From the Department of Laboratory Medicine and
Pathology, University of Minnesota, Minneapolis, Minnesota 55455, the
¶ Department of Orthopedic Surgery and Biochemistry, University of
Minnesota, Minneapolis, Minnesota 55455,
Athena Neuroscience
Inc., South San Francisco, California 94080, the ** Division of
Hematology, School of Medicine, University of Washington, Seattle,
Washington 98195, and the
Biomedical
Engineering Institute, University of Minnesota, Minneapolis,
Minnesota 55455
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ABSTRACT |
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We have previously reported that
4
1 (but not
5
1) integrin-mediated melanoma cell
adhesion is inhibited by removal of cell surface chondroitin sulfate
glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate
proteoglycan plays a role in modulating the adhesive function of
4
1 integrin. In the current study,
we demonstrated that
4
1 integrin binds to
CSGAG. We have identified a peptide from within
4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell
surface melanoma chondroitin sulfate proteoglycan, indicating that SG1
represents a CSGAG binding site within the
4 integrin
subunit. Soluble SG1 inhibits
4
1
integrin-mediated human melanoma cell adhesion to CS1. Polyclonal
antibody generated against the peptide inhibits melanoma cell adhesion
to CS1, and the inhibition is reversed by Mn2+ and an
activating monoclonal antibody anti-
1 (8A2).
Additionally, pretreatment of cells with anti-SG1 IgG inhibits the
expression of the monoclonal antibody 15/7 epitope in the presence of
soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand
binding by
4
1 integrin. These results
demonstrate that
4
1 integrin interacts
directly with CSGAG through SG1 site, and that this site can affect the
ligand binding properties of the integrin.
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INTRODUCTION |
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Tumor cells must adhere to a variety of extracellular matrix
(ECM)1 proteins and molecules
on other cells as they invade and metastasize. These interactions of
tumor cells have a profound effect on their phenotype (1, 2). Integrins
are a family of receptors that are fundamentally important for
mediating cell adhesion to ECM proteins (3).
4
1 integrin is important in tumor cell
invasion and metastasis, although its exact role is complex and not
completely understood. This integrin is expressed on many hematopoietic
malignancies and also on nonhematopoietic tumors, such as melanomas (4, 5). It is unusual among integrins in that it binds to both ECM
components (e.g. fibronectin (FN)) and the Ig superfamily adhesion receptor VCAM-1, which is expressed on activated endothelial cells and other cell types (4).
4
1
integrin has been implicated in tumor cell arrest and/or extravasation,
because agents that interfere with
4
1
integrin can inhibit tumor cell/endothelial cell interactions in
vitro or experimental metastasis and pulmonary retention of
injected tumor cells in mice (6, 7).
4
1
integrin also binds to itself and promotes homotypic cell adhesion (8, 9).
Various monoclonal antibodies (mAbs), divalent cations, or phorbol
12-myristate 13-acetate can regulate 4
1
integrin-mediated cell adhesion in lymphoid cell systems (10-13). This
regulation of integrin function results from conformational alterations
induced by the direct binding of these reagents to integrin subunits or by indirect (so called inside-out) effects of signaling pathways on
integrin conformation. Distinct conformational changes associated with
4
1 integrin activation or ligand binding
have been identified by using specific mAbs. One mAb, 15/7
(anti-
1 integrin), detects an epitope that is induced by
ligand binding or is exposed on activated integrins in lymphoid cells
(14). Although a role for
4
1 integrin has
clearly been established in influencing various aspects of tumor cell
biology, the mechanisms by which tumor cells control the functional
activity of this integrin merits further consideration.
Cell surface proteoglycans (PGs) are important in modulating cell adhesion and motility (15, 16). Many cell adhesion-promoting components in ECM or on cell surfaces (e.g. CD31 or PECAM) can bind PGs and support adhesion as a result of this interaction (17). PGs act as co-receptors for integrins and play a role in transducing signals. Cell surface proteoglycans interact with ECM proteins and often colocalize with integrins and/or may stimulate integrin-mediated signaling through outside-in/inside-out mechanisms. Syndecan-4 localizes in focal contacts (18-21), whereas syndecan-1 associates with stress fibers (22). Recently, it has also been reported that Raji cells transfected with syndecan-1 spread on substrata coated with fibronectin, whereas mock transfectants failed to spread (23). Furthermore, the transfectants adhered and spread on substrata coated with an anti-syndecan-1 core protein monoclonal antibody (23). These results suggest that the core protein of certain proteoglycans can modulate integrin-mediated cell spreading as a result of intracellular signaling events that are stimulated by clustering of the core proteins.
Previous studies have demonstrated that melanoma chondroitin sulfate
proteoglycan (MCSP) might play a role in melanoma cell adhesion and
invasion. For example, certain mAbs against MCSP will partially inhibit
melanoma cell adhesion to cultured endothelial cells (24). Harper
et al. (25, 26) have reported that mAb 9.2.27 inhibits human
melanoma cell adhesion and cytoplasmic spreading on ECM proteins, such
as collagen, or on collagen-fibronectin complexes and that this mAb can
also inhibit anchorage-independent cell growth. Additionally, at least
one anti-NG2 mAb (NG2, the rat homologue of human MCSP (27)) has been
demonstrated to inhibit melanoma cell invasion through reconstituted
basement membranes in vitro (28). Recently, human MCSP was
cloned by Pluschke et al. (29). We have also demonstrated
that clustering of MCSP stimulates 4
1
integrin-mediated cell spreading and focal contact formation in
melanoma cells (30), implying a role for the MCSP core protein in
modulating integrin function by an inside-out signaling mechanism.
Furthermore, we have previously shown that removal of cell surface
chondroitin sulfate glycosaminoglycan (CSGAG) by chondroitinase ABC or
inhibition of synthesis of chondroitin sulfate proteoglycan (CSPG) by
p-nitrophenyl
-D-xyloside inhibits
4
1 but not
5
1 integrin-mediated cell adhesion,
suggesting an important role for cell surface CSGAG in enhancing
4
1 integrin function (31).
In the current study, we demonstrated that
4
1 integrin can bind to CSGAG.
Furthermore, we used a synthetic peptide approach for identifying
potential CSGAG binding site(s) within the
4 integrin
subunit. One site, defined by synthetic peptide SG1 (KKEKDIMKKTI), directly binds to MCSP proteoglycan, and when used as a soluble antagonist, it inhibits melanoma cell adhesion to CS1. Polyclonal antibody generated against this peptide (anti-SG1 IgG) also inhibits
4
1 integrin-mediated melanoma cell
adhesion, and the inhibition can be reversed by Mn2+ and
mAb 8A2. Furthermore, anti-SG1 IgG prevents the CS1-induced exposure of
the mAb 15/7 epitope. These results demonstrate that
4
1 integrin interacts directly with CSGAG
through the SG1 site and that this interaction can regulate the ligand
binding properties of the integrin.
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MATERIALS AND METHODS |
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Cell Culture-- Highly metastatic human melanoma cells (A375SM), which were selected by in vivo experimental metastasis assays of parent A375P cells in nude mice, were kindly provided by Dr. I. J. Fidler (M. D. Anderson Cancer Center, University of Texas, Houston, TX) (32). SKMEL-2 was purchased from the American Type Culture Collection (Rockville, MD). The cells were maintained in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 50 µg/ml gentamycin, vitamin solution, and 1 mM sodium pyruvate. Ramos cells were kindly given by Dr. Tucker Lebien (Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. These cells were routinely used after fewer than 15 passages from frozen stocks.
mAbs--
P4C2 (anti-4), P1D6
(anti-
5), and P5D2 (anti-
1) were provided
by Dr. Elizabeth A. Wayner (University of Washington, Seattle, WA). The
specificity of these mAbs was demonstrated elsewhere (33-36). mAbs
15/7 (anti-
1) and 8A2 (anti-
1) were also
used (14, 37). Anti-MCSP core protein antibody (9.2.27) was kindly
given by Dr. Ralph Reisfeld (The Scripps Research Institute, San Diego, CA) (38). Anti-CD44 mAb (clone P1G12) was purchased from Chemicon International (Temecula, CA).
Protein Isolation-- Human plasma FN was purified as a by-product of factor VIII production by sequential ion-exchange and gelatin affinity chromatography as described previously (6). The tryptic/catheptic 33-kDa heparin binding fragment of FN A-chains was purified according to methods previously reported (6). Purity of FN or a tryptic, RGD-containing 75-kDa fragment was verified by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining.
Peptide Synthesis and Characterization-- All of the peptides used in this study were synthesized at the Microchemical Facility of the University of Minnesota (Minneapolis, MN) using a Beckman System 990 peptide synthesizer. The procedures used were based on the Merrifield solid phase system as described previously (30). Lyophilized crude peptides were purified by preparative reverse-phase high performance liquid chromatography on a C-18 column using an elution gradient of 0-60% acetonitrile with 0.1% trifluoroacetic acid in water. The purity and composition of the peptides were verified by high performance liquid chromatography analysis of hydrolysates prepared by treating the peptides under nitrogen in 6 N HCl overnight at 110 °C (30).
Generation and Purification of Polyclonal IgG against Peptides-- Synthetic peptides were coupled to keyhole limpet hemocyanin (Sigma) using L-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Sigma) as a coupling reagent. The conjugate was mixed with Freund's adjuvant and then used to immunize New Zealand White rabbits. IgG was purified from pooled immune sera by precipitation with ammonium sulfate followed by DEAE anion exchange chromatography as described previously (39). Purity of the IgG was determined by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of the gels. Specificity of the purified IgG was then determined by enzyme-linked immunosorbent assay as described previously (31). Fab fragments of the anti-SG1 IgG or normal rabbit IgG were obtained by incubating the purified IgG with immobilized papain (Pierce) for 5 h at 37 °C following the manufacturer's instructions. The digestion products were examined by SDS-polyacrylamide gel electrophoresis and were found to be free of intact IgG heavy chain.
Conjugation of the Peptide to Ovalbumin (OVA)--
Peptide was
chemically conjugated to OVA using EDC because previous studies have
shown that the coupling of synthetic peptides to larger carrier
proteins resulted in enhanced cell adhesion activities (30, 31).
Briefly, equal amounts (by weight) of peptide CS1 and OVA were
solubilized in water and mixed with a 10-fold excess (by weight) of EDC
dissolved in water. The sample was then mixed overnight at 4 °C on a
circular rotator. The coupled peptide was then dialyzed extensively
against PBS to remove the excess EDC and uncoupled peptide (Spectrapore
6, 10-kDa exclusion, Spectrum Medical Industries, Los Angeles, CA). The
conjugates were stored at 20 °C until use. The specificity of cell
adhesion to CS 1/OVA was verified by using mAbs against
4 and
1 as well as soluble peptide CS1 as
described under "Results."
CSGAG-affinity Chromatography--
Aggrecan was prepared as
described previously (40). The purified aggrecan (dry weight, 50 mg)
was digested with both 1 mg of trypsin (Sigma) and 1 mg of proteinase K
(Qiagen) in 50 mM Tris-HCl containing 100 mM
sodium acetate (pH 7.5) overnight at 37 °C and then mixed with an
equal volume of 50 mM Tris-HCl containing 8 M
guanidine. CsCl was added to the solution at a ratio of 6:10 (w/w) to
give a density of 1.56 and then centrifuged at 40,000 rpm for 48 h. The bottom of the solution, in which CSGAG was enriched, was
collected and extensively dialyzed against 0.25 M sodium
acetate following by PBS. The purified CSGAG was then coupled to
CNBr-activated sepharose (Sigma) as described in manufacture's
protocol. Under these experimental conditions, 1 mg (as uronic acid) of
CSGAG was immobilized on the matrix. Ramos cells were biotinylated and lysed in 50 mM Tris-HCl (pH 7.3) containing 50 mM -octylglucoside, 15 mM NaCl, 1 mM Ca2+, 1 mM Mg2+, 1 mM Mn2+, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 1 mM N-ethylmaleimide (NEM)
(lysis buffer) and then centrifuged at 36,500 rpm for 1 h at
4 °C. The lysates were precleared on a mock column and then applied
to the CSGAG column. The column was extensively washed with the lysis
buffer, and then the bound materials were eluted by the lysis buffer
containing 0.4 M NaCl (41). The proteins were separated by
SDS-polyacrylamide gel consisting of 7.5% running with 5% stacking
gels and then transferred onto an Immobilon-P membrane. Nonspecific
binding sites on the membranes were blocked with PBS containing 3%
bovine serum albumin (BSA) overnight at 4 °C. The membranes were
then incubated with peroxidase-conjugated streptavidin for 1 h at
room temperature. Immunoreactivity was visualized by using an enhanced
chemiluminescence systems (Boeringer Mannheim). Elutions from the
affinity column were immunoprecipitated with mAb anti-
4
(P4C2) or isotype-matched control antibody (FLOPC21). The proteins were
separated, transferred onto Immobilon-P membrane, and detected as
described above.
Cell Adhesion Assays-- Cell adhesion assays were performed as described, with minor modifications (30). Briefly, ligands were diluted in PBS, and 50-µl aliquots were dispersed in triplicate into Immulon-1 polystyrene microtiter wells. The wells were incubated at 37 °C overnight, and nonspecific binding sites on plastic were then blocked by treating the wells with 200 µl of PBS containing 2% BSA. Subconfluent A375SM human melanoma cells that had been radiolabeled overnight with [3H]thymidine (specific activity, 6.7 Ci/mmol; NEN Life Science Products) were harvested by rinsing with 1 mM EDTA, washed two times with Eagle's minimum essential medium/BSA (Eagle's minimum essential medium containing 2 mg/ml BSA and 0.15 mM Hepes, pH 7.2) and adjusted to a concentration of 105 cells/ml in the same medium. Aliquots of 100 µl of the cell suspension were dispensed into the wells, and the cells were incubated at 37 °C for 30 min. The assays were terminated by aspirating loosely bound and unbound cells from the wells, washing the wells three times, and solubilizing the bound cells in 0.5 N NaOH containing 1% SDS. Bound radioactivity, determined in a Beckman model 3801 liquid scintillation counter, was used to calculate the percentage of cells that remained adherent to each substratum. Unless otherwise indicated, the data represent the means of triplicate determinations.
SDS-Polyacrylamide Gel Electrophoresis and Western Blotting-- A375SM and SKMEL-2 cells were lysed in 80 mM Tris-HCl (pH 6.8) containing 3% SDS, 15% glycerol, and 0.01% bromphenol blue. The same amounts of proteins were separated by 7.5% running with 5% stacking gels and then transferred onto an Immobilon-P membrane. Nonspecific binding sites on the membranes were blocked with PBS containing 3% BSA overnight at 4 °C. The membranes were then incubated with primary IgGs (1 µg/ml) for 4 h at room temperature followed by an incubation with peroxidase-conjugated goat anti-rabbit IgG for 1 h at room temperature. Immunoreactivity was visualized by using an enhanced chemiluminescence system (Boeringer Mannheim).
Affinity Chromatography--
Peptide-affinity chromatography was
performed according to the methods described previously (31). Peptide
SG1 was immobilized on activated CH-sepharose 4B (Pharmacia Biotech
Inc.) and blocked according to the methods described in manufacturer's
instructions. Human melanoma cells were surface-radiolabeled with
125I using lactoperoxidase and extracted as described
previously (30). Cells were extracted with 50 mM Tris-HCl
(pH 7.3) containing 50 mM -octylglucoside, 15 mM NaCl, 1 mM Ca2+, 1 mM Mg2+, 1 mM Mn2+, 1 mM PMSF, and 1 mM NEM (extraction buffer) and
then centrifuged at 36,500 rpm for 1 h at 4 °C. The cell
lysates were first precleared by incubating with a mock column, on
which no peptide was immobilized, and then applied to an SG1 column.
The column was extensively washed with the extraction buffer, and then
bound proteins were eluted by a linear gradient of sodium chloride (15 mM to 1 M). Radioactivity in each fraction was
quantified using a
counter, and positive fractions were pooled and
immunoprecipitated as described below.
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RESULTS |
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4
1 Integrin Interacts with
CSGAGs--
To evaluate potential interactions between
4
1 integrin with CSGAGs, we first
prepared a CSGAG-affinity column as described under "Materials and
Methods." Aggrecan was used as a source of CSGAG chain because the
proteoglycan contains CSGAG as the major GAG. Biotinylated cell lysates
were applied to the CSGAG column and washed, and the bound proteins
were eluted with 0.4 M NaCl as described by Diamond
et al. (41). The proteins were separated on
SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P
membrane. Although several proteins eluted from CSGAG column under
these conditions, two prominent proteins migrated at 140 and 120 kDa
(Fig. 1, CSGAG). These
proteins were not eluted from mock column (Fig. 1, Mock),
indicating that they specifically interact with the GAG chain.
Anti-
4 integrin mAb (P4C2) specifically immunoprecipitated
4
1 integrin from the
CSGAG column eluants, whereas no detectable immunoprecipitated proteins
were detected from the mock column eluant, indicating that
4
1 specifically bound the CSGAG
column.
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Peptide SG1 Derived from 4 Integrin Inhibited
Melanoma Cell Adhesion to CS1--
In previous reports, we
demonstrated that removal of cell surface CSGAGs inhibits
4
1 integrin-mediated but not
5
1 integrin-mediated cell adhesion (31).
These results suggest that cell surface CSGAGs can enhance the adhesive
function of
4
1 integrin, possibly as a
result of directly interacting with the
4 integrin
subunit. To further explore this possibility, we selected several
positively charged, relatively hydrophilic synthetic peptides from the
4 integrin subunit sequence (Fig.
2A). One of these peptides,
termed SG1, is uniquely contained within the
4 integrin
subunit as described by Takada et al. (42), when comparing
integrin sequences that are aligned for maximum homology using CLUSTAL
W algorithm (43) (Fig. 2B).
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Anti-SG1 IgG Inhibits Melanoma Cell Adhesion to CS1--
To
further evaluate the biological activities of the SG1 site in the
context of 4
1 integrin, we generated
polyclonal antibodies against this and the other synthetic peptides and
tested the ability of these antibodies to inhibit
4
1 integrin-mediated melanoma cell
adhesion. Cells were preincubated with 100 µg/ml of anti-SG1 IgG,
anti-A4-107 IgG, or anti-A4-110 IgG for 15 min at room temperature prior to cell adhesion to substrata coated with 5 µg/ml CS1. Anti-SG1 IgG completely inhibited cell adhesion to CS1, however, neither anti-A4-107 nor anti-A4-110 polyclonal antibodies had any effect on
cell adhesion to the same substrata (Fig.
4), demonstrating that the SG1 site plays
a specific role in
4
1 integrin-mediated cell adhesion to CS1. As controls, mAbs anti-
4 integrin
(but not anti-
5 integrin) inhibited cell adhesion to CS1
(Fig. 4). Monovalent Fab fragments of anti-SG1 IgG were also generated
and tested for the ability to inhibit cell adhesion to CS1. Fab
fragments of anti-SG1 IgG inhibited cell adhesion to CS1 in a
concentration-dependent manner (Fig.
5), suggesting that the inhibitory effect
of anti-SG1 IgG does not occur because of antibody-induced
cross-linking of
4
1 integrin.
Furthermore, anti-SG1 IgG did not cross-react with peptide CS1 as
determined by enzyme-linked immunosorbent assay (not shown), indicating
that anti-SG1 IgG did not inhibit cell adhesion by binding to
peptide-coated surfaces.
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Anti-SG1 IgG Inhibits 4
1 Integrin
Interactions with CS1 Ligand--
To further evaluate the mechanism of
anti-SG1 IgG-mediated inhibition of
4
1
integrin function, we first tested the ability of agents that activate
integrin-mediated cell adhesion to counteract the inhibitory effects of
anti-SG1 IgG (Fig. 7). When anti-SG1 IgG
was used in the presence of Mn2+, it was ineffective in
inhibiting melanoma cell adhesion to CS1, suggesting that anti-SG1 IgG
may affect ligand binding by the integrin. Furthermore, the addition of
mAb 8A2, which activates
1 integrin-mediated cell
adhesion by stimulating high affinity ligand binding (37), also
reverses the inhibitory effects of anti-SG1 IgG on melanoma cell
adhesion.
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MCSP Proteoglycan Interacts Directly with Peptide SG1-- To evaluate possible interactions between peptide SG1 and melanoma CSPG, we next performed affinity chromatography using immobilized SG1. Iodinated total cell extracts were applied to an SG1 affinity column, and bound proteins were eluted by a linear gradient of NaCl (Fig. 9A). Almost all radioactivity (over 97%) was eluted from the SG1 peptide affinity column at 0.13-0.15 M NaCl. These fractions were pooled and immunoprecipitated using specific anti-MCSP or anti-CD44 mAbs because these cells express both core proteins as cell surface chondroitin sulfate proteoglycans.2 Furthermore, these cells express large amount of unmodified MCSP and CD44 core proteins (26, 30, 44). Chondroitinase ABC treatment of immunoprecipitates demonstrated that chondroitin sulfate-modified MCSP was the main core protein eluted from the SG1 column eluates (Fig. 9B). In contrast, CD44 chondroitin sulfate proteoglycan was not detected in the pooled fractions (Fig. 9B). To further characterize the nature of interaction between MCSP and the SG1 peptide, 35SO4-labeled MCSP/CSPG was purified according to the methods described previously (31) and applied to the SG1 affinity column. Purified MCSP was eluted at 0.13-0.15 M NaCl, indicating that MCSP and peptide SG1 interact directly (Fig. 9C). When purified MCSP/CSPG was treated with chondroitinase ABC, the core protein did not bind the SG1 peptide column (not shown), demonstrating that the interaction is mediated through CSGAG.
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DISCUSSION |
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Integrin-mediated adhesion to ECM components and to other cells is
tightly controlled by regulating functional properties of specific
integrin heterodimers. We have previously shown that MCSP cell surface
chondroitin sulfate proteoglycan can enhance the activity of
4
1 integrin on melanoma cells. The effect
of MCSP on integrin function is complex, involving contributions from
the core protein, as well as the glycosaminoglycans (45). The current
study focuses on understanding the role of CSGAG in the functional
activity of
4
1 integrin. The results show
that
4
1 integrin binds directly to CSGAG
and that the
4 integrin subunit contains at least one
CSGAG binding site, defined by synthetic peptide SG1. Furthermore, this
interaction directly affects
4
1 integrin
adhesive function. These results are consistent with a model in which
cell surface MCSP interacts directly with
4
1 integrin and enhances
4
1 integrin-mediated adhesion and
signaling.
Among several synthetic peptides tested from 4 integrin,
only peptide SG1 or antibodies against SG1 significantly inhibit
4
1 integrin-mediated (but not
5
1 integrin-mediated) cell adhesion. This
inhibition could be reversed by the addition of Mn2+ or mAb
8A2, indicating that anti-SG1 IgG inhibition is reversible and is
related to the ability of the antibody to inhibit ligand binding by
4
1 integrin. These results suggest that
anti-SG1 IgG may act by changing integrin conformation, leading to a
direct inhibition of ligand binding, although it is also possible that anti-SG1 IgG may act by mechanisms related to integrin clustering. It
is of interest that the SG1 site is not within the ligand binding site
of
4 integrin. Furthermore, SG1 is a unique site among
other
chains when sequences of
integrin subunits are aligned
and compared (Fig. 2B). Thus, SG1 may represent a specific
GAG binding site that promotes the interaction between cell surface
proteoglycans and
4
1 integrin.
Although this study demonstrates that MCSP but not CD44/CSPG binds SG1,
these results should be interpreted with caution. A375SM melanoma cells
express almost 50% of their MCSP as CSPG, whereas levels of CD44
expressed as a CSPG are much lower (only approximately 5% of total
CD44 is expressed as CSPG).3
In light of this discrepancy, it is possible that the apparent specificity of MCSP binding to SG1 may reflect the large excess of MCSP
in the samples. Previous studies in hematopoietic progenitor cells have
indicated that cell surface CD44, expressed as CSPG, may influence the
adhesive function of 4
1 integrin (46),
suggesting that
4
1 integrin interactions
may involve more than one type of cell surface proteoglycan. Further
work is necessary using MCSP deficient cell lines to determine whether
CD44/CSPG on melanoma cells can similarly effect
4
1 integrin-mediated adhesion.
GAGs have been implicated in modulation of integrin function,
although the exact mechanism by which they do this is not
known. Earlier work has demonstrated that heparan sulfate
plays a role in formation of focal contacts in fibroblasts (20,
21, 47-49). It has also been reported that cell adhesion to basement
membrane proteoglycan, perlecan, is influenced by the heparan sulfate
GAGs on the core protein (50). Recent results suggest that the I domain
of CD11b/CD18 directly interacts with heparin and/or heparan sulfate
GAG chains, which are expressed on endothelial cells and that this
interaction may facilitate leukocyte rolling (41). In this study, we
demonstrated that 4
1 integrin interacts
with CSGAGs by affinity chromatography and that removal of CS reduces
4
1 integrin-mediated melanoma cell
adhesion and ligand binding. Furthermore, we have observed that
clustering of
4
1 integrin by CS1 coated
beads causes accumulation of MCSP in close proximity with
4
1 integrin on human melanoma cells.
Importantly, this accumulation is prevented by pretreating cells with
chondroitinase ABC, indicating that CSGAG chains may promote the
colocalization of MCSP with
4
1 integrin
that has been clustered by substratum-bound ligand.4 These results
indicate that CSGAGs may be important for bringing cell surface CSPG
and
4
1 integrin into close proximity on
the cell surface and that binding of ligand by integrin may help to facilitate codistribution of the two receptors.
It remains to be determined whether 4
1
integrin/GAG interactions are specific for chondroitin sulfate.
Although CSGAGs are the primary GAG expressed by these
melanoma cells, previous work has shown that other melanoma cells can
also express heparan sulfate (51, 52). Although there are many examples
of protein binding GAGs with a high degree of selectivity for a
specific GAG or even a sequence within a GAG, the same is not true for
several cell surface proteins. Many of the receptors on the cell
surface that bind polyanionic molecules recognize, although with
different affinity, members of several different GAG families. For
example, CD44 binds both chondroitin sulfate and hyaluronic acid (53). L-Selectin, which binds several variants of sialyl Lewis
antigen, including fucosylated or sulfated forms in mucin and
glycolipids, will recognize related sulfated lactosaminoglycan
structures of keratan sulfate (54) and unrelated structures in heparan
sulfate (55). Because there is no clear precedent, this leaves open the
discussion of specificity, which will be pursued in studies using
quantitative assay systems.
Collectively, regulation of integrin function on cell surfaces has been studied to understand the molecular mechanisms of inflammation and tumor metastasis. In this regard, it is important to realize that cell surface proteoglycans act not only as a cell surface receptor but also as an important modulator of integrins. The fact that PG binding sites are often expressed in close proximity to integrin binding domains within ECM proteins or cell surface adhesion molecules suggests that cellular recognition of the ECM or counter-adhesion receptors on opposing cells might involve the formation of a receptor cluster on the plasma membrane that include both PGs and integrins (45). Understanding the nature of such interactions may help to explain cell type-specific behavior on ECM proteins that are often observed for integrins. Studying the molecular mechanisms of the cooperation between these two distinct receptors will lead to a better understanding of the complexity of integrin functions in the processes of migration and extravasation, which occur during development and in tumor invasion, angiogenesis, and diseases associated with inflammation.
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ACKNOWLEDGEMENT |
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We thank Dr. Gregg Fields for his help in peptide design and synthesis.
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FOOTNOTES |
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* This work was supported by Grant CA21463 from the National Institutes of Health and a Allen-Pardee professorship in Cancer Biology (to L. T. F.).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: Dept. of Laboratory Medicine and Pathology, Box 609, University of Minnesota Health Center, 420 Delaware St. S. E., University of Minnesota, Minneapolis, MN 55455.
1 The abbreviations used are: ECM, extracellular matrix; CSGAG, chondroitin sulfate glycosaminoglycan; MCSP, melanoma chondroitin sulfate proteoglycan; mAb, monoclonal antibody; FN, fibronectin; CSPG, chondroitin sulfate proteoglycan; GAG, glycosaminoglycan; OVA, ovalbumin; EDC, L-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PG, proteoglycan.
2 J. Iida, unpublished data.
3 J. Iida, unpublished observations.
4 A. M. L. Meijne, J. Iida, T. A. Yednock, N. L. Kovach, L. T. Furcht, and J. B. McCarthy, submitted for publication.
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REFERENCES |
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