* Center for Research in Neuroscience, McGill University, Montreal General Hospital Research Institute, Montreal,
Quebec H3G 1A4, Canada; and Chromos Molecular Systems, Inc., Burnaby, British Columbia V5A 1W9, Canada
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Abstract |
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-Dystroglycan (
-DG) is a laminin-binding
protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the
function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes
from two resulting clones (11F and 11E) had at least a
40-50% and 80-90% reduction, respectively, in
-DG
but normal or near normal levels of
-sarcoglycan, integrin
1 subunit, acetylcholine receptors (AChRs), and
muscle-specific kinase (MuSK) when compared with
parental C2 cells or three clones (11A, 9B, and 10C)
which went through the same transfection and selection
procedures but expressed normal levels of
-DG. Antisense DG-expressing myoblasts proliferate at the same
rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in
these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with
condensed chromatin and more nuclei labeled by the
TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as
would be expected with necrosis. Unlike parental C2
myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with
-DG on their surfaces but far
fewer LN/
-DG plaques were seen after LN addition to
11F or 11E myotubes. These results suggest that
-DG
is a functional LN receptor in situ which is required for
deposition of LN on the cell and, further, implicate
-DG in the maintenance of myotube viability.
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Introduction |
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-DYSTROGLYCAN (
-DG)1 is a component of the dystrophin associated glycoprotein complex (DGC) which, in
muscle, is thought to serve as a transmembrane link between the submembraneous cytoskeleton and the basal
lamina (Campbell and Kahl, 1989
; Yoshida and Ozawa, 1990
; Ervasti and Campbell, 1991
). Other components of
the complex include at least seven transmembrane glycoproteins,
-DG, sarcospan (Crosbie et al., 1997
),
-,
-,
-,
-, and
-sarcoglycan (SG; McNally et al., 1998
) as well as
the syntrophins, a family of three intracellular PDZ domain containing proteins (for review see Carbonetto and
Lindenbaum, 1995
).
- and
-DG are derived from cleavage of a common polypeptide precursor (Ibraghimov-Beskrovnaya et al., 1992
) and remain associated with one another on the cell surface (Bowe et al., 1994
).
-DG
associates with either dystrophin or its autosomal homologue utrophin via an SH3 domain-binding region in its
COOH terminus (Jung et al., 1995
). This region has also
been shown to interact with the adapter protein Grb 2 (Yang et al., 1995
) raising the possibility that modulation
of DG-cytoskeletal interactions may be mediated by a signaling pathway involving small GTP-binding proteins.
In contrast to the transmembrane protein -DG,
-DG
is a heavily glycosylated, mucin-like protein (Smalheiser
and Kim, 1995
) anchored on the extracellular surface of
the myotube. In vitro studies have demonstrated that
-DG can bind the extracellular matrix component laminin (LN) with high affinity (Smalheiser and Schwartz,
1987
; Douville et al., 1988
; Ibraghimov-Beskrovnaya et al.,
1992
; Gee et al., 1993
). LN is a heterotrimer of
,
, and
chains, each of which is a member of multigene families.
-DG binds to the last two globular domains in the
COOH-terminal extension of the LN
1 and
2 chains
(Gee et al., 1993
).
-DG has been shown to colocalize with
LN in skeletal and cardiac muscle (Klietsch et al., 1993
)
and a number of other cells including peripheral nerve (Yamada et al., 1994
), astrocytes, Purkinje neurons (Tian et
al., 1996
), and kidney epithelium (Durbeej et al., 1995
). During muscle development,
-DG upregulation coincides temporally with the onset of innervation and ECM
deposition (Leschziner, A., and S. Carbonetto, unpublished observations). These findings are consistent with
the hypothesis that
-DG is a receptor in situ linking LN
in the ECM to the subsarcolemmal cytoskeleton and thus
may be important in the organization of these extracellular and subplasmalemmal networks. The existence of this
putative transmembrane linkage is further supported by
genetic evidence. For example, naturally occurring mutations in mice and humans that alter the expression of dystrophin and secondarily the DGC give rise to Duchenne
and Becker muscular dystrophies, which are characterized by severe progressive damage to the sarcolemma (reviewed in Campbell, 1995
; Worton, 1995
). The phenotypes
of these lesions bear considerable resemblance to those resulting from mutations affecting the gene encoding the
2
chain of LN 2 (
2
1
1; merosin), which is enriched in the
ECM of skeletal muscle (Xu et al., 1994
; Sunada et al., 1995
), or mutations affecting expression of the SG complex (Roberds et al., 1994
; Bönnemann et al., 1995
; Jung
et al., 1996a
,b; Nigro et al., 1996
; Carrie et al., 1997
). This
convergence of biochemical and genetic evidence underscores the importance of interactions between the ECM
and the DGC in the maintenance of sarcolemmal integrity.
To date, no naturally occurring mutations in -DG have
been identified which would substantiate and elucidate its
role as an ECM receptor. Recently, targeted mutations
abolishing DG expression in mice have been shown to result in early embryonic lethality due to disruption of the
Reichert's membrane (Williamson et al., 1997
; Côté, P., M. Lindenbaum, and S. Carbonetto. 1997. Mol. Biol. Cell.
8:222a). This indicates that DG expression is absolutely required for embryonic survival and strongly implicates
DG in the maintenance of Reichert's basement membrane
but sheds little light on its function(s) in more differentiated tissues such as skeletal muscle.
To test the assertion that -DG is a LN receptor required for the elaboration of the ECM of muscle, we have
perturbed its expression with stable transfection of C2 myoblasts with an antisense DG expression construct. In the
process, we have generated two clonal cell lines 11F and
11E that express 40-50% and 10-20%, respectively, of the
levels of
-DG protein in parental C2 cells after differentiation. These cells maintain the ability to fuse and form
multinucleate myotubes and express normal or near normal levels of other DGC components including
-SG, but
have greatly reduced levels of LN expression on their surfaces relative to C2 cells. This reduction correlates well
with the level of
-DG in these clones. We also show that
the residual, unbound
-DG on the surface of 11F myotubes is redistributed upon addition of exogenous LN.
However, little or no binding by exogenous LN is seen in
11E myotubes, which express the lowest levels of
-DG. After transfer to fusion medium there is an increase in cell
death and increased numbers of apoptotic nuclei in 11F
and 11E cultures, which again correlates with levels of
-DG
expressed in these clones. In 11E cells the integrity of the
plasma membrane is not obviously compromised, as revealed by exclusion of the vital dye Trypan blue, and is
consistent with apoptotic, not necrotic, cell death. We conclude that
-DG serves as a LN receptor in muscle and
that interactions between the ECM and the DGC are required for maintenance of muscle viability.
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Materials and Methods |
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Materials
LN was purified from Engelbreth-Holm-Swarm (EHS) tumor by the
method of Timpl et al. (1982). SDS-PAGE of purified preparations shows
bands at ~215 kD (
and
chains) and ~400 kD (
chain). The latter was
not recognized by an antiserum to LN
2 chain (a gift from Dr. Peter
Yurchenco) but did react with an anti-LN antiserum produced by immunizing rabbits with purified EHS tumor LN. This antiserum recognizes all
three subunits of LN 1 (
1,
1, and
1), but does not cross react with agrin
or LN
2 chain. Anti-LN IgG was purified by chromatography on Affigel
Blue (BioRad Laboratories) according to the manufacturer's instructions.
An antiserum to LN
2 chain was raised against the recombinant G domain of this chain and does not cross-react with LN
1 chain. mAb 5D3 to
LN (Life Technologies) has been previously characterized (Abrahamson et al., 1989
). Antiserum to the integrin
1 subunit was generated by immunizing rabbits with purified rat integrin
1 subunit (Tawil et al., 1990
).
mAb IIH6 recognizes a unique carbohydrate epitope on
-DG while the
antiserum to fusion protein B recognizes a portion of the core protein of
both
- and
-DG. mAb NCL-
-DG is directed against the last 15 amino
acids of the COOH terminus of
-DG (Novocastra Laboratories Ltd.).
The antiserum to
-SG, raised against a peptide corresponding to the last
19 amino acids of the COOH-terminal domain of
-SG, specifically recognizes a single protein of ~50 kD in purified DGC from rabbit muscle and
in crude protein extracts from skeletal muscle and C2 cells. mAb HUC1-1
to muscle actin was purchased from ICN Pharmaceuticals.
Plasmid Construction
A 1.8-kb fragment of the mouse DG cDNA extending from approximately 100 bp (5' to the translation start site) to the HindIII site situated
at +1,725 bp was removed by digestion of a mouse DG cDNA subclone in
bluescript SK(
) with NotI-HindIII. This Not1-HindIII fragment was
then subcloned in the antisense orientation into the pRcCMV expression
vector (Invitrogen) using standard subcloning techniques (Sambrook et
al., 1989
). Before transfection, the plasmid was linearized by digestion
with BglII.
Cell Culture and Transfection
C2 cells were plated on 10-cm tissue culture plastic dishes (Falcon) maintained at 37°C, 8% CO2 atmosphere, in growth medium consisting of DMEM (low glucose; Life Technologies) supplemented with 20% FBS (heat inactivated; Life Technologies), 0.5% chick embryo extract (ultrafiltered; Life Technologies), and penicillin/streptomycin (Life Technologies).
Stable transfections were carried out by calcium phosphate coprecipitation after methods of Yoshihara and Hall (1993). In brief, when C2 myoblasts reached 70% confluence, they were harvested by treatment with
trypsin/EDTA, replated at a 1:20 dilution on fresh 10-cm dishes and allowed to attach overnight. On day 2, the medium was changed 3 h before
transfection and DNA/Ca2PO4 coprecipitate, prepared according to published procedures (Graham and Van der Eb, 1973
; Wigler et al., 1979
),
and was added directly to the culture medium (5 mg of linearized plasmid
per dish). Cells were returned to the incubator for 16 h, and on the following day, the medium and precipitate were removed. The cells were washed briefly with Dulbecco's PBS plus 0.5 mM EDTA to remove excess precipitate. Fresh medium was added and 24-36 h after the beginning of transfection it was replaced with selection medium consisting of growth medium supplemented with G418 (750 µg/ml active concentration; Life
Technologies). Selection was carried out for up to 10 d, or until all cells of
an equivalent, untransfected culture were killed. At that point, drug-resistant C2 cell colonies could easily be seen on transfected plates. Colonies
were then picked and expanded for further characterization. Stable clones
were maintained in growth medium supplemented with 70 µg/ml active
G418. Low (6-20) passage 11F, 11E, 9B, 10C, 11A, and control C2 cells
were cultured on tissue culture plastic dishes (Falcon) coated with 0.15%
gelatin and maintained in growth medium until confluent. Cultures were switched to fusion medium (DMEM high glucose, 1% horse serum) and
allowed to differentiate for an additional four days. Some cultures were
treated with 12 nM LN on the third day of fusion.
SDS-PAGE and Western Blotting
Control and transfected C2 clones differentiated into myotubes for 3-5 d
were washed three times with ice-cold Ca/Mg-free PBS, then extracted
into 0.2-ml/10-cm dish of 1× SDS sample buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol)
preheated to 85°C. The remaining extract was scraped and transferred to a
1.5-ml microcentrifuge tube, and heated at 95°C for 5 min. It was subsequently passed five times through a 30-gauge syringe needle and centrifuged at 16,000 g for 10 min to remove insoluble debris. A portion of the
lysate was precipitated with trichloroacetic acid/deoxycholate for protein determination (Peterson, 1977). For some experiments, cultured myotubes were subjected to subcellular fractionation generating soluble fractions and KCl-washed light and heavy microsomes (Ohlendieck et al.,
1991
). To assess expression of the integrin
1 subunit by Western blotting,
cells were scraped in ice-cold PBS and membrane proteins were detergent
extracted in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% (vol/vol) Triton X-100, 20 mM N-ethylmaleimide, Complete
protease inhibitor cocktail (Boehringer Mannheim) for 10 min on ice.
Protein concentration was determined (Peterson, 1977
) and extracts were
diluted in 5× SDS sample buffer without dithiothreitol. Cellular proteins
released into the culture medium were assayed by collecting medium from
two 10-cm dishes of differentiated cells. The medium was pooled, concentrated ~100-fold and partially purified using Centricon Plus-20 centrifugal
filter devices (Millipore). All samples were assayed for protein content (Peterson, 1977
) before electrophoresis on 7.5 or 10% SDS-polyacrylamide mini gels (0.75-mm thickness; BioRad Laboratories) at 20-mA constant current for 1 h. Fractionated proteins were electroblotted onto nitrocellulose membranes (BA-S 75; Schleicher and Schuell) under standard
conditions (100-V constant voltage for 1 h). Blots were stained with Ponceau-S red to assess transfer, then incubated in 10 ml of Blotto (10 mM
Tris, pH 7.5, 150 mM NaCl, 1% Tween 20, 5% dried skim milk powder) at
room temperature for 1 h and subsequently in an appropriate dilution of
primary antibody in 4-5 ml Blotto for 1 h at room temperature with constant agitation. Primary antibodies used in Western blotting included:
mAb IIH6 culture supernatant (1:10); antiserum to fusion protein B
(1:50); antiserum to
-SG (1:1,000); mAb to
-DG (1:350); antiserum to LN (1:100); antiserum to the integrin
1 subunit (1:5,000); mAb to sarcomeric actin (1:1,000). After incubation with the primary antibody, blots
were washed 4× 15 min in TBS-Tween (10 mm Tris, pH 7.5, 150 mM
NaCl, 1% Tween 20), then incubated with the appropriate HRP-labeled
secondary antibody diluted in Blotto for 1 h at room temperature with
constant agitation. Blots were washed in TBS-Tween (4× 15 min) and labeled bands were visualized by enhanced chemiluminescence (Mandel/
NEN Life Science Products) after exposure to x-ray film (Hyperfilm-ECL;
Amersham Life Sciences). Blots were often stripped in 0.2 M glycine,
0.1% (vol/vol) Tween 20, pH 2.5, for 30 min, rinsed in PBS and reprobed
with mAb HUC1-1 to muscle actin to take into account differences in the
levels of DGC and integrin expression due to differences in the proportion of differentiated myotubes among cultures (see text).
Microscopy and TUNEL Assay
Cultures of confluent myoblasts (day 0), or of cells allowed to differentiate for 2 or 4 d, were washed twice with PBS then fixed and stained with Coomassie blue (25% propanol, 10% acetic acid, 0.1% Coomassie brilliant blue) for 10 min at room temperature. Cultures were washed twice in PBS, air dried and visualized in brightfield on a Zeiss Axioskop.
For immunocytochemistry, myotube cultures were fixed with 2%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, for 20 min, rinsed
three times with PBS, blocked for 1 h in PBS + 1% horse serum, then incubated overnight at 4°C with the primary antibody. For immunostaining
of live cells, the primary antibody was added directly to the culture medium and cells were incubated for 30 min at 37°C in 8% CO2, then fixed
as described above. Cells were then washed and incubated with the appropriate biotinylated secondary antibody for 1 h, followed by fluorescein-conjugated streptavidin and rhodamine-conjugated -bungarotoxin
(
-BTX; Molecular Probes) for 20 min at room temperature. Staining was
visualized with epifluorescence illumination on a Zeiss Axioskop. Primary
antibodies used include: mAb IIH6 ascites, 1:100 dilution; antiserum to
LN 1, 1:50 dilution; mAb 5D3, 1:100 dilution; antiserum to LN
2 chain,
1:150 dilution; antiserum to the integrin
1 subunit, 1:100 dilution.
To assess the level of apoptosis in myotube cultures, cells were differentiated for 3 d, then fixed sequentially in 2% formaldehyde and 4% neutral-buffered formalin for 10 min each at room temperature and washed twice in PBS, pH 7.4. Cultures were permeabilized by incubation in either 0.1% Triton X-100 for 15 min at room temperature or in ethanol/acetic alcohol (1:4) for 5 min at -20°C, then processed for TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; Apoptag Plus In Situ Apoptosis Detection Kit; Oncor). Negative controls were run for each experiment by omitting the anti-digoxigenin antibody or the terminal deoxynucleotidyl transferase enzyme. Positive control slides were provided with the kit. Cultures were counterstained with 0.1 µg/ml DAPI to visualize the total number of nuclei present and to confirm by morphological criteria that TUNEL stained nuclei were not mitotic or necrotic. The number of apoptotic nuclei and the total number of nuclei were counted with a 63× objective on a Zeiss Axioskop. 20 fields were quantified per coverslip and three coverslips were analyzed per cell type and per experiment. Collapsed myotubes and masses of dead cells were not included in the quantification since the number of labeled nuclei could not be accurately determined.
Cell Loss, Proliferation, and Membrane Integrity Assays
C2, 11F, 11E, 9B, and 10C cells were cultured in 96-well plates (Falcon)
coated with 0.15% gelatin and assayed at day 0 (confluent myoblasts), and
days 2 to 4 in fusion medium. To determine cell number, plates were thoroughly washed with PBS, frozen at 80°C and labeled with the Blue DNA
assay kit (Molecular Probes). Hoechst fluorescence was measured with a
Cytofluor 2300 fluorometer using a 360 nm excitation/460 nm emission filter set. Empty wells coated with gelatin were used as controls for background fluorescence. Proliferating cells were labeled using the colorimetric cell proliferation ELISA kit (Boehringer Mannheim). In brief, cells
were incubated for 2 h in medium containing 10 µM bromodeoxyuridine
(BrdU), fixed for 30 min at room temperature, incubated for 2 h with the
anti-BrdU antibody, washed, and incubated for 5 min in the substrate solution. The reaction was stopped with 1 M sulfuric acid and the plates were immediately read with an ELISA plate reader at 450 nm using a reference wavelength of 690 nm. Wells where the BrdU was omitted were
used as control for nonspecific labeling of the antibody. Plates for the cell
loss and proliferation assays were prepared on the same day and from the
same aliquot of cells. Four replicate wells were prepared per cell type and
per plate. Proliferation is expressed as a ratio of the average number of
cells per well as determined by Hoechst staining on an age-matched plate.
To assay membrane integrity cells differentiated for 4 d on tissue culture dishes coated with gelatin were washed in serum-free DMEM and incubated for 1 min in 0.2% Trypan blue in DMEM. Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at room temperature, washed in PBS and then dehydrated in 70% and 95% ethanol for 1 min each. Cultures were counterstained with eosin (0.1% eosin in 95% ethanol, 2.8 µl/ml concentrated acetic acid) for 1 min then washed three times 1 min each in 100% ethanol. Cultures were visualized on a Zeiss Axioskop. Blue nuclei were counted with a 40× objective for three sets of 10-cm dishes (10-15 fields/dish) for C2 and 11E cells. Large aggregates of dead cells and cell debris were not included in the quantification.
Statistical Analyses
Statistical significance for cell loss, proliferation, and membrane integrity
assays was determined for three to four independent experiments using a
simple ANOVA test followed by the Fisher's t-test. The n represents the
individual experiments. For statistical analysis of the percentage of apoptotic nuclei, each experiment was evaluated separately since the permeabilization procedure used was not always the same. A simple ANOVA
test was also used followed by the Fisher's t-test. The n represents the
number of dishes used (three for each experiment). The correlation between cell number and -DG expression for C2, 11F, and 11E cells was determined using a simple regression analysis followed by Fisher's z-test
(null hypothesis: correlation coefficient = 0). The relative cell number was
obtained from quantification of Hoechst fluorescence and the average levels of
-DG expression for each cell type were assessed by densitometry
from seven individual Western blots. In each Western blot, extracts from
all three cell types had been assayed side by side.
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Results |
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Characterization of Stably Transfected C2 Clones
Expressing Antisense -DG cDNA
To assess the role of -DG in the deposition and organization of muscle ECM, the C2 muscle cell line was transfected with a DG cDNA fragment (Fig. 1 A) in the antisense orientation under the control of the cytomegalovirus
promoter (Yoshihara and Hall, 1993
). G418-resistant
clones were selected and screened by examining the levels
of
-DG expression relative to untransfected controls or
cells transfected with the vector alone. Five clones, 11E, 11F, 9B, 10C, and 11A were chosen for further characterization. Control C2 cells, and antisense clones were cultured to confluence then induced to fuse into myotubes
as described in Materials and Methods. After 4-5 d in culture, the myotubes were extracted directly in SDS sample
buffer and equal protein loads were fractionated by SDS
PAGE and transferred onto nitrocellulose membranes.
Fig. 1 B shows a representative blot probed with mAb
IIH6, which recognizes a carbohydrate epitope on
-DG
(Ervasti and Campbell, 1993
). Control C2 cells show a typically broad band of muscle
-DG extending from 130-160
kD. By contrast, only low levels of
-DG expression were
detected in 11F cells and even lower levels were detected in 11E cells (this blot is greatly overexposed to allow for
visualization of
-DG expression in the 11E line). Nearly
normal levels of
-DG expression were detected in the 9B,
10C, and 11A clones (Fig. 1 C) possibly as a result of low
expression of the antisense construct, and these clones
were used as additional controls for any nonspecific effects
resulting from the transfection or selection procedures. Densitometric analysis of films from Western blots exposed over a range of times to insure that the emulsion
was not overexposed, revealed a 50-60% decrease in
-DG
in 11F cultures and an 80-90% decrease in 11E cultures.
The blot in Fig. 1 B was also probed with an antiserum
raised against
-SG, one of the four transmembrane components of the SG complex which is associated with, but
distinct from, the DG complex (Yoshida et al., 1994
). No
reduction in the expression of this 50-kD protein was seen
in either
-DG antisense clone compared with C2 cells
(Fig. 1 B). A similar result was obtained for
-SG (not
shown). Since mutations affecting the expression of any
one member of the SG complex can lead to the loss of all other members from the sarcolemma (Roberds et al.,
1994
; Bönnemann et al., 1995
; Noguchi et al., 1995
; Jung et
al., 1996a
,b), we deduced that the SG complex is unaffected in the antisense clones. This result is also in agreement with the reported relative independence of the DG
and SG complexes (Roberds et al., 1994
; Yoshida et al., 1994
; Bönnemann et al., 1995
; Jung et al., 1996a
,b). In addition, Western blots of extracts probed for LN (Fig. 4 B),
collagen IV and perlecan (Montanaro, F., and S. Carbonetto, unpublished data), integrin
1 (Fig. 5 B) AChR
,
,
and
subunits, or for the MuSK receptor tyrosine kinase
(Jacobson et al., 1998
) showed no reduction in expression
of these proteins in 11F and 11E cells, nor in 9B, 10C, and
11A cells, confirming the specificity of the antisense construct.
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Since mAb IIH6 is known to recognize a carbohydrate
epitope on -DG involved in binding to LN (Ervasti and
Campbell, 1993
), it was important to demonstrate that the
defect in the antisense-expressing clones was not simply
due to aberrant glycosylation of
-DG. Therefore, similar
blots were probed with an antiserum to an
-DG fusion protein (anti-fusion protein B) which recognizes the DG
core protein (Ibraghimov-Beskrovnaya et al., 1992
). For
C2 cells as well as 11F and 11E antisense clones, the relative intensity of labeling by this antiserum is very similar,
albeit generally weaker (Fig. 1 D), to that observed with
mAb IIH6 (Fig. 1 B) suggesting that the decreased expression of
-DG in the 11E and 11F clones is due to decreased levels of
-DG polypeptide and not merely altered glycosylation. To confirm further that the expression
of membrane associated
-DG was similarly altered, C2,
11F, and 11E myotubes were fractionated into soluble and
KCl-washed light microsome fractions, then analyzed by
SDS-PAGE and Western blotting with mAb IIH6. The
decrease in membrane associated
-DG seen in the antisense clones relative to control C2 cells (Fig. 1 E) closely parallels that seen for the levels of total
-DG (Fig. 1 B).
Little, if any, expression of
-DG was detected in the soluble fraction (Fig. 1 E) or heavy microsome fraction (data
not shown) of either control C2 cells or antisense clones,
suggesting that the latter are not defective in the localization of
-DG to the cell membrane. Consistent with this
-DG, like its cotranscript
-DG, is also reduced by 70 and 88% in 11F and 11E cells, respectively, but not significantly in 9B, 10C, and 11A cells, when compared with C2
cells (Fig. 1 F). This confirms that DG synthesis is reduced in antisense-expressing cell lines and that the reduced levels of
-DG do not result simply from shedding into the medium.
Morphological Characterization of Antisense-expressing Myoblasts and Myotubes
-DG is a high affinity LN binding protein in vitro (Smalheiser and Schwartz, 1987
; Douville et al., 1988
; Ibraghimov-Beskrovnaya et al., 1992
; Gee et al., 1993
) and colocalizes with LN in muscle cells (Klietsch et al., 1993
;
Cohen et al., 1997
). Since LN isoforms have been reported
to play an important role in myoblast fusion (Foster et al.,
1987
; Öcalan et al., 1988
; von der Mark and Öcalan, 1989
;
Schuler and Sorokin, 1995
; Vachon et al., 1996
), we compared the ability of C2, 11F, 11E, 9B, 10C, and 11A myoblasts to differentiate into myotubes. In low density cultures 11F and 11E myoblasts appeared somewhat flatter
and more spread than control C2 myoblasts. However, the
proliferation rate and survival of 11F and 11E myoblasts in these cultures were indistinguishable from C2 myoblasts.
In cultures approaching confluence little morphological
difference could be seen between C2, 11F, and 11E myoblasts (Fig. 2). One day after transfer to fusion medium,
myoblasts assumed a more elongate shape and on the second day of differentiation numerous thin myotubes could
be seen in C2 as well as in 11F and 11E cultures (Fig. 2). After 4 d of differentiation, many large myotubes were
found in all cultures (Fig. 2) indicating that 11F and 11E
myoblasts can differentiate into myotubes with the same
time course as C2 cells although 11F and 11E cultures had
lower densities of myotubes (discussed below). 9B, 10C,
and 11A cells also differentiated normally within the same
time frame as C2 cells but did not show any appreciable
decrease in cell density with differentiation. 11A myoblasts seemed to replicate more slowly compared with the other clones and usually required 3 d to reach confluence
rather than 2 d like C2 cells and all the other clones, although, once confluence was reached, differentiation into
myotubes proceeded at the same rate as C2 cells. It should
be noted that after 4 d in fusion medium, cultures of 11F,
9B, 10C, and 11A cells had a very low proportion of myoblasts compared with C2 cells. This may be a reflection of
heterogeneity in the parental C2 line relative to the transfected myoblast clones. Thus, substantial depletion of
-DG in 11F and 11E cells does not obviously affect these
aspects of muscle cell differentiation in agreement with
studies implicating integrins as the major receptors mediating the effects of ECM on myoblast alignment and fusion (Chung and Kang, 1990
; MacCalman et al., 1992
;
Collo et al., 1993
; George-Weinstein et al., 1993
; Blaschuk
and Holland, 1994
; Sastry et al., 1996
; Blaschuk et al., 1997
). However, even in the 11E cell line
-DG expression
is not completely abolished and it remains possible that
the residual
-DG is sufficient to play a role in the differentiation of myoblasts into myotubes.
|
Expression of -DG, LN, and Integrin
1 Subunits in
Cultured C2 and Antisense-expressing Myotubes
In addition to -DG, several LN receptors of the integrin
superfamily such as the
7
1,
9
1, and
3
1 integrins
are expressed in skeletal muscle (Collo et al., 1993
; Palmer
et al., 1993
; de Melker et al., 1997
) raising questions as to
the relative contributions of integrins and
-DG in LN
deposition on myotubes. As a first step in exploring this issue, C2 cells were immunostained with mAb IIH6 to
-DG
or with antibodies recognizing several LN isoforms and
the distribution of these proteins was compared in DG-deficient clones. In C2, 11F, and 11E cells,
-DG immunoreactivity is punctate and uniformly distributed over the
myotube surface (Fig. 3). However, the intensity of staining is greatly decreased on the surfaces of 11F and even
more so on 11E myotubes relative to C2 myotubes (Fig. 3;
the top photomicrograph for C2 cells and the two corresponding ones for 11F and 11E cells were taken at the
same exposure to illustrate this point). A similar pattern
was obtained with live cultures labeled with this antibody
(data not shown) confirming that the residual
-DG is
properly targeted to the cell surface in the antisense cell
lines. In all cell types, dense patches of
-DG immunoreactivity were found to be associated with spontaneous AChR clusters (Montanaro et al., 1998
; unpublished observations). Thus, the antisense cDNA construct affects
the amount of
-DG on the surface of myotubes but not
its distribution.
|
In adult skeletal muscle, LN 2 and
1 chains are expressed both synaptically and extrasynaptically, LN
1
chain is excluded from synaptic regions, while LN
4,
5,
and
2 chains are only found at the NMJ (Sanes et al.,
1990
; Patton et al., 1997
). To map the distribution of most
LN heterotrimers expressed in C2 cells, we used an anti-LN antiserum to LN
1,
1, and
1 chains as well as the
rat mAb 5D3 (Abrahamson et al., 1989
) which recognizes mouse LN
1,
2,
1, and
1 chains. Both antibodies gave
similar results, showing a patchy distribution of LN on the
surface of C2 myotubes with some larger aggregates (Fig.
4 A). Since
-DG is diffusely distributed on the surface of
C2 myotubes and there appears to be a pool of
-DG unbound to LN, it is difficult to determine from their distributions alone whether LN and
-DG may be interacting
with one another. Nevertheless, decreased expression of
-DG in 11F and 11E cells leads to a dramatic reduction in LN immunoreactivity on the myotube surface (Fig. 4 A)
suggesting that LN is bound to
-DG. Occasionally, dense
accumulations of LN immunoreactivity were seen on the
surfaces of 11F and, much less frequently, on 11E myotubes and these often coincided with densities of AChRs
(Montanaro et al., 1998
). 11A, 9B, and 10C myotubes expressed LN on their surface in a pattern and amount similar to C2 myotubes (Fig. 4 A). Western blots of total extracts of C2, 11A, 9B, 10C, 11F, and 11E myotubes probed
with the anti-LN antiserum showed similar levels of LN
expression (Fig. 4 B) indicating that there is no defect in
the biosynthesis of LN per se in the DG-deficient clones.
Furthermore, very little LN is released into the culture
medium (Fig. 4 B), and no difference could be seen in the
pattern of LN staining between cultures immunolabeled
live or after fixation indicating that 11F and 11E myotubes
do not accumulate LN intracellularly. Rather, most of the
LN in 11F and especially in 11E cells is deposited on the
surface of the dish in large, irregularly shaped deposits
found between live cells (not shown). This paucity of surface LN and the extensive extracellular deposition of LN
observed in cultures of antisense-expressing myotubes
suggest a deficit in the ability of LN to bind to myotubes deficient in
-DG.
Because of the high expression of LN 2 chain in skeletal muscle and its involvement in some types of muscular
dystrophy in both humans and mice, we looked specifically
at the distribution of this LN chain in C2 cells and DG antisense clones. In C2, 11F, and 11E cells LN
2 chain immunoreactivity was detected only on the surface of myotubes and never on the culture dish. In contrast with its
abundance in mature skeletal muscle, LN
2 is rather sparse in C2 cells and could not be detected by Western
blot on crude protein extracts (data not shown) but has
been detected after enrichment by immunoprecipitation
(Vachon et al., 1996
). LN
2 immunoreactivity on the cell
surface was associated with AChR clusters (Fig. 5 A, arrows) in C2, 11F, and 11E myotubes. Outside of these clusters, LN
2 immunoreactivity was diffuse and faint.
Integrin localization in C2 cells was determined with an
antiserum to the 1 subunit which is common to all integrin heterodimers expressed in muscle that recognize LN
(Collo et al., 1993
; Palmer et al., 1993
; de Melker et al.,
1997
). In all cultures, myotubes were intensely labeled,
whereas myoblasts showed a much fainter punctate staining pattern. For C2 as well as 11F myotubes the integrin
1
subunit either had a diffuse punctate pattern or was found
in large, intensely immunoreactive, oval patches (Fig. 5 A).
In 11E cells, a strip of intense immunoreactivity occasionally ran the length of the myotube. In cultures double labeled with
-BTX, immunoreactivity for the integrin
1
subunit only rarely overlapped with AChR clusters (Montanaro et al., 1998
), indicating that this receptor is not
likely to be responsible for the presence of LN at these
sites. To further investigate any differences in the levels of
expression of this integrin subunit, we used Western blotting to simultaneously probe the same blot for expression of the integrin
1 subunit and sarcomeric actin (Fig. 5 B).
Expression of sarcomeric actin has been shown to increase
with muscle differentiation (Sawtell and Lessard, 1989
)
and is therefore an indicator of the relative proportion of
myotubes versus myoblasts in our cultures. Under these
conditions, Western blotting confirmed that all clones express similar levels of the integrin
1 subunit, indicating that
1 integrins are not upregulated to compensate for
decreased DG expression in 11F and 11E cells. Conversely, the integrin
1 subunit is not downregulated in
11F and 11E cells indicating that the decrease in LN immunoreactivity on the surface of 11F and 11E myotubes is
not due to a lack of integrin expression and that integrins
are unable to compensate for decreased
-DG expression. In summary, the level of surface LN correlates well with
-DG levels in C2, 11F, and 11E myotubes, suggesting
that
-DG is responsible for the assembly of an LN-rich
ECM on the surface of myotubes. However, the amount of
-DG staining appeared more diffuse and extensive than
that of LN, in C2 and, to a lesser extent, 11F myotubes,
possibly because a significant proportion of
-DG was either free or else bound to another ligand, such as agrin
(Bowe et al., 1994
; Campanelli et al., 1994
; Gee et al.,
1994
; Sugiyama et al., 1994
).
Effect of Exogenous LN Addition on -DG-deficient
Cell Lines
As a further test of the ability of -DG to function as a LN
receptor, we assessed whether excess surface
-DG was
capable of binding exogenously added LN and whether
such binding could redistribute
-DG on the myotube surface as shown previously for Xenopus myocytes (Cohen
et al., 1997
). C2 and antisense-expressing myotubes were
incubated overnight in the presence of 12 nM EHS purified LN 1 (
1
1
1), then fixed and immunostained for
-DG or LN. A frequent feature of the LN treated C2 myotubes was the presence of large plaques of
-DG immunoreactivity (Fig. 6; arrows) as well as similar plaques of
LN immunoreactivity (Fig. 6, arrows) suggesting that exogenous LN can bind to and reorganize the unbound pool
of
-DG on the myotube surface (Cohen et al., 1997
).
Plaques of
-DG and LN immunoreactivity were also seen
on the surface of LN-treated 11F myotubes but these were
of smaller size and much less frequent (Fig. 6, arrows). No
such plaques were found on LN-treated 11E myotubes,
nor did exogenous LN bind well to 11E myotubes, consistent with the relative abundance of
-DG in these clones.
This correlation of LN binding with
-DG expression and
the redistribution of
-DG by exogenous LN suggest
strongly that
-DG is a functional LN receptor. As noted
previously (Cohen et al., 1997
), the aggregation of
-DG
by LN may be an early step in ECM assembly, which, from
our data, appears to be dependent on the level of
-DG
expression.
|
-DG Is Involved in Cell Survival
Although antisense clones were able to differentiate normally (Fig. 2), a consistently higher degree of cell loss was
observed after differentiation in 11F and 11E cultures.
DAPI staining of cultures after 4 d in fusion medium confirmed a decreased number of nuclei in 11F and 11E cultures compared with C2 cultures (Fig. 7, DAPI). The degree and time of onset of cell loss in the antisense clones
was quantified spectrofluorometrically after staining DNA with Hoechst dye. For the first 24 hours in fusion medium,
C2, 11F, and 11E cultures have comparable numbers of
cells. Subsequently, significant drops in cell number compared with C2 cultures are first detected at day 2 in 11E
cultures and at day 3 in 11F cultures (Fig. 8 A; P < 0.0001)
and by day 4, cultures of 11F and 11E cells emit 45 and
65% less Hoechst fluorescence, respectively, when compared with C2 cultures (Fig. 7, DAPI, and Fig. 8 A). Statistical analysis revealed a linear correlation between cell
number in C2, 11F, and 11E cultures at day 4 and the
amount of -DG expressed (R2 = 0.80; P < 0.0001). In
contrast, 9B and 10C clones behaved essentially like C2
cells. 11A cells also showed an increase in cell number similar to C2 cells but were not included here because the
slightly slower proliferation rate of 11A myoblasts led to
an asynchronous differentiation of these cultures compared with the other clones rendering any comparison difficult. Thus the level of
-DG expression correlates with
the amount as well as the time of onset of cell loss during
differentiation of myoblasts into myotubes.
|
|
Since the number of cells increases with differentiation
in control cultures of C2, 9B, and 10C cells, it was important to establish whether the decreased cell number in
DG-deficient clones was due to actual cell loss or a deficiency in proliferation. BrdU incorporation, as measured
by a colorimetric assay, was used to determine proliferative activity. When relative proliferation rates were expressed as a ratio of BrdU incorporation over total cell
number as determined by Hoechst fluorescence, 11F and
11E cells did not have lower proliferation rates than C2,
9B, or 10C cells (Fig. 8 B). In fact the loss of cells at day 2 in 11E cultures (Fig. 8 A) seems to cause a drop in cell
density sufficient to stimulate proliferation of the remaining
myoblasts as indicated by the increase in BrdU incorporation in 11E cultures at day 3 (Fig. 8 B). This increased proliferation translates into a small increase in cell number at day
4 (Fig. 8 A). The reduction in cell number observed in
both -DG-deficient clones is therefore unlikely to be due to
decreased proliferation rates but reflects a genuine loss of cells
in these cultures due to increased apoptosis or necrosis.
Visual inspection of DAPI-labeled cultures revealed the
presence of a greater number of nuclei with condensed
chromatin in cultures of antisense clones compared with
control cells. Since apoptotic cell death leads to chromatin
condensation, we used the TUNEL method to assay for
apoptosis (Fig. 7, TUNEL). To verify the specificity of this
method in our assay, we counterstained the cultures with
the nuclear dye DAPI allowing us to also identify apoptotic cells by morphological criteria. We found that cells with marked condensation of chromatin and cytoplasm
(apoptotic cells) as well as cytoplasmic fragments with
condensed chromatin (apoptotic bodies) were intensely labeled by the TUNEL method. Generally, labeled cells
were in the latest stages of apoptotic cell death and had often assumed a round morphology. This method labeled
both individual, poorly attached cells and nuclei within aggregates. It was therefore difficult to determine whether
TUNEL-positive nuclei belonged to former myoblasts or
myotubes. More rarely, nuclei of adherent cells were labeled, albeit less intensely. These nuclei were often found
within myoblasts but were occasionally also seen in myotubes. Since in most cases TUNEL-positive nuclei could
not be definitely assigned to myoblasts or myotubes, we quantified the number of apoptotic nuclei without attributing them to a particular cell type (Fig. 8 C). A small proportion of nuclei was TUNEL-positive in differentiating
C2, 11A, 10C, and 9B cultures, while a larger number of
nuclei, often within rounded, poorly attached cells, were
labeled in day 2 and older cultures of 11F and 11E cells
(Fig. 7, TUNEL). At day 3, 11F, and 11E cultures have 3 and 11 times more TUNEL-positive nuclei compared with
cultures of C2 cells (Fig. 8 C). These data indicate that reduced -DG expression results in a persistent loss of
myoblasts after transfer to fusion medium and a correspondingly significant increase in apoptotic cell death in
both myoblasts and myotubes.
Decreased Levels of -DG Expression Are Not
Associated with Loss of Membrane Integrity
Studies of dy/dy and mdx mice suggest that loss of membrane integrity is linked to a loss of interaction between
dystrophin and the actin cytoskeleton, rather than a decreased cell surface expression of the DGC (Straub et al.,
1997). However, recent studies have provided evidence
that mutations affecting the expression of SGs can result
in a loss of membrane integrity (Duclos et al., 1998
; Hack
et al., 1998
). Since we perturbed the expression of DG
without affecting the SGs, we set out to determine the
role, if any, of DG in the maintenance of membrane integrity. We used the vital dye Trypan blue to visualize myoblasts or portions of myotubes where membrane integrity
was compromised. In our hands, Trypan blue stained nuclei much more intensely than the cell cytoplasm and allowed a direct visualization of the extent of membrane
damage on each multinucleated myotube. In cultures of
C2, 11F, and 11E cells maintained 3 d in fusion medium
cell death has peaked (Fig. 8 A) and Trypan blue labeled
the nuclei of some myoblasts (Fig. 9 B). At this time myotubes often had one or two blue nuclei, indicating a localized loss of membrane integrity and only rarely were all
nuclei in a myotube labeled. Notably, there were no significant differences between C2 and 11E cultures in either
the number of myotubes with one or more nuclei stained
with Trypan blue (Fig. 9 A) or in the proportion of myoblasts
versus myotubes with blue nuclei (Fig. 9 B). Therefore, a
substantial reduction of
-DG expression at the surface of
myotubes does not lead to a loss of membrane integrity.
|
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Discussion |
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In muscle, the DGC is thought to link two proteinaceous
matrices, the ECM and the subplasmalemmal cytoskeleton, providing structural support for the interposed plasma
membrane. According to this widely held model - and
-DG associate to form the core of the DGC with
-DG
bound to LN (Smalheiser and Schwartz, 1987
; Douville et
al., 1988
; Ibraghimov-Beskrovnaya et al., 1992
; Gee et al.,
1993
; Smalheiser and Kim, 1995
; Yoshida et al., 1994
) and
-DG to dystrophin (Suzuki et al., 1994
; Jung et al., 1995
). To investigate functional aspects of this model, we have
perturbed the expression of
- and
-DG by generating
muscle cell lines stably transfected with an antisense DG
cDNA expression construct. After several transfections
and screening of many stable lines we identified two
clones of C2 cells in which expression of DG was reduced
significantly i.e., 40-50% and 80-90%, respectively. The
two DG-deficient cell lines retain the ability to fuse and form myotubes, and express near normal levels of
- and
-SG, two other DGC proteins. Similarly, expression of
other membrane proteins such as the AChR, and the
MuSK receptor tyrosine kinase (Jacobson et al., 1998
) and
1 integrin (Fig. 4) are indistinguishable from parental C2
cells. Three other clones, 9B, 10C, and 11A, which were
subjected to the same transfection and antibiotic selection and have wild-type levels of
- and
-DG, fuse normally
and have no obvious cell loss indicating that the altered
phenotype of 11E and 11F cells is not a trivial outcome of
transfection and antibiotic selection. These observations
argue that 11E and 11F cells differ only in their abnormally low expression of DG. An alternative possibility, especially in light of the relatively small number of clones we
were able to isolate, is that an unidentified mutation in the
antisense-expressing cell lines affects the glycosylation of
-DG so that it is poorly detected by mAb IIH6 (which
recognizes, at least in part glycosylated epitopes; Ervasti
and Campbell, 1993
). In fact, we do see a slightly faster migration during SDS-PAGE of
-DG from 11F and 11E
myotubes (Fig. 1 B). This may well be due to altered
glycosylation since most of the apparent mass of
-DG
on SDS-PAGE is due to carbohydrate moieties (Ibraghimov-Beskrovnaya et al., 1992
; Smalheiser and Kim, 1995
;
Chiba et al., 1997
). However, an antibody directed against
the core protein (anti-fusion protein B antiserum) also reveals a decrease of at least 50-80% in
-DG levels, which
is equivalent to that seen with mAb IIH6 (Fig. 1 C). Thus,
-DG appears to be expressed at low levels in 11F and 11E cells. That the residual
-DG is recognized about
equally by a polyclonal antiserum to DG and by mAb
IIH6 which is to a binding site on
-DG (Ervasti and
Campbell, 1993
; Campanelli et al., 1994
; Gee et al., 1994
;
Sugiyama et al., 1994
; Durbeej et al., 1995
) suggests that
the small shift in electrophoretic mobility of
-DG in the
antisense clones does not affect the interaction of
-DG
with LN. The concomitant decrease of
-DG by 70-90%
in 11F and 11E cells compared with control C2 cells, further suggests that any possible defects in glycosylation do
not lead to excessive shedding of
-DG into the medium
of 11F and 11E cells.
-DG Is a Functional LN Receptor In Situ That Is
Involved in ECM Assembly on Muscle Cells
To date there have been no reports of any human or animal myopathy linked to mutations in the DG gene. Mice
null for the DG gene die very early in development (Williamson et al., 1997; Côté, P., M. Lindenbaum, and S. Carbonetto. 1997. Mol. Biol. Cell. 8:222a) suggesting that mutations which compromise its expression in tissues other
than skeletal muscle may lead to embryonic lethality in humans. Previous studies indicate that
-DG functions as
a receptor for two ECM proteins agrin (Gee et al., 1994
;
Campanelli et al., 1994
; Bowe et al., 1994
; Sugiyama et al.,
1994
; Cohen et al., 1995
) and LN. For LN, this includes observations on: (a) binding of LN to
-DG isolated from
muscle and nervous system (Smalheiser and Schwartz,
1987
; Ibraghimov-Beskrovnaya, 1992; Gee et al., 1993
; Yamada et al., 1994
, 1996
); (b) colocalization of LN with
-DG in muscle and other tissues (Klietsch et al., 1993
;
Yamada et al., 1994
; Durbeej et al., 1995
; Cohen et al.,
1997
); (c) inhibition of LN-dependent differentiation in
kidney by a mAb IIH6 to
-DG (Durbeej et al., 1995
); (d)
coprecipitation of LN with dystrophin in cultured muscle
cells (Dickson et al., 1992
); (e) disruption of Reichert's
basement membrane in mice rendered null for the DG
gene (Williamson et al., 1997
; Côté, P., M. Lindenbaum,
and S. Carbonetto. 1997. Mol. Biol. Cell. 8:222a). More recently, Henry and Campbell (1998)
have implicated DG in
basement membrane assembly in cultured embryoid bodies, though it is unclear whether this is mediated uniquely
by
-DG binding to LN or also to other ECM molecules
such as agrin (Bowe et al., 1994
; Campanelli et al., 1994
;
Gee et al., 1994
; Sugiyama et al., 1994
; Cohen et al., 1995
)
and perlecan (Peng et al., 1998
). In support of the hypothesis that
-DG is a LN receptor in muscle, we find that
lower
-DG levels in 11E and 11F cells result in a corresponding reduction in the level of exogenous LN bound to
the surfaces of these lines when compared with parental
C2 cells. Moreover, there is a clear decrease in the deposition of endogenous LN on the surface of myotubes. This is
not obviously a result of reduced synthesis and secretion
of LN since DG-deficient cells have normal levels of LN
(Fig. 4 C) and the ECM which forms on the culture substratum between the myotubes appears equivalently LN-rich in DG-deficient and parental C2 cells. The loss of surface LN in 11F and 11E myotubes does not appear to be a
secondary consequence of a general disruption of the
ECM since the distribution of collagen IV is not significantly affected (Montanaro, F., and S. Carbonetto, unpublished observations). Instead, DG deficiency leads to a
rather selective loss of LN from the myotube ECM implicating
-DG as a functional LN receptor necessary for
proper ECM assembly in skeletal muscle.
Vachon et al. (1997) have suggested recently that the
"de facto receptor" for LN 2 (
2
1
1) in skeletal muscle is
the
7
1 integrin. They report a decrease of the
7
1 integrin in human and mouse muscular dystrophies where mutations in the LN
2 chain lead to loss of LN 2 or expression of a truncated form. They further note that
-DG
expression is unaffected in these instances concluding that
it is not necessary for LN assembly in vivo. However in the dy/dy and dy2J mutant mice there is a compensatory upregulation of LN 8 (
4
1
1) which replaces LN 2 (Patton et
al., 1997
) and could be responsible for the maintained expression of the DGC at the cell surface. Alternatively, expression of the DGC might be more dependent on its association with dystrophin than with its extracellular ligands. In fact, muscle cells in culture have a pool of surface
-DG
that is not bound to LN (Fig. 5; Cohen et al., 1997
). Similarly, in the dystrophin mutant mdx mouse, the observation that LN is present in the muscle ECM in spite of the
dramatic decrease in the DGC at the cell surface, does not
eliminate
-DG as a LN receptor in skeletal muscle. For
example, although the
7
1 integrin is expressed at wild-type levels in mdx mice (Vachon et al., 1997
), a large fraction of LN in skeletal muscle in these mice and DMD patients is unusually soluble indicative of a weak binding to the muscle cell surface (Dickson et al., 1992
). Indeed, an
early sign of pathology in DMD is the separation of the
basement membrane from the muscle cell surface (Carpenter and Karpati, 1979
). Furthermore, no obvious colocalization of LN and the integrin
1 subunit is observed in
cultures of C2 cells or of primary myotubes from mouse or
human (Fig. 4; Dickson et al., 1992
) and in C2 myotubes
the distribution of the LN
2 chain (Fig. 4) does not match
that reported for the
7 integrin subunit (Vachon et al.,
1997
). More importantly, in mice null for the integrin
7
gene the distribution of LN in skeletal muscle appears normal and the progressive muscle degeneration seems to be
predominantly due to defects at the myotendinous junction (Mayer et al., 1997
). Thus, as Mayer et al. (1997)
, we
also propose integrins and
-DG may function as independent receptor complexes which together provide a link between LN and the muscle membrane that is necessary for
muscle homeostasis.
-DG Is Involved in Muscle Cell Viability in Culture
The DGC has been postulated to act as a superstructure
for the muscle cell surface, and its loss has been correlated
with disruption of the plasma membrane, necrosis, and in
some cases apoptosis (Rosalki, 1989; D'Amore et al., 1994
;
Matsuda et al., 1995
; Tidball et al., 1995
; Tews and Goebel,
1997
).
In vivo, apoptosis could be a secondary consequence of
the inflammation caused by the constant degeneration and
regeneration of muscle fibers that occurs in the absence of
dystrophin. Indeed, apoptotic cell death in skeletal muscle
of the mdx mouse appears to be mainly caused by activated inflammatory cells that infiltrate the muscle mass
and subsequent release of the cytotoxic protein perforin
(Spencer et al., 1997). However, our studies and those of
Vachon et al. (1996)
suggest that other apoptotic pathways might also be activated. Vachon et al. (1996)
have studied
the effect of LN 2 (
2
1
1; merosin) on myotube stability
in human and mouse muscle cell lines in culture. Several
spontaneous variants deficient in LN 2 expression were
cloned and found to have increased myotube degeneration
after fusion. Addition of exogenous LN 2 increased myotube numbers in all clones and transfection with a human
LN
2 chain cDNA decreased myotube degeneration and
the abnormally high level of apoptosis in these cell lines.
In our studies, decreased expression of
-DG disrupts LN
expression on the surface of myotubes, and we expected to
see a similar pattern of myotube degeneration. While
there are some apoptotic nuclei within adherent myotubes, most TUNEL-positive nuclei were found within
amorphous, loosely adherent masses, which could have
been detached or "collapsed" myotubes, or clumps of
dead myoblasts. Possibly, deficiency of
-DG could cause
myotubes to detach more readily than a reduction in the
expression of a single LN isoform. Indeed,
-DG-deficient cells would have impaired binding to most if not all
LN heterotrimers expressed by muscle cells, as well as
agrin (Bowe et al., 1994
; Campanelli et al., 1994
; Gee et al., 1994
; Sugiyama et al., 1994
; Cohen et al., 1995
) and possibly perlecan (Peng et al., 1998
). However, Henry and
Campbell (1998)
reported that absence of DG in embryonic stem cells affects basal lamina assembly but not adhesion to a LN-coated substratum. Similarly, preliminary results show no detectable difference in the ability of 11F
and 11E myoblasts to adhere to LN-coated dishes compared with control myoblasts. Vachon et al. (1996)
also reported that myoblasts from LN-deficient clonal variants
have a reduced ability to fuse. In our studies, 11E and 11F
cells fuse and differentiate normally indicating that
-DG
may not mediate the effects of LN on myoblast fusion and
that these are likely integrin mediated (Chung and Kang,
1990
; MacCalman et al., 1992
; Collo et al., 1993
; George-Weinstein et al., 1993
; Blaschuk and Holland, 1994
; Sastry et al., 1996
; Blaschuk et al., 1997
). However myoblast survival was compromised in both clones after serum withdrawal from the onset of differentiation (Fig. 8 A). Serum
withdrawal is typically associated with cell death, and we
observed a decrease in the number of cells in all cultures
within 24 h of switching to fusion medium. While C2 cells
and all control clones had completely recovered after 48 h,
11F and 11E cells continued to be lost. Although death of
DG-deficient myoblasts could be due to loss of adhesion,
we located some adherent myoblasts that were TUNEL-positive indicating that apoptotic cell death can precede
cell detachment. It is interesting to note that during differentiation of C2 myoblasts, the level of
-DG expression
increases dramatically and its glycosylation changes
(Leschziner, A., and S. Carbonetto, unpublished observations) suggesting that it might play a particular function at
this developmental stage. Our results suggest that DG is
important for myoblast survival during differentiation in
culture and we speculate that loss of DG in vivo might affect muscle regeneration by satellite cells.
Straub et al. (1997) have studied the permeability of
muscle fibers to the vital dye Evans blue in dystrophic
mice. In mdx mice skeletal myofibers have increased permeability to this hydrophilic dye most likely through disruptions in their plasma membranes which allow serum
proteins to enter these presumably necrotic cells (Straub
et al., 1997
). In contrast, dy/dy mice with mutations in the
LN
2 chain develop a more severe muscular dystrophy
with a minor loss in membrane integrity as reflected by a
relative impermeability to Evans blue and by normal levels of creatine kinase in the serum. In both dy/dy mice and
patients with merosin-deficient congenital muscular dystrophy skeletal myofibers are apoptotic (Miyagoe et al.,
1997
; Tews and Goebel, 1997
). Furthermore, recent studies have shown a strong correlation between loss of membrane integrity and lack of SGs at the muscle cell surface
(Duclos et al., 1998
; Hack et al., 1998
). In our studies, decreased expression of DG does not appear to affect the expression of
- and
-SG. We are currently investigating
whether these SGs are correctly expressed at the sarcolemma of DG-deficient myotubes as would be expected
from observations that membrane integrity is not compromised in myotubes from both 11F and 11E cells (Fig. 9).
Our results support the notion that the DG and SG complexes are expressed independently of one another in muscle cells and that they perform distinct functions vis à vis
membrane integrity.
Mechanism of -DG-mediated Apoptosis
The data presented here raise questions about how -DG,
a peripheral membrane protein, may transduce an extracellular signal to suppress apoptosis in either myoblasts or
myotubes.
-DG is bound tightly to its transmembrane
partner
-DG (Bowe et al., 1994
; Yoshida et al., 1994
)
which, in turn, can interact with the SH2/SH3 domain containing adapter protein Grb 2 (Yang et al., 1995
). An additional, second messenger, nitric oxide synthase, associates
with syntrophins (Brenman et al., 1995
, 1996
), equipping the DGC further for intracellular signaling. Thus, one can
envision a scenario wherein these signaling intermediates
are activated by LN binding to
-DG transmitting a signal
to the cell interior via
-DG. To our knowledge, however,
there is no precedent for an extracellular peripheral
membrane protein like
-DG transmitting signals in this manner.
Although no evidence is presently available for the involvement of the DG complex in the initiation of signaling, DG has been shown to be involved in the aggregation
of acetylcholine receptors and to act downstream of the
muscle specific tyrosine kinase receptor MuSK (Jacobson
et al., 1998). Apoptosis or its prevention is often associated
with activation of receptors to growth factors or ECM molecules. DG could be part of a cell surface signaling
complex and/or act downstream of other tyrosine kinase
receptors. LN/
-DG complexes on the myotube surface
are involved in the assembly of a heterogeneous basal lamina that would include constituents of the ECM (e.g., glial
growth factor, agrin, collagen) which directly activate receptor tyrosine kinases (Jo et al., 1994
; Glass et al., 1996
;
Shrivastava et al., 1997
; Vogel et al., 1997
) or act as coreceptors for them (e.g., perlecan; Aviezer et al., 1994
).
Their inclusion in a two dimensional array of ECM in
close proximity to the plasma membrane may facilitate activation of these or similar receptors inhibiting cell death.
Also, the assembly of an ECM of proteins occurs coincident with assembly of an intracellular network which may
"trap" and concentrate receptor tyrosine kinases activating them in a ligand-independent manner.
Integrins may offer another route for intracellular signaling after LN binding to -DG. Integrin expression
changes dramatically during muscle differentiation (Gullberg et al., 1995
), affecting the passage from a proliferative
to a quiescent state in myoblasts (Sastry et al., 1996
), and
promoting fusion (Rosen et al., 1992
; Sastry et al., 1996
).
In mature myotubes, Vachon et al. (1996)
speculate that
the LN receptors responsible for regulating apoptosis in
their studies are members of the integrin superfamily viz.
7
1. Integrins are well-known to be involved in apoptosis
in nonmuscle cells where loss of integrin-mediated adhesion results in the downregulation of Bcl 2 and upregulation of Bax expression (Zhang et al., 1995
; Stromblad et al.,
1996
), as well as activation of the ICE protease cascade
(Guan, 1997
), all of which are intermediates in apoptotic
cell death. Interestingly,
-DG accumulates at focal adhesions and colocalizes with the integrin
1 subunit when fibroblasts are grown on a LN substrate (Belkin and Smalheiser, 1996
). Furthermore, Yoshida et al. (1998)
recently
reported evidence for cross-talk between integrins and the
DGC. In their studies the
5
1 fibronectin receptor in L6
myoblasts can associate with dystrophin and the DGC. Fibronectin, or amino acid mimetics of fibronectin, stimulate phosphorylation of
- and
-SG. Perhaps, similar cross-talk occurs between
7
1 heterodimer, and the DGC.
-DG
binds to the last 2 globular domains in the LN
1 and
2
chains, a region distal to that of any known integrin-binding site so that LN bound to an integrin should be able to
bind
-DG or vice versa. Binding of LN to
-DG stimulates aggregation of
/
-DG on muscle cells (Fig. 5; Cohen
et al., 1997
) which may increase the chance of interacting
with an integrin. Thus,
-DG, in addition to its function as
a structural element of the cell surface may, by stimulating ECM assembly, enhance the interaction of ligands embedded in the ECM with integrins and other transmembrane
receptors that suppress apoptosis.
In conclusion, our data provide strong evidence that
-DG functions in muscle cells as a LN receptor which
mediates ECM assembly. Furthermore, they indicate that
the DGC regulates apoptosis in culture, which may have
important implications for novel functions of the DGC as
a signaling complex.
![]() |
Footnotes |
---|
Address correspondence to Salvatore Carbonetto, Center for Research in Neuroscience, McGill University, Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada. Tel.: (514) 937-6011, Ext. 4237. Fax: (514) 934-8265. E-mail: cy93{at}musica.mcgill.ca
Received for publication 6 April 1998 and in revised form 23 April 1999.
This research was supported by grants to S. Carbonetto from the Muscular Dystrophy Association (US), and the Medical Research Council
(Canada). F. Montanaro was the recipient of a studentship from the Canadian National Centers of Excellence.
The first two authors contributed equally to this work.
We thank Drs. Kevin Campbell (HHMI, University of Iowa) and Peter
Yurchenco (UMDNJ) for their generous gifts of antibodies to -DG and
LN-2, respectively. We also thank Chris Jacobson for generously contributing panel C of Fig. 1, and Cathy Lan for her technical assistance with
protein extraction and Western blotting.
![]() |
Abbreviations used in this paper |
---|
-DG,
dystroglycan;
AChR, acetylcholine receptor;
BrdU, bromodeoxyuridine;
BTX, bungarotoxin;
DAPI, 4',6-diamidino-2-phenylindole;
DGC, dystrophin associated glycoprotein
complex;
EHS, Engelbreth-Holm-Swarm;
LN, laminin;
SG, sarcoglycan;
TUNEL, terminal deoxylnucleotidyl transferase-mediated dUTP nick end
labeling.
![]() |
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