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INTRODUCTION |
The extracellular matrix
(ECM)1 of the corneal stroma
is synthesized and maintained by keratocytes. The matrix is primarily composed of collagen fibrils stacked in orderly lamellae surrounded by
proteoglycans. The organization of proteoglycans and collagen fibrils
in the stroma may be responsible for the optical and structural properties of the tissue (1).
The corneal stroma contains two major classes of proteoglycans, one
possessing keratan sulfate side chains and the other possessing chondroitin/dermatan sulfate side chains (2-5). Three corneal keratan
sulfate proteoglycans, lumican, keratocan, and mimecan, have been
cloned and sequenced (6-9). The gene for the corneal chondroitin/dermatan sulfate proteoglycan protein core has been cloned
from chick corneas and identified as decorin (10). The deduced amino
acid sequences of decorin, lumican, and keratocan identify them as
members of a group of small leucine-rich proteoglycans (5, 8).
The structural and biochemical properties of ECM molecules in the
corneal stroma are altered upon injury. Corneal wounds contain collagen
fibrils with abnormally large diameter and irregular interspacing (11,
12). Disruption of the fibrillar organization of collagen fibrils in
corneal wounds is thought to be attributed, in part, to alterations in
the proportion and chemical characteristics of specific proteoglycans.
Injured corneas contain unusually large chondroitin/dermatan sulfate
proteoglycans possessing glycosaminoglycan (GAG) side chains with
higher than normal sulfation and increased amounts of iduronic acid
(13). Keratan sulfate (KS) chains in corneal scars have increased size
and lower sulfation (14, 15). The ratio of chondroitin/dermatan sulfate
to keratan sulfate has been shown to increase after wounding, and
heparan sulfate (HS) has been detected in corneal scars (13, 16, 17).
Interestingly, both transforming growth factor-
(TGF-
) and basic
fibroblast growth factor are detected transiently in corneal wounds
coincident with the expression of heparan sulfate proteoglycans (HSPGs)
(16, 18).
TGF-
has been implicated as a regulatory agent in numerous cellular
and physiological processes, including proteoglycan expression (19).
This influence appears to be at the level of core protein synthesis and
GAG chain elongation (20). TGF-
has been detected in corneal wounds
and in corneal fibroblast cultures, suggesting that it plays a role in
regulating the synthesis of stromal ECM components (16, 21, 22).
Although TGF-
has been detected in vivo and in
vitro, the relationship between TGF-
and proteoglycan expression by corneal fibroblasts has not been fully elucidated.
Cultured corneal fibroblasts synthesize proteoglycans remarkably
similar to those in wounded corneas. Early reports indicated cultures
of rabbit corneal fibroblasts produce mainly chondroitin sulfate (CS)
and HS, with only low levels of KS (23-25). Hassell et al.
(26) reported human corneal fibroblasts in culture synthesize substantial amounts of decorin and perlecan (basement membrane HSPG).
Schrecengost et al. (27) reported reduced levels of a keratan sulfate proteoglycan containing truncated unsulfated keratan chains in chick corneal fibroblasts. Funderburgh et al. (28) reported that bovine corneal fibroblasts in culture synthesize keratan
sulfate proteoglycans with shorter KS chains and lower sulfation
compared with those in normal corneas. The altered properties of KS and
the increase in chondroitin sulfate proteoglycan (CSPG) and HSPG
synthesis suggests that the conditions of cell culture may recapitulate
some of the aspects of injury. To date, most studies of corneal
proteoglycans produced in vitro have been based upon
biochemical analysis of GAG chains, with only limited analysis of the
protein cores. Additionally, each of these studies employed different
culture techniques, making the results difficult to compare.
We developed a culture system to examine the regulation of proteoglycan
synthesis by corneal fibroblasts during injury. The major proteoglycans
synthesized by corneal fibroblasts were characterized and identified
after culture in a defined environment. Specifically, we evaluated the
effects of TGF-
1 and serum on the synthesis of specific GAGs and
protein cores. We found that corneal fibroblasts synthesized
predominantly CS and HS, with only trace amounts of an unsulfated form
of keratan. The major proteoglycan species secreted into the medium
were decorin and perlecan, and proteoglycan synthesis was mediated by
TGF-
1 and serum. This model will allow us to systematically examine
the relationship between specific growth factors and proteoglycan
expression using a defined culture system.
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EXPERIMENTAL PROCEDURES |
Materials--
Chondroitinase ABC (protease-free), keratanase
II, chondroitin sulfate B, keratan sulfate, and the mouse monoclonal
antibody 3G10 directed against unsaturated uronic acid residues arising from heparinase digestion of heparan sulfate were purchased from Seikagaku America, Inc. (Ijamsville, MD). Endo-
-galactosidase was
purchased from Boehringer Mannheim. Heparan sulfate, heparinase I,
heparinase III, phenylmethylsulfonyl fluoride, benzamidine, N-ethylmaleimide, and peroxidase-conjugated donkey
anti-sheep IgG antibodies were from Sigma. Q-Sepharose came from
Pharmacia Biotech Inc. (Uppsala, Sweden). Peroxidase-conjugated donkey
anti-rat IgG antibodies were purchased from Amersham Pharmacia Biotech . Ultrapure urea, sodium chloride, Tween-20, Tris-HCl, bovine serum
albumin, and EDTA were obtained from American Bioanalytical (Natick,
MA). TGF-
1 was obtained from R & D Systems (Minneapolis, MN). All
cell culture reagents were purchased from Life Technologies, Inc. The
mouse monoclonal antibody A7L6 directed against perlecan was obtained
from Upstate Biotechnology Inc. (Lake Placid, NY). The polyclonal sheep
antiserum directed against rabbit corneal decorin was a generous gift
from Dr. Charles Cintron (Schepen Eye Research Institute, Boston, MA).
The rabbit polyclonal antibody R36 that binds unsaturated uronic acid
residues resulting from chondroitinase ABC treatment was a generous
gift from Dr. John Couchman (University of Alabama, Birmingham, AL).
Corneal Fibroblast Isolation and Cell Culture--
Corneas were
excised from whole rabbit eyes purchased from Pel Freeze (Rogers, AR),
and the epithelium and endothelium were removed as described previously
(29). The corneas were washed two times with Dulbecco's modified
Eagle's medium (DMEM) containing 1000 units/ml penicillin, 1.0 mg/ml
streptomycin sulfate, and 20 units/ml nystatin. The corneas were minced
with a sterile razor blade and subsequently digested with collagenase A
(1.5 mg/ml) in DMEM containing 200 units/ml penicillin, 200 µg/ml
streptomycin, and 100 units/ml nystatin for 2-3 h with agitation at
37 °C. The digests were centrifuged at 1840 × g for
10 min, and the cells were suspended in DMEM supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 100 units/ml nystatin,
nonessential amino acids, and 10% fetal calf serum (FCS). Cell were
plated in 75-mm vented tissue culture flasks, and cultures were
maintained in DMEM supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 100 units/ml nystatin, nonessential amino acids
and 4% fetal calf serum. The cultures achieved confluency after 7-10
days, at which time cells were passaged 1:4 and cultured in 4% FCS for
3 days. All experiments were performed on confluent fibroblast cultures that had been passaged once.
Cell Treatment and Metabolic Labeling--
The synthesis of
sulfated glycosaminoglycans was followed by metabolically radiolabeling
corneal fibroblasts with [3H]glucosamine (18 µCi/ml)
and/or [35S]sulfate (36 µCi/ml). Proteoglycan core
proteins were metabolically labeled with
[35S]cysteine/methionine (50 µCi/ml). Corneal
fibroblasts in first passage were cultured until confluent (3 days) in
4% FCS. Upon confluence, corneal fibroblasts were treated as indicated
in the figure legends. Radioisotopes were added immediately after the addition of TGF-
1.
After the designated radiolabeling period, the medium was collected and
immediately combined with two volumes of 10 M urea containing 50 mM Tris-HCl, 10 mM EDTA, pH 7.0. Cell monolayers were washed with phosphate buffered saline (pH 7.4) and
ECM proteins were isolated by gently scraping cell monolayers in 1.0 M urea, 50 mM Tris-HCl, 50 mM EDTA,
pH 7.0. The resulting suspension was centrifuged (5520 × g) for 10 min, and the supernatant was collected and defined
as the ECM fraction. The cell pellet was extracted with TUT (8 M urea, 50 mM Tris-HCl, 0.1% Triton X-100, pH
7.0). The extract was clarified by centrifugation, and the supernatant was collected and defined as the cell fraction (30). Cell number was
determined by measuring acid phosphatase activity on a replicate set of
cultures (31). Total radiolabeled protein present in [35S]cysteine/methionine labeled samples was determined
by performing trichloroacetic acid precipitation on aliquots of medium
and cell fractions prior to proteoglycan purification and quantitating the radioactivity in a liquid scintillation counter (27).
Proteoglycan Purification--
Medium, cell, or ECM fractions
were mixed with 1.0 ml of a 70% Q-Sepharose suspension and rocked for
45 min. The slurries were poured into 5.0-ml disposable minicolumns
(Pierce), and the unbound fractions were discarded. The columns were
washed with 25 column volumes of TUE (8 M urea, 50 mM Tris-HCl, 50 mM EDTA, pH 7.0) and
subsequently washed with 25 column volumes TUE containing 0.2 M NaCl. Columns were eluted with 7 column volumes of TUE
containing 1.5 M NaCl. Salt fractions were exhaustively
dialyzed against Milli-Q water, using membranes with a molecular weight
cutoff of 25,000 (Spectropore, Laguna Hills, CA), and lyophilized.
Samples were resuspended in 2 mM sodium phosphate, 30 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, pH 7.4.
Selective Polysaccharidase Treatment--
Selective
polysaccharidases were used to identify and quantitate GAGs and
proteoglycan core proteins. Digestion conditions were optimized for
time, temperature, concentration, and pH. Enzymes were routinely tested
for activity and specificity using highly purified GAG standards
(Seikagaku, Tokyo, Japan) and the dimethyl methylene blue assay (32).
Purified proteoglycans were subjected to digestion for 3 h at
37 °C in 40 mM Tris-HCl. The pH of the digest was
adjusted to the optimum for each enzyme: chondroitinase ABC (1.0 unit/ml, pH 8.0), a mixture of heparinase I and heparinase III (10 and
20 units/ml respectively, pH 7.3), and a mixture of keratanase II and
endo-
-galactosidase (both enzymes 0.01 unit/ml, pH 5.9).
Glycosaminoglycans Analysis--
Specific GAGs were quantitated
by measuring the low molecular weight digestion products released after
polysaccharidase treatment. Purified GAGs co-radiolabeled with
[3H]glucosamine and
[35S]SO4 were treated with chondroitinase
ABC, a mixture of heparinase I and heparinase III, a mixture of
keratanase II and endo-
-galactosidase, or control with buffer
lacking enzyme. Digests were subjected to ultrafiltration (Microcon,
Millipore) to separate GAG digestion products from intact
proteoglycans. The radioactivity in the filtrate was determined using
liquid scintillation. A 10,000 molecular weight cut-off filter was used
to recover CS and KS digestion products, and a 30,000 molecular weight
cut-off filter was used to recover HS digestion products. The
radioactivity in the filtrate from the undigested control was
subtracted from the enzyme treated samples.
In experiments with a large number of samples, fractions containing
[35S]SO4-GAGs were analyzed, without any
prior purification. [35S]Glycosaminoglycans in medium,
cell, and ECM fractions were quantitated by dot-blotting samples onto
cationic nylon filters as described previously (30). Briefly, filters
(Zeta-probe; Bio-Rad) were prehydrated in TBS (50 mM
Tris-HCl, 0.15 M NaCl, pH 8.0). The filter was then placed
into a Bio-Dot apparatus (Bio-Rad) and washed once by drawing TBS
through each well with vacuum. Samples (100 µl) were applied to each
well and pulled through under vacuum and wells were washed with 0.6 ml
of TUT. The filter was washed twice with TBS followed by two additional
washes with Milli-Q water (Millipore, Bedford MA). The washed filter
was briefly immersed in 95% ethanol, and the area of the filter
containing each sample was removed and counted using liquid scintillation.
Heparan sulfate and CS were determined by treating a replicate filter
with nitrous acid, which selectively degrades HS chains. After sample
application, washed filters were treated twice with fresh nitrous acid
(0.48 M sodium nitrite combined with 3.6 M acetic acid) for 90 min followed by a wash with TBS containing 0.65 M NaCl. The difference between the radioactivity measured on the non-acid-treated and acid-treated filters was defined as HS. The
amount of radioactivity remaining after nitrous acid treatment was
defined as CS.
Proteoglycan Core Protein Analysis--
Proteoglycan core
proteins were identified and quantified using selective
polysaccharidases in conjunction with SDS-PAGE and/or Western blotting.
Proteoglycans radiolabeled with [35S]cysteine/methionine
were digested with either chondroitinase ABC, heparinases I and III, or
a mixture of chondroitinase ABC and heparinases I and III. Digests were
run on 5 or 10% SDS-PAGE gels under reducing conditions (33), and
loading was normalized to total radioactive protein present in
fractions prior to purification (trichloroacetic acid precipitation).
Gels were either processed for autoradiography or electrophoretically
transferred to polyvinylidene difluoride membrane (Millipore) using a
semi-dry transfer apparatus (integrated separations systems) in 25 mM Tris, 192 mM glycine, 20% methanol. The
membranes were blocked in 5% bovine serum albumin in TBS-T buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.2) and were incubated with either mouse monoclonal anti-perlecan (2 µg/ml), sheep polyclonal anti-rabbit corneal decorin (1:9000), mouse
anti-HS stub (1:1000), or rabbit anti-CS stub in 5% bovine serum
albumin in TBS-T at room temperature for 1 h. Blots were washed
with 1% bovine serum albumin in TBS-T and incubated with appropriate
secondary antibodies coupled to horseradish peroxidase (1:3000) for
1 h at room temperature. Proteins were visualized using
chemiluminescence (NEN Life Science Products). To distinguish between
chemiluminescence and radioactivity, a piece of transparent plastic was
placed between the membrane and the film, which was demonstrated to
block >95% of the radioactivity. After probing, the blots were
treated with 10% sodium salicylate in methanol and dried. Radiolabeled
proteins were detected using autoradiography.
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RESULTS |
Effects of TGF-
1 and Fetal Calf Serum on GAG
Synthesis--
Confluent corneal fibroblasts were cultured for 96 h in either 1 or 10% dialyzed fetal calf serum with or without daily
treatments with TGF-
1 (1 ng/ml). Cellular viability studies were
performed, and no significant differences were observed after 6 days of
culture in 0, 1, or 10% FCS (data not shown). To evaluate GAG
synthesis and sulfation, cells were metabolically labeled with both
[35S]SO4 and [3H]glucosamine.
Glycosaminoglycans in medium, cell, and ECM fractions were purified
using anion exchange chromatography on Q-Sepharose. The chromatographic
conditions were optimized to separate highly charged proteoglycans from
weakly charged glycoproteins and hyaluronic acid (16). Specific GAGs
were quantitated by selective digestion with polysaccharidases.
Chondroitin sulfate was determined using chondroitinase ABC.
Glycosaminoglycans susceptible to chondroitinase ABC are referred to as
CS because this enzyme does not distinguish between polymers containing
iduronate and glucuronate (34). Keratan sulfate was determined using a
mixture of keratanase II and endo-
-galactosidase, and HS was
determined using a mixture of heparinases I and III. Sulfation was
defined as the ratio of polysaccharidase-sensitive
[35S]SO4 to polysaccharidase-sensitive
[3H]glucosamine. Tables I
and II summarize the data obtained by enzymatic analysis of purified GAGs.
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Table I
[3H]Glucosamine incorporation into GAGs by corneal
fibroblasts cultured in 1 or 10% dFCS with or without TGF- 1
for 96 h
Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml), and GAGs were labeled with
[3H]glucosamine. Glycosaminoglycans were purified using
anion exchange chromatography on Q-Sepharose. Purified GAGs were
digested with selective polysaccharidases. Digests were subjected to
ultrafiltration (Microcon, Millipore), and the radioactivity in the
filtrate was counted. The radioactivity in the filtrate from an
untreated control was subtracted from the enzyme-treated samples.
Results are expressed as cpm per 1 × 103 cell ± S.E. (n = 3).
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Table II
[35S]SO4 incorporation into GAGs by corneal
fibroblasts cultured in 1 or 10% dFCS with or without TGF- 1 for 96 h
Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml), and GAGs were labeled with
[35S]SO4. Glycosaminoglycans were purified using
anion exchange chromatography on Q-Sepharose. Purified GAGs were
digested with selective polysaccharidases. Digests were subjected to
ultrafiltration (Microcon, Millipore), and the radioactivity in the
filtrate was counted. The radioactivity in the filtrate from the
undigested control was subtracted from the enzyme-treated samples.
Results are expressed as cpm per 1 × 103 cell ± S.E. (n = 3).
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Approximately 75-85% of the [3H]GAGs synthesized during
the 96-h incubation period were secreted into the medium with the
remaining present in the cell (16-21%) and ECM (2-3%) fractions.
Compositional analysis revealed that the majority of the
[3H]GAGs synthesized by corneal fibroblast in culture
were CS (53-80%) and HS (18-47%), with a trace amount of a
nonsulfated form of keratan (<1%). Corneal fibroblasts cultured in
10% dFCS showed an overall reduction in CS synthesis compared with
cells cultured in 1% dFCS. This was evident from both
[35S]SO4 and [3H]glucosamine
incorporation. A similar decrease in HS was observed when cells were
cultured in 10% dFCS compared with 1% dFCS. Treatment with TGF-
1
resulted in a reduction in the amount of
[35S]SO4 and
[3H]glucosamine-labeled CS recovered from cell and ECM
fractions from cells cultured in 1% dFCS. Corneal fibroblasts grown in
the presence of 10% dFCS showed increased sulfation of both CS and HS
compared with 1% dFCS (Table III).
Moreover, this increase in GAG sulfation must result from some
nonspecific alteration in GAG metabolism, as both extracellular and
cellular populations of CS and HS exhibited this response. TGF-
1 did
not significantly influence the sulfation of GAGs synthesized by cells
cultured in 1 or 10% dFCS.
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Table III
Sulfation of GAGs synthesized by corneal fibroblasts cultured in 1 or
10% dFCS with or without TGF- 1 for 96 h
Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml), and GAGs were labeled with
[35S]SO4 and [3H]glucosamine. Purified GAGs
were analyzed using selective polysaccharidases as described under
"Experimental Procedures." Data presented are the ratio of
[35S]sulfate cpm to [3H]glucosamine cpm. Values
were calculated from the results presented in Tables I and II.
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The decrease in GAG synthesis observed with culture in 10% dFCS
compared with 1% dFCS seems to be influenced by TGF-
1. Corneal fibroblasts exhibited smaller serum-associated decreases in
[3H]CS and [3H]HS isolated from cell and
ECM fractions when TGF-
1 was present (Table
IV). To evaluate the extent of this
response, GAGs synthesized by corneal fibroblasts cultured in 1 or 10%
dFCS with or without TGF-
1 (1 ng/ml) were monitored over a 96-h time
course using [35S]SO4 incorporation. Fig.
1 depicts the amount of GAGs synthesized by corneal fibroblasts cultured in 1% dFCS relative to 10% dFCS. TGF-
1 attenuated the increase in [35S]CS synthesis
observed when cells were cultured in medium containing 1% dFCS. This
change was apparent in cell and medium fractions by 39 h and in
ECM fractions after 96 h. TGF-
1 did not significantly alter the
ability of serum to suppress synthesis of either cell-associated or
secreted [35S]HS. The response to TGF-
1 was less
pronounced when measuring [35S]GAGs compared with
[3H]GAGs, presumably because of the increase in sulfation
observed with culture in high serum.
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Table IV
The influence of TGF- 1 on serum associated changes in GAG synthesis
by corneal fibroblasts cultured for 96 h
Glycosaminoglycans synthesized by corneal fibroblasts cultured for in 1 or 10% dFCS were compared in cells treated with or without TGF- 1.
Values are the fold differences in specific GAGs synthesized after
cultured in 1% dFCS compared to specific GAGs synthesized after
cultured in 10% dFCS.
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Fig. 1.
TGF- 1 modulates
serum-associated changes in GAG synthesis. Corneal fibroblasts
were metabolically radiolabeled with
[35S]SO4, and medium (A and
B), cell (C and D), and ECM (E and F)
fractions were dot blotted onto replicate pairs of cationic nylon
filters. One filter was treated with nitrous acid and the other was not
treated. [35S]GAG was determined by counting each filter
using liquid scintillation and the values were normalized to cell
number. Heparan sulfate was defined as [35S]GAG
susceptible to nitrous acid, and CS content was defined as
[35S]GAG resistant to nitrous acid. Serum associated
changes in CS (A, C, and E) and HS (B,
D, and F) synthesis after treatment with ( ) or
without TGF- 1 ( ) was examined over a 96 h time course. The
data are presented as the fold differences in specific GAGs synthesized
after culture in 1% dFCS compared with cultured in 10% dFCS ± S.E. (n = 3).
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Effects of TGF-
1 and Fetal Calf Serum on Protein Core
Synthesis--
Radiolabeled ([35S]Cys/Met) proteoglycans
were purified from the medium and cell fractions of confluent corneal
fibroblast cultures treated with 1 or 10% dFCS with or without
TGF-
1 for 24 or 96 h. Proteoglycan protein cores were detected
using Western blotting and autoradiography, and band intensities were
estimated using densitometry. Gel loading was normalized to total
radioactive protein present in prior to purification on Q-Sepharose columns.
Electrophoresis of proteoglycans from the medium on 10% SDS-PAGE gels
without prior enzyme treatment resulted in poorly resolved smears
migrating between Mr 140,000 and 200,000. After
digestion with chondroitinase ABC, these smears were no longer
apparent, and bands with Mr
45,000, Mr = 60,600, and Mr = 119,100 were present (Fig. 2.). A core
protein (Mr = 60, 600) was also released by
heparinase treatment. Digestion with both chondroitinase ABC and
heparinases I and III did not release any additional core proteins or
substantially alter the electrophoretic mobility of the protein cores.
A chondroitin/dermatan sulfate proteoglycan core
(Mr
45,000) protein migrated as a doublet
and was identified as decorin using Western blot analysis (Fig.
3.). After 24 h, decorin levels were
1.4-fold higher in cells cultured in 1% dFCS than in cells cultured in
10% dFCS. Cultures treated with TGF-
1 had decorin levels similar to
controls. After 96 h, decorin was 2.1-fold higher in cells
cultured in 1% dFCS than in cells cultured in 10%. TGF-
1 decreased
decorin by 32 and 19% in 1 and 10% dFCS respectively. The
Mr = 60,600 and Mr = 119,100 core proteins detected after 96 h in culture migrated with
Mr consistent with those of syndecan-1 and
betaglycan (35, 36). Syndecan-1 belongs to the class of transmembrane
proteins that undergo proteolytic cleavage and release their
ectodomains into the extracellular environment (37, 38). Betaglycan has
been reported to exist as a soluble form that is released by cells into
the medium and is found in the extracellular matrices and serum (36,
39).

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Fig. 2.
Analysis of low
Mr core proteins. Corneal fibroblasts
were cultured for 96 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml), and proteins were radiolabeled with [35S]Cys/Met.
Proteoglycans purified from the medium were digested with either
chondroitinase ABC, both chondroitinase ABC and heparinases I and III,
or buffer lacking enzyme. Digests were run under reducing conditions on
10% SDS-PAGE gels. Radioactive proteins were detected using
autoradiography. Sample loading was normalized to the amount total
radioactive protein present in medium (trichloroacetic acid
precipitation) prior to anion exchange chromatography. Data are
representative of at least three experiments.
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Fig. 3.
Analysis of decorin. Corneal fibroblasts
were cultured for 24 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml). Radiolabeled ([35S]Cys/Met) proteoglycans
purified from the medium were digested with chondroitinase ABC or
buffer lacking enzyme. Digests were run on 10% SDS-PAGE gels under
reducing conditions. Proteins were electrophoretically transferred to
polyvinylidene difluoride. A, blots were probed with
polyclonal antiserum raised against rabbit corneal decorin.
B, after probing, radioactive proteins were detected using
autoradiography. Sample loading was normalized to the amount total
radioactive protein present in medium (trichloroacetic acid
precipitation) prior to Q-Sepharose chromatography. Data are
representative of at least three experiments.
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Analysis of lyase-treated proteoglycans from the medium on 5% SDS-PAGE
gels revealed the presence of three high Mr
proteoglycan core proteins (Mr
375,000,
440,000, and
480,000) (Fig. 4). The Mr
375,000 protein core was released by
treatment with either chondroitinase ABC, heparinases I and III, or a
mixture of both lyases, indicative of a proteoglycan bearing either CS,
HS, or both CS and HS. The Mr
440,000 core
protein could only be resolved after treatment with heparinases I and
III or with both heparinases and chondroitinase ABC and was therefore
synthesized either as an HSPG bearing only HS chains or a hybrid
proteoglycan possessing both HS and CS chains. Both the
Mr
375,000 and Mr
440,000 core proteins containing either HS and/or CS chains reacted
with a monoclonal antibody directed against perlecan (Fig.
5). This heterogeneity observed in the
size of the perlecan core protein is suggestive of alternative
splicing, as has been reported in several species (40). Heparan sulfate
proteoglycan and CSPG forms of perlecan have been reported in the
Engelbreth-Holm-Swarm tumor matrix (41). The Mr
480,000 protein core was derived from a CSPG, as it was only
observed after treatment with chondroitinase ABC or both chondroitinase
ABC and heparinase treatment. The Mr
480,000, core protein did not react with the antibody directed against
perlecan. The smear between Mr
400,000 and
460,000 in both the heparinase-treated and untreated control lanes was
not present after treatment with chondroitinase ABC and likely
represents an intact CSPG.

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Fig. 4.
Analysis of high
Mr core proteins. Corneal fibroblasts
were cultured for 96 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml), and proteins were radiolabeled with [35S]Cys/Met.
Proteoglycans purified from the medium were digested with
chondroitinase ABC, both chondroitinase ABC and heparinases I and III,
or buffer lacking enzyme. Digests were run under reducing conditions on
5% SDS-PAGE gels. Sample loading was normalized to total radioactive
protein present in medium (trichloroacetic acid precipitation) prior to
purification. Radioactive proteins were detected using autoradiography.
Data are representative of at least three experiments.
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Fig. 5.
Analysis of perlecan. Corneal
fibroblasts were cultured for 24 h in 1 or 10% dFCS ± TGF- 1 (1 ng/ml). Radiolabeled proteoglycans
([35S]Cys/Met) purified from the medium were digested
with chondroitinase ABC, hepatinase I and III, or with both
chondroitinase ABC and heparinase I and III. Digests were run under
reducing conditions on 5% SDS-PAGE gels and electrophoretically
transferred to polyvinylidene difluoride. Sample loading was normalized
to total radioactive protein present in medium (trichloroacetic acid
precipitation) prior to purification. A, radioactive
proteins were detected using autoradiography. B, the blots
were probed with mAb A7L6. Data are representative of at least three
experiments.
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To determine the effects of TGF-
1 and serum on the secreted high
molecular weight core proteins, bands detected on autoradiographs after
combined chondroitinase ABC and heparinases I and III treatment were
compared by densitometry. A 1.6-fold increase in the amount of the
Mr
375,000 perlecan secreted by
TGF-
1-treated corneal fibroblasts cultured in 1% dFCS was observed
after 24 h, and a 2.0-fold increase was observed after 96 h.
This stimulatory effect of TGF-
1 did not depend on serum factors, as
similar increases were observed when cells were cultured in 10% dFCS.
After 96 h of culture in 10% dFCS, there was a decrease in the
amount of both the Mr
375,000 and the
Mr
440,000 forms of perlecan by 1.6- and
2.2-fold, respectively, compared with cells cultured in 1% dFCS.
Similar serum associated decreases were seen when TGF-
1 was present.
After 96 h, the levels of the Mr
440,000 core proteins were not significantly affected by TGF-
1
treatment. The pattern of expression of the Mr
480,000 protein core was similar to that of the
Mr
375,000 with respect to TGF-
1
treatment and serum. Densitometric analysis of the immunoblotted core
proteins released with combined chondroitinase ABC and heparinase
treatment (Fig. 5B) revealed substantial increases in both
the Mr
440,000 (2.2-fold) and
Mr
375,000 (2.1-fold) forms of perlecan with TGF-
1 treatment when cells were cultured in 1% dFCS for 24 h. Furthermore, culture in 10% dFCS for 24 h increased the levels of
the Mr
440,000 core by 2.3-fold with respect
to 1% dFCS.
Polysaccharidase-treated proteoglycans purified from cell fractions
were analyzed by Western blot analysis. Blots were probed with
antibodies that recognize CS stubs remaining after chondroitinase ABC
treatment (R36) or HS stubs remaining after heparinase treatment (mAb
3G10). Western blot analysis with R38 revealed the presence of a
Mr = 44,700 band that was released only with
combined chondroitinase and heparinase treatment (Fig.
6A). Interestingly, this band
did not react with mAb 3G10. It is conceivable that chondroitinase ABC
may have modified the epitope for mAb 3G10. Heparinases I and III
released a Mr = 51,600 protein core that reacted
with mAb 3G10 (Fig. 6B). This band was not detected after
combined chondroitinase ABC and heparinase treatment. The presence of
both CS and HS chains and the electrophoretic mobility suggest that these proteoglycans may be members of the syndecan family (35). After
96 h, both the Mr = 44,700 and
Mr = 51,600 protein cores were decreased in
cells cultured in 10% dFCS compared with 1% dFCS but were unchanged
by treatment with TGF-
1.

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Fig. 6.
Analysis of cell-associated CSPGs and
HSPGs. Corneal fibroblasts were cultured for 96 h in 1 or
10% dFCS ± TGF- 1 (1 ng/ml), and radiolabeled proteoglycans
([35S]Cys/Met) were purified from the cell fraction on
Q-Sepharose columns. A, immunoblot analysis of proteoglycans
digested with chondroitinase ABC, chondroitinase ABC and heparinase I
and III, or a buffer lacking enzyme. Blots were probed with antibody
R36 directed against the CS stub remaining after chondroitinase ABC
digestion. B, immunoblot analysis of proteoglycans digested
with heparinase I and III, chondroitinase ABC and heparinase I and III,
or a buffer lacking enzyme. Blots were probed with mAb 3G10 directed
against the HS stub remaining after heparinase treatment. Sample
loading was normalized to total radioactive protein present
(trichloroacetic acid precipitation) prior to purification. Data are
representative of at least three experiments.
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Effects of TGF-
1 on Proteoglycan Synthesis--
To examine the
effects of TGF-
1 in the absence of serum factors, corneal fibroblast
were cultured without dFCS for 18 h and subsequently treated with
0, 1, 5, or 10 ng/ml TGF-
1. Proteoglycans were radiolabeled with
[35S]SO4 for 0-24, 24-48, or 48-72 h, and
medium fractions were collected after the radiolabeling periods.
Aliquots of medium were treated with chondroitinase ABC or left
untreated, and resistant proteoglycans were isolated on Q-Sepharose
columns. Chondroitin sulfate was defined as GAG susceptible to
chondroitinase ABC. Heparan sulfate was defined as GAG resistant to
chondroitinase ABC.
Corneal fibroblasts cultured in the absence of serum exhibited
TGF-
1-dependent changes in GAG synthesis. During the
0-24 h labeling period, TGF-
1 treatment resulted in a
dose-dependent increase in both CS (up to 2.0-fold) and HS
(up to 2.3-fold) (Fig. 7, A
and B). From 24 to 48 h, the overall synthesis of CS
was not significantly affected by TGF-
1 treatment. However, cells treated with TGF-
1 continued to show increased HS (up to 3.0-fold) synthesis during the 24-48-h labeling period. During the 48-72-h labeling period, substantial decreases in GAG synthesis were observed after TGF-
1 treatment. Chondroitin sulfate synthesis decreased by as
much as 13.6-fold relative to control. During this period, HS
synthesis, although modestly elevated after treatment with 1 ng/ml
TGF-
1, exhibited a 5.7-fold decrease relative to control after
treatment with either 5 or 10 ng/ml TGF-
1.

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Fig. 7.
Analysis of proteoglycans synthesized
in response TGF- 1. Corneal fibroblast
were cultured without dFCS for 18 h and subsequently treated with
0, 1, 5, or 10 ng/ml TGF- 1. Proteoglycans were radiolabeled with
[35S]SO4 for 0-24, 24-48, or 48-72 h and
medium fractions were collected after the radiolabeling periods.
Aliquots of medium were treated ± chondroitinase ABC and
proteoglycans were isolated on Q-Sepharose columns. Chondroitin sulfate
was defined as GAG susceptible to chondroitinase ABC. Heparan sulfate
was defined as GAG resistant to chondroitinase ABC. Chondroitin sulfate
synthesis (A) and HS synthesis (B) are expressed
as CPM per 1 × 106 cell ± S.E.
(n = 3). Open columns, control;
stippled columns, 1 ng/ml; hatched columns, 5 ng/ml; filled columns, 10 ng/ml. Aliquots of medium from
TGF- 1-treated corneal fibroblasts radiolabeled with
[35S]SO4 were applied to 5% SDS-PAGE gels.
Labeled proteoglycans and proteins from 1.5 × 104
cells were visualized by autoradiography (C). Autoradiograph
is representative of three gels.
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The TGF-
1-dependent increase in GAG production seems to
involve the induction of specific proteoglycans. Aliquots of medium from TGF-
1-treated corneal fibroblasts radiolabeled with
[35S]SO4 were analyzed on 5% SDS-PAGE gels.
Bands exhibited broad size heterogeneity characteristic of proteoglycan
migration on SDS-PAGE gels (Fig. 7C). Three major
populations of proteoglycans were observed; a low
Mr proteoglycan (median
Mr
175,000), a high
Mr proteoglycan (median
Mr
420,000), and a second high Mr proteoglycan that barely entered the gel. To
quantitate TGF-
1-induced changes in secreted proteoglycans, bands
detected on autoradiographs were compared by densitometry. The second
high Mr proteoglycan was not sufficiently
resolved to be accurately quantitated. TGF-
1 treatment resulted in
dose dependent increases in the high Mr proteoglycans from 0 to 24 h (up to 4.7-fold) and from 24 to
48 h (up to 3.3-fold). In contrast, the low
Mr decreased by as much as 47% during the 0-24
h labeling period and by as much as 70% during the 48-72 h labeling
period. During the 48-72 h labeling period, TGF-
1 treatment
resulted in substantial decreases in both the low and high
Mr proteoglycans. The low
Mr proteoglycan decreased by 76% in 1 ng/ml
TGF-
1, relative to control, and was not detected at higher
concentrations of TGF-
1. The high Mr was not
significantly changed by 1 ng/ml TGF-
1, and was not detected at
higher concentrations of TGF-
1. These TGF-
1-dependent
decreases in proteoglycan synthesis from 48 to 72 h after
treatment were not the result of decreased cell viability as cells
treated with TGF-
1 exhibited similar levels of
[3H]thymidine incorporation into DNA during this period.
 |
DISCUSSION |
The current study was initiated to identify the major core
proteins and GAG chains synthesized by rabbit corneal fibroblasts in
culture. A defined culture system will allow the systematic examination
of the relationship between specific growth factors and proteoglycans
within the injured cornea in vitro. This system should
provide a useful model of corneal injury. Because TGF-
has been
detected in the corneal stroma after injury, we examined the effects of
TGF-
1 and serum on proteoglycan synthesis by corneal fibroblasts
(16, 21). The results of these studies showed that the synthetic
profile of proteoglycans produced by corneal fibroblasts in culture,
although significantly different from those of normal corneas, were
remarkably similar to those found in wounded corneas. Approximately
60% of the GAG in the normal corneal stroma is KS and 40% is
chondroitin/dermatan sulfate (2-4). Wounded corneal stromas synthesize
increased quantities of both CS and HS and have reduced KS content
(13-17). Corneal fibroblasts, in our culture system, synthesize
substantial quantities of CS and HS with negligible amounts of an
unsulfated form of keratan. Hassell et al. (13) reported
that wounded corneas synthesize unusually large CSPGs (13). We detected
substantial quantities of large proteoglycans bearing CS and HS chains
secreted into the culture medium. Several reports have documented
increased sulfation of CS and HS and decreased sulfation of KS after
wounding (14-15, 17). Keratan was not sulfated in our system, whereas the sulfation of CS and HS was significantly increased with increased serum. These results suggest that conventional cell culture and injury
induce similar phenotypic changes and that altered proteoglycan expression is a reflection of these changes.
In addition to the GAG chains, we extended the analysis of
proteoglycan production by characterizing and identifying core proteins
secreted into the medium and associated with the cells. Four major
species of protein core were observed in the medium of cultured corneal
fibroblasts. The smallest core protein (Mr
45,000) possessed only CS chains and was identified as decorin. Two
large core proteins were identified as perlecan by immunoblotting (Mr
375,000 and
440,000). An
additional CSPG containing a Mr
480,000 protein core was detected that did not react with the antibody directed
against perlecan. Furthermore, we demonstrated that serum and TGF-
1
influenced both the expression and glycanation of these core proteins
and altered the sulfation of their associated GAG chains.
Decorin is a member of the gene family of small leucine-rich
proteoglycans and a normal constituent of the corneal stroma (10).
There is increasing evidence that decorin is an important regulator of
a number of important physiological processes. Several studies suggest
an inhibitory role for decorin on cell proliferation through
TGF-
-dependent and TGF-
-independent mechanisms.
Overexpression of decorin inhibits cell growth in a number of different
cell types (42-45). Addition of exogenous recombinant decorin to
cultures of several tumor cell lines suppresses cell growth (43).
Decorin suppresses cell growth by activating the epidermal growth
factor receptor and elevating cytosolic Ca2+ in A431
squamous carcinoma cells (46-47). In addition, Yamaguchi et
al. (48) demonstrated that decorin specifically binds TGF-
and
suppressed its growth stimulatory activity in Chinese hamster ovary
cells. The ability of decorin to modulate the bioactivity of TGF-
1
has been demonstrated in a number of different systems (49-51). The
effects of TGF-
on decorin expression vary widely among different
fibroblast types. TGF-
down-regulates decorin expression in dermal
(52) and gingival fibroblasts (53), whereas TGF-
up-regulates
decorin expression in lung fibroblasts (54) and myocardial fibroblasts
(55). TGF-
has been shown to stimulate proliferation of corneal
fibroblasts through a mechanism that may involve the induction of basic
fibroblast growth factor (56). In our model, both TGF-
treatment and
culture in high serum decreased decorin production (Figs. 2 and 3).
This is consistent with studies showing a substantial induction of
decorin expression upon quiescence in a variety of fibroblasts (52, 57,
58). The observations that decorin is an inducer of quiescence and that
decorin is induced upon quiescence are suggestive of an autocrine
mechanism of cell growth control possibly involving TGF-
. It is
conceivable that the substantial quantities of decorin in the normal
corneal stroma limits TGF-
activity. In this manner, the proteolytic
degradation of decorin likely to occur within a corneal wound might
remove the restriction on TGF-
activity within this localized region.
In addition to decorin, corneal fibroblasts in our system synthesized
two large core proteins (Mr
375,000 and
440,000) that reacted with a mAb directed against perlecan. The
Mr
440,000 isoform was synthesized as either
an HSPG or a hybrid possessing both CS and HS. The
Mr
375,000 isoform was primarily glycanated with CS; however, forms bearing HS or potentially both CS and HS were
detected. Perlecan, initially identified as an HSPG (59), has also been
shown to be glycanated with CS or both CS and HS in a number of
different tissues and cell types (26, 41, 60-64). Perlecan isolated
from the culture media in our system has shown heterogeneity not only
in GAG chain substitution but also in the size of the core proteins.
Size variants of perlecan bearing HS chains have been detected in the
Engelbreth-Holm-Swarm tumor matrix (41). Several studies suggest that
perlecan variants may be generated by alternative splicing in human
(65-66) and mouse (67). The significance of this heterogeneity of
perlecan is not fully understood. However, when considering the
potential importance of HS and its interactions with growth factors
(68), it is conceivable that alternate glycanation could have a
substantial impact on the biological activity of perlecan. We find that
both TGF-
1 and serum induce substantial increases in perlecan
bearing both CS and HS chains at the early time point (Fig. 5). In
contrast, after extended periods in culture the amount of perlecan
bearing HS chains in the medium was unaffected by TGF-
1, and
decreased by serum. The levels of perlecan bearing CS chains were
elevated with TGF-
1 treatment and were decreased with serum (Fig.
6). These apparent differences with respect to culture duration and perlecan expression are indicative of an indirect response. The fact
that TGF-
can induce secondary effectors, such as growth factors and
matrix molecules, introduces further levels of complexity to the
mechanism through which TGF-
may influence proteoglycan synthesis.
Corneal fibroblasts secreted an additional large core protein linked to
CS (Mr
480,000) that did not react with a
mAb directed against perlecan. It is unlikely that the
Mr
480,000 protein core was a hybrid
possessing both CS and HS chains, as the band intensities of the cores
released with both chondroitinase ABC and heparinase were not
significantly higher than those released with chondroitinase ABC alone.
This core protein may be a perlecan variant lacking the epitope
recognized by mAb A7L6 or may represent a novel CSPG. Although the
identity of this proteoglycan is unclear, it may be important during
wound healing as it responded to both TGF-
1 and serum in culture.
Our results indicate that corneal fibroblasts in culture synthesize
predominantly CS and HS with trace amounts of an unsulfated keratan.
The major proteoglycan species secreted into the medium were decorin
and perlecan. Our analysis indicates that TGF-
1 and serum modulate
the GAG chains and protein cores of both of these proteoglycans. In
light of the influence that both decorin and perlecan exert on growth
factor activity, changes in the expression of either of these
proteoglycans could have important consequences on the cellular
response to injury. A well characterized model system should allow the
cooperation between proteoglycans and growth factors to be analyzed in
detail. These studies could ultimately provide important insight into
the mechanisms that control tissue remodeling after corneal stromal injury.