1 Institute for Molecular Science of Medicine, Aichi Medical University,
Nagakute, Aichi 480-1195, Japan
2 Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1
Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
* Author for correspondence (e-mail: kimata{at}amugw.aichi-med-u.ac.jp )
Accepted 27 May 2002
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Summary |
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Key words: Anti-adhesion, Chondroitin-sulfate-binding proteins, Cell attachment, CSPE, PG-M/versican
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Introduction |
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PG-M/versican, a large CS proteoglycan, is expressed preferentially in the
mesenchymal cell condensation area of chick limb bud at the prechondrogenic
stage (Kimata et al., 1986;
Shinomura et al., 1990
).
Almost all CS proteoglycans inhibit cell-substratum adhesion in vitro
(Rich et al., 1981
;
Brennan et al., 1983
;
Lewandowska et al., 1987
;
Yamagata et al., 1989
;
Winnemoller et al., 1991
;
Bidanset et al., 1992
). In
addition, immunohistochemical detection of CS chains revealed that they are
localized at the cell surface and are excluded from focal contacts of cultured
fibroblasts (Yamagata et al.,
1993
). Moreover, the reduction of the PG-M/versican level by
anti-sense RNA expression in osteosarcoma cells MG63, which express large
quantities of PG-M/versican, resulted in an increase in cell adhesion, the
formation of focal contacts and the elaboration of stress fibers. This
supports the notion that PG-M/versican, which is present in the extracellular
matrix near the cell surface, functions as an anti-adhesive molecule in many
cell types (Yamagata and Kimata, 1994). We previously showed that
PG-M/versican, when immobilized on tissue culture substrata, had a marked
inhibitory effect on the adhesion of cells to precoated proteins
(Yamagata et al., 1989
). The
inhibitory activity of CS proteoglycan was abolished by chondroitinase
digestion, and CS-conjugated serum albumin also inhibited cell adhesion. These
results suggest that the inhibitory activity is caused by CS chains
immobilized on the tissue culture plastic wells through their protein portions
and is independent of the core protein of proteoglycan. To investigate this
suggestion, we developed new molecules,
L-
-dipalmitoylphosphatidylethanolamine (PE)-derivatized
glycosaminoglycans (GAGs) (GAG-PEs), by chemically coupling the reducing
terminals of GAGs to the amino group of PE, which enables hydrophilic GAGs to
bind to hydrophobic tissue culture wells like native chondroitin sulfate
proteoglycans do. In fact, CS-derivatized PE (CSPE) mimics the inhibitory
activity of CS proteoglycans (Sugiura et
al., 1993
). These results support the idea that immobilized or
matrix-associated CS chains inhibit cell-substratum adhesion, whereas free CS
chains do not. They also indicate that this new molecule is a novel tool for
examining the effect of CS chains on cell adhesion.
Annexins are a family of proteins that have the ability to bind to acidic
phospholipids in the presence of Ca2+. All annexins possess
variable N-terminal domains followed by conserved core regions that impart
membrane-binding capabilities and usually contain four 70-80 amino-acid
repeats with an annexin consensus sequence. The core region of annexin 6,
however, contains eight such repeats. These conserved core regions are
responsible for the Ca2+- dependent binding of the proteins to
phospholipids. By contrast, the N-terminal domains of the annexins are highly
variable and may contribute to the specific functions of the various annexins
(Raynal and Pollard, 1994;
Edwards and Moss, 1995
). Some
of annexins bind to GAG in a Ca2+- dependent manner. Annexin 2 has
specific and high-affinity heparin-binding activity
(Kassam et al., 1997
). Annexin
4 binds to heparin, heparan sulfate and CS columns in a Ca2+-
dependent manner, annexin 5 to heparin and heparan sulfate columns in a
Ca2+-dependent manner and annexin 6 to heparin and heparan sulfate
columns in a Ca2+-independent manner and to CS columns in a
Ca2+-dependent manner
(Ishitsuka et al., 1998
).
These results suggest that some annexin species may function as recognition
elements for GAGs under some conditions.
In this study, we have further investigated the mechanism of anti-adhesive activity of CS chains. We first tested the possibility that a cell surface receptor for CS chains is involved in this activity using a cell attachment assay. We have successfully isolated a 68 kDa protein as a candidate receptor for CS chains and identified that protein as annexin 6. Moreover, taking advantage of A431 cells that do not express annexin 6 and transfecting them with exogenous annexin 6, we have demonstrated that annexin 6 is directly involved in the attachment of cells to CS chains and is expressed on cell surfaces.
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Materials and Methods |
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Cell and cell culture
Human osteosarcoma MG63 cells, human fibroblast IMR90, WI38, MRC5 and human
epidermoid carcinoma cell A431 cells were obtained from Japanese Cancer
Research Resources Bank (JCRB)-Cell, Tokyo, Japan. Chick embryonic fibroblasts
were established from 10-day-old chick embryos as described previously
(Yamagata et al., 1986). These
cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco)
supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences), 50 units/ml
penicillin and 50 µg/ml streptomycin.
The cell attachment assay
The centrifugation assay described previously
(Friedlander et al., 1988;
Ernst et al., 1995
) was
modified, and the procedure is summarized in
Fig. 1A. U-shaped wells in a
96-well polystyrene plates (Nunc-Immuno plate U) were coated with 100 µl of
5 µg/ml human fibronectin (FN) (Gibco), 8 µg/ml concanavalin A (ConA)
(Gibco) or 5 µg/ml CS conjugated to
L-
-dipalmitoylphosphatidyl-ethanolamine (CSPE) at 4°C overnight.
The wells were washed three times with PBS (-) and were blocked with 10 mg/ml
heated BSA in PBS (-) for 30 minutes. Cells cultured up to subconfluency were
rinsed twice with PBS (-) and harvested by treating cells with 0.05% trypsin
in PBS (-) at 37°C for 3 minutes. The cells were collected and washed
twice with 1mg/ml trypsin inhibitor in PBS (-) and resuspended with the growth
medium at 37°C for 10 minutes to allow cells to recover. Cells were then
washed once with serum-free DMEM and suspended in the same medium. A portion
of the cell suspension (5x103 cells/100 µl) was added into
each well and incubated for 10 minutes. The plate was then centrifuged at 200
g for 1 minute. Each well was photographed under a microscope.
On a non-adhesive substratum such as BSA, the centrifugal force predominates,
so that the cells form a pellet at the bottom of the well. When cells attach
to the substratum, cells bind in a uniform distribution both on the sides and
at the bottom of the well. At the intermediate level of attachment, cells
distribute in a ring, and the size of the ring depends upon the balance
between the centrifugal force and the adhesion. For the assessment of the cell
attachment activity, cells on each photograph were counted from the center of
the well and the areas with 4x102 cells were determined.
Values were obtained in triplicate independent experiments. From these values,
we used an `attachment index' to quantify the cell-substratum attachment using
the following calculation: attachment
index=(
substratum-
BSA)/
BSA, where
substratum and
BSA are the areas when a test sample and BSA were used as a substratum,
respectively.
|
Purification and identification of receptor molecules for chondroitin
sulfate chains
Human fibroblast WI38 cells and chick embryonic fibroblasts were cultured
in 150 mm culture dishes (Falcon). Cells from 20 dishes were rinsed twice with
ice-cold PBS (-) and harvested with a cell scraper. Cells were collected by
centrifugation. The pellet was suspended in 0.25 M sucrose, 0.5 mM
CaCl2, 0.02 M Tris-acetate, pH 7.2, and gently homogenized with a
glass-teflon homogenizer at 4°C. The homogenate was centrifuged at 2,000
g at 4°C for 10 minutes. The supernatant solution was
centrifuged at 105,000 g at 4°C for 1 hour. The
precipitate was obtained as the membrane fraction, and the membrane fraction
was extracted with TNE buffer (10 mM Tris-HCl, pH 7.8, 1% NP-40, 0.15 M NaCl,
10 mM EDTA, 1 mM PMSF, 1 mg/ml leupeptin) at 4°C for 1 hour. The extract
was centrifuged at 10,000 g for 25 minutes to remove the
debris and then subjected to PD-10 (Amersham Pharmacia Biotech.), which was
equilibrated with the affinity buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1%
NP- 40, 1 mM PMSF, 1 mg/ml leupeptin, 10 mM ß-mercaptoethanol, 5 mM
CaCl2, 5 mM MgCl2). The sample in the affinity buffer (3
ml) was subjected to the CS-conjugated Sepharose CL-6B gel column. After the
column was washed with 20 ml of the affinity buffer, the bound proteins
containing the expected receptor molecules for CS chains were eluted with the
elution buffer (20 mM Tri-HCl, pH 7.4, 0.15 M NaCl, 1% NP-40, 5 mM EDTA, 1 mM
PMSF, 1 mg/ml leupeptin). Subsequently, further elution was performed with 4 M
urea. The eluted proteins were precipitated with trichloroacetic acid,
dissolved in SDS-buffer and subjected to SDS-PAGE. The protein bands on the
gel were digested with V8 protease and then subjected to the second SDS-PAGE.
Some of the peptide bands were transferred to PVDF membrane and subjected to
the Applied Biosystems model 476A sequencer and Applied Biosystems model 120A
HPLC apparatus as described previously
(Zhao et al., 1995).
Construction of human annexin 6 and the deletion mutants
Full-length human annexin 6 cDNA was obtained by RT-PCR using the sequence
data (Crompton et al., 1988).
Total cellular RNA was isolated from IMR90 cells using the Trizol reagent
(Life Technologies). The first strand of cDNA was prepared using Ready-to-Go
T-Primed First-Strand kit (Amersham Pharmacia biotech). A human annexin 6 PCR
fragment was amplified by 25 cycles using Pfu DNA polymerase (Stratagene) and
the following primers: forward1 5'-TGCGTCCGTCTGCGACCCGAG-3'
(corresponding to the position of -70 to -30 in human annexin 6 cDNA);
reverse5 5'-GCGTTTCCTAAGCTCCACTGAAG-3' (corresponding to the
position of 2157 to 2179 in human annexin cDNA). The full-length cDNA was
directionally inserted into the EcoRV site of pIRES1neo (Clontech
Lab., Inc. USA). The deletion mutants of human annexin 6 were created by 20
cycles using Pfu DNA polymerase and the primers indicated in
Fig. 6A. Wild-type (full-length
human annexin 6) was amplified using the set of primers, forward 1 and reverse
4:5'-TTAAGCATAATCTGGAACATCATATGGATAGTCCTCACCACCACAGAGAG-3' (this
primer contains the sequence corresponding to a hemagglutinin A epitope, stop
codon, and the sequence corresponding to the amino acid sequence of ALCGGED of
human annexin 6). ALT (the alternative splicing form) was created by combining
the following two DNA fragments: the DNA fragment that was amplified with
forward1 and reverse3 primer, 5'-CTGGGCATCTTCCCGTGCCT-3'
(corresponding to the amino acid sequence of QAREDAQ) and the DNA fragment
that was amplified with forward3, 5'-GAAATAGCAGACACACCTAG-3'
(corresponding to the amino acid sequence of EIADTPS) and reverse4 primer. N
(the N-terminal annexin-6-specific domain and the N-terminal half of the
annexin 6-core region) was amplified using forward1 and reverse2 primer,
5'-AGCAGCATCATCATCTCC-3' (corresponding to the amino acid sequence
of GDDDAA). C+ (the N-terminal annexin-6-specific domain and the C-terminal
half of the annexin 6-core region) was created by combining the following two
DNA fragments: the DNA fragment that was amplified with the set of forward1
and reverse1 primer, 5'-CCGGTACTTGGCACCCTGTG-3' (corresponding to
the amino acid sequence of AQGAKYR) and the DNA fragment that was amplified
with forward2, 5'-GGAACTGTGCGCCCAGCCAA-3' (corresponding to the
amino acid sequence of GTVRPA) and reverse4 primer. C(the N-terminal
annexin 6-specific domain and the C-terminal half without the alternative
splicing domain) was created by replacing the BamHI/EcoRI
fragment of ALT with the BamHI/EcoRI fragment of C+. Each
cDNA fragment was directionally inserted into the EcoRV site of
pIRES1neo.
|
Transfection of A431 cells with human annexin 6 cDNA and its deletion
mutants
Exponentially growing A431 cells were cultured at 1x105
cells in 60 mm dishes for 24 hours and transfected with the cDNAs; they
continued to be cultured for another 24 hours. Transfection was achieved using
Trans IT polyamine transfection reagents (Pan Vera corp., WI).
Geneticin-resistant colonies were isolated two weeks after the transfection
and examined for the annexin 6 expression by western blotting. Control
transfection (transfected with the vector alone) were also isolated.
Western blotting
For the direct detection of expressed proteins, cells were extracted with
the lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 0.1% SDS). 50 µg
protein from the cell lysate was electrophoresed in a 10% SDS-polyacrylamide
gel. After electrophoresis, protein bands were electrophoretically transferred
onto nitrocellulose membranes. The proteins on the membrane sheets were
incubated with antibodies against annexin 6 (rabbit polyclonal or mouse
monoclonal) and annexin 4 (mouse monoclonal). After the incubation with
HRP-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG, detection was
performed using ECL western blotting detection reagents (Amersham Pharmacia
Biotech, USA). The blotting of the deletion mutants of annexin 6 was performed
with anti-hemagglutinin-A rabbit polyclonal antibody.
Flow cytometry analysis
Both the annexin 6 stably expressing transfectant, Anx#1, and the mock
vector transfectant, mock#1, were cultured in DMEM supplemented with 10% FBS,
50 units/ml penicillin, 50 µg/ml streptomycin and 0.2 mg/ml Geneticin
(Sigma). Cells cultured up to subconfluency were rinsed twice with PBS (-) and
harvested by treatment with 0.05% trypsin in PBS (-) at 37°C for 3
minutes. Cells were collected, washed twice with 1 mg/ml trypsin inhibitor in
PBS (-) and resuspended in the growth medium at 37°C for 10 minutes to
allow cells to recover. Cells (1x106) were suspended in 0.5
ml of DMEM containing 1% BSA (FACS buffer) in 1.5 ml microcentrifuge tubes and
cooled to 4°C. The rabbit anti-annexin 6, rabbit anti-focal adhesion
kinase (FAK), rabbit anti-ß-1 integrin or various anti-annexin 4
antibodies were added to each tube. Cells were incubated with the primary
antibody at 4°C for 1 hour, washed twice with FACS buffer and resuspended
in the FACS buffer containing a secondary mouse FITC-conjugated anti-rabbit or
anti-goat IgG (1:1000 dilution) and 5 µg/ml propidium iodide at 4°C for
30 minutes (Martin et al.,
1995). Cells were then washed three times with FACS buffer,
resuspended in 0.5 ml medium at 4°C and subjected to flow cytometric
analysis using ABI model FACScan flow cytometer
(Chung and Erickson, 1994
;
Tressler et al., 1994
).
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Results |
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IMR90 cells weakly but significantly attached to CSPE substratum (Fig. 2B). When CSPE substratum was treated with chondroitinase ABC, IMR90 cells were not able to attach to this substratum (Fig. 2B), indicating that CS chains are essential for the attachment and that the PE portion is not implicated in the cell-CSPE substratum interaction. These results suggest that the attachment of cells to CSPE substrata is caused by the direct interactions between cells and CS chains. Further, the attachment of cells to the CSPE substrata was altered by the addition of free CS chains (from shark cartilage and whale cartilage) to the assay medium, but not by addition of hyaluronan or chondroitin (Fig. 2B). Dermatan sulfate produced the same interfering effect at almost the same concentration, but heparin caused cell-cell aggregation at even lower concentrations (data not shown). Thus, alteration of cell-attachment to CSPE substratum seemed to be dependent on the concentration of disengaged CS (data not shown). It is likely, therefore, that the attachment of cells to CSPE substratum is not caused by direct and specific interactions between cells and CS chains; instead, it suggests that there is a receptor for CS chains on the cell surface.
The addition of EGTA to the assay medium containing 1.8 mM Ca2+ decreased the attachment of cells to the CSPE substratum, and the further addition of Ca2+ recovered the attachment in a stoichiometric manner (Fig. 2C), indicating the involvement of Ca2+ in the attachment of cells to CSPE substratum.
Purification and characterization of chondroitin-sulfate-binding
proteins
We then embarked on the characterization of molecules that bind to CS
chains in a Ca2+-dependent manner on the cell surface. We prepared
the membrane extract from cultured human fibroblast WI38 cells. The extract
was then subjected to the CS affinity column, and the bound proteins were
eluted as described in the Materials and Methods. The eluted fraction was
concentrated and subjected to SDS-PAGE in 10% polyacrylamide gels. The
proteins were transferred onto a PVDF membrane and visualized by Coomassie
brilliant blue staining (Fig.
3A). The two protein bands of 68 kDa and 35 kDa were detected in
the affinity fraction (the elution buffer). CS affinity column chromatography
using the membrane extract of cultured chick embryonic fibroblasts revealed
only the 68 kDa band in the affinity fraction
(Fig. 3A). The 68 kDa band from
human fibroblast WI38 cells was analyzed for the amino-acid sequence. Direct
sequencing was unsuccessful, probably because of an N-terminal modification.
Three internal sequences, ELKWGTDXAQFI, LSAXARVXLK and EDXQVQAA, were obtained
from the digests of the 68 kDa band with V8 protease. These partial sequences
correspond to human annexin 6 (Fig.
3B). In addition, the human 68 kDa band was specifically
recognized using anti-annexin antibodies
(Fig. 3C). We also identified
the 68 kDa protein from chick fibroblasts by amino-acid sequencing, and we
found it to be the partial sequence of chicken annexin 6
(Fig. 3B)
(Cao et al., 1993). We
concluded that the 68 kDa bands in both the affinity fractions were annexin 6
and suggested that it might be a candidate receptor for binding to CS chains
on the cell surface. We also characterized the 35 kDa protein band in the
human sample. Sequence analyses of peptide fragments following V8 protease
digestion showed that they were human annexin 4 fragments (data not shown).
These results suggest that annexin 4, as well as annexin 6, is a receptor for
CS chains in human cells. However, since annexin 6 was identified in both
human and chicken cells, we focused on this molecule.
|
Possible role of annexin 6 as a receptor for chondroitin sulfate on
the cell surfaces
We then examined whether annexin 6 acts as a receptor for CS chains on the
cell surface using the cell attachment assay. A431 cells did not attach to the
CSPE substratum, whereas IMR90, MG63, MRC5 and WI38 cells did
(Fig. 4A). Interestingly, the
A431 cell line is so far only the cell line known to lack endogenous annexin 6
(Theobald et al., 1994), and
we confirmed this lack of expression by western blotting
(Fig. 4B). Annexin 4 was
expressed in all of the cell lines examined. The results suggest that annexin
6, but not annexin 4, participates in the binding of cells to the CSPE
substratum. To confirm the function of annexin 6 in binding to CS chains, we
expressed it in A431 and examined whether attachment of these cells to the
CSPE substratum was recovered. We transfected the full-length human annexin 6
cDNA into A431 cells (Fig. 5A).
Two A431 cell lines, Anx#1 and Anx#2, that were stably expressing annexin 6
were obtained by transfection of the full-length human annexin 6 cDNA. The
transfectants attached to CSPE substrata, but the parent A431 cells and the
mock transfectants did not (Fig.
5B). Moreover, the extent of the attachment to CSPE substratum was
increased with higher levels of expression of annexin 6
(Fig. 5A,B). These results
strongly suggest that specific attachment can occur on CSPE substratum via
annexin 6.
|
|
In contrast to other annexins, which have a structural motif of four
repeats in the central core region, annexin 6 has eight repeats
(Raynal and Pollard, 1994;
Edwards and Moss, 1995
)
(Fig. 6A). Analysis of the
crystal structure of annexin 6 also indicates that it is uniquely organized
into two lobes, the N-terminal half (from repeat one to four) and the
C-terminal half (from repeat five to eight) of the molecule (Kawasaki et al.,
1994; Benz et al., 1994), and each lobe has convex and concave sides and a
hydrophilic pore surrounded by the four repeats that might be involved in GAG
interactions. In vitro, the C-terminal half, prepared by digestion with V8
protease of bovine brain annexin 6, bound to chondroitin sulfate in a
Ca2+-dependent manner (Ishitsuka et al., 1996). Furthermore, exon
21 was alternatively spliced, giving rise to two annexin 6 isoforms that
differ with respect to a six amino-acid insertion at the starting site of
repeat seven (Smith et al.,
1994
). Considering these unique structures of annexin 6, we
examined which region of annexin 6 is essential for the unique cell-CS binding
and whether or not the alternatively spliced form has the same activity. We
made five different deletion mutants of annexin 6 and examined the ability of
cells with these mutants to attach to CS chains
(Fig. 6A). The transfectant
expressing the alternative splicing variant form of annexin 6 (ALT#5) attached
to CSPE substrata as well as those expressing wild-type annexin 6 (WT#4) did.
The transfectant expressing either the N-terminal or the C-terminal half (N#3,
C+#2, C-#5) hardly attached to CSPE substratum
(Fig. 6B). The results suggest
that both lobes of annexin 6, the N- and C-terminal halves of the molecule,
are necessary for specific attachment of the annexin-6-expressing cells to CS
chains.
Occurrence of annexin 6 on cell surfaces
Annexin family proteins, including annexin 6, are cytoplasmic proteins.
However, if annexin 6 functions as a receptor for CS chains, it must be
exposed on the external cell surface membrane. Some reports have described the
extracellular expression of annexins on the outer plasma membrane
(Kirsch and Pfaffle, 1992;
Yeatman et al., 1993
;
Chung and Erickson, 1994
;
Tressler et al., 1994
). We,
therefore, examined whether annexin 6 was actually located on the outer
surface of cell membranes. We also measured the population of damaged or leaky
cells using propidium iodide or FAK staining. By flow cytometry analysis,
Anx#1 was positively stained with anti-annexin 6 polyclonal antibodies. Under
the same condition, less than 5% of cells were stained with polyclonal
antibodies to an intracellular antigen, focal adhesion kinase (FAK) or with
propidum iodide (Fig. 7A). When
the cells were incubated with anti-annexin 4 polyclonal antibodies, very few
cells were stained. To avoid the possibility that the epitopes were not
exposed to the antibodies, other antibodies to annexin 4 (two different
monoclonal antibodies) were also used. Again, only a few cells were stained.
These results indicate that a significant amount of annexin 6 is exposed on
the external cell surface membrane. Thus, it is likely that annexin 6
functions as a receptor for CS chains and is involved in the anti-adhesive
activity of CS proteoglycans.
|
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Discussion |
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Although annexin 6 is a cytoplasmic protein, we and another group have
shown that annexin 6 also exists on the cell surface
(Fig. 7) (Tressler et al., 1994). A
number of recent papers have reported that cytoplasmic proteins can be
secreted into the extracellular phase by an unknown mechanism; cytoplasmic
proteins can also be found on the cell surface as receptors for various
proteins. For example, annexin 5 was identified as a receptor for type II
collagen, which used to be called anchorin CII
(Kirsch and Pfaffle, 1992
).
Annexin 2 is a receptor for the alternatively spliced segment of FN type III
domains in tenascin-C (Chung and Erickson,
1994
) and for plasminogen and tissue plasminogen activator
(Hajjar et al., 1994
). In
addition, calreticulin is a widely expressed Ca2+-binding protein
found mainly in the endoplasmic reticulum but also in other cellular
compartments and could be functional as a receptor for thrombospondin
(Goicoehea et al., 2000
).
These reports also support the present notion that annexin 6 exists on cell
surfaces and is a receptor for CS.
In the present study, we have shown that human A431 cells devoid of annexin
6 expression were not able to attach to the CSPE substratum. A431 cells would,
therefore, be insensitive to the inhibitory effect of CS proteoglycans or
immobilized CS on cell-substratum adhesion if annexin 6 were the receptor.
However, A431 cells could not adhere to and spread on FN-CSPE substratum (data
not shown). We suggested that annexin 4 as well as annexin 6 might be a
receptor for CS chains in human cells (Fig.
3). In addition, although annexin 4 has an affinity for CS, as
observed using the in vitro system, its binding to CS is also Ca2+
dependent (Ishitsuka et al.,
1998). Therefore, the observed sensitivity of A431 cell to the
inhibitory effect of CS-immobilized CS on cell-substratum adhesion might be
explained by this redundancy.
The molecular mechanisms of anti-adhesive activities of other proteins have
been characterized recently. For example, tenascin is an anti-adhesive
substratum molecule, and the active region that induces focal adhesion
disassembly has been identified as the alternatively spliced FN type III
repeats of tenascin (TNfnA-D)
(Murphy-Ullrich et al., 1991).
TNfnA-D when added to confluent endothelial cells reduced the number of
positive focal adhesions on the cell by 50%, and this activity was
substantially blocked by an affinity-purified annexin 2 antibody
(Chung et al., 1996
).
Moreover, the ability of TNfnA-D to stimulate loss of focal adhesions was
blocked by Rp-8-Br-cGMPS and KT5823 at the concentrations that selectively
inhibit cyclic GMP-dependent protein kinase. These results indicate that
annexin 2 is a receptor for TNfnA-D and that cyclic GMP-dependent protein
kinase mediates focal adhesion disassembly triggered by tenascin
(Murphy-Ullrich et al., 1996
).
However, inhibitors of signaling, as far as we have tested with those that are
commercially available, did not significantly effect the anti-adhesive
activity of CS proteoglycans and immobilized CS chains (data not shown). These
results suggest that the inhibitory mechanism for CS chains may be different
from that for tenascin. However, the anti-adhesive mechanism of immobilized CS
chains is hardly known. It is interesting to speculate how CS chains inhibit
cell spreading. Some signaling molecules that interact with annexin 6 have
been described. Recent reports indicate that the interaction of annexin 6 and
protein kinase C
is dependent on the presence of Ca2+ and
phosphatidylserine (Schmitz-Peiffer et
al., 1998
). Additionally, annexin 6 makes a complex with
p120GAP (Ras GTPase-activating protein), Fyn (Src family kinase)
and Pyk2 (focal adhesion kinase family member). In addition, annexin 6
directly binds to p120GAP and Fyn
(Chow et al., 2000
). It may be
possible that the interaction of CS chains and annexin 6 causes the state
change of these signaling molecules and induces the anti-adhesion
activity.
In most eukaryotic cell surfaces, there are specialized lipid microdomains,
rafts, that are plasma-membrane assemblies enriched in cholesterol and
glycosphingolipids and that are involved in cell signaling at the plasma
membrane. A variety of signaling molecules
glycosyl-phosphatidylinositol-anchored protein, tyrosine kinases, GTP-binding
proteins are concentrated in rafts
(Anderson, 1998). This
localization increases the concentration and stability of signaling molecule
complexes, with a direct enhancing effect on signaling levels
(Kholodenko et al., 2000
).
Annexin 6 interacts with rafts at elevated intracellular Ca2+
concentrations (Babiychuk et al.,
1999
). Owing to the interaction of annexin 6 with CS chains on the
cell surfaces, the membrane characteristics might be changed. This
modification of membrane properties influences lipid microdomain organization
and/or protein-membrane association, and as a result, might inhibit cell
spreading. Our present findings raise many interesting questions about the
mechanisms of the anti-adhesive activity of CS proteoglycans on
cell-extracellular-matrix interactions.
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Acknowledgments |
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