(Received for publication, September 13, 1996, and in revised form, March 3, 1997)
From the ¶ Department of Neurology and Neuroscience, Teikyo
University School of Medicine, Tokyo 173, Japan, Department of
Glycobiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan, the ** University School of Neuroscience and Muscular Dystrophy
Group Laboratories, Regional Neuroscience Centre, Newcastle General
Hospital, Newcastle-upon-Tyne NE4 6BE, United Kingdom, the
Department of Neurology, Institute of Brain Research,
University of Tokyo Faculty of Medicine, Tokyo 113, Japan, and the
¶¶ Howard Hughes Medical Institute and Department of
Physiology and Biophysics, University of Iowa College of Medicine,
Iowa City, Iowa 52242
Dystroglycan is encoded by a single gene and
cleaved into two proteins - and
-dystroglycan by
posttranslational processing. Recently,
-dystroglycan was
demonstrated to be an extracellular laminin-binding protein anchored to
the cell membrane by a transmembrane protein
-dystroglycan in
striated muscle and Schwann cells. However, the biological functions of
the dystroglycan-laminin interaction remain obscure, and in particular,
it is still unclear if dystroglycan plays a role in cell adhesion. In
the present study, we characterized the role of dystroglycan in the
adhesion of schwannoma cells to laminin-1. Immunochemical analysis
demonstrated that the dystroglycan complex, comprised of
- and
-dystroglycan, was a major laminin-binding protein complex in the
surface membrane of rat schwannoma cell line RT4. It also demonstrated
the presence of
-dystroglycan, but not
-dystroglycan, in the
culture medium, suggesting secretion of
-dystroglycan by RT4 cells.
RT4 cells cultured on dishes coated with laminin-1 became spindle in
shape and adhered to the bottom surface tightly. Monoclonal antibody
IIH6 against
-dystroglycan was shown previously to inhibit the
binding of laminin-1 to
-dystroglycan. In the presence of IIH6, but
not several other control antibodies in the culture medium, RT4
cells remained round in shape and did not adhere to the bottom surface.
The adhesion of RT4 cells to dishes coated with fibronectin was not
affected by IIH6. The known inhibitors of the interaction of
-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan,
dextran sulfate, heparin, and sialic acid, also perturbed the adhesion
of RT4 cells to laminin-1, whereas the reagents which do not inhibit
the interaction, including dextran, chondroitin sulfate, dermatan
sulfate, and GlcNAc, did not. Altogether, these results support a role
for dystroglycan as a major cell adhesion molecule in the surface
membrane of RT4 cells.
There is now mounting evidence that the intracellular signal
transduction pathways activated by the adhesion of cells to other cells
or the extracellular matrix (ECM)1 play
crucial roles in cellular differentiation, migration, and proliferation. The prototypical cell adhesion molecules are the cell
surface receptors for the ECM glycoproteins. Dystroglycan, originally
identified as a member of the sarcolemmal glycoproteins complexed with
dystrophin, is encoded by a single gene and cleaved into two proteins
- and
-dystroglycan by posttranslational processing (1, 2).
-Dystroglycan is an extracellular glycoprotein anchored to the cell
membrane by a transmembrane glycoprotein
-dystroglycan, and the
complex comprised of
- and
-dystroglycan is called the
dystroglycan complex (2-4). In striated muscle,
-dystroglycan binds
the ECM components laminin-1 and -2 in a Ca2+-dependent manner (3, 5, 6). On the
cytoplasmic side of the sarcolemma,
-dystroglycan is anchored to the
cytoskeletal proteins dystrophin or its homologues (7, 8). Besides this structural role,
-dystroglycan is also proposed to play a role in
signal transduction, based on the finding that its cytoplasmic domain
contains a phosphotyrosine consensus sequence and several proline-rich
regions that could associate with SH2 and SH3 domains of signaling
proteins (1). Dystrophin deficiency causes a drastic reduction of the
dystroglycan complex in the sarcolemma and, thus, the loss of the
linkage between the subsarcolemmal cytoskeleton and the ECM, eventually
leading to muscle cell death in Duchenne muscular dystrophy and its
animal model mdx mice (for reviews, see Refs. 9 and 10).
The dystroglycan complex is also expressed in non-muscle tissues (1, 3,
11-13). In the peripheral nervous system, it is expressed in the
Schwann cell membrane, and the Schwann cell -dystroglycan binds not
only laminin-1 but also laminin-2, a major component of the
endoneurium, in a Ca2+-dependent manner
(13-15). Recently, laminin-2 was shown to be deficient in congenital
muscular dystrophy and its animal model dy mice, which are
characterized by peripheral dysmyelination as well as muscular
dystrophy (16-22). These findings have suggested roles for the
dystroglycan-laminin interaction in not only the maintenance of
sarcolemmal architecture but also peripheral myelinogenesis.
Despite these recent developments, the biological functions of the dystroglycan complex remain obscure, and in particular, it has not yet been established if the dystroglycan complex is indeed involved in the process of cell adhesion. In the present study, we have identified the dystroglycan complex as a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4 and characterized its role in RT4 cell adhesion to laminin-1.
Rat schwannoma cell line RT4 was kindly provided by Drs. A. Asai (University of Tokyo) and Y. Kuchino (National Cancer Center, Tokyo) (23). RT4 cells were grown in Dulbecco's modification of Eagle's medium containing 10% fetal calf serum, 16.7 mM glucose, 2 mM glutamine, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin. Culture medium was changed every 3 days.
Identification of the Dystroglycan Complex in RT4 CellsFor
immunocytochemical analysis, RT4 cells were grown on cover glasses,
fixed in methanol cooled to 20 °C for 10 min, and then
immunostained as described previously (13, 15). Specimens were observed
and fluorescent images were obtained on a Zeiss confocal laser scanning
microscope (cLSM) model LSM 310, employing an argon ion laser (
= 488 nm), as described previously (13, 14).
For immunochemical analysis, RT4 cells were grown to confluency on three 10-cm diameter culture dishes, scraped using a rubber policeman, homogenzied in 1.5 ml of phosphate-buffered saline (PBS), and centrifuged at 140,000 × g for 30 min at 4 °C. The pellets were homogenized in 1.5 ml of buffer A (50 mM Tris-HCl, pH 7.4, 0.75 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 0.7 µM pepstatin A, 76.8 nM aprotinin and 1.1 µM leupeptin) containing 1% digitonin and 0.5 M NaCl, extracted for 1 h at 4 °C, and then centrifuged at 140,000 × g for 30 min at 4 °C. After the supernatants were diluted 10-fold using buffer A, CaCl2 and MgCl2 were added to the final concentration of 1 mM each. The diluted supernatants were incubated overnight at 4 °C with 100 µl of laminin-1-Sepharose (2.5 mg/ml), which was blocked for 2 h at 4 °C with buffer A containing 0.1% digitonin, 50 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 3% bovine serum albumin (BSA). After washing with buffer A containing 0.1% digitonin, 50 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2, the laminin-1-Sepharose was eluted with 300 µl of buffer A containing 0.1% digitonin, 50 mM NaCl, and 10 mM EDTA.
Identification ofThe RT4 cell culture medium was collected by decanting slowly when RT4 cells grew to near confluency on 10-cm diameter culture dishes. After brief centrifugation, the RT4 cell culture medium (80 ml) was circulated over 5 ml of wheat germ agglutinin (WGA)-Sepharose (Pharmacia Biotech Inc.) at 4 °C overnight in the presence of 0.5 M NaCl. The WGA-Sepharose was washed with buffer A containing 0.5 M NaCl and then eluted with 15 ml of buffer A containing 0.3 M N-acetylglucosamine (GlcNAc). 100 µl of laminin-1-Sepharose was blocked with buffer A containing 1 mM CaCl2, 1 mM MgCl2, and 3% BSA at 4 °C for 2 h, and then washed with buffer A containing 1 mM CaCl2 and 1 mM MgCl2. The GlcNAc eluates of WGA-Sepharose were incubated with laminin-1-Sepharose at 4 °C overnight in the presence of 1 mM CaCl2 and 1 mM MgCl2. After washing with buffer A containing 1 mM CaCl2 and 1 mM MgCl2, the laminin-1-Sepharose was eluted with 300 µl of buffer A containing 10 mM EDTA.
Assay of Adhesion of RT4 Cells to Laminin-1As shown in
Fig. 3, we have found that RT4 cells synthesize laminin themselves. For
cell adhesion assay, however, culture dishes were coated with laminin-1
as follows, to ensure homogeneous distribution of laminin on the dish
surface. A solution of mouse EHS sarcoma laminin-1 (Biomedical
Technologies) (5 µg/ml in PBS) was added to the 11.3-mm diameter
wells of a 48-well plate, incubated at 37 °C overnight, and then
aspirated. 200 µl of 3% BSA in PBS were added to the wells,
incubated at room temperature for 1 h, and then aspirated. 50,000 RT4 cells were added to the wells and incubated, in the presence or
absence of antibodies or inhibitors, at 37 °C in a CO2
incubator. The shape and adhesion of the cells were observed after
1 h or overnight incubation. For quantification of cell adhesion,
the medium, and the unattached cells were removed by pipetting after
1 h of incubation. The number of the attached cells was quantified
by means of the endogenous enzyme hexosaminidase as described by
Landegren (24): after PBS wash, 120 µl of 3.75 mM
p-nitrophenyl-N-acetyl--D-glucosaminide
(substrate) in 50 mM citrate buffer, pH 5.0, 0.25% Triton
X-100 was added to each well and incubated at 37 °C for 1 h.
After a suitable interval, the color reaction was developed, and enzyme
activity was blocked by the addition of 180 µl of 50 mM
glycine, pH 10.4, containing 5 mM EDTA per well. Absorbance
was measured at 405 nm.
Antibodies
Affinity-purified sheep antibody against
dystroglycan fusion protein D (Anti-FPD) was characterized previously
(1-3). Monoclonal antibodies IIH6 and VIA41 against
-dystroglycan, 8D5 against the C terminus of
-dystroglycan, A1C
and XIXC2 against dystrophin, and DRP1 against utrophin
were characterized previously (2, 3, 25, 26). IIH6, VIA41,
and XIXC2 are IgM isotypes, while 8D5 and A1C are IgG
isotypes. For inhibition experiments of RT4 cell adhesion to
laminin by monoclonal antibodies, hybridoma supernatants containing
IIH6, VIA41, A1C, and XIXC2 were
concentrated 10-fold using a Centricon concentrater (Amicon), and one
part of the concentrates were added to nine parts of culture
medium.
Laminin-1-Sepharose was prepared as described
previously using mouse EHS sarcoma laminin-1 (13-15).
Immunohistochemistry, SDS-polyacrylamide gel electrophoresis, and
immunoblotting were performed as described previously (13-15, 26).
Laminin blot overlay was performed using mouse EHS sarcoma laminin-1,
and the laminin-1 which bound to -dystroglycan on the nitrocellulose
transfer was detected using antibody against mouse EHS sarcoma
laminin-1 (Sigma), as described previously (13-15).
Previously, we have demonstrated the expression
of the dystroglycan complex and utrophin, a dystrophin homologue, in
the surface membrane and cytoplasm of Schwann cells, respectively (13,
14). In addition, we have also found the expression of these proteins in human schwannomas by immunohistochemistry (Fig. 1).
These findings prompted us to look at the expression of the
dystroglycan complex and utrophin in the schwannoma cell line RT4. Fig.
2 shows the results of immunocytochemical analysis using
cLSM. Similar to Schwann cells, both - and
-dystroglycan were
localized in the surface membrane of RT4 cells, while utrophin was
localized in the cytoplasm diffusely (Fig. 2). To see if these proteins
are complexed to function as a laminin receptor, we performed laminin affinity chromatography of the digitonin extracts of RT4 cells (Fig.
3). By immunoblot analysis,
-dystroglycan, with a
molecular mass of approximately 160 kDa, was detected in the EDTA
eluates of laminin-1-Sepharose (Fig. 3). RT4 cell
-dystroglycan was
confirmed to bind laminin-1 in blot overlay (Fig. 3).
-Dystroglycan,
with a molecular mass of approximately 40 kDa, was also detected in the
EDTA eluates of laminin-1-Sepharose (Fig. 3). However, RT4 cell
-dystroglycan did not bind laminin-1 in blot overlay (Fig. 3). These
results indicate that, similar to striated muscle and Schwann cells,
-dystroglycan is complexed with laminin-binding
-dystroglycan in
RT4 cells (3, 14). On the other hand, utrophin was not detected in the
EDTA eluates of laminin-1-Sepharose (not shown). In addition, it should
also be noted that
-dystroglycan and possibly laminin were the only
proteins that bound laminin-1 in blot overlay (Fig. 3). All together,
these results indicate that the dystroglycan complex, comprised of
-
and
-dystroglycan, is a major laminin-binding protein complex in the
surface membrane of RT4 cells. They also indicate that the major
fraction of utrophin is localized in the cytoplasm of RT4 cells and not
associated with the dystroglycan complex in the cell membrane.
Thus far, secretion of -dystroglycan by cells has been proposed as
an intriguing possibility, but this has never been confirmed (11). By
immunoblot analysis,
-dystroglycan, but not
-dystroglycan, was
detected in the RT4 cell culture medium after successive WGA and
laminin affinity chromatographies (Fig. 3). Taken together with the
fact that
-dystroglycan is an extracellular protein anchored to the
cell membrane by a transmembrane protein
-dystroglycan (3, 14),
these results indicate that a fraction of RT4 cell surface
-dystroglycan is dissociated from the cell membrane
-dystroglycan and released into the culture medium. Although this dissociation may
partially be due to lysis of the cells, these findings suggest a
possibility of active secretion of
-dystroglycan by RT4 cells.
Figs. 4a, 5, 6, 7, 8a show the shape of RT4 cells grown overnight on culture
dishes coated with laminin-1 in the presence or absence of various
reagents in the culture medium. In the absence of inhibitors, RT4 cells
became spindle in shape after 1 h of incubation (not shown) and
remained so after overnight incubation (Fig. 4a). Spindle
cells adhered to the bottom surface of the culture dishes tightly and
did not oscillate when the dishes were shaken rigorously by hands.
Monoclonal antibody IIH6 against -dystroglycan was demonstrated
previously to inhibit the binding of laminin-1 to
-dystroglycan (3,
27-29). When IIH6 was present in the culture medium, RT4 cells
remained round in shape and did not take the characteristic spindle
shape, even after overnight incubation (Fig. 4a). Round
cells oscillated, and many of them drifted when the dishes were shaken
rigorously by hand. When the wells were pipetted after 1 h of
incubation, the spindle cells in the control wells were not detached,
but the round cells in the wells with IIH6 were removed almost
completely (not shown). This was confirmed by the quantification
procedure described under "Experimental Procedures" (Fig.
4b). Monoclonal antibody VIA41 against
-dystroglycan, which does not inhibit the binding of laminin-1 (3,
29), did not have such effects, neither did monoclonal antibodies A1C and XIXC2 against dystrophin (Fig. 4). We compared the
effects of IIH6 on the adhesion of RT4 cells to fibronectin with those to laminin-1. In contrast to dishes coated with laminin-1, the adherence of RT4 cells to dishes coated with fibronectin was not affected by IIH6 (Fig. 5).
Since the interaction of -dystroglycan with laminin-1 is
Ca2+-dependent (1, 3, 11-13), we tested
Ca2+ dependence of RT4 cell adhesion to laminin-1. When
EDTA was added to the culture medium, RT4 cells remained round in shape
and were easily detached by pipetting (Fig. 6).
Furthermore, the antiadhesive effects of EDTA were reversed by the
addition of excess amount of Ca2+, indicating
Ca2+ dependence of RT4 cell adhesion to laminin-1 (Fig.
6).
Finally, we asked if the known inhibitors of the interaction of
-dystroglycan with laminin-1 would affect RT4 cell adhesion to
laminin-1. It was demonstrated previously that sulfatide, fucoidan, and
dextran sulfate were potent inhibitors of the interaction of
-dystroglycan with laminin-1, whereas heparin was less potent (30,
31). When sulfatide, fucoidan, or dextran sulfate was added to the
culture medium, RT4 cells remained round in shape and were easily
detached by pipetting (Fig. 7). The antiadhesive effects
of heparin were less than those of sulfatide, fucoidan, or dextran
sulfate (Fig. 7). On the other hand, the addition of dextran,
chondroitin sulfate, or dermatan sulfate, which were demonstrated
previously not to inhibit the interaction of
-dystroglycan with
laminin-1 (30), did not affect the shape or adhesion of RT4 cells to
laminin-1 (Fig. 7). We have demonstrated previously that the
interaction of
-dystroglycan with laminin-1 is inhibited by a
relatively high concentration of sialic acid but not GlcNAc (14). In
the presence of sialic acid, but not GlcNAc, in the culture medium, RT4
cells remained round in shape and were easily detached by pipetting
(Fig. 8). These results, altogether, point to a role for
-dystroglycan in the adhesion of RT4 cells to laminin-1.
-Dystroglycan, which is an extracellular peripheral membrane
glycoprotein anchored to the cell membrane by a transmembrane glycoprotein
-dystroglycan, binds laminin and agrin in striated muscle, neuromuscular junction, and Schwann cells (1-6, 13-15, 27,
28, 31-34). On the other hand, the cytoplasmic domain of
-dystroglycan contains a phosphotyrosine consensus sequence and several proline-rich regions that could associate with SH2 and SH3
domains of signaling proteins (1, 35). Indeed, Grb2, an adaptor protein
in the signal transduction pathways, was recently demonstrated to bind
to the cytoplasmic proline-rich regions of
-dystroglycan via the two
SH3 domains (35). These findings have suggested a possible role for the
dystroglycan complex, comprised of
- and
-dystroglycan, as a
signaling receptor involved in the maintenance of sarcolemmal
architecture, peripheral synaptogenesis, and myelinogenesis. In
addition, the dystroglycan complex has also been implicated in kidney
epithelial development, although its ECM ligand in kidney has not yet
been identified (29). Despite these recent developments, the
precise roles of the interaction of the dystroglycan complex with
ECM ligands in these specialized biological processes remain obscure,
and in particular, it has not yet been confirmed if the dystroglycan
complex plays a role in cell adhesion.
Under these circumstances, identification of cell lines that express
the dystroglycan complex in the surface membrane would provide us
useful tools for testing the proposed functions of the dystroglycan
complex in vivo. In the present study, we have demonstrated,
by immunochemical analyses, that the dystroglycan complex, comprised of
- and
-dystroglycan, is a major laminin-binding protein complex
in the surface membrane of rat schwannoma cell line RT4. Similar to
Schwann cells (14), utrophin, which has the binding capacity for the
cytoplasmic domain of
-dystroglycan (7, 8, 36), was localized
diffusely in the cytoplasm of RT4 cells and not associated with the
dystroglycan complex. Thus, the putative membrane-associated
cytoskeletal protein anchoring the dystroglycan complex to the
underlying submembranous cytoskeleton remains to be elucidated.
In the present study, we have also tested the role of -dystroglycan
in RT4 cell adhesion to laminin-1. When RT4 cells were cultured on
laminin-1, they became spindle in shape immediately and adhered to the
bottom surface tightly. However, when RT4 cells were cultured on
laminin-1 in the presence of the known inhibitors of the interaction of
-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan,
dextran sulfate, heparin, and sialic acid, they remained round in shape
and did not adhere to the bottom surface. Because these reagents may
also perturb the interaction of laminin-1 with the cell surface
adhesion molecules other than
-dystroglycan, such as the members of
the integrin family for instance, we have looked at the effects of
monoclonal antibody IIH6 against
-dystroglycan, which inhibits the
interaction of
-dystroglycan with laminin-1, and found that this
antibody drastically reduces the adhesion of RT4 cells to laminin-1.
Furthermore, IIH6 did not perturb the adhesion of RT4 cells to
fibronectin. Together with the previous demonstration of high affinity
binding of laminin-1 to
-dystroglycan (1, 3), these results indicate
a role for
-dystroglycan as a major cell adhesion molecule in the
surface membrane of RT4 cells and suggest that the dystroglycan complex may play an important role in cell adhesion in vivo.
Finally, we have demonstrated the results which suggest the secretion
of
-dystroglycan by RT4 cells. In the future, it would be
interesting to see if
-dystroglycan is secreted in vivo
and if the secreted
-dystroglycan has inhibitory, and potentially
regulatory, effects on the interaction of the cell surface
-dystroglycan with the ECM ligands.
The mechanism by which the dystroglycan complex may mediate such
diverse and specific biological processes as sarcolemmal stabilization,
epithelial morphogenesis, synaptogenesis, and myelinogenesis remains
unclear. For instance, it has been disputed if the dystroglycan complex
is actively involved in the acetylcholine receptor clustering in the
neuromuscular junction as a signaling receptor of agrin (31, 33, 34,
37-43). Among others, our results seem consistent with at least two
possibilities. First, the dystroglycan complex may function as a helper
protein in these processes; the initial and high affinity binding of
the ECM ligands to the dystroglycan complex may enable the more
specific and functional cell surface receptors, such as the members of
the integrin family or the putative myotube-associated specificity
component (MASC), which was recently proposed to work in concert with
the receptor tyrosine kinase MuSK in the neuromuscular junction
formation, to interact with these ligands (31, 33, 34, 37, 38, 42).
Second, the dystroglycan complex may function as a structural protein
in the maturational stages of these processes. In this scenario, it
would be intriguing to postulate that the binding of the ECM ligands to
the dystroglycan complex may trigger the reorganization of the
submembranous dystrophin/utrophin-cytoskeleton and lead to the
stabilization of the cell membrane (44). The fact that the binding
sites for dystrophin/utrophin and Grb2 overlap in the C terminus of
-dystroglycan raises a possibility that this process may be mediated
by Grb2 and other signaling/adaptor proteins (8, 35).