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INTRODUCTION |
The differentiation of skeletal muscle starts when myoblasts
withdraw from the cell division cycle and is characterized
morphologically by the alignment, elongation, and fusion of
mononucleated myoblasts into multinucleated myotubes. Myogenesis is
largely controlled by the myogenic basic helix-loop-helix family of
transcription factors (MyoD, myogenin, myf5, and MRF4), and the myocyte
enhancer factor-2 (1, 2), which control the expression of many
muscle-specific genes, such as the myosin heavy chain and creatine
kinase. Cell division is prevented during muscle differentiation by the
induction of cyclin-dependent protein kinase inhibitors (3,
4).
Skeletal muscle differentiation is negatively influenced either by
treating myoblasts with serum or growth factors (e.g. basic fibroblast growth factor-2, transforming growth factor
1) or by
oncogenes, such as c-myc, c-jun,
c-fos, Ha-ras, and E1a (1, 5, 6), and
differentiation can be induced by switching the cells to medium
containing low concentrations of serum. Some differentiation inhibitors
activate the classical mitogen-activated protein kinase (MAPK)1 pathway, which has
been implicated in regulating proliferation and differentiation in a
variety of cells (7, 8). For this reason, the role of the MAPK pathway
in regulating skeletal muscle differentiation has been studied
extensively, but the results have been controversial. For example, the
compound PD 098059 (which prevents the activation of MAPK by inhibiting
the activation of MAPK kinase-1 (9)) was reported to increase the rate
of myotube formation in the presence of insulin-like growth factor-1
(IGF-I) in L6 (10) and 23A2 cells (11), suggesting a negative role for
MAPK in this process. In contrast, another laboratory reported that
MAPK plays a positive role in myogenesis (12). They reported that MAPK
activity is induced concomitantly with muscle differentiation in either
C2 myoblasts or muscle cells derived from MyoD-expressing 10T1/2
fibroblasts. They also reported that PD 098059 partially inhibited the
fusion of myoblasts to multinucleated myotubes and the induction of
MyoD expression that normally takes place during terminal
differentiation, without affecting the expression of certain
muscle-specific proteins. Another group reported that MAPK has a
negative role at the early stages of myogenesis because myoblasts
overexpressing MAPK phosphatase-1 (MKP1) showed enhanced production of
muscle-specific genes. However, overexpression of MKP1 inhibited
myotube formation at later times, and it was suggested that the MAPK
pathway plays positive, as well as negative roles in muscle
differentiation (13). Other investigators reported that PD 098059 did
not affect differentiation of C2 myoblasts, implying that the MAPK
pathway is not required for this process (14). They also reported that
wortmannin, an inhibitor of phosphatidylinositide 3-kinase, inhibits
the IGF-I-dependent differentiation of C2 myoblasts.
MKP1 is not specific for MAPK but can also inactivate other MAPK family
members in vitro, such as SAPK2a/p38
(15) and SAPK1/JNK (16). SAPK2a/p38
and SAPK1/JNK lie in distinct signal transduction pathways and, in many cells, become activated in response to cellular stresses (such as the protein synthesis inhibitor anisomycin), lipopolysaccharide, or proinflammatory cytokines (reviewed in Ref. 17).
The effects of MKP1 on muscle differentiation (13) could therefore be
explained by the inhibition of other MAPK cascades. For this reason, we
have investigated the potential role of SAPK2/p38 in skeletal muscle
differentiation by studying the effect of SB 203580, a specific
inhibitor of both SAPK2a/p38
and its close relative SAPK2b/p38
(collectively referred to as SAPK2/p38) (18-21). We demonstrate that
the SAPK2/p38 pathway is activated during C2C12 cell differentiation
and that SB 203580 (but not PD 098059) blocks myotube formation and the
expression of muscle-specific genes. Our results demonstrate for the
first time that SAPK2/p38 is required for C2C12 myogenesis triggered by
low concentrations of serum.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture reagents, myelin basic protein
(MBP) and human IGF-1 were purchased from Life Technologies, Inc.
(Paisley, UK). Protein G-Sepharose and GSH-agarose were from Amersham
Pharmacia Biotech (Milton Keynes, UK). The N-terminal 194 residues of
c-Jun (cJun-(1-194)), provided by Dr. R. Treisman (Imperial Cancer
Research Fund, London), was expressed in Escherichia coli as
a glutathione S-transferase (GST) fusion protein and
purified on GSH-agarose. Rapamycin, LY 294002, and SB 203580 were
purchased from Calbiochem. PD 098059 was a gift from Dr. Alan Saltiel
(Parke-Davis Pharmaceuticals, Ann Arbor, MI). The anti-myosin heavy
chain antibody, the creatine kinase assay kit, anisomycin, and phorbol
12-myristate 13-acetate (PMA) were from Sigma (Poole, UK). Anti-p21 and
anti-myogenin antibodies were from Santa Cruz Biotechnology. Anti-p42
MAPK, anti-MAPK-activated protein kinase-2 (MAPKAP-K2), anti-p70 S6 kinase, and anti-SAPK3 antibodies were raised and purified as described
elsewhere (9, 20, 22, 23). Other reagents were of analytical grade or
better and purchased from BDH Chemicals or Sigma.
Cell Culture--
The mouse C2C12 myoblasts (American Type
Culture Collection) were cultured at 37 °C (in an atmosphere of 5%
CO2) in Dulbecco's modified Eagle's medium supplemented
with 20% (v/v) fetal calf serum (FCS), 0.5% (v/v) chick embryo
extract, and antibiotics.
Treatment with Specific Inhibitors--
To analyze the effect of
different inhibitors of signal transduction pathways on C2C12
differentiation, confluent cells were washed with Dulbecco's modified
Eagle's medium and differentiation medium was added without or with 10 µM SB 203580 (19) or 100 nM rapamycin (24),
10 µM LY 294002 (10), or 50 µM PD 098059 (9). All the inhibitors were dissolved in dimethyl sulfoxide (Me2SO) to give concentrations of 1-50 mM, and
1 µl was added per 1 ml of culture medium. The equivalent volume of
Me2SO was added to control cultures in every experiment.
The medium was replaced with fresh medium (with or without inhibitors)
every 24 h.
At various times, C2C12 cells were lysed in 50 mM Tris
acetate, pH 7.5, 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM orthovanadate, 10 mM
sodium
-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1% (w/v) Triton X-100, 0.01%
(w/v) Brij-35, 0.1% (v/v) 2-mercaptoethanol, 1 mM
benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, and
leupeptin (5 µg/ml). The extracts were centrifuged for 5 min at
13,000 × g, and the supernatant (termed lysate) was removed and either used immediately or quick frozen in liquid nitrogen
and stored at
80 °C.
Immunoprecipitation and Assay of Protein Kinases--
MAPKAP-K2
(22), p70 S6 kinase (25), protein kinase B
(PKB
) (26), and p42
MAPK (27) were immunoprecipitated from 0.1 mg of C2C12 lysates as
described previously for other cell lines. After immunoprecipitation,
MAPKAP-K2 was assayed using the peptide KKLNRTLSVA (28), p70 S6 kinase
using the specific peptide substrate KKRNRTLTV (29), PKB
using
Crosstide (26), and p42 MAPK using MBP (27). To assay SAPK1/JNK in
C2C12 extracts, 10 µg of GST-c-Jun-(1-194) bound to GSH-agarose
beads was incubated with cell lysate (150 µg) for 3 h at 4 °C
to allow SAPK1/JNK to bind to its substrate GST-c-Jun-(1-194). The
beads were washed, and SAPK1/JNK activity was assayed as described
previously (30). One unit of protein kinase is that amount which
catalyzes the phosphorylation of 1 nmol of peptide or protein substrate
in 1 min.
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RESULTS |
Activation of Different Protein Kinase Cascades during the
Differentiation of C2C12 Cells--
C2C12 myoblasts were grown to
80-90% confluency and then induced to differentiate by changing the
medium from 20% FCS to 5% horse serum and left for up to 6 days.
During this period, the activities were measured of five protein
kinases that lie in distinct signal transduction pathways, namely
MAPKAP-K2, p70 S6 kinase, p42 MAPK, SAPK1/JNK, and PKB
.
We found that MAPKAP-K2, an in vivo substrate of SAPK2/p38
(15, 19), was gradually activated during differentiation (half-time 1.5 days) and became maximally activated after 6 days as shown by the
finding that no further activation occurred upon stimulation with
anisomycin (Fig. 1A).
Similarly, p70 S6 kinase, whose activation is prevented by rapamycin,
was gradually activated during differentiation (half-time 1 day), being
maximally activated after 3 days as shown by the failure of PMA to
increase activity further (Fig. 1B). These experiments also
indicated that the expression of MAPKAP-K2 and p70 S6 kinase do not
alter during differentiation.

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Fig. 1.
Activity of different kinases during C2C12
differentiation. Myoblasts were cultured in 20% FCS until
80-90% confluency, changed to differentiation medium containing 5%
horse serum, and lysed at the times indicated, and MAPKAP-K2
(A), p70 S6 kinase (B), p42 MAPK (C),
SAPK1/JNK (D), or PKB (E) activities were
measured. Cells in the differentiation medium for 0 or 6 days were
stimulated for 15 min with 300 ng/ml PMA or 100 ng/ml IGF-1 or for 30 min with 10 µg/ml anisomycin before lysis. The results are presented
as mean ± S.E. for three experiments. In panel C, PD
098059 (50 µM) was added 1 h before PMA.
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In contrast to MAPKAP-K2 and p70 S6 kinase, p42 MAPK of the classical
MAPK cascade was not activated at all during differentiation but was
activated 6-fold in response to PMA (Fig. 1C). SAPK1/JNK was
not activated significantly up to the first 3 days of differentiation, at which stage multinucleated myotubes predominated, but was slightly activated (1.8-fold) at 6 days when the myotubes were fully formed (Fig. 1D). The stimulation of myoblasts (day zero) or
myotubes (day six) with anisomycin caused a four-fold activation of
SAPK1/JNK (Fig. 1D). PKB
, which is activated in
vivo via a phosphatidylinositide (PI)
3-kinase-dependent pathway, was slightly activated up to 6 days, but activation was only 5-10% of that which occurred
after stimulation for a few minutes with IGF-1 (Fig.
1E).
Effect of Inhibitors of Different Protein Kinase Cascades on the
Differentiation of C2C12 Cells--
To further investigate the
mechanisms involved in differentiation, we studied the effects of
several inhibitors of particular signal transduction pathways on C2C12
cell morphology during differentiation. During the first 24 h of
differentiation, the myoblasts elongated and aligned with each other
while, in the following 48 h, multinucleated contractile myotubes
were formed (Fig. 2B). When 10 µM SB 203580 was added to the differentiation medium,
cells remained quiescent, and a delay in the elongation and alignment
of myoblasts was observed after 3 days (Fig. 2C).
Furthermore, even after 6 days in differentiation medium (data not
shown), SB 203580 prevented the fusion of C2C12 myoblasts into
multinucleated myotubes. Myotube formation was also largely (but not
completely) blocked when 100 nM rapamycin or 10 µM LY 294002 (an inhibitor of PI 3-kinase) was present in the differentiation medium (Figs. 2, D and E).
Consistent with the MAPK pathway not becoming activated during
differentiation, 50 µM PD 098059 did not affect the rate
of myotube formation (Fig. 2F) although the PMA-induced
activation of MAPK was strongly suppressed by PD 098059 (Fig.
1C).

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Fig. 2.
Effect of the differentiation medium and
inhibitors of different signaling pathways on C2C12 cell
morphology. A, myoblasts were cultured in a medium
containing 20% FCS and allowed to grow to approximately 80-90%
confluency. To induce differentiation, cells from panel A
were switched to differentiation medium containing 5% horse serum and
allowed to differentiate for 3 days in the absence (B) or in
the presence of either 10 µM SB 203580 (C), 10 µM LY 294002 (D), 0.1 µM
rapamycin (E), or 50 µM PD 098059 (F).
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We next studied the effects of inhibitors on the expression or activity
of several proteins whose levels rise during muscle differentiation
(Fig. 3). SB 203580, rapamycin, and LY
294002 severely inhibited the induction of creatine kinase, myogenin, and myosin heavy chain after 3 days. In contrast, the induction of p21
was only partially suppressed by SB 203580 or rapamycin but was
prevented by LY 294002. PD 098059 did not inhibit the induction of any
of the four differentiation markers studied but actually appeared to
increase creatine kinase activity. The level of p42 MAPK, which is not
a muscle-specific protein, did not change during C2C12 differentiation
and was not affected by any of the inhibitors (Fig. 3).

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Fig. 3.
Effect of inhibitors on the expression and
activation of some muscle differentiation markers. C2C12 myoblasts
were cultured to 80-90% confluency (day 0) and allowed to
differentiate for 3 days (Fig. 2), in the absence or presence of 10 µM SB 203580 (SB), 0.1 µM
rapamycin (Ra), 10 µM LY 294002 (LY), or 50 µM PD 098059 (PD). Cell
lysates were prepared at the times indicated, and aliquots (25 µg of
protein) were electrophoresed on 10 or 15% SDS-polyacrylamide gels and
immunoblotted with anti-myosin heavy chain, anti-myogenin, anti-p21, or
anti-p42 MAPK antibodies. Creatine kinase was assayed using a kit
(Sigma); 1 unit of activity was that amount which catalyzes the
formation of 1 µmol of NADPH in 1 min. The results are presented as
mean ± S.E. for two separate experiments.
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Effect of Rapamycin and SB 203580 on the Activation of SAPK2/p38
and p70 S6 Kinase and on the Expression of Creatine Kinase in C2C12
Cells--
The lack of myotube formation in the presence of 10 µM SB 203580 or 100 nM rapamycin and the
strong activation of MAPKAP-K2 and p70 S6 kinase during the conversion
of C2C12 myoblasts to myotubes indicated that SAPK2/p38 and a
rapamycin-sensitive pathway are required for differentiation. As
expected from previous work in other cell types, SB 203580 inhibited
the activation of MAPKAP-K2, and rapamycin inhibited the activation of
p70 S6 kinase (Fig. 4). Unexpectedly
however, rapamycin also prevented the activation of MAPKAP-K2, whereas
SB 203580 prevented the activation of p70 S6 kinase during the
differentiation process (Fig. 4). In contrast, rapamycin had no effect
on the acute activation of MAPKAP-K2 induced by anisomycin (Fig.
5A), and SB 203580 had no
effect on the acute activation of p70 S6 kinase by PMA, in myotubes
(Fig. 5B). Consistent with the effects of SB 203580 and
rapamycin on the activation of MAPKAP-K2 and p70 S6 kinase,
respectively, these inhibitors also prevented the induction of creatine
kinase activity (Fig. 4C). The activation of MAPKAP-K2 and
p70 S6 kinase, and the induction of creatine kinase during C2C12
differentiation, were also suppressed by LY 294002 (Fig. 4).

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Fig. 4.
The activation of SAPK2/p38 and p70 S6 kinase
during C2C12 cell differentiation is inhibited by rapamycin or SB
203580. C2C12 cells were incubated in differentiation medium in
the absence (closed circles) or in the presence of 10 µM SB203580 (open circles), 100 nM
rapamycin (open triangles), or 10 µM LY 294002 (closed triangles) for the times indicated in the figure.
Cells were lysed, and the MAPKAP-K2 (A), p70 S6 kinase
(B), and creatine kinase (C) activities were
assayed. The results are presented as averages (mean ± S.E.) for
three separate experiments.
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Fig. 5.
The acute activation of MAPKAP-K2 by
anisomycin is not blocked by rapamycin, and the PMA-induced activation
of p70 S6 kinase is not blocked by SB 203580. C2C12 myoblasts were
incubated for 30 min at 37 °C in the absence or presence of 10 µM SB203580 or 100 nM rapamycin and then
stimulated for 30 min with 10 µg/ml anisomycin (A) or for
15 min with 300 ng/ml PMA (B) in the continued presence or
absence of these compounds. The cells were lysed, and MAPKAP-K2
(A) or p70 S6 kinase (B) were immunoprecipitated
from the lysates and their activities assayed as described under
"Experimental Procedures." The results are presented as mean ± S.E. for three separate experiments.
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The elevation in creatine kinase activity (Fig. 4C) during
differentiation paralleled the rise in the activation of MAPKAP-K2 (Fig. 4A) and p70 S6 kinase (Fig. 4B). The
activation of MAPKAP-K2 and the induction of creatine kinase were both
blocked by SB 203580 with IC50 values of <1
µM (Fig. 6A).
The conversion of C2C12 myoblasts to myotubes was suppressed by SB
203580 at a similar concentration (Fig. 6B).

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Fig. 6.
Inhibition of C2C12 cell
differentiation at different SB 203580 concentrations.
A, effect of SB 203580 on MAPKAP-K2 activation and creatine
kinase activity during C2C12 differentiation. MAPKAP-K2 (open
circles) and creatine kinase (closed circles) were
assayed in the lysates of C2C12 cells that had been incubated for 3 days in differentiation medium containing the indicated concentrations
of SB 203580. B, effect of SB 203580 on C2C12 cell
morphology during differentiation. C2C12 cells were incubated for 3 days in differentiation medium containing the indicated concentrations
of SB 203580.
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Expression of SAPK3 during C2C12 Differentiation--
SAPK3 (also
termed ERK6 (31) and p38
(32)) is a MAPK family member that, when
overexpressed in cell lines, can be activated by the same cellular
stresses and proinflammatory cytokines as SAPK2/p38 (20). Moreover,
SAPK3 mRNA expression is induced during the differentiation of
C2C12 cells, and overexpression of SAPK3 in C2C12 cells enhances the
fusion rate of the myoblasts (31). In the present study, we confirmed
that the level of SAPK3 increases during C2C12 myotube formation (Fig.
7A) and also showed that induction is prevented by either SB 203580 or rapamycin (Fig. 7B) but not by PD 098059 (data not shown).

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Fig. 7.
SAPK3 is induced during muscle
differentiation. A, C2C12 myoblasts were differentiated
for the times indicated. Cell lysates (30 µg of protein) were
electrophoresed on 10% SDS-polyacrylamide gels and transferred to
nitrocellulose, and the samples were immunoblotted with anti-SAPK3 or
anti-p42 MAPK antibodies. B, effect of rapamycin
(Ra) or SB 203580 (SB) on the expression of SAPK3
and p42 MAPK. C2C12 myoblasts were differentiated for 3 days in the
absence or presence of 10 µM SB203580 or 0.1 µM rapamycin. Cell lysates were subjected to
SDS-polyacrylamide gel electrophoresis and immunoblotted as in
panel A.
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DISCUSSION |
Previous work suggested both positive and negative roles for the
classical MAPK cascade in the differentiation of myoblast cell lines to
multinucleated myotubes (see the Introduction), but in this study, we
failed to find any evidence for a role for p42 MAPK in C2C12
myogenesis. The differentiation of C2C12 cells was not accompanied by
any activation or inhibition of p42 MAPK (Fig. 1C).
Moreover, PD 098059, which prevents activation of the MAPK pathway, had
no apparent effect on myotube formation (Fig. 2F) or on the
expression of differentiation markers such as p21, myogenin, or myosin
heavy chain (Fig. 3). Instead, SB 203580, a specific inhibitor of
SAPK2/p38, which is a MAPK family member that lies in a distinct
protein kinase cascade, blocked myotube formation (Fig. 2C)
and the expression of muscle-specific proteins and at concentrations
similar to those that suppressed SAPK2/p38 activity in vivo
(Figs. 4C and 6). SB 203580 also prevented the expression of
SAPK3, a MAPK family member that is highly expressed in skeletal
muscle. It is possible that the suppression of myotube formation that
occurs when the MAPK phosphatase MKP1 is overexpressed in C2C12 cells
(13) results from the inactivation of SAPK2/p38 and/or SAPK3, rather
than inactivation of MAPK (see the Introduction).
Myotube formation and muscle-specific gene expression is also blocked
by rapamycin in C2C12 (Figs. 2 and 3) or L6A1 myoblasts (10). Rapamycin
interacts with FK506-binding protein (FKBP), and the rapamycin·FKBP
complex then inhibits the protein kinase termed mammalian target of
rapamycin (mTOR) (33). mTOR phosphorylates 4EBP1, which may stimulate
protein synthesis by triggering its dissociation from eIF4E (34). mTOR
is also required for the activation of p70 S6 kinase (35), but whether
it phosphorylates p70 S6 kinase directly or suppresses the activity of
a p70 S6 kinase phosphatase is unclear.
LY 294002 is best known as an inhibitor of PI 3-kinase (24), but it
also inhibits other members of the PI 3-kinase superfamily. This
includes mTOR, which is inhibited by LY 294002 with an IC50 of 5 µM (36). The blockade of C2C12 myogenesis by 10 µM LY 294002 could therefore be explained by the
inhibition of mTOR, rather than the inhibition of PI 3-kinase. This
would be consistent with the slight activation of PI 3-kinase during
differentiation, which is indicated by the minute activation of PKB
(Fig. 1E). However, LY 294002 completely prevents the
induction of p21, whereas rapamycin only has a partial effect (Fig. 3).
LY 294002 must therefore suppress the induction of p21, at least in
part, by a rapamycin-insensitive pathway.
SAPK2/p38 is not inhibited by rapamycin (data not shown), and rapamycin
does not prevent the acute activation of SAPK2/p38 by anisomycin (Fig.
5). It was therefore surprising that the slow (but strong) activation
of SAPK2/p38 during C2C12 differentiation is suppressed by rapamycin
(Fig. 4). This suggests that a rapamycin-sensitive pathway may induce
the expression of a factor that triggers the activation of SAPK2/p38.
Another unexpected observation was that the slow (but strong)
activation of p70 S6 kinase during differentiation is not only blocked
by rapamycin but also by SB 203580. SB 203580 does not inhibit mTOR
because phorbol ester-induced activation of p70 S6 kinase (which is
blocked by rapamycin) (Fig. 5) is unaffected by SB 203580. This
suggests that the activation of SAPK2/p38 induces the expression of a
factor that triggers the activation of mTOR and p70 S6 kinase. An
intriguing possibility is that SAPK2/p38 and mTOR are both required to
induce the same factor(s), which then activates both pathways creating
a positive feedback loop that drives differentiation to completion.
Whatever the mechanism, our results strongly suggest that SAPK2/p38 and
mTOR both play essential roles in the differentiation of C2C12
myoblasts to myotubes.