|
|
|
|||||||
|
|||||||||
(Received for publication, July 21, 1994; and in revised form, December 12,
1994) From the
Because syndecans are present at sites of cell-cell contact in vivo it has been hypothesized that they play a role in
mediating cell-cell adhesion. However, there has been no direct
evidence to support this notion. To address this question, B lymphoid
(ARH-77) cells were transfected with the cDNA for murine syndecan-1.
Unlike the parental cells, the transfectants form large multicellular
aggregates in suspension cultures and stain intensely for syndecan-1 at
sites of cell-cell contact. Using rotation-mediated aggregation assays,
we find that aggregation of syndecan-1-transfected cells is dependent
on divalent cations and is inhibited by the following: (i) addition of
heparin and heparin-like glycosaminoglycans, (ii) removal of heparan
sulfate from the cell surface, or (iii) addition of exogenous purified
syndecan-1. Mixing of syndecan-1-transfected and control-transfected
cells results in aggregates containing both cell types indicating that
aggregation occurs through a heterophilic adhesion mechanism in which
heparan sulfate chains bind to a counter-receptor present on these
cells. Importantly, syndecan-4-transfected cells also aggregate in a
heparan sulfate-dependent manner, while in contrast,
betaglycan-transfected cells aggregate poorly. Thus, syndecans may be
important mediators of cell-cell adhesion, but this function may not be
common to all transmembrane heparan sulfate-bearing proteoglycans.
Although there is extensive evidence that cell surface heparan
sulfates participate in cell-matrix
adhesion(1, 2, 3) , it has only recently been
demonstrated that heparan sulfates can interact with cell adhesion
receptors thereby mediating cell-cell adhesion. For example, the
homophilic interaction between N-CAMs on adjacent cells appears in some
instances to require the presence of heparan sulfate as a
co-receptor(4) . Recently, it was shown that both L-selectin
and PECAM-1 can bind glycosaminoglycans, and PECAM-1 can mediate
cell-cell adhesion by binding to heparin-like ligands present on
adjacent cell surfaces(5, 6) . These interactions, at
least in the case of N-CAM and PECAM-1, are apparently mediated by
protein domains within these adhesion receptors that contain consensus
sequences for glycosaminoglycan
binding(6, 7, 8, 9) . Furthermore,
glycosaminoglycan-ligand interactions may have important physiological
consequences as suggested by a recent study in which an acute
inflammatory response was inhibited following intravenous injection of
a heparin preparation known to bind L- and P-selectins(10) .
Given the ubiquitous distribution of glycosaminoglycans at the cell
surface, the interactions of proteoglycans with cell adhesion molecules
may represent an important and widely distributed mechanism for
cell-cell adhesion. The syndecans are a family of transmembrane
proteoglycans that regulate cell behavior by binding cells to
extracellular matrix and by binding growth factors (reviewed in (11) ). Syndecan-1, via its heparan sulfate chains, binds to a
variety of extracellular matrix macromolecules including interstitial
collagens(12, 13, 14) , fibronectin (15) , thrombospondin(16) , and tenascin(17) .
In addition, it has been widely speculated that syndecans participate
in cell-cell adhesion. This is due to a number of reports indicating
that syndecan-1, as well as other members of the syndecan family, are
often found at sites of cell-cell
contact(18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .
For example, in adult mice, syndecan-1 is present on the basal surface
of simple epithelia, consistent with its role in binding interstitial
matrix. It is also present on the lateral surface of simple epithelia
and over the entire surface of stratified epithelia, consistent with a
role in cell-cell adhesion(18) . However, there has been no
direct evidence demonstrating that syndecan-1 or other members of the
syndecan family can mediate cell-cell adhesion. The present study
was initiated upon discovery that cells of a line derived from a human
plasma cell leukemia form large aggregates in culture following their
transfection with a cDNA for murine syndecan-1. Aggregation of the
syndecan-1-transfected cells is mediated by heparan sulfate, is
dependent on the presence of divalent cations, and occurs via
syndecan-1 interaction with a heparan sulfate-binding ligand present on
adjacent cells. Transfection of cells with a cDNA for syndecan-4 also
promotes cell aggregation. This is in contrast to another transmembrane
heparan sulfate proteoglycan, betaglycan, which promotes aggregation
poorly.
For syndecan-4, ARH-77
cells were transfected with the pcDNA3 vector containing the full
coding region for rat syndecan-4 (kindly provided by Dr. John
Gallagher). Transfected cells were selected in G418, cloned by limiting
dilution, and screened by Northern blotting. For betaglycan, the
full-length rat betaglycan cDNA containing a human c-myc epitope (35) (kindly provided by Dr. Joan
Massagué) was cloned into the pBK-CMV phagemid
expression vector (Stratagene). Prior to transfection, the pBK-CMV
vector was modified by removal of the lac promotor. Transfected cells
were selected in G418, and cells expressing betaglycan were isolated by
flow cytometry using antibody 9E10 (ATCC CRL 1729) (36) that
recognizes the c-myc epitope (EQKLISEEDL) present in the betaglycan
extracellular domain.
For inhibition
experiments, inhibitors were added to the cell suspension immediately
after cells were placed in the 24-well plate. All glycosaminoglycans
were from Sigma including dextran sulfate (catalog no. D-6001),
chondroitin sulfate A (C-8259), chondroitin sulfate B (C-2413),
chondroitin sulfate C (C-4384), heparin (H-3393), heparan sulfate from
bovine intestine (H-7641), heparan sulfate from bovine kidney (H-7640),
and hyaluronic acid (H-1751). For removal of glycosaminoglycans from
the surface of cells prior to aggregation assays, approximately 1.7
For
aggregation assays in which two different cell lines were mixed
together, experiments were performed as above except equal numbers of
syndecan-1-transfected and control-transfected cells were added to
wells. Prior to addition of cells to wells, one of the two cell lines
was labeled with PKH-26 fluorescent dye (Zynaxis Cell Science, Malvern,
PA) according to the manufacturer's directions. This dye
selectively partitions into the cell membrane allowing examination of
labeled cells by fluorescence microscopy(38) . To confirm that
results in the mixing experiments were not caused by nonspecific cell
adhesion due to labeling cells with PKH-26, experiments were also
performed using cells labeled intracellularly with Cell Tracker
Cells growing in
suspension were harvested by centrifugation, resuspended, dispersed
into a single cell suspension by pipetteting, and placed within wells
of a 24-well plate. Following rotation for 60 min at 37 °C, both
the parental ARH-77 cells and control transfectants form only a few
small aggregates (Fig. 1, A and C). In
contrast, the syndecan-1-transfected cells form numerous aggregates
ranging in size from a few cells to many cells (>30 cells/aggregate) (Fig. 1B). Quantification of the percent of cells in
aggregates reveals that 60% of the syndecan-1-transfected cells are
incorporated into aggregates composed of four or more cells (Table 1; ARH
Figure 1:
Cells
expressing syndecan-1 form large aggregates in rotation-mediated
aggregation assays. Cells were removed from suspension cultures,
dispersed into single cell suspensions, and rotated for 60 min at 37
°C as described under ``Experimental Procedures''. A, the syndecan-1-negative ARH-77 cells do not form large
aggregates. B, in contrast, syndecan-1-transfected ARH-77
cells form numerous large aggregates. The cells shown are from
transfected clone A5P3. C, control-transfected cells do not
aggregate extensively. Bar = 100
µm.
To determine if divalent cations are
required for aggregation of the syndecan-1-transfected cells, we
analyzed aggregation in buffer containing 1 mM EDTA and no
calcium or magnesium. Under these conditions, cell aggregation is
virtually abolished (Table 1;
ARH
Figure 2:
Syndecan-1 is present at sites of
cell-cell contact. Aggregates of syndecan-1-transfected cells were
fixed in suspension, stained with antibody 281.2 that is specific for
the syndecan-1 core protein(39) , and viewed by
immunofluorescence microscopy. Intense staining for syndecan-1 is
present at sites of cell-cell contact (two adjacent fields are shown).
Observation of these aggregates at several different planes of focus
revealed that in addition to the bright staining in regions of
cell-cell contact, syndecan-1 is also present over the entire cell
surface in a broadly dispersed punctate staining pattern (not shown). Bar = 50 µm.
Addition of heparin to the assay buffer
inhibits cell aggregation in a concentration-dependent manner and
almost completely abolishes cell aggregation even at low concentrations
(0.250 µg heparin/ml) (Fig. 3). However, other exogenously
added glycosaminoglycans fail to abolish aggregation, even when the
glycosaminoglycans are present at 10 µg/ml (Fig. 4), a
40-fold higher concentration than that required for complete inhibition
by heparin (Fig. 3). Only bovine intestinal heparan sulfate and
the highly sulfated synthetic polymer dextran sulfate substantially
reduce cell aggregation, while bovine kidney heparan sulfate and
chondroitin sulfates reduce cell aggregation to a lesser degree (Fig. 4). The non-sulfated glycosaminoglycan hyaluronic acid has
no effect. The observation that heparan sulfate from porcine intestine
inhibits cell aggregation better than heparan sulfate from bovine
kidney may reflect structural differences between these two
preparations. Similar to our findings, intestinal heparan sulfate is
reported to inhibit the binding of human trophoblastic cells to uterine
epithelial cells better than does heparan sulfate from
kidney(42) .
Figure 3:
Cell aggregation is inhibited by low
concentrations of heparin. Heparin at the indicated concentrations was
added to the aggregation buffer, and assays were performed on
syndecan-1-transfected cells as described under ``Experimental
Procedures.'' Results are presented as means ± S.E. from
three separate experiments.
Figure 4:
Effect of glycosaminoglycans on
aggregation of syndecan-1-transfected cells. Aggregation assays were
performed in the presence of 10 µg/ml of the indicated
glycosaminoglycan or following removal of glycosaminoglycans from the
cell surface with either heparitinase or chondroitinase ABC. Control
experiments were performed without added glycosaminoglycan or enzyme
treatment. Two different preparations of heparan sulfate were used;
heparan sulfate (I) from bovine intestine, and heparan sulfate (K) from bovine kidney. In one experiment, (denoted heparin*) cells were incubated with heparin for 30 min,
unbound heparin was washed away, and cells rotated for an additional 30
min. Results are presented as means ± S.E. from three separate
experiments.
Results of these inhibition experiments suggest
that heparin or heparan sulfate may be binding to a receptor on the
cell surface. To test this, cells were incubated with heparin for 30
min followed by removal of unbound heparin by washing cells three times
with aggregation buffer. These washed cells fail to aggregate following
rotation for 60 min at 37 °C (see Fig. 4, heparin*), thus providing further support for the notion that
the exogenous heparin binds to a receptor on the cell surface, thereby
blocking cell aggregation. To confirm the role of heparan sulfate in
aggregation of the syndecan-1-transfected cells, heparan sulfate chains
were removed from the cell surface by treatment with heparitinase prior
to the aggregation assay. Cells treated in this manner exhibit a
drastically reduced ability to form aggregates as compared to
non-heparitinase treated controls (12 versus 72% of cells in
aggregates, respectively) (Fig. 4). Treatment of cells with
chondroitinase ABC has no inhibitory effect on cell aggregation
indicating that the partial inhibition of cell aggregation by
exogenously added chondroitin sulfates (Fig. 3) may be due to
nonspecific charge interactions between chondroitin sulfate and other
molecules at the cell surface. Taken together, these results
demonstrate that aggregation of the syndecan-1-transfected cells is
dependent on the presence of heparan sulfate at the cell surface.
Figure 5:
Exogenous syndecan-1 inhibits aggregation
of syndecan-1-transfected cells. Aggregation assays were carried out in
the absence of exogenous syndecan-1 (A) or in the presence of
approximately 0.70 µg/ml of purified syndecan-1 ectodomain (B). Quantification of aggregation reveals that in wells where
syndecan-1 was added, less than 10% of the cells are incorporated into
aggregates while in wells without exogenous syndecan-1, 60-70% of
the cells are within aggregates. Bar = 100
µm.
Figure 6:
Syndecan-1-mediated cell aggregation is
heterophilic. A, aggregation assays were performed by mixing
equal numbers of syndecan-1-transfected and control-transfected cells.
In this experiment the syndecan-1-transfected cells are unlabeled, and
the control-transfected cells are fluorescein-labeled with the membrane
labeling compound PKH-26. Shown in A is one large
representative field and two inserts of adjacent fields containing
aggregates. B, identical to panel A except the
syndecan-1-transfected cells were treated with heparitinase prior to
the aggregation assay. No significant aggregation is present. C, identical to panel A except control-transfected
cells were treated with heparitinase prior to the aggregation assay.
Both syndecan-1-transfected and control-transfected cells are present
in the aggregates. Bar = 40
µm.
To
determine if aggregation was specifically mediated by syndecans or if
any heparan-sulfate bearing proteoglycan would suffice, ARH-77 cells
were transfected with a cDNA for betaglycan. Betaglycan is a
transmembrane heparan sulfate/chondroitin sulfate proteoglycan similar
to syndecan-1 although its core protein structure is unrelated to
syndecan-1(44) . Betaglycan is known to bind to transforming
growth factor- The present work provides the first direct evidence that
syndecans participate in cell-cell adhesion. This role is supported by
the following observations: (i) cells aggregate spontaneously in
suspension cultures following their transfection with the cDNA for
syndecan-1; (ii) within the cell aggregates, syndecan-1 localizes to
sites of cell-cell contact; (iii) cell aggregation is dependent on the
presence of heparan sulfate; and (iv) exogenous syndecan-1 inhibits
aggregation of the syndecan-1-transfected cells. Aggregation of the
syndecan-1-transfected cells is dependent on the presence of divalent
cations, and mixing experiments indicate that aggregation occurs via a
heterophilic mechanism suggesting that a counter-receptor for
syndecan-1 is present on the aggregating cells. Furthermore, we show
that syndecan-4 can also mediate aggregation of ARH-77 cells while
another transmembrane heparan sulfate proteoglycan, betaglycan promotes
aggregation poorly. Thus, while different members of the syndecan
family may participate in cell-cell adhesion, this may not necessarily
be a function common to all transmembrane heparan sulfate
proteoglycans. The finding that the syndecans mediate cell-cell
interactions is consistent with previous work demonstrating their
presence at sites of cell-cell contact in vivo. For example,
during embryogenesis, syndecan-1 is found at sites of cell-cell contact
in the unhatched blastocyst and in the embryonic ectoderm and mesoderm
of the early postimplantation embryo(47) . During
epithelial-mesenchymal interactions syndecan-1 expression is acquired
on the mesenchymal cells as they condense and aggregate in
tooth(19) , kidney(20, 21) , limb (22) , uterus(24) , and the optic, vibrissal, nasal,
and otic anlage (23) . Additionally, when induced mesenchymes
are disaggregated in vitro, syndecan-1 is intensely expressed
by those cells that reaggregate leading to speculation that syndecan-1
mediates this aggregation(25) . Syndecan-1 is also present at
sites of cell-cell contact in mature tissues. It is present on the
lateral surfaces of simple epithelia and over the entire cell surface
of stratified epithelia(18) . Staining for syndecan-1 is
intense between the keratinocytes within the spinous and lower granular
layers of the skin (18) . During keratinocyte differentiation
and stratification, a time when cell-cell adhesion is strengthened, the
amount of syndecan-1 present at the cell surface increases as compared
to non-stratified cells(26, 27) . Thus, both the
temporal and spatial expression of syndecan-1 are consistent with its
role as a cell-cell adhesion molecule. In addition, immunohistochemical
studies with antibodies to syndecans -2, -3, and -4 have demonstrated
the presence of these syndecans at sites of cell-cell
contact(28, 29, 31) , and it was recently
reported that Drosophila syndecan expression is markedly
enhanced at sites of cell-cell contact(48) . Thus, there is
circumstantial evidence that all the syndecans may participate in
cell-cell adhesion. Further supporting a role for syndecans in
cell-cell adhesion is the observation that syndecan-1, like other
cell-cell adhesion molecules such as the cadherins(41) , is
lost from cells prior to changes in their shape or location. For
example, epithelial cells undergoing changes in shape lose syndecan-1
expression (19) and, within healing cutaneous wounds,
syndecan-1 expression is absent from the leading edge of the
wound(49) . Upon malignant transformation, syndecan-1 is
sometimes lost from the cell surface (27, 50) and
transfection of malignant cells with the cDNA for syndecan-1 restores
their normal cell morphology and growth characteristics(51) .
Taken together, these findings indicate that loss of syndecan-1
expression and subsequent weakening of cell adhesion may be required
prior to normal cell movement and tumor cell invasion. The fact that
syndecans are present at numerous sites of cell-cell contact does not
confirm that they are always mediating cell-cell adhesion at these
sites. Syndecans differ in their glycosaminoglycan composition between
tissue and cell types(11, 52) , and structural
differences in the heparan sulfate chains of syndecan-1 can affect
their affinity for ligands (40, 53) and determine the
adhesive capacity of cells(40) . Furthermore, the adhesion seen
in the present study is apparently dependent on a receptor for heparan
sulfate. Thus, successful cell-cell adhesion via the syndecans is
likely dependent on both the appropriate heparan sulfate structure and
the presence of a receptor. Indeed, in testing numerous human myeloma
cell lines, we have found that some lines express syndecan-1 but do not
self-aggregate suggesting that they lack the receptor for syndecan
heparan sulfate (not shown). Similarly, it has been reported that Raji
cells (a human lymphoblastoid cell line) do not undergo cell-cell
adhesion following their transfection with syndecan-1(54) . Although we do not yet know the mechanism by which syndecans mediate
cell aggregation, the present study does provide some clues.
Aggregation apparently is not via a homophilic interaction of syndecan
molecules. Nor is it due to self-association of heparan sulfate, either
between syndecan molecules or between syndecans and another cell
surface proteoglycan bearing heparan sulfate. This is supported by the
observation that even after the control-transfected
(syndecan-1-negative) cells are treated with heparitinase, they adhere
to the syndecan-1-transfected (syndecan-1-positive) cells (Fig. 6C). Taken together, the mixing experiments
indicate that syndecan-1-mediated aggregation occurs via a heterophilic
mechanism suggesting that a counter-receptor for syndecan-1 is present
on the surface of some cells. Furthermore, the finding that heparan
sulfate from kidney and intestine differ in their inhibitory effect on
the aggregation of syndecan-1-transfected cells (Fig. 4)
supports the notion that the putative counter-receptor for syndecan
preferentially recognizes certain structural features within heparan
sulfate. Several adhesion molecules are known to interact with
heparan sulfate-bearing ligands and could be potential ligands for
syndecan. Both L-selectin and PECAM-1 can interact with heparin-like
glycosaminoglycans(5, 6) . In common with our
findings, the L-selectin and PECAM-1 interactions with
glycosaminoglycans are calcium-dependent. Another adhesion receptor,
N-CAM, mediates cell-cell adhesion via a homophilic mechanism, and
apparently, in some instances, this requires the presence of a heparan
sulfate proteoglycan as a co-factor(4) . Although N-CAM is
expressed by some human B lymphoid tumors(55, 56) , it
is not the ligand for syndecan-1 on our cells because neither the
ARH-77 parental nor syndecan-1-transfected cells have detectable levels
of N-CAM on their surface as determined by flow cytometry of cells
stained with antibody to N-CAM. ( Clearly there are numerous heparan sulfate-bearing proteoglycans on
the surface of cells (i.e. syndecans, glypicans, and
betaglycans). However, it is not known if these different proteoglycans
bear structurally similar or different heparan sulfate chains on the
same cell type. The present studies indicate that both syndecans-1 and
-4, which have very similar core proteins, bear functionally similar
heparan sulfate chains in regard to their ability to mediate cell
aggregation. In contrast, betaglycan, which has a core protein that is
dissimilar to the syndecans, mediates cell aggregation poorly, raising
the possibility that betaglycans bear heparan sulfate chains that
differ functionally from those present on the syndecans.
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5077-5083
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
SYNDECANS-1 AND -4 MEDIATE INTERCELLULAR ADHESION FOLLOWING THEIR
TRANSFECTION INTO HUMAN B LYMPHOID CELLS (*)
Cell Lines and Preparation of Transfected
Cells
ARH-77 cells (The American Type Culture Collection,
Rockville, MD) were established from the peripheral blood of a patient
with plasma cell leukemia(32) . These cells do not express
syndecan-1 (33) and have low levels of detergent-extractable
heparan sulfate proteoglycan. Syndecan-1-transfected cells were
established by electroporation of ARH-77 cells in the presence of the
pMAMneo plasmid containing a cDNA for murine syndecan-1 (cDNA was
kindly provided by Dr. M. Bernfield). Control-transfected cells contain
the pMAMneo plasmid without the syndecan-1 insert. The procedures
employed to establish and maintain stable transfectants and
characterization of the syndecan-1-expressing clones used in this study
have been previously described(34) .Cell Aggregation Assays
A variation of the
rotation-mediated aggregation assay of Takeichi (37) was
utilized. Briefly, cells growing in suspension cultures were harvested
by centrifugation, washed twice, and resuspended at a concentration of
3-4 
10
cells/ml in Hank's balanced salt
solution containing 1 mM calcium chloride, 1 mM magnesium sulfate, 1% bovine serum albumin, and 10 mM HEPES (aggregation buffer). Cell aggregates were dispersed by
pipetteting and 0.5 ml of the cell suspension placed in wells of a
24-well cell culture plate (Corning) that had been previously incubated
with 0.5 ml of aggregation buffer overnight at 37 °C to inhibit
cell binding to the plate surface. The culture plate was then rotated
on a gyratory shaker (100 revolutions/min) at 37 °C for 60 min.
Cells were removed from wells by gentle pipetteting, and wells were
washed with 200 µl of aggregation buffer to ensure all cells were
removed from the wells. The cells and wash were combined and gently
pipetted five times through a glass Pasteur pipette to disperse any
weakly aggregating cells. 8 µl of the cell suspension were placed
on a microscopic slide and contained within a 15-mm diameter area. The
number of single cells and the number of cells in aggregates lying
within or associated with a central rectangular area of 6.2 mm
were counted. In some experiments the aggregates contained too
many cells to accurately access their numbers. In these instances, the
number of cells in aggregates was determined by subtracting the total
number of single cells present at the completion of the assay from the
total number of cells present at the beginning of the assay. Aggregates
were defined as containing four or more cells. The percent of total
cells present in aggregates was calculated for each sample. Photos were
taken using an inverted Nikon phase microscope. 10 cells were resuspended in complete medium
containing either 1 milliunit/ml heparitinase (Seikagaku, Rockville,
MD) or 50 milliunits/ml chondroitinase ABC (Seikagaku). Following
incubation with enzyme for 30 min, an equal amount of enzyme was added
to the cells and the digestion continued for another 30 min.
Orange
(5-(and-6-)-(((chloromethyl)benzoyl)amino)-tetramethylrhodamine;
Molecular Probes, Eugene, OR). Aggregates were examined using a Nikon
Labophot microscope equipped for fluorescence and photos taken using
Ilford HP5+ film.Immunofluorescence Staining of Syndecan-1
Cell
aggregates were pipetted into microcentrifuge tubes and fixed for 30
min at room temperature in Zamboni's solution composed of 2%
paraformaldehyde and 0.18% picric acid in 0.1 M phosphate
buffer, pH 7.4. Cells were washed several times in PBS (0.01 M phosphate, 0.15 M NaCl, pH 7.4) and blocked in PBS
containing 1% normal rabbit serum. Cells were then incubated for 30 min
at room temperature with 20 µg/ml of monoclonal antibody 281.2 (39) or antibody to L3T4 (Pharmingin, San Diego, CA), an
isotype matched control monoclonal antibody. Following washing in PBS,
cells were incubated for 30 min at room temperature with PBS containing
5 µg/ml biotinylated rabbit anti-rat IgG. After washing in PBS,
cells were incubated with avidin fluorescein isothiocyanate (Vector)
for 10 min, washed again in PBS, and mounted using Vectashield mounting
medium (Vector).Purification of Syndecan-1
For experiments in
which exogenous syndecan-1 was added to inhibit aggregation, the
syndecan-1 ectodomain was purified from medium conditioned by
syndecan-1-transfected cells. Medium was brought to 6 M urea,
50 mM sodium acetate, pH 4.5, and boiled for 10 min.
Syndecan-1 was then purified using DEAE beads followed by 281.2
affinity chromatography as described(40) . Prior to using the
purified syndecan-1 ectodomain in aggregation assays, it was
equilibrated in aggregation buffer by elution over an Excellulose G5
column (Pierce).
Cells Transfected with Syndecan-1 Form Multicellular
Aggregates
We have recently isolated a series of
syndecan-1-expressing clones following the stable transfection of
murine syndecan-1 cDNA into the human plasma cell leukemia line ARH-77 (34) . Upon routine examination of cell cultures, a striking
difference between control-transfected and syndecan-1-transfected cells
was evident: Control-transfected cells were present predominantly as a
single cell suspension, while all of the syndecan-1-transfected clones
contained numerous large aggregates of cells (not shown). To assess
this aggregation, we employed a short term aggregation assay similar to
that used by Takeichi and colleagues (37, 41) for
studying cadherins. For the present study, a syndecan-1-transfected
clone designated A5P3 was used because cells of this clone are known to
express syndecan-1 that bears heparan sulfate chains and the intact
syndecan-1 proteoglycan is nearly identical in size to that isolated
from other B lymphoid cells(34) .
). This is over 4-fold greater than
the percentage of cells in aggregates of the syndecan-1-negative
parental ARH-77 cells and 20-fold greater than the control
transfectants (Table 1). In addition to the dramatic difference
in the percentage of cells within aggregates, the aggregates formed by
syndecan-1-transfected cells are on average much larger in size than
those formed by ARH-77 and control transfectants (Fig. 1).
Similar results were obtained in aggregation assays using the
syndecan-1-transfected cell clone B3P3 (not shown) that expresses
relatively low levels of syndecan-1(34) . Thus, aggregation of
the syndecan-1-transfected cells is not likely due to overexpression of
syndecan-1 at the cell surface.
/EDTA
). Subsequent removal of
buffer containing EDTA and addition of buffer containing cations
restores cell aggregation (ARH/EDTA
).
In separate assays, it was also found that addition of calcium alone
was as effective in promoting aggregation, as was addition of both
calcium and magnesium (not shown).Syndecan-1 Is Present at Sites of Cell-Cell
Contact
Immunostaining of cell aggregates with antibody 281.2
indicates that syndecan-1 is present at sites of cell-cell contact and
often forms an intense band of staining in these regions (Fig. 2). In contrast, cell surfaces not in contact with
adjacent cells usually display syndecan-1 in a punctate staining
pattern without large focal accumulations. Aggregates were negative for
staining when a control isotype-matched monoclonal antibody was
substituted for antibody 281.2 (not shown).
Aggregation of Syndecan-1 Transfectants Is Dependent on
Heparan Sulfate
We next performed assays in the presence of
heparin to determine if the aggregation of syndecan-1-transfected cells
is mediated by heparan sulfate. Heparin was used initially because it
is a functional analogue of heparan sulfate and recent studies indicate
that syndecan-1 can bind some types of collagen with an affinity
similar to that of sub-populations of low molecular weight
heparin(14) . Therefore, the heparan sulfate of syndecan-1 may
contain ``heparin-like'' regions that promote high affinity
interactions with ligands.
Exogenous Syndecan-1 Inhibits Cell Aggregation
The
finding that cells aggregate following transfection and expression of
syndecan-1 indicates that syndecan-1 may be directly involved in
promoting the adhesive interactions between cells. However, it is also
possible that expression of syndecan-1 by the transfectants results in
changes in other adhesion molecules at the cell surface that could
promote aggregation independent of syndecan-1. Therefore, to determine
if syndecan-1 plays a direct role in mediating cell aggregation, we
performed assays in the presence of exogenous purified syndecan-1
ectodomain. The ectodomain includes the extracellular portion of the
syndecan-1 core protein and the attached glycosaminoglycan chains. In
the absence of syndecan-1, large aggregates of syndecan-1-transfected
cells are formed (Fig. 5A). However, in the presence of
approximately 0.70 µg/ml of exogenous syndecan-1, there is a
striking inhibition of cell aggregation (Fig. 5B).
Quantification reveals that the control samples routinely have
60-70% of cells in relatively large aggregates while in those
wells where syndecan-1 was added, less than 10% of the cells are
incorporated into aggregates of four or more cells. In addition, the
few aggregates that do form remain small in size (Fig. 5B). Addition of significantly smaller amounts of
syndecan-1 to the aggregation buffer results in partial inhibition of
cell aggregation. For example, following addition of 0.20 µg/ml of
syndecan-1, approximately 37% of the cells were present within
aggregates (not shown). The inhibition of aggregation by exogenous
syndecan-1 supports the notion that syndecan-1 plays a direct role in
mediating aggregation of these cells.
Heterophilic Adhesion Suggests a Counter-receptor for
Syndecan-1
The discovery that syndecan-1 mediates cell
aggregation raises the question as to whether syndecan-1 on one cell
interacts with syndecan-1 on an adjacent cell (homophilic interaction)
or with a different cell surface receptor (heterophilic interaction).
Both are viable possibilities because heparan sulfates can
self-associate in a manner that may promote homophilic
adhesion(43) , and heparan sulfates can also interact with
other adhesion molecules such as PECAM-1 thereby promoting adhesion in
a heterophilic fashion (6) . To address this question we
performed aggregation assays using fluorescein-labeled
control-transfected cells mixed with unlabeled syndecan-1-transfected
cells. After rotating for 1 h, cells were removed and examined by
fluorescence microscopy. Clearly, labeled cells are incorporated into
aggregates containing unlabeled cells indicating that syndecan-1 does
not have to be present on both cell types for aggregates to form
(heterophilic interaction; Fig. 6A). As expected,
treatment of syndecan-1-transfected cells with heparitinase prior to
the assay inhibits aggregation (Fig. 6B), indicating
that heparan sulfate is required for aggregation of the two cell types.
However, treatment of control-transfected cells with heparitinase did
not affect their binding to syndecan-1-transfected cells (Fig. 6C), indicating that their co-aggregation is not
dependent on the interaction of syndecan-1 heparan sulfate with heparan
sulfate from a different (non-syndecan-1) proteoglycan present on
control-transfected cells. To ensure that fluorescence cell labeling
did not influence adhesion between the cells, two controls were
performed. First, the syndecan-1-transfected cells were labeled rather
than the control-transfected cells. Second, separate experiments were
performed using two different fluorescein-labeling compounds, PKH-26
which labels cell membranes and Cell Tracker Orange which
labels cells intracellularly. All controls gave results identical to
those shown in Fig. 6(not shown). In a separate experiment,
labeled syndecan-1-transfected cells were mixed with
heparitinase-treated unlabeled syndecan-1-transfected cells. Again, the
aggregates that formed contained both the heparitinase-treated and
-untreated cells (not shown). Taken together these mixing experiments
support the conclusion that aggregation mediated by syndecan-1 is via a
heterophilic mechanism, wherein the syndecan-1 proteoglycan binds to a
counter-receptor on adjacent cells.
Syndecan-4-transfected Cells Also Aggregate Extensively
But Betaglycan-transfected Cells Aggregate Poorly
To determine
if other syndecans could act to mediate cell aggregation, we
transfected ARH-77 cells with the cDNA for syndecan-4. These cells
behave similarly to cells transfected with syndecan-1; they form large
aggregates in culture and aggregate extensively in rotation-mediated
aggregation assays (Table 2). Aggregation is greatly diminished
in the presence of 10 µg/ml heparin, although inhibition of
aggregation of syndecan-4-transfected cells was not as great as that of
syndecan-1-transfected cells. Additionally, aggregation of
syndecan-4-transfected cells was almost completely inhibited when cells
were pretreated with heparitinase prior to the assay (not shown).
via its core protein and to basic fibroblast growth
factor via its heparan sulfate chains(45) . However, it is not
known if betaglycan plays a role in mediating cell adhesion. In
contrast to syndecans-1 and -4, cells transfected with betaglycan fail
to aggregate extensively (Table 2). Only 16.5% of the
betaglycan-transfected cells are incorporated into aggregates, similar
to the parental cells in which 14% of the cells aggregate (Table 1). The intact betaglycan extracted from the transfected
cells migrates as a broad species above 200 kDa and contains both
heparan sulfate and chondroitin sulfate glycosaminoglycan chains as
determined by enzyme digestions prior to Western blotting (not shown).
This is consistent with the molecular characteristics of betaglycan
present on other cell types(44, 46) . Also, because
the transfected cells used for experiments in Table 2stain
brightly for cell surface betaglycan (ratio of median fluorescence
intensity of 5.9 relative to control cells as determined by flow
cytometry), their failure to aggregate is not due to low levels of
betaglycan expression. However, when a series of transfected clones
were examined, several that expressed very high levels of betaglycan
did aggregate (not shown). This is in contrast to syndecan-1-mediated
aggregation which occurs between cells even when they express
relatively low levels of syndecan-1 (see above), indicating that the
heparan sulfate on syndecan-1 may have a significantly higher affinity
for its counter-receptor than does the heparan sulfate on betaglycan.
)However, we cannot rule
out the possibility that syndecan-1 acts as a co-factor for another
adhesion molecule thereby promoting cell aggregation in a manner
similar to the association of heparan sulfate proteoglycan with N-CAM.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
ABSTRACT
This article has been cited by other articles:
|
|
 |
|
  R Joshi, D Horncastle, K Elderfield, I Lampert, A Rahemtulla, and K N Naresh Bone marrow trephine combined with immunohistochemistry is superior to bone marrow aspirate in follow-up of myeloma patients J. Clin. Pathol., February 1, 2008; 61(2): 213 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I Abiatari, J Kleeff, J Li, K Felix, M W Buchler, and H Friess Hsulf-1 regulates growth and invasion of pancreatic cancer cells J. Clin. Pathol., October 1, 2006; 59(10): 1052 - 1058. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Houston, M. A. Julien, S. Parthasarathy, and E. L. Chaikof Oxidized linoleic acid regulates expression and shedding of syndecan-4 Am J Physiol Cell Physiol, February 1, 2005; 288(2): C458 - C466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yang, V. MacLeod, M. Bendre, Y. Huang, A. M. Theus, H.-Q. Miao, P. Kussie, S. Yaccoby, J. Epstein, L. J. Suva, et al. Heparanase promotes the spontaneous metastasis of myeloma cells to bone Blood, February 1, 2005; 105(3): 1303 - 1309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Deepa, S. Yamada, M. Zako, O. Goldberger, and K. Sugahara Chondroitin Sulfate Chains on Syndecan-1 and Syndecan-4 from Normal Murine Mammary Gland Epithelial Cells Are Structurally and Functionally Distinct and Cooperate with Heparan Sulfate Chains to Bind Growth Factors: A NOVEL FUNCTION TO CONTROL BINDING OF MIDKINE, PLEIOTROPHIN, AND BASIC FIBROBLAST GROWTH FACTOR J. Biol. Chem., September 3, 2004; 279(36): 37368 - 37376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-J. Lee, S. Kadambi, C. David, A. D. Cooper, and S. Y. Choi Association of hepatic lipase with proteoglycans stimulates the production of proteoglycans in vivo and in vitro J. Lipid Res., July 1, 2004; 45(7): 1266 - 1271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kelly, H.-Q. Miao, Y. Yang, E. Navarro, P. Kussie, Y. Huang, V. MacLeod, J. Casciano, L. Joseph, F. Zhan, et al. High Heparanase Activity in Multiple Myeloma Is Associated with Elevated Microvessel Density Cancer Res., December 15, 2003; 63(24): 8749 - 8756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yang, M. Borset, J. K. Langford, and R. D. Sanderson Heparan Sulfate Regulates Targeting of Syndecan-1 to a Functional Domain on the Cell Surface J. Biol. Chem., April 4, 2003; 278(15): 12888 - 12893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zako, J. Dong, O. Goldberger, M. Bernfield, J. T. Gallagher, and J. A. Deakin Syndecan-1 and -4 Synthesized Simultaneously by Mouse Mammary Gland Epithelial Cells Bear Heparan Sulfate Chains That Are Apparently Structurally Indistinguishable J. Biol. Chem., April 4, 2003; 278(15): 13561 - 13569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. W. B. Derksen, R. M. J. Keehnen, L. M. Evers, M. H. J. van Oers, M. Spaargaren, and S. T. Pals Cell surface proteoglycan syndecan-1 mediates hepatocyte growth factor binding and promotes Met signaling in multiple myeloma Blood, February 15, 2002; 99(4): 1405 - 1410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Lacy and R. D. Sanderson Sperm protein 17 is expressed on normal and malignant lymphocytes and promotes heparan sulfate-mediated cell-cell adhesion Blood, October 1, 2001; 98(7): 2160 - 2165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Matsuda, H. Maruyama, F. Guo, J. Kleeff, J. Itakura, Y. Matsumoto, A. D. Lander, and M. Korc Glypican-1 Is Overexpressed in Human Breast Cancer and Modulates the Mitogenic Effects of Multiple Heparin-binding Growth Factors in Breast Cancer Cells Cancer Res., July 1, 2001; 61(14): 5562 - 5569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. van der Voort, R. M.J. Keehnen, E. A. Beuling, M. Spaargaren, and S. T. Pals Regulation of Cytokine Signaling by B Cell Antigen Receptor and CD40-controlled Expression of Heparan Sulfate Proteoglycans J. Exp. Med., October 9, 2000; 192(8): 1115 - 1124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Borset, O. Hjertner, S. Yaccoby, J. Epstein, and R. D. Sanderson Syndecan-1 is targeted to the uropods of polarized myeloma cells where it promotes adhesion and sequesters heparin-binding proteins Blood, October 1, 2000; 96(7): 2528 - 2536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yamashita, K. Oritani, E. K. Miyoshi, R. Wall, M. Bernfield, and P. W. Kincade Syndecan-4 Is Expressed by B Lineage Lymphocytes and Can Transmit a Signal for Formation of Dendritic Processes J. Immunol., May 15, 1999; 162(10): 5940 - 5948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Iba, R. Albrechtsen, B. J. Gilpin, F. Loechel, and U. M. Wewer Cysteine-Rich Domain of Human ADAM 12 (Meltrin {alpha}) Supports Tumor Cell Adhesion Am. J. Pathol., May 1, 1999; 154(5): 1489 - 1501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Borghesi, Y. Yamashita, and P. W. Kincade Heparan Sulfate Proteoglycans Mediate Interleukin-7- Dependent B Lymphopoiesis Blood, January 1, 1999; 93(1): 140 - 148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Langford, M. J. Stanley, D. Cao, and R. D. Sanderson Multiple Heparan Sulfate Chains Are Required for Optimal Syndecan-1 Function J. Biol. Chem., November 6, 1998; 273(45): 29965 - 29971. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Hoffman, M. Nomizu, E. Roque, S. Lee, D. W. Jung, Y. Yamada, and H. K. Kleinman Laminin-1 and Laminin-2 G-domain Synthetic Peptides Bind Syndecan-1 and Are Involved in Acinar Formation of a Human Submandibular Gland Cell Line J. Biol. Chem., October 30, 1998; 273(44): 28633 - 28641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. McFall and A. C. Rapraeger Characterization of the High Affinity Cell-binding Domain in the Cell Surface Proteoglycan Syndecan-4 J. Biol. Chem., October 23, 1998; 273(43): 28270 - 28276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Liu, E. D. Litwack, M. J. Stanley, J. K. Langford, A. D. Lander, and R. D. Sanderson Heparan Sulfate Proteoglycans as Adhesive and Anti-invasive Molecules. SYNDECANS AND GLYPICAN HAVE DISTINCT FUNCTIONS J. Biol. Chem., August 28, 1998; 273(35): 22825 - 22832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. V. Dhodapkar, E. Abe, A. Theus, M. Lacy, J. K. Langford, B. Barlogie, and R. D. Sanderson Syndecan-1 Is a Multifunctional Regulator of Myeloma Pathobiology: Control of Tumor Cell Survival, Growth, and Bone Cell Differentiation Blood, April 15, 1998; 91(8): 2679 - 2688. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |