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J Biol Chem, Vol. 274, Issue 43, 30756-30763, October 22, 1999
From the Institute for Drug Discovery Research, Yamanouchi
Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba,
Ibaraki 305-8585, Japan
The extra domain-A (EDA), present in fibronectin
(FN) molecules arising from alternatively spliced transcripts, appears
only during specific biological and pathogenic processes. However, its
function is poorly understood. To define the physiologic role of this
domain in joint connective tissue, the biological effects on rabbit
cartilage explants, chondrocytes, and synovial cells were studied. A
recombinant EDA protein (rEDA) increased proteoglycan release
(3.6-fold) in cartilage explant cultures and markedly induced
production of matrix metalloproteinase (MMP)-1 in chondrocytes. In
addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These
effects were elicited only by rEDA, while its neighboring type III
repeats, III11 or III12, scarcely had any
such effects. Interestingly, reorganization of F-actin stress fibers
accompanied MMP-1 expression in synovial cells treated with rEDA,
suggesting alteration of cellular phenotype. Subsequent Northern
blotting revealed expression of pro-inflammatory cytokines, including
interleukin (IL)-1 Fibronectin (FN)1 is a
multifunctional glycoprotein abundant in plasma and widely distributed
in the extracellular matrix (1). It is a dimer of subunits cross-linked
by disulfide bonds. Each FN monomer is comprised of three types of
repeating units designated type I, II, and III (2). Some of these
repeats bind to cell surface and extracellular matrix components such
as integrins, collagens, heparin, and fibrin. Several of these binding
activities have been assigned to the motif sequences in FN, including
the Arg-Gly-Asp (RGD) motif in the type III10 domain (3),
the Pro-His-Ser-Arg-Asn (PHSRN) motif in the type III9
domain (4), and the CS-1 sequence in the III-CS region (5).
Consequently, this multifunctional glycoprotein mediates a variety of
cellular functions including cell adhesion, cell migration, and cell differentiation.
FN molecules have multiple isoforms generated from a single gene by
alternative splicing of combinations of 3 exons: extra domain-A (EDA),
extra domain-B (EDB), and III-CS. Both EDA and EDB exons are type III
repeating units (6). Plasma fibronectin (pFN), produced by hepatocytes
and abundant in plasma, lacks both the EDA and EDB domains. However,
cellular FNs (cFNs), many of which are insoluble and incorporated into
the pericellular matrix, contain the EDA and EDB segments in various
combinations. The EDA domain is present in FN molecules produced during
embryonic development (7). However, its presence in adults is minimal except in some disease states such as rheumatoid arthritis (8), wound
healing (9), epithelial fibrosis (10), and vascular intimal
proliferation (11). Thus, the highly regulated splicing of the EDA
domain into transcripts suggests significantly different FN functions.
It was reported that pFN and cFN differ in structural and physical
properties such as glycosylation (12) and solubility (13) as well as
presence of EDA domain. However, the biological functions the EDA
domain confers upon these molecules are still poorly understood
(7).
In this study, the biological functions of the FN EDA domain were
studied in synovial cells and chondrocytes, which are integral components of joint connective tissue metabolic regulation. The results
show induction of MMPs and pro-inflammatory cytokines occurs in
response to exposure to the EDA domain. This observation, that a
distinct domain of an extracellular matrix protein can trigger gene
expression, has profound implications on biological and pathologic processes.
Preparation of Recombinant FN Type III Repeat
Proteins--
cDNAs encoding individual human fibronectin type III
repeats were amplified by reverse transcription-polymerase chain
reaction (RT-PCR) of human liver mRNA
(CLONTECH, Palo Alto, CA), using Takara RNA PCR kit
(AMV) version 2.1 (Takara, Kyoto, Japan) according to the
manufacturer's instructions. The PCR profile consisted of 40 cycles of
94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.
After the final cycle, the reaction was maintained at 72 °C for an
additional 7 min. The primers for EDA (from Asn1600 through
Thr1689) are the sense primer EDA-s,
5'-CCATATGAACATTGATCGCCCTAAAGGACT-3' and the antisense
primer EDA-a,
5'-AGCGGCCGCTGTGGACTGGGTTCCAATCAGGGG-3'. The primers
for III11 (from Glu1510 through
Thr1599) are the sense primer III11-s,
5'-CCATATGGAAATTGACAAACCATCCCA-3' and the antisense primer
III11-a, 5'-AGCGGCCGCGGTTACTGCAGTCTGAACCA-3'. The primers for III12 (from Ala1690 through
Thr1779) are the sense primer III12-s, 5'-
CCATATGGCTATTCCTGCACCAACTGA-3' and the antisense primer
III12-a, 5'- AGCGGCCGCAGTGGTGACAACACCCTGAG-3'. The NdeI site on the sense primers and the
NotI site on the antisense primers are underlined. Amino
acids are numbered from the amino-terminal pyroglutamate of the mature
protein (GenBank accession number X02761). cDNAs encoding FN
fragment proteins with several permutations of type III repeats
consisting of III11, EDA, and III12, as
depicted in Fig. 9A, were also amplified by RT-PCR as
described above. PCR was performed using primer sets,
III11-s and EDA-a for the III11-EDA protein,
and EDA-s and III12-a for the EDA-III12
protein. cDNAs encoding the III11-EDA-III12
protein and the III11-III12 protein were
amplified using the common primer set, III11-s and III12-a, and they were then separated on agarose
electrophoresis according to their molecular sizes. PCR-amplified
cDNA was subcloned into the pCRII plasmid (Invitrogen,
Carlsbad, CA), digested with NdeI and NotI, then
subcloned into the bacterial expression vector pET-22b (Novagen,
Madison, WI), which enables expression of fusion proteins carrying six
additional histidine residues (6×His tags) at the carboxyl terminus.
Individual FN type III protein genes with 6×His tags were expressed in
Escherichia coli BL21(DE3) and purified by TALON Metal
Affinity Resin column (CLONTECH). Endotoxin levels
in the highest rEDA concentration used were under 0.2 ng/ml, which are
insufficient to affect MMP biosynthesis in the culture systems
subsequently described.
Proteoglycan Degradation Assay--
Rabbit articular cartilage
was obtained as described previously (14). The cartilage was cut into
slices, whose weight was adjusted to approximately 5 mg and placed in
the wells of 24-well plates. Explants were cultured in 0.5 ml of
Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine
serum (FBS) for 48 h, with or without exogenous recombinant FN
type III proteins. Glycosaminoglycan in the culture supernatants and
papain-digested cartilage were determined by the dimethylmethylene blue
assay (15). Results are expressed as the percentage of
glycosaminoglycan in the culture supernatant to the sum of
glycosaminoglycan contents in the culture supernatants and cartilage
digested with papain.
Cell Culture--
Synovial cells and chondrocytes were prepared
from Japanese white rabbits. Synovial cells were isolated from synovial
tissue incubated with 0.4% collagenase A (Roche Molecular
Biochemicals, Indianapolis, IN) for 3 h at 37 °C followed by
incubation with 0.25% trypsin and 1 mM EDTA for 1 h
at 37 °C. Chondrocytes were isolated from cartilage slices incubated
with 0.25% trypsin and 1 mM EDTA for 1 h at 37 °C
followed by incubation with 0.15% collagenase A for 3 h at
37 °C. Cells were cultured to a subconfluent monolayer in DMEM
containing 10% FBS. To assess effects of rEDA, cells were stimulated
with different recombinant FN type III proteins in DMEM containing 5%
FBS. Synovial cells were used for assays after just 1 passage.
Chondrocytes were used without passage.
Measurement of Marix Metalloproteinase-1 (MMP-1) by Enzyme-linked
Immunosorbent-Assay (ELISA)--
MMP-1 in the culture supernatants was
measured using a double-antibody sandwich ELISA system that recognizes
pro and active forms of MMP-1, as described previously (14). Mouse
polyclonal antibodies generated against rabbit pro-MMP-1 were used as
both the primary trapping antibody and the secondary biotinylated
antibody. Briefly, plates were coated with the first antibody and
blocked with 1% bovine serum albumin. The plates were then incubated
with the culture supernatants of rabbit synovial cells, followed by incubation with the secondary biotinylated antibody. The plates were
developed using peroxidase-conjugated streptavidin (Amersham Pharmacia
Biotech) at a dilution rate of 1:2000, followed by addition of
tetramethylbenzidine. Absorbance at 450 nm was determined using a plate reader.
Western Blot Analysis of MMP-1 and MMP-3--
The culture
supernatants of rabbit synovial cells were heat-denatured with
2-mercaptoethanol and subjected to SDS-PAGE. Proteins were transferred
onto a polyvinylidene difluoride membrane and blocked with Block Ace
(Snow Brand, Sapporo, Japan). The membrane was then incubated with 1 µg/ml mouse anti-rabbit pro-MMP-1 polyclonal antibodies or the
mouse monoclonal antibody MP1807, which was generated against rabbit
MMP-3 as described previously (16). Both antibodies recognize pro and
active forms. After washing, the membrane was incubated with
peroxidase-conjugated rabbit anti-mouse IgG antibodies
(Zymed Laboratories Inc., San Francisco, CA) at a
dilution rate of 1:2000, followed by staining using an enhanced chemiluminescent detection kit (NEN Life Science Products Inc., Boston,
MA) according to the manufacturer's instructions.
Gelatin Zymography--
The culture supernatants of rabbit
synovial cells were applied without reduction to a 10% polyacrylamide
slab gel impregnated with 1 mg/ml gelatin. Gel electrophoresis was
performed at 4 °C. After electrophoresis, the gel was incubated in
2.5% (v/v) Triton X-100 for 1 h and then for 18 h at
37 °C in 50 mM Tris-HCl, pH 7.5, containing 200 mM NaCl, 10 mM CaCl2, 10 µM ZnCl2, and 0.02% Brij-35. The gel was
then stained with Coomassie Brilliant Blue.
Northern Blotting--
Northern blotting was performed as
described previously (17). Total RNA was isolated from rabbit synovial
cells using an ISOGEN kit (WAKO, Osaka, Japan), according to the
manufacturer's instructions. Total RNA (20 µg) was separated on
agarose gels, transferred to Hybond-N nylon membrane(Amersham Pharmacia
Biotech) and hybridized to 32P-labeled cDNA probes.
Probes for rabbit MMP-1, rabbit MMP-3, rabbit MMP-2, rabbit MMP-9, and
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were prepared using a
BcaBEST Labeling Kit (Takara, Kyoto, Japan), as described previously
(18). The probe for rabbit vascular endothelial cell growth factor
(VEGF) was prepared from cDNA fragments obtained according to the
method of Maniscalco et al. (19). Other probes were
generated from cDNA fragments corresponding to nucleotide 6-496
for rabbit interleukin-1 Cytohistochemistry--
Rabbit synovial cells were cultured
using a Lab-Tek Chamber Slide system (Nunc, Naperville, IL) in DMEM
containing 10% FBS. Subconfluent synovial cells were treated with
different recombinant FN type III proteins for 48 h. Slides were
then washed with phosphate-buffered saline, fixed with 3.7%
paraformaldehyde for 20 min, permeabilized with ice-cold acetone, and
stained with fluorescein-phalloidin (Molecular Probes, Eugene, OR) and
propidium iodide (WAKO, Osaka, Japan). The coverslips were then mounted
using a SlowFade-Light Antifade kit (Molecular Probes) and examined
with confocal laser scanning microscopy (Olympus, Tokyo, Japan).
Preparation and Characterization of FN Proteins and an FN Protein
Fragment--
pFN was purified from human plasma as described
previously (17). cFN, obtained from Upstate Biotechnology (Lake Placid, NY), was treated with the Kurimover II (Kurita, Tokyo, Japan) column
according to the manufacturer's instructions to remove endotoxin. The
EDA-positive FN protein fragment was prepared from human placentas.
Placental tissue was homogenized and extracted in 4 M urea
in 20 mM Tris-HCl (pH 7.5) containing 1 mM
phenylmethylsulfonyl fluoride according to the method of Laine et
al. (20). The extract was diluted 20-fold with 20 mM
Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride and
applied to a Gelatin-Sepharose CL-4B column. The gelatin-column was
washed with 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl (TBS), 1 mM phenylmethylsulfonyl fluoride, then eluted
with 1.5 M KBr-TBS, followed by 8 M urea-TBS.
The EDA-positive FN fragment was found in fractions eluted with 8 M urea by Western blotting using rabbit anti-EDA polyclonal
antibodies, which were raised against rEDA. These fractions were
dialyzed against 10 mM phosphate buffer (pH 7.0) and
further purified by hydroxyapatite chromatography (CHT-10, BioLogic
system, Bio-Rad) with a 10-500 mM linear gradient of
phosphate buffer (pH 7.0).
The binding activities of anti-EDA monoclonal antibodies (mAbs) against
different epitopes of EDA-positive FN fragment were determined by
ELISA, as follows. Plates were coated with a single type of
EDA-positive FN fragment, blocked with 1% bovine serum albumin, then
were incubated with the anti-FN mAb being tested, followed by
horseradish peroxidase-conjugated rabbit anti-mouse IgG
(Zymed Laboratories Inc., San Francisco, CA). Anti-FN
mAbs, FN9-1, FNC4-4, FN12-8, FNH3-8, and FN8-12, which were obtained from Takara (Kyoto, Japan), specifically recognize the amino-terminal heparin/fibrin-binding domain, the gelatin-binding domain, the cell-binding domain, the second heparin-binding domain, and the COOH-terminal fibrin-binding domain, respectively.
Cartilage Catabolism Is Enhanced by Recombinant EDA
Protein--
The recombinant human FN type III repeat proteins,
illustrated in Fig. 1A, were
expressed in E. coli as 6×His fusion proteins. Each protein
was then purified on a TALON Metal Affinity Resin column and gave a
single band on SDS-PAGE (Fig. 1B). The purified recombinant
protein encoding the EDA domain (rEDA) was added to rabbit cartilage
explant cultures to determine whether the EDA domain affects cartilage
catabolism. rEDA increased proteoglycan release 3.6 times more than the
unstimulated control level (Fig. 2).
However, its adjacent type III repeat, III11, did not
significantly increase proteoglycan release. This result suggests that
rEDA facilitates cartilage catabolism, possibly by inducing or
modulating protease expression.
rEDA Induces Expression of MMPs--
Rabbit chondrocyte and
synovial cell monolayers were treated for 48 h with rEDA and other
recombinant type III repeats. MMP-1 levels in the culture supernatants
were then assessed by ELISA to determine whether rEDA affects protease
levels or not. In chondrocyte cultures, Fig.
3A shows MMP-1 levels markedly
increased to more than 1.5 µg/ml after treatment with rEDA
concentrations ranging from 30 to 300 nM. In contrast,
MMP-1 levels were negligible in unstimulated control cultures (Fig.
3A). This induction was specific for rEDA, since treatment
with either of its adjacent type III repeats III11 or
III12 did not increase MMP-1 levels. Similar increased
MMP-1 levels due to rEDA treatment were observed in synovial cell
cultures (Fig. 3B). In addition to ELISA experiments, Western blotting analysis detected pro-MMP-1 (55 and 57 kDa) and pro-MMP-3 (53 kDa) in the culture supernatants of synovial cells treated with rEDA, but not in cell culture supernatants of unstimulated cultures or cultures treated with III11 or
III12 (Fig. 4, A
and B). These data are consistent with previous reports
that MMP-1 and MMP-3 pro-enzymes were detected in stimulated
synovial cell cultures (21) and chondrocyte cultures (22). In addition, gelatin zymography showed that gelatinase activity corresponding to
pro-MMP-9 increased slightly in synovial cell cultures after treatment
with rEDA, but was negligible in unstimulated control cultures (Fig.
4C). Another gelatinase activity corresponding to pro-MMP-2,
which is constitutively expressed (22), was not affected by rEDA
treatment (Fig. 4C). Thus, it is unlikely that the increased production
of MMP-1, -3, and -9 by rEDA is due to a general induction of protein
synthesis. Northern blotting analysis of MMP transcripts in rabbit
synovial cells was performed to determine whether rEDA induces
transcription of MMPs. As shown in Fig.
5, rEDA treatment increased MMP-1
mRNA level. However, neither III11 nor
III12 protein treatment significantly increased MMP-1
mRNA level. These observations are in good agreement with the
protein results. rEDA also induced transcription of MMP-3
and MMP-9 mRNA. Any change was scarcely observed in the
level of MMP-2 mRNA, but its doublet signals are
consistent with a previous report (18). Thus, results from protein and
mRNA experiments show that FN EDA domain treatment promotes
expression of MMPs at the transcription level.
rEDA Induces Cytoskeletal Reorganization--
Because rabbit
synovial cell MMP genes responded differently to treatment
with rEDA or other recombinant FN type III repeats, cell morphology was
examined to determine whether rEDA causes any changes to cell shape or
cytoskeletal structure. F-actin microfilaments in synovial cell
monolayers were stained with fluorescein-labeled phalloidin after
treatment with rEDA for 48 h (Fig.
6). In untreated cultures, cell
morphology was fibrous, with F-actin stress fibers aligned parallel to
the long axis of the cell (Fig. 6, A and D). This
is consistent with the fibroblast-like shape of synovial cell
monolayers previously reported (23). In contrast, cells treated with
rEDA showed a marked change to a more rounded morphology, with partial
disassembly of F-actin stress fibers (Fig. 6, B and E). No morphologic change or change in F-actin fibers was
observed in cells treated with III11 protein (Fig. 5,
C and F). Thus, rEDA appears to cause
reorganization of F-actin stress fibers, leading to morphologic change
of synovial cells.
rEDA Induces Pro-inflammatory Cytokine Gene Expression--
Since
morphologic change in synovial cells was induced by rEDA, an
examination of other genes possibly induced by rEDA was undertaken.
Cytokine gene expression was analyzed by Northern blotting of mRNA
from synovial cells treated with rEDA. IL-1 IL-1 Mediates rEDA-induced MMP-1 Expression--
A
Northern blot time course shows MMP-1 gene
expression is first detected 8 h after stimulation with rEDA (Fig.
7B), which is late compared with expression of
IL-1 Structures Surrounding the EDA Domain Required for MMP-1 Inducing
Activity--
To examine the effects of surrounding structures on
MMP-1 inducing activity of EDA, recombinant FN fragment proteins with several permutations of type III repeats consisting of
III11, EDA, and III12, were generated as
depicted in Fig. 9A. Neither III11-EDA-III12 nor
III11-III12 induced MMP-1 production. In
contrast, III11-EDA and EDA-III12 could induce
MMP-1 production although they were less potent than rEDA (Fig.
9B). These results may suggest that exposure of either the
NH2 or COOH terminus of EDA domain releases the inducing
activity. In agreement with these results, cFN did not promote MMP-1
expression, even at concentrations up to 300 nM (Fig.
9B). pFN, which does not have an EDA domain, did not induce
MMP-1 expression from the basal level either (Fig. 9B), which is consistent with the report of Arner et
al. (22).
EDA Domain Activities in FN Fragments Purified from Placental
Tissue--
Purification of FN fragments from human placenta was
performed to explore the existence of FN fragments containing the EDA domain in vivo. One candidate FN fragment was positive in
Western blotting using anti-rEDA antibodies and migrated to 160 kDa in SDS-PAGE analysis under nonreducing conditions (Fig.
10B). The smaller apparent
molecular weight than intact FN suggests the absence of several
domains. In order to determine which domain was included in this
fragment, ELISA was performed using several anti-FN mAbs that recognize
the particular FN regions as shown in Fig. 10A. The results
show the 160-kDa EDA-positive FN fragment was recognized by FN9-1,
FNC4-4, and FN12-8, but not by FNH3-8 and FN8-12 (Fig. 10D),
whereas pFN was recognized by all of these mAbs (Fig. 10C).
In addition, protein sequencing found the NH2-terminal sequence of this fragment was blocked, just like an intact FN molecule.
Thus, the epitope mapping suggests this fragment contains the
NH2 terminus through the EDA domain but not the following III12 domain or COOH terminus, which is in agreement with
the molecular weight indicated by SDS-PAGE. This fragment induced MMP-1
production in synovial cells (Fig. 10E). These results may suggest the existence of a potent EDA-positive FN fragment in vivo.
The present study demonstrated that treatment with rEDA protein
facilitates cartilage catabolism and markedly induces expression of MMP
in chondrocytes and synovial cells. The induction of MMP-1 was specific
for rEDA among adjacent FN type III proteins tested. The physical
properties of the cartilage matrix, which distribute loads over bone
surfaces and provide a low-friction surface over which bones can move,
are regulated through the biosynthesis and degradation of extracellular
matrix by chondrocytes and synovial cells (25). In both healthy and
pathologic conditions, the biological functions of synovial cells and
chondrocytes are regulated through interactions with the extracellular
matrix molecules, including FN, collagen, and other glycoproteins, and
various mediators such as cytokines (22, 26). Indeed, cellular
interaction with structural components of the extracellular matrix
influences expression of many proteases, including MMP-1 induction by
type I and III collagens (27), urokinase-type plasminogen activator
induction by laminin (28), and MMP-2 induction by vitronectin (29).
However, this study is the first demonstration that MMP expression of
synovial cells and chondrocytes is regulated by the FN EDA domain.
Induction of MMP, which is implicated in biological processes such as
inflammation (30) and angiogenesis (31), may result in matrix
degradation and remodeling of extracellular matrix structure, since the
MMP family degrades extracellular matrix proteins (32). Thus, the finding that rEDA induces MMPs may indicate a role for the EDA domain
in regulating functions of chondrocytes and synovial cells in joint
connective tissue.
rEDA treatment causes cell rounding with accompanying F-actin stress
fiber reorganization of synovial cells in addition to induction of
MMPs. Recently, it has been reported that anti-integrin The examination of the effects that flanking sequences may have on EDA,
using recombinant FN fragments encoding several permutations of type
III repeats consisting of III11, EDA, and
III12, shows connection with an adjacent type III repeat on
either the NH2 or COOH side of EDA (III11-EDA,
EDA-III12) reduced the potency of EDA to induce MMP-1
production. Moreover, the flanking sequences on both sides of EDA
(III11-EDA-III12) abolished the induction ability, which is in good agreement with the failure to detect the
potency in full-length cFN. It is known that cleavage of extracellular matrix proteins can mediate cellular responses to changes in the microenvironment (37), such as cleavage of laminin-5 that induces cell
migration (38) and cleavage of plasminogen that generates angiostatin
(39). Therefore, these results suggest that the activity of EDA to
induce MMP-1 is masked by adjacent type III repeats and disclosed by
removal of them. The failure to detect MMP-1-inducing potency in cFN is
not consistent with the report that the effect of EDA domain was
detected in cFN on activation of lipocytes (40). However, the recent
study showed that cFN did not affect According to the results of permutated FN fragments, the existence of
potent FN fragments containing EDA was preliminarily explored in
vivo to suggest their biologic relevance. A 160-kDa EDA-positive
FN fragment was found, which induced MMP-1 production. The COOH
terminus of the 160-kDa FN fragment was missing just posterior to the
EDA domain as determined by mAb epitope mapping. Therefore, the
exposure of COOH terminus of EDA domain by proteolytic cleavage would
give the 160-kDa EDA-positive FN fragment this potency. In synovial
fluids of patients with arthritis, EDA-positive FN proteins were
detected as fragments weighing 220, 200, 180, 170, 110, and 100 kDa
(42). Expression of FN containing the EDA domain is increased in
cartilage and synovium of patients with arthritis (8, 42). In addition,
proteases including MMPs increase in synovial fluids of patients with
arthritis (43). Taken together, increase of the EDA domain and
proteases may result in generation of the potent FN fragments
containing EDA domain, leading to further deterioration of
pathogenesis, by induction of proinflammatory cytokines and subsequent MMPs.
In summary, treatment with rEDA protein facilitated cartilage
catabolism and markedly induced expression of MMP in chondrocytes and
synovial cells. In addition, rEDA induced cytoskeletal reorganization and expression of IL-1 in synovial cells, leading to subsequent expression of MMPs in an autocrine manner. This activity of EDA domain
was masked by connection with adjacent type III repeats and revealed by
exposure of either side of the EDA domain. Consistently, MMP-1
production was not induced by full-length cFN, but a 160-kDa EDA-positive fragment did induce MMP-1 production. These findings suggest that the EDA domain in FN fragments triggers alterations of
cell physiology and plays a role in matrix degradation in joint connective tissue.
We thank Steven E. Johnson for editing the
manuscript. We also thank Dr. Jun Takasaki for helpful discussions.
*
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.
The abbreviations used are:
FN, fibronectin;
EDA, extra domain-A;
EDB, extra domain-B;
pFN, plasma fibronectin;
cFN, cellular fibronectin;
MMP, matrix metalloproteinase;
IL-1, interleukin-1;
VEGF, vascular endothelial cell growth factor;
IL-1RA, interleukin-1 receptor antagonist;
FBS, fetal bovine serum;
ELISA, enzyme-linked immunosorbent assay;
RT-PCR, reverse
transcription-polymerase chain reaction;
DMEM, Dulbecco's modified
Eagle's medium;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel
electrophoresis;
G3PDH, glyceraldehyde-3-phosphate dehydrogenase.
The Fibronectin Extra Domain A Activates Matrix Metalloproteinase
Gene Expression by an Interleukin-1-dependent
Mechanism*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IL-1
, was induced by rEDA prior to MMP-1
expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s
promote MMP-1 expression in an autocrine manner. This hypothesis is
supported by the reduction of EDA-induced MMP-1 production by IL-1
receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the
NH2 or COOH side of EDA and was abolished by connection on
both sides of EDA, suggesting that exposure of either the
NH2 or COOH terminus of EDA domain by proteolytic cleavage
releases the inducing activity. In agreement with these results,
full-length cellular FN did not induce MMP-1 production. Furthermore, a
160-kDa EDA-positive FN fragment, which was purified from human
placental tissue and corresponds to the region from NH2
terminus through the EDA, induced MMP-1 production. Taken together,
these results suggest that the EDA in FN fragments triggers alterations
of cell physiology and plays a role in matrix degradation in joint
connective tissue.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IL-1) (GenBank accession number X02852)
and nucleotide 227-778 for rabbit IL-1 (GenBank accession number
M26295).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of recombinant FN type III repeat
proteins. A, the modular structure of FN is shown
schematically. The three alternative spliced domains (EDA, EDB, and
IIICS) are stippled. Recombinant FN type III repeats,
III11, EDA, and III12 were expressed as fusion
proteins with 6×His tags at their carboxyl-terminal ends.
B, SDS-PAGE results showing recombinant FN type III repeat
proteins. Recombinant FN type III repeat proteins, III11,
EDA, and III12 were expressed in E. coli and
proteins were purified by TALON Metal Affinity Resin column
chromatography. Two micrograms of each protein were analyzed by
SDS-PAGE under reducing conditions. Lane 1,
III11; lane 2, rEDA; lane 3,
III12. The positions and sizes (in kDa) of marker proteins
are indicated on the left.
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Fig. 2.
rEDA enhances proteoglycan release from
rabbit cartilage explant culture. Rabbit cartilage explants in
DMEM containing 5% FBS were treated with rEDA or III11 for
48 h. The percentages of proteoglycan release into the media were
determined using the dimethylmethylene blue assay and expressed as the
mean and standard deviation of quadruplicate cultures. *,
p < 0.005 versus unstimulated control
cultures (Student's t test).
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Fig. 3.
Dose-related induction of MMP-1 by rEDA.
Cells were treated with increasing concentrations (10, 30, 100, and 300 nM) of rEDA or other type III repeats for 48 h. The
amounts of MMP-1 in the culture supernatants were measured by ELISA and
expressed as the mean standard deviation of triplicate cultures.
A, rabbit chondrocytes; B, rabbit synovial
cells.
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Fig. 4.
rEDA induces MMP-1, MMP-3, and MMP-9 in
rabbit synovial cells. Western blotting analysis of: A,
MMP-1 and B, MMP-3. Rabbit synovial cells were treated with
300 nM rEDA or other recombinant type III repeats for
48 h. The culture supernatants were analyzed by Western blotting
using mouse anti-rabbit MMP-1 polyclonal antibodies (A) or
the mouse anti-rabbit MMP-3 monoclonal antibody, MP1807 (B).
The positions of standard pro-MMP-1 and pro-MMP-3 are indicated on the
right of each figure. The positions and sizes (in kDa) of
marker proteins are indicated on the left. C, gelatinase
activities released from rabbit synovial cells. Gelatinase activities
in the culture supernatants were analyzed using gelatin zymography. The
positions of standard pro-MMP-2 and pro-MMP-9 are indicated on the
right. Lane 1, control; lane 2, rEDA; lane
3, III11; lane 4, III12 .
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Fig. 5.
rEDA induces MMP mRNA
levels in rabbit synovial cells. Total RNA (20 µg) from rabbit
synovial cells unstimulated (lane 1) or treated with 300 nM rEDA (lane 2), III11 (lane
3), or III12 (lane 4) for 16 h was
blotted and then hybridized to 32P-labeled cDNAs
encoding MMP-1, MMP-3, MMP-9, MMP-2, and G3PDH.
View larger version (72K):
[in a new window]
Fig. 6.
rEDA induces reorganization of F-actin stress
fibers in rabbit synovial cells. Rabbit synovial cells were
untreated (A and D) or treated with 300 nM rEDA (B and E) or
III11 (C and F) for 48 h.
D, E, and F are images of the fields in A,
B, and C magnified three times, respectively. F-actin
and nucleic acid were stained with fluorescein-phalloidin and propidium
iodide, respectively, then examined with confocal laser scanning
microscopy. Scale bars, 20 µm.
and
IL-1 genes were induced by rEDA, but not by its adjacent type III repeats III11 or III12, as measured by
increases in mRNA levels (Fig.
7A). Transcripts of these
cytokines were detected as early as 1 or 3 h after treatment, with
expression peaking at 8 h (Fig. 7B). In contrast, the
VEGF gene, whose product has been reported in the synovium
of rheumatoid arthritis patients with arthritis but not healthy
individuals (24), was expressed in unstimulated synovial cells but was
not induced by rEDA. Thus, rEDA induces IL-1 gene expression
in addition to MMP gene expression.
View larger version (42K):
[in a new window]
Fig. 7.
Cytokine mRNA expression in rabbit
synovial cells treated with rEDA. A, total RNA (20 µg) from rabbit synovial cells unstimulated or treated with 300 nM rEDA, III11 or III12 for 16 h was blotted and then hybridized to 32P-labeled cDNAs
encoding IL-1 , IL-1, VEGF, and G3PDH. B, time course
of rEDA-induced gene expression. Total RNA (20 µg) from rabbit
synovial cells treated with rEDA (300 nM) for 0, 1, 3, 8, or 16 h was blotted and then hybridized to 32P-labeled
cDNAs encoding IL-1, IL-1, VEGF, MMP-1, and G3PDH.
and IL-1. This delayed expression
suggests that MMP-1 expression is a secondary response provoked by
rEDA. In addition, cytokine gene expression prior to MMP-1
gene expression indicates the possibility that rEDA-induced cytokine
production subsequently promotes MMP expression. To test this
hypothesis, the IL-1 receptor antagonist (IL-1RA) (R & D Systems,
Minneapolis, MN) was added to rabbit synovial cell cultures treated
with rEDA to suppress the activity of the induced IL-1 and IL-1.
The addition of 100 ng/ml IL-1RA resulted in inhibition of rEDA-induced
MMP-1 production by 4.3-fold (Fig. 8).
These results suggest that EDA-induced MMP-1 expression is mediated, at
least in part, by IL-1 cytokines.
View larger version (19K):
[in a new window]
Fig. 8.
IL-1RA inhibits rEDA-induced MMP-1
expression. Rabbit synovial cells were cultured with rEDA (300 nM) alone or in combination with increasing concentrations
(3, 10, 30, and 100 ng/ml) of IL-1RA for 48 h. MMP-1 released into
the culture supernatants was measured by ELISA and expressed as the
mean and standard deviation of quadruplicate cultures. *,
p < 0.005 versus rEDA alone (Student's
t test).
View larger version (23K):
[in a new window]
Fig. 9.
Effects of flanking structures on the EDA
domain's ability to induce MMP-1 production. A,
recombinant FN proteins, III11-EDA-III12,
III11-EDA, EDA-III12, rEDA, and
III11-III12 are shown schematically. These
molecules were expressed as fusion proteins with 6×His tags at their
carboxyl-terminal ends. B, rabbit synovial cells were
cultured in media containing one of the recombinant FN proteins, pFN or
cFN for 48 h. The amounts of MMP-1 in the culture supernatants
were measured by ELISA and expressed as the mean and standard deviation
of triplicate cultures.
View larger version (27K):
[in a new window]
Fig. 10.
Characterization of a 160-kDa EDA-positive
FN fragment. A, schematic diagram of FN structure
showing anti-FN monoclonal antibodies epitopes. B, SDS-PAGE
and Western blot analysis of the 160-kDa EDA-positive placental FN
fragment. An EDA-positive FN fragment was purified from human placenta
as described under "Experimental Procedures," then analyzed by
SDS-PAGE under nonreducing conditions along with pFN, followed with
Coomassie Brilliant Blue (CBB) staining (lanes 1 and 2) or Western blotting (lanes 3 and
4) using rabbit anti-rEDA polyclonal antibodies.
C and D, reactivities of anti-FN monoclonal
antibodies with pFN (C) and the 160-kDa EDA-positive FN
fragment (D). Plates coated with either pFN or the 160-kDa
EDA-positive FN fragment were blocked with 1% bovine serum albumin and
then incubated with several monoclonal antibodies against different
epitopes of FN as indicated in A. The plates were then
incubated with horseradish peroxidase-conjugated anti-mouse IgG,
followed with tetramethylbenzidine. NC-IgG is negative control IgG.
E, activity of the 160-kDa EDA-positive FN fragment. Rabbit
synovial cells were cultured with increasing concentrations of the
160-kDa EDA-positive FN fragment, pFN, or rEDA for 48 h. The
amounts of MMP-1 in the culture supernatants were measured by ELISA and
expressed as the mean and standard deviation of triplicate
cultures.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
51 antibody triggers cytoskeletal
reorganization by a small GTP-binding protein
Rac1-dependent mechanism, resulting in induction of MMP-1
(26). Thus, modulation of the extracellular matrix by the EDA domain
might influence cytoskeletal structure, leading to MMP-1 expression.
Furthermore, regulation of cell shape by the extracellular matrix
triggers diverse signaling pathways and expression of many genes
(33-35). As the data indicate, rEDA triggers gene expression of
pro-inflammatory cytokines, including IL-1 and
IL-1. Expression of IL-1 genes increased as
early as 1 or 3 h after rEDA treatment, while MMP-1
gene expression required 8 h. Delay of MMP-1 gene
expression suggests that it is a secondary event in response to rEDA
treatment. IL-1 may subsequently induce MMP-1 expression, supported by
the finding that IL-1 (14) induces MMP-1, -3, and -9, similar to
induction by rEDA. This hypothesis is strongly supported by the partial
reduction of MMP-1 production by IL-1RA. Taken together, these results
indicate that modulation of the extracellular matrix by rEDA and
subsequent cytoskeletal reorganization activate an autocrine loop of
IL-1 (36), leading to expression of MMP genes.
-smooth muscle actin expression
in fibroblasts, whereas rEDA protein modulated expression of -smooth
muscle actin and type I collagen during myofibroblastic phenotype
induction by transforming growth factor-1 (41). Inability of
exogenous cFN to affect -smooth muscle actin expression might be
consistent with the present study with synovial fibroblasts. The effect
of the EDA domain on fibroblasts might be regulated by a slightly different mechanism than lipocyte activation.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Product Planning
Dept., Yamanouchi Pharmaceutical Co., Ltd., 3-17-1, Hasune, Itabashi, Tokyo 174-8612, Japan. Tel.: 81-3-5916-5543; Fax:
81-3-5916-5616.
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