 |
INTRODUCTION |
Dystroglycan (DG)1
consists of
DG and
DG, the two subunits yielded by proteolytic
cleavage of a single precursor protein.
DG is a highly glycosylated
extracellular protein with a molecular mass of 120-190 kDa, and
DG
is a 43-kDa transmembrane protein (1, 2). On the cell surface,
DG is
anchored to
DG by noncovalent bonds (3, 4).
DG is the central component of the dystrophin-associated glycoprotein
complex, and its physiological roles have been extensively studied in
skeletal muscle, from which it was originally isolated. In the skeletal
muscle sarcolemma, DG forms a physical link between the extracellular
matrix and intracellular cytoskeleton by the binding of
DG and
DG
with laminin-2 in the matrix and dystrophin in the cytoskeleton,
respectively (5, 6). The membrane stability of the sarcolemma depends
on this link as evidenced by the progressive muscle degeneration in
Duchnne's muscular dystrophy that is caused by an abnormal dystrophin
gene (7). A specialized form of the DG complex is found in the
neuromuscular junction, where
DG and
DG associate with agrin in
the matrix and utrophin in the cytoskeleton, respectively (8-10). This
protein complex is critically involved in agrin-induced clustering of
acetylcholine receptors (11).
DG is expressed in various tissues and cell lines, and evidence for its
functions in non-muscle tissues has been accumulating in recent years.
In the peripheral nervous system, DG is expressed by Schwann cells and
is involved both in Schwann cell adhesion to the extracellular matrix
and in myelinogenesis (12-15). In the central nervous system, DG is
expressed both by glial cells and by certain groups of neurons and is
suggested to be involved in blood brain barrier and synapse formation
(16-18). Outside the nervous system, DG has been shown to be involved
in epithelial morphogenesis during embryogenesis (19, 20).
Immunohistochemical studies using human brain sections indicated the
expression of DG by vascular endothelial cells (21, 22). An
immunocytochemical study indicated the expression of DG by cultured
human umbilical endothelial cells as well (23). The expression of DG by
vascular endothelial cells, however, is still controversial because of
the negative immunostaining of anti-DG antibodies found in brain
capillary endothelial cells (16). Most recently, Durbeej et
al. (24) suggested that the anti-DG immunostaining in some blood
vessels emanates not from the endothelial cells, but from the smooth
muscle cells, which are a rich source of DG.
Vascular endothelial cells undergo drastic morphological and functional
changes during angiogenesis, and it is well established that the
behavior of the cell is critically influenced by interaction with the
extracellular matrix in their milieu. Laminin is the major constituent
of the vascular endothelial basement membrane (25), and it is generally
accepted that the principal endothelial cell-surface receptor that
recognizes laminin is the integrin family of cell adhesion molecules
(26-28). Several lines of evidence, however, suggested the existence
of non-integrin types of laminin receptors expressed by vascular
endothelial cells whose identity remains unclear (29, 30). Given the
laminin-binding capacity of DG, the purposes of this study were to
confirm the expression of DG by primary cultured bovine aortic
endothelial (BAE) cells and to establish the role of DG as a
non-integrin type of laminin receptor involved in BAE cell adhesion to
the extracellular matrix.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium (DMEM),
fetal calf serum, mouse Engelbreth-Holm-Swarm sarcoma laminin-1, and
bovine plasma fibronectin were from Gibco BRL (Tokyo, Japan).
Glycosaminoglycans (heparin, dextran sulfate, fucoidan, sulfatide,
chondroitin sulfate, and dextran) and oligosaccharides
(N-acetylgalactosamine and N-acetylneuraminic acid) were from Wako (Tokyo). [
-32P]CTP (220 TBq/mmol)
was from Amersham International (Buckinghamshire, United Kingdom).
EZ-link sulfo-NHS-SS-biotin was from Pierce. A mouse monoclonal
anti-human
DG antibody was from Novo CASTRA (Clarement Place, United
Kingdom). A rabbit polyclonal antibody against platelet endothelial
cell adhesion molecule-1 (PECAM1) was a kind gift from Dr. K. Fujiwara
(National Cardiovascular Research Institute, Suita, Japan). The
cDNA clone for rabbit DG was a kind gift from Dr. K. P. Campbell (Howard Hughes Medical Institute and Department of Physiology
and Biophysics, University of Iowa College of Medicine). All other
chemicals were of reagent grade and were obtained commercially.
Cell Culture and Adhesion Assay--
A primary culture of BAE
cells was obtained as described (31). The cells were routinely
maintained in DMEM and 10% fetal calf serum at 37 °C in a
humidified atmosphere containing 5% CO2. They were
passaged once a week at a ratio of 1:5 to keep an exponentially growing
state. All cells used were between passages 5 and 10. For the cell
adhesion assay, 96-well plates were incubated with laminin-1 or
fibronectin (both at 10 µg/ml in phosphate-buffered saline) for
1 h at 37 °C. Both laminin-1- and fibronectin coating of the
substratum caused a dose-dependent enhancement of BAE cell adhesion, and a 10 µg/ml concentration of either substance was a
supramaximal concentration for the effect (data not shown). Residual
protein-binding sites were blocked by incubating the plates in 2% BSA
overnight at 4 °C. Cells were seeded at 5 × 104
cells/well in 100 µl of DMEM and 0.2% BSA and then kept in a CO2 incubator. Because a time course analysis showed a
linear increment of the attached cell number at least for 2 h
(data not shown), the incubation time was set to 2 h in the
following experiments. After the incubation, unattached cells were
removed by washing the plates with phosphate-buffered saline. The
attached cells were lysed in 100 µl of Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl)
containing 0.2% Tween 20. The number of the attached cells was
estimated by the lactate dehydrogenase activity in the lysate measured
with a lactate dehydrogenase cytotoxic assay kit (Wako) according to
the manufacturer's instructions. This assay gave a linear relationship
between the cell number and the measured A560 up
to the cell number of 16 × 104/well. Under the
control condition after 2 h of incubation, (1.8 ± 0.1) × 104 (n = 8; ~36% of the cells seeded)
cells attached to laminin-1-coated dishes, and (4.8 ± 0.1) × 104 (n = 8; ~96% of the cells seeded)
cells attached to fibronectin-coated dishes.
cDNA Cloning and Northern Blotting--
cDNA was
synthesized from the total BAE RNA by SuperScript II reverse
transcriptase (Gibco BRL) and used as a template for polymerase chain
reaction (PCR). The sequences of the PCR primers were 5'-
GCCCTGGAGCCTGACTTTAAGGC-3' (sense) and 5'-TCGTCCAGCTCGTCTGCAAAGA-3' (antisense). These sequences correspond to nucleotide sequences 2252-2274 and 2565-2577 of rabbit DG (1), respectively. The PCR
product was labeled with [32P]dCTP (110 TBq/mmol;
Amersham International) using a random primer DNA labeling system
(Gibco BRL) and was used as a probe for hybridization screening of a
gt11 cDNA library from BAE poly(A)+ RNA. A single
positive clone was extracted from the plaque, and the EcoRI
fragment of the phage was subcloned into pBlueScript to give pBSK/DG.
The nucleotide sequence of the pBSK/DG insert was determined using a
BcaBEST dideoxy sequencing kit (Takara, Otsu, Japan). The probe for
Northern blotting was prepared by labeling the EcoRI
fragment of pBSK/DG with [32P]dCTP as described above.
Blotting procedures were as described (32). The blot was visualized for
radioactivity with a BAS2000 image analyzer (Fujitsu, Tokyo).
Western Blotting--
The trichloroacetic acid precipitate of
the whole cell lysate was used for Western blotting with the anti-
DG
antibody. In brief, the cells were harvested in phosphate-buffered
saline by scraping and lysed by sonication, and the cellular protein
was precipitated by incubation at 4 °C for 1 h in 10%
trichloroacetic acid. After centrifugation at 3000 × g
for 1 h at 4 °C, the pellet was dissolved in Laemmli's sample
buffer and subjected to SDS-10% polyacrylamide gel electrophoresis and
Western transfer to polyvinylidene difluoride membranes. Immunostaining
of the blots was done as described (33). The blots were developed using
an ABC immunodetection kit (Vector Labs, Inc., Burlingame, CA) and
Konica immunostain HRP-1000.
Preparation of a Recombinant Laminin
5 Fragment--
An
~3-kilobase pair fragment of the mouse laminin
5 (nucleotides
7855-11007; GenBankTM/EBI accession number
U37501 (34)) was subcloned into the XhoI/NotI
site of pET28b (Novagen, Madison, WI). This fragment encodes the
globular domains of laminin
5 (35). The plasmid was transformed into
Escherichia coli BL21(DE3) (Novagen), and the recombinant
His6-laminin
5 protein was purified by TALON metal
affinity resin chromatography (CLONTECH).
Biotin-labeled Laminin Overlay Assay--
The trichloroacetic
acid precipitate of the whole cell lysate or the soluble proteins in
the concentrated conditioned medium were subjected to SDS-10%
polyacrylamide gel electrophoresis and Western transfer as described
above. Laminin-1 and the recombinant laminin
5 fragment were
biotinylated using EZ-link sulfo-NHS-SS-biotin according to the
manufacturer's instructions. After blocking in Block-Ace (Yukijirushi,
Tokyo) at 4 °C overnight, the blot was incubated in overlay buffer
(10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
and 3% BSA) containing biotin-labeled laminin-1 or laminin
5 (both
at a concentration of 1 µg/ml) at room temperature for 2 h. The
blot was extensively washed with the buffer, and the bound
biotin-laminin was detected with an ABC immunodetection kit and Konica
immunostain HRP-1000.
Transient Expression of DG--
The entire coding sequence of
rabbit DG was subcloned into the mammalian expression vector pREP9
(Invitrogen, Carlsbad, CA) to give pREP9/DG. BAE cells cultured in
100-mm plates were transfected with 2 µg of pREP9/DG or with an empty
vector plasmid using 10 µl of LipofectoAMINE (Gibco BRL) according to
the manufacturer's instructions. After a recovery period of 16 h
in DMEM and 10% fetal calf serum, the cells were incubated in
serum-free DMEM for 24 h. The conditioned medium was collected,
filtered through a 0.2-µm pore filter, and then concentrated for
100-fold using Centriprep-50 (Amicon, Inc., Beverly, MA).
Immunocytochemistry--
Cells cultured on
poly-L-lysine-coated glass-bottom dishes were used for
immunostaining. All procedures were done at room temperature. The cells
were rinsed with a buffer (10 mM Mes, pH 6.1, 150 mM NaCl, 5 mM EGTA, 5 mM
MgCl2, and 5 mM glucose), fixed with 4%
paraformaldehyde in the same buffer for 10 min, and then permeabilized
with 0.1% Triton X-100 for 1 min. After blocking with 1% BSA and 2%
goat serum, they were incubated with the anti-
DG antibody (1:200
dilution in Tris-buffered saline and 0.1% BSA) for 30 min. After
washing, bound antibody was detected with Cy2-conjugated anti-mouse IgG
(Amersham International). For double staining, the rabbit polyclonal
anti-PECAM1 antibody (1:5000 dilution) was applied together with the
anti-
DG antibody, and the bound anti-PECAM1 and anti-
DG
antibodies were detected with Cy3-conjugated anti-rabbit IgG and
Cy2-conjugated anti-mouse IgG, respectively. Fluorescent images of the
cells were obtained using an Axiovert 25 fluorescent microscope (Carl
Zeiss, Inc.) or an MRC1024 confocal microscope (Bio-Rad).
Statistical Analysis--
Where necessary, statistical analysis
was done by analysis of variance.
 |
RESULTS |
Expression of DG by Cultured BAE Cells--
To examine whether BAE
cells expressed DG, we conducted Northern and Western blotting. The
bovine DG cDNA probe for Northern blotting was prepared by reverse
transcription-PCR followed by hybridization screening of a BAE cDNA
library in
gt11 using the PCR product as a probe. The primers for
the PCR were designed based on the rabbit DG cDNA sequence (1). In
this process, we eventually isolated a full-length cDNA encoding
bovine DG (GenBankTM/EBI accession number
AB009079). Northern blotting of BAE mRNA using the whole coding
sequence of the DG cDNA as a probe detected a major band at 5.4 kilobase pairs and a minor band at 5.0 kilobase pairs (Fig.
1a). On Northern blotting of
bovine multiple-tissue mRNAs, we found a ubiquitous distribution of
the 5.4-kilobase pair band (data not shown). For Western blotting, we
used a monoclonal anti-human
DG antibody. This antibody recognizes
an epitope in the carboxyl-terminal 10-amino acid sequence of human
DG that is identical to that of bovine
DG. The antibody detected
a single band at 43 kDa in the BAE cell lysate (Fig. 1b).
Immunofluorescent staining of the confluent cell layer revealed that
almost all of the cells expressed both
DG and the endothelial
cell-specific cell-cell adhesion molecule PECAM1 (36) (Fig.
1c). These results indicated that the DG mRNA and
protein detected in our primary culture derived from endothelial cells,
but not from contaminating smooth muscle cells.

View larger version (79K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of DG by cultured BAE cells.
a, Northern blotting. Two µg of mRNA was loaded on the
lane. The blot was probed with full-length bovine DG cDNA labeled
with 32P. Molecular sizes are given in kilobase pairs.
b, Western blotting. Five µg of membrane proteins was
loaded on the lane. The blot was probed with a monoclonal anti- DG
antibody. Molecular sizes are given in kilodaltons. c,
immunofluorescent staining. Confluent cells were doubly stained with
the indicated antibodies as described under "Experimental
Procedures." Shown are the fluorescent images of Cy2 and Cy3 obtained
with an Axiovert 25 microscope. The anti- DG antibody stained
dot-like clusters in the cell body, and the anti-PECAM1 antibody
stained cell-cell attachment sites.
|
|
Subcellular Localization of DG in Migrating BAE
Cells--
Immunofluorescent staining of
DG in subconfluent cells
revealed a distinct difference in the staining patterns between the resting and migrating cells (Fig. 2). In
the resting cells, the antibody stained multiple plaques located on the
basal side of the cells as revealed by confocal microscopy. The plaques
appeared to be diffusely distributed in the central portion of the cell floor (Fig. 2a). In the migrating cells, the patchy staining
was obscured, and instead, strong immunostaining was observed in the trailing edge (Fig. 2b). The trailing edge was retracted
when the cells were detached and actually moved (Fig.
2c).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
Subcellular localization of DG in migrating
BAE cells. The fixed cells were immunostained with the anti- DG
antibody, and the bound antibody was detected with Cy2-conjugated
anti-mouse IgG. Shown are the confocal images of the horizontal planes
of the cell floor. a, a resting cell; b, a cell
that began to move with the trailing edge still attached; c,
a cell that detached from the substratum and was moving in space.
Arrows indicate the direction of cell migration.
|
|
Biotin-Laminin-1 Binding to
DG--
The interaction of
DG
with laminin-1 was characterized by biotin-laminin-1 overlay on the
Western blot of BAE membrane proteins. Biotin-laminin-1 detected a
major band at 130 kDa (Fig.
3b) and a minor band at 110 kDa (Figs. 3b and 5b, lane 4) on the
blot. The same assay of the soluble proteins contained in the
conditioned medium from pREP9/DG- or vector-transfected cells verified
the presence of the soluble form of
DG and its overexpression by pREP9/DG transfection (Fig. 3a). The conditioned medium from
the pREP9/DG-transfected cells, but not that from the
vector-transfected cells, displaced the binding of biotin-laminin-1 to
the 130-kDa BAE membrane protein (Fig. 3b), indicating the
identity of this protein as
DG. The minor band at 110 kDa was also
abolished by the conditioned medium from pREP9/DG-transfected cells,
suggesting that the 110-kDa protein also derived from
DG. The
appearance of the 110-kDa minor band was not as consistent as
that of the 130-kDa band on the blot of membrane proteins, and it was
not detected on the blot of soluble proteins. Therefore, it is most likely that the 110-kDa minor band represents immature
DG with less
glycosylation.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 3.
Detection of DG by
biotin-laminin-1 overlay assay. Soluble proteins in the
conditioned medium (a) or membrane proteins (b)
were separated by SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membranes. In both cases, 10 µg of protein was loaded on a lane. The membranes were probed with
biotin-laminin-1 as described under "Experimental Procedures."
a, the conditioned medium was from cells transfected with
vector plasmid (lane 1) or pREP9/DG (lane 2).
b, the membrane strip was incubated with biotin-laminin-1 in
the absence (lane 1) or presence of the concentrated
conditioned medium (10%, v/v) from cells transfected with vector
plasmid (lane 2) or pREP9/DG (lane 3). Molecular
sizes are given in kilodaltons.
|
|
The binding to the 130-kDa protein was totally abolished in the
presence of EDTA (Fig. 4) as expected
from the Ca2+ dependence of the
DG-laminin interaction
(1).
DG is heavily glycosylated, and its mucin-like carbohydrate
moiety is required for binding with laminin (37, 38). Therefore, we
tested the ability of a series of glycosaminoglycans to inhibit the
biotin-laminin-1 binding to BAE cell
DG and found that the binding
was inhibited by heparin, dextran sulfate, and fucoidan, but not by
sulfatide, chondroitin sulfate, and dextran (Fig. 4). We also tested
two oligosaccharides, N-acetylgalactosamine and
N-acetylneuraminic acid and found that neither of them could
displace the binding (Fig. 4).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibition of biotin-laminin-1 binding
to DG by glycosaminoglycans. BAE membrane
proteins were separated by SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membranes. The membrane strips
were incubated with biotin-laminin-1 in the absence (None)
or presence of EDTA (5 mM), glycosaminoglycan, or
oligosaccharide as indicated. ChS, chondroitin sulfate;
Dex, dextran; DexS, dextran sulfate;
Fuc, fucoidan; Hep, sodium heparin;
Sul, sulfatide; NAN,
N-acetylneuraminic acid; NAG,
N-acetylgalactosamine. The
glycosaminoglycans/oligosaccharides were used at 2 mg/ml.
|
|
Binding of Recombinant Laminin
5 to
DG--
Because of the
recent identification of laminin
5 as a major subtype of laminin
chains present in the basement membrane of endothelial cells (35, 39,
40), we tested the binding capacity of a recombinant laminin
5
fragment for
DG. The ~110-kDa fragment that includes the globular
domains of laminin
5 was expressed in E. coli, purified
(Fig. 5a), and labeled with
biotin. The biotin-labeled laminin
5 protein detected both the 130- and 110-kDa bands on the Western blot of BAE membrane proteins (Fig. 5b, lane 1). These bindings were abolished in the
presence of a high concentration of unlabeled laminin-1 (lane
2) and also in the presence of the conditioned medium from
pREP9/DG-transfected cells (lane 3). In another set of
experiments, unlabeled laminin
5 protein disrupted the binding of
biotin-laminin-1 to the 130/110-kDa proteins (lanes 4 and
5).

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 5.
Binding of recombinant laminin
5 fragment to DG.
a, Coomassie Blue staining of the 110-kDa recombinant 5
fragment (arrow) expressed in E. coli. The
protein was expressed with (lane 1) or without (lane
2) isopropyl- -D-thiogalactopyranoside induction.
b, biotin-laminin overlay assay. Membrane strips were cut
from a Western blot of BAE membrane proteins. They were incubated with
the biotin-labeled laminin 5 fragment (1 µg/ml) in the absence
(lane 1) or presence of unlabeled laminin-1 (1 mg/ml)
(lane 2) or the concentrated conditioned medium from
pREP9/DG-transfected cells (10%, v/v) (lane 3). In another
set of experiments, the strips were incubated with biotin-labeled
laminin-1 (1 µg/ml) in the absence (lane 4) or presence
(lane 5) of the unlabeled laminin 5 fragment (1 mg/ml).
Molecular masses (in kilodaltons) are indicated.
|
|
Adhesion of BAE Cells to Laminin-1 Mediated by DG--
The
immunocytochemical localization of DG on the basal side of BAE cells
and the interaction of
DG with laminin in the ligand overlay assay
suggested the involvement of DG in BAE cell adhesion to the
extracellular matrix. To obtain direct evidence for it, we examined
whether 1) the soluble
DG in the conditioned medium from
pREP9/DG-transfected cells and/or 2) the glycosaminoglycans that
inhibited the biotin-laminin-1 binding to
DG in the overlay assay
could inhibit the BAE cell adhesion to laminin-1-coated dishes. To test
the substratum specificity, we also conducted the same assay using
fibronectin-coated dishes.
The conditioned medium from pREP9/DG-transfected cells, but not that
from vector-transfected cells, caused significant inhibition of the
cell adhesion to laminin-1-coated dishes (Fig.
6). It caused no further decrement of the
adhesion in the presence of heparin. In contrast to its effects on
laminin-1-coated dishes, the conditioned medium caused no effect on BAE
cell adhesion to fibronectin-coated dishes (Fig. 6). Of the six
glycosaminoglycans, heparin, dextran sulfate, fucoidan, and sulfatide
caused significant inhibition of the cell adhesion to laminin-1 (Fig.
7). Chondroitin sulfate and dextran
failed to inhibit the adhesion and so did the two oligosaccharides
tested, N-acetylgalactosamine and
N-acetylneuraminic acid. Of the four effective
glycosaminoglycans, sulfatide was by far the most effective compared
with the modest inhibition by the other three. None of the
glycosaminoglycans/oligosaccharides caused any effects on BAE cell
adhesion to fibronectin-coated dishes, except for sulfatide. Sulfatide
again caused a drastic inhibition of the adhesion to fibronectin-coated
dishes (Fig. 7).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of soluble
DG on BAE cell adhesion. The same number of
cells in 100 µl of cell suspension was seeded on plastic plates
coated with laminin-1 or fibronectin. The wells contained 90 µl of
DMEM and 0.2% BSA with or without heparin (2 mg/ml) as indicated and
10 µl of the concentrated conditioned medium from vector-transfected
cells (open bars) or from pREP9/DG-transfected cells
(closed bars). After 2 h of incubation, the number of
attached cells was estimated by the lactate dehydrogenase activity in
the cell lysate as described under "Experimental Procedures." The
results are expressed relative to the values in the absence of heparin
and the conditioned medium (100%). Shown are the mean ± S.E. of
three determinations, each done in triplicate. *, p < 0.01 (significantly different from the values in the presence of the
concentrated conditioned medium from vector-transfected cells).
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of glycosaminoglycans and
oligosaccharides on BAE cell adhesion. The same number of cells in
100 µl of cell suspension was seeded on plastic plates coated with
laminin-1 or fibronectin. The wells contained 100 µl of DMEM and
0.2% BSA with or without the glycosaminoglycan or oligosaccharide as
indicated. The glycosaminoglycans/oligosaccharides were used at 2 mg/ml. Shown are the mean ± S.E. of three determinations, each
done in triplicate. *, p < 0.01 (significantly
different from the control values in the absence of the
glycosaminoglycan/oligosaccharide). ChS, chondroitin
sulfate; Dex, dextran; DexS, dextran sulfate;
Fuc, fucoidan; Hep, sodium heparin;
Sul, sulfatide; NAN,
N-acetylneuraminic acid; NAG,
N-acetylgalactosamine.
|
|
 |
DISCUSSION |
In this study, expression of DG by cultured BAE cells was
confirmed by cDNA cloning from a BAE cDNA library, Northern
blotting of mRNA, and Western blotting of membrane proteins (Fig.
1, a and b). Coexpression of
DG and PECAM1 by
the cells confirmed the endothelial cell origin of the DG mRNA and
protein detected (Fig. 1c). The length of the deduced amino
acid sequence of bovine DG (895 amino acids) was exactly the same as
those of the rabbit, mouse, and human versions, and 91-93% of the
amino acid sequence was identical to the three reported sequences (6).
Together with the similar size of the mRNA and its ubiquitous
distribution on the Northern blot, these results indicate high
conservation of the DG precursor gene among mammalian species.
Immunocytochemical analysis revealed localization of DG on the basal
side of BAE cells and a drastic change in the localization associated
with cell migration (Fig. 2). In the resting cells, DG was confined in
multiple plaques, the morphology of which is apparently close to that
of the laminin/agrin-induced DG plaques in skeletal muscle cells (41).
The plaques were obscured in migrating cells, in which the trailing
edge was most intensely stained. The trailing edge was then retracted
when the cells actually moved in space. A straightforward explanation
for these observations is that DG, with its tight association with the
extracellular matrix, is left in the last part of the cell that
detaches from the substratum when it moves. Although it is necessary to
monitor the location of DG in live cells to verify this scenario, it is at least clear that the subcellular localization of DG in migrating BAE
cells is quite different from that of integrins that have been shown to
be recruited to the frontal portion of migrating leukocytes (42).
Two lines of biochemical evidence were presented in the this study that
indicated the role of DG as a non-integrin laminin receptor involved in
BAE cell adhesion to the extracellular matrix. The first is the
inhibition of BAE cell adhesion to laminin-1 by soluble
DG contained
in the conditioned medium from DG cDNA-transfected cells (Fig. 6).
Soluble
DG could also inhibit the biotin-laminin-1 binding to
DG
in the dissociated membrane proteins (Fig. 3). Secretion of
DG to
the medium was first indicated in RT4 schwannoma cells (15), and we
have confirmed the finding in cultured BAE cells. The molecular
mechanism that generates the soluble form and its physiological role
must be the subjects in a future study.
The second line of biochemical evidence is the inhibition of BAE cell
adhesion to laminin-1 by a set of glycosaminoglycans (heparin, dextran
sulfate, fucoidan, and sulfatide) (Fig. 7). Of the four
glycosaminoglycans, sulfatide was by far the most effective; however,
it did not inhibit the
DG-laminin-1 interaction in the ligand
overlay assay (Fig. 4), and it also inhibited the BAE cell adhesion to
fibronectin (Fig. 7). Therefore, it is plausible to conclude that the
other three, but not sulfatide, inhibited BAE cell adhesion to
laminin-1 by specifically disrupting the
DG-laminin-1 interaction.
The mechanism for sulfatide inhibition is still unknown. The three
glycosaminoglycans (heparin, dextran sulfate, and fucoidan) have been
shown to inhibit the adhesion of RT4 schwannoma cells to laminin-1 (15)
as well as agrin-induced acetylcholine receptor clustering in myotubes
(43), suggesting that the similar carbohydrate moiety on
DG was
involved in the binding to laminin-1/agrin in these cell lines.
Together with the failure of oligosaccharides to inhibit the adhesion
of RT4 (15) and BAE (Fig. 7) cells, these results indicated the
importance of the high anionic charge and polymeric structure of the
glycosaminoglycans in interrupting the
DG-laminin-1 interaction. The
soluble
DG in the conditioned medium caused no further decrement of
the cell adhesion in the presence heparin (Fig. 6), providing
supportive evidence for the interruption of the
DG-laminin-1
interaction by heparin.
Of the three glycosaminoglycans, heparin has been known for its
activities to stimulate both endothelial cell proliferation (44) and
migration (45). Polyanions such as heparin bind to a wide variety of
glycoproteins, including extracellular matrix proteins, growth
factors, and protease inhibitors, and the mechanism of action of
heparin to modulate endothelial cell behavior is still unknown. Studies
are underway in our laboratory to test the hypothesis that the cell
behavior modulation by heparin is due to the inhibition of the
DG-laminin interaction.
Both in the cases of the soluble
DG and the three
glycosaminoglycans, inhibition was not observed in fibronectin-coated
dishes, suggesting the laminin specificity of the DG-mediated adhesion. Laminin is a group of heterogeneous proteins, and care must be taken to
interpret the results obtained with a purified protein. The mouse
Engelbreth-Holm-Swarm laminin-1 used in this study is composed of the
three subunits,
1,
1, and
1. BAE cells have been shown to
express
1 and
1, but not
1 (40). Recent histochemical analysis
indicated the presence of
5, but not
1, in the vascular basal
laminae of murine heart (23). The laminin
5 in the vascular basal
laminae may be produced by the underlying smooth muscle cells because
neither BAE nor mouse aortic endothelial cells produce laminin
5,
but only laminin
4 (46). Therefore, it is at least clear that
laminin-1 is unlikely to be an in vivo ligand for DG in
vascular endothelial cells. We have, however, shown in the ligand
overlay assay that the
5 protein expressed in E. coli bound
DG and that it could disrupt the binding of laminin-1 to
DG
(Fig. 5). The recombinant
5 protein immobilized on the dishes promoted the BAE cell adhesion (data not shown). These results support
the idea that endothelial DG works as a laminin receptor in
vivo. Further studies with laminin
5 purified from tissues or
gene knockout experiments will be required to test the idea directly.
In conclusion, we have confirmed the expression of DG by cultured BAE
cells and presented evidence for the role of DG as a non-integrin
laminin receptor involved in BAE cell adhesion to the extracellular
matrix. These findings expand our knowledge on the physiological roles
of DG in non-muscle tissues. The distinct subcellular localization of
DG in BAE cells and the essential role of the carbohydrate moiety in
the
DG-laminin interaction suggest the influence of DG-mediated cell
adhesion that is quite different from that of integrin-mediated cell
adhesion. The control of cell behavior by the DG-laminin interaction
must be the subject for future studies.