CD44 binds a chondroitin sulfate proteoglycan, aggrecan
Takashi Fujimoto,
Hiroto Kawashima,
Toshiyuki Tanaka,
Mayumi Hirose,
Noriko Toyama-Sorimachi2,,
Yuji Matsuzawa1, and
Masayuki Miyasaka
Department of Bioregulation, Biomedical Research Center, and
1 Department of Internal Medicine and Molecular Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan
2 Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Hon-Komagome, Bunkyo-ku, Tokyo 113-8613, Japan
Correspondence to:
M. Miyasaka
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Abstract
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Here we report that CD44 binds a chondroitin sulfate (CS) proteoglycan, aggrecan, a major component of cartilage. Soluble CD44IgG and CD44+ cells bound to aggrecan from rat chondrosarcoma and bovine cartilage, immobilized on microtiter plates. In both cases, binding was blocked by a neutralizing anti-CD44 mAb or by the pretreatment of aggrecan with chondroitinase, but not hyaluronidase or keratanase, indicating that CD44 binds aggrecan in a manner dependent on CS side chains of aggrecan and that hyaluronic acid is not involved in the binding. Structural analysis showed that glycosaminoglycans of aggrecan from rat chondrosarcoma and bovine articular cartilage consist of mainly CS A and a mixture of CS A and C respectively. When immobilized on microtiter plates, both CS A and C bound CD44IgG, and the reaction was specifically inhibited by an anti-CD44 mAb. In addition, aggrecan augmented apoptosis in cells expressing CD44Fas chimeric molecules in synergy with a non-blocking anti-CD44 mAb IRAWB14.4, suggesting that CD44aggrecan interaction can induce oligomerization of the chimeric molecules. These results suggest that aggrecan interacts with CD44 to mediate cell adhesion and to trigger oligomerization of CD44 molecules, which may lead to intracellular signaling.
Keywords: aggrecan, CD44, chondroitin sulfate, extracellular matrix, proteoglycan
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Introduction
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CD44 is an integral membrane glycoprotein of a polymorphic family expressed on a wide variety of cell types, including lymphoid cells, myeloid cells, fibroblasts, epithelial cells and endothelial cells (14). This molecule has many isoforms that bear a variety of inserts in the membrane-proximal portion. CD44 binds some extracellular matrix components, such as hyaluronic acid (HA) (5), fibronectin (6), and collagen types I and VI (79), as well as a secretary granule proteoglycan, serglycin (10).
CD44 has been reported to play important roles in various immunological events: it mediates the release of tumor necrosis factor-
and IL-1ß (11), co-stimulates T cell proliferation (12,13), mediates lymphocyte rolling along the endothelium (14), and mediates the migration of activated T cells into inflamed sites (15). Of note is that CD44 is strongly up-regulated in inflamed synovial tissue (16), and anti-CD44 mAb treatment results in a marked reduction in tissue swelling and in leukocyte infiltration into arthritic joints in mice (17,18). Cross-linking of CD44 on synovial cells from rheumatoid arthritis (RA) patients with fragmented HA or treatment of these cells with appropriate anti-CD44 mAb up-regulates the expression of VCAM-1 (19). These facts have suggested that the interaction between CD44 and its ligands in the arthritic joints may play an important role in the pathogenesis of RA.
We have previously reported that CD44 binds a chondroitin sulfate (CS) proteoglycan, serglycin, that is secreted from the mouse T cell line CTLL-2 (10,20) and that enhances the release of granzyme A from CD44-expressing cytotoxic T cells (10). Since CD44 interacts with CS side chains of serglycin, we have been trying to determine if CD44 can interact with other CS proteoglycans as well. We have recently found that CD44 interacts with an extracellular matrix CS proteoglycan, versican, secreted from a renal adenocarcinoma cell line (21).
As an extension of these studies, we herein examined whether CD44 interacts with aggrecan, a major component of the cartilage matrix. Aggrecan is a CS proteoglycan with a core protein of high mol. wt (~230 kDa) encoded by a single gene. The core protein has two N-terminal globular domains, G1 and G2, linked by a short segment called the interglobular domain, and most of the keratan sulfate (KS) and CS side chains are attached to an extended segment between G2 and a C-terminal globular domain, G3 (22). In the course of arthritis, aggrecan is cleaved from the cartilage extracellular matrix by several proteinases, such as aggrecanases (23, 24) and other matrix metalloproteinases (25), and released into the synovial fluid.
In this report, we show that aggrecan interacts with CD44, and that this binding mediates cell adhesion and CD44 clustering, which may play an important role at inflammatory sites in vivo, such as in joints that have been damaged by arthritis.
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Methods
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Cells
A murine thymoma cell line, AKR1, provided by Dr Jayne Lesley (The Salk Institute, La Jolla, CA), was maintained in DMEM containing 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% (v/v) x100 non-essential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µM 2-mercaptoethanol (complete medium). AKR1 cells expressing murine standard form CD44 (A-CD44WT) or a chimeric protein made up of murine standard form CD44 and the cytoplasmic domain of human Fas (A44/F-4) have been described previously (26).
mAb and reagents
Rat anti-mouse CD44 mAb IRAWB14.4 (27) and KM201 (28) were purified by Protein GSepharose (Pharmacia Biotech, Uppsala, Sweden) from hybridoma culture supernatant. Anti-human CD44 mAb BRIC235 (29) was purchased from International Blood Group Reference Laboratory (Bristol, UK). Rat IgG1,
was purchased from Zymed (San Francisco, CA). Mouse IgG2b was purchased from Charles River Pharmservices (Southbridge, MA). Human IgG1, HA purified from rooster comb and bovine aggrecan purified from articular cartilage were purchased from Sigma (St Louis, MO). HA purified from human umbilical cord was purchased from ICN (Costa Mesa, CA). Rat aggrecan derived from a chondrosarcoma cell line, CS A (chondroitin-4-sulfate), CS C (chondroitin-6-sulfate), chondroitinase ABC from Proteus vulgaris, hyaluronidase from Streptomyces hyalurolyticus, and keratanase from Pseudomonas species were purchased from Seikagaku Kogyo (Tokyo, Japan). Alkaline phosphatase-conjugated streptavidin was purchased from Promega (Madison, WI). BluePhos phosphatase substrate was purchased from Kirkegaard & Perry (Gaithersburg, MD).
Neoglycolipids coupled with glycosaminoglycans were prepared by conjugating glycosaminoglycans to dipalmitoyl phosphatidylethanolamine, according to the method of Mizuochi et al. (30) with some modifications. Briefly, each glycosaminoglycan was dissolved in distilled water (10 mg/ml, 50 µl), then mixed with dipalmitoyl phosphatidylethanolamine that was dissolved in chloroform:methanol 1:1 (5 mg/ml, 95 µl); the glycosaminoglycanphosphatidylethanolamine mixture was sonicated for 10 min and incubated at 60°C for 2 h. After the incubation, sodium cyanoborohydride dissolved in methanol (10 mg/ml, 100 µl) was added and the mixture was incubated again at 60°C for 16 h. The reaction mixture was then lyophilized and dissolved in 2 M NaCl in 15% ethanol and spun to remove the insoluble material. The sample was precipitated with five equivalent volumes of ethanol, and the precipitate was dissolved in 0.2 M NaCl and applied to a TSKgel Phenyl Toyopearl 650M (Tosoh, Tokyo, Japan) column equilibrated with 0.2 M NaCl. After being eluted from the column with 30% methanol, the lipid-linked glycosaminoglycan was lyophilized and redissolved in distilled water before use.
CD44IgG ELISA
CD44IgG, which consists of the extracellular domain of human CD44 and the Fc portion of human IgG1, was prepared as described previously (5). Rooster comb HA (100 µg/ml in PBS, 50 µl/well) or aggrecan (1 µg/ml in PBS, 50 µl/well) was applied to the wells of 96-well flat-bottom microtiter plates (MS-8596F; Sumitomo Bakelite, Tokyo, Japan). After incubation overnight at 4°C, the wells were filled with PBS containing 3% BSA and incubated for 2 h at room temperature to block non-specific binding sites. Glycosaminoglycan-degrading enzymes were used as follows: chondroitinase ABC (10 mU/ml) in 0.1 M Trisacetic acid (pH 8.0) was applied to the wells and incubated at 37°C for 30 min. At this concentration and reaction time, chondroitinase ABC did not degrade HA, if at all. Hyaluronidase (40 turbidity reducing units/ml) in 0.1 M sodium acetate (pH 6.0) was applied to the wells and incubated at 37°C for 2 h. Both enzymes were used in the presence of 1 mM PMSF. After the enzyme treatment, human IgG1 or CD44IgG fusion protein (1 µg/ml) with or without mAb BRIC235 for blocking, control mouse IgG2b (20 µg/ml) or 5 mM EDTA was added, and the plate was incubated for 2 h at room temperature. The plates were then washed and incubated with alkaline phosphatase-conjugated goat anti-human IgG (American Qualex, San Clemente, CA) at a final concentration of 0.5 µg/ml and incubated for 1 h at room temperature. After the addition of the BluePhos substrate to the wells, the plates were read with a microplate reader NJ-2300 (Nalge Nunc International, Rochester, NY). Each experiment was run in triplicate.
For the competition assay using biotinylated aggrecan, CD44IgG (0.75 µg/ml, 25 µl/well) was applied to the wells of 96-well flat-bottom microtiter plates (3690; Costar, Corning, NY) and the plates were incubated overnight at 4°C. The wells were then filled with 3% BSA in PBS() and incubated for 2 h at room temperature to block non-specific binding sites. In some wells, mAb BRIC235 (20 µg/ml, 20 µl/well) was applied and pre-incubated at room temperature for 30 min. Biotinylated bovine aggrecan (0.5 µg/ml, 20 µl/well) was then added to the wells with or without various concentrations of unlabeled bovine aggrecan or rooster comb HA, and the plates were incubated at room temperature for 2 h. After washing, alkaline phosphatase-conjugated streptavidin (diluted 1:500, 25 µl/wells) was applied to the wells and incubated at room temperature for 1h. After washing, the BluePhos substrate was added to the wells and the plates were read with a microplate reader.
Neoglycolipids of CS A, CS C, KS and human umbilical cord HA (30 µg/ml, 250 µl/well) were immobilized on microtiter plates by drying at 60°C for 5 h. After blocking with 3% BSA in PBS, a binding assay with CD44IgG or human IgG1 was performed as described above.
Disaccharide analysis
After the treatment of aggrecan with chondroitinase ABC (100 mU/ml, 37°C overnight), unsaturated disaccharide analysis was carried out according to the method of Sugahara et al. (31,32). HPLC analysis was performed with the GULLIVER system (Nippon Bunko Engineering, Tokyo, Japan) on a YMC-Pack PA-03 column (YMC, Wilmington, NC; 4.6 mmx250 mm), with a programmed linear gradient elution from 16 to 578 mM NaH2PO4 over 60 min at a flow rate of 1.0 ml/min.
Cell adhesion assay
The cell adhesion assay was performed as described previously (33). Immobilization of HA or aggrecan on microtiter plates and enzyme treatment of the wells were done as described above. A-CD44WT cells were washed and resuspended in DMEM containing 5 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). After incubation for 40 min at 37°C, the cells were washed with and resuspended in serum-free DMEM. Cells were then stimulated with an anti-CD44 mAb IRAWB14.4 (5 µg/ml, for 30 min at 37°C), which enhances HA binding (27,34). The labeled cells (1x105 cells/well) were applied to the microtiter plates and incubated for 30 min in the presence or absence of anti-CD44 mAb. Non-adherent cells were removed by inverting the plate. Adherent cells were solubilized with 1% NP-40 in PBS and the fluorescence intensity of each well was measured with a Fluoroskan II (Labsystems Japan, Tokyo, Japan). Each experiment was run in triplicate. The background level, defined as the amount of binding to BSA, was subtracted from all values to yield the specific signal.
Apoptosis induction assay
A-CD44WT or A44/F-4 cells were resuspended in serum-free DMEM containing IRAWB14.4 (5 µg/ml). Cells (1x104/well) were seeded into each well of microtiter plates with or without 25 µg/ml rooster comb HA, rat aggrecan, CS A, CS C or KS and incubated for 7 h in a CO2 incubator at 37°C in 5% CO2. Apoptosis was detected by Cell Death Detection ELISA Plus (Roche Digagnostics, Mannheim, Germany). In brief, cells were lysed and the lysates were placed into a streptavidin-coated plate. A mixture of biotin-labeled anti-histone mAb and peroxidase-labeled anti-DNA mAb was then added, and the plate was incubated for 2 h. After washing the wells, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was added and the plate was read at 405 nm. The value thus obtained represents the amount of peroxidase-labeled anti-DNA mAb bound to the DNA component of the nucleosomes that were released from apoptotic cells.
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Results
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CD44 specifically binds aggrecan
First, we examined the binding of soluble human CD44IgG to aggrecan immobilized on plastic plates. CD44IgG bound both aggrecan from rat chondrosarcoma (rat aggrecan) and from bovine articular cartilage (bovine aggrecan) (Fig. 1A
), whereas control human IgG, L-selectinIgG or P-selectinIgG did not (Fig. 1A
and data not shown). The binding of CD44IgG to aggrecan was independent of the presence of divalent cations and was specifically inhibited by an anti-human CD44 blocking mAb, BRIC235 (Fig. 1A
). Since BRIC235 also inhibited the binding of CD44IgG to HA, this result suggests that the link protein homology domain of CD44 interacts with aggrecan, which also binds HA. This notion was supported by two lines of observation. First, unlabeled bovine aggrecan inhibited the binding of biotinylated rooster comb HA to immobilized CD44IgG in a dose-dependent manner (data not shown). Second, as shown in Fig. 1
(B), unlabeled rooster comb HA inhibited the binding of biotinylated bovine aggrecan to CD44IgG by 50% at a concentration of 100 ng/ml, while unlabeled bovine aggrecan needed ~3040 times higher concentration to inhibit the binding by 50%. Since the mol. wt of aggrecan and rooster comb HA are in a similar range (35,36), this result suggests that the binding affinity of CD44 to aggrecan is lower than that to HA, while CD44 uses the same or overlapping regions to recognize these molecules.

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Fig. 1. CD44 specifically binds aggrecan. (A) HA, rat aggrecan or bovine aggrecan was immobilized on microtiter plates, and the binding of human IgG1 or CD44IgG with or without EDTA, control antibody or BRIC235 was determined. (B) Biotinylated bovine aggrecan was applied to CD44IgG-coated wells, with or without varying concentrations of unlabeled bovine aggrecan (solid line) or rooster comb HA (broken line). The binding of labeled aggrecan was determined as described in Methods. All results are presented as means ± SD of experiments performed in triplicate.
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CS side chains on aggrecan are necessary for CD44 binding
In a previous study, we showed that CD44 binds a CS proteoglycan, serglycin, by interacting with CS side chains of serglycin (10). To determine whether CD44 binds CS or some other glycosaminoglycan side chain on aggrecan, we next examined the effects of glycosaminoglycan-degrading enzymes on the interaction between CD44IgG and aggrecan. Pretreatment of aggrecan with chondroitinase ABC markedly inhibited CD44IgG binding to immobilized aggrecan, whereas hyaluronidase or keratanase had little if any effect (Fig. 2
). The dose of keratanase we used was sufficient to block the binding of the anti-KS mAb to immobilized KS (data not shown). Similarly, hyaluronidase effectively abrogated the binding of CD44 to HA with the dose used in this series of experiments. These results indicate that CD44 recognize the CS side chain on aggrecan.

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Fig. 2. The binding of CD44 to aggrecan is dependent on the CS chains of aggrecan. Immobilized HA and aggrecan were treated with keratanase, hyaluronidase or chondroitinase ABC. After treatment with the enzymes, the binding of CD44IgG was examined as described in Methods. Results are presented as means ± SD of experiments performed in triplicate.
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To characterize the CS side chain involved in the recognition by CD44, the two kinds of aggrecan were digested with chondroitinase ABC and the disaccharides obtained were subjected to HPLC analysis. The major digest of rat aggrecan was eluted at the position of unsaturated 4-sulfated disaccharides (Fig. 3A
), and the major digest of bovine aggrecan eluted at the position of unsaturated 6- and 4-sulfated disaccharides (Fig. 3B
). These results indicate that the glycosaminoglycan chain of aggrecan consists primarily of CS A alone or mixture of CS A and CS C, and that these CS side chains are recognized by CD44.

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Fig. 3. Disaccharide analysis of glycosaminoglycans of aggrecan by HPLC. The oligosaccharide sample prepared by chondroitinase ABC treatment of aggrecan was fractionated on a YMC-Pack PA-03 column. The elution positions of authentic unsaturated chondro-disaccharides are indicated. (A) Oligosaccharides obtained from rat aggrecan. (B) Oligosaccharides obtained from bovine aggrecan. Disaccharide standards used were: Di-0S, 4,5-HexA( 13)GalNAc; Di-6S, 4,5-HexA( 13)GalNAc(6-O-sulfate); Di-4S, 4,5-HexA( 13)GalNAc(4-O-sulfate); Di-diSD, 4,5-GlcA(2-O-sulfate)(§13)GalNAc(6-O-sulfate); DidiSE, 4,5-HexA-( 13) GalNAc(4,6-O-sulfate); Di-triS, 4,4-HexA(2-O-sulfate)( 13) GalNAc(4,6-O-disulfate).
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To verify whether these glycosaminoglycans could directly be recognized by CD44, we next prepared neoglycolipids of these glycosaminoglycans and examined binding of CD44. As shown in Fig. 4
(A), neoglycolipids of CS A, CS C and HA allowed the binding of soluble CD44IgG, while the neoglycolipid of KS did not. The binding was inhibited by BRIC235 but not by an isotype-matched antibody (Fig. 4B
). Although the data are not shown, the efficiency of immobilization was comparable among the glycosaminoglycans used. Collectively, these results show that CD44 can directly interact with both CS A and CS C chains.

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Fig. 4. Direct binding of soluble CD44IgG to the immobilized glycosaminoglycans. (A) CD44IgG or human IgG1 was applied to wells that had been coated with lipid alone or with the lipid-linked CS A, CS C, KS or HA. The binding was determined as described in Methods. (B) CD44IgG binding to the immobilized lipid-linked CS A and CS C was also examined in the presence or absence of anti-CD44 mAb BRIC235 or the control antibody. Results are presented as means ± SD of experiments performed in triplicate.
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CD44aggrecan interaction mediates cell adhesion
We next examined whether the interaction of CD44 on the cell surface with immobilized aggrecan could mediate cell adhesion. As shown in Fig. 5
, AKR1cells expressing murine CD44 bound both immobilized rat aggrecan and HA in a divalent cation-independent manner, and this binding was almost completely inhibited by the anti-murine CD44 mAb KM201, which blocks CD44 binding to HA. No binding was observed with CD44 AKR1 cells (data not shown). CD44+ cells also bound to bovine cartilage aggrecan, albeit less efficiently than to rat chondrosarcoma aggrecan, and the binding was completely blocked by KM201 (data not shown). Chondroitinase ABC pretreatment of rat aggrecan strongly inhibited cell binding, indicating that CD44+ cells bind aggrecan in a CS-dependent manner.

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Fig. 5. Interaction of cell-surface CD44 with aggrecan mediates cell adhesion. HA and rat aggrecan immobilized on microtiter plates were untreated or treated with hyaluronidase or chondroitinase. CD44 transfected AKR1 cells were labeled with BCECF-AM, stimulated with IRAWB14.4 and added to the HA- or aggrecan-coated wells with or without KM201 or control antibody. The ratio of bound cells to the total number of cells added to the well is shown. Results are presented as means ± SD of experiments performed in triplicate.
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Aggrecan augments the apoptotic signal mediated by a CD44Fas chimeric protein
To evaluate the functional significance of the CD44aggrecan interaction, we next performed several functional assays with which signals mediated through CD44 have been successfully detected previously. First, we attempted to detect the release of cytokines such as MIP-1
and IL-12 from mouse peritoneal exudate cells or mouse alveolar macrophage cell line MH-S upon stimulation via CD44 with HA or aggrecan (37,38). However, the cytokine release was detectable subsequent to stimulation with HA but not aggrecan (data not shown). Similarly, the proliferation of mouse spleen cells (39) was observed upon stimulation of the cells with HA but not aggrecan (data not shown). Significant changes in Ca2+ influx in mouse T lymphoma cell line BW5147 (40) or induction of CD44 shedding from the cell surface of BW5147 or mouse T lymphoma cell line EL-4 (17,41) was not detectable with either HA or aggrecan (data not shown). The only assay with which we could detect the signal mediated by the interaction between CD44 and aggrecan was a death induction assay using cells expressing murine CD44human Fas chimeric protein (26). In this assay apoptosis was used as a parameter of CD44 oligomerization in cells expressing the chimeric CD44Fas, subsequent to stimulation of cells via the CD44-derived extracellular domain. As shown in Fig. 6
, the anti-murine CD44 mAb IRAWB14.4 induced apoptosis in CD44Fas-expressing AKR1 cells (26), which was enhanced by the addition of rat or bovine aggrecan, while the addition of HA and CS had no, or minimal, effect. Since oligomerization of Fas is essential for Fas-mediated apoptotic signals (42), our results suggest that aggrecan can augment apoptosis in cells expressing CD44Fas chimeric proteins through the oligomerization of these molecules.

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Fig. 6. Aggrecan augments apoptosis of CD44Fas-expressing cells induced by anti-CD44 mAb IRAWB14.4. Wild-type CD44 transfectants (A-CD44WT) or CD44Fas transfectants (A44/F-4) stimulated with IRAWB14.4 were incubated for 7 h with or without HA, rat aggrecan, bovine aggrecan, CS A, CS C or KS. Apoptosis was quantified as described in Methods. Results are presented as means ± SD of experiments performed in triplicate.
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Discussion
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In this study we report that CD44 binds aggrecan derived from rat chondrosarcoma and bovine articular cartilage, and that the binding was dependent on CS chains of aggrecan. The CD44aggrecan interaction is apparently independent of HA, since hyaluronidase had no effect on the binding (Figs 2 and 5
), even though the dose of hyaluronidase used was sufficient to block CD44HA interactions. The KS side chains of aggrecan were also apparently uninvolved in the interaction with CD44. The binding site of aggrecan on the CD44 molecule appears to overlap with, or at least to be very close to, the hyaluronate-binding site in the link protein homology domain, since the aggrecan binding to human CD44 was blocked with mAb BRIC235, which can block the binding of hyaluronate by recognizing the epitope 2a in the link protein homology domain of human CD44 (43). Similarly, the binding of murine CD44 to aggrecan was blocked with mAb KM201, which can also block CD44hyaluronate binding by recognizing an epitope in the link homology domain of mouse CD44. We also demonstrated that the binding of soluble biotinylated aggrecan to immobilized CD44IgG was inhibited by soluble unlabeled HA in a dose-dependent manner (Fig. 1b
). These results strongly indicate that the CD44aggrecan interaction is mediated by the link protein homology domain of CD44 and the CS GAG chain of aggrecan.
Disaccharide analysis indicated that the CS side chains of rat chondrosarcoma aggrecan are exclusively CS A, and those of bovine cartilage aggrecan are CS A and CS C (Fig. 3
). We have previously reported that CD44 binds a CS proteoglycan serglycin, that is modified with either CS A or CS C, or a mixture of both (depending on the cell type), in a CS-dependent manner (10,20), and that depletion of these CS chains from serglycin results in the loss of binding by CD44. To further investigate whether CD44 can directly interact with the CS A and CS C chains, we used neoglycolipid preparations of these GAG chains and found that CD44Ig can specifically bind both CS A and CS C (Fig. 4
). Note, however, that previous attempts to detect the interaction between CD44 and GAG chains in the absence of core protein have yielded conflicting results (5,4446), and that we ourselves were also unable to detect significant binding of CD44 with CS chains previously (10). The reason why we could clearly detect binding of CD44 with CS A and CS C in the present study (Fig. 4
) might have been partly due to the fact that we could immobilize these GAG chains more efficiently than in previous studies by attaching a lipid tail to them. According to the fact that CD44 directly binds CS A and CS C, we suggest that CD44 should be able to interact with other proteoglycans modified with these CS chains in general. In accord with this implication, we recently demonstrated that CD44 also binds another CS proteoglycan, versican, bearing at least CS C (21). However, the simple presentation of the appropriate CS chains on proteoglycans may not be sufficient to allow binding to CD44. As seen with the selectins (4749), certain properties, such as the extent to which the sugar chains are clustered, their numbers, sulfation and other modifications, may also affect the binding. The exact requirements for binding need to be investigated further.
To examine whether CD44aggrecan interaction can signal cells, we have employed an apoptosis assay using cells expressing a CD44Fas chimeric protein. Our results showed that aggrecan can augment apoptotic cell death in CD44Fas-expressing cells in synergy with a non-blocking anti-CD44 mAb IRAWB14.4, suggesting that aggrecan induces oligomerization of the chimeric molecules, which may lead to intracellular signaling. Aggrecan alone, without IRAWB14.4, could not generate apoptotic signals (data not shown), which may be explained by the fact that CD44Fas chimera-expressing cells do not have sufficient ligand-binding ability without IRAWB14.4 stimulation (data not shown). In this assay, however, a mere oligomerization of CD44Fas chimeric protein may not to be sufficient to induce apoptosis into cells, since certain mAb cannot induce apoptosis even after further cross-linking with secondary antibody (26). Thus, it seems possible that not only oligomerization of CD44Fas chimeric protein with aggrecan, but also some other factors, e.g. stimulation of specific epitopes in the extracellular domain of CD44, may be involved in apoptosis induction by aggrecan. The reason why CD44-binding glycosaminoglycans, HA, CS A and CS C had very little effect to augment apoptosis in synergy with anti-CD44 mAb could have been due to the extent of sugar chain clustering, in the absence of core protein, which could affect the extent of CD44Fas chimera cross-linking.
Unfortunately we were unable to demonstrate that aggrecan induces cytokine release, cell proliferation, Ca2+ mobilization or CD44 shedding. It is currently unclear whether these negative results were due to the assay conditions we used or to inherent characteristics of aggrecan. The signal-transducing activity of CD44aggrecan interaction should be examined in more detail in the future, probably by a more direct approach such as the examination of changes in the tyrosine phosphorylation of Src kinases (5052), activation of Rho-like GTPases (53) or activation of NF-
B (54), which have been reported to take place subsequent to the appropriate ligation of CD44.
CD44 is hardly expressed in normal synovial tissue (16) where aggrecan is selectively localized and hence it is unlikely that aggrecan interacts with CD44 in vivo, unless certain pathological responses are initiated in situ, such as the mechanical disruption of the synovial tissue and/or the invasion of CD44+ leukocytes into the synovium, which can occur in arthritis of various etiologies. Consistent with this premise, aggrecan in the extracellular matrix of cartilage is actually degraded in the course of arthritis by aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-11), which cleave it at the N-terminal interglobular region between the G1 and G2 domains (23,24). As a result, the C-terminal fragment of aggrecan with its CS-rich domain is released from the cartilage matrix and detected in synovial fluid of the arthritic joint (5557). A separate study by us indicates that aggrecan is also degraded by another ADAMTS member, ADAMTS-1, which cleaves a site in the CS attachment domain (58). Therefore, soluble degradation products of aggrecan generated by these enzymes contain CS side chains in any case, and, like the degraded low-mol.-wt HA generated in the inflamed synovium (19,38), they are potentially active in stimulating CD44+ cells. Concurrently, strong up-regulation of CD44 is observed in infiltrating leukocytes and also in endothelial cells in inflamed synovial tissues (16). Hence, a future study should be directed to examine the functional ability of the aggrecan degradation products to interact with CD44, which may promote further understanding of the biological significance of the CD44aggrecan interaction in vivo.
In summary, we have demonstrated in the present study that CD44 interacts with aggrecan in a manner dependent on the CS chains of aggrecan. Stimulation of CD44 with aggrecan appears to trigger oligomerization of CD44 molecules, which may lead to intracellular signaling. Thus, similar to hyaluronate, aggrecan may activate CD44-bearing cells in vivo, although where and when such interactions can occur in vivo remains to be experimentally verified.
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Acknowledgments
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This work was supported in part by a Grant-in-Aid for COE Research and Scientific Research on Priority Areas: Sugar Remodeling and Cellular Communications from the Ministry of Education, Science and Culture, Japan, grants from the Science and Technology Agency, Japan, and Ono Pharmaceutical Co.
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Abbreviations
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BCECF-AM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester |
CS chondroitin sulfate |
HA hyaluronic acid |
KS keratan sulfate |
RA rheumatoid arthritis |
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Notes
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Transmitting editor: M. Taniguchi
Received 3 October 2000,
accepted 6 December 2000.
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