From the Departments of Prosthetic Dentistry,
¶ Orthodontics, and
Biochemistry, Hiroshima University
School of Dentistry, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan and the § Department of Biochemistry, Institute of
Endemic Disease, Norman Bethune University of Medical Sciences,
Changchun 1300-21, China
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ABSTRACT |
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Cartilage matrix protein (CMP; also known as
matrilin-1), one of the major noncollagenous proteins in most
cartilages, binds to aggrecan and type II collagen. We examined the
effect of CMP on the adhesion of chondrocytes and fibroblasts using
CMP-coated dishes. The CMP coating at 10-20 µg/ml enhanced the
adhesion and spreading of rabbit growth plate, resting and articular
chondrocytes, and fibroblasts and human epiphyseal chondrocytes and
MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes
was synergistically increased by native, but not heated, type II
collagen (gelatin). The monoclonal antibody to integrin
Cartilage matrix protein
(CMP1/matrilin-1) was
originally isolated as a protein that binds to aggrecan and thereafter
was shown to bind to type II collagen (1-3). CMP is synthesized in a
cartilage-specific manner, except that eye tissues, notochord, and
tendon express CMP at low levels (4-7). The role of CMP is unknown,
but it may have a structural role, modulating physical properties of
cartilage, or may be involved in matrix-cell interactions.
CMP exists in vivo as a homotrimer of 148 kDa, as measured
by sedimentation equilibrium centrifugation, although estimates of the
molecular mass of this protein by SDS-polyacrylamide gel electrophoresis (PAGE) yield a higher value. CMP migrates at positions corresponding to 215 and 60 kDa under nonreducing and reducing conditions, respectively, during SDS-PAGE (2, 8). The subunits of CMP
are connected in the C-terminal region by disulfide bonding and the
presence of the coiled-coil Numerous adhesion proteins are recognized by integrins, which are
heterodimeric proteins with two ( In this study, we examined the effect of coating culture dishes with
CMP on cell adhesion and investigated whether CMP interacts with
integrins using 125I-CMP and various anti-integrin
antibodies. In addition, we examined whether collagen modulates the
effect of CMP on cell adhesion. The results show that CMP markedly
enhances the adhesion and spreading of chondrocytes, particularly in
the presence of type II collagen, and that integrin
Antibodies--
Mouse neutralizing monoclonal antibodies (mAbs)
to human integrins CMP and Anti-CMP Antiserum--
CMP was purified from a collagen
fiber-rich fraction of bovine cartilage as described previously (8).
SDS-PAGE analysis showed that purified CMP migrated as a single band at
a position corresponding to 215 and 60 kDa under nonreducing and
reducing conditions, respectively (Fig.
1), as expected from previous studies (2).
Phosphate-buffered saline (PBS; calcium- and magnesium-free) containing
pure CMP (14 µg; 0.08 ml/mouse) was mixed with Ribi adjuvant (0.14 ml/mouse; Ribi ImmunoChemical Research, Hamilton, MT) and injected
subcutaneously into two female BALB/cAnnNCrj mice once every 2 weeks.
The serum was obtained 5 weeks after the first injection.
Immunostaining of CMP--
Rib cartilage obtained from
10-day-old pigs was fixed with periodate/lysine/paraformaldehyde for
24 h at 4 °C, washed with water for 12 h, dehydrated, and
then embedded in paraffin wax as described previously (24). Sections (6 µm) were incubated with 250 units/ml hyaluronidase (Sigma) at
37 °C for 30 min and then with 4% skim milk at room temperature for
30 min. After being washed with PBS, the sections were incubated with
nonimmune serum or anti-CMP serum (300-fold dilution) at room
temperature for 30 min, washed with PBS containing 0.05% Tween 20, and
then incubated with fluorescein-conjugated goat IgG fraction to mouse
IgG F(ab')2 at room temperature for 30 min in the absence
of light. After washing with PBS containing 0.05% Tween 20, the
histology was observed under a confocal laser microscope (LSM410, Carl
Zeiss, Inc., Oberkochen, Germany).
Cells--
Chondrocytes were isolated from the femur articular
cartilage at knee joints and the rib growth plate of 4-week-old
Japanese White rabbits or from the epiphyseal cartilage of human
embryos as described previously (25). Human embryonic epiphyseal
cartilage was obtained from the Department of Pathology, Norman Bethune University of Medical Sciences (Changchun, China) (25). Rabbit fibroblasts were isolated from soft connective tissue of the rabbits described above (26). A human embryonic lung fibroblast line (MRC5) was
obtained from RIKEN (Tsukuba, Japan). All cells were seeded at a
density of 5 × 105 cells/10-cm plastic tissue culture
dish and maintained in Coating Dishes with CMP--
CMP, bovine type II collagen
(acid-soluble, pepsin-resistant; Koken, Osaka, Japan), or both at
various concentrations were incubated in 50 µl of PBS containing 10 mM NaHCO3 in 6-mm plastic microwells
(Falcon-3072, Becton Dickinson, or Sumilon MS-8096, Sumitomo Bakelite,
Osaka) at 4 °C for 18 h. The substrata were washed three times
with PBS and then incubated with 50 µl of PBS containing 10 mg/ml
bovine serum albumin (BSA; Sigma) at room temperature for 2 h to
block nonspecific cell attachment.
Cell Adhesion--
When the cultures became 80% confluent, the
chondrocytes or fibroblasts were preincubated with 10 µg/ml
cycloheximide for 2 h and then harvested with PBS containing 0.1%
trypsin and 0.1% EDTA. The cells were seeded at 5 × 103 cells/6-mm plastic tissue culture microwell
(Falcon-3072) coated with CMP and incubated at 37 °C for 15 min to
2 h in 0.1 ml of
In some experiments, cells were seeded at a density of 5 × 104 cells/6-mm plastic non-tissue culture microwell
(Sumilon MS-8096) coated with CMP and then incubated at 37 °C for
1-2 h in 0.1 ml of medium A. Nonadherent cells were removed by gentle
rinsing, and the number of adherent cells in each microwell was then
quantified using an aqueous soluble tetrazolium/formazan assay
(Promega, Madison, WI) (27).
Labeling of CMP with 125I--
CMP was labeled with
125I using chloramine T (ICN Biomedicals, Costa Mesa, CA).
CMP (0.1 mg) was dissolved in 0.25 ml of 0.1 M sodium
phosphate (pH 7.5), mixed with 10 µl of Na125I solution
(100 mCi/ml in 0.1 M sodium phosphate (pH 7.5)), and then
incubated with 2 µl of chloramine-T solution (1 mg/ml in 0.1 M sodium phosphate buffer (pH 7.5)) for 1 min. The reaction was terminated by the addition of 2.5 µl of
Na2S2O5 solution (1 mg/ml in 0.1 M sodium phosphate (pH 7.5)). The reaction mixture was
applied to a Sephadex G-10 column (1 ml) that was equilibrated with 0.1 M sodium phosphate (pH 7.5). SDS-PAGE analysis showed that
this preparation of 125I-CMP migrated at a position
corresponding to 60 kDa even under nonreducing conditions.
Preparation of Lysates from Isolated Single Cells--
When
human fibroblasts (MRC5) were grown to 80% confluence, the cells were
incubated for 5 h with PBS containing 0.5 mM
CaCl2, 2.5 mM N-ethylmaleimide, and
0.8 mg/ml pure bacterial collagenase (type VII, Sigma) at 37 °C. The
cells were washed twice with PBS and then incubated with PBS containing
0.1% EDTA and 0.1% trypsin for 3 min. The dispersed cells were
transferred to small plastic tubes, washed three times with PBS, and
then incubated for 30 min with RIPA buffer (1% Nonidet P-40 and 10 mM Tris-HCl (pH 7.4) containing 0.1% sodium deoxycholate,
0.1% SDS, 0.15 M NaCl, 1 mM MgCl2,
and 1 mM phenylmethanesulfonyl fluoride) at 4 °C.
Insoluble materials were removed by centrifugation at 13,000 × g for 20 min at 4 °C. The supernatant was precleared by
protein G-Sepharose beads (Pharmacia, Uppsala, Sweden) to remove
proteins that nonspecifically bound to the beads. Lysates were analyzed
for protein using a protein assay kit (Bio-Rad).
Precipitation of 125I-CMP in the Presence of Cell
Lysates with Anti-integrin Antibodies--
The MRC5 cell lysate (100 µl, 450 µg/ml protein) was incubated at 4 °C with
125I-CMP (2000 cpm, 150 ng of protein in 1 µl of 0.1 M sodium phosphate (pH 7.5)) for 2 h and then mixed
with rabbit antiserum to integrin ( Surface Labeling with Biotin and Immunoprecipitation--
When
MRC5 fibroblasts were grown to 80% confluence, the cells were
harvested with PBS containing 0.1% EDTA and 0.1% trypsin. Cells in
suspension were incubated with 10 ml of PBS containing 10 µg/ml
NHS-LC-biotin (Pierce) on ice for 90 min and then washed twice with
PBS. The cells were dissolved with RIPA buffer and triturated
intermittently for 30 min. Insoluble materials were removed by
centrifugation at 13,000 × g for 20 min at 4 °C.
The supernatant was precleared by protein G-Sepharose beads. Lysates were analyzed for protein using the Bio-Rad protein assay kit mentioned above.
The biotin-labeled cell lysates (100 µl) were incubated with 1 µl
of rabbit anti-integrin Localization of CMP--
Conventional immunohistochemistry showed
that CMP was present in the territorial and interterritorial matrixes
in fetal bovine rib cartilage (10). The amount of CMP in the
interterritorial matrix was less than that in the territorial matrix
(10). In human arthritic articular cartilage, CMP was present near or
within chondrocytes, but was barely detectable in the interterritorial matrix (8). In this study, we examined CMP in the resting cartilage (Fig. 2A) and the
matrix-forming (prehypertrophic) zone of the growth plate (Fig.
2B) of newborn pigs, using the anti-CMP antiserum and
confocal microscopy. In these cartilages, CMP was concentrated near the
cell surface (Fig. 2, A and B). No stain was
observed with the control serum (Fig. 2C).
Stimulation of Cell Adhesion and Spreading by CMP--
CMP is
present in the pericellular matrix (Fig. 2). In addition, CMP has type
A-like domains that may be involved in cell adhesion (13). We therefore
hypothesized that CMP may be an adhesion factor. To test this
hypothesis, we seeded rabbit articular chondrocytes on plastic tissue
culture dishes coated with various concentrations of CMP and incubated
them at 37°C for 2 h in the presence of
cycloheximide, an inhibitor of protein synthesis. Round and spread
cells were separately counted using a phase-contrast microscope. The
chondrocytes spread rapidly on the CMP-coated dishes, whereas only a
few cells spread on the dishes not coated with CMP (Fig. 3A,
inserts a and b).
The percentage of spread cells to total cells on the CMP (20 µg/ml)-coated dishes was 42% compared with 3% on the control dishes
(Fig. 3A). This stimulation of spreading was induced when
dishes were preincubated with CMP at 2 µg/ml and became almost
maximal at 20 µg/ml (Fig. 3A). In another experiment,
adherent cells were quantified using an aqueous soluble
tetrazolium/formazan assay. CMP enhanced the adhesion of chondrocytes
in a similar dose-dependent fashion (Fig. 3B).
Fig. 4 shows that CMP was effective in
stimulating the spreading of rabbit growth plate chondrocytes
(rGC), rabbit articular chondrocytes (rAC), human
embryonic chondrocytes (hEC), rabbit fibroblasts
(rFB), and human MRC5 fibroblasts (hFB).
Synergism between CMP and Type II Collagen--
Since CMP binds to
type II collagen (2), we examined whether type II collagen modulates
the effect of CMP on the spreading of chondrocytes. A low concentration
of CMP (0.5 µg/ml) enhanced the spreading of chondrocytes in the
presence, but not absence, of type II collagen (3 µg/ml) (Fig.
5A). This synergism between CMP and collagen was observed 15 min after cell seeding and was sustained for at least 60 min (Fig. 5A). The concentration
of CMP required for cell spreading was 20-200-fold higher in the absence of type II collagen than in its presence (Fig. 5B).
At high concentrations (3-10 µg/ml), type II collagen alone
stimulated cell spreading (Fig. 5C). CMP at 0.5 µg/ml
increased this effect of type II collagen by 3-4-fold. The synergism
between CMP and collagen was not observed with denatured type II
collagen (gelatin) that was boiled for 5 min at pH 3.0, although
gelatin alone had a greater effect on cell spreading than type II
collagen alone (Fig. 5D).
Effects of EDTA and Divalent Cations on CMP-mediated Cell
Spreading--
Although CMP plus collagen produced a synergistic
stimulation of cell spreading, our subsequent studies of
CMP-recognizing integrins were carried out without collagen because
collagen alone modulates integrin activity. The presence of collagen
makes the interpretation of the data difficult.
Since integrins require divalent cations to bind ligands, we examined
whether EDTA inhibits cell adhesion to CMP. The addition of EDTA at 2 mM (but not 1 mM) to Inhibition of Cell Adhesion and Spreading on CMP-coated Dishes by
Anti-integrin Antibodies--
Since antibodies to rabbit integrins are
rather difficult to obtain, we used antibodies to human integrins.
Unless otherwise specified, a human fibroblast cell line (MRC5) was
used because it was difficult to obtain human chondrocytes in primary cultures.
The spreading of MRC5 fibroblasts on CMP-coated dishes was suppressed
by the mAb against integrin Binding of CMP to Integrins--
We next examined whether
125I-CMP added to the cell lysates is coprecipitated with
integrin subunits using antibodies to the
In the presence of increasing concentrations of 125I-CMP
and the antiserum to integrin The biological role of CMP is not known, although it is one of the
major noncollagenous proteins in growth plates, tracheal cartilage, and
other cartilages (4, 8, 29). It constitutes up to 5% of the wet weight
of tracheal cartilage (4). CMP may stabilize the cartilage matrix and
alter the tensile strength and elasticity of the matrix by binding to
collagen and aggrecan. Although articular chondrocytes do not usually
synthesize CMP, CMP synthesis is markedly enhanced in arthritic joints
(8), suggesting that CMP may be involved in the destruction and/or remodeling of cartilage.
Our immunohistochemical analyses showed that unlike aggrecan and type
II collagen, CMP is concentrated near the chondrocyte surface in
vivo. This observation, as well as the presence of the type A-like
domain on CMP, suggested that CMP could be involved in the matrix-cell
interaction. We tested this hypothesis and showed for the first time
that CMP is an adhesion protein for chondrocytes and fibroblasts.
The physiological significance of the CMP-induced spreading of
fibroblasts is not known. However, CMP may function as an adhesion factor for fibroblast cells in eye tissues, notochord, and tendon that
contain CMP at low levels (5-7).
Biochemical studies have shown that CMP binds to type II collagen
in vitro (2), and immunostained transmission electron microscopy has shown that CMP binds to the exterior of the collagen fibril in vivo (2). CMP distributes along type II collagen fibers with a periodicity of 59 nm (2). However, the biological significance of the CMP-type II collagen complex is not known. We
showed here that native, but not denatured, type II collagen increases
the effect of CMP on the spreading of chondrocytes. This finding
suggests that CMP attached on type II collagen fibers more efficiently
concentrates CMP receptors/integrins at adhesion plaques than CMP alone
attached uniformly on a plastic or gelatin surface. We also observed
that fibronectin and laminin did not produce a synergistic stimulation
of cell spreading in the presence of type II collagen (data not shown),
although fibronectin and laminin bind to collagen; this distinguishes
the role of CMP in cartilage from those of fibronectin and laminin.
CMP forms two types of filamentous networks in the pericellular matrix:
one that contains type II collagen and another that does not contain
type II collagen (30). The collagen-independent CMP filaments may also
serve as a scaffold for cell adhesion in vivo because CMP at
high concentrations (10-20 µg/ml) enhanced cell spreading in the
absence of type II collagen in vitro.
In tracheal cartilage, CMP binds to aggrecan noncovalently and
covalently, and the covalent cross-linking to the aggrecan core protein
increases with age (3). The CMP-aggrecan complex is unlikely to enhance
the adhesion or spreading of chondrocytes because aggrecan suppresses
cell adhesion in vitro (31).
In this study, we examined whether integrins are involved in the
CMP-induced cell adhesion. In our assays, the CMP-induced cell
spreading required Mg2+ or Mn2+.
Ca2+ had less effect on the cell spreading than
Mg2+ and Mn2+. This is consistent with results
from extensive studies of the effect of divalent cations on integrin
activity (32, 33). The mAb to human integrin Of the various antibodies to integrin subunits examined here, only the
antibody to integrin Integrin Chondroadherin is also an adhesion protein prominently expressed in
cartilage and binds to integrin Whether CMP has a special role in cartilage in the presence of other
adhesion proteins is not known. It is noteworthy, however, that genetic
variation at the CMP gene locus was found to be significantly associated with hip radiographically evident osteoarthritis in 55-65-year-old men, whereas a significant association between hip or
knee radiographically evident osteoarthritis in men or women and the
cartilage link protein gene was not observed (35). Typically, hip
radiographically evident osteoarthritis is most frequently present in
men in the 55-65-year age group. Radiographically evident
osteoarthritis particularly of the hip is often considered to arise due
to anatomic abnormalities. During endochondral bone formation, the
length and shape of the bone are determined. If CMP protein plays a
distinctive role in the matrix-cell interaction during endochondral
bone formation, genetic variation at the CMP gene locus may alter the
shape of the skeleton. In addition, CMP may modulate the matrix-cell
interaction in articular cartilage of osteoarthritic joints (8).
cDNAs encoding CMP-like proteins (matrilin-2 and -3) were recently
cloned (36-38). Matrilin-2 is expressed in a variety of organs, but
not in cartilage (36), whereas matrilin-3 is expressed in a
cartilage-specific manner (37, 38). Matrilin-3 and CMP form
disulfide-linked hetero-oligomers in bovine epiphyseal cartilage (39).
These proteins may also function as adhesion factors since their
modular structure is similar to that of CMP.
In conclusion, CMP was found to be an adhesion factor for fibroblasts
and chondrocytes. CMP is the first protein observed to synergistically
increase the effect of collagen on adhesion and spreading. Integrin
1 or
1 abolished CMP-induced cell
adhesion and spreading, whereas the antibody to integrin
2,
3,
5,
2,
5
1, or
V
5
had little effect on cell adhesion or spreading. The antibody to
integrin
1, but not to other subunits, coprecipitated
125I-CMP that was added to MRC5 cell lysates, indicating
the association of CMP with the integrin
1 subunit.
Unlabeled CMP competed for the binding to integrin
1
with 125I-CMP. These findings suggest that CMP is a potent
adhesion factor for chondrocytes, particularly in the presence of type
II collagen, and that integrin
1
1 is
involved in CMP-mediated cell adhesion and spreading. Since CMP is
expressed almost exclusively in cartilage, this adhesion factor, unlike
fibronectin or laminin, may play a special role in the development and
remodeling of cartilage.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical assembly domain (9, 10). The
monomer has two type A-like (von Willebrand factor-like or I) domains
connected with an epidermal growth factor-like domain in addition to
the short C-terminal domain (9, 11, 12). A type A-like domain is
present in several proteins such as complement factors B and C2; type
VI, XII, XIV, and XVI collagens; and
subunits of several integrins
(13). Some members of this protein family are involved in cell
adhesion. However, whether CMP is an adhesion factor remains unknown.
and
) membrane-spanning subunits. The extracellular domain of the
subunit has divalent cation-binding sites (14). The
subunits play crucial roles in
determining the ligand specificity. Chondrocytes express integrins
1
1,
2
1,
3
1,
5
1,
V
3, and
V
5
(15, 16), but their ligands in cartilage have not been fully defined.
1
1 plays a pivotal role in the
CMP-mediated cell adhesion.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (FBI2),
2 (P1E6)
(17),
3 (ASC-6) (18),
5 (P1D6) (19),
2 (P4H9-A11) (20),
5
1
(JBS5), and
V
5 (P1F6) (21); rabbit
antisera to human integrins
1,
2,
V,
1, and
5; and rabbit
antiserum to collagen type I were purchased from Chemicon International, Inc. (Temecula, CA). Mouse neutralizing mAbs to human
integrin
1 (5E8D9) (22) and
1 (DE9) (19)
were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Mouse neutralizing mAbs to human integrin
2 (P1E6) (23)
and
3 (P1B5) (23) were purchased from Becton Dickinson
(Lincoln Park, NJ). Fluorescein-conjugated goat IgG fraction to mouse
IgG F(ab')2 was purchased from Organon Teknika (Durham, NC).
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Fig. 1.
SDS-PAGE profiles of purified bovine
CMP. CMP was resolved by SDS-PAGE in the presence (left
lane) or absence (right lane) of 1%
-mercaptoethanol (
-ME) and stained with silver
nitrate.
-modified Eagle's medium containing 10%
fetal bovine serum (Mitsubishikasei, Tokyo, Japan), 25 µg/ml ascorbic
acid, 32 units/ml penicillin, and 60 µg/ml kanamycin. Cultures were
incubated in an atmosphere of 5% CO2 in a humidified incubator.
-modified Eagle's medium containing 1 mg/ml
BSA, 25 µg/ml ascorbic acid, 32 units/ml penicillin, 60 µg/ml
kanamycin, and 10 µg/ml cycloheximide (medium A) or in 0.1 ml of 10 mM Hepes (pH 7.4) containing 0.9% NaCl, 1 mg/ml BSA, and
10 µg/ml cycloheximide. All cells dropped to the bottom of the dishes
within 15 min after seeding if they did not attach to the culture
surface. Spread cells were distinguishable by their cellular
projections. Round and spread cells were separately counted under a
phase-contrast microscope. At least 60-80 cells were evaluated, and
the percentage of spread cells to total cells was calculated.
1,
2,
V,
1, or
5), rabbit
antiserum to human type I collagen, or rabbit nonimmune serum (1 µl)
and a 1:1 suspension of protein G-Sepharose in RIPA buffer (40 µl).
The suspension was incubated for 2 h at 4 °C. The protein
G-Sepharose beads with bound immune complexes were washed five times
with RIPA buffer and then boiled in Laemmli buffer (40 µl). Laemmli
buffer-resolved proteins were fractionated by SDS-PAGE. The gel with
the fractionated proteins was dried and exposed to x-ray film (Eastman
Kodak Co.).
1 antiserum or rabbit nonimmune serum at 4 °C for 2 h. Immune complexes were then incubated
with a 1:1 suspension of protein G-Sepharose in RIPA buffer (40 µl) at 4 °C for 2 h. The Sepharose beads with bound immune
complexes were washed five times with RIPA buffer and then boiled in
Laemmli buffer (40 µl). Laemmli buffer-resolved proteins were
fractionated by SDS-PAGE and transferred to a nitrocellulose membrane
(28). The membrane was blocked with BSA and incubated for 1 h in
streptavidin-conjugated horseradish peroxidase (Amersham International,
Buckinghamshire, United Kingdom). Biotinylated proteins were visualized
using enhanced chemiluminescence (Amersham International) and then
exposed to x-ray film.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Localization of CMP in pig rib
cartilage. Immunohistochemistry was done with the antiserum to
bovine CMP. The presence of CMP is clearly observed around the cells in
resting cartilage (A) and the matrix-forming zone of the
growth plate (B). A control section of the growth plate was
treated with nonimmune serum at the same dilution (C).
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Fig. 3.
Adhesion and spreading of rabbit articular
chondrocytes on CMP-coated dishes. A, 6-mm wells were
incubated with CMP at various concentrations. Rabbit articular
chondrocytes were seeded and incubated in the wells for 2 h, and
the percentage of spread cells to total cells was calculated. The
appearance of cells on CMP-free and CMP-coated dishes is shown in
insets a and b, respectively. B, 6-mm
wells were incubated with CMP solutions at various concentrations.
Chondrocytes were seeded and incubated in the wells for 2 h. After
gentle washing, adherent cells were quantified using an aqueous soluble
tetrazolium/formazan assay. The values are the means ± S.D. of
triplicate determinations.
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Fig. 4.
Spreading of various chondrocytes and
fibroblasts on CMP-coated dishes. Rabbit growth plate chondrocytes
(rGC), rabbit articular chondrocytes (rAC), human
embryonic epiphyseal chondrocytes (hEC), rabbit fibroblasts
(rFB), and human MRC5 fibroblasts (hFB) were
seeded and incubated for 2 h on uncoated dishes (open
bars) or on dishes coated with 20 µg/ml CMP (closed
bars). The values are the means ± S.D. of triplicate
determinations.
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Fig. 5.
Synergism between CMP and type II
collagen. A, 6-mm wells were not incubated (open
circles) or were incubated with CMP at 0.5 µg/ml (open
squares), type II collagen at 3 µg/ml (open triangles), or CMP
plus type II collagen (closed triangles). Rabbit articular
chondrocytes were allowed to adhere on the wells for 15-60 min.
B, 6-mm wells were incubated with CMP at various
concentrations in the absence (open squares) or presence
(closed triangles) of type II collagen at 3 µg/ml.
Chondrocytes were seeded and incubated in the wells for 1 h.
C, 6-mm wells were incubated with type II collagen at
various concentrations in the absence (open triangles) or
presence (closed triangles) of CMP at 0.5 µg/ml.
Chondrocytes were seeded and incubated in the wells for 1 h.
D, 6-mm wells were not incubated (open bars) or
were incubated with CMP at 0.5 µg/ml (closed bars) in the
presence of type II collagen (coll. II) or boiled type II
collagen (gelatin) at 3 µg/ml. Chondrocytes were seeded
and incubated in the wells for 1 h. The values are the means ± S.D. of triplicate determinations.
-modified Eagle's
medium suppressed the spreading of chondrocytes on CMP-coated dishes
(Fig. 6A). The concentrations
of Mg2+ and Ca2+ in
-modified Eagle's
medium are 0.8 and 1.8 mM, respectively. When chondrocytes
were suspended in 10 mM Hepes (pH 7.4) containing 0.9%
NaCl, few cells were attached on CMP-coated dishes. However, the
addition of Mg2+ to the buffer markedly enhanced the cell
spreading on CMP-coated dishes (Fig. 6B). This was induced
at a Mg2+ concentration of 1-2 mM and
increased dose-dependently at least until 5 mM.
Mn2+ had a greater effect on cell spreading on CMP-coated
dishes than Mg2+ at 1-2 mM (Fig.
6C). Ca2+ had less effect on cell spreading than
Mg2+ and Mn2+(Fig. 6D).
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Fig. 6.
Effects of EDTA and divalent cations on
spreading of chondrocytes on CMP-coated dishes. A,
rabbit articular chondrocytes were incubated for 2 h with EDTA
(0-5 mM) in medium A on 20 µg/ml CMP-coated dishes
(closed triangles) or on uncoated dishes (open
triangles). B-D, rabbit articular chondrocytes were
incubated for 1 h with Mg2+, Mn2+, or
Ca2+, respectively, at various concentrations in 10 mM Hepes, pH 7.4, containing 0.9% NaCl, 1 mg/ml BSA, and
10 µg/ml cycloheximide on 20 µg/ml CMP-coated dishes (hatched
bars) or on uncoated dishes (open bars). The values are
the means ± S.D. of triplicate determinations.
1 or
1 (Fig.
7). The mAb against
2,
3,
5,
2,
5
1, or
V
5,
as well as control IgG, had little effect on cell spreading on
CMP-coated dishes. The inhibition of the CMP-induced cell adhesion by
the mAb to integrin
1 or
1, but not other
mAbs, was observed with human chondrocytes using an aqueous soluble
tetrazolium/formazan assay (Fig. 8). These findings suggest that the integrin
1 and
1 subunits were involved in the cell adhesion and
spreading on CMP-coated dishes.
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Fig. 7.
Inhibition of spreading of human MRC5
fibroblasts on CMP-coated dishes by mAbs to human integrins. MRC5
fibroblasts were incubated for 2 h on dishes coated with CMP in
medium A containing mouse IgG or various mAbs to human integrins at 10 µg/ml. The values are the means ± S.D. of three to nine
determinations.
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Fig. 8.
Inhibition of adhesion of human embryonic
chondrocytes to CMP by mAbs to human integrins. Human embryonic
chondrocytes were incubated for 2 h on dishes coated with CMP in
medium A containing 3 or 10 µg/ml mouse IgG (open circles)
or mAb to human integrin 1 (5E8D9) (closed
circles),
2 (P1E6) (open triangles),
3 (P1B5) (closed triangles), or
1 (DE9) (open squares). After gentle washing,
adherent cells were quantified using an aqueous soluble
tetrazolium/formazan assay. The values are the means ± S.D. of
triplicate determinations.
1,
2,
V,
1, and
5 subunits. After iodination, CMP did not form trimers
even under nonreducing conditions during electrophoresis in the
presence of SDS, as described under "Experimental Procedures." However, 125I-CMP enhanced cell adhesion at 10 µg/ml.2 The lysates of MRC5
cells that were dispersed with bacterial collagenase and trypsin were
incubated with 125I-CMP in the presence of rabbit antiserum
to a human integrin subunit (
1,
2,
V,
1, or
5), rabbit
antiserum to human type I collagen, or nonimmune rabbit serum, and then
the material precipitated with protein G-Sepharose was analyzed by
SDS-PAGE. Of these sera, only the antiserum to integrin
1 precipitated 125I-CMP in the lysates (Fig.
9A, panel a). No
125I-CMP directly bound to the antibodies in the absence of
the lysates (Fig. 9A, panel b). The antiserum to
integrin
1 precipitated the 200-kDa integrin
1 subunit in MRC5 lysates (Fig. 9B). The molecular mass of the integrin
1 subunit in human
chondrocytes is ~200 kDa under nonreducing conditions (15). In other
experiments, the mAb to integrin
1 also coprecipitated
125I-CMP in the cell lysates, although the
125I-CMP level precipitated with this mAb was lower than
the precipitated level with the polyclonal antibodies to integrin
1 (data not shown).
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Fig. 9.
Precipitation of 125I-CMP and the
integrin 1 subunit using the
antiserum to integrin
1.
A, 125I-CMP was incubated with rabbit antisera
(1%) to various human integrins or human type I collagen or to
nonimmune serum (1%) in the presence (panel a) or absence
(panel b) of the lysates of MRC5 fibroblasts pretreated with
pure collagenase. The proteins bound to protein G-Sepharose were
resolved by Laemmli buffer. Laemmli buffer-resolved proteins were
fractionated by SDS-PAGE. The gel with the fractionated samples was
dried and exposed to x-ray film. B, MRC5 cells were labeled
with biotin. The lysates of biotin-labeled cells were incubated with
the antiserum (1%) to human integrin
1 or the control
serum. Immune complexes were then incubated with protein G-Sepharose
beads. Laemmli buffer-resolved proteins were fractionated by SDS-PAGE
under nonreducing conditions and transferred to nitrocellulose
membranes. The membranes were blocked with BSA and incubated for 1 h in streptavidin-conjugated horseradish peroxidase. Biotinylated
proteins were visualized using enhanced chemiluminescence.
1, the precipitation of
125I-CMP was detectable at 15 ng/ml 125I-CMP
and increased with the increase in 125I-CMP, at least until
1.5 µg/ml (Fig. 10A). In
contrast, the control serum did not precipitate 125I-CMP
even at the highest concentration of 125I-CMP (1.5 µg/ml)
(Fig. 10A). Next, the cell lysates were incubated in the
presence of 1.5 µg/ml 125I-CMP, the antiserum to the
integrin
1 subunit, and increasing concentrations of
unlabeled native CMP. The native CMP decreased the precipitated level
of 125I-CMP dose-dependently (Fig.
10B).
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[in a new window]
Fig. 10.
Interaction between 125I-CMP and
the integrin 1 subunit in the
absence or presence of increasing concentrations of native CMP
trimers. A, increasing concentrations of
125I-CMP were incubated with the antiserum to integrin
1 or nonimmune serum in the presence of MRC5 cell
lysates. B, the cell lysates were incubated with 1.5 µg/ml
125I-CMP, the antiserum (1%) to the integrin
1 subunit, and increasing concentrations of unlabeled
native CMP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 or
1 suppressed the adhesion and spreading of human
chondrocytes and human MRC5 fibroblasts on dishes coated with CMP. The
other tested mAbs to various integrins had a marginal effect on cell
adhesion or spreading on CMP-coated dishes. These findings suggest that
integrin
1
1 plays a pivotal role in the
CMP-mediated cell adhesion and spreading.
1 coprecipitated
125I-CMP in cell lysates. Unlabeled native CMP competed for
the binding to integrin
1 with 125I-CMP.
Although the anti-integrin
1 mAb inhibited the effect of
CMP on cell adhesion, this mAb (data not shown), as well as the
antiserum to integrin
1, did not consistently
precipitate 125I-CMP in cell lysates. Under these
conditions, the antibody to integrin
1 may coprecipitate
the
1 subunit only at low levels. In addition, the
affinity of integrin
1 for CMP may decrease in the
absence of the intact plasma membrane. In any case, our findings
suggest that CMP selectively binds to the integrin
1 subunit even in the presence of detergents (Nonidet P-40 and SDS).
1
1, as well as
2
1, binds to collagen under some
experimental conditions. However, the precipitation of CMP with anti-integrin
1 antibodies is not due to the binding of
CMP to collagen because 125I-CMP used in this study did not
bind to type I, II, III, IV or V
collagen.3 Furthermore, the
lysates were prepared after cells were dispersed from the cell layer
with bacterial pure collagenase at a high concentration (0.8 mg/ml) and
trypsin. No type I collagen was detected in the cell lysates by
immunoblotting.3
2
1 (34).
Chondroadherin can promote cell adhesion, but not spreading (34),
whereas CMP enhances both cell adhesion and spreading. These findings
suggest that CMP and chondroadherin have different roles in cartilage.
1
1 seems to be involved in the
CMP-induced cell adhesion. The cartilage-specific adhesion protein CMP
may play an important role in the development and repair of skeletal tissues.
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ACKNOWLEDGEMENT |
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We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for use of the facilities.
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FOOTNOTES |
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* This work was supported in part by a grant from the Ministry of Education, Science, Sports, and Culture of Japan.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. Fax: 81-82-257-5629; E-mail: ykato{at}ipc.hiroshima-u.ac.jp.
2 The percentage of spread articular chondrocytes to total cells on dishes coated with 125I-CMP (10 µg/ml) at 2 h was 13 ± 2% compared with 4 ± 2 or 18 ± 3% on dishes not coated or coated with native CMP (10 µg/ml), respectively. The values are the means ± S.D. of triplicate determinations.
3 S. Makihira, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CMP, cartilage matrix protein; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RIPA, radioimmune precipitation assay.
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REFERENCES |
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