Department of Cell and Molecular Biology, Section for Connective Tissue Biology, Lund University, S-221 00 Lund, Sweden
Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate
adhesion of isolated chondrocytes. In the present study
we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was
dependent on Mg2+ or Mn2+ but not Ca2+. Adhesion
was partially inhibited by an antibody recognizing 1
integrin subunit. Chondroadherin-binding proteins
from chondrocyte lysates were affinity purified on
chondroadherin-Sepharose. The
1 integrin antibody
immunoprecipitated two proteins with molecular mass
~110 and 140 kD (nonreduced) from the EDTA-eluted
material. These results indicate that a
1 integrin on
chondrocytes interacts with chondroadherin. To identify the
integrin subunit(s) involved in interaction of
cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified
2
1 as a
chondroadherin-binding integrin. These results are in
agreement with cell adhesion experiments where antibodies against the integrin subunit
2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since
2
1 also is a receptor
for collagen type II, we tested the ability of different
antibodies against the
2 subunit to inhibit adhesion of
T47D cells to collagen type II and chondroadherin. The
results suggested that adhesion to collagen type II and
chondroadherin involves similar or nearby sites on the
2
1 integrin. Although
2
1 is a receptor for both
collagen type II and chondroadherin, only adhesion of
cells to collagen type II was found to mediate spreading.
THE cartilage extracellular matrix is highly specialized in its composition and organization to adapt to
and withstand mechanical forces. A number of the
matrix molecules are found predominantly or exclusively
in cartilage (20). The major matrix components are collagens and proteoglycans (19), with collagen type II representing ~95% of the collagens (11) and aggrecan ~95% of the proteoglycans (16). Collagen type II fibers provide
tensile strength to the tissue, whereas aggrecan, bound to
hyaluronan, provides resilience. The interplay between
these molecules is essential for cartilage function (33).
Several other matrix components are involved in maintaining the specific cartilage properties, where some have
primarily structural roles and others are associated with
the chondrocytes and are likely to be involved in monitoring matrix properties and mediating signals to the cells (20). The chondrocytes, being the only type of cell in cartilage, have a key function in cartilage homeostasis. Their
roles include controlling normal turnover of matrix molecules, depositing molecules into a functioning matrix, and
responding to alterations in load with appropriate remodeling.
Chondroadherin (CHAD)1, originally described as a 36-kD protein, is a prominent noncollagenous extracellular
protein in cartilage (31). Although the protein has been
detected in extracts from cartilage and bone (31), recent
data show very low expression of CHAD mRNA in bone
while it is prominently expressed in certain zones of cartilage in young rats (Shen, Z., D. Heinegård, and Y. Sommarin, unpublished results). CHAD contains only a short oligosaccaride lacking sialic acid and hexosamines on
serine 122 (31, 35). More recently its sequence was determined, both at the protein and cDNA level, showing that
CHAD is a unique member of the leucine-rich repeat
(LRR) protein family (35). Other members of this diverse
family include the small cartilage proteoglycans biglycan
(12), decorin (28), fibromodulin (36), lumican (2), and
keratocan (6), as well as PRELP (1).
It has been shown earlier that isolated chondrocytes adhere to chondroadherin immobilized on plastic culture
dishes (44) indicating that one function of this protein is to
mediate interactions between the chondrocytes and the
extracellular matrix. Fibroblasts and osteoblasts also adhered to CHAD (44), suggesting that a cell surface protein
common to several cell types may be the receptor for the
protein.
Integrins, a family of membrane glycoproteins, are of
prime importance for adhesion of most cells to extracellular matrix proteins (22, 25, 37). They consist of two subunits, Antibodies
Monoclonal antibodies against the human integrin subunits Cell Isolation and Culture
Bovine chondrocytes were isolated by collagenase (CLS1; Worthington
Biochemical Corp., Lakewood, NJ) digestion of articular cartilage from
4-6-month old calves as described elsewhere (43). Briefly, cartilage slices
were digested by collagenase in EBSS (Earle's balanced salt solution;
GIBCO BRL, Gaithersburg, MD) for 15-16 h at 37°C. The cells were filtered through a 100 µm nylon filter, washed three times in Dulbecco's
modified PBS (GIBCO BRL), and used immediately after isolation. Human chondrocytes from knee joint cartilage were isolated by pronase
(Calbiochem, La Jolla, CA) digestion for 1 h followed by collagenase
(Boehringer Mannheim, Indianapolis, IN) digestion for 15-18 h as described by Häuselmann et al. (17). The cells were filtered and washed as
described above. Human lung carcinoma fibroblasts (A549) and human
mammary tumor cells (T47D) obtained from the American Type Culture
Collection (Rockville, MD) were cultured in Dulbecco's minimal essential
medium (DMEM) supplemented with 10% fetal calf serum, 50 UI penicillin, and 50 µg/ml streptomycin (GIBCO BRL). Human chondrocytes
were cultured in DMEM and F12 (1:1) supplemented with 10% fetal calf
serum, 25 µg/ml ascorbic acid, 50 UI penicillin, and 50 µg/ml streptomycin
(GIBCO BRL). To harvest cells, the culture dish was washed three times
with Ca/Mg-free PBS, and the cells were incubated with 0.5% trypsin and
1 mM EDTA (GIBCO BRL) in PBS ( Cell Adhesion
Tissue culture-treated, 48-well dishes (Nunclon, Nunc, Denmark) were
coated overnight at room temperature with 5 or 10 µg/ml CHAD in 4M
guanidine-HCl, 50 mM Tris-HCl, pH 7.6, or 10 µg/ml collagen type II
(CII) in PBS and blocked with 0.25% BSA (Serva Feinbiochemica, Wichita Falls, TX; Sigma Chemical Co.) in PBS. Collagen type II was isolated
from nasal cartilage by pepsin digestion (34). The dishes were rinsed four
times with PBS before the experiment. When the effects of divalent cations were studied, the cells were washed three times in Ca/Mg-free PBS
and resuspended in the same buffer supplemented either with 1 mM CaCl2, 1 mM MgCl2, 50 µM MnCl2, 1 mM of both CaCl2 and MgCl2 or supplemented with all the divalent cations. The cells suspended in PBS containing 0.1% BSA were added to the wells at a concentration of 100,000/well of bovine chondrocytes and 50,000/well of human chondrocytes, A549, or
T47D-cells. Cells were allowed to adhere for 1 h at 37°C. When the effect
of antibodies on adhesion was investigated, the cells were suspended in
PBS (+Ca and Mg) and incubated with antibodies for 20 min at room
temperature before plating of the cells. The monoclonal antibodies Cell Spreading
Chamber slides (8 chamber; Lab-Tek®, Nunc Inc., Naperville, IL) were
coated with 5 µg/ml of CHAD in 4M guanidine-HCl, 50 mM Tris-HCl, pH
7.5, or 5 µg/ml of collagen type II in PBS and blocked with 0.25% BSA in
PBS. T47D-cells (20,000/well) were added to the chambers, allowed to adhere, and spread for 3 h at 37°C in the absence or in the presence of
10 Surface Labeling with 125I
Bovine chondrocytes or human lung carcinoma fibroblasts A549 were suspended in 1 ml of PBS containing 1 mg/ml glucose. 125I (1 mCi; Nordion
Inc., Kanata, ON, Canada) was added to the cells together with 4 U of lactoperoxidase (Sigma Chemical Co.; 120 U/mg) and 0.05 U of glucose oxidase (Sigma Chemical Co.; 1010 U/ml) prepared fresh in PBS-glucose.
The cells were kept on ice for 15 min, whereafter the reaction was stopped
by adding 10 ml of Dulbecco's culture medium. The cells were then
washed three times with PBS and lysed for 1h on ice in 2 ml of 1% Triton
X-100, 100 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 1 mM
PMSF (Sigma Chemical Co.), 1 mM MnCl2, 1 mM MgCl2 in 10 mM Tris-HCl, pH 7.4. Cell lysates were centrifuged at 10,000 rpm for 30 min at 4°C,
and the pellets were discarded.
Isolation and Coupling of CHAD to Agarose
CHAD was purified from bovine tracheal cartilage essentially according
to the published procedure (31). For coupling, CHAD (2.5 mg) was solubilized in 0.5% SDS and coupled to 2 ml of Mini-Leak agarose (Biocarb
Chemicals, Lund, Sweden) according to the manufacturer's instructions.
The control agarose was treated in a similar manner but in the absence of
protein.
Affinity Purification of CHAD-binding Protein
The CHAD agarose (0.5 ml) and the control agarose (0.5 ml) were packed
in mini-columns (Bio Rad, Hercules, CA) and equilibrated with at least 20 vol of 0.1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 1 mM MnCl2, and 1 mM
MgCl2. The lysates from the 125I-labeled cells were passed over the control
agarose two times and then incubated with the CHAD agarose in the
mini-columns for 2-3 h with continuous end-over-end mixing. The CHAD
agarose was washed with 15 vol of the equilibration buffer containing 75 mM NaCl, and the column was then eluted with 20 mM EDTA, 1 mM
PMSF, 10 mM Tris-HCl, pH 7.4. The eluted protein peak was passed over a desalting column (PD-10; Pharmacia Fine Chemicals, Uppsala, Sweden)
equilibrated with 50 mM Tris, pH 7.4, 0.3 M NaCl, 1% Triton X-100, 0.1%
BSA, 1 mM CaCl2, 1 mM MgCl2, and 1 mM PMSF. Samples of the affinity-purified proteins were then either precipitated by methanol/chloroform (48) or immunoprecipitated by antibodies followed by separation on
4-12% SDS-PAGE and visualized by autoradiography or image analysis
using the BioImaging Analyzer Bas2000 (Fuji Photo Film Co., Tokyo,
Japan).
Immunoprecipitation
Radiolabeled proteins were immunoprecipitated from cell lysate and from
affinity-purified material. In experiments where lysates were immunoprecipitated they were passed over a desalting column (PD-10; Pharmacia
Fine Chemicals) equilibrated and eluted with 50 mM Tris, pH 7.4, 0.3 M
NaCl, 1% Triton X-100, 0.1% BSA, 1 mM CaCl2, 1 mM MgCl2, 1 mM
PMSF. The cell lysate or the affinity-purified samples were incubated with
continuous end-over-end mixing overnight with 5 µl/ml of monoclonal antibodies ( Statistics
Results are presented as means ± SD. Student's t test was used to determine statistical significance.
Adhesion of Cells to Chondroadherin
CHAD, immobilized on culture dishes, mediated adhesion
of cells in a dose-dependent manner (Fig. 1). Maximal adhesion was seen at a coating concentration of 1.2 µg/ml.
Adhesion of bovine chondrocytes to CHAD was dependent on divalent cations such that Mg2+ or Mn2+ but not
Ca2+ was required (Fig. 2). Only a low number of cells adhered to the control BSA (Fig. 2). The adhesion to CHAD
decreased by 2/3 in the presence of 100 µg/ml of a polyclonal rat
Affinity Chromatography of CHAD-binding Cell
Surface Proteins
To identify integrins with affinity for CHAD, Triton X-100-
solubilized, 125I-labeled cell surface proteins from bovine
chondrocytes were affinity purified on CHAD coupled to
agarose. As shown in Fig. 4, two proteins with molecular
weight ~110 and 140 kD (nonreduced) were eluted from
the CHAD column with EDTA. These proteins were immunoprecipitated with a polyclonal antibody against
Inhibition of Cell Adhesion to CHAD by
Integrin Antibodies
Fibroblasts were adhered to CHAD in cell adhesion experiments in the presence of antibodies against various integrin subunits. As shown in Fig. 7 antibodies against the
To study whether
Since
Spreading of Cells on Collagen Type II or CHAD
It has earlier been shown (44) that chondrocytes adhered
to CHAD appeared to stay round, while chondrocytes immobilized on collagen type II spread on the substratum.
We found, similarly, that T47D cells (Fig. 10 and Table I)
and fibroblasts (data not shown) spread when they were
adhered to collagen type II but not to CHAD. The average cell area of T47D cells that were adhered to CHAD
for 3 h was ~2/3 of those adhered to collagen type II (Table I). Addition of PMA (10
Table I.
Spreading of T47D Cells on CII or CHAD in the
Absence or in the Presence of PMA
and
, where the extracellular domain of the
subunits has several divalent, cation-binding sites. The integrins
1
1,
2
1,
3
1,
5
1, and
6
1
v
3 and
v
5
have been found on chondrocytes (8, 50; Holmvall, K., L. Camper, and E. Lundgren-Åkerlund, unpublished results),
but their ligands in cartilage have not been fully defined. Integrins
1
1 and
2
1 have been found to mediate binding
to collagen type II (8, 24) and
5
1 mediates binding to fibronectin (38). In the present study we investigated the interaction of cells with the cartilage matrix protein CHAD to
identify the cellular receptor that is involved.
Materials and Methods
1 (P4C10),
2 (P1E6),
3 (P1B5),
5 (P1D6), and
v (VNR147) (unpurified ascites
fluid) were from Life Technologies Inc. (Grand Island, NY). Monoclonal
antibody against the human integrin
3 (RUU-PLF12, purified IgG) were
purchased from Becton Dickinson (Bedford, MA). Monoclonal antibodies against the human integrins
v
5 (P1F6) and
v
3 (LM609) (purified
IgG) were from Chemicon International, Inc. (Temecula, CA). The monoclonal antibodies against the human integrin subunits
1 (TS2/7; hybridoma supernatant) and
2 (P1H5; hybridoma supernatant) and rabbit
polyclonal antibodies against rat
1 integrin were kind gifts from Drs.
William Carter, (Fred Hutchinson Cancer Research Center, Seattle, WA;
3), Timothy Springer (Boston Blood Center, Boston, MA; 23), and
Kristofer Rubin (Uppsala University, Uppsala, Sweden; 15), respectively.
The monoclonal antibodies Gi9, Gi14, Gi19, and Gi26 (hybridoma supernatant), recognizing human
2 integrin subunit, were kind gifts from Dr. Sentot Santoso (Justus-Liebig University, Giessen, Germany; 39).
Ca and Mg) for 5 min. Detached
cells were suspended in medium containing 10% FCS or in PBS containing 1 mg/ml trypsin inhibitor (Sigma Chemical Co., St. Louis, MO) and
then washed in PBS.
1,
2
(P1E6),
3,
5,
v, and
v
5 (unpurified ascites fluid) were diluted 1:100,
P1H5 (hybridoma supernatant) was diluted 1:25, and Gi9, Gi14, Gi19, and
Gi26 (hybridoma supernatant) were diluted 1:10. The monoclonal antibodies
3 and
v
3 were used at a concentration of 10 µg/ml. After 1 h of
incubation the wells were gently rinsed with PBS to remove nonadherent
cells. Adhesion was determined by measuring lysosomal hexosaminidase as described by Landegren (29).
8 M phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.). Nonadherent cells were removed by washing, and the adherent cells were
fixed with 2% paraformaldehyde in PBS and stained with Mayers's hematoxylin and 0.3% erythrosin. Spreading was visualized by light microscopy, and mean cell area was calculated by image analysis using the Zeiss
Software KS400/V2.00 (Zeiss, Inc., Oberkochen, Germany).
1,
3,
1,
2 (P1E6),
3,
5,
v, and
v
5) or 50 µg/ml of the
polyclonal antibody (
1) followed by addition of 75 µl anti-mouse IgG-agarose (Sigma Chemical Co.) or 100 µl protein A-Sepharose (Pharmacia
Fine Chemicals) and incubation for 2 h. The beads were centrifuged for 4 min at 4,000 rpm, washed three times with 1% Triton X-100, 0.5 M NaCl, and 10 mM Tris-HCl, pH 7.4. All steps were performed at 4°C. SDS-PAGE sample buffer (100 µl) was added to the washed immunoprecipitates, and the samples were boiled for 5 min with or without 2-mercaptoethanol (5%). The immunoprecipitated proteins were separated by
SDS-PAGE (4-12%) and visualized by autoradiography or by using the
phosphoimager.
Results
1 integrin antibody compared to adhesion in
the absence of antibody (Fig. 3). This indicated that
1 integrins are involved in the adhesion of chondrocytes to
CHAD. The control antibody had only a minor effect on
the adhesion.
Fig. 1.
Adhesion of T47D cells to dishes coated with CHAD.
Culture dishes (48 well) were coated with various concentrations
of CHAD and blocked for nonspecific binding with BSA
(0.25%). T47D cells (50,000/well) were allowed to adhere for 1 h
at 37°C. Nonadherent cells were removed by washing, and adhesion was determined by analyzing lysosomal hexosaminidase.
The results presented are the mean adhesion in duplicates from
one of two experiments.
[View Larger Version of this Image (12K GIF file)]
Fig. 2.
Divalent cation-dependent adhesion of chondrocytes to
CHAD. Culture dishes (48 well) were coated with CHAD (5 µg/
ml) and blocked for nonspecific binding with BSA (0.25%). Bovine chondrocytes were allowed to adhere to the dishes for 1 h at
37°C in the absence of divalent cations or in the presence of Ca2+
(1 mM), Mg2+ (1 mM), or Mn2+ (50 µM). Nonadherent cells were
removed by washing, and adhesion was determined by analyzing
lysosomal hexosaminidase. Adhesion is expressed as a percentage of the total number of cells added to the dish. The numbers
represent the mean adhesion from three wells ±SD from one of
three experiments.
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
1 integrin-dependent adhesion of chondrocytes to
CHAD. Culture dishes (48 well) were coated overnight with
chondroadherin (5 µg/ml) and blocked for nonspecific binding
with BSA (0.25%). Bovine chondrocytes were allowed to adhere
to the dishes for 1 h at 37°C in the presence of various concentrations of a polyclonal antibody against the rat
1 integrin subunit
or control IgG. Nonadherent cells were removed by washing, and
adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control,
and the numbers represent mean of duplicate adhesion from one
of three experiments.
[View Larger Version of this Image (13K GIF file)]
1
integrin. As shown in the figure, this
1 integrin showed
two bands migrating corresponding to 120 (
1 chain) and
150 kD (
chain) upon SDS-PAGE under reducing conditions. In addition, a protein band with mobility corresponding to 100 kD was found, which may represent a degradation product of the
1 integrin. We were not able to
further identify the
chain from the chondrocyte integrin,
since available antibodies against human integrins showed
too low cross-reactivity to bovine integrins. To identify the
1-associated
chain with affinity for CHAD, Triton
X-100-solubilized, 125I-labeled cell surface proteins from
human fibroblasts were affinity purified on the CHAD
column. Proteins eluted from the CHAD affinity purification experiments were immunoprecipitated with monoclonal antibodies against the human integrin subunits
1,
1,
2,
5, and
v. Fig. 5 shows that the antibodies against
the integrin subunits
1 and
2 immunoprecipitated an integrin dimer of similar appearance, while antibodies against
1,
5, and
v did not specifically immunoprecipitate integrins from the EDTA eluate. In a control experiment (Fig.
6) it was shown that these cells express a number of different integrins. Taken together, these results strongly indicate that the integrin
2
1 is a receptor for CHAD.
Fig. 4.
Affinity purification of
CHAD-binding cell surface proteins. Bovine chondrocytes were
125I-labeled and lysed with 1% Triton X-100, 100 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin
A, 1 mM PMSF, 1 mM MnCl2, and
1 mM MgCl2 in 10 mM Tris-HCl,
pH 7.4. The lysate was passed over
control agarose followed by CHAD
agarose. Proteins with affinity for
CHAD were eluted by EDTA (20 mM), passed over a desalting column (PD-10) equilibrated, and
eluted with 0.3 M NaCl, 1% Triton X-100, 0.1% BSA 1 mM CaCl2, 1 mM MgCl2, 1 mM PMSF, in 50 mM
Tris-HCl, pH 7.4. An aliquot of the
protein peak was immunoprecipitated with the polyclonal rat 1
integrin antibody. Proteins in the eluate (E) and in the immunoprecipitate (
1) were separated by 4-12% SDS-PAGE under reducing (R) or nonreducing (NR) conditions.
[View Larger Version of this Image (41K GIF file)]
Fig. 5.
Immunoprecipitation of CHAD-binding integrins from
human fibroblasts. 125I-labeled A549 fibroblasts were lysed with
1% Triton X-100, 100 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml
pepstatin A, 1 mM PMSF, 1 mM MnCl2, and 1 mM MgCl2 in 10 mM Tris-HCl, pH 7.4. The lysate was passed over control agarose
followed by CHAD agarose. Proteins with affinity for CHAD
were eluted by EDTA (20 mM), passed over a desalting column
(PD-10) equilibrated, and eluted with 0.3 M NaCl, 1% Triton
X-100, 0.1% BSA, 1 mM CaCl2, 1 mM MgCl2, 1 mM PMSF in 50 mM Tris-HCl, pH 7.4. Aliquots of the protein peak were immunoprecipitated with monoclonal antibodies against the integrin
subunits 1 (P4C10),
1 (TS2/7),
2 (P1E6),
5 (P1D6), and
v
(VNR147). The immunoprecipitated proteins were separated by
SDS-PAGE (4-12%) under nonreducing conditions and visualized by autoradiography.
[View Larger Version of this Image (76K GIF file)]
Fig. 6.
Immunoprecipitation of integrins from human fibroblasts. 125I-labeled A549 fibroblasts were lysed with 1% Triton
X-100, 100 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin
A, 1 mM PMSF, 1 mM MnCl2, and 1 mM MgCl2 in 10 mM Tris-HCl, pH 7.4. Aliquots of the lysate were immunoprecipitated
with monoclonal antibodies against the integrin subunits 1
(P4C10),
3 (RUU-PLF12),
1 (TS2/7),
2 (P1E6),
3 (P1B5),
5 (P1D6),
v (VNR147), and
v
5 (P1F6). The immunoprecipitated proteins were separated by SDS-PAGE (4-12%) under
nonreducing conditions and visualized by autoradiography.
[View Larger Version of this Image (105K GIF file)]
2 or
1 integrin subunits inhibited the cell adhesion to
>50% while antibodies against
3,
5,
v,
v
3, or
v
5
had no or only a minor effect on the adhesion. In contrast
to the other antibodies, the
3 antibody stimulated the adhesion of fibroblasts to CHAD. In agreement with the affinity purification experiments, these results show that
2
1 is a CHAD-binding integrin.
Fig. 7.
Inhibition of fibroblast adhesion to CHAD by integrin
antibodies. Culture dishes (48 well) were coated with CHAD (5 µg/ml) and blocked for nonspecific binding with BSA (0.25%).
Human A549 fibroblasts were allowed to adhere to the dishes for
1 h at 37°C in the presence of monoclonal antibodies against the
human integrin subunits 1 (P4C10),
3 (RUU-PLF12),
2
(Gi9),
3 (P1B5),
5 (P1D6),
v (VNR147),
v
3 (LM609), and
v
5 (P1F6). Nonadherent cells were removed by washing, and
adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control,
and the numbers represent the mean of duplicate adhesion from
three individual experiments ±SD. *P < 0.05; **P < 0.01; P
1 = 0.002; P
2 = 0.011; P
3 = 0.021.
[View Larger Version of this Image (29K GIF file)]
2
1 is involved in the adhesion of
chondrocytes to CHAD, human chondrocytes were adhered to immobilized CHAD in the absence or in the presence of an antibody against the integrin subunit
2. The
antibody partially inhibited the adhesion of chondrocytes
in a dose-dependent manner (Fig. 8). Around 30% of the
adhesion was inhibited at the highest antibody concentration. This result confirmed that
2
1 is a CHAD-binding integrin on chondrocytes.
Fig. 8.
Inhibition of human chondrocyte adhesion to CHAD
by 2 integrin antibodies. Culture dishes (48 well) were coated
with CHAD (5 µg/ml) and blocked for nonspecific binding with
BSA (0.25%). Human chondrocytes were allowed to adhere to
the dishes for 1 h at 37°C in the presence of various concentrations of the monoclonal antibody against the human integrin subunit
2 (Gi9). Nonadherent cells were removed by washing, and
adhesion was determined by analyzing lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control,
and the numbers represent the mean adhesion ±SD from three
wells in one of two experiments.
[View Larger Version of this Image (37K GIF file)]
2
1 is a receptor for both collagen type II (24)
and CHAD, we investigated the interaction of T47D-cells
(cells that express the
2 but not the
1 subunit) with
these two substrates, using various antibodies to the
2 integrin subunit (Fig. 9). The
2 antibodies inhibited cell adhesion to collagen type II and CHAD in a similar manner,
although they were somewhat less effective in the CHAD
experiment. Higher concentrations of the antibodies did
not change the inhibition pattern (data not shown). This
indicates that similar or nearby sites on the
2
1 integrin are binding to the two substrates.
Fig. 9.
Inhibition of adhesion of T47D cells to CHAD (a) and to collagen type II (CII; b) by various 2 antibodies. Culture dishes (48 wells) were coated with 5 µg/ml of CHAD or CII and blocked for nonspecific binding with BSA (0.25%). T47D cells were allowed to
adhere to the dishes for 1 h at 37°C in the absence or in the presence of monoclonal antibodies against the integrin subunits
1 (P4C10),
3 (RUU-PLF12), or various
2 antibodies. Nonadherent cells were removed by washing, and adhesion was determined by analyzing
lysosomal hexosaminidase. The adhesion is expressed as a percentage of the control, and the numbers represent the mean of duplicate
adhesion from three individual experiments ±SD. *P < 0.05; **P < 0.01; (a) P
1 = 0.004; PGi9 = 0.006; PGi19 = 0.026. (b) P
1 = 0.000;
PP1E6 = 0.001; PP1H5 = 0.001; PGi9 = 0.000; PGi26 = 0.047.
[View Larger Versions of these Images (35 + 32K GIF file)]
8 M) to the adhered cells
stimulated spreading and increased the cell area with
~40% on both CHAD and collagen type II (Fig. 10 and
Table I).
Fig. 10.
Spreading of T47D
cells on collagen type II (CII)
or CHAD. Chamber slides
(eight chambers) were coated
with 5 µg/ml of CII (A and
B) or CHAD (C and D) and
blocked for nonspecific binding with BSA (0.25%). Human T47D cells (20,000/
well) were plated onto the
chambers and allowed to adhere and spread for 3 h at 37°C in the absence (A and
C) or in the presence (B and
D) of 108 M PMA. Nonadherent cells were removed by
washing, and the adherent cells were fixed with 2%
paraformaldehyde in PBS
and stained with Meyer's hematoxilin and erythrosin.
Spreading was visualized by light microscopy, and mean
cell area (Table I) was calculated by image analyses using
the Zeiss software KS400/
V2.00.
[View Larger Version of this Image (120K GIF file)]
In the present investigation we show that CHAD, a relatively abundant noncollagenous protein in cartilage extracellular matrix, interacts with 1 integrins on bovine chondrocytes. This interesting finding identifies CHAD as a
candidate for mediating signals between the chondrocytes
and the cartilage matrix.
CHAD is a member of the LRR protein family (35). Among other members in this family are the small cartilage proteoglycans biglycan, decorin, fibromodulin, and lumican. These proteoglycans are all known to interact with collagen (18, 41, 47), but it is not known if CHAD interacts with collagen or other matrix molecules.
Chondrocyte adhesion to CHAD was partially inhibited
by a rat polyclonal antibody against 1 integrin. Species
differences between cells and antibodies may explain why
the inhibition was not total. Alternatively, other receptors
than
1 integrins may also be involved in the adhesion to
CHAD. We found that adhesion of chondrocytes to
CHAD was dependent on Mg2+ or Mn2+ but not on Ca2+.
This is consistent with results from extensive studies of
regulation of integrin activity by divalent cations. The activity of several integrins, including
2
1, is stimulated by
Mg2+ or Mn2+ and inhibited by Ca2+ (14).
Available antibodies did not immunoprecipitate the 1-associated
integrin subunit from bovine chondrocytes
that mediated the adhesion to CHAD. The most likely explanation for this is that antibodies raised against human
integrin subunits show weak or no cross-reactivity to many
of the bovine chondrocyte integrins. From the molecular
weight of the CHAD-binding
integrin subunit (140 kD
nonreduced and 150 kD reduced), the fact that the apparent size increased upon reduction and that the adhesion
was Mg2+ dependent, it is likely that the
subunit involved is
2. Since we know from FACS® analysis that isolated human primary chondrocytes from articular cartilage
have relatively small amounts of the
2
1 integrin (Holmvall, K., L. Camper, and E. Lundgren-Åkerlund, unpublished results), we chose to investigate CHAD-binding integrins on human fibroblasts. These cells express
2
1 as
well as other integrins (Fig. 6). In affinity purification experiments we were able to show that the integrin
2
1 indeed is a CHAD-binding integrin. Antibodies against
5
and
v immunoprecipitated orders of magnitude-lower amounts of their respective integrins from the CHAD-agarose eluate. We further found that monoclonal antibodies against the subunits
1 or
2 inhibited the adhesion
of cells to culture dishes coated with CHAD, while antibodies against the other integrin subunits had minor or no
effect on the adhesion. In contrast to the lack of effects of
the other integrin antibodies, the
3-integrin antibody appeared to stimulate the adhesion to CHAD. The findings
corroborated further that the integrin
2
1 mediates the
interaction between cells and CHAD. To confirm a participation of
2
1 integrins also in chondrocyte adhesion, we
studied the adhesion of human chondrocytes to CHAD in
the presence of the
2 antibody Gi9. We found that the
antibody partially inhibited adhesion of cultured chondrocytes to CHAD (Fig. 8), which confirms that the integrin
2
1 indeed is involved in adhesion of chondrocytes to
this substrate. In agreement with the fibroblast experiment
the Gi9 antibody only partially inhibited the adhesion of
human chondrocytes. This may indicate that another receptor in addition to
2
1 is involved in the adhesion of
cells to CHAD. It is also possible that the immobilized
CHAD mediate a high degree of nonspecific binding.
In previous experiments, it has been shown that adhesion of chondrocytes and chondrosarcoma cells to collagen
type II was mediated by the integrins 1
1 and
2
1 (24).
Since
1
1 is present on both chondrocytes (24) and fibroblasts (Fig. 5) and since this integrin appeared not to interact with CHAD in the affinity chromatography experiments
(Figs. 4 and 5), it is unlikely that collagen contaminants in
the CHAD preparation were mediating the cell binding.
Since integrin 2
1 is also a receptor for collagen type II
(24) we asked whether adhesion to collagen type II and to
CHAD were mediated by similar mechanisms. One observation indicating that there is a difference in the
2
1 integrin binding to these ligands is that chondrocytes (37),
T47D-cells (Fig. 10), and fibroblasts (data not shown) all
spread on immobilized collagen type II, while adhesion to
CHAD did not promote spreading. One explanation may
be that different sites on the
2 chain are involved in adhesion to collagen type II and CHAD. To study this, we adhered T47D cells to the two substrates in the presence of
various
2 antibodies. The monoclonal antibodies P1E6,
P1H5, and Gi9 are known to block adhesion of cells to collagen. The monoclonal antibodies Gi19 and Gi29 have
some inhibitory effect on adhesion of platelets to collagen,
while Gi14 does not inhibit adhesion. (Santoso, S., personal communication) The T47D cells do not express collagen type II binding integrins other than
2
1 and were
therefore particularly informative in these studies (45;
Camper, L., and E. Lundgren-Akerlund, unpublished results). We found that the different
2 antibodies inhibited
adhesion to collagen type II and CHAD in a similar manner, indicating that these ligands bind to similar or nearby
sites on the
2
1 integrin (Fig. 9). Further experiments using
2 integrin antibodies recognizing other epitopes on
the
2 subunit will be needed to elucidate the binding
sites. Another explanation is that the binding of collagen
type II and CHAD is regulated differently. It has previously been shown that
2
1 integrins from different cell
types show different ligand specificity.
2
1 on platelets and melanoma cells bind collagen (27, 46), while
2
1 on
other cell types binds both collagen and laminin (9, 30).
Several factors including divalent cations, proteoglycans,
and phospholipids have been suggested to modulate integrin activity, and it has been suggested that the degree of
activation may regulate their ligand specificity (4). Since
Mn2+ has been shown to increase the affinity between integrins and their ligands in affinity chromatography (13)
and cell adhesion (10, 32), we tested the possibility that
Mn2+ could stimulate cell spreading. However, Mn2+ appeared not to stimulate spreading of T47D cells on immobilized chondroadherin (data not shown). Phorbol esters
such as PMA are known to mimic the effect of several different integrin-activating stimuli and to induce clustering
of integrins (7, 42, 49). Protein kinase C may therefore be
an important regulator of the integrin affinity and ligand
specificity. Our finding that cells showed some spreading
on CHAD in the presence but not in the absence of PMA
(Fig. 10 and Table I) indicates that spreading of cells may
require activation and altered affinity of the integrins. It
also lends strong support to the involvement of integrins in the cell attachment.
It is likely that integrin 2
1, being a receptor for two
different proteins in cartilage, has an important function in
mediating signals between the chondrocytes and the cartilage matrix. We and others (8, 24, 50) have found that isolated chondrocytes express relatively small amounts of
2
1 integrin. The collagen type II binding integrin
1
1,
on the other hand, is one of the major integrins on isolated
chondrocytes. It is possible that
2
1 is a more dynamic
integrin that is upregulated during changes in cell-matrix
interactions such as matrix turnover, remodelling, or mechanical stress. We have found that the integrin subunit
2
was upregulated in chondrosarcoma cells during mechanical stress while the expression of
1 was not changed (24).
It has also been shown that the integrin subunit
2, but not
1, is upregulated in fibroblasts in contracting collagen
gels (26, 40) during reorganization of the collagen matrix.
This supports the idea that the integrin
2
1 can respond
to changes in the extracellular matrix. Furthermore, it has
also been shown that growth factors such as TGF
(21)
and EGF (5) stimulate expression of the integrin
2
1, indicating that growth factors can regulate the
2
1-mediated interactions with the extracellular matrix. However, further investigations are needed to understand the functional role of the specific interaction of
2
1 with CHAD
in cartilage and to elucidate the differences between interaction of
2
1 with CHAD and collagen type II.
Received for publication 31 October 1996 and in revised form 6 May 1997.
Please address all correspondence to Dr. Evy Lundgren-Åkerlund, Department of Cell and Molecular Biology, Section for Connective Tissue Biology, Lund University, P.O. Box 94, S-221 00 Lund, Sweden. Tel.: (46) 46-222-3311; FAX: (46) 46-211-3417.We are grateful to Dr. Arnoud Sonnenberg for valuable advice and for the generous gift of T47D cells.
1. |
Bengtsson, E.,
P.J. Neames,
D. Heinegard, and
Y. Sommarin.
1995.
The
primary structure of a basic leucine-rich repeat protein, PRELP, found in
connective tissue.
J. Biol. Chem.
270:
25639-25644
|
2. |
Blochberger, T.C.,
J.-P. Vergnes,
J. Hempel, and
J.R. Hassell.
1992.
cDNA
to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in
muscle and intestine.
J. Biol. Chem.
267:
347-352
|
3. |
Carter, W.,
E. Wayner,
T. Bouchard, and
P. Kaur.
1990.
The role of integrins ![]() ![]() |
4. |
Chan, B.M.C., and
M.E. Hemler.
1993.
Multiple functional forms of integrin VLA-2 can be derived from a single ![]() ![]() |
5. |
Chen, J.D.,
J.P. Kim,
K. Zhang,
Y. Sarret,
K.C. Wynn,
R.H. Kramer, and
D.T. Woodley.
1993.
Epidermal growth factor (EGF) promotes human
keratinocyte locomotion on collagen by increasing the ![]() |
6. |
Corpuz, L.M.,
J.L. Funderburgh,
M.L. Funderburgh,
G.S. Bottomley,
S. Prakash, and
G.W. Conrad.
1996.
Molecular cloning and tissue distribution of keratocan.
J. Biol. Chem.
271:
9759-9763
|
7. | Danilov, Y.N., and R.L. Juliano. 1989. Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event. J. Cell Biol. 108: 1925-1933 [Abstract]. |
8. |
Dürr, J.,
S. Goodman,
A. Potocnik,
H. von der Mark, and
K. von der Mark.
1993.
Localization of ![]() |
9. | Elices, M.J., and M.E. Hemler. 1989. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others. Proc. Natl. Acad. Sci. USA. 86: 9906-9910 [Abstract]. |
10. | Elices, M.J., L.A. Urry, and M.E. Hemler. 1991. Receptor functions for the integrin VLA-3: fibronectin, collagen, and laminin binding are differentially influenced by ARG-GLY-ASP peptide and by divalent cations. J. Cell Biol. 112: 169-181 [Abstract]. |
11. | Eyre, D.R., J.-J. Wu, and P. Woods. 1992. Cartilage-specific collagens. Structural studies. In Articular Cartilage and Osteoarthritis. K.E. Kuettner, R. Schleyerbach, J.G. Peyron, and V.C. Hascall, editors. Raven Press, Ltd., New York. 119-131. |
12. |
Fisher, L.W.,
J.D. Termine, and
M.F. Young.
1989.
Deduced protein sequence of bone small proteoglycan I (biglycan) shows homolgy with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species.
J. Biol. Chem.
264:
4571-4576
|
13. |
Gailit, J., and
E. Ruoslahti.
1988.
Regulation of the fibronectin receptor affinity by divalent cations.
J. Biol. Chem.
263:
12927-12932
|
14. | Garratt, A.N., and M.J. Humphries. 1995. Recent insights into ligand binding, activation and signalling by integrin adhesion receptors. Acta Anat. 154: 34-45 |
15. |
Gullberg, D.,
L. Terracio,
T.K. Borg, and
K. Rubin.
1989.
Identification of
integrin-like matrix receptors with affinity for interstitial collagens.
J.
Biol. Chem.
264:
12686-12694
|
16. | Hardingham, T.E., A.J. Fosang, and J. Dudhia. 1992. Aggrecan, the chondroitin sulfate/keratan sulfate proteoglycan from cartilage. In Articular Cartilage and Osteoarthritis. K.E. Kuettner, R. Schleyerbach, J.G. Peyron, and V.C. Hascall, editors. Raven Press, Ltd., New York. 5-20. |
17. | Häuselmann, H.J., M.B. Aydelotte, B.L. Schumacher, K.E. Kuettner, S.H. Gitelis, and E.J.-M.A. Thonar. 1992. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix. 12: 116-129 |
18. |
Hedbom, E., and
D. Heinegård.
1993.
Binding of fibromodulin and decorin
to separate sites on fibrillar collagens.
J. Biol. Chem.
268:
27307-27312
|
19. | Heinegård, D., and M. Paulsson. 1987. Cartilage. In Methods in Enzymology. Structural and Contractile Proteins. Part E. Extracellular Matrix. L.W. Cunningham, editor. Academic Press Inc., Orlando, FL. 336-363. |
20. |
Heinegård, D., and
Å. Oldberg.
1989.
Structure and biology of cartilage
and bone matrix noncollagenous macromolecules.
FASEB (Fed. Am.
Soc. Exp. Biol.) J.
3:
2042-2051
|
21. |
Heino, J., and
J. Massagué.
1989.
Transforming growth factor-![]() |
22. | Hemler, M.E.. 1990. VLA-proteins in the integrin family: structure, functions, and their role on leukocytes. Annu. Rev. Immunol. 8: 365-400 |
23. |
Hemler, M.E.,
F. Sanchez-Madrid,
T.J. Flotte,
A.M. Krensky,
S.J. Burakoff,
A.K. Bhan,
T.A. Springer, and
J.L. Strominger.
1984.
Glycoproteins
of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.
J. Immunol.
132:
3011-3018
|
24. | Holmvall, K., L. Camper, S. Johansson, K. Rubin, J.H. Kimura, and E. Lundgren-Åkerlund. 1995. Chondrocyte and chondrosarcoma cell integrins with affinity for collagen type II and their response to mechanical stress. Exp. Cell Res. 221: 496-503 |
25. | Hynes, R.O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25 |
26. |
Klein, C.E.,
D. Dressel,
T. Steinmayer,
C. Mauch,
B. Eckes,
T. Krieg,
R.B. Bankert, and
L. Weber.
1991.
Integrin ![]() ![]() |
27. |
Kramer, R.H., and
N. Marks.
1989.
Identification of integrin collagen receptors on human melanoma cells.
J. Biol. Chem.
264:
4684-4688
|
28. | Krusius, T., and E. Ruoslahti. 1986. Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc. Natl. Acad. Sci. USA. 83: 7683-7687 [Abstract]. |
29. | Landegren, U.. 1984. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase. Application to detection of lymphokines and cell surface antigens. J. Immunol. Methods. 67: 379-388 |
30. |
Languino, L.R.,
K.R. Gehlsen,
E. Wayner,
W.G. Carter, and
E. Engvall.
1989.
Endothelial cells use ![]() ![]() |
31. |
Larsson, T.,
Y. Sommarin,
M. Paulsson,
P. Antonsson,
E. Hedbom,
M. Wendel, and
D. Heinegård.
1991.
Cartilage matrix proteins. A basic 36-kDa protein with a restricted distribution to cartilage and bone.
J. Biol.
Chem.
266:
20428-20433
|
32. | Lundgren, E., E. Berger, and K.-E. Arfors. 1992. Effect of divalent cations on adhesion of polymorphonuclear leukocytes to matrix molecules in vitro. J. Leukocyte Biol. 51: 603-608 [Abstract]. |
33. | Maroudas, A. 1979. Physicochemical properties of articular cartilage. In Adult Articular Cartilage. M.A.R. Freeman, editor. Pittman, London. 215-290. |
34. | Miller, E.J.. 1972. Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry. 11: 4903-4909 |
35. |
Neame, P.J.,
Y. Sommarin,
R.E. Boynton, and
D. Heinegård.
1994.
The
structure of a 38-kDa leucine-rich protein (chondroadherin) isolated
from bovine cartilage.
J. Biol. Chem.
269:
21547-21554
|
36. | Oldberg, Å., P. Antonsson, K. Lindblom, and D. Heinegård. 1989. A collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin). EMBO (Eur. Mol. Biol. Organ.) J. 8: 2601-2604 [Abstract]. |
37. | Ruoslahti, E., and M.D. Pierschbacher. 1987. New perspectives in cell adhesion: RGD and integrins. Science (Wash. DC). 238: 491-497 |
38. | Salter, D.M., D.E. Hughes, R. Simpson, and D.L. Gardner. 1992. Integrin expression by human articular chondrocytes. Br. J. Rheumatol. 31: 231-234 |
39. |
Santoso, S.,
R. Kalb,
M. Walka,
V. Kiefel,
C. Mueller-Eckhardt, and
P.J. Newman.
1993.
The human platelet alloantigens Bra and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin
subunit ![]() |
40. |
Schiro, J.A.,
B.M.C. Chan,
W.T. Roswit,
P.D. Kassner,
A.P. Pentland,
M.E. Hemler,
A.Z. Eisen, and
T.S. Kupper.
1991.
Integrin ![]() ![]() |
41. |
Schonherr, E.,
P. Witsch-Prehm,
B. Harrach,
H. Robenek,
J. Rauterberg, and
H. Kresse.
1995.
Interaction of biglycan with type I collagen.
J. Biol.
Chem.
270:
2776-2783
|
42. |
Shimitzu, Y.,
G.A. Van Seventer,
K.J. Horgan, and
S. Shaw.
1990.
Regulated expression and binding of three VLA (![]() |
43. | Sommarin, Y., and D. Heinegård. 1983. Specific interaction between cartilage proteoglycans and hyaluronic acid at the chondrocyte cell surface. Biochem. J. 214: 777-784 |
44. | Sommarin, Y., T. Larsson, and D. Heinegård. 1989. Chondrocyte-matrix interactions. Attachment to proteins isolated from cartilage. Exp. Cell Res. 184: 181-192 |
45. |
Sonnenberg, A.,
C.J.T. Linders,
P.W. Modderman,
C.H. Damsky,
M. Aumailley, and
R. Timpl.
1990.
Integrin recognition of different cell-binding
fragments of Laminin (P1, E3, E8) and evidence that ![]() ![]() ![]() ![]() |
46. | Staaz, W.D., S.M. Rajpara, E.A. Wayner, W.G. Carter, and S.A. Santoro. 1989. The membrane glycoprotein Ia-IIIa (VLA-2) complex mediates the Mg2+-dependent adhesion of platelets to collagen. J. Cell Biol. 108: 1917-1924 [Abstract]. |
47. | Vogel, K., M. Paulsson, and D. Heinegard. 1984. Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem. J. 223: 587-597 |
48. | Wessel, D., and U. I. Flügge. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138: 141-143 |
49. |
Wilkins, J.A.,
D. Stupack,
S. Stewart, and
S. Caixia.
1991.
![]() |
50. | Woods, V.L. Jr, P.J. Schreck, D.S. Gesink, H.O. Pacheco, D. Amiel, W.H. Akeson, and M. Lotz. 1994. Integrin expression by human articular chondrocytes. Arthritis Rheum. 37: 537-544 |