Versican Interacts with Chemokines and Modulates Cellular
Responses*
Jun
Hirose
,
Hiroto
Kawashima
,
Osamu
Yoshie§,
Kei
Tashiro¶, and
Masayuki
Miyasaka
From the
Department of Bioregulation, Biomedical
Research Center, Osaka University Graduate School of Medicine, 2-2,
Yamada-Oka, Suita 565-0871, the § Department of
Microbiology, Kinki University School of Medicine, Ohno-Higashi,
Osaka-Sayama, 589-8511, and the ¶ Center for Molecular Biology and
Genetics, Kyoto University, Shogo-in, Sakyo-ku,
Kyoto 606-8507, Japan
Received for publication, August 18, 2000, and in revised form, November 8, 2000
 |
ABSTRACT |
We previously reported that versican, a large
chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma
cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding
was strongly inhibited by the chondroitinase digestion of versican or
by the addition of soluble chondroitin sulfate (CS) B, CS E, or heparan
sulfate. Furthermore, these glycosaminoglycans (GAGs) could bind
directly to the chemokines that bind versican. Thus, versican appears
to interact with chemokines via its GAGs. We next examined if versican
or GAGs affect secondary lymphoid tissue chemokine (SLC)-induced
integrin activation and Ca2+ mobilization in lymphoid
cells expressing a receptor for SLC, CC chemokine receptor 7. Interestingly, whereas heparan sulfate supported both
4
7 integrin-dependent binding
to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-IgG and
Ca2+ mobilization induced by SLC, versican or CS B
inhibited these cellular responses, and the extent of inhibition was
dependent on the dose of versican or CS B added. These findings suggest that different proteoglycans have different functions in the regulation of chemokine activities and that versican may negatively regulate the
function of SLC via its GAG chains.
 |
INTRODUCTION |
Proteoglycans are proteins that carry glycosaminoglycans
(GAGs).1 The common GAGs are
heparin, heparan sulfate (HS), chondroitin sulfate (CS), dermatan
sulfate, and hyaluronic acid. These GAGs have a large number of sulfate
or carboxyl groups and, hence, have a strong negative charge, which
makes it possible for proteoglycans to bind through their GAG chains
various positively charged molecules such as certain growth factors,
cytokines, and chemokines (1, 2). This interaction in the extracellular
matrix or on the cell surface has been suggested to play important
roles in vivo in the formation of immobilized gradients of
these humoral factors, in their protection from proteolytic
degradation, and in their presentation to specific cell-surface
receptors (3-5). There is an increasing body of evidence indicating
that HS proteoglycans are particularly important in promoting the
oligomerization of chemokines and in facilitating their presentation to
specific receptors (6-8). Sporadic reports indicate that CS
proteoglycans may also have this function (9, 10). For instance, CS
proteoglycans on human neutrophils specifically bind platelet factor 4 and are involved in the activation of neutrophils (9). An artificial proteoglycan modified with CS binds RANTES via its GAGs, which also
induces the activation of T cells expressing this molecule on their
surface (10). Thus, not only HS but also CS proteoglycans appear to
bind chemokines and to play important roles in the regulation of
chemokine functions.
Versican is a large CS proteoglycan (11, 12) expressed in various
tissues (13). We have recently reported that versican derived from a
renal adenocarcinoma cell line, ACHN, interacts through its CS chains
with leukocyte adhesion molecules such as L-selectin and CD44 (14, 15),
both of which have been implicated in leukocyte trafficking (16, 17).
Since versican has abundant CS side chains and is secreted into the
extracellular matrix, we speculated that it might also interact with
certain chemokines to help form haptotactic gradients in the tissues
where chemokines and versican are colocalized. In the present study we
examined the ability of versican to bind a large panel of chemokines
and the biological consequences of such binding. Our results indicate that versican can bind specific chemokines through its CS chains and
that the binding tends to down-regulate the chemokine function. This
raises the possibility that different proteoglycans have different
functions in the regulation of chemokine activities and that certain
proteoglycans, such as versican, may negatively regulate chemokine functions.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Versican was isolated from the culture supernatant
of a human renal adenocarcinoma cell line, ACHN, as described
previously (14). Epithelial-derived neutrophil attractant 78 (ENA-78), growth-related gene (Gro-
), interleukin-8,
interferon-inducible protein 10 (IP-10), platelet factor 4, lymphotactin, MCP-1, -2, -3, MIP-1
, MIP-1
, and RANTES were
purchased from Pepro Tech, Inc. (Rocky Hill, NJ). Recombinant human
SDF-1
(18) was purchased from Genetics Institute. Inc. (Cambrige,
MA). Recombinant human liver and activation-regulated chemokine (LARC),
pulmonary and activation-regulated chemokine (PARC), and thymus and
activation-regulated chemokine (TARC) were produced by using a
baculovirus expression system and purified as described
previously (19-21). EBI1-ligand chemokine was produced in
Escherichia coli and purified as described previously (22).
SLC and goat anti-human SLC polyclonal antibody were purchased from
DAKO Japan Co. Ltd (Tokyo, Japan). Anti-versican monoclonal antibody
(mAb) 2B1 (23), anti-CS mAb CS56 (24), chondroitinase ABC (EC
4.2.2.4), chondroitinase B (EC 4.2.2), keratanase (EC 3.2.1.103),
chondroitin, CS A (whale cartilage), CS B (pig skin), CS C (shark
cartilage), CS D (shark cartilage), CS E (squid cartilage), and heparan
sulfate (bovine kidney) were all purchased from Seikagaku Kogyo (Tokyo,
Japan). Rat MAdCAM-1-IgG (25) was kindly provided by Dr. Toshihiko
Iizuka (Tokyo Medical and Dental University).
Cells--
The murine T cell line TK-1 (26) and murine pre B
cell line L1.2 (27), kindly provided by Drs. Berhhard Holzmann
(Technische Universität Munich) and Eugene Butcher (Stanford
University School of Medicine), respectively, were cultured in RPMI
1640 supplemented with 10% fetal calf serum (FCS), 10 mM
HEPES, 2 mM L-glutamine, 1 mM
sodium pyruvate, 1% (v/v) 100× nonessential amino acids, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 50 µM
2-mercaptoethanol (RPMI-FCS). The human acute T cell leukemia Jurkat
cells were also cultured in RPMI-FCS. The L1.2 cells stable-expressing
transfected human CCR7 (L1.2/CCR7) was generated as described
previously (21) and was cultured in RPMI-FCS containing 0.8 mg/ml
Geneticin (G418, Sigma).
Dot Blot Analysis--
Various concentrations of chemokines were
spotted onto a Hybond C nitrocellulose membrane (Amersham Pharmacia
Biotech) and air-dried for 5 h. The membrane was blocked overnight
with 3% BSA, 0.1% NaN3 in PBS at 4 °C. After blocking,
the membrane was washed with PBS containing 1 mM
CaCl2, 1 mM MgCl2 (PBS+) containing 0.05% Tween 20, and 0.1% BSA. Subsequently, the membrane was
incubated with biotinylated versican (3 µg/ml) in the presence or
absence of various GAGs (10 µg/ml) for 1 h at room temperature.
After washing, ABC reagent (Vector Laboratories Inc., Burlingame, CA) was applied, and bound versican was detected using ECL (Amersham Pharmacia Biotech).
ELISA--
Versican (20 µg/ml) or GAGs (50 µg/ml) were
coated on 4-mm-diameter 96-well EIA/RIA plates (Costar, Corning
Inc.) overnight at 4 °C. The wells were washed with PBS+, and
blocked with FCS for 2 h at room temperature. SLC was added to the
versican- or GAG-coated wells and incubated for 2 h. After
washing, the wells were incubated with biotinylated goat anti-human SLC
(1 µg/ml, DAKO) in PBS+ containing 0.05% Tween 20 and 1% FCS for
1 h at room temperature. After washing, the wells were incubated
with streptavidin-conjugated alkaline phosphatase (1:500, Promega, Madison, WI) for 1 h. To quantify the reaction, Blue PhosTM
(Kirkegaard Perry Laboratories, Gaithersburg, MD) was added, and the
optical density was read at 620 nm in a microtiter plate reader
(InterMed, Tokyo, Japan). To determine the binding of MCP-1 to
versican, versican (20 µg/ml) was coated on 4-mm-diameter 96-well
EIA/RIA plates (Costar, Corning Inc.) overnight at 4 °C. The
wells were washed with PBS+ and blocked with 3% BSA in PBS+ for 2 h at room temperature. MCP-1 (0-3 µg/ml) was added to the
versican-coated wells and incubated for 2 h. After washing, the
wells were incubated with biotinylated goat anti-human MCP-1 (1 µg/ml, Santa Cruz, Biotechnology Inc. Santa Cruz, CA) and then
with peroxidase-conjugated streptavidin in PBS+ containing 0.1% BSA
and 0.05% Tween 20 for 1 h at room temperature. To quantify the
reaction, o-phenylenediamine (0.4 mg/ml) was used as the
substrate, and the optical density at 490 nm was read in a microtiter
plate reader. In some experiments, immobilized versican was treated
with chondroitinase ABC (0.6 units/ml, pH 8.0), chondroitinase B (0.06 units/ml, pH 8.0), or keratanase (0.6 units/ml, pH 8.0) overnight at
37 °C. After blocking, SLC (1 µg/ml) was added to the wells and
incubated for 1 h at room temperature. After washing, bound SLC
was detected with streptavidin-conjugated alkaline phosphatase and Blue
PhosTM as described above. To examine the efficacy of the enzyme
digestion, either mAb 2B1 (3 µg/ml) or CS56 (1:500 dilutions) was
added to the wells after the enzyme treatment. Peroxidase-conjugated
anti-mouse IgG or anti-mouse IgG + IgM was then added to detect the
binding of the primary antibodies. The binding was quantified by the
o-phenylenediamine reaction as described above.
Cell Adhesion Assay--
Cell adhesion to MAdCAM-1 induced by
SLC was determined as follows. SLC (0-3 µg/ml) and MAdCAM-1-IgG (100 ng/well) or human IgG (100 ng/well) were coimmobilized on 4-mm-diameter
96-well EIA/RIA plates (Costar, Corning Inc.) overnight at
4 °C. After washing twice with PBS, plates were blocked with FCS.
TK-1 cells labeled with 2',7'-bis-(2-carboxyethyl)-5-(and
6)carboxyfluorescein acetoxymethyl ester (BCECF-AM) (2 µM) were then added to the wells (1.2 × 105 cells/well) in triplicate. After a 20-min incubation at
37 °C, the wells were filled with RPMI-FCS and sealed with Parafilm. The plates were inverted for 30 min at 37 °C to allow nonadherent cells to detach from the bottom surface of the wells, after which the
medium containing unbound cells was removed by suction. The remaining
bound cells were lysed with 0.1% Nonidet P-40, and the fluorescence
intensity was read in a Fluoroskan II microplate fluorometer
(Labsystem, Tokyo, Japan). The results are expressed as the percentage
of cells bound and represent triplicate determinations.
Cell adhesion to MAdCAM-1 induced by SLC that had been immobilized on
versican or GAGs was determined as follows. Various combinations of
versican (300 ng/well), CS A, CS B, or HS (50 µg/ml) and MAdCAM-1-IgG
(100 ng/well) or human IgG (100 ng/well) were coimmobilized in the
wells overnight at 4 °C. After blocking with FCS, SLC (0-3 µg/ml)
was added to the wells for 2 h at room temperature. Unbound SLC
was removed by washing with PBS. Cell adhesion to each well was
determined as described above.
Ca2+ Mobilization Assay--
The Ca2+
mobilization assay was carried out according to the method of Nagira
et al. (28). In brief, L1.2, L1.2/CCR7, and Jurkat cells
were incubated at 37 °C for 1 h with 2 µM
Fura-2/AM (Sigma) at 1.0 × 106 cells/ml in RPMI
without FCS in the dark. Cells were washed twice and resuspended at
1.0 × 106 cells/ml in PBS+ containing 1% FCS. A
1.5-ml sample of cell suspension was placed in a cuvette and set into a
spectrofluorophotometer (RF1500, Shimadzu, Japan) with constant
stirring. SLC (1 µg/ml), SDF-1
(1 µg/ml), or leukotriene D4
(Sigma, 0.2 µg/ml) was preincubated with versican (0.1, 0.2, or 0.4 µg/ml) or GAGs (50 µg/ml) for 10 min at room temperature and added
to the cell suspension. Emission fluorescence intensity at 510 nm was
measured upon excitation at 340 nm (F340) and 380 nm (F380). Data were
presented as the ratio of F340 divided by F380 (R340/380). After each
measurement, R340/380 in the presence or absence of excess calcium was
determined by the sequential addition of 100 µl of 1% Triton X-100
with or without 100 µl of 0.5 M EDTA.
Binding of Radiolabeled SLC to CCR7 Transfectant Cells--
The
recombinant SLC was radioiodinated using Bolton-Hunter reagent
(Amersham Pharmacia Biotech) according to the manufacturer's instruction. L1.2 or L1.2/CCR7 cells (5 × 105 cells)
were incubated with 125I-labeled SLC (10 ng/ml) alone or in
the presence of unlabeled SLC (2 µg/ml) or increasing concentrations
of versican (0.001-10 µg/ml), CS A, CS B, or HS (0.5, 5, or 50 µg/ml) for 1 h. After incubation, cell suspensions were placed
on a mixture of dibutyl phthalate/olive oil (4:1) and separated from
unbound radiolabeled SLC by centrifugation (8,000 × g,
4 °C, 5 min). The binding of 125I-labeled SLC was
counted in a
counter. All determinations were made in triplicate.
 |
RESULTS |
Versican Binds Various Chemokines--
The binding of versican to
18 different chemokines was examined by dot blot analysis. Various
chemokines were spotted onto a nitrocellulose membrane, biotinylated
versican was added, and after washing, its binding was determined.
Anti-versican core protein mAb 2B1 and anti-CS mAb CS56 were also
spotted on the membrane to serve as positive controls for versican
binding. As shown in Fig. 1A,
biotinylated versican bound the CXC chemokines
interferon-inducible
protein 10 (IP-10), platelet factor 4, and SDF-1
, the C chemokine
lymphotactin, and the CC chemokines MCP-1, MCP-2, RANTES, liver and
activation-regulated chemokine (LARC), and SLC. In contrast,
biotinylated versican failed to bind interleukin-8 or MIP-1
, which
are known to bind HS (29-31).

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Fig. 1.
Versican binds various chemokines.
A, binding of versican to chemokines. Anti-versican mAb 2B1,
anti-CS mAb CS56, or chemokines (6.25-100 ng/spot) were spotted onto a
nitrocellulose membrane. After blocking, biotinylated versican (3 µg/ml) was applied to the membrane. Binding of biotinylated versican
was detected as described under "Experimental Procedures."
B, binding of SLC or MCP-1 to immobilized versican.
Closed ( ) and open circles ( ) indicate the
binding of chemokines to versican and blocking reagents, respectively.
Serial dilutions of SLC (0-3 µg/ml) or MCP-1 (0-3 µg/ml) were
added to the versican (300 ng/well)-coated wells or wells treated with
blocking reagents and incubated for 2 h. After washing, the
binding of chemokines to versican was measured by ELISA.
ENA-78, epithelial-derived neutrophil attractant 78;
Gro- , growth-related gene; IL-8,
interleukin-8; IP-10, interferon-inducible protein 10;
PF-4, platelet factor 4; LARC, liver and
activation-regulated chemokine; PARC, pulmonary and
activation-regulated chemokine; TARC, thymus and
activation-regulated chemokine; ELC, EBI1-ligand
chemokine.
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We then performed a reverse assay, where the ability of immobilized
versican to bind soluble chemokines was tested by ELISA. As shown in
Fig. 1B, versican bound SLC and MCP-1 in a
dose-dependent manner. Similarly, immobilized versican also
bound SDF-1
and lymphotactin in a dose-dependent manner
(data not shown).
Versican Binds Chemokines in a GAG-dependent
Manner--
To examine whether the chemokine binding is mediated by
the GAG chains on versican, the effects of GAG-degrading enzymes on the
binding of SLC to versican were examined. As shown in Fig. 2, top panel, both
chondroitinase ABC and chondroitinase B inhibited the binding of
versican to SLC, whereas neither of these enzymes affected the
reactivity of versican with anti-versican core protein mAb 2B1 (Fig. 2,
middle panel). In addition, chondroitinase ABC as well as
chondroitinase B almost completely abrogated the reactivity of versican
with the anti-CS mAb CS56, whereas keratanase failed to alter the
reactivity of versican with mAb CS56 (Fig. 2, bottom panel).
These results suggest that the GAGs on versican play an important role
in chemokine binding. However, the possibility remains that the core
protein of versican is also involved in SLC binding, since treatment
with chondroitinases did not completely inhibit the binding of SLC to
versican.

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Fig. 2.
Versican binds SLC through glycosaminoglycan
chains. Versican (300 ng/well) was coated onto the wells of an
ELISA plate. After blocking, the wells were untreated or treated with
chondroitinase ABC, chondroitinase B, or keratanase overnight at
37 °C. After washing, binding of SLC (1 µg/ml), anti-versican core
protein mAb 2B1 (3 µg/ml), or anti-CS mAb CS56 (1:500 dilution) was
detected as described under "Experimental Procedures." Values are
expressed as the percentage of specific binding compared with the
control.
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To further investigate the involvement of GAGs in the chemokine
binding, we examined whether exogenously added soluble GAGs could
inhibit the binding of chemokines to versican. Interestingly, CS B, CS
E, or HS almost completely abolished the binding of most of the
chemokines examined, whereas CS C and CS D inhibited the binding of a
restricted set of chemokines, and chondroitin and CS A were only
slightly inhibitory (Fig. 3A).
Given that CS B, CS E, and HS were extremely potent inhibitors of the
chemokine binding to versican, we speculated that these GAGs might have particularly high chemokine binding abilities compared with other types
of GAGs. To further examine this issue, various GAGs were immobilized
on plastic wells, and the binding of SLC was determined by ELISA. As
shown in Fig. 3B, SLC bound strongly to CS B, CS E, and HS
in a dose-dependent manner but bound other GAGs only weakly
or not at all, consistent with the results shown in Fig. 3A.
SLC did not bind to a nonsulfated type of GAG, chondroitin. Collectively, these results indicate that the GAG chains of versican are instrumental in chemokine binding and that the sulfation of GAG
chains may affect the binding.

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Fig. 3.
Certain GAGs are involved in chemokine
binding to versican. A, binding of versican to
chemokines is inhibited by certain GAGs. Chemokines were spotted onto
nitrocellulose membranes (50 ng/spot). After blocking, biotinylated
versican was applied to each chemokine spot in the presence or absence
of 10 µg/ml GAGs. In the case of SLC, GAGs were added at a
concentration of 30 µg/ml. The amount of bound versican was
quantified using a densitometer. Values are expressed as the percentage
of specific binding compared with the control without GAGs.
B, SLC directly binds certain GAGs. Chondroitin
(CH), CS A, CS B, CS C, CS D, CS E, or HS (50 µg/ml) was
coated onto the wells of an ELISA plate. After blocking, SLC (0.01, 0.03, 0.1, or 0.3 µg/ml) was added to the wells and incubated for
1 h. The binding of SLC to GAGs was assessed by ELISA. The values
shown have been corrected by subtracting the background. Representative
results from three separate experiments are shown. IP-10,
interferon-inducible protein 10; PF-4, platelet factor
4; LARC, liver and activation-regulated chemokine.
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Versican Down-regulates the
4
7
Integrin-mediated Cell Adhesion Induced by SLC--
We next
investigated the possible functional consequences of the
versican-chemokine interaction. First, we examined whether versican
could influence the SLC-induced activation of integrins using
4
7 integrin- and CCR7-expressing TK-1
mouse T cells. SLC, which we found to bind versican strongly, can
facilitate the adhesion of
4
7
integrin-positive cells to their ligand, MAdCAM-1 (32). To examine the
effect of versican on this chemokine action, SLC was coimmobilized in
wells with MAdCAM-1-IgG alone or with MAdCAM-1-IgG and versican. To
evaluate nonspecific binding, human IgG was used instead of
MAdCAM-1-IgG. As shown in Fig.
4A, left panel, SLC enhanced the binding of TK-1 cells to MAdCAM-1-IgG, showing a bell-shaped dose-response pattern in the absence of versican. This
up-regulated adhesion was apparently mediated by the interaction between
4
7 integrin and MAdCAM-1, since
the effect was completely abrogated by anti-
7 mAb and anti-MAdCAM-1
mAb (data not shown). By contrast, SLC immobilized on versican
up-regulated the cell adhesion only marginally, if at all (Fig.
4A, right panel). The lack of strong
up-regulation of TK-1 adhesion by SLC complexed with versican was not
simply due to the reduced availability of SLC to TK-1, because the
actual amount of SLC bound to the versican-coated wells would have been
sufficient to up-regulate cell adhesion, at least when SLC was
immobilized at a concentration of 1 µg/ml or higher (see Fig.
4B and its legend).

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Fig. 4.
Versican and CS B inhibit SLC-induced
adhesion of TK-1 cells to MAdCAM-1-IgG. A, effects of
versican on the SLC-induced adhesion of TK-1 cells to MAdCAM-1. The
left panel shows the adhesion of TK-1 cells stimulated with
immobilized SLC (0-3 µg/ml) to MAdCAM-1-IgG (100 ng/well ( )) or
human IgG (100 ng/well ( )). The right panel shows the
adhesion to MAdCAM-1-IgG ( ) or human IgG ( ) of TK-1 cells
stimulated with SLC that had been immobilized on versican. Cell
adhesion was determined as described under "Experimental
Procedures." B, binding of SLC to versican or other
immobilized samples. Binding of SLC to wells coated with versican
( ), versican, and MAdCAM-1-IgG ( ), versican and human IgG ( ),
MAdCAM-1-IgG ( ), or human IgG ( ) was determined by ELISA as
described under "Experimental Procedures." The open
circles ( ) indicate the control in which SLC was directly
coated on the plastic wells. Note that the amount of SLC bound to
versican-coated wells at a concentration of 1 µg/ml was comparable
with that of SLC bound to uncoated wells at 0.3 µg/ml, which could
effectively up-regulate cell adhesion (see A, left
panel). This indicates that the lack of strong up-regulation of
TK-1 adhesion in the presence of versican shown in A
(right panel) is not due to the insufficient availability of
SLC to TK-1 cells, since little up-regulated adhesion was observed even
when the SLC concentration was increased to 1 µg/ml or higher in
versican-coated wells (A). C, effects of GAGs (CS
A, CS B, or HS) on the SLC-induced adhesion of TK-1 cells to MAdCAM-1.
The experimental design was identical to that for A, except
that 50 µg/ml GAGs were used instead of versican. Adhesion of TK-1
cells to MAdCAM-1-IgG (100 ng/well ( )) or human IgG (100 ng/well
( )) was determined as described under "Experimental Procedures."
Results were expressed as the percentage of bound cells, where the
input cell number into each well was defined as 100%.
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We then examined whether the apparent inhibitory effect of versican was
mediated by its GAG chains. For this purpose, we used GAGs instead of
versican in the above-mentioned assay and compared their effects with
that of versican. As shown in Fig. 4C, the SLC·CS B
complex induced the adhesion of TK-1 cells to immobilized MAdCAM-1-IgG
only minimally, similar to the SLC-versican complex, whereas the
SLC·HS complex strongly up-regulated the cell adhesion (Fig.
4C), in agreement with previous reports that the
chemokine-HS complexes effectively activate integrins on lymphocytes
(7, 8). Little or no chemokine-induced cell adhesion was observed in CS
A-coated wells, probably because of the inability of CS A to bind SLC
(Fig. 3B). In addition, inhibitory effects of versican and
CS B appear specific for SLC-induced activation of integrins, since
versican or CS B did not inhibit phorbol 12-myristate
13-acetate-induced TK-1 adhesion (data not shown). These results
demonstrate that a certain type of CS, similar or identical to CS B,
negatively regulates SLC-induced cell adhesion, suggesting that
versican exerts its suppressive effect on chemokine function at least
partly through its GAG chains. In a separate study, we showed that
versican is indeed modified at least partly with CS B (15).
Versican or a Certain Type of GAG Can Down-regulate the
Chemokine-induced Intracellular Ca2+ Response--
We next
evaluated another effect of versican on chemokine functions by
examining the intracellular Ca2+ response elicited by SLC
and SDF-1
in L1.2/CCR7 (22) and Jurkat cells (33), respectively. As
shown in Fig. 5A, leukotriene
D4, SLC, or SDF-1
induced a rapid and transient intracellular
Ca2+ response. However, versican inhibited the
chemokine-induced intracellular Ca2+ response in a
dose-dependent manner. Versican did not affect the
leukotriene D4-induced intracellular Ca2+ response (Fig.
5A). When GAGs were used instead of versican, CS B was again
inhibitory on SLC-induced intracellular Ca2+ response,
whereas CS A and HS were not (Fig. 5B). These results further suggest that versican inhibits the ability of chemokines to
elicit intracellular signals, and it does so, at least in part, via its
GAG chains.

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Fig. 5.
Versican and CS B inhibit Ca2+
mobilization induced by chemokines. L1.2 cells stably transfected
with CCR7 (L1.2/CCR7) and Jurkat cells were loaded with Fura-2-AM and
stimulated with stimulators that had been preincubated for 10 min with
the indicated concentrations of versican (A) or 50 µg/ml
GAGs (B). The arrowhead indicates the time of
application of the stimulators. The intracellular calcium concentration
was monitored by measuring the fluorescence ratio (F340/F380).
Representative results from three separate experiments are shown. The
scale is shown as a bar on the right.
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How Does Versican Inhibit the Effects of SLC?--
To investigate
the mechanism by which versican inhibits chemokine functions, we
examined whether versican affects the binding of SLC to CCR7-expressing
cells. As shown in Fig. 6A,
the binding of 10 ng/ml 125I-SLC to L1.2/CCR7 cells was
inhibited by a 200-fold excess amount of unlabeled SLC, which reduced
the binding to the background level. In contrast, SLC binding was not
significantly inhibited by even 10 µg/ml versican, even though the
intracellular Ca2+ response induced by 1 µg/ml SLC in
L1.2/CCR7 cells was inhibited by 0.4 µg/ml versican (Fig. 5). Taking
the molecular size of SLC (15 kDa) and versican (1, 600 kDa) into
account, this means that the intracellular Ca2+ response
was inhibited at a molar ratio of 1:0.0037 (SLC:versican), whereas the
binding of SLC to its receptor was uninhibited, even at a molar ratio
of 1:9.37 (SLC:versican). Thus, the down-regulation of chemokine
function by versican is unlikely to be due to the inhibition of SLC
binding to its receptor. We next examined if GAGs affect the binding of
SLC to L1.2/CCR7 cells. The binding of 10 ng/ml 125I-SLC to
L1.2/CCR7 cells was inhibited by 50 µg/ml but not by lower doses of
CS B or HS (5 and 0.5 µg/ml), whereas, as shown in Fig. 5, the
intracellular Ca2+ response induced by 1 µg/ml SLC was
inhibited by 50 µg/ml of CS B. Taking the molecular size of CS B
(11-25 kDa) into consideration, this result implies that the
SLC-induced intracellular Ca2+ response was inhibited by CS
B at a molar ratio of 1:30.0-68.2 (SLC:CS B), whereas the binding of
125I-SLC to L1.2/CCR7 cells was inhibited only at much
higher molar ratios (1:3,000-6,820).

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|
Fig. 6.
Versican does not inhibit the binding of SLC
to its receptor. L1.2 cells (open bars) or L1.2/CCR7
cells (closed bars) were incubated with 125I-SLC
(10 ng/ml) in the presence or absence of unlabeled SLC (2 µg/ml) or
increasing concentrations of versican (A) or CS A, CS B, or
HS (B). The assay was performed in triplicate. Data are
shown as mean ± S.D.
|
|
 |
DISCUSSION |
It is generally accepted that proteoglycans interact with small
molecular weight humoral mediators such as chemokines that are produced
in the same tissue. These interactions protect the small molecules from
degradation, help them form concentration gradients in situ,
and help to present them to specific receptors on the cell surface
(1-5). However, it is not known whether proteoglycans always regulate
chemokine function positively, let alone the exact identity of the
proteoglycans involved, and it is also unclear whether this interaction
is a prerequisite for chemokine function. In the present study, we
showed that a large CS proteoglycan, versican, which is derived from a
human renal carcinoma cell line, binds a variety of chemokines through
its CS side chains. This binding was inhibited by the addition of
soluble CS B, CS E, or HS or by treatment with chondroitinases. We also
showed that, in the absence of a core protein, certain GAG chains such
as CS B, CS E, and HS could directly interact with a CC chemokine, SLC. Furthermore, we found that versican or certain types of CS can inhibit
the function of SLC; i.e. SLC that was complexed with versican or CS B was significantly less efficient than SLC alone in
inducing
4
7 integrin activation as well
as the intracellular Ca2+ response, and the extent of
inhibition by versican or CS B was dose-dependent. In
contrast, SLC that was complexed with HS was as efficient as SLC alone
in its chemokine activities. These findings suggest that the
interaction between proteoglycans and chemokines results in different
biological consequences, depending on the proteoglycans involved, and
that certain proteoglycans or their GAGs alone may negatively regulate
the function of chemokines in certain situations.
Although CS proteoglycans bear negatively charged GAG chains that can
interact with positively charged residues of chemokines, a nonspecific
electrostatic interaction may not be the sole factor determining the
interaction between CS proteoglycans and chemokines. The results shown
in Fig. 3 that nonsulfated chondroitin failed to interact with SLC
indicates that sulfation of the GAG chains is important in the
interaction. However, CS E but not CS D could interact with SLC even
though both are composed of disulfated disaccharides, indicating that
sulfation at a specific position is critical. In addition, CS B and CS
E, but not CS A, interacted with SLC even though all of these GAGs have
4-sulfation on N-acetylgalactosamine residues, suggesting
that 4-sulfation on N-acetylgalactosamine residues is not
sufficient but that sulfation of another position(s) and/or the
structure of uronic acid residues is critical for chemokine binding.
Based on the observations that binding of the CC chemokine SLC to
versican was inhibited by chondroitinase B, which specifically cleaves
the CS B chain, and that CS B itself bound SLC directly, it is likely
that CS B or a GAG containing a CS B-type structure on versican is
actually involved in the chemokine binding. This hypothesis is
supported by our GAG composition analysis using FACE
(fluorophore-assisted carbohydrate electrophoresis) and immunological analysis using anti-GAG mAbs, which indicated that versican is modified
with at least CS B and CS C (15). However, we do not know currently
whether versican is modified with CS E or a similar moiety, and further
structural analysis will be required to understand the exact
carbohydrate structure responsible for SLC binding.
The mechanism by which versican or a specific GAG chain (versican/GAG)
inhibits SLC function remains unclear, but several possibilities can be
considered: (i) versican/GAG inhibits SLC binding to specific chemokine
receptors; (ii) versican/GAG competes with endogenously expressed
cell-surface proteoglycans that may help present SLC to its receptors;
(iii) versican/GAG transduces a negative signal by binding to molecules
different from chemokine receptors; (iv) versican/GAG allows SLC to
bind its receptor even after forming a complex with SLC but inhibits an
appropriate signal to be transduced.
The first possibility, that versican inhibits the binding of SLC to
CCR7, appears unlikely since versican inhibited the SLC-induced intracellular Ca2+ responses (Fig. 5) at a dose that did
not affect the binding of SLC to its receptor (Fig. 6A). The
observations that the SLC-induced intracellular Ca2+
response was inhibited by CS B at a molar ratio of 1:30-68 (Fig. 5)
and that the binding of 125I-SLC to CCR7-expressing cells
was not inhibited at this dose range (Fig. 6B) suggest that
CS B may also exert its inhibitory function without inhibiting the
binding of SLC to its receptor. Concerning the second possibility, that
versican/GAG competes with endogenous proteoglycans that may help
present SLC to its specific receptor CCR7 and, thus, inhibit SLC
signaling, our experiments showed that SLC specifically bound to a
single class of high affinity receptors expressed on L1.2/CCR7 cells
with a Kd of 2.26 nM (data not shown),
indicating that endogenous cell-surface proteoglycans are not involved
in SLC binding in this cell line. In addition, our preliminary
experiments showed that the binding of SLC to L1.2/CCR7 cells was not
altered by treating the cells with chondroitinase or heparitinase (data
not shown). In line with this notion, it was recently reported that
cell-surface GAG chain expression is not necessary for chemokines such
as MIP-1
, MIP-1
, and RANTES to exert their biological effects
(34). Thus, the second possibility also appears unlikely. Concerning
the third possibility, that versican/GAG transduces a negative signal
by binding to a nonchemokine receptor type molecule, neither the TK-1
cells nor the L1.2/CCR7 cells used in this study bound to versican
immobilized in plastic wells (data not shown). Furthermore, these cells
do not express L-selectin or the active form of CD44, to which
ACHN-derived versican binds (14, 15). Thus, versican on its own does
not appear to bind to the cells used in this study, and hence, the
third possibility seems unlikely. The fourth possibility, that the SLC that is complexed with versican or certain GAGs is unable to transduce an appropriate chemokine signal after binding to CCR7, is currently difficult to exclude and may, at least in part, be involved in the
mechanism of inhibition. Consistent with this idea, it was recently
reported that soluble complexes of RANTES and GAG chains can bind to an
appropriate chemokine receptor but cannot induce intracellular
Ca2+ signaling (35). Collectively, the available data
suggest that versican/GAG does not inhibit SLC signaling through
inhibiting SLC binding to CCR7. Rather, the inhibition of SLC signaling
is probably at least partly due to inhibition of the chemokine signal after SLC binds its receptor. Still other possible mechanisms remain to
be completely excluded.
Versican binds certain chemokines but not all of them. It is notable
that versican tends to preferentially bind chemokines that attract
mononuclear leukocytes. Preliminary analysis of the amino acid
sequences of versican-reactive and nonreactive chemokines has failed to
identify any conserved amino acid residues or peptide regions that may
be involved in versican binding (data not shown). Other factors, such
as the tertiary structure, may explain the differences in binding affinity.
Although versican is found in the skin, brain, kidney, and aorta (13),
SLC is principally found in secondary lymphoid tissues (36). One might
thus argue that this particular combination of proteoglycan and
chemokine, i.e. versican and SLC, is not physiological. However, it should be pointed out that versican also binds SDF-1
that is expressed in normal human skin as well as in inflammatory infiltrates of autoimmune skin diseases (37). In the inflamed skin, a
large number of mononuclear cells expressing the receptor for SDF-1
,
CXCR4, selectively accumulates where SDF-1
is localized (37),
suggesting the possibility that SDF-1
is involved in the
infiltration of mononuclear cells into the inflamed skin. To inhibit
excessive infiltration of mononuclear cells, however, an excess amount
of SDF-1
produced in inflammatory infiltrates may have to be
eliminated locally. In this sense, our result showing that
versican-SDF-1
interaction results in down-regulation of SDF-1
activity (Fig. 5A) is interesting, pointing to the
possibility that versican expressed in the inflamed skin interacts with
SDF-1
to modulate the infiltration of mononuclear cells by
down-regulating SDF-1
activity, although this has yet to be
evidenced experimentally. In addition, several CS proteoglycans,
i.e. L-selectin-reactive CS proteoglycans of 150 and >200
kDa (38) and endoglycan (39), have been reported to be expressed in
lymph node high endothelial venules where SLC is produced (36). It
would be interesting to examine whether these CS proteoglycans can
interact with SLC to regulate the function of SLC in
situ.
Given that only specific kinds of GAGs can bind chemokines (Fig. 3), it
is likely that only versican species modified with specific GAGs can
bind chemokines. We previously showed that versican derived from ACHN
binds via its GAG chains, a leukocyte adhesion molecule, L-selectin
(14), and that certain GAGs such as CS B, CS E, and HS bind L-selectin
directly (15). This specificity of binding is quite similar to what we
observed for chemokine binding (Fig. 3B); i.e.
those GAGs that bind L-selectin also bind certain chemokines, and those
GAGs that do not bind L-selectin fail to bind the chemokines. Thus, we
speculate that versican species modified with L-selectin-reactive GAGs
also bind chemokines, which may be of particular importance in certain
in vivo situations such as in inflammatory responses, where
L-selectin and chemokines are concomitantly involved. The biological
significance of the interaction between versican and chemokines is an
important subject for further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. T. Imai (KAN Research Institute)
for helpful advice and Drs. T. Tanaka and T. Murai (Osaka University
Graduate School of Medicine) for helpful discussions and critical
reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid for Center
of Excellence Research and Scientific Research on Priority
Areas, Sugar Remodeling and Cellular Communications from the Ministry of Education, Science, and Culture, Japan, a grant from the Science and
Technology Agency, Japan, and a grant from Ono Pharmaceutical Co.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. Tel.:
81-6-6879-3972; Fax: 81-6-6879-3979; E-mail:
mmiyasak@orgctl.med.osaka-u.ac.jp.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M007542200
 |
ABBREVIATIONS |
The abbreviations used are:
GAG, glycosaminoglycan;
MAdCAM-1, mucosal addressin cell adhesion
molecule-1;
CCR7, CC chemokine receptor 7;
SLC, secondary
lymphoid-tissue chemokine;
HS, heparan sulfate;
CS, chondroitin
sulfate;
MCP, monocyte chemoattractant protein;
MIP, macrophage
inflammatory protein;
RANTES, regulated on activation, normal T
cell-expressed and -secreted;
SDF-1, stromal cell-derived factor 1;
mAb, monoclonal antibody;
FCS, fetal calf serum;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked immunosorbent
assay.
 |
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