From the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
Received for publication, November 19, 2002
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ABSTRACT |
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Binding of fibronectin to the small proteoglycan
decorin plays an important role in cell differentiation and cell
migration. The cartilage-specific (V+C) Fibronectin (FN)1 is a
major constituent of extracellular matrices in many tissues. The FN
protein is folded into a series of globular homologous repeats of three
distinct types (I, II, and III) composed of approximately 45, 60, and
90 amino acids, respectively (Fig.
1A) (1). It is secreted as a
dimer with two interchain disulfides formed at the C terminus (2, 3). Although there are multiple isoforms of FN produced by different cell
types, a single gene encodes all of these variants (4). The molecular
heterogeneity of FN protein results primarily from alternative splicing
of its pre-mRNA and different dimerization patterns (5). The
(V+C) fibronectin
isoform, in which nucleotides that normally encode the protein segments
V, III15, and I10 are spliced out, is one of
the major splice variants present in cartilage matrices. Full-length and truncated cDNA constructs were used to express recombinant versions of fibronectin. Results demonstrated that the
(V+C)
isoform has a higher affinity for decorin.
Dissociation constants for decorin and fibronectin interaction were
calculated to be 93 nM for the V+C+
isoform and 24 nM and 223 nM for
(V+C)
fibronectin. Because heparin competed with decorin
competitively, binding of decorin to fibronectin likely occurs at a
heparin-binding region. We propose that alternative splicing of the V
and C regions changes the global conformation of fibronectin in such a
way that it opens an additional decorin-binding site. This
conformational change is responsible for the higher affinity of the
(V+C)
fibronectin isoform for decorin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform lacks protein segments V,
III15, and I10 and forms primarily homodimers
(6, 7).
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Fig. 1.
Schematic representation of fibronectin and
mininectin constructs. A, full-length (V+C)
and V+C+ FN. The shaded areas
indicate regions of alternative splicing. EDA and EDB are spliced in or
out in their entirety. V region splice variants occur in plasma and
other tissues. The (V+C)
FN isoform, however, is created
when the V and C regions are removed entirely by alternative splicing,
and a new splice junction joining III14 with
I11 is created. Expression of this isoform is
cartilage-specific. B, mininectin constructs. MNs are
truncated FN constructs that originate at three different sites within
the Hep II region and extend part of the way into segment
I12. Thus, they all lack the major
5
1 integrin site in III10, a
GAG-binding site in III5, and a putative cryptic binding
site for decorin near III10. They also lack interchain
C-terminal disulfide bonds that are essential for dimerization and
therefore are monomeric. The MN3 series, which delete V,
III15, and I10, are truncated versions of the
(V+C)
isoform and were derived from canine cartilage RNA.
MN3-III12 includes the major heparin-binding site in III13.
MN3-III14 lacks the major heparin-binding site in III13 but
includes putative GAG and
4
1 integrin
sites in III14. The structure of type III repeats consists
of seven linked
-sheets, designated by convention as A-G from the N
to the C terminus. The MN3-III14fg construct originates in the junction
between
-sheets E and F in III14.
Studies of FN in cartilage and other tissues have revealed that
expression of (V+C) FN is tightly linked to the
cartilaginous phenotype (8). In articulator cartilage, the
(V+C)
isoform represents 55-80% of total tissue FN.
RNase protection assays determined that steady-state levels of canine
(V+C)
FN mRNA range from 11% of total FN in the
nucleus pulpous to 71% in the rib, with expression of this isoform
rapidly lost if chondrocytes are cultured as monolayers and allowed to
de-differentiate (8). These data suggest that the (V+C)
isoform plays an important role in cartilage matrix organization and
perhaps even in regulating or maintaining the differentiated phenotype
of chondrocytes.
The protein structure of FN can be related to its known functions. In
different tissues, FN has a role in cell adhesion and differentiation,
wound healing, blood clotting, cell migration, and spreading (9, 10).
The high affinity heparin-binding site (Hep II) is located within the
C-terminal half of the molecule in domains III12-14 with a
Kd close to 10 nM, although reported
dissociation constants vary widely (11-14). Small proteoglycans (PG)
have been reported to bind to plasma FN, presumably at the heparin-binding sites. The 4
1
integrin facilitates cell binding to two sites within the V region of
FN (15). More recently, Sharma et al. (16) demonstrated the
presence of two additional
4
1
integrin-binding sites as follows: one at the junction of III13-III14 containing the amino acid sequence
IDAPS, and the other in the III14 segment (PRARI motif
homologous to PHSRN in III9). Only the former site appears
to be accessible to the integrin. Moreover, Moyano et
al. (17) uncovered a site in the III5 segment for
cooperative binding of chondroitin sulfate proteoglycan and
4
1 integrin. The cooperative binding at
III5 segment appears to be important for FN matrix assembly
(17). Close proximity of heparin and
4
1
integrin-binding sites suggests that coordinated interactions of
4
1 integrin and chondroitin sulfate
proteoglycans with FN may be an important theme in FN biology.
Consistent with this model, FN fragments with mutations in the Hep II
domain adjacent to a functional V region decreased pp125FAK
protein levels downstream of integrin signaling and regulated apoptosis
in fibroblasts. These effects were mediated via cell surface
chondroitin sulfate (18).
Decorin is a member of a family of small leucine-rich PGs. Depending on
the species, it can have chondroitin- or dermatan sulfate
glycosaminoglycan (GAG) chains attached. Its role in cartilage matrix
biology is unclear. Data substantiate that decorin controls the
diameter of type I collagen fibrils (19), and it is assumed to have a
similar role for type II collagen organization in cartilage. Decorin
may modulate transforming growth factor- signaling pathways by
binding and sequestering transforming growth factor-
(20).
The cartilage-specific (V+C) isoform is structurally
different from the widely expressed V+C+ and
V
C+ FN variants. In (V+C)
FN,
the V, III15, and I10 segments adjacent to the
Hep II site are spliced out. The omission of a large part (257 amino
acids) near the heparin/PG/
4
1
integrin-binding site may result in altered function. In this study, we
demonstrate that (V+C)
FN retains its ability to bind
both heparin and chondroitin sulfate PGs. Moreover, we show that the
(V+C)
FN contains a high affinity decorin-binding site
not found in V+C+ FN.
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EXPERIMENTAL PROCEDURES |
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Materials--
35SO-Ketoglutarate, papain, N-acetylcysteine,
EDTA-tetrasodium salt, were obtained from Sigma. Calcium chloride was
purchased from Mallinckrodt (Phillipsburg, NJ). L-Ascorbic
acid phosphate was purchased from Wako Pure Chemical Industries
(Dallas, TX). ITSTM Premix (insulin, transferrin, selenious
acid, bovine serum albumin, and linoleic acid) was obtained from BD
Biosciences. Spodoptera frugiperda (Sf21)
insect cells were obtained from Invitrogen. Murine monoclonal anti-rat
FN antibody (IC3) and the pVL1392 baculovirus cloning vector
(Invitrogen) containing cDNA encoding V+C+
FN were generous gifts from Dr. Jean Schwarzbauer (Princeton, NJ).
EX-CELL 400 serum-free insect medium was purchased from JRH Biosciences
(Lenexa, KS). Gelatin-Sepharose was purchased from Amersham
Biosciences.
Cartilage Culture and Labeling--
Canine articular cartilage
was collected from knee, hip, and shoulder joints at necropsy from
2-year-old Labrador Retriever dogs, raised as part of a colony
maintained at the James A. Baker Institute for Animal Health. Cartilage
explants were washed with Gey's balanced salt solution and cultured
for 48 h in Ham's F-12 complete medium supplemented with calcium
chloride (485 µg/ml), Hepes buffer (25 mM),
-ketoglutarate (30 µg/ml), gentamicin (20 µg/ml), penicillin
G/streptomycin sulfate (20 units/ml, 20 µg/ml), L-glutamine (0.3 mg/ml), ITS-CR+,
L-ascorbic acid phosphate (0.05 mg/ml),
35SO
Purification of Small PGs from Cartilage-- Proteoglycans were separated from other proteins in the cartilage extract by DEAE chromatography. The cartilage extract was transferred by dialysis into DEAE column buffer (2 M urea, 0.05 M Tris-HCl, 10% PI, pH 8.0) and applied to a DEAE column at gravity flow (22). Proteins were eluted with the addition of 0.2 M NaCl to the DEAE column buffer and discarded. Proteoglycans were eluted with 4 M guanidine HCl, 0.1 M Na2SO4, 0.05 M sodium acetate, 0.1% Triton X-100, 10% PI, pH 6.1, and further purified by gel filtration chromatography on Sepharose CL-6B in order to separate the small PGs from aggrecan.
Decorin was separated from fibromodulin and biglycan by hydrophobic chromatography on octyl-Sepharose as described elsewhere (23), except that the material bound to the column was batch-eluted from the gel with increasing concentrations of guanidine HCl. Decorin was present in the flow-through and wash fractions. Samples were then re-purified by DEAE chromatography to remove any aggrecan spillover. Decorin was eluted with 0.3 M NaCl.
Gel Electrophoresis and Western Blot Analyses-- All fractions were analyzed by 5-15% SDS-PAGE in Tris/glycine buffer, pH 8.6 (7), and transferred to Hi-Bond N+ membranes (Amersham Biosciences) (22, 24). Blots were probed with rabbit polyclonal antibodies against decorin, fibromodulin, and biglycan, followed by peroxidase-linked goat anti-rabbit IgG. Peroxidase activity was detected by enhanced chemiluminescence (ECL Western blotting detection system, Amersham Biosciences). Dried blots were also exposed to a PhosphorImager plate to detect radioactive bands. The plate was read on a Fuji PhosphorImager and analyzed by MacBas software.
Expression and Purification of Recombinant FN and Recombinant MN
Constructs--
Construction of recombinant baculovirus containing
full-length rat V+C+ and (V+C) FN
cDNA has been described previously (25-27). Both isoforms were expressed following infection of Sf-21 insect cells according to the
manufacturer's instructions. Fibronectin proteins were purified by
sequential gelatin affinity and heparin affinity chromatography steps
in a modification of the method proposed by Poulouin et al.
(28). Fractions were analyzed by SDS-PAGE followed by silver staining
to determine protein purity. Fibronectin concentration was quantified
by a gelatin affinity enzyme-linked immunosorbent assay as described
previously (29).
Bacterial expression constructs for truncated canine FN cDNA
fragments termed mininectins (MNs) were produced using the pTrcHis-TOPO system (Invitrogen) according to the manufacturer's instructions (Fig.
1B). Total RNA was purified from articular cartilage as described earlier (6). The cDNA fragments of interest were amplified by RT-PCR using the primer pairs listed in Table
I. The three MN3 constructs represent the
(V+C) isoform (Fig. 1B). The amplified
cDNA fragments were ligated into the pTrcHis-TOPO vector containing
a 6-histidine tag at the N terminus and transfected into TOP-10
bacterial cells. Mininectins were purified on ProBond nickel resin
(Invitrogen) according to the manufacturer's protocol. Protein purity
was assessed by SDS-PAGE followed by a silver stain. Recombinant
proteins (1 nmol/column for full-length FNs and 7 nmol/column for MNs)
were immobilized in duplicate on the CNBr-activated Sepharose-4B.
Similarly, purified rat plasma FN was immobilized and used as a
positive control, and BSA was immobilized and used as a negative
control.
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Binding of Small PGs to FNs-- Purified small PGs in 30 NTE (30 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH 7.3) buffer were applied to the columns with immobilized FN and to the column with immobilized BSA and allowed to bind for 15 min. The optimal binding time was determined empirically. Columns were washed with 30 NTE buffer until no radioactivity above background was detected. Columns were then eluted with 1 M NaCl in NTE buffer to determine total PGs bound. Fractions were analyzed for radioactivity by scintillation counting in order to quantitate bound PGs as a percentage of the total. Bound and unbound (free) fractions were resolved on 5-15% SDS-PAGE, stained with toluidine blue dye, dried, and exposed to a PhosphorImager plate. The plate was read by a Fuji PhosphorImager and analyzed with MacBas software.
Competition between Heparin and Small PGs for Binding to FNs-- Small PGs were mixed with heparin (0.2-25 µg) to test for competitive binding to FN. Samples containing heparin were applied to the columns with immobilized FN and analyzed as described above. Binding of decorin was compared with maximum binding in the absence of heparin, and the data were fitted to a curve for competitive inhibition using GraphPad PRISM3 software (GraphPad Software, San Diego, CA).
Determination of Dissociation Constants for Decorin-FN Interactions-- A range of concentrations of purified decorin in a standard volume of 1 ml were applied to columns containing either immobilized FN or BSA, followed by elution with 1 M NaCl. The molar concentration of free decorin was plotted as a function of the molar concentration of bound decorin, and curve fitting was performed using the Kaleidagraph software to Equations 1 and 2,
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(Eq. 1) |
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(Eq. 2) |
Binding of Decorin GAGs and Core Protein to FNs--
Equivalent
amounts of decorin were either digested with chondroitinase ABC to
produce free core protein or digested with papain to produce free GAGs.
The free GAG components and the free decorin core protein components
were independently applied to the FN columns as above. Bound and free
fractions were analyzed by scintillation counting for the presence of
radioactivity. The percentages of bound core protein and GAGs were
calculated and plotted.
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RESULTS |
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(V+C) FN Binds Significantly More PGs Than Does
V+C+ FN--
Analysis of small PG fractions
bound to recombinant (V+C)
FN, recombinant
V+C+ FN, and plasma FN revealed that the
cartilage-specific isoform was able to bind small PGs despite the
absence of protein segments V, III15, and I10
near the Hep II-binding domain. Fig.
2A shows an SDS-PAGE
separation of both bound and free fractions of small PGs. In fact,
(V+C)
FN bound significantly more PGs than equivalent
amounts of V+C+ or plasma FN (p < 0.0004 and p < 0.0001, respectively) (Fig. 2B).
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Further analysis indicated that all three small PGs, decorin, biglycan, and fibromodulin, bound to FN, and all three exhibited increased binding to the cartilage-specific FN isoform. Decorin and biglycan were identified by sensitivity to chondroitinase ABC and by reactivity with anti-decorin and anti-biglycan antibodies, respectively. Decorin and fibromodulin (67-78 and 84-90 kDa, respectively) were not resolved by SDS-PAGE. Therefore, to determine whether fibromodulin bound to FNs, small PGs were first digested with chondroitinase ABC to remove chondroitin/dermatan sulfate GAG chains. This reduced decorin and biglycan to their core proteins. The small PG with a molecular mass of 84-90 kDa that remained after this chondroitinase ABC digestion was identified by immunoblot analysis as the keratan sulfate containing small, leucine-rich PG, fibromodulin.
Cartilage-specific (V+C) FN Contains Two
Decorin-binding Sites--
The above results indicated that the
(V+C)
FN isoform bound more small PGs than the
V+C+ isoform. To assess if this reflected an
increased binding affinity, we determined dissociation constants
(Kd) for the binding of purified decorin. Decorin
was separated from fibromodulin and biglycan by octyl-Sepharose
chromatography, as described under "Experimental Procedures," and
applied to the immobilized full-length FNs at a range of
concentrations. Results were plotted, and Kd values
were calculated (Fig. 3). The binding
profile indicates two decorin-binding sites for (V+C)
FN
with dissociation constants of 23 and 224 nM. Only one
decorin-binding site is suggested for V+C+ FN
with a dissociation constant of 93 nM.
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GAG Chains of Decorin and Not the Core Protein Are Responsible for
Binding to FN--
There are conflicting reports with regard to what
part of the PG molecule is responsible for interactions with FN.
Barkalow and Schwarzbauer (30) showed that in 75 mM NaCl,
binding of PGs is mainly due to GAG chains. Schmidt et al.
(31) have demonstrated that in 150 mM NaCl, 94% of
interactions are due to the core protein moiety of decorin. Our results
in 30 mM NaCl indicate that binding of decorin to all three
FN isoforms is due mainly to the GAG chains and not to the core
protein. Binding of GAGs alone accounted for 70% of total binding by
intact decorin, whereas core protein accounted for less than 5% (Fig.
4).
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Binding of Decorin to MNs Requires Both III13 and
III14 Repeats of the Hep II Domain--
Three FN
constructs, termed MNs (Fig. 1B), were produced in bacteria
in order to elucidate the domain responsible for binding of decorin to
FNs. MN3-III12 contained segments III12 through I12 for the (V+C) isoform. MN3-III14
contained segments III14 through I12, and MN3-III14fg contained the C-terminal part of III14
(
-sheets F and G) through I12. All constructs lacked the
disulfide dimerization domain near the C terminus. MN3-III12 was able
to bind decorin, but further truncation to eliminate the
III12-13 domains reduced decorin binding to background
levels (Fig. 5).
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Heparin Inhibits Binding of Small PGs to FNs--
Heparin has a
higher affinity for FN than do small PGs (30). There are at least three
known heparin-binding sites identified on FN (32). The reported high
affinity site is located in repeats III13-III14
(Hep II) (Fig. 1A). Another site is located at the N
terminus of the molecule. A third site or sites is within the first six
type III repeats C-terminal to the gelatin-binding site. To determine
whether the small PGs interact with FNs at or near a heparin-binding
site, a competition binding experiment was conducted. Fig.
6, A and B, shows
that all FN isoforms bound heparin and that binding of small PGs to FN
was almost completely abolished in the presence of excess heparin.
Increasing concentrations of unlabeled heparin inhibited the binding of
decorin to FN in a dose-dependent manner. Interestingly,
half-maximal inhibition of binding of equimolar amounts of decorin
required three to four times as much heparin for (V+C) FN
than was required for V+C+ FN (Fig.
6C). The data fit theoretical curves for competitive inhibition with R2 values of 0.97.
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DISCUSSION |
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In this report, we demonstrate that the cartilage-specific
(V+C) FN isoform interacts with leucine-rich small PGs.
Interactions of decorin, biglycan, and fibromodulin with FN isoforms
were studied in a solid-phase binding assay at low ionic strength (30 mM NaCl). Binding of small PGs to cartilage-specific
(V+C)
FN was quantitatively different from binding to the
other FN isoforms examined. (V+C)
FN consistently bound
up to 50% more small PGs than did the V+C+
isoform. The dissociation constants for binding of decorin PG to
recombinant (V+C)
FN suggest the presence of two binding
sites: a high affinity site (Kd = 24 nM)
and a low affinity site (Kd = 223 nM).
Only one binding site was detected on the V+C+
FN with a Kd of 93 nM.
It was determined that decorin GAG chains were responsible for the
binding to FN isoforms. Our data are in general agreement with Barkalow
and Schwarzbauer (30), who found interactions of
V+C+ and VC+ FNs with
chondroitin sulfate GAGs at low ionic strength (75 mM), which were eliminated at physiological salt concentrations (150 mM). Schmidt et al. (31) observed binding of
plasma FN to decorin in 150 mM NaCl. They showed that
decorin core protein, but not the GAG chains, was responsible for
binding. Only very low levels of binding between decorin core protein
and FNs was seen in the current study. A potential reason for why our
results differ from that of Schmidt et al. (31) is that by
working at 150 mM NaCl, their experimental conditions
likely excluded binding to the GAG chain of decorin. One might question
whether interactions between FN and chondroitin/dermatan sulfate GAGs
are relevant in vivo because, in contrast to heparin, they
only occur at less than the assumed physiological salt concentration.
However, considerable evidence exists that chondroitin sulfate PGs,
often cooperatively with
4
1 integrins,
contribute to cell adhesion to FN (discussed in Refs. 14, 30, 33).
The major heparin-binding site is thought to be located within repeats
III12-III14. This domain also supports cell
adhesion and spreading through five peptides composed of basic residues that, in vitro, have been shown independently to bind
heparin and/or chondroitin sulfate (33-36). Repeat III14
contains three of the five basic peptides, one immediately N-terminal
to the V region which may be inaccessible in the native molecule (37, 38). X-ray crystallography studies by Sharma et al.
(16) confirmed the prediction that a cluster of six basic residues from
one or more of these peptides come together to generate a positively charged "cradle" on one side of the molecule that forms the major heparin-binding site in III13. Structural analysis revealed
a similar cluster of five basic residues in the putative additional heparin-binding site in III14. Based on this information,
we hypothesized that altered conformation of the (V+C) FN
isoform, created by deletion of V, III15, and
I10 through alternative RNA splicing, enhances exposure of
a heparin-binding site in III14, making it more accessible
to decorin and contributing to the increase in binding affinity between
decorin and (V+C)
FN. To test this hypothesis, MN
constructs were created. The largest construct, MN3-III12, contains an
intact heparin-2 binding domain. The next construct, MN3-III14, is
further truncated to remove domains III12 and
III13 but retains an intact III14 domain. The
smallest construct, MN3-III14fg, retains only the f and g
-sheets of
the III14 domain. The hypothesis predicts that MN3-III14 will bind decorin. If truncation of MN3-III14 disrupts the integrity of
the binding site, then MN3-III14fg will fail to bind decorin. The data
indicate, however, that only the MN containing domains III12-14 was able to bind decorin. The other two
constructs did not bind decorin above background. Heparin inhibited
binding of decorin to both FNs and MNs. These results suggest that
binding of decorin to the (V+C)
and
V+C+ FN isoforms requires the heparin-binding
domain in III13, but the heparin-binding site in
III14 does not bind decorin independent of the site in
III13. Barkalow and Schwarzbauer (11) showed that both
III13 and III14 domains bind to heparin, but
binding is greater if both are present. They also showed that
chondroitin sulfate GAGs bind at identical or overlapping sites in both
III13 and III14 albeit with lower affinity
(30). Our results are in partial agreement with this but do not support
an independent GAG-binding site in III14. Interestingly,
recent results reported by Sachchidanand et al. (39) do not
support binding of heparin to III14.
Although an additional heparin-binding site in III14 may
not explain the increased binding affinity to decorin, nevertheless, the protein segment immediately adjacent to the Hep II domain appears
to be important for the function of this region. Santas et
al. (40) showed that the amino acids adjacent to III14
influence cell spreading on FN and FN fibrillogenesis. Ten amino acids
from the N terminus of the V region were sufficient to make
fibrillogenesis and cell spreading responsive to interactions of cell
surface heparin sulfate proteoglycans with the Hep II domain. Replacing these 10 amino acids with 6 amino acids from the III15
domain abolished this dependence. The altered binding capacity and
affinity for small proteoglycans observed with (V+C) FN
further support the concept that native alternative splicing patterns,
which position different amino acids immediately C-terminal to
III14, alter functional properties of the Hep II region.
(V+C) FN represents from 50 to 80% of total FN in adult
canine and equine articular cartilage, respectively (8). During postnatal development, steady-state (V+C)
FN mRNA in
cartilage increases slightly, then roughly doubles to adult levels
during puberty (10 months of age in dogs (8)). By contrast, the
splicing pattern that generates ED-A+ FN is turned off as
mesenchymal cell precursors differentiate into chondrocytes.
Interestingly, cell migration (41) and FN-induced cell cycle
progression appear to be enhanced by ED-A+ FN (42). The
onset of decorin expression is also correlated with differentiation of
mesenchymal stem cells into chondrocytes occurring along with increases
in aggrecan synthesis and just prior to the appearance of type II
collagen (43). The migration of cells on FN is inhibited by decorin
(44, 45). It has been suggested that the interaction of decorin with
extra cellular matrix components such as FN and collagens supports
formation of a pericellular matrix that stabilizes cellular
differentiation (46). The higher affinity of (V+C)
FN for
decorin appears to support this model, enhancing the interaction of
extracellular matrix components that may help to stabilize the
differentiated phenotype of chondrocytes.
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ACKNOWLEDGEMENTS |
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We thank Drs. Paul Scott (Edmonton, Alberta,
Canada), Peter Roughley (Montreal, Quebec, Canada), and Gabriella
Cs-Szabo (Chicago, IL) for generous gifts of anti-proteoglycan
antibodies. We thank Dr. Jean Schwarzbauer (Princeton, NJ) for gifts of
V+C+ FN constructs from which
(V+C) FN constructs were derived. We are grateful to Dr.
Robert Oswald (Cornell University) for helpful discussions and advice
in analyzing the binding data. We acknowledge the valuable technical
contributions of Michael Jackson and Alma Jo Williams and the
secretarial assistance of Dorothy Scorelle.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AR44340 and the Arthritis Foundation.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.
To whom correspondence should be addressed. Tel.: 607-256-5651;
Fax: 607-256-5608; E-mail: niw1@cornell.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211799200
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ABBREVIATIONS |
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The abbreviations used are: FN, fibronectin; PG, proteoglycans; GAG, glycosaminoglycan; BSA, bovine serum albumin; PI, protease inhibitor; MN, mininectin.
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