Skeletal muscle cell hypertrophy induced by inhibitors of
metalloproteases; myostatin as a potential mediator
Clotilde
Huet1,2,
Zhi-Fang
Li1,
Hai-Zhen
Liu1,
Roy A.
Black3,
Marie-Florence
Galliano1,4, and
Eva
Engvall1
1 The Burnham Institute, La Jolla, California 92037;
2 Institut National de la Santé et de la Recherche
Médicale-Institut National de la Recherche Agronomique U418,
Communications Cellulaires et Différenciation, Hôpital
Debrousse, 69322 Lyon, France; 3 Research Administration,
Immunex Corporation, Seattle, Washington 98101; and 4 Centre
National de la Recherche Scientifique UPR 2163, Physiologie
Moléculaire et Cellulaire, Centre Hospitalier Universitaire
Purpan, 31059 Toulouse Cedex 03, France
 |
ABSTRACT |
Cell growth
and differentiation are controlled in many tissues by paracrine
factors, which often require proteolytic processing for activation.
Metalloproteases of the metzincin family, such as matrix
metalloproteases and ADAMs, recently have been shown to be involved in
the shedding of growth factors, cytokines, and receptors. In the
present study, we show that hydroxamate-based inhibitors of
metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion
of C2C12 myoblasts and provoke myotube
hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that
have previously been shown to regulate muscle growth in vitro, such as
insulin-like growth factor-I, calcineurin, and tumor necrosis factor-
. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in
C2C12 cells treated with HIMPs, as suggested by
the presence of nonprocessed myostatin precursor only in hypertrophic
myotubes. Myostatin is a known negative regulator of skeletal muscle
growth, belonging to the transforming growth factor-
/bone
morphogenetic protein superfamily. These results indicate that
metalloproteases are involved in the regulation of skeletal
muscle growth and differentiation, that the proteolytic maturation of
myostatin in C2C12 cells may be directly or
indirectly linked to the activity of some unidentified HIMP-sensitive
metalloproteases, and that the lack of myostatin processing on HIMP
treatment may be a mediator of myotube hypertrophy in this in vitro model.
metalloendopeptidases; protease inhibitor; growth and
differentiation factor-8
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INTRODUCTION |
DIFFERENTIATION AND
REGENERATION of skeletal muscle are controlled by many factors,
including insulin-like growth factors (IGFs), hepatocyte growth factor,
fibroblast growth factor-2, platelet-derived growth factor, and some
members of the transforming growth factor-
(TGF-
) superfamily
(reviewed in Refs. 13, 17, and 25). The
biological activity of many such growth and differentiation factors is
regulated by proteolysis, such as shedding of membrane-bound growth
factors and their receptors (42, 45), activation of secreted latent growth factors (46), and degradation of
growth factor binding proteins (12). Metalloproteases are
known to regulate many developmental events (for review see Refs.
49, 66, and 75). In particular, members
of the ADAM (a disintegrin and metalloprotease) family of
membrane-bound metalloproteases are believed to activate growth factors
and cytokines (for review see Refs. 6, 7, and
62), including tumor necrosis factor-
(TNF-
) (5, 44,
54, 59), TNF-
receptor II, L-selectin, TGF-
,
heparin-binding epidermal growth factor (26), Notch
receptor (8, 52) and its ligand delta (56),
amyloid protein precursor (9, 30, 31, 70), and IGF binding
protein-3 (68).
The C2C12 cell line of mouse myoblasts derived
from satellite cells is frequently used to study skeletal muscle
differentiation in vitro and is considered to be a good model for
myogenesis and muscle regeneration. High fetal calf serum concentration
is used to keep C2C12 cells in an
undifferentiated, proliferating myoblast stage, but in low
concentration of horse serum, C2C12 cells stop proliferating and differentiate into multinucleated myotubes. This
indicates that high concentrations of factors present in the bovine
serum, probably including TGF-
, exert an inhibitory effect on
skeletal muscle cell differentiation and that muscle differentiation
requires finely tuned regulation of growth factor activation, possibly
involving proteases. In the present study, we investigated the role of
metalloproteases in skeletal muscle cell differentiation. Little is
known about the possible role of metalloproteases in modulating
differentiation factors in skeletal muscle cells. An exception is ADAM
12, also called meltrin-
, which has been shown to affect skeletal
muscle differentiation (78). ADAM 12 appears to play a
dual role in myogenesis; its disintegrin and cysteine-rich domains are
promyogenic (20, 78), whereas an antimyogenic activity is
observed when its metalloprotease domain is present (78).
However, other ADAMs and metalloproteases are expressed in
C2C12 cells (19), and ADAM 12 may
not be the only metalloproteases involved in differentiation of
C2C12 cells.
We show here that hydroxamate-based inhibitors of metalloproteases
(HIMPs), added at an early stage of differentiation, stimulate muscle
cell fusion and myotube growth and ultimately provoke myotube hypertrophy. HIMP treatment was accompanied by a reduction in the
proteolytic maturation of myostatin, a member of the TGF-
/bone morphogenetic protein (BMP) superfamily and known as a negative regulator of myogenesis. Thus our results indicate that the inhibition of metalloproteases by HIMPs triggers skeletal muscle hypertrophy by a
mechanism yet to be determined but that seems to involve a lack of
myostatin processing.
 |
MATERIALS AND METHODS |
Reagents.
HIMPs, IC-3 [also called TAPI (tumor necrosis factor-
processing
inhibitor)], and BB-3103 were obtained from Immunex (Seattle, WA) and
British Biotech (Oxford, UK), respectively. Stock solutions of TAPI and
BB-3103 were prepared at 5 mM in water and DMSO, respectively. GM-6001
(Ilomastat) was purchased from Chemicon (Temecula, CA) and stored at
2.5 mM in DMSO. 1-10-Phenanthroline was purchased from Sigma and
stored at 200 mM in DMSO. Cyclosporin was purchased from Calbiochem (La
Jolla, CA) and stored at 42 mM in ethanol. TNF-
was obtained from
Calbiochem and stored at 10 µg/ml in PBS containing 0.1% BSA.
Culture of C2C12 cells.
C2C12 cells (American Type Culture Collection,
Manassas, VA) were cultured on gelatin-coated dishes in proliferation
medium composed of DMEM, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 U/ml penicillin and containing 10% fetal calf serum. When cells reached confluence (day 0),
differentiation was induced by culture in differentiation medium
composed of DMEM, glutamine, sodium pyruvate, streptomycin, and
penicillin and containing 2% horse serum. The medium was changed every
1 or 2 days depending on the type of experiment.
Quantification.
For morphological analysis and measurement of diameter of myotubes,
cells were fixed with 0.5% glutaraldehyde, permeabilized with 0.1%
Triton X-100, and stained with hematoxylin and eosin. For
quantification of nuclei, cells were fixed with 4% paraformaldehyde and stained with eosin and Hoechst 33258. 1) To quantify the
effect of HIMPs on myotube size throughout differentiation, we
attempted to measure the maximum myotube diameter reached in control
and HIMP-treated conditions at various stages of culture. We randomly selected 10 microscopic fields from 3 independent culture wells, and
the diameter of the 4 largest myotubes in each field (40 myotubes in
total) was measured (Fig. 1B). 2) To define
myotubes according to their number of nuclei, 5 microscopic fields were
randomly selected from 3 independent culture wells, and the number of
nuclei per myotube was measured in 20 randomly selected myotubes per field (100 myotubes in total). Myotubes were then defined on the basis
of the number of nuclei (2-9, 10-19, 20-29, 30-49,
55-99, and >100). Data (Fig. 4A) are shown as the
percentage distribution of myotubes in control and HIMP-treated
cultures. 3) The cultures also were characterized by how
many nuclei could be maximally found in myotubes after various
treatment protocols. The number of nuclei in the 10 apparently largest
myotubes found in each of 3 culture wells (30 myotubes in total) was
measured (Fig. 4B). Statistical analyses of the results of
quantification were performed by one-way ANOVA followed by the
Newman-Keuls multiple comparison test. Results were considered
significantly different when P < 0.05.

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Fig. 1.
Effect of hydroxamate-based inhibitors of
metalloproteases (HIMPs) on C2C12 cell
differentiation. A: tumor necrosis factor- processing
inhibitor (TAPI) induces C2C12 myotube
hypertrophy. Parental C2C12 cells were cultured
as described in MATERIALS AND METHODS. After
differentiation was induced at day 0, cells were treated
with various concentrations of TAPI from day 2. Cells were
fixed at day 7 and stained with hematoxylin and eosin. Scale
bar, 100 µm. B: BB-3103 increases the diameter of
myotubes. C2C12 clone 18 cells were
cultured as described in MATERIALS AND METHODS. After
differentiation was induced at day 0, cells were treated
with DMSO or various concentrations of BB-3103 from day 1.
Cells were fixed and stained at different stages of differentiation,
and the diameter of the 4 largest myotubes present within 10 fields was
measured. Data represent means ± SD (n = 40).
*P < 0.05; **P < 0.001, significant
differences between DMSO- and BB-3103-treated cells at the same
stage.
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Generation of C2C12 cell clones.
Parental C2C12 cells were grown at very low
density to generate colonies. Cells from individual colonies were
collected with a pipette tip and cultured in separate microplate wells.
Eighteen individual clones were expanded and used up to four passages.
Gelatin and casein substrate zymography.
Ten microliters of culture medium from differentiating
C2C12 cells were subjected to SDS-PAGE in 10%
gelatin zymogram or 12% casein zymogram gels (Novex; Invitrogen,
Carlsbad, CA) under nonreducing conditions at 4°C. After
electrophoresis, gels were incubated in 2.5% Triton X-100 in distilled
water for 1 h at room temperature to renature proteins. Gels were
then incubated overnight at 37°C in 50 mM Tris · HCl (pH 7.7)
containing 5 mM CaCl2 to allow proteinases to digest the
substrate in the gel. Gels were then stained with Coomassie blue
(G-250). Finally, gels were rinsed in 30% methanol-5% glycerol and
dried. Areas of proteolysis appeared as clear zones against a blue background.
Ligand blotting with biotinylated IGF-I.
Recombinant human IGF-I (rhIGF-I; kindly provided by Dr. P. Monget,
Nouzilly, France) was biotinylated as previously described (18,
51). Briefly, 0.1 mg of rhIGF-I was incubated for 1 h at
room temperature with 0.32 mg of EZ-Link sulfo-NHS-LC-biotin (Pierce,
Rockford, IL) in a total volume of 100 µl of 0.23 M
NaHCO3 (pH 9.2). The reaction was stopped by adding 200 µl of 1M Tris (pH 7.4). Biotinylated IGF-I was dialyzed against PBS
containing 0.05% sodium azide for 24 h in Slide Lyser CO 3,500 (Pierce). Biotinylated IGF-I was then stored at 4°C at a
final concentration of 60 µg/ml in PBS containing 1% BSA.
Ten microliters of medium, conditioned by C2C12
cells for 48 h, were subjected to SDS-PAGE in 16% polyacrylamide
gels under nonreducing conditions. Proteins were transferred onto a
nitrocellulose filter. Filters were washed with Tris-buffered saline
(TBS), and proteins were renatured for 30 min in TBS-3% Nonidet P-40
(NP-40) at room temperature. Filters were then treated with
TBS-0.1% Tween 20 (TBS-T) containing 1% BSA for 1 h at room
temperature, washed with TBS-T, and incubated with biotinylated rhIGF-I
(0.6 µg/ml) in TBS-T for 1 h at room temperature. After being
washed with TBS-T, filters were incubated with horseradish peroxidase
(HRP)-coupled Immunopure NeutrAvidin (Pierce) diluted 1:5,000 in TBS-T
for 45min at room temperature. Filters were washed extensively, and the signal was detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) on XAR Kodak autoradiography film.
Immunoblotting.
Skeletal muscle tissues (tibialis anterior, soleus, and plantaris) were
collected from an adult IRC male mouse and homogenized with a
Tissue-Tearor (Biospec Products, Bartlesville, OK) in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). C2C12 cells
were lysed on ice in lysis buffer and collected with a cell scraper.
Samples were briefly centrifuged, and the supernatant was stored at
20°C until use. Protein concentration in each lysate was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). Samples were
subjected to 4-20% gradient SDS-PAGE (Novex; Invitrogen) under
reducing conditions. After electrophoresis, proteins were transferred
onto nitrocellulose membranes. Membranes were treated for 1 h at
room temperature with TBS containing 10% nonfat dry milk, incubated
for 1 h at room temperature with a rabbit antiserum directed
against myostatin NH2-terminal portion (kindly provided by
Dr. James G. Tidball) or a goat antiserum directed against myostatin
COOH-terminal portion (C-20; Santa-Cruz Biotechnology, Santa Cruz,
CA) diluted 1:500 in TBS containing 1% BSA, and finally incubated for
1 h at room temperature with HRP-conjugated goat anti-rabbit IgG
or rabbit-anti-goat IgG antibody (Calbiochem) diluted 1:2,000 in TBS
containing 10% nonfat dry milk. The signal from ECL (Amersham
Pharmacia Biotech) was detected on XAR Kodak autoradiography film.
 |
RESULTS |
HIMPs induce hypertrophy of C2C12 myotubes.
To test the possible involvement of metalloproteases in skeletal muscle
cell proliferation and differentiation, we cultured C2C12 cells in the presence of various
concentrations of HIMPs. Treatment of differentiating
C2C12 cells with TAPI or BB-3103 had a dramatic
effect on myoblast fusion and myotube morphology. Myotubes resulting
from C2C12 cells differentiating in the
presence of TAPI or BB-3103 (added from day 2 to day
7) were much larger (hypertrophic) and exhibited frequent
branching (Fig. 1A). The effects of TAPI and BB-3103 were indistinguishable from one another and
were dose dependent, with a maximum effect at 5-15 µM and no
effect at 0.15 µM or lower concentrations. Treatment with DMSO (0.1%
vol/vol) had no effect. Quantification showed that BB-3103 dramatically
increased the diameter of C2C12 myotubes during
differentiation (Fig. 1B). GM-6001, another HIMP, also
induced C2C12 myotube hypertrophy at 5 µM
(not shown). The zinc chelator 1-10-phenanthroline, an inhibitor
of metalloproteases with broad specificity, was also tested, but the
results obtained were inconclusive because of its lower specific
activity and higher toxicity to C2C12 cells.
Treatment of subconfluent C2C12 myoblasts with
TAPI (not shown) or BB-3103 up to 5 µM in proliferation medium had no
significant effect on C2C12 number or on
C2C12 cell survival (Fig.
2).

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Fig. 2.
Effect of BB-3103 on proliferation and survival of
C2C12 cells. C2C12
clone 18 cells were cultured in 96-well dishes at low
density in proliferation medium containing DMSO or various
concentrations of BB-3103 for 2 days. After trypsinization, the number
of live cells (open bars) and dead cells (solid bars) per well was
determined in 6 independent culture wells. Cell viability was assessed
with trypan blue dye. Data represent means ± SD
(n = 6).
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HIMPs enhance myoblast fusion.
As previously described by us and others, C2C12
cells are genetically unstable and usually consist of a heterogeneous
population of cells. Cell lines with different fusion abilities and
myotube shapes can be obtained by cloning (74). We took
advantage of this variation in C2C12 cells to
investigate the response to TAPI and BB-3103 of 18 clonal cell lines
derived from the parental cells. As expected, the 18 cell lines showed
large variation in their ability to fuse in the differentiation medium
and also variation in the shape of the resulting myotubes. We divided
the 18 clones into 5 groups, each characterized by a distinct fusion
phenotype: fusion incompetent (n = 1), fusion
inefficient (n = 6), fusion competent with normal
myotube shape (n = 5), fusion competent with myosacs
(n = 2), fusion competent with thinner myotubes
(n = 4). When treated with HIMPs, cells from
the clones within each group responded in a characteristic and
reproducible manner, as described below. Clonal cells with fusion
capacity similar to the parental C2C12 cells
(fusion competent with normal myotube shape) responded to HIMPs in a
way similar to the parental C2C12 cells. There
was a significant acceleration in fusion during the first few days of
differentiation and hypertrophy of the resulting myotubes at a later
stage. Cells from this group differentiated even faster and fused even
more efficiently than the parental cells, probably because of the
homogeneity of the cloned cells. In this group, clone 18 was
chosen for later experiments (see below). Clonal cells with inefficient
fusion also responded to HIMPs by an increase in fusion efficiency,
characterized by the appearance of a larger number of multinucleated
cells and an increase in the size of the myotubes (Fig.
3, clone 6). Clonal cells
showing a defect in myotube elongation (fusion competent with myosacs) responded to HIMPs by an increase in both fusion and elongation, as
shown by an increase in size and length of the multinucleated cells
(Fig. 3, clone 17). Cells from one clone with a complete inability to fuse (fusion incompetent) showed no response to HIMPs (Fig. 3, clone 8). Finally, some clonal cells with
abnormally thin myotubes (fusion competent with thinner myotubes) did
not respond to treatment with HIMPs (Fig. 3, clone 2).

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Fig. 3.
Effect of BB-3103 on the differentiation of
C2C12 clonal cell lines.
C2C12 clonal cell lines were obtained and
cultured as described in MATERIALS AND METHODS. After
differentiation was induced at day 0, cells were treated
with DMSO or 5 µM BB-3103 from day 1. Images show cells
after 6 days of differentiation. Bar, 100 µm.
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Noting that HIMPs increased the fusion efficiency of
C2C12 clonal lines with inefficient fusion, we
subsequently investigated whether myotube hypertrophy was associated
not only with an increased diameter of myotubes but also with an
increase in the number of nuclei per myotube. In these experiments, we
used the C2C12 clone 18 cells, which
are more homogeneous but differentiate in a manner similar to parental
C2C12 cells. Figure
4A shows the range of nuclei
within myotubes in control and HIMP-treated conditions at day
5 of differentiation. The results clearly show that HIMP treatment
is accompanied by an increase in the number of nuclei per myotube.

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Fig. 4.
Effect of BB-3103 on cell fusion. A: BB-3103
increased the number of nuclei per myotube.
C2C12 clone 18 cells were cultured
as described in MATERIALS AND METHODS. After
differentiation was induced at day 0, cells were treated
with DMSO (open bars) or 5 µM BB-3103 (filled bars) from day
1. Cells were fixed and stained at day 5. The number of
nuclei per myotube was measured on 20 myotubes randomly selected in 5 fields (n = 100). B: the effect of BB-3103
on C2C12 cell fusion is stage and time
dependent. C2C12 clone 18 cells were
cultured as described in MATERIALS AND METHODS. After
differentiation was induced at day 0, cells were treated
with DMSO or 5 µM BB-3103 at different stages of differentiation and
for various periods of time. Cells were fixed and stained at day
5. The number of nuclei per myotube was measured in the 10 largest
myotubes of 3 independent culture wells. Data represent means ± SD (n = 30). *P < 0.05;
**P < 0.001, significant difference between DMSO- and
BB-3103-treated cells. Data in A and B correspond
to separate culture experiments.
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We then investigated whether the hypertrophy observed at late stages of
differentiation resulted from an early increase in fusion at the onset
of differentiation. Cells were treated with 5 µM BB-3103 at different
stages of differentiation and for variable periods of time, and the
resulting hypertrophy was assessed on day 5 by measuring the
number of nuclei within the largest myotubes (Fig. 4B).
Continuous presence of BB-3103 (from day 1 to day
5) provoked a dramatic hypertrophy of the myotubes on day
5 (see Fig. 7, control). The average number of nuclei within the
hypertrophic myotubes at day 5 was more than threefold
higher than in the control myotubes at the same stage (Fig.
4B). When the cells were treated with BB-3103 from day
2, 3, or 4 and up to day 5,
myotubes showed progressively less hypertrophy on day 5 (not
shown), and the number of nuclei per myotube on day 5 decreased (Fig. 4B). A short and early treatment with
BB-3103, started on day 1 and terminated on day
2, 3 or 4, appeared to be sufficient to
produce hypertrophic myotubes on day 5. These results
indicate that the hypertrophy induced by BB-3103, and most obvious at
day 5 of differentiation, may result from events triggered
by BB-3103 at early stages of differentiation.
Possible targets of HIMPs.
HIMPs are efficient inhibitors of metalloproteases of the metzincin
family including matrix metalloproteases (MMPs) and ADAMs. They act by
chelating the zinc within the catalytic site of these enzymes. We
previously showed that C2C12 cells express
several ADAMs, including ADAM 9, 10, 12, 15, 17, and 19 (19). We now tested for expression of MMPs in
C2C12 cells. Differentiated
C2C12 clone 18 cells expressed two
gelatinases of 96 and 75 kDa, likely corresponding to MMP-9 and MMP-2.
These two gelatinases are mainly present as precursors, as shown in
gelatin gel zymography (Fig. 5). No
changes in the level of expression or in the activation state of MMP-2 and MMP-9 were observed in the culture medium of
C2C12 cells regardless of treatment with HIMPs
(Fig. 5). Casein gel zymography did not reveal any band (not shown),
suggesting that differentiated C2C12 clone 18 cells do not express MMP-1, MMP-3, MMP-7, MMP-10,
or plasminogen activators in their medium.

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Fig. 5.
Detection of gelatinases in the medium of
C2C12 cells. Parental
C2C12 cells were cultured and either treated or
not with 5 µM TAPI from day 1 to day 5.
Gelatinases secreted into the culture medium between day 3 and day 5 were analyzed by gelatin-substrate zymography. Two
major bands were detected at 96 and 75 kDa, representing the latent
forms of matrix metalloprotease (MMP)-9 and MMP-2, respectively.
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Mechanism of action of HIMPs in C2C12 cell
differentiation.
To elucidate the mechanisms by which HIMPs increase
C2C12 cell fusion and provoke myotube
hypertrophy, we investigated whether some of the factors known to be
involved in muscle hypertrophy or atrophy could be mediating the effect
of HIMPs.
IGFs play a critical role in skeletal muscle growth and differentiation
(17) and have been implicated in skeletal muscle hypertrophy both in vivo (15, 34) and in vitro (48,
64, 65). Recent reports have shown that IGF-I-transfected
myoblasts differentiate into hypertrophic myotubes in a
calcineurin-dependent manner (47, 48, 65) reminiscent of
the pathway involved in cardiac muscle hypertrophy (43,
71). ADAM 12 (68) and other metalloproteases
(32) have been shown recently to have the capacity to
cleave the IGF binding proteins (IGFBPs) and may thus regulate the
bioavailability of IGF-I. Therefore, we investigated whether treatment
with HIMPs would be associated with changes in levels of IGFBPs in the
culture medium of differentiating C2C12 cells. As shown in Fig. 6,
C2C12 cells expressed high amounts of
IGFBP-5 and small amounts of IGFBP-4 at late stage of differentiation. Fetal calf serum also contained IGFBPs, mainly IGFBP-2 but
also some IGFBP-3 and IGFBP-4. Unidentified high-molecular-weight
binding proteins were found in both fetal calf serum and horse serum. No significant changes in the levels of any of these IGFBPs were detected in the medium of C2C12 cells, treated
or untreated with TAPI.

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Fig. 6.
Detection of insulin-like growth factor-I (IGF-I) binding
proteins (IGFBP) in the medium of C2C12 cells.
Media were subjected to 16% SDS-PAGE, and IGFBPs were detected by
ligand blotting with biotinylated recombinant human IGF-I prepared as
described in MATERIALS AND METHODS. Left:
nonconditioned differentiation and proliferation media. HS, horse
serum; FCS, fetal calf serum. Right: media conditioned by
C2C12 cells for 2 days and collected at
day 0, 1, 3, and 7. Three
bands at 40, 35, and 24 kDa were detected in proliferation media
containing 10% FCS, likely corresponding to IGFBP-3, IGFBP-2, and
IGFBP-4, respectively. Media conditioned by
C2C12 cells also contained a major band at 29 kDa and a minor band at 24 kDa, likely corresponding to IGFBP-5 and
IGFBP-4, respectively. Two additional high-molecular-weight bands
corresponding to unidentified binding proteins (unlabeled arroweads)
were detected in all differentiation media containing 2% HS.
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We then tested the effects of IGF-I and the calcineurin inhibitor
cyclosporin A (CsA) in our system (Fig.
7). IGF-I at 25 ng/ml
did not provoke hypertrophy when added to C2C12
cells at day 1 of differentiation, in agreement with
previous reports (64, 65). In addition, IGF-I did not
affect the BB-3103-induced hypertrophy at this concentration. In the
presence of 250 ng/ml of IGF-I, the number of myotubes increased.
However, this effect was not inhibited by 1 µM CsA. In fact, CsA by
itself induced a moderate hypertrophy of C2C12
myotubes when used at 1 µM (Fig. 7) and 10 µM (not shown), and
BB-3103 had an additive effect on the size of hypertrophic myotubes.
Together, these results suggest that HIMP-induced hypertrophy does not
involve an IGF pathway and occurs in a calcineurin-independent manner.

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Fig. 7.
Effect of BB-3103, cyclosporin A (CsA), IGF-I, and
tumor necrosis factor- (TNF- ) on C2C12
clone 18 differentiation. C2C12
clone 18 cells were cultured as described in MATERIALS
AND METHODS. Cells were treated with the indicated combinations
of BB-3103 (5 µM), CsA (1 µM), IGF-I (25 or 250 ng/ml), and TNF-
(1 ng/ml) from day 1. Cells were fixed and stained with
hematoxylin and eosin at day 5. Bar, 100 µm.
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TNF-
has been shown previously to reduce
C2C12 cell differentiation and to induce
protein loss in muscle in vitro (35, 41) and in vivo
(22, 50). Because TNF-
is activated by HIMP-sensitive
metalloproteases such as ADAM 10 (59), ADAM 17 (5, 44), and MMP-17 (16), inhibition of these ADAMs
could block the activation of endogenous TNF-
, thereby obliterating
its negative effect on C2C12 cell
differentiation and myotube growth. Supplying soluble active TNF-
would thus be expected to counteract the effect of HIMPs. In our
system, TNF-
induced widespread apoptosis at 10 ng/ml, and
when used at subapoptotic doses such as 1 ng/ml, TNF-
did not
inhibit the effect of BB-3103 on myotube hypertrophy (Fig. 7).
Moreover, although skeletal muscle is a source of TNF-
in humans
(60), the levels of TNF-
produced by
C2C12 cells were undetectable by ELISA (not
shown). Together, our data suggest that TNF-
does not play any role
in HIMP-induced C2C12 myotube hypertrophy.
We next investigated whether myostatin, another known negative
regulator of muscle growth that requires proteolysis for its activation, could be involved in our hypertrophy model. Absence of
functional myostatin (4, 21, 33, 39, 40) or overexpression of a dominant negative form of myostatin (81) has indeed
been shown to provoke skeletal muscle hypertrophy in vivo. A reduction of the amount of active myostatin in our system would be expected to
provoke myotube hypertrophy. Expression analysis by cDNA array (not
shown) indicated that myostatin was indeed expressed in differentiating C2C12 cells and that its level of expression
was not significantly different in HIMP-treated
C2C12 cells compared with untreated cells.
Therefore, we investigated whether the proteolytic processing of
myostatin would be affected upon treatment with HIMPs. As with other
members of the TGF-
/BMP superfamily, myostatin is synthesized as a
50-kDa precursor protein. During its maturation, a proteolytic process
generates a COOH-terminal 15-kDa peptide corresponding to the bioactive
growth factor and a 37-kDa fragment of unknown function also called
latency-associated peptide (LAP) (39). Immunoblotting
analysis of myostatin in cells indicated that myostatin was produced
and fully processed in C2C12 cells and in
various mouse muscles, as shown by the presence of the 37-kDa LAP in
cell and tissue lysates (Fig. 8).
Notably, the expression of LAP was high in undifferentiated
C2C12 cells, decreased at the onset of differentiation, and progressively increased as differentiation progressed. This result agrees with a recent RT-PCR analysis of myostatin expression in C2C12 cells
(57) and with the pattern of expression of myostatin
previously described during embryonic muscle development in the chicken
(29). Interestingly, whereas the 50-kDa myostatin
precursor was undetectable in control C2C12 cells as an indicator of a complete constitutive processing, a result
that also agrees with recent data from Rios et al. (57), the precursor was clearly present in C2C12
cells treated with BB-3103 as shown with antisera directed against both
the COOH- and the NH2-terminal portions of the protein. The
processed COOH-terminal 15-kDa bioactive portion of myostatin was never
detected in tissues and cell lysates with the anti-COOH-terminal
antibody. The difficulty to detect the 15-kDa COOH-terminal peptide in
cell and tissue lysates is possibly due to the instability of the
active myostatin peptide or to its diffusion into the extracellular
milieu. The 37-kDa LAP may be detected because it is presumably
complexed to a latent TGF-
binding protein (LTBP)-like molecule and
stored in the extracellular matrix, as previously shown for other
TGF-
family members (72).

View larger version (27K):
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|
Fig. 8.
Effect of BB-3103 on myostatin expression and processing.
Myostatin was detected by immunoblotting with antibodies against the
NH2-terminal portion (anti-Nterm) or the COOH-terminal
portion (anti-Cterm) of myostatin. Samples correspond to lysates
prepared from C2C12 clone 18 cells
at various stages of differentiation, treated with DMSO or 5 µM
BB-3103, and lysates prepared from plantaris (pl), soleus (so), and
tibialis anterior (ta) muscles, as described in MATERIALS AND
METHODS. A 37-kDa band, corresponding to the latent-associated
peptide of myostatin, was detected in C2C12
cells and muscles with the anti-Nterm antibody. A 50-kDa band,
representing the nonprocessed precursor of myostatin, was detected with
both antibodies only in cells treated with BB-3103.
|
|
Together, our results suggest that the HIMP-induced myotube hypertrophy
results from a mechanism independent of IGF-I, calcineurin, and TNF-
and rather implicates lack of myostatin activation as the mechanism of action.
 |
DISCUSSION |
Proteases targeted by HIMPs.
HIMPs are specific and powerful inhibitors of the metzincin family of
metalloproteases, including MMPs and ADAMs. HIMPs show some degree of
specificity toward different members of the metzincin family, depending
on the chemical nature of their lateral groups (1, 2, 11,
79). HIMPs seem to inhibit MMPs efficiently at concentrations in
the nanomolar range and ADAMs in the micromolar range
(11). Whether such differences in the sensitivity of MMPs and ADAMs toward HIMPs are always true or depend on the nature of the
system used to assess their activity (cell-containing vs. cell-free
systems) remains unclear (3). Here we show that skeletal muscle cell hypertrophy was induced by both BB-3103 and TAPI at 5 µM
concentrations and higher, indicating that in our system ADAMs may be
the targets, rather than MMPs. As shown here,
C2C12 cells produce only a few MMPs, and their
level of expression or activation state did not significantly change
with cell differentiation, suggesting that MMPs may not play a critical
role in this in vitro model of myogenesis. Moreover, ADAMs, as
membrane-bound metalloproteases, are more likely candidates than MMPs
to participate in the local activation and release of growth factors
and receptors. Although unknown effects of HIMPs are possible, we thus
speculate that myotube hypertrophy induced by treatment with HIMPs
might result from the inhibition of one or several endogenous ADAM-type
metalloproteases. This suggestion is in agreement with the effect of
various processed forms of ADAM 12 on muscle cell differentiation
(20, 78). In a previous study, we showed that
C2C12 cells express mRNA for ADAM 9, 10, 12, 15, 17 and 19 (19), all of which have proven or predicted
catalytic activities and are thus potential targets for HIMPs in this system.
Myostatin in HIMP-induced skeletal muscle hypertrophy.
In a series of experiments, we ruled out the involvement of IGFs,
calcineurin, and TNF-
, all factors previously shown to regulate
skeletal muscle growth, in HIMP-induced myotube hypertrophy. Instead,
we found that the proteolytic activation of myostatin is affected in
HIMP-treated myotubes.
Myostatin, also called growth and differentiation factor-8, is a
negative regulator of skeletal muscle growth. Its absence or mutation
in mice and bovines leads to skeletal muscle hypertrophy and
hyperplasia (33, 39, 40). Myostatin is variably expressed during degeneration and regeneration of different types of muscles (10, 28, 61, 67, 76, 80, 81) and has been detected in
C2C12 cells (73). Here we found
that C2C12 cells expressed myostatin mRNA and
protein and that hypertrophy induced by HIMPs is associated with
changes in myostatin processing in differentiating C2C12 cells. Indeed, the full-length precursor
form of myostatin was detected in C2C12 cells
only upon treatment with HIMPs, suggesting that the proteolytic
maturation of myostatin was reduced or prevented in HIMP-treated cells.
Therefore, the reduced maturation of myostatin might be at least a part
of the mechanism by which HIMPs induce skeletal muscle cell
hypertrophy. The mechanism by which myostatin negatively regulates
muscle growth is not clear. Its total absence in mice leads to both
hypertrophy and hyperplasia, suggesting that myostatin controls both
the number and size of the myofibers (39). Two recent
publications (57, 73) described that myostatin, overexpressed or provided as a recombinant protein, reduces
proliferation and enhances survival of C2C12
myoblasts. On the other hand, mice expressing a mutant, dominant
negative form of myostatin in the already formed muscle, with the use
of the creatine kinase promoter, displayed muscle hypertrophy without
hyperplasia (81). Together, these results suggest that
myostatin may be involved both in controlling the number of myoblasts
before fusion and in controlling the degree of fusion during muscle
development and/or regeneration. The absence of clear effect of HIMPs
on C2C12 cell proliferation in our study could
be explained by the presence of saturating levels of protease inhibitors in high serum conditions.
The processes of synthesis, maturation, and activation of myostatin are
yet unknown. For other members of the TGF-
/BMP superfamily, the
polypeptide chains initially dimerize and are cleaved inside the cell
by furin proteases to release a latent growth factor consisting of the
bioactive growth factor noncovalently bound to its LAP. The latent
growth factor, once secreted, can be stored in the matrix by
interacting with other proteins such as LTBP. The growth factor has to
be activated by a process that may involve various proteolytic or
nonproteolytic events (for review see Refs. 38,
46, 55, and 72). A recent report
(81) showed that mice expressing a mutant,
processing-deficient myostatin in skeletal muscle exhibited skeletal
muscle hypertrophy. The mutant myostatin protein acted in a dominant
negative fashion in that it reduced the processing of the endogenous
wild-type myostatin protein. Together with our results, this
observation strongly suggests that the reduction in myostatin
processing occurring in HIMP-treated cells could be one of the causes
of the resulting hypertrophy. A study from Wells and Strickland
(77) showed that aprotinin, an inhibitor of serine
proteases, stimulates skeletal muscle cell differentiation by
decreasing the extracellular activation of latent TGF-
in the
culture medium. The production of active TGF-
/BMPs may therefore
require the successive actions of several proteases with different
substrate specificity.
The link between metalloprotease inhibition by HIMPs and the reduction
in myostatin processing remains to be determined. The possibility that
myostatin would be directly processed by a HIMP-sensitive ADAM is
theoretically possible but would require that 1) myostatin and active ADAM proteases coexist in the same cellular compartment and
that 2) HIMPs such as TAPI and BB-3103 can cross cellular membranes. Both requirements have in fact been validated by previous reports. Although nonprocessed precursor forms of myostatin have been
detected extracellularly in a model of transfected Chinese hamster
ovary cells (39), it is believed that myostatin is
processed intracellularly, possibly by furins, similarly to other
members of the TGF-
/BMP superfamily. Although ADAMs are known as
plasma membrane-bound proteases that shed extracellular substrates,
some ADAMs may function as intracellular proteases (14, 23, 27, 37, 63). ADAMs are activated by furins or by autocatalysis in
the Golgi apparatus (24, 58) and may exert their
proteolytic activity in the same organelle (70). For
instance, ADAM 10 and/or ADAM 17 has been identified as a protein
kinase C-regulated
-secretase responsible for the nonconstitutive
cleavage of amyloid protein precursor in the Golgi network
(70). ADAM 19 has been shown to cleave Neuregulin
along its secretory pathway (69). Finally, the ability of
HIMPs to penetrate cellular membranes to reach intracellular targets
has been suggested by previous studies (36, 53, 70).
Although our results show that myostatin processing is influenced by
the activity of one or more HIMP-sensitive metalloproteases, further
experiments are necessary to provide conclusive evidence that myostatin
is directly processed by one of these proteases.
 |
ACKNOWLEDGEMENTS |
We thank Dr. James Tidball for the antiserum to myostatin, Immunex
Corporation for TAPI, and British Biotech for BB-3103.
 |
FOOTNOTES |
This work was supported in part by the National Institutes of Health
and by the Muscular Dystrophy Association.
Address for reprint requests and other correspondence: E. Engvall, The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla,
CA 92037 (E-mail: eengvall{at}burnham.org).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 February 2001; accepted in final form 11 July 2001.
 |
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