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J Biol Chem, Vol. 275, Issue 18, 13918-13923, May 5, 2000
From the Keratocytes of the corneal stroma produce a
specialized extracellular matrix responsible for corneal transparency.
Corneal keratan sulfate proteoglycans (KSPG) are unique products of
keratocytes that are down-regulated in corneal wounds and in
vitro. This study used cultures of primary bovine keratocytes to
define factors affecting KSPG expression in vitro. KSPG
metabolically labeled with [35S]sulfate decreased during
the initial 2-4 days of culture in quiescent cultures with low serum
concentrations (0.1%). Addition of fetal bovine serum, fibroblast
growth factor-2 (FGF-2), transforming growth factor The corneal stroma is a disc of connective tissue that constitutes
about 90% of the mammalian cornea. This tissue consists of a unique
transparent extracellular matrix populated by keratocytes, flattened
mesenchymal cells responsible for production and maintenance of this
matrix. In healing corneal wounds keratocytes become activated, begin
mitosis, and migrate to the wound location, where they secrete nontransparent scar components (1, 2). Cells in the healing wound are
characterized by secretion of pro-inflammatory cytokines such as
interleukin-1 The extracellular matrix of the normal corneal stroma is characterized
by a unique class of molecules known as the corneal keratan sulfate
proteoglycans (KSPG).1 These
consist of three structurally related proteins modified with
N-linked keratan sulfate chains (6). The three proteins (lumican, keratocan, and mimecan) are found in a number of connective tissues but are expressed at much higher levels in the cornea compared
with noncorneal tissues (7-9). In noncorneal tissues, these proteins
are not modified with keratan sulfate (10). The high level of
expression of these three proteins combined with a specialized
glycosylation is a property unique to keratocytes and constitutes an
essential feature of the role of the keratocyte in maintenance of
corneal transparency. This conclusion is supported by the recent
demonstration that mice bearing null mutations in the lumican gene lose
corneal transparency, whereas mice with a similar mutation in the gene
for decorin, the major corneal chondroitin/dermatan sulfate
proteoglycan, maintain clear corneas (11, 12).
Expression of KSPG appears closely linked to the stromal environment.
During corneal development, secretion of KSPG commences very soon after
migrating neural crest cells enter the primary corneal stroma (13). A
number of early studies reported that when removed from the stroma,
keratocytes rapidly lose the ability to secrete keratan sulfate
(14-21). We examined this in vitro down-regulation of KSPG
using cultured bovine keratocytes and reported that these cells
continue to secrete KSPG proteins, but the proteins are modified with
truncated under-sulfated keratan sulfate chains (22). A recent study
has shown that primary bovine keratocytes in low-serum or serum-free
media maintain KSPG secretion for several days in culture, whereas
growth in 10% fetal bovine serum resulted in decreased KSPG synthesis
(23). The apparent association between cell division and loss of KSPG
secretion both in vivo and in vitro suggests that
mitosis per se may be involved in KSPG down-regulation. In
the current study we examined the response of freshly isolated bovine
keratocytes to cytokines and serum to better define the cellular
environment that determines maintenance and/or loss of expression of
the KSPG differentiation marker. We report that keratocyte mitosis is
not directly linked to KSPG down-regulation but that subculture after
trypsin treatment, particularly in the presence of mitogen-rich fetal
bovine serum, results in irreversible loss of KSPG synthesis. The
presence of FGF-2, however, supports KSPG synthesis by induction of
core protein biosynthesis.
Cell Culture--
Central portions of fresh corneas from
slaughter-aged steers were incubated in 0.2% trypsin in a 1:1 mixture
of Dulbecco's modified Eagle's medium and Ham's F-12 medium
(DMEM/F-12; Sigma, D6905) containing antibiotics (penicillin, 100 units/ml; streptomycin 100 µg/ml; gentamicin, 50 µg/ml;
amphotericin B, 2.5 µg/ml) for 16 h at 4 °C followed by 30 min at 37 °C. Epithelial and endothelial cells were removed by
scraping with a plastic spatula (Cell Lifter; Fisher 08-773-1) in cold
saline. The tissue was rinsed, minced into 2-3-mm cubes with razor
blades, and shaken at 37 °C, 70 rpm for 2-3 h in a volume of 1 ml/corneal stoma of 1% (w/v) collagenase (Sigma C8176 L) in DMEM/F-12
with antibiotics until complete disruption of the tissue was achieved.
The cell suspension was passed through a 70-µm nylon mesh (Cell
Strainer, Falcon 2350), washed twice by centrifugation in DMEM/F-12
with antibiotics, and plated at 3-4 × 104
cells/cm2 in polystyrene tissue culture vessels (Costar
3506 or Corning 25100) in DMEM/F-12 with antibiotics containing insulin
5 µg/ml, selenous acid, 5 ng/ml, and transferrin 5 µg/ml (ITS,
Collaborative Research Inc., Lexington, MA) and 0.1% platelet-poor
horse serum (Sigma) as described previously (22). In some experiments
the serum concentration of this medium was altered as noted. Media were
changed after 24 h of culture and subsequently at 48-h intervals. At termination of cultures, cells were rinsed twice with saline, and
cell number was estimated by lysis of the attached cell layer at
37 °C for 30 min in 10 mM NaOH, 1% SDS. Cell number was
calculated from absorbance at 260 nm of the lysate based on a standard
curve obtained by hemocytometer counting of cells released by trypsin.
Proteoglycan Purification--
Sulfated proteoglycans were
labeled with 100 µCi/ml of carrier-free
H235SO4 (ICN Radiochemicals 640405)
or with 50 µCi/ml [35S]methionine/cysteine (TransLabel,
ICN 51006). 48 h after labeling, the culture medium from each well
was collected and combined with a rinse of the cell layer with saline.
This soluble fraction contained >95% of the detectable proteoglycan
and was used as the source for KSPG purification and analysis.
Analysis of total [35S]methionine-labeled secreted
proteins was carried out by repeated concentration of the labeled
culture medium in 0.1 M Tris-HCl, pH 6.8, using centrifugal
ultrafiltration with a 3-kDa cut-off to remove unincorporated label,
followed by separation of the proteins by SDS-PAGE and autoradiography. Purification of proteoglycans from the media was achieved by passing the culture medium and rinse through SPEC-3 ml-NH2
microcolumns (Ansys, Inc., 531-07-20) preactivated with methanol and
distilled water as per manufacturer's instructions. The columns were
rinsed with 3 ml of 6 M urea, 0.2 M NaCl, 0.02 M Tris-HCl, pH 8. Proteoglycans were eluted from the column
with 0.4 ml of 4 M guanidine HCl, 0.02 M
Tris-HCl, pH 8.
Solid Phase Chondroitinase Assay--
Incorporation of sulfate
into KSPG and chondroitin/dermatan sulfate proteoglycan was assessed by
adsorption of aliquots of the purified proteoglycans to polyvinylidene
difluoride membrane in Millipore Multiscreen 96-well plates (MAIP
N4510, Millipore Corp). The membranes were rinsed in water and blocked
with 50 µl of 1% (w/v) bovine serum albumin in 0.1 M
Tris-acetate, pH 8. Duplicate sets of samples were treated with 50 µl
in the same solution with or without 0.1 unit/ml affinity-purified
chondroitinase ABC (Sigma). The plate was covered and incubated in a
humidified chamber at 37 °C for 16 h. The membranes were rinsed
with TBS (0.02 M Tris-HCl, pH 7.4, 0.15 M NaCl,
0.01% (w/v) thimerosal), and bound radioactivity was determined by
excising the filters and scintillation counting. Immobilized
proteoglycan radioactivity sensitive to chondroitinase was
representative of the amount of chondroitin/dermatan sulfate
proteoglycan in the sample, and chondroitinase-resistant radioactivity
approximated the amount of KSPG.
SDS-PAGE Analysis--
Column eluates were dialyzed against 0.1 M Tris-acetate, pH 8, followed by digestion with
chondroitinase ABC 0.1 unit/ml or keratanase II (Seikagaku) and/or
endo- Immune Precipitation--
Immune precipitation of labeled KSPG
was carried out using a minor modification of our previously published
procedure (25, 26). Affinity purified polyclonal anti-KSPG antibody
reactive with all three KSPG proteins (6) or monoclonal antibody
against sulfated epitopes on the keratan sulfate chain (25) was
incubated with immobilized protein G (Sigma P-2169) for 2 h then
washed by centrifugation in TTBS (TBS with 0.02% Tween 20). The
immobilized antibody was incubated with dialyzed proteoglycan eluate at
4 °C for 16 h in 1% bovine serum albumin in TTBS. The bound
proteoglycan was rinsed once in 1% Triton X-100 in TTBS at room
temperature and rinsed three additional times in TTBS for 5 min. Bound
proteoglycans were released from pellets at 70 °C for 30 min in
SDS-PAGE sample buffer (24) and separated by SDS-PAGE as described
above. Control experiments using purified iodine-labeled KSPG (27)
added to culture medium demonstrated that the amount of KSPG
precipitated was proportional to the amount present in the medium and
that >80% of the added radioactivity was recovered in a single round of precipitation.
In an effort to define factors involved in KSPG regulation we
compared KSPG secretion by primary keratocytes isolated by collagenase (23) to that from cells obtained by outgrowth corneal explants (22)
using SDS-PAGE and immunodetection by monoclonal antibody I22, which
recognizes sulfated regions of keratan sulfate chains (25). Fig.
1A (lane 1) shows
that primary cells secreted a heterogeneous high molecular weight
material reacting with the antibody, similar in size to purified bovine
corneal KSPG (lane 3). Culture medium from the outgrowth
cells (lane 2) had virtually no KSPG reacting with this
antibody. Active secretion of a high molecular weight KSPG by the
primary cultures was confirmed by metabolically labeling with
[35S]sulfate (Fig. 1B). Approximately 20-25%
of the labeled proteoglycan was resistant to digestion with
chondroitinase ABC (Fig. 1B, lane 2). This
chondroitinase-resistant proteoglycan had a size distribution similar
to the KSPG identified with antibody I22 and was >90% sensitive to
keratanase digestion (Fig. 1B, lane 3). Molecules with properties similar to normal corneal KSPG, therefore, appear to
make up a significant proportion of the proteoglycans secreted by these
cells in culture. Confirmation of the chondroitinase-resistant 35S-labeled proteoglycans as KSPG was demonstrated by
immunoprecipitation using antibody I22. As shown in Fig. 1C,
the macromolecular components of the immunoprecipitated proteoglycans
were sensitive to endo- KSPG secreted by quiescent primary keratocytes is larger and more
highly sulfated than that we described previously in cells that had
undergone extensive cell division (22), suggesting the possibility that
active cell division may be involved in modulation of KSPG expression.
We examined this hypothesis by treatment of the primary cells with
mitogens. The primary keratocytes divided rapidly in the presence of
several cytokines and sera (Fig.
2A), but KSPG secretion
exhibited a mixed response to these agents, Fig. 2B. As we
found previously (23), FBS reduced the amount of sulfated KSPG, however
other mitotic agents such as platelet-derived growth factor and
interleukin 1
Fibroblast Growth Factor-2 Promotes Keratan Sulfate Proteoglycan
Expression by Keratocytes in Vitro*
,,,,, and Division of Biology, Kansas State
University, Manhattan Kansas, 66506-4901 and the
§ Department of Ophthalmology, University of Pittsburgh
Medical School, Pittsburgh, Pennsylvania 15213-2588
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, or platelet
derived growth factor all stimulated cell division, but only FGF-2
stimulated KSPG secretion. Combined with serum, FGF-2 also prevented
serum-induced KSPG down-regulation. KSPG secretion was lost during
serial subculture with or without FGF-2. Expression of KSPG core
proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and
steady state mRNA pools for these proteins, particularly keratocan,
were significantly increased by FGF-2 treatment. KSPG expression
therefore is supported by exogenous FGF-2 and eliminated by subculture
of the cells in presence of serum. FGF-2 stimulates KSPG core protein
expression primarily through an increase in mRNA pools.
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and proteolytic enzymes involved in tissue remodeling,
collagenase, gelatinase, and stromelysin (3, 4). This remodeling
(fibroblastic) phenotype is simulated in vitro when
keratocytes are cultured in medium containing fetal bovine serum and
subcultured by trypsinization (5).
MATERIALS AND METHODS
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-galactosidase (Sigma) each at 0.02 unit/ml for 1 h at
37 °C, followed by separation on a 3-12% acrylamide gel as
described previously (24). The separated proteoglycans were
electrophoretically transferred to Millipore Immobilon polyvinylidene
difluoride membranes, standards were detected by staining with Ponceau
S, and radioactivity was detected on x-ray film using BioMax low energy
transcreen (Millipore Corp.) at 
70 °C.
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-galactosidase and specifically competed with
unlabeled purified KSPG. A 50 kDa band generated by
endo--galactosidase (Fig. 1C, lane 2) is the previously determined size of KSPG core proteins. Labeling of this band
may result from tyrosine sulfation of these proteins (7).
View larger version (21K):
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Fig. 1.
KSPG secretion by primary keratocytes.
A, purified proteoglycans from 2-day primary keratocyte
cultures (lane 1) or from passaged stromal cell cultures
similar to those previously reported (22) (lane 2) or 5 µg
of KSPG extracted from bovine cornea (lane 3) were separated
on a 3-12% SDS-PAGE gel, and keratan sulfate was detected by
immunoblotting with monoclonal antibody I22. B,
[35S]sulfate-labeled proteoglycans from 4-day primary
cultures were separated on 3-12% SDS-PAGE gels without pretreatment
(lane 1), after digestion with chondroitinase ABC
(lane 2), or after digestion with chondroitinase ABC and
keratanase II (lane 3). Labeled proteoglycans were detected
by autoradiography as described under "Materials and Methods."
C, [35S]sulfate-labeled proteoglycan from
primary keratocyte cultures was immunoprecipitated with monoclonal
antibody I22 and separated on 3-12% SDS-PAGE gels without
pretreatment (lane 1), after digestion with
endo- -galactosidase (lane 2), or when precipitation was
carried out the presence of 100 µg/ml purified bovine KSPG
(lane 3).
had little effect. One mitogen, FGF-2 (also known as
basic fibroblast growth factor) clearly increased KSPG secretion in
comparison with the other treatments. Immunoprecipitation of the KSPG
from these cultures with anti-keratan sulfate antibodies (Fig.
2C) provided results similar to the solid phase assay of keratan sulfate and also showed that KSPG secreted in the presence of
transforming growth factor or FBS is somewhat smaller in size than
that from control cultures.
View larger version (37K):
[in a new window]
Fig. 2.
Response of primary keratocytes to
cytokines. A, primary keratocytes were cultured for 6 days in the presence of 10 ng/ml FGF-2 (FGF), 10 ng/ml
platelet-derived growth factor (PDGF), 0.5 ng/ml interleukin
1 (IL1 alpha), 1 ng/ml transforming growth factor (TGF beta), or 10% FBS. Bars show the mean and
standard deviation of cell numbers in triplicate cultures normalized
with respect to untreated control. B, primary cells treated
with cytokines under conditions similar to those in A were
[35S]sulfate labeled during days 4-6 of culture and KSPG
estimated by a solid phase assay as described under "Materials and
Methods." Bars show the means and standard deviations of
triplicate cultures normalized with respect to untreated control.
C, 35S-labeled proteoglycans combined from
triplicate cultures similar to those described in B were
immune precipitated using anti-keratan sulfate antibody I22 as
described under "Materials and Methods." Immune precipitated KSPG
was separated on a 3-12% SDS-PAGE gel and detected by
autoradiography.
Stimulation of KSPG by FGF-2, as shown in Fig.
3A, required a minimum of 2-4
days of exposure. Without FGF-2, KSPG secretion dropped about 50-70%
during a 6-day culture. period, but in the presence of FGF-2, KSPG
secretion was stimulated after a 2-day lag period. At the end of the
6-day period sulfated KSPG secretion was as much as 10-fold greater
than cultures without FGF-2. The amount of stimulation was a function
of FGF-2 concentration and below 1 ng/ml closely matched the increase
in cell number (Fig. 3B). At FGF-2 concentrations above 1 ng/ml, however, the KSPG per cell secretion increased slightly in
comparison to control. Concentrations greater than 10 ng/ml FGF-2 were
difficult to assay because these resulted in loss of cell attachment
(not shown).
|
Previously, culture of primary cells in 10% FBS was found to result in
a significant reduction in KSPG secretion (23). When FGF-2 was combined
with FBS, however, KSPG secretion on a per cell basis was maintained at
the level of the cultures in 0.1% horse serum (Fig.
4A). We showed earlier that
keratocytes subcultured using trypsin and grown in FBS secrete very
little KSPG (22). This conclusion was confirmed in Fig. 1A
(lane 2). When serial subculture was carried out in FGF-2
and 1% FBS, keratocytes also lost KSPG secretion (Fig. 4B),
albeit more gradually than cultures without FGF. This loss of KSPG
secretion was not reversed by removal of serum from the cultures
(data not shown).
|
Sulfate is incorporated primarily into the keratan sulfate chains of
the KSPG, and previously we found that in passaged cells, keratan
sulfate chains were severely truncated (22). Keratan sulfate chain
length, examined by gel filtration chromatography, of protease-treated
KSPG showed that keratan sulfate in untreated primary cultures (Fig.
5B) eluted later, indicating a
somewhat smaller molecular mass compared with keratan sulfate made by
intact bovine corneas (Fig. 5A). The most abundant keratan
sulfate chains after FGF-2 treatment (Fig. 5C) were similar
in size to those in untreated cultures; however, more label eluted in
earlier (high molecular weight) fractions after FGF-2 treatment than in
the control cultures, indicating an increased proportion of longer keratan sulfate chains after FGF-2 treatment. Keratan sulfate from
cells maintained in 10% FBS (Fig. 5D) were clearly smaller than those from untreated cultures.
|
The moderate changes in keratan sulfate chain length shown in Fig. 5
indicate that the dramatic alterations in sulfate-labeled KSPG in
response to FGF-2 seen in Figs. 2 and 4 cannot be explained completely
in terms of keratan sulfate chain length. These data suggest that an
increase in the secretion of the proteins to which the keratan sulfate
chains are attached may be involved. This hypothesis was supported by
the observation that FGF-2 clearly stimulated incorporation of
[35S]methionine into secreted KSPG (Fig.
6A). To assess whether these KSPG core proteins were stimulated on a per cell basis, equal amounts
of nonfractionated methionine-labeled proteins from FGF-2-treated and
control cultures were separated before or after treatment with
endo--galactosidase to release proteins bound to keratan sulfate
chains (Fig. 6B). KSPG core proteins released by
endo--galactosidase exhibiting an apparent molecular mass of 48-50
kDa could be clearly differentiated from other labeled proteins in the
culture media (Fig. 6B, arrow). Densitometric
analysis of the autoradiograms indicated a 4.5-fold increase in KSPG
proteins in FGF-2 treated cultures on a per cell basis (Fig.
6C).
|
Pool sizes of mRNA of the KSPG proteins were examined with a
ribonuclease protection assay. As seen in Fig.
7A, levels of all three
mRNAs increased in the presence of FGF-2, but levels of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA did not change.
In Fig. 7B, densitometric analysis of the individual
protected bands found that lumican transcripts increased 2-3-fold,
mimecan showed only a slight increase, and the keratocan mRNA
increased 8-10-fold during 6 days of treatment in FGF-2. Summing all
three KSPG mRNAs showed an approximate 3-fold increase as a result
of FGF-2 treatment.
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This report examines factors that affect in vitro secretion of corneal KSPG proteins bearing macromolecular keratan sulfate chains. The lability of corneal keratan sulfate synthesis has been well established in the literature (14, 16, 18-21, 28), yet it is only recently that the mechanism of this down-regulation has begun to be investigated in detail. A report from our laboratory showed that passaged bovine keratocytes express the KSPG proteins in culture but observed these to be modified with oligolactosamine chains bearing minimal sulfation, rather than the fully sulfated keratan sulfate chains typical of normal corneas (22). In 1996 Jester et al. (29) found that primary keratocytes under serum-free conditions maintained a dendritic morphology typical of keratocytes in the normal cornea in vivo. More recently, Beales et al. (23) reported that primary keratocytes in low serum and serum-free conditions secrete KSPG similar to that made in vivo, providing an initiation point for the current investigation. The Beales study also demonstrated dermatan sulfate-containing proteoglycans to be regulated independently of those with keratan sulfate. Thus in the current work we have avoided expressing keratan sulfate synthesis in terms of its relative abundance compared with other proteoglycans (a frequent means of assessing glycosaminoglycan secretion), choosing rather to examine the rate of synthesis of KSPG components (proteins and keratan sulfate) on a per cell basis. This provides a measure of KSPG regulation that is independent of other proteoglycans.
Most previous studies of KSPG secretion used keratocytes cultured in the presence of fetal bovine serum, and most also had been expanded by trypsinization and subculturing. Both of these culture conditions result in loss of fully sulfated KSPG secretion in bovine keratocytes (Figs. 2, 4, and 5). Our current data suggest that this well documented down-regulation of KSPG in keratocytes may involve alterations in both KSPG protein expression and in the glycosylation of these proteins with keratan sulfate chains. The action of FGF-2 to promote secretion of KSPG appears to result mainly from increased expression of KSPG proteins, particularly of keratocan. In primary cultures, reduction of KSPG secretion by fetal bovine serum was largely reversed by FGF-2, suggesting that the initial effect of serum may also be related to protein secretion levels. FBS treatment, however, also altered keratan sulfate chain size (Fig. 5D). The current data show that KSPG secretion in subcultured cells was not reversed by FGF-2, and our previous study showed that passaged keratocytes do not secrete fully sulfated keratan sulfate chains (22). These findings suggest that KSPG loss may also involve down-regulation of enzymes involved in keratan sulfate sulfation or elongation. This hypothesis is supported by the data of Nakazawa et al. (36), which showed a dramatic loss of keratan sulfate glucosamine sulfotransferase activity in chick keratocytes cultured in serum for 7 days. Full understanding of the cellular mechanisms that control KSPG secretion will clearly require more study. Our current findings, however, demonstrate a useful experimental system with which we can begin the unraveling of the long-standing enigma of KSPG biosynthesis.
The current study is not the first to examine the effects of FGF on
keratocytes. Nakazawa et al. (30) reported a modest increase
of both keratan sulfate and dermatan sulfate as a result of a 16-h
FGF-2 treatment of primary chick keratocytes in collagen gels. These
results are not inconsistent with our findings (from Fig. 3) that 2-4
days is required before the most significant effects on KSPG can be
observed in primary bovine keratocytes. A report by Jester et
al. (29) found that 7 days of FGF-2 treatment of primary
keratocytes reversed the induction of -smooth muscle actin, a
myofibroblastic marker, in response to serum or transforming growth
factor . These results suggest that FGF may alter a range of
keratocyte cellular functions. That FGF-2 may be a generalized differentiation factor for corneal stroma is not unlikely. Action of
FGF-2 during embryonic development is important in the differentiation of numerous tissues including lens and retina (31, 32). FGF-2 has also
been implicated as a survival factor for a variety of quiescent or
terminally differentiated cells (33). Smooth muscle cells in culture
maintain differentiated characteristics via autocrine expression of
FGF-2 and enter apoptosis when this secretion is blocked (34).
Keratocytes express both mRNA for FGF-2 and for its receptor (35).
It seems reasonable to hypothesize, therefore, based on our current
data, that autocrine FGF-2 production by keratocytes may be involved in
the maintenance of the differentiated keratocyte phenotype in
vivo.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants EY09368 (to J. L. F.) EY08098 (Core grant to the University of Pittsburgh, Ophthalmology), and EY00952 (to G. W. C.), by NASA Grant NASA-NAGW 2368 (to G. W. C.), and by funds from Research to Prevent Blindness and the Eye and Ear Foundation of Pittsburgh.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Jules and Doris Stein Research to Prevent Blindness Professor. To whom correspondence should be addressed: Dept. of Ophthalmology, University of Pittsburgh, 203 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-647-3853; Fax: 412-647-5880; E-mail: jlfunder@pitt.edu.
1 The abbreviations used are: KSPG, keratan sulfate proteoglycan; FBS, fetal bovine serum; FGF-2, fibroblast growth factor-2; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM/F-12, Dulbecco's modified Eagle's medium and Ham's F-12 medium, 1:1.
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|
|---|
| 1. | Cintron, C., Schneider, H., and Kublin, C. (1973) Exp. Eye Res. 17, 251-259[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Cintron, C., and Kublin, C. L. (1977) Dev. Biol. 61, 346-357[CrossRef][Medline] [Order article via Infotrieve] |
| 3. |
West-Mays, J. A.,
Sadow, P. M.,
Tobin, T. W.,
Strissel, K. J.,
Cintron, C.,
and Fini, M. E.
(1997)
Invest. Ophthalmol. Vis. Sci.
38,
1367-1379 |
| 4. | Girard, M. T., Matsubara, M., Kublin, C., Tessier, M. J., Cintron, C., and Fini, M. E. (1993) J. Cell Sci. 104, 1001-1011[Abstract] |
| 5. |
Fini, M. E.,
and Girard, M. T.
(1990)
J. Cell Sci.
97,
373-383 |
| 6. |
Funderburgh, J. L.,
Funderburgh, M. L.,
Mann, M. M.,
and Conrad, G. W.
(1991)
J. Biol. Chem.
266,
14226-14231 |
| 7. |
Funderburgh, J. L.,
Funderburgh, M. L.,
Brown, S. J.,
Vergnes, J. P.,
Hassell, J. R.,
Mann, M. M.,
and Conrad, G. W.
(1993)
J. Biol. Chem.
268,
11874-11880 |
| 8. |
Funderburgh, J. L.,
Corpuz, L. M.,
Roth, M. R.,
Funderburgh, M. L.,
Tasheva, E. S.,
and Conrad, G. W.
(1997)
J. Biol. Chem.
272,
28089-28095 |
| 9. |
Corpuz, L. M.,
Funderburgh, J. L.,
Funderburgh, M. L.,
Bottomley, G. W.,
Prakash, S.,
and Conrad, G. W.
(1996)
J. Biol. Chem.
271,
9759-9763 |
| 10. |
Funderburgh, J. L.,
Funderburgh, M. L.,
Mann, M. M.,
and Conrad, G. W.
(1991)
J. Biol. Chem.
266,
24773-24777 |
| 11. |
Chakravarti, S.,
Magnuson, T.,
Lass, J. H.,
Jepsen, K. J.,
LaMantia, C.,
and Carroll, H.
(1998)
J. Cell Biol.
141,
1277-1286 |
| 12. |
Danielson, K. G.,
Baribault, H.,
Holmes, D. F.,
Graham, H.,
Kadler, K. E.,
and Iozzo, R. V.
(1997)
J. Cell Biol.
136,
729-743 |
| 13. | Funderburgh, J. L., Caterson, B., and Conrad, G. W. (1986) Dev. Biol. 116, 267-277[CrossRef][Medline] [Order article via Infotrieve] |
| 14. | Conrad, G. W., and Dorfman, A. (1974) Exp. Eye Res. 18, 421-433[CrossRef][Medline] [Order article via Infotrieve] |
| 15. | Coster, L., Cintron, C., Damle, S. P., and Gregory, J. D. (1983) Exp. Eye Res. 36, 517-530[CrossRef][Medline] [Order article via Infotrieve] |
| 16. | Klintworth, G. K., and Smith, C. F. (1981) Lab. Invest. 44, 553-559[Medline] [Order article via Infotrieve] |
| 17. | Dahl, I. M., and Coster, L. (1978) Exp. Eye Res. 27, 175-190[CrossRef][Medline] [Order article via Infotrieve] |
| 18. | Dahl, I. M., and Laurent, T. C. (1982) Exp. Eye Res. 34, 83-98[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Yue, B. Y., and Baum, J. L. (1976) Biochem. J. 158, 567-573[Medline] [Order article via Infotrieve] |
| 20. |
Yue, B. Y.,
Baum, J. L.,
and Silbert, J. E.
(1978)
Invest. Ophthalmol. Vis. Sci.
17,
523-527 |
| 21. | Gnadinger, M. C., and Schwager-Hubner, M. E. (1975) Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 196, 9-19[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Funderburgh, J. L.,
Funderburgh, M. L.,
Mann, M. M.,
Prakash, S.,
and Conrad, G. W.
(1996)
J. Biol. Chem.
271,
31431-31436 |
| 23. |
Beales, M. P.,
Funderburgh, J. L.,
Jester, J. V.,
and Hassell, H. R.
(1999)
Invest. Ophthalmol. Vis. Sci.
40,
1658-1663 |
| 24. |
Funderburgh, J. L.,
Hevelone, N. D.,
Roth, M. R.,
Funderburgh, M. L.,
Rodrigues, M. R.,
Nirankari, V. S.,
and Conrad, G. W.
(1998)
Invest. Ophthalmol. Vis. Sci.
39,
1957-1964 |
| 25. | Funderburgh, J. L., Stenzel-Johnson, P. R., and Chandler, J. W. (1982) Curr. Eye Res. 2, 769-776[Medline] [Order article via Infotrieve] |
| 26. |
Jost, C. J.,
Funderburgh, J. L.,
Mann, M.,
Hassell, J. R.,
and Conrad, G. W.
(1991)
J. Biol. Chem.
266,
13336-13341 |
| 27. |
Funderburgh, J. L.,
and Conrad, G. W.
(1990)
J. Biol. Chem.
265,
8297-8303 |
| 28. |
Hassell, J. R.,
Schrecengost, P. K.,
Rada, J. A.,
SundarRaj, N.,
Sossi, G.,
and Thoft, R. A.
(1992)
Invest. Ophthalmol. Vis. Sci.
33,
547-557 |
| 29. | Jester, J. V., Barry-Lane, P. A., Cavanagh, H. D., and Petroll, W. M. (1996) Cornea 15, 505-516[Medline] [Order article via Infotrieve] |
| 30. |
Nakazawa, K.,
Morita, A.,
Nakano, H.,
Mano, C.,
and Tozawa, N.
(1996)
J. Biochem. (Tokyo)
120,
117-125 |
| 31. | Bosco, L., Venturini, G., and Willems, D. (1997) Development 124, 421-428[Abstract] |
| 32. | Araki, M., Yamao, M., and Tsudzuki, M. (1998) Dev. Growth Differ. 40, 167-176[CrossRef][Medline] [Order article via Infotrieve] |
| 33. | Shaw, R., Cianchetti, R., Pleasure, D., and Kreider, B. (1997) J. Neurosci. Res. 47, 400-404[CrossRef][Medline] [Order article via Infotrieve] |
| 34. |
Fox, J. C.,
and Shanley, J. R.
(1996)
J. Biol. Chem.
271,
12578-12584 |
| 35. | Li, D. Q., and Tseng, S. C. (1995) J. Cell. Physiol. 163, 61-79[CrossRef][Medline] [Order article via Infotrieve] |
| 36. | Nakazawa, K., Takahashi, I., and Yamamoto, Y. (1998) Arch. Biochem. Biophys. 359, 269-282[CrossRef][Medline] [Order article via Infotrieve] |
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