Enhancement of Cell Adhesion and Spreading by a Cartilage-specific Noncollagenous Protein, Cartilage Matrix Protein (CMP/Matrilin-1), via Integrin alpha 1beta 1*

Seicho MakihiraDagger , Weiqun Yan§, Shigeru Ohno, Takeshi Kawamotoparallel , Katsumi Fujimotoparallel , Akinobu Okimura, Eri Yoshidaparallel , Mitsuhide Noshiroparallel , Taizo HamadaDagger , and Yukio Katoparallel **

From the Departments of Dagger  Prosthetic Dentistry,  Orthodontics, and parallel  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 alpha 1 or beta 1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha 2, alpha 3, alpha 5, beta 2, alpha 5beta 1, or alpha Vbeta 5 had little effect on cell adhesion or spreading. The antibody to integrin alpha 1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha 1 subunit. Unlabeled CMP competed for the binding to integrin alpha 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 alpha 1beta 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

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 alpha -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 alpha  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.

Numerous adhesion proteins are recognized by integrins, which are heterodimeric proteins with two (alpha  and beta ) membrane-spanning subunits. The extracellular domain of the alpha  subunit has divalent cation-binding sites (14). The alpha  subunits play crucial roles in determining the ligand specificity. Chondrocytes express integrins alpha 1beta 1, alpha 2beta 1, alpha 3beta 1, alpha 5beta 1, alpha Vbeta 3, and alpha Vbeta 5 (15, 16), but their ligands in cartilage have not been fully defined.

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 alpha 1beta 1 plays a pivotal role in the CMP-mediated cell adhesion.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Antibodies-- Mouse neutralizing monoclonal antibodies (mAbs) to human integrins alpha 1 (FBI2), alpha 2 (P1E6) (17), alpha 3 (ASC-6) (18), alpha 5 (P1D6) (19), beta 2 (P4H9-A11) (20), alpha 5beta 1 (JBS5), and alpha Vbeta 5 (P1F6) (21); rabbit antisera to human integrins alpha 1, alpha 2, alpha V, beta 1, and beta 5; and rabbit antiserum to collagen type I were purchased from Chemicon International, Inc. (Temecula, CA). Mouse neutralizing mAbs to human integrin alpha 1 (5E8D9) (22) and beta 1 (DE9) (19) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse neutralizing mAbs to human integrin alpha 2 (P1E6) (23) and alpha 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).

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).


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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% beta -mercaptoethanol (beta -ME) and stained with silver nitrate.

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 alpha -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.

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 alpha -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.

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 (alpha 1, alpha 2, alpha V, beta 1, or beta 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.).

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 alpha 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

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).


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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).

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).


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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.

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).


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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.

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).


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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.

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 alpha -modified Eagle's medium suppressed the spreading of chondrocytes on CMP-coated dishes (Fig. 6A). The concentrations of Mg2+ and Ca2+ in alpha -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.

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 alpha 1 or beta 1 (Fig. 7). The mAb against alpha 2, alpha 3, alpha 5, beta 2, alpha 5beta 1, or alpha Vbeta 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 alpha 1 or beta 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 alpha 1 and beta 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 alpha 1 (5E8D9) (closed circles), alpha 2 (P1E6) (open triangles), alpha 3 (P1B5) (closed triangles), or beta 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.

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 alpha 1, alpha 2, alpha V, beta 1, and beta 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 (alpha 1, alpha 2, alpha V, beta 1, or beta 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 alpha 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 alpha 1 precipitated the 200-kDa integrin alpha 1 subunit in MRC5 lysates (Fig. 9B). The molecular mass of the integrin alpha 1 subunit in human chondrocytes is ~200 kDa under nonreducing conditions (15). In other experiments, the mAb to integrin alpha 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 alpha 1 (data not shown).


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Fig. 9.   Precipitation of 125I-CMP and the integrin alpha 1 subunit using the antiserum to integrin alpha 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 alpha 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.

In the presence of increasing concentrations of 125I-CMP and the antiserum to integrin alpha 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 alpha 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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Fig. 10.   Interaction between 125I-CMP and the integrin alpha 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 alpha 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 alpha 1 subunit, and increasing concentrations of unlabeled native CMP.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1 or beta 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 alpha 1beta 1 plays a pivotal role in the CMP-mediated cell adhesion and spreading.

Of the various antibodies to integrin subunits examined here, only the antibody to integrin alpha 1 coprecipitated 125I-CMP in cell lysates. Unlabeled native CMP competed for the binding to integrin alpha 1 with 125I-CMP. Although the anti-integrin beta 1 mAb inhibited the effect of CMP on cell adhesion, this mAb (data not shown), as well as the antiserum to integrin beta 1, did not consistently precipitate 125I-CMP in cell lysates. Under these conditions, the antibody to integrin beta 1 may coprecipitate the alpha 1 subunit only at low levels. In addition, the affinity of integrin beta 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 alpha 1 subunit even in the presence of detergents (Nonidet P-40 and SDS).

Integrin alpha 1beta 1, as well as alpha 2beta 1, binds to collagen under some experimental conditions. However, the precipitation of CMP with anti-integrin alpha 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

Chondroadherin is also an adhesion protein prominently expressed in cartilage and binds to integrin alpha 2beta 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.

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 alpha 1beta 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.

    ACKNOWLEDGEMENT

We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for use of the facilities.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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