(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
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.
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 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.
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). 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.
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/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).
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- 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.
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. ()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.
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.