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Identification of fibronectin neoepitopes present in human osteoarthritic cartilage.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 9, September 2006, pp 2912–2922
DOI 10.1002/art.22045
© 2006, American College of Rheumatology
Identification of Fibronectin Neoepitopes Present in
Human Osteoarthritic Cartilage
Marc D. Zack, Elizabeth C. Arner, Charles P. Anglin, James T. Alston,
Anne-Marie Malfait, and Micky D. Tortorella
C-terminus VRAA271 were detected following cytokine
treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA
cartilage.
Conclusion. Human OA cartilage contains fibronectin fragments with catabolic activity and a major
cleavage site within fibronectin. This study is the first to
characterize fibronectin neoepitopes in OA cartilage,
suggesting that they may represent a novel biomarker of
arthritis.
Objective. Fibronectin fragments are present at
high concentrations in the cartilage of patients with
rheumatoid arthritis and patients with osteoarthritis
(OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and
progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in
human OA cartilage and confirm their ability to elicit
cartilage catabolism, 2) identify the cleavage sites in
fibronectin and generate the corresponding neoepitope
antibodies, and 3) explore the utility of fibronectin
neoepitopes as biomarkers.
Methods. Fibronectin fragments were purified
from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing.
Bovine nasal cartilage was treated with affinity-purified
fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans
and the aggrecan neoepitope 1771AGEG. Fibronectin
neoepitopes were detected by Western blotting in
cytokine-treated media of human cartilage explants,
and by immunohistochemical analyses of human OA
cartilage.
Results. Multiple fibronectin fragments were isolated from human OA cartilage, and all contained
the N-terminus 272VYQP. These fragments induced
aggrecanase-mediated cartilage catabolism in bovine
cartilage explants. Fibronectin fragments with the
N-terminus 272 VYQP and fragments with the
Fibronectin is a multimeric glycoprotein found in
human plasma and tissues. Alternative splicing of fibronectin messenger RNA reveals many different species of fibronectin monomers that comprise plasma and
tissue fibronectin (1,2). The plasma form of fibronectin
is a soluble dimer composed of 200–220-kd subunits
bound together through C-terminal disulfide bonds,
yielding a total molecular weight of ⬃440 kd. Tissue
fibronectin exists in the form of large insoluble multimers within the extracellular matrix (ECM) bound together through disulfide bonding, as well as interactions
through self-association domains (2). Monomers contain
functional domains that can bind various integrins,
heparin, fibrin, and collagen/gelatin (for review, see ref.
2). As a result of the activity of these many functional
domains, fibronectin is involved in a wide range of
biologic processes, such as cell migration, wound healing, angiogenesis, and cell differentiation (3–6).
In normal cartilage, fibronectin is a minor component of the ECM. However, osteoarthritic (OA) cartilage explants can contain up to 10-fold more fibronectin than is present in normal cartilage (7,8). This is due
to an increase in both synthesis and accumulation of
fibronectin (9). Increases in cartilage fibronectin are
accompanied by a corresponding accumulation of fibronectin in the synovial fluid of both rheumatoid
arthritis (RA) and OA patients (10). Fibronectin can be
Marc D. Zack, MS, Elizabeth C. Arner, MS, Charles P.
Anglin, MS, James T. Alston, BS, Anne-Marie Malfait, MD, Micky D.
Tortorella, BS: Pfizer Global Research & Development, Chesterfield,
Missouri.
Address correspondence and reprint requests to Marc D.
Zack, MS, Pfizer Global Research & Development, BB334-K, 700
Chesterfield Parkway, Chesterfield, MO 63017. E-mail: marc.d.zack
@pfizer.com.
Submitted for publication February 6, 2006; accepted in
revised form May 16, 2006.
2912
FIBRONECTIN NEOEPITOPES IN OA CARTILAGE
proteolysed into smaller fragments by multiple enzymes,
and fragmentation is seen in the synovium and cartilage
of both patients with OA and patients with RA (11,12).
Xie et al (12) have shown that synovial fluid fibronectin
fragments can reach levels higher than 1 ␮M, which is
ascribed to the increase in fibronectin production and
the concomitant elevation of proteolytic enzymes in
these disease states.
It is well established that the products of fibronectin degradation, and not intact fibronectin, are
able to induce a catabolic phenotype in chondrocytes
and cartilage explants. Fragments have been shown to
induce metalloproteinases, including matrix metalloproteinase 3 (MMP-3) (13,14), MMP-13, and aggrecanases
(13), as well as serine proteinases such as urokinase
plasminogen activator (14). These proteases have the
ability to break down the major cartilage matrix components, including type II collagen and aggrecan, which is
a hallmark of OA. It has also been shown that prolonged
exposure of cartilage to high levels of fibronectin fragments can suppress matrix synthesis (15), thus limiting
the anabolic reparative response to cartilage damage. In
addition, fibronectin fragments have been demonstrated
to up-regulate proinflammatory mediators such as
interleukin-1␤ and inducible nitric oxide synthase (16),
which are thought to play roles in the onset and progression of OA and RA. Therefore, degradation of fibronectin, and the resulting fibronectin fragments, may play an
important role in the initiation and progression of
arthritic disease.
To date, only fibronectin fragments present in the
synovial fluid have been characterized. Peters et al (17)
observed that several fibronectin fragments, ranging in
molecular weight from 47 kd to 170 kd, contained an
intact N-terminus, in both patients with RA and those
with OA. These studies focused on the structural and
ligand-binding properties of the fragments that contained the N-terminal heparin-binding domain of fibronectin. Although these experiments helped characterize the components of fibronectin associated with
arthritic diseases, the cleavage sites within fibronectin
were not determined. Furthermore, these experiments
analyzed the presence of fragments in the synovial fluid,
which may not accurately reflect that in cartilage.
The present study was conducted to 1) isolate
fibronectin fragments from human OA cartilage using
affinity chromatography, in order to determine whether
these fragments are capable of inducing catabolism in
cartilage explant cultures, 2) identify the N-terminus of
the fibronectin fragments and generate antibodies to
both the N-terminal and the C-terminal neoepitopes,
2913
and 3) begin to explore these fragments as potential
biomarkers of cartilage breakdown.
PATIENTS AND METHODS
Materials. All common laboratory chemicals, the silver
staining kit (ProteoSilver Plus), and rabbit polyclonal antifibronectin antibody (F-3468) were purchased from Sigma (St.
Louis, MO). Protease inhibitor cocktail tablets, purchased
from Roche Diagnostics (Indianapolis, IN), were used to
inhibit a broad spectrum of proteases, including cysteine,
serine, aspartyl, and metalloproteinases. Gelatin–Sepharose
4-B was purchased from Amersham Biosciences (Upsala,
Sweden). Recombinant human oncostatin M (OSM) and recombinant human tumor necrosis factor ␣ (TNF␣) were
purchased from R&D Systems (Minneapolis, MN). All electrophoresis reagents, cell culture media, and supplements were
purchased from Invitrogen (Carlsbad, CA). Alkaline
phosphatase–conjugated anti-rabbit IgG antibodies (Promega,
Madison, WI) were used according to the manufacturer’s
directions. Deglycosylating enzymes keratanase I, keratanase
II, and chondroitinase were purchased from Seikagaku (East
Falmouth, MA). Chemicals and reagents used for N-terminal
protein sequencing were purchased from Applied Biosystems
(Foster City, CA).
Human cartilage extraction. The human OA cartilage
used for extraction, identification, and Western blotting of the
fibronectin fragments was obtained from OA patients at the
time of total knee replacement (a gift from Dr. Kosei Ijiri,
Kagoshima University, Kagoshima, Japan). All donors were
men between the ages of 64 and 82 years. Cartilage was
removed from the femoral condyles and tibial plateau and then
minced into small pieces with a scalpel. Thereafter, all steps
were performed in the presence of protease inhibitor (used as
suggested by the manufacturer). Proteins were extracted from
human OA cartilage at 4°C with constant inversion for 7 days
in extraction buffer (4M guanidine HCl, 50 mM sodium
acetate, pH 6.8; 10 ml per 1 gm cartilage). Samples were then
subjected to cesium chloride density-gradient centrifugation.
Supernatants were adjusted to a density of 1.5 gm/ml with
cesium chloride, centrifuged at 39,400 revolutions per minute
for 72 hours at 4°C, and the D4 fraction (top 25%) of the
sample was retained and extensively dialyzed (molecular
weight cutoff point of 10 kd) against Tris buffered saline (TBS)
(50 mM Tris, pH 7.6, 100 mM NaCl) in the presence of
protease inhibitors.
Fibronectin fragments were quantified using Lowry’s
modified protein assay according to the manufacturer’s directions (Pierce, Rockford, IL). Silver staining was used to assess
the purity of the fragments, with analyses carried out by
electrophoresis of 30 ␮l dialyzed extract under reducing conditions. Molecular weight was estimated by Western blotting
with molecular weight standards (Bio-Rad, Hercules, CA).
Human cartilage explant cultures. Macroscopically
normal cartilage was obtained postmortem from the femoral
condyles of 2 male donors (National Disease Research Interchange, Philadelphia, PA). Cartilage was cut into ⬃100-mg
sections and allowed to equilibrate at 37°C in 5% CO2 for 2
days in serum-free Dulbecco’s modified Eagle’s medium
(DMEM)–F12 media (1:1 ratio) supplemented with 100
units/ml penicillin, 100 ␮g/ml streptomycin, and 250 ng/ml
2914
amphotericin B. Following equilibration, cartilage was incubated in DMEM–F12 (100 mg cartilage/1.5 ml media) containing TNF␣ and OSM at 10 ng/ml each. Supernatants were
collected after 14 days of culture. For time-course experiments,
cartilage from a single donor was divided into separate wells
for independent cultures over 4, 6, 8, and 10 days, without
media change.
Neoepitope antibody generation. Polyclonal neoepitope antibodies (Quality Controlled Biochemicals, Hopkinton, MA) were prepared against the peptide sequences
PFTDVRAA and VYQPQPHP, representing the C-terminus
and N-terminus, respectively, of the fibronectin fragments
that were generated by cleavage at the Ala271/Val272 bond.
Antibodies recognizing the spanning peptide (PFTDVRAAVYQPQPHP) were removed by passing sera through a
spanning peptide agarose column. Antibodies flowing through
the spanning peptide column were then positively selected
based on their ability to bind C-terminal (PFTDVRAA) or
N-terminal (VYQPQPHP) peptides conjugated to agarose.
Antibodies were further characterized using Western blotting.
Purification of fibronectin fragments. After dialyzing
into TBS, fibronectin and fibronectin fragments were isolated
using gelatin chromatography. Gelatin–Sepharose was equilibrated with 5-column volumes of TBS. Extracts were loaded
onto the column and washed with 5-column volumes of TBS,
and gelatin-binding proteins were eluted with TBS containing
8M urea. Fractions were then dialyzed against TBS and
subjected to Western blot analysis. In some cases, fractions
were concentrated from 1 ml to ⬃50 ␮l, using centrifugal
concentrations (molecular weight cutoff of 10 kd). Elution of
fibronectin fragments with 8M urea ensured that ADAMTS-4
and ADAMTS-5 were not copurified with the fibronectin
fragments, since neither of these enzymes refold efficiently
(⬍7%) after denaturation with 8M urea (Tortorella MD, et al:
unpublished observations). Silver staining provided additional
confirmation of the purity of these fibronectin fragments
(results not shown).
Western blotting. Samples (30 ␮l) were subjected to
electrophoresis in denaturing and reducing conditions (NuPage lithium dodecyl sulfate [LDS] 4⫻ sample buffer, 2.5%
␤-mercaptoethanol) using 10% Bis-Tris 10-well gels (Invitrogen). Proteins were then transferred to 0.2-␮m polyvinylidene
difluoride membranes and probed with antibody. For analysis
of aggrecan fragments, samples were enzymatically deglycosylated, prior to electrophoresis, with chondroitinase ABC (0.1
unit/50 ml media), keratanase (0.1 unit/50 ml media), and
keratanase II (0.002 unit/50 ml media) for 3 hours at 37°C in
buffer containing 50 mM sodium acetate, 0.1M Tris HCl, pH
6.5 (18). After digestion, the aggrecan was precipitated with 5
volumes of acetone and reconstituted in 30 ␮l 4⫻ LDS sample
buffer (Invitrogen) containing 2.5% ␤-mercaptoethanol, and
heated for 10 minutes at 95°C. Fibronectin neoepitope antibodies were used at 2 ␮g/ml, and antifibronectin antibodies
(Sigma) were diluted 1:5,000.
Aggrecan neoepitope antibodies, as described by Tortorella et al (18), were diluted 1:1,000. The AGEG neoepitope
measures a preferred aggrecanase cleavage site within aggrecan and is much more reactive than other antibodies to the
other 4 aggrecanase cleavage sites. Thus, this antibody is more
likely to detect early levels of aggrecanase-mediated aggrecan
breakdown. Incubation and detection were performed with the
ZACK ET AL
Western Breeze alkaline phosphatase detection system (Invitrogen) using protocols provided by the manufacturer.
N-terminal sequencing. Proteins for N-terminal sequencing were stained using 0.025% Coomassie R-250 stain in
40% methanol, and destained with 50% methanol to allow
visualization of proteins. Bands were then cut from the membrane and sequenced via the Edman degradation method,
using a Procise-cLC (Applied Biosystems) N-terminal sequencer. Results were analyzed using SequencePro software
(Applied Biosystems).
Bovine nasal cartilage cultures. Cartilage disks (1
mm ⫻ 8 mm) from bovine septa (Covance, Princeton, NJ)
were incubated for 6 days in DMEM containing 5% heatinactivated fetal bovine serum and penicillin, streptomycin,
and amphotericin B (100 units/ml, 100 ␮g/ml, and 0.25 ␮g/ml,
respectively). Disks were cut into 8 slices of ⬃12–15 mg each.
Each piece was weighed, placed into a 96-well plate, and
allowed to equilibrate for 2 days in 180 ␮l serum-free DMEM
(containing antibiotics). After equilibration, media were replaced with 180 ␮l media containing human OA fibronectin
fragments. Treatments were performed in duplicate. Fibronectin fragments were diluted in DMEM and sterile filtered (0.2
␮m) before application to explant cultures.
Detection of sulfated glycosaminoglycan (GAG) by
1,9-dimethylmethylene blue (DMMB assay). Aggrecan degradation was monitored by assessing the release of sulfated GAG
into the media, using the DMMB assay with shark chondroitin
sulfate as a standard. This assay was adapted from the method
described by Farndale et al (19). Results are expressed as
micrograms of GAG per milligram of wet cartilage.
Histology and immunohistochemistry. Human OA
cartilage obtained at the time of knee replacement from a
57-year-old female patient (Washington University Department of Orthopedics, St. Louis, MO) was fixed overnight with
10% neutral buffered formalin and processed for paraffin
embedding. Cartilage was graded using the macroscopic Collins score (20). Sections were stained with Safranin O for
histologic evaluation.
Slides prepared for immunohistochemistry were
deparaffinized, rehydrated through graded ethanols, and then
pretreated with 0.1 units/ml chondroitin ABC lyase (MP
Biomedicals, Aurora, OH). Slides were then washed in TBST
(50 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH
8.0). Endogenous peroxidase was blocked using Peroxidase
Block (DakoCytomation, Carpinteria, CA), and nonspecific
antibody binding was blocked using Protein Block (DakoCytomation). Slides were incubated with primary antibody (antiVYQP at 1 ␮g/ml or anti-VRAA at 1 ␮g/ml). Antibody binding
was detected using the Envison Plus horseradish peroxidase
system (DakoCytomation). Sections were counterstained with
hematoxylin. Negative controls contained no primary antibody
but an equivalent dilution of rabbit serum. As antibodyspecificity controls, the primary antibody was preabsorbed with
its immunizing or blocking peptide at 50⫻ molar excess.
RESULTS
Purification of fibronectin fragments present in
OA cartilage. Fibronectin was purified from OA knee
cartilage extracts using gelatin–Sepharose chromatogra-
FIBRONECTIN NEOEPITOPES IN OA CARTILAGE
2915
Figure 1. Purification of fibronectin fragments from osteoarthritic (OA) cartilage. A, Silver staining of isolated fibronectin fragments from OA
cartilage extracts assessed using gelatin chromatography. A comparison of staining pre– and post–gelatin chromatography demonstrates that the
majority of the protein in the extract did not bind gelatin. Four bands were detected in the eluted fraction, and 3 of the 4 bands were visualized after
transfer to a polyvinylidene difluoride membrane and subsequent Coomassie staining. Asterisks denote bands that were N-terminally sequenced. The
N-terminus of each fragment was 272VYQPQP. The diagram adjacent to the gel predicts the fibronectin modules contained in the fibronectin
fragments, based on molecular weight. B, Structure of intact fibronectin monomer and known cleavage sites. A major site of cleavage within
fibronectin is predicted to be at Ala271/Val272. The known cleavage sites of other mammalian fibronectin-degrading enzymes are also denoted.
Squares represent type I modules (labeled 1–12). Circles represent type II modules (labeled 1 and 2). Ovals represent type III modules (labeled
1–15). Shaded ovals represent type III modules found in splice variants of fibronectin. EDA ⫽ extra domain A; EDB ⫽ extra domain B; CS ⫽
variable connecting segment; MMP-2 ⫽ matrix metalloproteinase 2.
phy. Eluates contained multiple gelatin-binding proteins
with molecular weights ranging from 50 kd to 85 kd, as
revealed by silver staining (representative gel in Figure
1A). After concentration, 2 of these proteins (⬃60 kd
and ⬃85 kd) were identified as fibronectin fragments by
N-terminal sequence analysis. Both of the fragments
contained the amino-terminus 272VYQPQPHP, suggesting a site of cleavage within fibronectin at Ala271/Val272.
This was repeated on knee cartilage extracts from 4
different OA donors, and fibronectin fragments containing this N-terminus were seen at molecular weights
ranging from ⬃50 kd to ⬃85 kd (results not shown). The
N-terminus 272VYQPQPHP is found in the spacer region between the fifth and sixth type I modules within
the intact molecule (Figure 1B). All of the fragments
purified contained an intact gelatin-binding domain near
the N-terminus, thus explaining the affinity for gelatin.
Because of the relatively low abundance of the other
gelatin-binding proteins that were purified from OA
cartilage, N-terminal sequencing and identification of
these could not be achieved.
Characterization of neoepitope antibodies to fibronectin fragments. Neoepitope antibodies were raised
against the new N-terminus 272VYQP and the new
2916
ZACK ET AL
Figure 2. Detection of fibronectin fragments generated by cleavage at Ala271/Val272 with a neoepitope antibody to 272VYQP and to VRAA271.
Cartilage extract (30 ␮l) was separated on a 10% Bis-Tris gel and analyzed for fibronectin fragments containing the N-terminus 272VYQP and
C-terminus VRAA271 by Western blot analysis using the 272VYQP (A) and VRAA271 (B) neoepitope antibody in the absence or presence of 10 ␮M
immunizing peptide (IP).
C-terminus VRAA271 generated following cleavage of
intact fibronectin at Ala271/Val272. Analysis of a human
OA knee cartilage extract from a 64-year-old male
donor using the anti-272VYQP antibody revealed multiple bands ranging in molecular weight from ⬃50 kd to
⬃100 kd (Figure 2A). This antibody did not detect intact
fibronectin, and inhibition of reactivity with the corresponding blocking peptide (VYQPQPHP) confirmed
antibody specificity. The presence of numerous fibronectin fragments having the identical N-terminus,
272
VYQP, suggests that there are multiple cleavage sites
downstream from Val272.
The anti-VRAA271 antibody detected a distinct
30-kd fragment (Figure 2B). Less intense staining was
also found at 250 kd, suggesting that this antibody may
recognize the intact molecule when present at high
concentrations. Inhibition of reactivity with the corresponding blocking peptide confirmed that this antibody
was specific for fibronectin fragments with the free
C-terminal sequence VRAA271 (Figure 2B). The blocking peptide PFTDVRAA was able to block immunoreactivity of the 250-kd band, and since this antibody did
not react with other fibronectin fragments, this may
indicate that the N-terminal VRAA271 30-kd fibronectin
fragment is bound to intact fibronectin, even under
denaturing and reducing conditions.
Stimulation of aggrecan catabolism in bovine
nasal cartilage by isolated OA fibronectin fragments.
Endogenous fibronectin fragments were purified from
human OA knee cartilage by gelatin chromatography, as
described above. Western blot analysis using the
272
VYQP neoepitope antibody confirmed that these
FIBRONECTIN NEOEPITOPES IN OA CARTILAGE
2917
monitor fibronectin degradation, macroscopically normal human knee cartilage explants were incubated with
and without TNF␣ and OSM (10 ng/ml each) for 2
weeks. Media were collected from treated and untreated
cartilage explants and probed for total fibronectin and
for the fibronectin neoepitopes VRAA271 and 272VYQP
by Western blot analysis.
Media samples collected from stimulated ex-
Figure 3. Induction of aggrecanase-mediated glycosaminoglycan
(GAG) release in bovine nasal cartilage by isolated osteoarthritic
(OA) fibronectin (Fn) fragments (Fn-f). Top, Bovine nasal cartilage
explants were treated for 4 days with either isolated OA Fn fragments
(purified by gelatin chromatography) or with intact Fn, and aggrecan
catabolism was monitored with the use of 1,9-dimethylmethylene blue
assay to measure GAG release into the media. Bars show the results in
individual wells. Replicate assays could not be performed due to the
limited amount of fibronectin fragments that could be isolated from
human OA cartilage. Bottom, Detection of the aggrecanase-generated
aggrecan neoepitope 1771AGEG. Detection of the neoepitope was
achieved by analyzing 30 ␮l of media on a 10% Bis-Tris gel followed by
Western blot analysis using the 1771AGEG neoepitope antibody.
fragments were generated through cleavage at the
Ala271/Val272 bond (results not shown). These isolated
OA fibronectin fragments, at a concentration of ⬃1 ␮M,
induced the release of GAG in bovine nasal cartilage
after 4 days of stimulation; this was not seen in cultures
treated with 1 ␮M intact fibronectin (Figure 3). Media
with bovine nasal cartilage were also analyzed for aggrecan fragments generated by aggrecanase-mediated
cleavage of the aggrecan core protein at the Glu1771/
Ala1772 bond, assessed using Western blotting with the
anti-1772AGEG antibody as previously described (18).
The AGEG Western blot showed increased production,
over control levels, of a 120-kd aggrecan fragment
(Figure 3, bottom), suggesting that the observed fibronectin fragment–induced catabolism was mediated
by aggrecanases.
Presence of fibronectin neoepitopes VRAA271 and
272
VYQP in human cartilage stimulated with TNF␣/
OSM. In an effort to determine whether detection of the
neoepitopes VRAA271 and 272VYQP may be used to
Figure 4. Release of fibronectin (Fn) fragments from normal human
knee cartilage in cultures stimulated with tumor necrosis factor ␣
(TNF␣) and oncostatin M (10 ng/ml each) for 14 days. Supernatant (30
␮l) was analyzed for Fn fragments by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis under reducing conditions on a
10% Bis-Tris gel. Western blotting was used to detect A, total Fn by
using a Fn polyclonal antibody, B, Fn fragments containing the
N-terminus 272VYQP using the 272VYQP neoepitope antibody, and C,
Fn fragments containing the C-terminus VRAA271 using the VRAA271
neoepitope antibody.
2918
ZACK ET AL
Presence of fibronectin neoepitopes VRAA271 and
VYQP in human OA cartilage at sites of cartilage
degradation. Localization of the fibronectin neoepitopes
272
VYQP and VRAA271 in cartilage was determined by
immunohistochemistry using neoepitope antibodies.
Macroscopically normal (Collins score 0) human knee
cartilage (Figures 6a–c) with very mild aggrecan depletion at the surface (Figure 6a) was compared with
severely affected (Collins score IV) OA knee cartilage
(Figures 6d–f). In normal cartilage, small amounts of
VRAA271 were detected in the regions of aggrecan
depletion (Figure 6b), whereas no 272VYQP staining was
observed (Figure 6c). In contrast, both neoepitopes were
abundantly present in OA cartilage, with the VRAA271
epitope primarily detected in the ECM, especially in the
fibrillated areas at the cartilage surface (Figure 6e). The
272
VYQP neoepitope was also seen in the cartilage
matrix and at sites of fibrillation (Figure 6f). In addition,
272
VYQP staining was very strong at the surface of
chondrocytes (Figure 6i). Specificity of the neoepitope
antibodies was confirmed by demonstrating that the
respective immunizing peptide blocked staining (Figure
6g for VRAA271 and Figure 6h for 272VYQP).
272
Figure 5. Release of fibronectin fragments containing the C-terminal
neoepitope VRAA271 from human osteoarthritic knee cartilage over
time. Cartilage explants were treated with tumor necrosis factor ␣
(TNF␣) and oncostatin M (10 ng/ml each) for 10 days. Media were
collected every other day on days 4–10. Supernatant (30 ␮l) was run
under reducing conditions on a 10% Bis-Tris gel and analyzed for
fibronectin fragments by Western blotting using the VRAA271 neoepitope antibody.
plants contained more total fibronectin than did control
explants. Moreover, stimulated samples contained a
greater abundance of degraded fibronectin when compared with controls (Figure 4A). An ⬃45-kd OA fibronectin fragment with the N-terminus 272VYQP (Figure 4B) was detected in 3 of 5 treated samples (weak
detection in the first lane), but was not detected in any of
the untreated samples. The anti-VRAA271 antibody
revealed an ⬃30-kd fibronectin fragment in all treated
samples, but not in any of the untreated samples
(Figure 4C).
Time-dependent increase in OA fibronectin fragments with TNF␣/OSM treatment. Our preliminary
experiments showed production of both of the OA
fibronectin neoepitopes, VRAA271 and 272VYQP, after
14 days of stimulation with TNF␣ and OSM. Since this
was a relatively long incubation time, the experiment was
repeated and the media were monitored for neoepitopes
on days 4, 6, 8, and 10. The fibronectin fragments with
the C-terminus VRAA271 were detected by Western
blotting by day 6, and increased in a time-dependent
manner at days 8–10 (Figure 5). Fibronectin fragments
with the N-terminus 272VYQP were not detected at
these time points. The apparent absence of this fragment
could be due to lower sensitivity of the 272VYQP
antibody. However, it could also be due to further
C-terminal degradation of 272VYQP-containing fibronectin fragments, which would dilute the neoepitope
among multiple species. In addition, the 272VYQPcontaining fibronectin fragments have multiple cell binding sites, and may be retained in the cartilage ECM and
not readily released into the media.
DISCUSSION
Cartilage degradation is the hallmark of arthritic
disease. It occurs when steady-state homeostasis of the
main structural cartilage macromolecules, aggrecan and
type II collagen, is altered, disturbing the fine balance
between anabolism and catabolism of the ECM. Accelerated breakdown of cartilage is an early and sustained
feature of OA and can be ascribed to proteolytic enzymes, including the aggrecanases, which mediate aggrecan breakdown, and the collagenases, which mediate
collagen breakdown. Accumulating evidence shows that
fragments of fibronectin function as a major stimulus
responsible for the induction of these proteolytic enzymes (14,21–23). Thus, identifying the fibronectin fragments responsible for stimulating catabolism in cartilage
could lead to a better understanding of arthritic diseases.
In this study we have shown that several fibronectin fragments found in cartilage contain the N-terminus
271
VYQPQPHP, suggesting a major cleavage site within
fibronectin at Ala271/Val272. The Ala271/Val272 bond is
located in the middle of a spacer region that separates
the fifth type I module from the sixth type I module. The
sixth type I module demarks the beginning of the
gelatin-binding domain (Figure 2). Thus, as expected,
these fragments were retained on a gelatin–Sepharose
column (Figure 1). This region within fibronectin con-
FIBRONECTIN NEOEPITOPES IN OA CARTILAGE
2919
Figure 6. Immunohistochemical analyses of fibronectin neoepitopes. Serial sections of macroscopically normal (Collins
score 0) (top) and severely osteoarthritic (Collins score IV) (middle) cartilage were prepared and stained for loss of
aggrecan with Safranin O (SAF O) (a and d), probed for the fibronectin fragment neoepitope VRAA271 in the absence
(b and e) or presence (g) of blocking peptide (immunizing peptide [IP]), or probed for the fibronectin fragment
neoepitope 272VYQP in the absence (c and f) or presence (h) of blocking peptide (IP). A higher magnification view of
f is shown in i (original magnification ⫻ 10), highlighting the cell-associated staining of this epitope.
tains a stretch of hydrophobic aliphatic amino acids that
confer linearity to this spacer domain. In addition, this
region of the molecule is particularly susceptible to
proteolytic attack, since it has been reported that multiple enzymes can hydrolyze fibronectin in the region
between amino acids Ser254 and His286. Proteinases that
have been shown to cleave fibronectin within this region
include bacterial subtilisin, meprins, plasmin, thrombin,
and trypsin (24–27).
Fibronectin fragments generated by cleavage at
Ala271/Val272 have been identified by N-terminal sequencing and by Western blot analysis using neoepitope
antibodies that detect the new C-terminus VRAA271 and
the new N-terminus 272VYQP. The 30-kd fibronectin
fragments containing the C-terminus VRAA271 were
present in multiple OA donors, as determined by Western blotting and immunohistochemistry. This particular
fragment may be the most abundant fragment found in
OA cartilage, because it is relatively small in size and has
a compact modular structure that may be less prone to
further proteolytic degradation. Ingham et al (28) have
shown that similar fragments generated by neutrophil
elastase digestion are thermally stable. Interestingly, this
fragment is very similar to the N-terminal heparinbinding fragment investigated by several research groups
and shown to be a potent inducer of cartilage catabolism
(29). The N-terminal fragment that was described by
Homandberg and Erickson is generated from thrombin
digestions to produce a 29–30-kd fragment with the
C-terminus FTDVR269 (26). However, fibronectin fragments with this neoepitope or the corresponding
N-terminal neoepitope 270AAVY have not been investigated in human cartilage to date, and we did not identify
this neoepitope at any time in the studies presented
2920
herein. The OA fibronectin fragment that we identified
is ⬃30 kd with a C-terminal sequence of DVRAA271.
Thus, nonphysiologic fragments commonly used to elicit
catabolic responses in human cartilage differ by only 2
amino acids from the physiologic fibronectin fragment
that we identified.
The gelatin-binding fibronectin fragments that
were isolated from human OA cartilage explants, which
included fragments containing the 272VYQP neoepitope, stimulated cartilage catabolism at physiologically relevant concentrations in bovine nasal explant
cultures after 4 days of stimulation. Aggrecan breakdown, as measured by GAG release, was mediated by
the aggrecanases, as indicated by the release of the
1771
AGEG aggrecan neoepitope. Thus, it is hypothesized that fibronectin fragments produced by cleavage
of fibronectin at Ala271/Val272 are able to induce the
production and/or activation of the aggrecanases
ADAMTS-4 and ADAMTS-5. The role of fibronectin
fragments in inducing aggrecanase-mediated aggrecan
catabolism is supported by findings from Stanton et al
(13), who demonstrated that a 45-kd fibronectin fragment stimulated the production of the neoepitope
ITEGE373, which is generated when aggrecanase cleaves
aggrecan at Glu373/Ala374. However, this particular fibronectin fragment was generated by cathepsin D and
trypsin digestion, and was therefore different from those
that we have identified in OA cartilage. Nevertheless,
the ability of the 45-kd fibronectin fragment used by
Stanton and colleagues to induce cartilage catabolism
suggests a common feature shared by fibronectin fragments generated by in vitro digestion and those isolated
from human OA cartilage.
We have also shown that the OA fibronectin
neoepitopes were generated in healthy human cartilage
explants after stimulation with a combination of the
catabolic cytokines TNF␣ and OSM. These cytokines
are frequently used to induce cartilage degradation in
bovine and human explant cultures. In these systems,
TNF␣ in combination with OSM has been shown to
induce MMP-1, MMP-3, and MMP-13, which degrade
multiple ECM components (30). OSM alone has been
shown to increase fibronectin production in human
breast cancer cell lines (31), consistent with the possibility that these cytokines may promote cartilage degradation through a fibronectin fragment–mediated pathway.
Fibronectin fragments containing the C-terminus
VRAA271 were released and detected in explant media
by day 6. However, proteolysis of fibronectin may occur
before this time, and the delay in detection may simply
be a function of diffusion of fibronectin fragments from
ZACK ET AL
the cartilage. In fact, we have previously demonstrated
that the majority of fibronectin fragments in nonstimulated OA cartilage are retained in the matrix even after
14 days of culture (Tortorella MD, et al: unpublished
observations). Thus, locally generated fibronectin fragments may stimulate cartilage catabolism for a prolonged period of time.
The apparent lack of the 272VYQP neoepitope in
the media of cytokine-treated cultures may have been
due to 1) the decreased ability of the fragment to diffuse
out of the cartilage because of increased cell binding
mediated by an intact central cell binding domain,
and/or 2) increased susceptibility of the 272VYQPcontaining fibronectin fragments to further proteolytic
cleavage, resulting in a lack of detection of multiple
species by Western blot analysis.
The consistent detection of fragments containing
the C-terminus VRAA271 in cytokine-stimulated cartilage cultures may have been due to the stability of this
particular fibronectin fragment. The 30-kd fragment is
smaller in size and is composed entirely of type I
modules. Because type I modules each contain 2 disulfide bonds, this N-terminal fragment is predicted to be
more stable than larger fragments containing the
272
VYQP N-terminus, which contain (at most) 7 type I
modules, 2 type II modules (2 pairs of disulfide bonds),
and 15 type III modules (no disulfide bonds). Therefore,
the 30-kd fibronectin fragment is predicted to be more
stable and less susceptible to further proteolytic cleavage, and thus to be more abundant in cartilage.
In these studies, fibronectin fragments with an
affinity for gelatin were purified from OA cartilage, but
we cannot exclude the possibility that other fibronectin
fragments exist in OA cartilage that are important in the
initiation and progression of disease. Future studies will
focus on the isolation of fibronectin fragments that do
not bind gelatin. However, at this point, antibodies that
detect the fibronectin neoepitopes VRAA271 and
272
VYQP may be valuable biomarkers of cartilage degradation in OA and RA. Immunohistochemical analysis
showed that both neoepitopes localize in areas of aggrecan depletion and fibrillation. Since fibronectin fragments induce catabolic factors that lead to the destruction of cartilage, detection of the VRAA271 and
272
VYQP neoepitopes may be useful for identifying the
initiation of arthritis prior to the onset of significant
cartilage destruction, and may be used to monitor more
established disease.
Our data suggest that fibronectin fragments containing the N-terminal neoepitope 272VYQP may be
more readily retained in the cartilage matrix when
FIBRONECTIN NEOEPITOPES IN OA CARTILAGE
compared with fibronectin fragments containing the
C-terminal VRAA271 neoepitope, which is more likely to
diffuse into the synovial fluid and bloodstream. Future
studies will determine if these neoepitopes are found in
the synovial fluid, blood, and urine of patients with OA
and those with RA. Moreover, in future studies, we
should be able to characterize the stability of the neoepitopes in these types of fluids.
The results presented herein raise the question as
to which enzyme is responsible for generating the fibronectin fragments found in OA. Bacterial subtilisin is
the only enzyme known to cleave fibronectin at Ala271/
Val272, bringing up the possibility that the human proteinases responsible for this cleavage are structurally
related. Thermolysin, plasmin, and thrombin have all
been shown to cleave fibronectin within the spacer
domain located between the fifth and sixth type I
fibronectin modules (24,26). N-terminal sequence analysis of thrombin digests, which are commonly used to
study the effects of fibronectin fragments, revealed a
major cleavage site at Arg269/Ala270 within fibronectin
(26). Thus, thrombin is not likely to be a contributor to
the pool of fibronectin fragments in OA.
Urokinase has also been shown to cleave at the
same site as thrombin, as well as at Arg2309/Thr2310 (27).
Recently, it was demonstrated that meprin-␣ preferentially cleaves at Tyr273/Gln274 and Asn689/Thr690, while
meprin-␤ cleaves at Leu701/Val702 and Arg883/Ser884
(13). MMP-2 has been shown to cleave fibronectin near
the C-terminus at Gly1786/Val1787 (24), and Wilhelm et al
(32) have shown that MMP-3 can degrade fibronectin in
both neutral and acidic conditions. However, the major
MMP-3 site of cleavage was identified as Pro700/Leu701,
which generates fibronectin fragments with molecular
weights of 160–180 kd (26). Many other mammalian
enzymes have also been shown to cleave fibronectin, but
thus far none have been shown to cleave at Ala271/
Val272. The identification of the human proteinase responsible for cleavage of fibronectin at Ala271/Val272,
currently referred to as endogenous fibronectinase, will
be critical for understanding its role in fibronectin degradation and in the pathogenesis and progression of OA.
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