots were then incubated with a 1:1000 dilution of
horseradish peroxidase–conjugated anti-rabbit or anti-mouse antibody
(Amersham) at room temperature for 45 minutes After they were washed
three times in PBS-Tween, the blots were developed by using chromogenic
substrate solution (diaminobenzidine substrate; Boehringer Mannheim).
Prestained molecular weight standards (SeeBlue) were purchased from
Novex.
Enhanced Expression of Fibronectin, Type I Collagen, and α-SMA
mRNAs in Human LECs of Anterior Polar Cataracts
To characterize transdifferentiated LECs in anterior polar
cataracts at the mRNA level, we compared the steady state mRNA amounts
of aberrant extracellular matrix proteins and a cytoskeletal component
specific for myofibroblasts in human nuclear and anterior polar
cataracts. In our preliminary experiments, we compared different types
of human cataractous LECs and found that LECs obtained from
noncataractous lenses and cortical cataracts showed similar patterns of
mRNA expression to LECs obtained from nuclear cataracts. LECs of
nuclear cataracts retained an epithelial morphology, displaying normal
polygonal arrays of cells, whereas those of anterior polar cataracts
were changed into fibroblast-like cells, showing strong
immunoreactivity to type I collagen, according to our microscopic and
immunohistochemical analysis (data not shown). As shown in
Figure 1 A, expression of fibronectin, type I collagen, and α-SMA mRNA was
prominent in LECs of anterior polar cataracts but negligible in nuclear
cataracts. The amount of β-actin product, an internal control for PCR
amplification, was similar in epithelial cell samples from both types
of cataract.
To confirm the production of the corresponding proteins, we
prepared cell lysates from the LECs attached to the anterior capsules
and performed western blot analysis with anti-fibronectin and anti-SMA
antibodies. As shown in
Figures 1B and 1C , the results correlated with
the RT-PCR data, demonstrating abundant expression of fibronectin and
SMA in anterior polar cataracts. SMA was not detected in nuclear
cataractous LECs by western blot analysis. These data were consistent
with those in a previous study in which strong immunostaining for SMA
was shown in spindle-shaped cells in human anterior subcapsular
cataract.
25 Abundant fibronectin was observed in
SDS-soluble and urea-soluble fractions of protein extract of anterior
polar cataracts. By contrast, fibronectin was barely detected in LECs
obtained from lenses with nuclear cataracts. Fibronectin has been
detected in subcapsular plaques in rat lenses cultured with TGF-β2
and in LECs transformed into mesenchyme-like cells within type I
collagen gel.
18 20 Taken together, our results demonstrate
that increased fibronectin accumulation is an additional pathologic
marker of human anterior subcapsular cataracts.
Enhanced mRNA Expression of TGF-β, TGF-βR, CTGF in Human LECs
of Anterior Polar Cataracts
Next, we determined mRNA levels of various growth factors and
growth factor receptors in nuclear and anterior polar cataracts.
Figure 2 A indicates that expression of mRNA for TGF-β1, TGF-β2, type II
TGF-βR (TβRII), and CTGF was greater in epithelial cells from
lenses with anterior polar cataracts compared with epithelial cells
from nuclear cataracts. In comparison, levels of mRNA for epidermal
growth factor (EGF), FGF-2, epidermal growth factor receptor (EGFR),
FGF receptor-1, and the β-actin internal standard were similar in
LECs from both types of cataract. Similar elevation of the levels of
TGF-β, TGF-β receptor, and CTGF in anterior polar cataracts was
observed when compared with those in noncataractous LECs
(Fig. 2B) .
Induction of mRNA Expression of Fibronectin, Type I Collagen, andα
-SMA by TGF-β1 in LECs
The above results suggest that TGF-β enhances the expression of
mRNA encoding extracellular matrix proteins and SMA in LECs of anterior
polar cataracts. Because human LECs have a limited availability and
proliferative potency, we used animal lenses to examine the production
of mRNAs for fibrotic marker proteins in response to treatment with
TGF-β1.
Figure 3 A shows that TGF-β1 mediated marked accumulation of mRNA for
fibronectin and type I collagen in bovine lens epithelial explants
within 24 hours. SMA mRNA was also significantly upregulated by
TGF-β1 in rabbit lens culture
(Fig. 3C) . FGF-2, a potent inducer of
lens fiber differentiation
26 did not induce SMA mRNA
expression. Similar data were reported previously at the protein
level.
27 Western blot analysis confirmed that treatment
with TGF-β1 led to increased accumulation of fibronectin in bovine
LECs
(Fig. 3B) and of SMA in rabbit LECs
(Fig. 3D) .
Previous studies have demonstrated that LECs of anterior
subcapsular cataracts and anterior polar cataracts are
transdifferentiated into fibroblast-like cells and produce a large
amount of extracellular materials not normally present in the lens
capsules.
14 17 28 Similar changes in the production of
extracellular matrix components are also seen in posterior capsular
opacification after cataract surgery (after-cataract). After-cataract
is thought to be caused by transdifferentiation of LECs remaining after
cataract surgery.
15 TGF-β has been proposed as the most
important factor driving the transdifferentiation and pathologic
fibrosis of LECs that occur in anterior subcapsular cataracts and after
cataract surgery. In cultured rat lenses, treatment with TGF-β leads
to the formation of distinct anterior opacities accompanied by the
appearance of spindle-shaped cells, capsule wrinkling, and deposition
of abnormal extracellular matrix components, such as type I
collagen.
22 The spindle-shaped cells induced by TGF-β
accumulate α-SMA, a specific marker for myofibroblastic
cells.
21 23 27 According to a recent report, transgenic
mice exhibiting lens-specific expression of active TGF-β1 driven by
the αA-crystallin promoter also developed anterior subcapsular
cataracts.
24 Focal plaques of spindle-shaped cells in
lenses of these transgenic mice showed strong reactivity to α-SMA.
Although the potential effects of TGF-β on cataract induction have
been studied in vitro and in animal models as described herein, the
expression level of TGF-β in clinical situations has not yet been
explored. In the present study we characterized LECs of anterior polar
cataracts by examining changes in gene expression of fibrotic markers,
various growth factors, and growth factor receptors at mRNA level. We
demonstrated that LECs from anterior polar cataracts had elevated
levels of TGF-β1, TGF-β2 and TGF-β receptor II mRNAs when
compared with specimens from age-related nuclear cataracts. Our
findings further support the concept that TGF-β is responsible for
the transdifferentiation of LECs in these cataracts. This is supported
by previous studies showing that TGF-β promotes
epithelial–mesenchymal transformation in other cell
systems.
3 4 Our data are also in agreement with the
reports indicating elevated levels of TGF-β mRNA and TGF-β protein
in fibrotic areas of lung, kidney, and skin.
7 29 30 Increased levels of mRNAs for TGF-β and TGF-β receptor shown in
this report suggest the existence of an autocrine pathway by which
TGF-β could modulate transdifferentiation and extracellular matrix
production of LECs. Induction of TGF-β mRNA by TGF-β has been
demonstrated in target cells in vitro.
31 32
TGF-β is secreted in a latent form noncovalently associated with
latency-associated peptide, which is often disulfide-linked to another
protein, latent TGF-β–binding protein. Activation of TGF-β from
this latent complex is a key regulatory step controlling the
bioactivity of this growth factor.
33 Our results showing
the presence of TGF-β message in noncataractous LECs and LECs with
nuclear cataracts may support the previous idea that the endogenous
TGF-β in the ocular media mostly exists in a latent form and that the
crucial step of pathogenesis is the inappropriate activation of latent
TGF-β.
24 48 It is complicated, however, to
estimate the amount of active TGF-β accessible to LECs in vivo and to
compare the magnitude of latent TGF-β activation between nuclear
cataracts and anterior polar cataracts in quantitative assays.
Nevertheless, the initial cause that leads to latent TGF-β activation
in intraocular environments during the pathogenesis of anterior polar
cataract remains to be studied. The mechanisms implicated in
physiological activation of latent TGF-β are not completely
elucidated. Results to date showed that latent TGF-β were activated
by denaturing treatments, plasmin, calpain, reactive oxygen species,
and thrombospondin in vitro.
33 34 35 36 Among these,
thrombospondin-1 was suggested to be a major activator of latent
TGF-β in vivo.
37 Moreover, there is emerging evidence
suggesting physiological roles of integrins in regulating TGF-β
bioactivity. It has been shown that integrin αvβ6 can lead to
TGF-β activation by interaction with latent TGF-β
complex.
38 39 Integrin αvβ6 is upregulated by injury
and inflammation,
40 and thrombospondin is positively
regulated by growth factors such as TGF-β and
FGF-2.
41 42
Thus, various cytokines, growth factors, or some other factors released
in response to certain ocular diseases or injury may stimulate these
and other related physiological activator(s) to trigger the activation
of latent TGF-β in the ocular media and set the resultant active
TGF-β to influence lens cells. Although further studies are
needed to determine how TGF-β mRNA overexpression is initiated and
sustained in the LECs of anterior polar cataracts, it is probable that
the described events stimulate latent TGF-β to become activated, and
once activated, this growth factor induces matrix deposition and its
own expression from LECs. In limited injury, the production of TGF-β
is transiently increased and then terminated when tissue repair is
complete. As previously proposed, however, repeated injury could lead
to continuous autocrine induction of TGF-β overriding the termination
signals, which results in overproduction of TGF-β and extracellular
matrix.
5 This could consist of an underlying mechanism for
sustained gene expression of TGF-β and extracellular matrix in
anterior polar cataract documented in this article.
TGF-β signals by simultaneously contacting two transmembrane
proteins, the type I (TβRI) and type II (TβRII) receptors. The
cytoplasmic kinase domains of both receptors are essential for signal
initiation.
43 44 Previously, type I and type II TGF-β
receptors were detected by immunohistochemistry in adult rat
LECs.
45 Because TGF-β receptors determine cellular
responsiveness to their ligands, the presence of cells overexpressing
the TGF-β receptor in anterior polar cataract suggests that anterior
polar cataract development involves the transformation of LECs toward
an increased TGF-β responsiveness. Enhanced expression of mRNA for
TGF-β receptors has been noted in pancreatic tissue samples from
patients with acute necrotizing pancreatitis, suggesting that TGF-βs
may be involved in tissue remodeling and the fibrotic reaction that
occurs in the pancreas after necrosis.
46
TGF-β consists of three isoforms, TGF-β1, 2, and 3, that are
structurally and functionally related to one another.
44 47 Although the relative roles of TGF-β1 and TGF-β2 in the
transdifferentiation and fibrotic process could not be determined in
the present studies, Gordon–Thomson et al.
48 reported
that all three isoforms could induce cataractous changes in cultured
rat lenses and lens epithelial explants. According to their results,
TGF-β2 and TGF-β3 were more potent than TGF-β1 in inducing
cataract. Furthermore, all three isoforms or their mRNAs were detected
in the lens, suggesting the availability of these polypeptides in vivo.
TGF-β has been detected in high concentration in the aqueous humor
from several mammalian species,
49 50 and its concentration
in this fluid was altered after experimental cataract
surgery.
51 The sources of TGF-β in the anterior chamber
have not completely been identified. A recent report showed that LECs
secrete TGF-β2 in vitro, suggesting that the lens may be one source
of TGF-β in vivo.
52 In this regard, it may be worthwhile
to examine in a future study whether the observed enhancement of mRNA
amount for TGF-β1 and TGF-β2 in LECs with anterior polar cataract
is consequently reflected in increased TGF-β levels in the
corresponding aqueous humor.
CTGF mRNA increased in a coordinate fashion with the mRNA for TGF-β,
suggesting its potential role in the aberrant extracellular matrix
production seen in fibrotic cataract. TGF-β is known to induce CTGF
expression.
10 Thus, enhancement of CTGF mRNA expression in
the clinical samples of anterior polar cataract indicates that this
growth factor may be increased by TGF-β in LECs. CTGF levels may
increase during wound healing, where this growth factor stimulates
fibroblasts to proliferate and secrete increased amounts of
extracellular matrix materials.
53 Our unpublished
results (1998) showed that CTGF message was present in the
human B-3 lens epithelial cell line and in fresh-isolated bovine LECs.
Taken together, these are the first results demonstrating the presence
of CTGF message in LECs. Further studies are required to test whether
CTGF protein is expressed in LECs and whether TGF-β increases CTGF
levels in these cells.
TGF-β increased collagen and fibronectin mRNA accumulation in LECs,
consistent with results shown in a variety of cell
types.
54 55 56 57 Previous studies have shown that rat lens
epithelial explants accumulate SMA protein when cultured in the
presence of TGF-β.
27 TGF-β significantly increases SMA
protein levels in bovine LECs when epithelia are cultured in a collagen
gel, whereas FGF-2 decreases it.
58 In our experiments
TGF-β1 induced SMA mRNA expression in rabbit LECs in intact lenses.
This result is in accord with those in previous work showing
TGF-β–mediated induction of SMA mRNA in other cultured
cells.
59 60 When we initially analyzed mRNA expression for
the previously mentioned myofibroblastic markers in fresh bovine lens
epithelial explants, messages for these proteins were not detected in
LECs in intact bovine lenses. We observed that a substantial amount of
SMA mRNA but not of fibronectin and type I collagen mRNA appeared in
the explants cultured for 24 hours without treatment with TGF-β1
(data not shown). This was similar to a previous study, which showed
that rat lens epithelial explants cultured for 3 days without the
addition of any growth factors showed some cells immunoreactive for
SMA.
27 It was thought that contact of the cells with
plastic may induce SMA expression. This led us to examine SMA mRNA
regulation by TGF-β in intact lenses.
In summary, this report represents the first study that analyzed mRNAs
for proteins associated with pathologic fibrosis in LECs obtained from
patients with anterior polar cataracts. TGF-β, TGF-β receptor, and
CTGF mRNAs were elevated in these LECs, suggesting a role for these
growth factors in the pathologic accumulation of extracellular matrix.
Our results also show that TGF-β increased the production of mRNAs
encoding extracellular matrix proteins in the lens epithelium in vitro.
Therapeutic approaches antagonizing the actions of TGF-β have already
been attempted in fibrotic disease models involving major
organs.
6 Recent studies point to CTGF as a new target for
the development of effective drug therapies. In this regard, the
present work may provide an insight into the cellular and molecular
mechanisms involved in the pathogenesis of cataract and after-cataract
and may lead to the development of therapies to inhibit
cataractogenesis and prevent after-cataract.
The authors thank David Beebe, Washington University School of
Medicine, St. Louis, Missouri, and John McAvoy at The University of
Sydney, Australia, for their valuable comments and critical review of
the manuscript; and Jong-Tak Kim and Young Seomun for their technical
assistance.