| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* CNR-Unit for Muscle Biology and Physiopathology; and
Department of Biomedical Sciences, University of Padova, Padova, Italy
1Correspondence: CNR-Unit for Muscle Biology and Physiopathology, c/o Department of Biomedical Sciences, University of Padova, via G.Colombo 3, 35121 Padova, Italy. E-mail: lgorza{at}civ.bio.unipd.it
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Here we show
that C2C12 cells up-regulate GRP94 during differentiation and,
similarly to primary cultures of murine skeletal myocytes, specifically
display GRP94 immunoreactivity on the cell surface. Stable transfection
of C2C12 cells with grp94 antisense cDNA shows lack of myotube
formation in clones displaying >40% reduction in GRP94 amount. The
same result is obtained after in vivo injection of
grp94-antisense myoblasts. Conversely, GRP94 overexpression is
accompanied by accelerated myotube formation. Analyses of BrdU
incorporation, p21 nuclear translocation, and muscle-gene expression
show that muscle differentiation is not apparently affected in
grp94-antisense clones. In contrast, cell-surface GRP94 is greatly
reduced in grp94-antisense clones, as shown by immunocytochemistry and
precipitation of cell-surface biotinylated proteins. Thus, cell-surface
expression of GRP94 is necessary for maintenance of fusion competence.
Furthermore, differentiating C2C12 cells grown in the presence of
anti-GRP94 antibody show decreased myotube number suggesting that
cell-surface GRP94 is directly involved in myoblast fusion
process.—Gorza, L., and Vitadello, M. Reduced amount of the
glucose-regulated protein GRP94 in skeletal myoblasts results in loss
of fusion competence.
Key Words: heat-shock protein • skeletal-muscle growth and differentiation • antisense-elements physiology • cell fusion • gene expression and regulation
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3
4)
. Furthermore, they belong to the stress protein
family, because their synthesis is enhanced in response to ER stress,
namely, when extracellular glucose is low or absent, intracellular
Ca2+ stores are depleted, or glycosylation is
inhibited (4)
. Although these proteins are considered to
be ubiquitously and constitutively expressed, we recently reported that
GRP94 expression in rabbit skeletal muscle myofibers appears to be
developmentally regulated, being easily detectable in muscle myocytes
of the fetal and newborn rabbits, whereas it is not any more detectable
at both mRNA and protein level in adult skeletal muscle fibers
(5)
. The developmentally restricted expression of grp94
gene in skeletal muscle myofibers is apparently not accompanied by
corresponding changes in GRP78 amount, which does not vary between
newborn and adult mammalian skeletal muscles (6)
. The
disappearance of both GRP94 transcript and protein in myofibers of
adult skeletal muscles cannot be reversed by exposure to bacterial
lipopolysaccharide (5)
, which, in contrast, is effective
in increasing GRP94 expression in lymphocytes in vitro
(7)
and in cardiac myocytes in vivo
(5)
. The lack of grp94 gene responsiveness in adult
skeletal muscle myofibers suggested to us that GRP94 accumulation in
developing skeletal muscle is devoted to important events occurring
during muscle differentiation.
Several functions have been recognized for GRPs: GRP78 and GRP94 act as
molecular chaperones involved in protein translocation into the ER, in
their subsequent folding and assembly, and in regulating protein
secretion (8)
. Secondly, they play a functional role in
cell survival because they exert a specific protection against
Ca2+ depletion stress (4
, 9
, 10)
.
Furthermore, GRP94 has been shown to participate in antigen
presentation (11
, 12)
. Finally, GRP94 is a low
affinity-high capacity Ca2+- binding protein, a
property which, like chaperoning and involvement in cell protection, is
in common with other ER resident proteins, such as calreticulin and
protein disulfide isomerase (13
, 14)
. Despite such a
multiplicity of functions, little is known concerning the functional
role of GRPs, and of stress proteins in general, during cell
differentiation (15)
. Until now, knocking-out of grp78 and
grp94 genes has been so far limited by the fact that a certain basal
level of protein is required for cell survival (4)
, and,
indeed, strategies aimed to reduce transcript accumulation have been
successful in demonstrating loss of protection against various stresses
(9
, 10)
.
Given that postnatal maturation of skeletal muscle is characterized by down-regulation of the grp94 gene, we asked whether reduced amounts of GRP94 in myoblasts may affect muscle maturation. Thus, we chose to reduce GRP94 levels in a skeletal muscle cell line (C2C12, 16) by stable transfection with a vector containing rabbit grp94 cDNA sequence in antisense orientation driven by a RSV promoter. Although >40% reduction in GRP94 amount does not interfere with cell cycle withdrawal and muscle-specific gene expression, it affects myotube formation. In contrast, GRP94 overexpression accelerates myotube formation.
In addition we report that control C2C12 cells and cultured primary myoblasts, but not fibroblasts isolated from the same newborn skeletal muscles, show the presence of GRP94 on the outer surface of the cell membrane. Cell surface GRP94 is strongly reduced in grp94 antisense clones; furthermore, incubation of C2 cells with anti-GRP94 antibody delays myotube formation suggesting that GRP94 localization at the cell surface plays an essential role in myotube formation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
.
Cell culture
Primary cultures of skeletal myocytes were prepared by pooling
muscles excised from newborn mice of the same litter. In brief, muscles
were minced and the cells were dispersed by treatment with 0.25 mg/ml
trypsin (Sigma, St. Louis, Mo.) at 37°C. The cells were resuspended
in proliferation medium [DMEM, containing 10% fetal calf serum,
L-glutamine, and antibiotics (Sigma, Milan)] and preplated 20 min to
remove nonmuscle cells. Unattached 1 x 105
muscle cells were seeded on gelatinated round coverslips in 24-well
Falcon plates (Becton Dickinson, Bedford, Mass.). After 24 h,
cells were switched to differentiation medium [DMEM, containing 2%
horse serum and 0.5 µg/ml insulin, L-glutamine, and antibiotics
(Sigma)].
C2C12 murine myoblast cells were cultured in proliferation medium (see above). To avoid spontaneous differentiation, the cells were not allowed to reach confluency. Differentiation was induced by replacing medium, as described for primary myoblast cultures. Stable transfectants were selected in the same media as for C2C12 added with 500 µg/ml G418 (Boehringer, Mannheim, Germany).
For immunofluorescence, cells were seeded on gelatinated coverslips in 24-well plates at a density of 10,000 cells per well and grown in proliferation medium. After 24 h, cells were switched to differentiation medium for 48 h and then processed as described below.
For incubation with antibodies, 1 x 103 C2C12 cells were seeded in 96-well culture plates (Becton Dickinson) in 150 µl proliferation medium. 24 h later, medium was replaced with differentiation medium added twice a day with either 10, 5, or 2 µg/well of 3C4 purified immunoglobulins or, alternatively, of comparable amounts of mouse nonimmune immunoglobulins (Sigma). Experiments were performed in triplicate and wells were examined for the presence of myotubes after 3–4 days.
For RNA extraction or homogenate preparation, cells were seeded at a density of 450,000 cells/10-cm petri dish (Becton Dikinson) in proliferation medium. Cells were either maintained in proliferation medium or switched to differentiation medium 24 h after plating, as described below, before utilization.
Probe and construct preparation
We have previously described 2.5 and 2.52 grp94 rabbit cDNA
(5)
. Clone 2.5 was used for generation of cRNA probes for
Northern blotting. Clone 2.52 was used to prepare constructs suitable
for eukaryotic expression studies. In brief, sense or antisense mRNA
expression was achieved ligating the 5' XbaI fragment of the
2.52 insert (1.1 kb in length) either in antisense or sense orientation
to the NheI site of the RSV promoter of the pBK-RSV phagemid
(Stratagene, La Jolla, Calif.). Antisense and sense orientation of the
insert in pBK-RSV 2.5X plasmids was checked by digestion with
appropriate restriction enzymes. Note that the mRNA transcribed from
the sense- oriented insert lacks translational recognition signal and
does not give origin to any protein.
Overexpression of GRP94 was achieved in the following way: a cDNA
sequence with an open reading frame coding for a rabbit-mouse GRP94
chimeric protein of 727 amino acids was obtained by adding 3' to the
RSV promoter a 75 bp fragment containing the Kozak consensus
translation initiation sequence (18)
and the signal
peptide of mouse GRP94 sequence (19)
(MRVLWVLGLCCVLLTFGFVRADA) obtained by PCR amplification, and thereafter
the insert of the longest rabbit grp94 cDNA (2.52 clone) ablated of the
3'UTR.
Furthermore, in order to visualize the transfected GRP94, a sequence
corresponding to a stretch of 13 amino acids containing the VSV-G
epitope (20)
was inserted downstream the signal peptide
(pBK-RSV94-TAG8). The resulting protein is 76 amino acids shorter at
the amino terminus than murine GRP94, whose length predicted by cDNA
translation is 803 amino acids (19)
. Sequence of
pBK-RSV94-TAG8 was checked with the dideoxychain termination method as
described previously (5)
. An eukariotic expression
construct containing the ß-gal cDNA (17)
was also
prepared using the pBK-RSV phagemid as a vector.
Transfection and selection
Approximately 1 µg of pBK-RSV 2.5X sense or antisense plasmids
and 1.5 µg of pBK-RSV94-TAG8 were linearized with DraIII
and trasfected by electroporation into C2C12 cells. Linearized pBK-RSV
2.5X sense or antisense plasmids were cotransfected with 2.8 µg of
the NsiI-NaeI fragment of pBK RSV-ßgal (not containing
neo-resistance). The 2 x 106 cells were
grown in proliferation medium and detached from plastic with trypsin.
After centrifugation (10 min at 200g) cells were resuspended
in 500 µl of proliferation medium, mixed with the appropriate plasmid
DNAs, and electroporated at 250 mV and 960 µF. Electroporated cells
were seeded in 96-well culture plates with proliferation medium. After
24 h, cells were transferred in selection medium (DMEM
supplemented with 10% FCS and 500 µg/ml G418). The medium was
changed every 48 h. Drug resistant cells were cloned by limiting
dilution.
Northern blotting
Total RNA was isolated from C2C12 cultures following the
procedure described by Chomczynski and Sacchi (21)
,
electrophoresed, transferred, and hybridized either with
32P-labeled grp94 antisense cRNA and 18S RNA
probes, as described previously (5)
.
Antibodies
Anti-GRP94 monoclonal antibody 3C4 was obtained after
immunization with a recombinant polypeptide as described previously
(5)
. Monoclonal anti-desmin and anti-VSV-G antibodies were
purchased from Roche (Montclair, N. J.). Specificity of monoclonal
anti-troponin T antibodies RV-C2 and BN-59 and anti-troponin I TI-4 has
been previously described (22
, 23)
. Polyclonal anti-p21
and anti-erbB2 antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif.). Polyclonal anti-desmin antibodies were
purchased from Sigma. Monoclonal anti-integrin ß3 subunit was
obtained from Transduction (Lexington, Ky.). Peroxidase and fluorescein
conjugated antibodies were provided from Dako (Glostrup, Denmark);
rhodamine conjugates were provided by Cappel (Eschwege, Germany).
Western blotting
Tissue samples were homogenized in electrophoresis sample
buffer, heated for 5 min in boiling water and centrifuged
(15,000g) for 15 min at 4°C. For quantitative analyses,
cells were homogenized in the same buffer without bromphenol blue and
ß-mercaptoethanol, and protein concentration was determined as
described by Lowry et al. (24)
using bovine serum albumin
as standard. Equal amounts of samples were run either in 6–12%
gradient or in 10% linear polyacrylamide gel together with
commercially available preparations of molecular weight standards
(Bio-Rad, Richmond, Calif.) at constant amperage (5 mA), transferred to
nitrocellulose, saturated with ovalbumin, and subsequently incubated
with the primary antibody. Unbound antibody was removed after extensive
rinses with TBST and filters were incubated with appropriate
anti-immunoglobulins conjugated with peroxidase. Peroxidase activity
was revealed using diaminobenzidine, as described previously
(5)
.
Analysis of samples obtained by streptavidin-agarose precipitation were performed on nitrocellulose blots saturated with 1% blocking reagent (Roche) in TBST using antibodies as specified in Results. Chemiluminescent substrate (Renaissance Plus; Dupont-Nen, Boston, Mass.) was used to demonstrate bound peroxidase activity in these assays.
Immunocytochemistry
Tissue cultures were fixed for 10 min at room temperature with
4% freshly prepared buffered paraformaldehyde, rinsed twice for 15 min
with phosphate-buffered saline (PBS), permeabilized with cold 0.1%
Triton X-100 for 5 min, and incubated with 3C4 mAb diluted 1:500 with
0.5% bovine serum albumin in PBS. Incubation was carried out in a
humidified chamber at room temperature for 30 min. After 3 x 10
min rinses in PBS, sections were incubated either with appropriate
dilutions of secondary antibodies coupled with peroxidase and using
diaminobenzidine as a substrate or with antibodies coupled with
rhodamine. After immunofluorescence staining and in order to label
nuclei, sections were mounted with glycerol buffered with PBS and
containing 2 µg/ml 4,6-diamino-2-phenylindole (DAPI; Sigma).
For surface labeling, incubation with the first antibody was carried out on unfixed cell cultures for 3 h at 4°C. Rinsing and incubation with the secondary antibody were performed at the same temperature. Cultures were then fixed with paraformaldehyde and mounted with DAPI. Control stainings were performed using in the first step nonimmune mouse immunoglobulins (1 µg/ml).
For double immunofluorescence staining, cells were incubated first with
3C4 followed by goat anti-mouse immunoglobulins conjugated with
rhodamine; subsequently, they were reacted with anti-desmin followed by
swine anti-rabbit immunoglobulins conjugated with fluorescein and
preabsorbed with mouse nonimmune immunoglobulins to eliminate
interspecies cross-reactivity, as described previously
(25)
.
Cell proliferation assay
Cell proliferation assays were performed by the
5-bromo-2'-deoxy-uridine Labeling and Detection Kit III (Roche).
Briefly, 1 x 103 cells (this density was
chosen in order to avoid confluence) were seeded in 96-well culture
plates (Becton Dickinson) in 100 µl proliferation medium. 24 h
later, medium was changed; half of the cells were switched to
differentiation medium, while the remaining cells received fresh
proliferation medium. After 48 h, BrdU was added to the cells
(with the exclusion of blanks) at 10 µM final concentration and
incubated for additional 6 h. Thereafter, BrdU incorporation was
determined by anti-BrdU-POD Fab as indicated by the manufacturer.
Extinction of the samples was measured in a Bio-Rad Novapath Microplate
Reader, at 405 nm with a reference wavelength at 490 nm.
Cell surface biotinylation, precipitation, and blot assay
Cells were grown in proliferation medium to subconfluence in 10
cm petri dishes and then switched to differentiation medium for 48–72
h. After three rinses with ice-cold PBS, plates were incubated with 3
ml PBS containing 1 mg/ml of Sulfo-NHS-LC-biotin
[sulfosuccinimidyl-6-(biotinamido)hexanoate; Pierce, Rockford, Ill.]
for 1–3 min at RT. The plates were rinsed three times with cold
PBS and lysed with RIPA buffer (0.9 ml/plate of 1% Nonidet P-40, 0.5%
sodium deoxycholate in PBS) supplemented with antiprotease inhibitors
(0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml each of pepstatin,
leupeptin, and aprotinin; Sigma) and centrifuged. Supernatant was
precleared for 1 h at 4°C using 20 µl of anti-mouse
immunoglobulins conjugated with agarose (Sigma).
Equal amounts of total protein lysates (120 µg) were incubated overnight at 4°C with 100 µl of streptavidin-agarose (Pierce). The beads containing biotinylated proteins were rinsed four times with PBS in the presence of antiprotease inhibitors, then bound biotinylated proteins were eluted from the beads by boiling for 5 min in electrophoresis sample buffer and processed for gradient gel electrophoresis and Western blotting as described above. Cell lysates from nonbiotinylated samples were processed in parallel.
Statistical analysis
Quantitative densitometry was performed on Northern and Western
blots of samples obtained from normal and transfected C2C12 clones.
Autoradiographic and diaminobenzidine positive bands were analyzed
using a Shimadzu chromatoscanner CS-930 at wavelengths of 600 and 530
nm, respectively. Densitometric profiles were cut from paper and
weighed. For RNA samples, values were normalized to the corresponding
amount in 18S RNA. For Western blot experiments, where identical
amounts of sample total protein were used, variability among different
experiments was compensated using a sample from untransfected C2C12
cells as internal reference. Statistical analysis was performed
utilizing the unpaired Student’s t test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
.
GRP94 expression was then investigated in vitro using both
primary cultures from mouse newborn skeletal muscles and the mouse cell
line C2C12. Specificity of our rabbit antisense cRNA probe for mouse
grp94 mRNA was assessed by Northern blot analysis performed on C2 cell
total RNA, which shows hybridization with a single mRNA species of an
approximate size of 3000 nt (Fig. 1A, B
). Northern blot analysis shows that grp94 mRNA is
detectable in C2 myoblasts grown in proliferative medium and switching
to differentiation medium is accompanied with an ~75% increase in
grp94 mRNA (Fig. 1A, B
and Table 1
).
|
|
Western blot analysis performed with the monoclonal antibody 3C4, which
was raised using a recombinant rabbit GRP94 polypeptide
(5)
, decorates in C2C12 cells a single 99 kDa polypeptide
corresponding to mouse GRP94 (Fig. 1C
). Change in protein
accumulation detected in differentiating C2 myoblasts 48 h after
medium replacement parallels the increased accumulation of grp94 mRNA
(Fig. 1C, D
and Table 1
).
Immunohistochemistry performed on fixed permeabilized cells shows
strong punctate immunoreactivity of GRP94, which is often concentrated
in proximity of the nucleus, in both growing and differentiating C2 and
primary myoblasts (Fig. 2A, B
). In primary culture no apparent difference in the
cytoplasmic distribution of the staining can be observed between
myocytes and fibroblasts (Fig. 2B
, compare m with
f), the latter being desmin-negative in double
immunostaining (not shown). Positive immunoreactivity for GRP94 is also
observed on the surface of unfixed nonpermeabilized myoblasts (Fig. 2C, G
). Immunoreactivity has a granular appearance, which
appears to be distributed rather homogeneously. Strikingly, no apparent
immunoreactivity is detectable on cell surface of fibroblasts in
primary cultures (Fig. 2G
). Control labeling of unfixed,
nonpermeabilized C2 and primary skeletal muscle cells with nonimmune
mouse immunoglobulins does not reveal any reactivity (Fig. 2E
).
|
To confirm that the immunostaining of GRP94 at the cell surface
is not because of the presence of a cross-reactive protein, C2 cells
maintained in differentiation medium were biotinylated and labeled
proteins were precipitated with streptavidin-agarose. Western blotting
analysis shows that the streptavidin-bound fraction is enriched in
membrane proteins such as erb-B2 and also displays GRP94
immunoreactivity, whereas no apparent immunoreactivity for cytoplasmic
proteins like troponin I or T subunits is detectable in the
streptavidin-bound fraction (Fig. 3
).
|
Antisense GRP94 mRNA affects GRP94 expression and myotube formation
To investigate more directly the role of GRP94 in developing
muscle cells, C2C12 cells were stably transfected with a G418
permissive vector containing a 1,000 bp fragment of grp94 cDNA in
antisense orientation under the control of the RSV promoter. Control
clones were transfected with a construct containing the same grp94 cDNA
in sense orientation, which could be transcribed but not translated.
Relative GRP94 levels, quantified by Western blotting analysis, were
determined in five different antisense and in two control clones grown
in differentiation medium and normalized to GRP94 amounts observed in
untransfected C2 cells (Fig. 4
and Table 2
). Four out of five antisense clones show >40%
reduction in GRP94 amount, whereas no significant change is detectable
in the control clones. In every antisense clone displaying <60% GRP94
amount, no myotube formation is observed after permanence in
differentiation medium, despite the fact that cells were confluent
(Table 2
and Fig. 5
). Such a finding is still observed 12 days after switching to
differentiation medium. Conversely, already after 5 days in culture in
differentiation medium, both untransfected C2 cells, control clones,
and the antisense clone AS4D4, which contains near normal levels of
GRP94, display a consistent degree of fusion, cultures being
represented by a complete layer of myotubes (Fig. 5A
).
|
|
|
Furthermore, although growth of grp94 antisense clones in media
without G418 shows the appearance of rare myotubes that probably derive
from spontaneously revertant cells (not shown), no formation of
myotubes was observed after in vivo transfer of two
antisense clones (AS3A12 and AS1G11) (Fig. 6B
). These two clones were chosen together with the control
clone S1H2 because of the additional stable integration of the RSV-lacZ
construct. We injected 3 x 105 cells of
each clone in tibialis anterior of CH3/HeJ adult female mice. After
either 6 or 7 days, muscles were removed and stained for the
histochemical demonstration of ß-galactosidase. Blue myotubes could
be easily detected in muscles injected with the control clone (Fig. 6A
), conversely, only weakly ß-gal positive myoblasts were
detected in muscles injected with each antisense clones (Fig. 6B
).
|
Reduced GRP94 level does not interfere with myoblast cycle
withdrawal and differentiation
We then investigated whether other fundamental steps of skeletal
muscle cell differentiation such as cycle withdrawal and
muscle-specific gene expression are affected in GRP94 antisense clones.
In cell proliferation assays, BrdU incorporation after 48 h growth
in differentiation medium was measured for untransfected C2 cells, two
control clones (S1C8 and S1H2), and four grp94 antisense clones
(AS3A12, AS1G11, AS1F4, and AS4F1), and values were normalized to the
amount of BrdU incorporation in proliferation medium determined for
each clone. Switching to differentiation medium significantly reduced
BrdU incorporation in both control and grp94 antisense clones (Fig. 7)
.Thus, antisense clones are responsive to the well-known inhibitory
activity on DNA synthesis caused in skeletal muscle cells by serum
deprivation. Furthermore, immunolabelling with anti-p21 antibodies
shows that the large majority of antisense and control clone myoblasts
display positive nuclear p21 staining after 2 days of growth in
differentiation medium (Table 2)
.
|
The presence of differentiation was then evaluated considering the
decrease in integrin ß3 subunit (26)
, the increase in
desmin accumulation (27)
, and the expression of
muscle-specific genes, such as troponin T (22)
. After 4
days of exposure to differentiation media, no decrease in integrin ß3
subunit amount is observed in C2C12 cells (Fig. 8
), at variance with what has been reported for human differentiating
myoblasts (26)
. The same result is observed in antisense
(Fig. 8)
and control clones (not shown). Conversely, increased desmin
accumulation with respect to the levels displayed in proliferating
medium is observed in both untransfected C2 cells (Fig. 8)
, control
clones (not shown), and antisense clones. Furthermore, cardiac troponin
T, which is expressed by embryonic skeletal muscle (22)
,
accumulates in untransfected cells, antisense, and control clones only
after switching to differentiation medium (Fig. 8
and not shown).
Although variations in the total amount of desmin and cardiac troponin
T were observed in different antisense clones (compare clone AS1G11
with clones AS2A2 and AS3A12), no obvious relationship apparently
exists with the absence of fusion competence (see Table 2
).
|
Overexpression of GRP94 is accompanied by accelerated
myotube formation
We then investigated whether GRP94 overexpression
influences positively myotube formation. C2C12 cells were transfected
with the same G418 permissive vector modified in order to obtain the
expression of a mouse-rabbit GRP94 chimera protein, which lacks <10%
of the amino acid sequence of murine GRP94. Expression of recombinant
GRP94 was visualized in permeabilized cells by mean of the anti-VSV-G
epitope antibody (Fig. 9B
), and in three out of seven overexpressing clones GRP94
levels were significantly increased with respect to values observed in
the sense clone S1C8 (83D11, P<0.003; 81A9,
P<0.03; 85D9, P<0.005; Table 2
). In GRP94
overexpressing clones myotube formation can be occasionally observed
still in proliferation medium and even at low seeding density and is
greatly accelerated with respect to C2C12 cells after exposure to
differentiation medium (Fig. 9
compare A and D).
The presence of myotubes was evaluated counting the number of nuclei in
terminally differentiated myoblasts identified by immunofluorescence
staining for troponin T (28
, 29)
, in parallel cultures of
untransfected, control, and overexpressing clones seeded at the same
density (Table 3
). Data show that the percentage of myotubes is higher
in the GRP94 overexpressing clone 81A9, whereas no apparent difference
is detectable between control and untransfected C2 cultures.
|
|
Fusion competence is related to surface GRP94 expression
Immunofluorescence analysis of cell-surface GRP94 immunoreactivity
in grp94 antisense clones shows reduced signals with respect to
untransfected C2 cells (Fig. 10
); conversely, increased cell surface immunostaining is detectable on
GRP94 overexpressing clones (not shown). To evaluate more precisely
whether the relative amount of GRP94 localized at the cell surface in
antisense clones is significantly changed with respect to the amount
displayed by untransfected C2 cells, GRP94 immunoreactivity of
untransfected and two different grp94-antisense clones was quantified
after streptavidin-agarose fractionation of cell-surface biotinylated
samples. Results show that cell-surface GRP94 of both the
grp94-antisense clone AS3A12 (Fig. 11
) and clone AS1G11(not shown) is strongly reduced. Densitometric
analysis indicates an ~40-fold decrease in clone AS3A12 (2.5%±1.2
SE) with respect to untransfected C2C12 cells (98%±20.3
SE). An additional evidence underscores the possible direct
involvement of cell-surface GRP94 in muscle cell fusion. Untransfected
C2C12 cells were incubated in differentiation medium with the addition
of the anti-GRP94 antibody 3C4. Results show reduced myotube formation
in the presence of repeated addition (twice a day for three and a half
days) of 5–10 µg/ml of anti-GRP94 antibody, whereas no change in the
percentage of myotubes is detectable when different amounts of
nonimmune mouse immunoglobulins were added to parallel cultures
(Fig. 12
and Table 4
).
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. We now
provide evidence that this protein is directly involved in muscle
maturation, because a reduction in cell-surface GRP94 expression is
followed by hampered myotube formation. Indeed, this evidence adds a
new item to the long list of functions that have been attributed to
GRP94 and its cognate proteins (4)
and points to the fact
that GRP94 is necessary for some tissue-specific or definite biological
functions for which it can not be substituted by any other related
protein.
GRP94 expression and subcellular localization in skeletal myoblasts
The presence of GRP94 characterizes immature myofibers with
respect to adult skeletal myocytes in vivo (5)
,
and, as described by this study, switching from proliferative to
differentiation medium of untransformed C2 cells regulates positively
grp94 mRNA accumulation and protein synthesis. The relative delay with
which both GRP94 mRNA and protein accumulate after medium replacement
indicates that these changes are independent from a stress-response
secondary to serum deprivation; consistent with what is reported for
other cell types, GRP94 cellular levels are not apparently influenced
by medium composition, at variance with other ER proteins such as
protein-disulfide isomerase (30)
. Rather, changes in GRP94
levels appear to be related to defined functional requirements, such as
immunoglobulin production (30)
, thyroglobulin folding and
transport (31)
, and processing of lysosomal enzymes such
as 
-L-iduronidase (9)
. Furthermore, in the present
study we demonstrate GRP94 localization at the cell surface of cultured
muscle cells by means of immunofluorescence and separation of
biotinylated cell-surface GRP94. Such a localization has been
convincingly shown for GRP94 only in neoplastic cells
(32)
, and it appears to be shared with other ER resident
proteins such as calreticulin (33)
. It is known that the
retention signal present at the carboxyterminus of many ER resident
proteins may be overcome in stress conditions and followed by secretion
of the protein (34)
. However, this does not appear to be
the case for our primary skeletal muscle cultures; in fact, if GRP94 is
secreted, labeling would be detected on the surface of any cell. Such a
possibility does not explain why muscle fibroblasts, which also contain
GRP94 in the ER (this work and ref 5
), do not display any
signal for GRP94 on the outer cell membrane. The possibility exists
that post-translational modifications such as
NH2-terminal or carboxyl-terminal truncations may
favor escape from the ER (1)
; however, biotinylated GRP94
did not apparently differ in molecular size from the intracellular
form. Although the precise mechanism that regulates intracellular GRP94
redistribution remains to be determined, our results indicate that both
GRP94 amount and targeting at the cellular surface appear to be crucial
for differentiating muscle cells.
GRP94 expression and muscle differentiation
Myotube formation represents the final step of a highly ordered
sequence of events that occur during myogenesis, beginning with cell
cycle withdrawal of proliferative myoblasts and followed by the
sequential activation of muscle-specific genes (28)
. Our
results show that the reduction in GRP94 amount observed in grp94
antisense clones does not apparently affect any of these stages, except
the formation of multinucleated myotubes. No significant difference in
cell cycle arrest, measured as the relative decrease in
bromodeoxyuridine incorporation, was observed between grp94 antisense
and control clones or untransfected C2C12 cells. Similarly, no
difference was observed in the percentage of the cells expressing
nuclear localization of p21 after 2 days of permanence in
differentiation medium. The lack of myotube formation is certainly the
more common evidence reported when describing the effects of
engineering antisense muscle-specific cDNAs in muscle cells in
vitro, like desmin (27)
, or of knocking-out
muscle-regulatory genes in vivo, like myogenin (35
, 36)
. In both cases, hampered myotube formation is associated
with the impairment of muscle-specific gene expression, an event that
should precede myoblast fusion (28)
. After switching from
proliferation medium to differentiation medium, both control and
antisense GRP94 clones display signs of muscle-specific gene
activation, like the increase in accumulation of desmin
(27)
and the expression of cardiac troponin T
(22)
, albeit to variable extent. Conversely, antisense
GRP94 clones showing a significantly reduced GRP94 amount do not form
multinucleated myotubes either in vitro or in
vivo despite of being confluent. If normal GRP94 levels are
necessary for the maintenance of fusion competence, the increase of
GRP94 obtained in overexpressing clones is conversely associated with
accelerated myotube formation, which could be observed even at low
seeding density.
Involvement of cell-surface GRP94 in muscle cell fusion
Another compelling piece of evidence concerning the involvement of
GRP94 in the preservation of fusion competence is provided by the
dramatic decrease of cell-surface GRP94 in antisense clones in
comparison with control clones. Furthermore, the experiment that shows
delayed myotube formation of untransfected C2 cells cultured in the
presence of anti-GRP94 antibody strongly suggests a direct involvement
of cell-surface GRP94 in myotube formation. Although the molecular
details of the surface attachment of GRP94 are still unknown, GRP94
stays there either through hydrophobic interactions (1)
or
in connection with transmembrane proteins, some of which could also
participate to myoblast fusion. Thus, our results indicate cell-surface
GRP94 as a necessary component for myoblast fusion both in
vitro and in vivo.
Such a conclusion may be weakened by the role played by GRP94 as
molecular chaperone. Indeed, the decrease in GRP94 amount may have
consequences on protein folding, favoring protein retention within the
ER and eventually influencing the turn-over of unfolded proteins as
well as gene expression. However, the effects on myoblast fusion
because of decreased GRP94 expression cannot be explained with reduced
externalization of N-cadherin or ß1 integrin,
because in vitro knock-out of N-cadherin and
null-ß1 integrin muscle cells in chimeric mice
have proven not to perturb muscle cell fusion (37
, 38)
.
Similarly, changes in GRP94 amount could affect intracellular transport
of proteases such as calpain, -meltrin, or cathepsin B
(39
40
41)
, which exert their proteolytic activity on the
extracellular matrix and are mechanistically involved in myotube
formation. However, our in vivo transplantation experiments
showed no appearance of myotubes from grp94 antisense clones indicating
that environmental cues cannot rescue the grp94 antisense phenotype. In
any case, the role played by GRP94 as a molecular chaperone does not
appear to be compensated by other ER proteins. In contrast to what one
would expect as a consequence of the unfolded protein response,
previous observations showed that reduced up-regulation of GRP78
accompanies the lack of GRP94 increase after exposure to stress of a
tumor cell line transfected with a grp94 targeted-ribozyme
(9)
. Comparable evidence for reduced GRP94 levels was
described in cells engineered with grp78 antisense vectors
(42)
. Also, our grp94 antisense clones show a parallel
reduction in the amount of GRP78 (L. Gorza and M. Vitadello,
unpublished results); however, our result showing decreased myotube
formation after exposure to anti-GRP94 antibody rules out a possible
role for GRP78 in muscle fusion. Furthermore, at variance with GRP94,
GRP78 expression in skeletal muscle fibers does not vary during
development to adulthood (5
, 6)
. Although adult skeletal
myofibers can fuse with transplanted myoblasts, as previously reported
(43)
, here we show that adult skeletal myofibers, which
express GRP78 but not GRP94, can not rescue fusion of grp94 antisense
myoblasts.
The direct involvement of GRP94 in myoblast fusion implies a
ligand-binding function of this protein. Recent evidence obtained
investigating immune response against tumors indicates that
externalized GRP94 can interact with cell receptors on dendritic cells,
which internalize GRP94-peptide complexes and process them to the class
I antigen pathway, probably using a mannose receptor that binds to the
high mannose content of GRP94 (11
, 12)
. Although we have
not identified so far a partner for GRP94 involved in myotube
formation, there is evidence that skeletal muscle cells express a
mannose receptor, which corresponds to the insulin-like growth factor
II receptor and appears to mediate the uptake of lysosomal enzymes,
such as N-acetylgalactosamine 4-sulfatase and acid alpha-glucosidase,
as shown by experiments with skeletal myoblasts engineered to correct
lysosomal storage diseases (44
, 45)
.
In conclusion, our data reveal a new and unexpected role for GRP94 in muscle cell maturation, because reduction in GRP94 expression deeply influences myoblast competence for fusion; furthermore, we identify the cell surface as the crucial site where GRP94 participates in myotube formation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
vß3 integrin levels are controlled by transcriptional regulation of expression of the ß3 subunit and down-regulation of the ß3 subunit expression is required for skeletal muscle cell differentiation. Dev. Biol. 184,266-277[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. V. Pajcini, J. H. Pomerantz, O. Alkan, R. Doyonnas, and H. M. Blau Myoblasts and macrophages share molecular components that contribute to cell-cell fusion J. Cell Biol., March 5, 2008; 180(5): 1005 - 1019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wanderling, B. B. Simen, O. Ostrovsky, N. T. Ahmed, S. M. Vogen, T. Gidalevitz, and Y. Argon GRP94 Is Essential for Mesoderm Induction and Muscle Development Because It Regulates Insulin-like Growth Factor Secretion Mol. Biol. Cell, October 1, 2007; 18(10): 3764 - 3775. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. TARRICONE, A. GHIRARDELLO, S. ZAMPIERI, R. M. ELISA, A. DORIA, and L. GORZA Cell Stress Response in |
HindIII DNA markers
(Pharmacia) is indicated on the left. B) A region of the
same blot hybridized for 18S RNA. C, D) Western blots
with anti-GRP94 monoclonal antibody 3C4 and Coomassie blue-stained
gels, respectively, of whole homogenates of C2C12 cells maintained in
proliferation medium (p) and for 48 h in differentiation medium
(d). Migration of protein Mr standard is
indicated on the right (200,000, 116,250, 97,400, 66,200, 45,000;
Bio-Rad).
