From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Haifa 31096, Israel
Received for publication, November 7, 2002
, and in revised form, February 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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Although the function of insulin-like growth factors (IGFs) as inducers of muscle survival has been known for a long time, the intracellular signaling pathways have only recently begun to emerge (6). Two classes of intracellular pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs) are involved in different aspects of IGF-facilitated muscle differentiation (7, 8, 9, 10). Recent studies have focused mostly on the function of the PI3K pathway in the survival of differentiating myoblasts. By manipulating different kinases and using inhibitors of this pathway, it was demonstrated that it played a major role in protecting differentiating myoblasts from undergoing cell death (11, 12, 13).
A second signaling pathway induced by IGF-MAPK might also protect muscle cells from apoptotic cell death (10). It was recently shown that transient transfection of constitutively active Mek1, a specific activator of extracellular regulated kinases (ERKs), maintained myoblast viability in the absence of growth factors (14).
Several authors (15, 16) reported that the activity of ERK was significantly induced with the onset of myoblast terminal differentiation. We suggested that this activation is an intrinsic property of muscle cells. It is now well established that the MAPK pathway that was commonly regarded as mitogenic, can also induce withdrawal from the cell cycle and survival of cells depending on the magnitude and length of the signal and the specific cell type (17, 18, 19). We decided to investigate the role played by the MAPK pathway in the commitment and differentiation of myoblasts. Our results show that the activity of the MAPK pathway reduces the number of differentiating myoblasts undergoing apoptotic cell death. The MAPK pathway also induces the accumulation of the p21WAF1 protein by prolonging its half-life in differentiating cells. Reduction of p21WAF1 protein by antisense expression interferes with the antiapoptotic function of the MAPK pathway. We conclude that the MAPK pathway regulates the survival of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.
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EXPERIMENTAL PROCEDURES |
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Plasmids
p21-Luc was described before
(20). Retroviral vectors
pBP3Raf1DD:ER and pBP3
Raf301:ER were a generous gift
from Dr. M. McMahon (18,
21). The retroviral vector
encoding for MD:ER was a generous gift from Dr. S. Tapscott
(22). The pBABE-GFP retroviral
vector was constructed by replacing the puromycin coding sequence with the
EGFP coding sequence, which was PCR-amplified and cloned into the
ClaI-HindIII sites of pBABE-puro. The mouse p21
cDNA fragment was cloned in the EcoRI site of pBABE-GFP, and a clone
that contained the antisense orientation of p21 relative to the
promoter was used for further studies.
Generation of Stable C2 Clones
C2 cells were a gift from Dr. D. Yaffe
(23). Infection of C2
myoblasts with replication-defective retroviruses was used to generate C2 cell
lines expressing the different chimera proteins. Retroviruses expressing the
different proteins were generated by transfection of retroviral vectors and an
expression vector of vesicular stomatitis virus, the glycoprotein, into viral
packaging cells, 293gp, expressing the gag and pol genes
(24). The medium of
transfected 293gp cells containing retroviruses was used to infect C2 cells.
Forty-eight hours later, cells were selected with puromycin (3 µg/ml).
Resistant clones were pooled together a week later. The expression of the
chimera proteins was determined in Western blots with an antibody to estrogen
receptor.
Cell Culture
Cell lines were maintained in Dulbecco's modified Eagle's medium
supplemented with 15% calf serum (HyClone), penicillin, and streptomycin
(growth medium, GM). To induce differentiation, we used Dulbecco's modified
Eagle's medium supplemented with 10 mg of insulin per ml and 10 mg of
transferrin per ml (differentiation medium, DM). Differentiation of C2 cell
lines expressing the fusion ER proteins was induced by the addition of DM.
-Estradiol (10-6 M) was added to DM at different
time periods as indicated, and U0126 (10 µM) was added to DM
after 24 h.
Transient Transfection Assays
Transfections were performed by calcium phosphate precipitation as
described (25) or using the
TransFast reagent of Promega according to the recommended protocol. Myoblasts
in 6-cm TC dishes (Corning) were transfected with a total amount of 10 mg (or
5 mg, using TransFast) of luciferase reporter plasmid DNA and a control
reporter gene expressing Renilla under the constitutive
cytomegalovirus promoter. Following transfection, the medium was replaced with
DM for another 2448 h. -Estradiol was added to the cells as
indicated. Protein extracts were prepared and used to measure luciferase and
Renilla activities using the Luciferase Assay system from Promega.
Luciferase activity was divided by Renilla activity of each reaction
to correct for the transfection efficiency.
Immunohistochemical Staining
Cells were fixed and immunostained as described previously
(16). The primary antibodies
used were anti-phospho-ERK (Sigma), anti-p21WAF1, anti-cleaved
caspase 3 (Transduction Laboratories), and monoclonal anti-MHC (MF-20). The
immunochemically stained cells were viewed at x200 magnification under a
fluorescence microscope (Olympus, model BX50) and photographed with a digital
camera.
RNA Analysis
RNA was extracted using TRITM reagent (MRC Inc.) and analyzed by
Northern blotting as described previously
(16). Blots were hybridized
with probes for MLC2 (PVZLC2), p21WAF1 (pCDNA-Waf1), and
glyceraldehyde-3-phosphate dehydrogenase (pMGAP).
Western Analysis
Cells were lysed, and whole cell extracts were collected as described
(16). Equal amounts of
extracted proteins (30100 µg) were loaded and separated by 10%
SDS-PAGE and transferred to nitrocellulose membranes. For detecting the
different forms of pRb, proteins were separated over 7.5% SDS-PAGE before
being transferred to membranes. Membranes were incubated in blocking buffer
(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, 2%
w/v nonfat dry milk) and then with the same buffer with the first and
secondary antibodies. Between the second and third incubations, membranes were
washed three times in 0.1% Tween 20 and 1x TBS (20 mM
Tris-HCl, pH 7.4, 150 mM NaCl). Immunoblotting was conducted with
the following antibodies: anti-ERK and anti-phospho-ERK (Cell Signaling),
1:1000; anti-pRb (Pharmingen), 1:1000; anti-p21WAF1, 1:1000;
anti-MHC, 1:2.5; and anti-caspase 9, 1:1000. Proteins were visualized using
the enhanced chemiluminescence kit by Pierce Inc.
Bromodeoxyuridine Staining
Bromodeoxyuridine (BrdUrd) was added to cell media at 10 µM.
After 23 h the cells were washed with PBS, fixed with methanol (10
min), and permeabilized in 0.25% Triton X-100 (10 min). Following a PBS wash,
the cells were incubated in 2 N HCl solution for 1 h. The solution
was neutralized by washing the cells three times in 0.1 M borate
buffer (pH 8.5). Subsequently, the cells were incubated with 6 mg/ml
anti-BrdUrd antibody in PBS containing 0.1% bovine serum albumin for 1.5 h.
The remainder of the procedure was identical to the immunohistochemical
staining of cells described earlier
(16).
Apoptotic Cell Death Assays
TUNEL AssayThe assay kit was purchased from Roche Applied
Science. The assay was performed according to the manufacturer's
instructions.
Hoechst StainingAfter washing with PBS, the cells were incubated with the DNA dye bisbenzimidine (Hoechst 33258) (10 µg/ml) for 30 min. Nuclear morphology was observed at x200 magnification under an upright fluorescence microscope (Olympus, model BX50) and photographed with a digital camera. The percentage of cells with condensed DNA was calculated.
DNA Fragmentation AssayAfter washing with PBS, cells were collected and then resuspended in extraction buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA, pH 8.0, 20 µg/ml RNase A, 0.5% SDS). Samples were incubated at 37 °C for 1 h. Proteinase K (100 mg/ml) was added, and incubation was continued at 50 °C for 3 more h. DNA was then extracted with phenol/chloroform and precipitated with ethanol. Following a 70% ethanol wash, genomic DNA was resuspended in TE (10 mM Tris, 1 mM EDTA, pH 8.0). An aliquot of 30 µg of DNA was analyzed by electrophoresis in 1.8% agarose gels containing ethidium bromide.
Antisense Expression
Replication-defective retrovirus expressing mouse p21 antisense
and the green fluorescent protein (GFP) or control retroviruses expressing the
GFP protein were used to infect C2 cells. One day following infection, the
medium was replaced by differentiation medium. -Estradiol was added to
medium 24 h later, and cells were stained with Hoechst after 48 h in DM. Cells
were viewed for the Hoechst and green fluorescence staining under the above
fluorescence microscope.
In Vitro Kinase Assay for ERK
The assay was performed as described in Gredinger et al.
(16).
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RESULTS |
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Several studies suggest that the activation of MAPK inhibits muscle
differentiation (26,
27,
28). To study whether MAPK
affected muscle differentiation, -estradiol was added to cells together
with the differentiation medium (0 h), during myoblast proliferation, or 36 h
following the addition of differentiation medium (36 h). ERK was
phosphorylated in each case of Raf1 activation (not shown). First, we
investigated the structure of myotubes after growing them for 72 h in DM by
immunostaining with an antibody to myosin heavy chain and found that ERK
activation affected the size of myotubes
(Fig. 1D). Early
activation of ERK (0 h) usually reduced the size of myotubes relative to
control cells (Fig.
1D, middle panel). Late activation of ERK (36 h)
was followed by the appearance of larger myotubes with more nuclei per myotube
relative to control cells (Fig.
1D, right panel). The number of nuclei per
myotube was calculated (Fig.
1D, histogram). Early activation of Raf1
slightly reduced the average number of nuclei per myotube, whereas late
activation increased this number by 2-fold relative to control cells. To find
out whether these differences in myotube structure were also reflected in the
expression of the structural protein MHC, the levels of MHC were analyzed at
several time points following early or late activation of Raf1
(Fig. 1E). Early
activation of Raf1 (0 h) mildly reduced (<2-fold) MHC expression after 8,
24, and 48 h of growth in DM relative to untreated cells (lanes
27). However, after 72 h of growth in DM, MHC levels were similar
between treated and untreated cells (lanes 9 and 10). Late
activation (36 h) of Raf1 did not affect the expression levels of MHC at 48
and 72 h of growth in DM relative to untreated cells (compare lanes
68 and 911). Thus, differences in the structure of
myotubes, especially those resulted from late Raf1 activation are probably not
due to any significant changes in muscle-specific expression.
MAPK Does Not Induce Proliferation of Differentiating
MyoBlastsOne obvious consequence of Raf1 activation was the higher
density of nuclei in -estradiol-treated cultures relative to untreated
cultures (Fig. 1D,
compare the left panel to the middle and right
panels). This difference raised the possibility that MAPK could promote
myoblast proliferation during these stages. The percentage of cells in S phase
was analyzed by the bromodeoxyuridine labeling assay
(Fig. 2A). Activation
of Raf1, at different times after myoblasts were induced to differentiate in
DM, did not induce any significant proliferation beyond the levels observed in
control cells grown in DM for the same period of time
(Fig. 2A). The
phosphorylation state of the retinoblastoma protein can serve as an indicator
for the proliferation state of muscle cells. Two major phosphorylated forms of
pRb exist in replicating myoblasts, whereas only one underphosphorylated form
is found in postmitotic cells. The phosphorylation of pRb is expected if
resting muscle cells are induced to re-enter the cell cycle. We analyzed how
the activation of MAPK affected the phosphorylation status of pRb
(Fig. 2B). The two
forms of pRb were present in proliferating myoblasts, whereas only the
underphosphorylated form was found in myoblasts growing in DM (lanes
1 and 2). Activation of MAPK in myoblasts growing in DM for
short (24 h) or long (72 h) periods did not change the phosphorylation pattern
of pRb; namely, the protein remained in its underphosphorylated form,
indicating that resting myoblasts did not re-enter the cell cycle (lanes
3 and 5). On the whole, the results presented in
Fig. 2 suggest that activation
of the MAPK pathway does not reverse the withdrawal of myoblasts from the cell
cycle.
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Activation of MAPK Prevents Apoptotic Cell Death of Differentiating MyoblastsA high proportion of myoblasts undergoes programmed cell death (PCD) during in vitro differentiation (1). First, we wanted to validate that, in the C2 cell line, myoblasts were undergoing PCD at these stages. For that purpose, we immunostained cells grown in DM for 48 h with an antibody to cleaved caspase 3 (active form) to detect ongoing apoptosis and with an antibody to MHC to detect myotubes (Fig. 3A). Most (above 95%) of the cells that were stained for the expression of cleaved caspase 3 did not stain for myosin heavy chain (Fig. 3A, see "Merge"). A higher magnification of a portion of the microscopic field presented in Fig. 3A shows that staining of caspase 3 was in most cases cytoplasmic (Fig. 3B). Occasionally, staining of cells appeared nuclear, although it could reflect false identification of cells found in advance stages of apoptosis with their cytoplasm collapsed and structure deformed. We can conclude that at these stages the majority of cells undergoing PCD are myoblasts and not myotubes. We calculated the number of cells positively stained for cleaved caspase 3 relative to the total number of myoblast nuclei and found that 29% of the cells were undergoing PCD.
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To investigate the role of MAPK in preventing apoptotic cell death of
myoblasts, we asked how activation or repression of the pathway affected cell
viability by Hoechst and by TUNEL staining of nuclei
(Fig. 4A). A
significant percentage of myoblasts growing in DM for 48 h undergo apoptosis
as can be seen by chromatin condensation observed by the dense staining of DNA
(Fig. 4A). Addition of
-estradiol to C2-
Raf1DD:ER cells after 24 h of
myoblasts growth in DM for an additional period of 24 h largely prevented cell
death. Conversely, treatment of cells with Mek inhibitor, U0126 increased
dramatically cell death (Fig.
4A). The addition of
-estradiol to the control
C2-
Raf301:ER cells did not change the number of cells undergoing PCD,
suggesting that the antiapoptotic effect was specific to Raf activity
(Fig. 4A). Apoptotic
cell death was also analyzed by TUNEL staining of fragmented DNA
(Fig. 4B). Activation
of the
Raf1DD:ER protein reduced while the addition of U0126
increased PCD to similar values observed in the Hoechst staining.
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Fragmentation of genomic DNA that appears as a typical ladder in gel
electrophoresis can serve as a hallmark of PCD. Identical amounts of genomic
DNA were separated over an agarose gel. No ladder was observed in DNA from
proliferating myoblasts, however, a ladder was noticeable after growth of
myoblasts in DM for 72 h (Fig.
4C, compare lanes 1 and 2). If the same
cells were treated with U0126 for 48 h, after 24 h of myoblasts growth in DM
the staining intensity of the ladder was increased significantly
(Fig. 4C, lane
3). Treatment with -estradiol to increase MAPK activity during the
same period of time decreased the intensity of the DNA ladder relative to
untreated cells (compare lanes 2 and 4).
During the process of apoptotic cell death certain caspases are being activated by proteolytic cleavage (29, 30). We analyzed the possible involvement of caspase 9 in PCD of myoblasts and searched whether the MAPK pathway affected its activation. In proliferating myoblasts the inactive pro-caspase was the only noticeable form (Fig. 4D, lane 1). During differentiation of myoblasts, in addition to the inactive form of caspase 9, the active shorter form was observed after 24 h in DM, and its amount was increased after 48 h (Fig. 4D, lanes 2 and 3). Myoblasts growing for 48 h in DM and treated with U0126 expressed more of the active form than control cells not treated with the inhibitor (compare lanes 3 and 4). On the other hand, induction of Raf1 activity reduced the levels of active caspase 9 (compare lanes 3 and 5). These results suggest that the MAPK pathway regulates the activation of caspase 9 in the apoptotic pathway of myoblasts.
Activation of Raf1 in Muscle Cells Induces the Expression of
p21WAF1It was shown before that the expression of
cyclin-dependent kinase inhibitor, p21WAF1, was necessary to
prevent PCD of myoblasts (2).
Next we tested whether MAPK was involved in the expression of
p21WAF1 in muscle cells. Addition of -estradiol to
C2-
Raf1DD:ER cells growing in GM induced protein levels of
p21WAF1, whereas treatment of the same cells with Mek inhibitor,
U0126, reduced its levels as observed by Western blotting
(Fig. 5A). By
immunostaining of cells treated with
-estradiol, we could observe that
myoblasts expressing the phosphorylated form of MAPK, also expressed
p21WAF1 in their nuclei (Fig.
5B). Hence, MAPK is involved in the expression of
p21WAF1 in proliferating myoblasts. The cdk inhibitor
p21WAF1 is normally induced during early stages of muscle
differentiation as myoblasts exit the cell cycle
(20,
31). We followed the
expression profile of p21WAF1 protein during the differentiation of
C2-
Raf1DD:ER cells (Fig.
5C). Levels of p21WAF1 protein were low in
proliferating myoblasts, induced after 24 h in DM, and gradually declined
during their further growth in DM (Fig.
5C, lanes 15). Addition of
-estradiol after growth of 36 h in DM induced the levels of
p21WAF1 protein observed at later periods of cell growth (compare
lanes 4 and 5 to lanes 6 and 7), whereas
U0126 added to cells after growth of 36 h in DM reduced p21WAF1
protein to almost undetectable levels after further growth of cells (compare
lanes 4 and 5 to lanes 8 and 9). These
results indicate that MAPK could be involved in the expression of
p21WAF1 in differentiating myoblasts in addition to other
factors.
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Activation of Raf1 Extends the Half-life of the p21WAF1
ProteinIn muscle cells, the MyoD protein functions to induce the
transcription of p21WAF1 during differentiation
(20). To find out how MAPK
affected the expression of p21WAF1, we analyzed the transcript
levels of p21WAF1 following activation or repression of MAPK
(Fig. 6A). The levels
of p21WAF1 were affected neither by the activation of MAPK caused
by adding -estradiol to myoblasts growing for 24 h in DM nor by its
inhibition following treatment with U0126, suggesting that MAPK did not affect
transcription of p21WAF1 in muscle cells
(Fig. 6A, lanes
2 and 3, Northern blot). However, in the same experiments, the
levels of the p21WAF1 protein were changed by the MAPK pathway
(Fig. 6A, Western
blot). To further test whether MAPK could regulate the transcription of the
p21 gene, a reporter gene containing the promoter sequences of
p21 was transfected into C2 cells expressing
Raf1DD:ER (Fig.
6A, graph).
-Estradiol was added following
24 h of growth in DM to induce and U0126 to repress MAPK activity, and
luciferase activity was analyzed after growth of 72 h in DM. The activity of
MAPK did not change in any significant way the expression levels of the
p21 promoter-reporter gene (Fig.
6A, graph). These results suggest that MAPK
functions post-transcriptionally to induce the levels of the
p21WAF1 protein. The changes in the levels of the
p21WAF1 protein could reflect alterations in protein stability. The
half-life of the p21WAF1 protein was analyzed in a pulse-chase
labeling experiment that was performed in
Raf1DD:ER-expressing cells grown for 48 h in DM. The
half-life of p21WAF1 was about 30 min in cells grown in the absence
of
-estradiol (Fig.
6B). However, the level of p21WAF1 did not
change when cells were grown in the presence of
-estradiol even after
120 min of chase (Fig.
6B). In contrast, incubating the cells with U0126 reduced
p21WAF1 half-life to less than 10 min (not shown). We conclude that
the MAPK pathway contributes to the stability of the p21WAF1
protein in muscle cells. Next we wanted to find out whether activation of
RafDD:ER had a general effect on protein stability or that
it stabilized p21WAF1 in a specific manner. For that purpose we
analyzed the protein levels of another cdk inhibitor family member,
p27KIP1, following the induction or inhibition of MAPK
phosphorylation with
-estradiol or U0126, respectively
(Fig. 6C, lower
panel). The steady-state levels of p27KIP1 were not affected,
whereas those of p21WAF1 were changed as expected. We conclude that
the MAPK pathway affects the protein levels of p21WAF1 but not of
another family member, p27KIP1.
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To investigate the possible interplay between transcriptional regulation by
MyoD and post-transcriptional regulation by MAPK in determining the levels of
p21WAF1 protein, we generated C2 myoblast cells that in addition to
the endogenous MyoD, expressed an inducible form of the MyoD protein
(C2-MyoD:ER). Addition of -estradiol to these cells induced higher
transcript levels of p21WAF1, suggesting that the MyoD:ER protein
induced the transcription of the p21 gene (data not shown). Induction
of the exogenous MyoD:ER protein also increased the amount of
p21WAF1 protein above its level in control cells
(Fig. 6D, compare
lanes 1 and 3). In contrast, treatment of cells with U0126,
significantly reduced the level of the p21WAF1 protein
(Fig. 6D, lane
2). However, inhibition of MAPK with U0126 did not reduce
p21WAF1 protein if during that time,
-estradiol was added to
induce the activity of the MyoD:ER protein
(Fig. 6D, lane
4). The amount of cells undergoing apoptotic cell death was inversely
correlated with the expression levels of p21WAF1
(Fig. 6D,
graph). Myoblasts expressing high levels of p21WAF1
resulting from the activation of MyoD:ER showed reduced PCD (lane 3),
whereas those treated with Mek inhibitor expressing very low levels of
p21WAF1 showed enhanced PCD (lane 2). Myoblasts expressing
intermediate levels of p21WAF1 protein, obtained from the
simultaneous activation of MyoD:ER and suppression of Mek, exhibited
intermediate levels of PCD (lane 4). Taken together, these results
suggest a possible interplay, between the transcriptional and
post-transcriptional activities of MyoD and MAPK, respectively, in
establishing the protein levels of p21WAF1 during muscle
differentiation, may exist and that p21WAF1 levels correlate
directly with the survival of differentiating myoblasts.
The Antiapoptotic Function of the MAPK Pathway in Muscle Cells Is
Mediated by p21WAF1To determine whether
p21WAF1 was required for MAPK-mediated cell survival, we infected
the C2-Raf1DD:ER cells with a retrovirus encoding for an
antisense p21 mRNA (p21WAF1AS) and the marker protein EGFP
or with a control retrovirus encoding for EGFP only. Infected cells were grown
in differentiation medium, and Raf1 was induced 12 h later. After 36 h in DM,
the percentage of apoptotic cells that were positive for EGFP expression was
analyzed by Hoechst staining (Fig.
7A). Some myoblasts infected with the control virus
underwent apoptotic cell death, however, the majority of cells survived. In
contrast, the majority of myoblasts, infected with the virus expressing
antisense p21, underwent apoptotic cell death regardless of whether
-estradiol was added to induce the
Raf1DD:ER protein
or not (Fig. 7A). In
addition to the condensed chromatin, these cells lost their normal elongated
structure and adopted a small spherical structure. Immunostaining of
differentiating myoblasts indicated that the expression of the antisense
p21-encoding virus prevented the expression of endogenous
p21WAF1 in most of the infected cells
(Fig. 7B). These
results indicate that MAPK promotes muscle cell survival by inducing the
protein levels of p21WAF1.
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DISCUSSION |
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How can one explain the different effects of the MAPK pathway during myogenesis? Several models can be suggested to explain the multiple responses mediated by the MAPK pathway: 1) the cellular response is dictated by the cellular context. This model suggests that one pathway may affect many processes by regulating different sets of transcription factors that are available in different tissues or at a given time point in a certain tissue (36); 2) the combined activities of different signal transduction pathways determine the biological response. For example, transformation of fibroblasts depends on the activities of several pathways, including MAPK and phosphoinositol 3-kinase (PI3K), whereas cell cycle arrest is induced by the activation of the MAPK pathway only (37); 3) quantitatively different levels of MAPK activity elicit different responses. For example, transient activation of MAPK in PC12 cells induces proliferation, whereas prolonged activation of MAPK triggers neurite outgrowth (38). These models, in total and individually, can explain the conflicting roles of MAPK in myogenesis. The basal phosphorylation and activity of MAPK is low in proliferating C2 myoblasts relative to its sustained and significant induction in myoblasts and myotubes growing in differentiation medium. Therefore, levels of MAPK induction as well as the cell context and additional signaling pathways may explain the differences in the way that the same signaling pathway affects cell growth at different stages of differentiation. Interestingly, significant activation of MAPK both during early and late phases of differentiation did not induce any proliferation of myoblasts (Fig. 2). This result is in conflict with other studies (27, 28) and may be explained in the following two ways: (a)We induced MAPK under serum starvation conditions that promoted withdrawal from the cell cycle. Under these conditions, cell context and signaling pathways may cooperate with MAPK in arresting cell cycle. In other studies, the MAPK pathway was inhibited in myoblasts growing in high serum. Under these conditions the pathway was involved in cell proliferation. (b)In muscle cells, high levels of MAPK activity induce withdrawal, whereas lower levels induce cell cycle progression. These possibilities deserve further investigation.
Antiapoptotic Activity of MAPK during Myoblast DifferentiationIn cell cultures many myoblasts undergo PCD under conditions that promote differentiation (1). Based on several different approaches we find that the MAPK pathway is involved in protecting myoblasts from undergoing PCD (Fig. 4).
Signals that induce apoptosis culminate in the activation of caspases, which are the ultimate effectors of PCD. To find out whether MAPK could affect the proteolytic activation of caspases, we followed the activation of caspase 9 (Fig. 4D). The processed form was detected during myoblast differentiation. Ectopic activation of Raf1 reduced the relative amount of the processed form, whereas inhibition of Mek with U0126 increased its relative levels. Therefore, MAPK affects the process at this stage or at stages that precede caspase activation. Recent studies suggested that in Rat1 fibroblast cells the MAPK pathway conferred protection against apoptosis at the level of cytosolic caspase activation and not in the earlier stage of cytochrome c release from the mitochondria (39, 40). Another study showed that MAPK promoted cell survival of neurons by phosphorylation of the proapoptotic protein BAD and the transcription factor CREB (41). The role of MAPK in cell survival was also explored in Drosophila, known to express a group of proteins, REAPER, HID, and GRIM, that activate caspase processing and other proteins known as inhibitors of apoptosis (IAPs) that inhibit caspase processing. The latter group can directly bind to activated caspases and block the proteolytic chain reaction. The first group of proteins binds directly to IAP and antagonizes its activity, thereby allowing the proteolytic activation of caspases and apoptosis to proceed. In Drosophila, MAPK phosphorylates the HID protein and inhibits its interaction with IAP and consequently its proapoptotic activity (42, 43). Although the functional mammalian homologue of Hid has not yet been identified, its existence was suggested (44, 45). One might speculate also that in mammalian cells, including muscle cells, MAPK may directly affect an HID-like protein in preventing the proteolytic activation of caspases.
MAPK Functions Independently of the PI3K Pathway in Protecting Myoblasts from PCDRecently, several works demonstrated the involvement of the phosphoinositol 3-kinase (PI3K) pathway in the survival of differentiating myoblasts (11, 13). It was suggested that Akt, a kinase in the pathway, phosphorylated the mitochondrial BAD protein known to protect cells from undergoing PCD (46, 47). In light of our results it is of interest to know whether the PI3K and the MAPK pathways exhibit cross-talk in their antiapoptotic functions. We found that modulation of MAPK activity did not affect the phosphorylation state of Akt during muscle differentiation.2 Therefore, it is likely that the antiapoptotic function of MAPK is not mediated by Akt. Our conclusion is further supported by a recent study that suggested IGF-I- and platelet-derived growth factor-induced myoblast survival via two independent signaling pathways (14). According to this work, IGF-I induced the PI3K pathway, whereas platelet-derived growth factor induced the MAPK pathway, and each of the pathways was sufficient to promote muscle cell survival.
The activities of PI3K and MAPK do not overlap in the differentiation process. Moreover, Akt was shown to phosphorylate and inhibit Raf1 in muscle cells (48). Whereas PI3K and Akt are induced at early stages of differentiation, MAPK activation occurs at later stages.2 Therefore, it is possible that each pathway functions independently to protect myoblasts from PCD at different stages of the differentiation process.
Several Factors, Including the MAPK Pathway, Maintain the Expression of
p21WAF1 in Differentiating MyoblastsMAPK induces the
expression of p21WAF1 in many cellular systems and causes cell
cycle arrest (49). The
induction of p21WAF1 expression during muscle differentiation plays
at least two fundamental roles in the withdrawal of myoblasts from the cell
cycle and in their survival
(1). MyoD is involved in the
transcriptional induction of p21
(20). In the present work we
asked whether MAPK contributed to the expression of p21WAF1, in
view of its role as a myoblast survival factor
(2). In proliferating myoblasts
where the levels of phosphorylated MAPK and p21WAF1 are low,
activation of Raf1 induced the expression of the p21WAF1 protein
(Fig. 5, A and
B). After 24 h of growth of C2 cells in DM the level of
p21WAF1 protein increased but gradually declined during further
growth (Fig. 5C). This
happens despite the normal induction of MAPK occurring during differentiation.
Further activation of MAPK via the exogenously expressed
Raf1DD:ER protein during these stages induced higher levels
of p21WAF1 protein, whereas inhibition of Mek with U0126 reduced
its levels. Therefore, MAPK plays a role in maintaining the levels of
p21WAF1 during late phases of differentiation. Some studies
indicate that other factors such as MyoD and the PI3K pathway also affect the
expression of p21WAF1
(13,
20). Thus, the balance between
different factors, including the MAPK pathway, may determine the absolute
levels of p21WAF1 in differentiating myoblasts. As the activities
of MyoD and PI3K are reduced during later stages of differentiation, enhanced
activity of MAPK may substitute for these proteins in maintaining the
expression of p21WAF. The vital role of p21WAF1 in cell
cycle withdrawal and myoblast survival may explain the multiple pathways and
factors involved in its expression.
MAPK Stabilizes the p21WAF1 ProteinIn some cellular systems MAPK regulates p21 transcriptionally (50, 51, 52), whereas in others it also affects the post-transcriptional processes (53). We studied the regulation of p21 by MAPK in muscle cells and found that the activation or repression of the pathway did not affect the transcripts levels or the promoter activity of p21. Nevertheless, MAPK significantly affected the protein levels of p21WAF1, suggesting changes in protein synthesis or breakdown. The finding, that activation of Raf1 dramatically extended while inhibition of Mek with U0126 significantly reduced the half-life of the p21WAF1 protein, indicates that the MAPK pathway regulates the stability of the p21WAF1 protein. The p21WAF1 protein is degraded by the proteasome in a process that does not involve ubiquitination of p21WAF1 (54). Recent studies have emphasized that the turnover of p21WAF1 protein is regulated by several signaling pathways affecting cell growth. Rac1/CDC42 activates the degradation of p21 (55), whereas p38 MAPK, JNK1 (56), and protein kinase B/Akt (57) stabilize the protein by phosphorylating several of its residues. One study suggested that the ERK MAPK pathway is required to stabilize p21 mRNA and p21WAF protein during the withdrawal of primary hepatocytes from the cell cycle (53). All in all, these studies suggest that the stability of the p21WAF1 protein is affected by multiple signaling events, implicating it as a major mechanism regulating the levels of p21WAF1 protein in cells.
MAPK Induces Hypertrophic Growth of MyotubesIn the present study we observed that MAPK was also involved in the determination of myotube size and the number of nuclei per myotube (Fig. 1). Similar observations were reported by others (10, 58). In those studies, as well as in ours, MAPK activity increased the size of myotubes and prevented the collapse that usually occurs after several days of growth in DM.2 Presently we don't know if this is a direct or indirect effect of the MAPK pathway. A direct activity of MAPK could target the translational apparatus and induce hypertrophic growth (59) or, alternatively, induce fusion of myoblast cells to multinucleated myotubes. An indirect effect could for example be a result of the better survival of myoblasts induced by MAPK that allows the recruitment of more competent myoblasts to fuse and form differentiated myotubes. These possibilities deserve further studies.
Interestingly, from studies with Rb-/- mice it is apparent that pRb protein may affect muscle growth in a way similar to MAPK (5). These mice die after birth with specific muscle defects, including increased myoblast PCD prior to myocyte fusion; the surviving myotubes are shorter, have less nuclei, and express reduced levels of late muscle-specific genes. The similarities between pRb and MAPK and the direct effect of MAPK on p21WAF1 protein suggest that these proteins may share the same pathway affecting muscle differentiation and survival.
Recently, we observed that the MAPK pathway was absolutely necessary for the differentiation of skeletal muscle during early development of Xenopus laevis (60). In this model system, MAPK affected the expression of late markers of differentiation. We could also demonstrate that activated Mek induced the levels of the MyoD protein in explants from injected embryos. Therefore, during early development of Xenopus, distinctly from the cell culture system used in the present study, MAPK directly affects myogenesis through the MyoD protein. However, it is possible that, like in the tissue culture model, MAPK functions to augment skeletal muscle differentiation by preventing myoblast cell death during Xenopus development.
A Model for the Antiapoptotic Activity of the MAPK Pathway in MuscleAs myoblasts undergo their terminal differentiation, many of the cells that cannot complete this process successfully are eliminated by PCD (Fig. 8). The cell cycle machinery and, specifically, the p21WAF1 cdk inhibitor regulate this process (61). p21WAF1 may serve as an indicator for the "decision" of myoblasts whether to proceed with the differentiation process or to undergo cell death. The protein levels of p21WAF1 are induced and maintained by several factors and signaling pathways during differentiation. MyoD, whose activity is the first to be induced during muscle differentiation, activates the transcription of the p21 gene. During these early stages, the PI3K-Akt pathway is also activated in a transient fashion to further induce the levels of p21WAF1. Later, as PI3K-Akt activity drops, the MAPK pathway is induced and functions to maintain the p21WAF1 protein during a later phase of differentiation.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 972-4-8295-287; Fax:
972-4-8553-299; E-mail:
bengal{at}tx.technion.ac.il.
1 The abbreviations used are: cdk, cyclin-dependent kinase; PI3K,
phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; pRb,
retinoblastoma; IGF, insulin-like growth factor; ERK, extracellular
signal-regulated kinase; GFP, green fluorescent protein; EGFP, enhanced GFP;
GM, growth medium; DM, differentiation medium; ER, estrogen receptor; MHC,
myosin heavy chain; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline;
TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling;
PCD, programmed cell death; IAP, inhibitors of apoptosis; JNK1, c-Jun
NH2-terminal kinase 1; DAPI,
4',6-diamidino-2-phenylindole.
2 O. Ostrovsky and E. Bengal, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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