From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel
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ABSTRACT |
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Differentiation of muscle cells is regulated by
extracellular growth factors that transmit largely unknown signals into
the cells. Some of these growth factors induce mitogen-activated
protein kinase (MAPK) cascades within muscle cells. In this work we
show that the kinase activity of p38 MAPK is induced early during
terminal differentiation of L8 cells. Addition of a specific p38
inhibitor SB 203580 to myoblasts blocked their fusion to multinucleated myotubes and prevented the expression of MyoD and MEF2 family members
and myosin light chain 2. The expression of MKK6, a direct activator of
p38, or of p38 itself enhanced the activity of MyoD in converting
10T1/2 fibroblasts to muscle, whereas treatment with SB 203580 inhibited MyoD. Several lines of evidence suggesting that the
involvement of p38 in MyoD activity is mediated via its co-activator
MEF2C, a known substrate of p38, are presented. In these experiments we
show that MEF2C protein and MEF2-binding sites are necessary for the
p38 MAPK pathway to regulate the transcription of muscle creatine
kinase reporter gene. Our results indicate that the p38 MAPK pathway
promotes skeletal muscle differentiation at least in part via
activation of MEF2C.
The development of skeletal muscle is a multistep process in which
pluripotent mesodermal cells give rise to myoblasts that subsequently
withdraw from the cell cycle and differentiate into myotubes. These
stages are driven by the expression and activity of myogenic
transcription factors from the MyoD family (1). Two members of the MyoD
family, Myf5 and MyoD, are expressed in dividing myoblasts that are
committed to the myogenic lineage. However, these proteins are not
functional in myoblasts, and their activities are induced only at a
subsequent stage to allow withdrawal from the cell cycle and terminal
differentiation (2). Induction of the activity of MyoD and Myf5
proteins may be a result of the activity of extracellular growth
factors like insulin and insulin-like growth factors known to promote
terminal differentiation of myoblasts (3, 4). Insulin and insulin-like
growth factors are involved in the activation of phosphatidylinositol
3-kinases and mitogen-activated protein kinases
(MAPK)1 via tyrosine kinase
receptors within many cell types including muscle (5, 6). One MAPK
induced by insulin is p38 MAPK (6). p38 is also activated by exposure
of cells to environmental stress or by treatment of cells with
pro-inflammatory cytokines (7, 8). The affectors regulating p38 are
only partly explored. However, the direct intracellular activators of
p38 are MKK3 and MKK6 (MAPK kinase) (9, 10). The role of p38 in
skeletal muscle differentiation is not known. Several recent findings
suggest that p38 MAPK may be involved in skeletal muscle
differentiation: 1) Transcripts of MKK6 gene are most abundant in
skeletal muscle (10). 2) Insulin that promotes skeletal muscle
differentiation also induces p38 activity (6). 3) p38 pathway regulates
the expression of glucose transporters in skeletal muscle cells (11). 4) p38 activates directly the transcription factor MEF2C in
inflammation (12). Together with the MyoD family, members of the MEF2
family are necessary for the differentiation of myoblasts (13). 5) p38
pathway plays a role in specific gene expression and cell hypertrophy
of the related cardiac muscle lineage (14). In this report we show that
1) the p38 pathway plays an essential role in the in vitro
differentiation of myoblasts and 2) that this role may be mediated via
the MEF2C transcription factor.
Materials
SB 203580 was a product of Calbiochem. Goat polyclonal antibody
raised against amino acids 341-360 of mouse p38 was obtained from
Santa Cruz Biotechnology. Rabbit polyclonal antibody that detects p38
MAPK only when activated by dual phosphorylation at Thr180
and Tyr182 was purchased from New England Biolabs.
Antibodies to MEF2A and C proteins and to GAL4 (amino acids 94-147)
were purchased from Santa Cruz Biotechnology. Protein A-Sepharose was
supplied by Sigma.
Plasmids
pEMSV-MyoD was described by Tapscott et al. (15). The
4R-tk-CAT reporter gene was described by Weintraub et al.
(16). pe+AT-CAT and p(+enh110)-80MCK-CAT were
described by Buskin and Hauschka (17). For MEF2-80MCK-CAT, three
copies of a double-stranded oligonucleotide of the MEF2 site (30 base
pairs) from the MCK enhancer were inserted upstream to a minimal MCK
promoter ( Cell Culture
L8 cells were a gift of Dr. David Yaffe (20). 10T1/2 cells were
obtained from ATCC. 10T1/2 cells that expressed the MyoD-estrogen receptor (ER) chimera protein were described by Hollenberg et al. (21). Cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 15% calf serum (Hyclone),
penicillin, and streptomycin (growth medium). To induce
differentiation, we used Dulbecco's modified Eagle's medium
supplemented with 10 µg of insulin/ml and 10 µg of transferrin/ml
(differentiation medium, DM). Differentiation of 10T1/2 cells that
expressed MyoD-ER protein was induced by the addition of DM that
contained 10 Growth of Cells in the Presence of SB 203580
SB 203580 was dissolved in Me2SO to a concentration
of 10 mM and was added directly to the differentiation
medium to a final concentration of 10-20 µM as
indicated. Control cells were incubated with the same volumes of
Me2SO without SB 203580. The medium was replaced every
12 h with medium containing fresh SB 203580.
Transfections
Transfections were performed by calcium phosphate precipitation
as described (22). Cells in 6-cm TC dishes (Corning) were transfected
for 12 h with a total amount of 10 µg of the following plasmid
DNA: 1 µg of pCMV-LacZ, 3 µg of CAT reporter gene, 3 µg of MyoD
expression plasmid, 3 µg of MEF2C expression vector, and 3 µg of
expression vector of wt MKK6 or activated form of MKK6b (MKK6b(E)).
Following transfection, the medium was replaced with either growth
medium or DM for another 24-48 h. The efficiency of transfections were
tested in soluble 5-bromo-4-chloro-3-indolyl Immunohistochemical Staining
Cells were fixed and immunostained as described (25). The
primary antibodies used were monoclonal anti-MyoD (5.8A) and polyclonal anti-MHC (Sigma). The immunochemically stained cells were viewed at
×200 magnification in a fluorescence microscope (Olympus, model BX50).
In Vitro Kinase Assays
Expression of GST Fusion Proteins--
The GST-ATF2 and
GST-MEF2C (128-467) proteins were expressed from the bacterial
expression vectors in BL21 strain of Escherichia coli and
purified from extracts on glutathione beads as described (26).
Preparation of Cell Extracts--
Cell extracts were prepared as
described (25).
In Vitro Kinase Assay for p38--
Cells were extracted in lysis
buffer (described above). Equal amounts of protein from each time point
of differentiation were rotated with 6 µl of anti-p38 antibody (Santa
Cruz) for 2.5 h at 4 °C. The immunoprecipitation procedure and
the kinase assay were performed as described for ERK (25). However, the
substrate in the present assay was GST-ATF2 (20 µg).
In Vitro Kinase Activity of p38 Using GST-MEF2C as a
Substrate--
Equal amounts of cell extracts were collected at
different times after the initiation of cell differentiation and were
mixed with GSH-agarose beads to which GST or GST-MEF2C proteins were bound. The mixtures were processed, and the kinase assay was performed as described by Hibi and colleagues for the JNK kinase assay (26). Results were quantified by PhosphorImager.
RNA Analysis
RNA was extracted and analyzed by Western blotting as described
(25). Blots were hybridized with probes for MEF2C (pEMSV-MEF2C), MyoD
(pEMSV-MyoD), myogenin (pEMSV-myogenin), MLC2 (PVZLC2), p21 (pCDNA-Waf1), and GAPDH (pMGAP).
Western Analysis
Cells were lyzed as described for the kinase assays, and equal
amounts of extracted proteins were loaded and separated by SDS-PAGE and
transferred to nitrocellulose filters. Immunoblotting was conducted
with the following two antibodies: anti-p38 (Santa Cruz) 1:100 and
anti-phospho-p38 antibody (New England Biolabs) 1:100. Proteins were
visualized using the enhanced chemiluminescence kit of Amersham
Pharmacia Biotech.
Metabolic Labeling of Cells and Immunoprecipitation of MEF2C
Labeling with [35S]Methionine--
Cells were
incubated in methionine-free Dulbecco's modified Eagle's medium and
dialyzed calf serum for 40 min and then incubated with 100 µCi/ml
[35S]methionine for 3 h before proteins were
extracted in RIPA buffer (50 mM Tris, pH 7.9, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5 mM
dithiothreitol, 0.1 mM Na3VO4, 2 µg/ml leupeptin, 20 mM p-nitrophenyl phosphate, and 100 µg/ml phenylmethylsulfonyl fluoride).
Labeling with [32P]Orthophosphate--
L8 cells
were incubated in Dulbecco's modified Eagle's medium without sodium
phosphate to which [32P]orthophosphate was added at 0.75 mCi/ml for 3 h before proteins were extracted in RIPA buffer.
Immunoprecipitation--
Equal amounts of labeled proteins were
incubated with antibody for 2 h and then for an additional hour
with protein A-Sepharose. The protein A-Sepharose beads were washed
four times with RIPA containing 0.5 M NaCl and once in RIPA.
p38 Activity Is Induced during Differentiation of L8
Cells--
The in vitro kinase activity of p38 was studied
using extracts of L8 muscle cells from different stages of
differentiation. Kinase activity of p38 was analyzed by
immunoprecipitation of p38, which was used to phosphorylate its
substrate, ATF2 (Fig. 1A). The
kinase activity of p38 was present in dividing myoblasts and was
gradually induced during the differentiation to multinucleated myotubes. Kinase activity was specific; it was induced by UV
irradiation and inhibited by the p38-specific inhibitor SB 203580 added
to L8 cells (Fig. 1A). The amount of the phosphorylated form
of p38 that reflects the active kinase was studied in L8 cells. An
antibody that recognizes the active dual phosphorylated form of p38 was used with the same extracts in a Western analysis. Phosphorylated forms
of p38 accumulated during the differentiation of cells in DM (Fig.
1B). The level of total p38 protein in differentiating cells
was not constant; however, the calculated percentage of phosphorylated
p38 in cells growing in DM for 48 h was 2-3-fold higher than that
of dividing myoblasts (Fig. 1B). Next, we assayed p38 kinase
activity with a second substrate, GST-MEF2C. p38 was recently
demonstrated to associate and directly phosphorylate MEF2C (12). In
addition, MEF2C is an essential transcription activator of
muscle-specific genes (13). Phosphorylation of GST-MEF2C substrate was
increased during the differentiation of L8 myoblasts to multinucleated
myotubes (Fig. 1C). p38 was probably the kinase that
phosphorylated GST-MEF2C because kinase activity was significantly
inhibited in cells treated with SB 203580 (Fig. 1C,
lanes 3 and 4). The increased activity of p38
during muscle differentiation suggests that it might have a role in
this process. Four isoforms of p38 ( Treatment of L8 Cells with SB 203580 Inhibits Their
Differentiation--
To evaluate the role of p38 in myogenesis, we
used the p38 inhibitor SB 203580 (27) that was added to L8 cells at
concentrations that blocked most of the p38 activity (Fig.
1A). The inhibitor was added concomitantly with the
induction of differentiation (differentiation medium, see
"Experimental Procedures"). After 36 h in that medium, cells
not treated with the drug fused to form developed myotubes, whereas
cells treated with the drug did not form myotubes (Fig.
2). Moreover, the expression of muscle specific genes like myogenin and MLC2 was completely inhibited in cells
treated with the drug (Fig.
3A). The expression of MEF2 that participates with the MyoD family in the differentiation process
was also abolished in the drug-treated cells (Fig. 3A). Induction of the cyclin/cyclin-dependent kinase inhibitor p21 (Waf-1)
participating in the exit of myoblasts from the cell cycle was
inhibited in SB 203580-treated cells. Consequently, the drug blocked
differentiation completely. We noticed dead cells in both untreated and
treated cells grown in the serum-free medium (DM), which probably
underwent programmed cell death under conditions of mitogen deprivation
(Fig. 2) (28). However, the drug was most probably not toxic to cells
because the percentage of living cells was not changed in drug-treated
culture, and these cells recovered and differentiated following removal
of the drug (Figs. 2 and 3B).
Activation of p38 with MKK6 Induces Muscle Differentiation by
MyoD--
The dramatic effect of p38 inhibitor on the expression of
MyoD and MEF2 family members prompted us to investigate whether p38
affected also the activity of the myogenic regulator MyoD. The ectopic
expression of MyoD converts 10T1/2 fibroblasts to muscle (29). The
effect of p38 on MyoD activity was studied by transiently transfecting
10T1/2 fibroblasts with expression vectors of MyoD and wild type MKK6,
the direct activator of p38. Most 10T1/2 fibroblasts transfected with
MyoD expression vector also expressed the differentiation marker MHC
when grown for 24 h in differentiation medium (Fig.
4). However, if the transfected cells
were continuously grown in high serum-containing medium (15% bovine
calf serum), only 28% of the cells that expressed MyoD expressed also
MHC (Fig. 4). Under these conditions, cells that were co-transfected
with MKK6 exhibited a significantly higher proportion of MHC staining
in the cytoplasm of MyoD-expressing cells (69% of the cells) (Fig. 4).
On the other hand, addition of SB 203580 reversed the effect of MKK6
and completely inhibited MyoD, i.e. only about 5% of the
cells that expressed MyoD also expressed MHC. In a parallel experiment,
we studied the effects of MKK6 and SB 203580 on the in vitro
kinase activity of p38 in extracts from transfected 10T1/2 cells (Fig.
4C). As expected, MKK6 induced whereas SB 203580 inhibited
p38 kinase activity. Like MKK6, ectopic expression of p38 isoforms,
mainly p38
Another approach was used to investigate whether p38 affected the
activity of MyoD. We used a cell line expressing an estrogen receptor-MyoD chimera protein (21). The chimera protein remains inactive in the cytoplasm of cells. Treatment of cells with estradiol induces the chimera protein that migrates to the nucleus and activates muscle-specific transcription. In this cell line, myogenesis is initiated by the activity of the chimera protein. Therefore, inhibition of MyoD in these cells is expected to abolish myogenesis. Activity of
the chimera protein was induced by the addition of estradiol to cells
that were grown in the presence or absence of SB 203580. Cells were
grown in the presence of estradiol for 24 h, at which time
mRNA levels of several muscle-specific genes were induced to
significant levels (Fig. 5, lane
3) (30). If cells were treated with SB 203580 during that period,
the induction of MEF2, myogenin, and MLC2 expression was significantly
inhibited, although expression of chimeric MyoD-ER remained unchanged
(Fig. 5, lanes 4 and 5). Therefore, the inhibitor
either inactivated the chimera MyoD protein or affected other
transcription factors or co-activators that may function in concert
with MyoD to initiate myogenesis.
MKK6 Augments Transcription of MCK via the MEF2-binding
Site--
To further analyze the effect of MKK6 on the transcriptional
activity of MyoD, expression vectors of these proteins were
co-transfected with a reporter gene that contained a minimal promoter
and four MyoD-binding sites (4R-tk-CAT). Surprisingly, the activity of MyoD in the induction of this reporter gene was almost not affected by
co-transfection of MKK6 (Fig.
6A, lanes 9-12).
However, MyoD activity was increased more significantly by MKK6 in
activating the transcription of a reporter gene whose expression was
driven by MCK regulatory sequences (pe+AT-CAT) (Fig.
6A, lanes 1-4). This reporter gene contained two MEF2-binding sites within the MCK enhancer element that could contribute to the effect of MKK6 (12). Co-expression of MEF2C did not
further potentiate the transcriptional activity of MyoD on this
promoter (not shown). These results may be explained by previous
results that indicated that endogenous MEF2 expression was stimulated
by the expression of myogenin in fibroblast cells (31). For this
reason, we did not co-transfect a MEF2C expression vector in the
subsequent experiments that analyzed MCK regulatory sequences. To study
the possible role of the MEF2 sites in the stimulation of MyoD activity
by MKK6, a similar transfection experiment was done with an MCK
reporter gene that contained the MyoD-binding sites and only one MEF2
site (p110-MCK-CAT) (17). Induction of this reporter gene by MyoD was
only mildly affected by the co-expression of MKK6 (Fig. 6A,
lanes 5-8). To prove MKK6 activated MCK via the MEF2 site,
we generated a reporter gene that contained multiple MEF2 sites of the
MCK enhancer and a basal promoter of MCK (MEF2-MCK-CAT). Indeed, this
promoter was induced by the expression of MEF2C and was further induced
by MKK6 (Fig. 6A, lanes 13-15). Expression of
MKK6 alone was sufficient to induce this promoter in a significant
fashion (Fig. 6A, lane 16). We conclude that MEF2
sites probably play a role in the stimulation of MCK transcription observed in the presence of MKK6. Addition of the p38 inhibitor, SB
203580, significantly blocked the transcriptional activity of MyoD in
the induction of the two MCK enhancer reporter genes, (pe+AT-CAT and p110-MCK-CAT) (Fig. 6B,
lanes 1-4), and more modestly affected its activity on the
minimal promoter driven by E boxes (4R-tk-CAT) (Fig. 6B,
lanes 5 and 6). The transcriptional activity of
MEF2 on a basal promoter driven by MEF2 sites was blocked by SB203580
(Fig. 6B, lanes 9 and 10). All in all,
these results suggest that p38 modulates the transcriptional activity
of the MCK reporter gene via the MEF2-binding sites.
A Transcriptionally Inactive MEF2C Protein Abrogates the Effects of
MKK6 on the MCK Enhancer--
To study the role of MEF2 in mediating
the effects of p38 on the transcription of the MCK reporter gene, we
generated a transcriptionally inactive MEF2C protein (see
"Experimental Procedures"). This protein did not contain a large
part of its transactivation domain that included two phosphorylation
sites of p38 (MEF2C- The p38 Pathway Affects the MEF2C Protein but Not the MyoD
Protein--
Although the results presented in Fig. 6 suggest that the
p38 pathway operates via MEF2 sites, we could still notice an effect on
MyoD (Fig. 6, A, lanes 9-12, and B,
lanes 5 and 6). This effect on MyoD may result
from its association with MEF2, which is known to function as a
co-activator of MyoD (18). To study the effect of p38 on MyoD
independently of MEF2, we used the GAL4 activator/reporter system. The
transcription activators in this system were composed of MyoD fragments
that were fused to the DNA-binding domain of yeast transcription factor
GAL4. The chimeric activators are expected to bind to the GAL4
DNA-binding sites of a reporter gene and activate transcription via the
transactivation domain of MyoD. MEF2 was suggested to interact with the
DNA-binding domain of MyoD-E12 heterodimers (18). Two MyoD proteins
that were not expected to interact with MEF2 were used; one that did
not contain the HLH domain (GAL- MEF2C Is Phosphorylated by p38 in Muscle Cells--
To demonstrate
that GAL4-MEF2C protein is phosphorylated in cells by p38, 293 cells
were transfected with expression vector of GAL4-MEF2C alone or
GAL4-MEF2C with MKK6 (Fig.
9A). In a duplicate set of
transfected plates, the cells were treated with SB 203580. Proteins
were metabolically labeled with [35S]methionine, and
GAL4-MEF2C was immunoprecipitated. The mobility of the GAL4-MEF2C
protein in the gel indicated its phosphorylation status. MEF2C was
phosphorylated by p38 in cells because it migrated faster in cells
treated with SB 203580 (Fig. 9A, compare lanes 1 and 2). Ectopic expression of MKK6 in the transfected cells resulted in a smear of slower migrating GAL-MEF2C species (Fig. 9A, lane 3). Phosphorylation of MEF2C was
confirmed by treatment of the immunoprecipitates with alkaline
phosphatase that compressed the smear to a tight faster migrating band
(Fig. 9A, lanes 5-7). Unlike GAL4-MEF2C, the
migration of transfected GAL4 protein was not affected by the
co-expression of MKK6 (Fig. 9A, lanes 8 and 9). Therefore, both the expression of MKK6 and treatment
with SB 203580 affected the phosphorylation state of transfected
GAL4-MEF2C protein in cells.
To find out if MEF2 proteins are phosphorylated in muscle cells, we
immunoprecipitated endogenous MEF2 proteins from L8 cells (Fig.
9B). MEF2 proteins were detected mainly in differentiated myotubes (Fig. 9B, lanes 1 and 2).
Differentiated cells were phosphate-labeled, and MEF2 proteins were
isolated to learn whether these proteins were phosphorylated in
myotubes. MEF2 proteins are phosphorylated in muscle cells (Fig.
9B, lane 6). Phosphorylation of MEF2 proteins is
partly inhibited by treatment of cells with SB 203580 (Fig. 9B, lane 8). Consequently, MEF2 proteins are
phosphorylated by p38 in L8 muscle cells.
In this report we suggest for the first time the possible
involvement of p38 in skeletal muscle differentiation. We show that the
activity of p38 was induced in L8 myoblasts during the formation of
multinucleated myotubes. Interference with p38 activity by the specific
inhibitor SB 203580 completely abolished muscle fusion and expression
of all myogenic markers that were tested. We studied the effects of p38
MAPK on muscle-specific transcription and found that its activator,
MKK6, stimulated the expression of muscle-specific genes, and the
effect was mediated by MEF2C. The latter was demonstrated before to be
an essential transcription activator in myogenesis (13, 33, 34).
Members of the MEF2 and MyoD families act within a regulatory network
that establishes the differentiated phenotype of skeletal muscle (13).
MyoD and MEF2 family members work in concert to activate the expression
of many muscle-specific genes including their own. Therefore,
inhibition of the activity of either MEF2 or MyoD family members
results in complete inhibition of skeletal muscle differentiation. p38
MAPK is a potential activator of MEF2C and as part of the MEF2-MyoD
circuit it is expected to control muscle differentiation.
That MEF2 family members played a part in mediating p38 function in the
activation of MCK transcription and myogenesis was suggested by the
following. 1) Induction of MCK reporter gene by MyoD was augmented by
MKK6 only if MEF2 sites were present at the regulatory sequences. MKK6
did not significantly affect the activation of MyoD from promoters that
did not contain MEF2 site (Fig. 6). 2) The effect of MKK6 was
specifically abolished by the expression of a transcriptionally
inactive MEF2C protein (Fig. 7). 3) The transcriptional activity of
GAL4-MEF2C fusion proteins was induced, whereas the activity of
GAL4-MyoD proteins was not changed by MKK6 (Fig. 8).
MEF2C is not the only member of the family that is expressed in muscle.
In fact MEF2A protein accumulates before MEF2C, which is expressed
later during the differentiation of myoblasts (35, 36). For that reason
we should expect that other members of the family may be similarly
regulated by p38. Interestingly, MEF2A contains a serine at the
position equivalent to Ser387 found in MEF2C (37),
suggesting that it may be regulated similarly. Indeed, recent studies
suggest that MEF2A is phosphorylated by p38.2
We noticed that inhibition of p38 in muscle cells also abolished the
expression of MEF2 (Figs. 3A and 5). Inhibition of MEF2 expression may be a result of the repression of MEF2C that functions to
induce its own transcription and/or the repression of other transcription factors such as MyoD or Myf5. Our results do not rule out
the possibility that MyoD was directly affected by p38, because the
reporter gene regulated by MyoD only, 4R-tk-CAT, was mildly affected by
MKK6 or SB 203580 (Fig. 6). Other transcription factors activated by
p38, like ATF2, CREB, and Elk-1 may participate in the differentiation
process induced by p38.
Four isoforms of p38 are known ( Recently, we reported that ERK MAPK activity was induced during the
differentiation of C2 cells and that this activity promoted muscle
differentiation (25). The similarities in the pattern of activities of
p38 and ERK in muscle cells imply that these two distinct pathways have
similar effects on muscle differentiation. However, our data suggest
that the two pathways also exhibit distinct activities. Firstly,
inhibition of ERK with PD 098059 only partially prevented the fusion of
myoblasts and did not inhibit the expression of muscle-specific
markers, whereas inhibition of p38 with SB 203580 prevented fusion of
myoblasts and expression of muscle-specific markers. Secondly, the
activities of ERK and p38 are differentially induced; p38 is induced
earlier than ERK in cells grown in differentiation medium (Ref. 25 and
present work). These differences suggest that the two pathways perform
distinct functions and that their combined activity may be required for
the differentiation process.
Studies of different cell lineages suggest that a distinct balance
between the different MAPK pathways is essential for the survival of
these cells. During maturation of HL-60 cells to the neutrophil
phenotype, the activity of JNK was reduced, whereas the activity of p38
was induced (41). In rat pheochromocytoma PC-12 cells that serve as a
model for neuronal differentiation, induced activity of ERK MAPK and
reduced activities of JNK and p38 pathways were critical for the
survival of these cells (42). The induction of p38 in myocardial cells
served to protect them from apoptosis in one study (43) or to increase
apoptosis in another study (44). In these cellular models, cell
survival or programmed cell death happens as a result of a balance
between the activities of ERK, JNK, and p38 MAPKs. Therefore, it is
possible that the induction of ERK and p38 MAPKs during skeletal muscle differentiation plays a role in preventing programmed cell death as
well as in the activation of muscle-specific genes.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
80 to +7 relative to the transcription start site).
pGEX-ATF2 plasmid was a gift from Dr. Ami Aronheim and Dr. Michael
Karin. The wild type (MKK6) and activated (MKK6b(E)) alleles of MKK6
were described by Han et al. (10). The MEF2C expression
vector (pCDNAI-MEF2C) was a generous gift of Dr. E. Olson (18). The
inactive form of MEF2C was generated by deleting a BglII
fragment that removed amino acids 202-321 of MEF2C. GAL4-MEF2C
constructs were described by Han et al. (12), and GAL4-MyoD
constructs were described by Weintraub et al. (19). pGEX-MEF2C was constructed by inserting a polymerase chain reaction fragment encoding amino acids 128-467 of MEF2C into the pGEX-2T vector.
7 M estradiol.
-D-galactopyranoside assays as described (23), and the
amount of extracts used for the CAT assays were adjusted accordingly (24).
RESULTS
,
,
, and
) are
presently known. To study the relative contributions of p38 isoforms in
the phosphorylation of MEF2C, each isoform was immunodepleted from
muscle extracts. Differential depletion of p38 isoforms affected the
phosphorylation of MEF2C (Fig. 1C, lanes 6-11).
p38
contributed most of p38 kinase activity (40% inhibition),
whereas p38
and
were less active (25 and 10% inhibition,
respectively). Depletion of p38
did not inhibit the phosphorylation
of MEF2C (Fig. 1C, lane 10).
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Fig. 1.
p38 kinase activity is induced during
differentiation of L8 cells. A, the in vitro
kinase activity of p38 was studied using extracts of L8 muscle cells
grown in differentiation medium for different time periods as
indicated. Kinase activity of p38 was analyzed by immunoprecipitation
of p38. The immunocomplex was used to phosphorylate the substrate,
GST-ATF2 protein, as described under "Experimental Procedures."
Proteins were separated over SDS-PAGE, and the amount of phosphorylated
GST-ATF2 was quantified using the PhosphorImager and plotted in the
histogram. Maximal kinase activity was set to 100 units. Average
results from two independent experiments are presented. Bars
represent standard errors. B, the same protein extracts from
L8 cells as in A were separated using SDS-PAGE, and
phosphorylated forms of p38 were identified by Western blotting with an
antibody that recognized the dual phosphorylated form of p38
(upper bands). The same blot was exposed also to an antibody
that recognized all forms of p38 (lower bands). The control
lanes (Cont.): , extract of C6 cells; +, extract of
anisomycine-treated C6 cells. The asterisk represents a
nonspecific band. C, GST-MEF2C protein was phosphorylated
in vitro as described under "Experimental Procedures"
using extracts of L8 cells grown in differentiation medium for
different time periods as indicated. In one case, L8 cells were grown
for 48 h in DM in the presence of 20 µM SB 203580. Isoforms of p38 were depleted from extracts in the following way:
antibodies to the different isoforms of p38 were added to extracts
followed by an immunoprecipitation procedure. p38-depleted extracts
were used to phosphorylate GST-MEF2C. Control lane, GST
protein was used in a kinase assay with extract of L8 cells grown for
48 h in DM.
View larger version (109K):
[in a new window]
Fig. 2.
The p38 inhibitor, SB 203580, prevents the
fusion of L8 myoblasts to multinucleated myotubes. The p38
inhibitor, SB 203580 (20 µM), was added to L8 cells
immediately after the addition of DM when cells were about 80%
confluent. SB 203580 was replaced with fresh drug every 12 h, and
cells were grown under these conditions for 36 h (middle
panel). In a control plate, L8 cells were grown only in the
presence of DM for 36 h (top panel). In a third plate,
cells were treated for 36 h with DM and SB 203580 and for an
additional period of 24 h with DM only (bottom panel).
Arrowheads point at multinucleated myotubes. Cells were
photographed at 200× magnification.
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Fig. 3.
SB 203580 inhibits the expression of
muscle-specific markers in L8 cells. A, L8 cells were
grown in DM and in the presence or absence of SB 203580 for different
periods of time as indicated. Total RNA was extracted from cells,
separated over an agarose gel, and blotted onto a filter. Specific
transcripts were determined by hybridizing the filter to different
labeled DNA probes as indicated. Hybridization to GAPDH was used to
control for loading of RNA on the gel. B, L8 cells were
grown in DM in the presence or absence of SB 203580 as indicated. In
one plate, cells were treated for 48 h with DM and SB 203580 and
for an additional period of 48 h with DM only (lane 4).
Total RNA was extracted and processed as described for
A.
and
, in 10T1/2 cells strongly induced the ability of
MyoD to activate endogenous MHC (data not shown). Therefore we conclude
that activation of p38 MAPK pathway appears to contradict the
inhibitory effects of serum on the function of MyoD as judged by the
activation of endogenous MHC expression.
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Fig. 4.
MyoD activity is up-regulated by MKK6 and
down-regulated by treatment of cells with SB 203580. A,
10T1/2 fibroblasts were transiently transfected either with MyoD
expression vector or with expression vectors of MyoD and MKK6. In one
case, cells that were transfected with MyoD and MKK6 were treated with
20 µM of SB 203580. Transfected cells were grown in the
presence of high serum (growth medium) for 24 h after which they
were fixed and double-stained for MHC (cytoplasmic staining) and for
MyoD (nuclear staining). B, quantification of the results
presented in A. Transfected cells were grown as described in
A, except in one case in which cells were grown in DM for
24 h. The percentage of differentiation was calculated by dividing
the number of double-stained cells (MyoD and MHC) by the total number
of MyoD-stained cells (single- and double-stained). Each bar
in the histogram represents the results of counting of about 100 transfected cells. C, the in vitro kinase
activity of p38 was analyzed using extracts from transfected cells as
described under "Experimental Procedures."
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Fig. 5.
Treatment with SB 203580 prevents muscle
differentiation of cells expressing a conditional MyoD-ER chimera
protein. 10T1/2 cells that constitutively express a chimera
protein of MyoD and estrogen receptor hormone-binding domain were
studied. MyoD activity was induced by the addition of estradiol and
differentiation medium to cells. Some cells were treated with SB 203580 at different concentrations added together with estradiol and DM, and
RNA was extracted 24 h later. RNA was separated over agarose gel
and analyzed by Northern blotting. Blot was repeatedly hybridized with
labeled probes of MyoD, MEF2, myogenin, MLC2, and GAPDH, which was used
to control for loading of RNA. RNA was underloaded in lane 2 as can be noticed by the appearance of a weak GAPDH band.
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Fig. 6.
p38 affects the transcription of MCK reporter
genes via the MEF2-binding sites. A, 10T1/2 fibroblasts
were transiently transfected with expression vectors of MyoD or MEF2C
with or without the direct activator of p38, MKK6. Several reporter
genes were used; pe+AT-CAT carried a regulatory sequence of
MCK enhancer that contained two MEF2-binding sites and MCK promoter;
p110 MCK CAT carried a smaller MCK enhancer that contained one
MEF2-binding site and MCK promoter; 4R-tk-CAT that contained four
MyoD-binding sites in the promoter region and MEF2-MCK-CAT that
contained three MEF2-binding sites and MCK promoter. All reporter genes
contained the CAT reading frame. Protein extracts of the transfected
cells were used in a CAT assay according to the transfection efficiency
that was measured as described under "Experimental Procedures." The
chloramphenicol products were separated by thin layer chromatography
and quantified by phosphor imaging (Fuji). For each reporter gene, the
maximal CAT activity was set to 100 units. Average results from three
independent experiments are presented. Error bars represent
standard errors. B, the same constructs described in
A were used to transfect 10T1/2 cells with the difference
that some plates were treated immediately after transfection with 10 µM SB 203580. Also, the expression vector of GAL4-VP16
was co-transfected with the pGAL-CAT reporter gene. For each reporter
gene, the maximal CAT activity was set to 100 units. Average results
from three independent experiments are presented. Error bars
represent standard errors.
) (12). A similar MEF2A protein that functioned
as a dominant negative was recently described by Ornatsky and
colleagues (32). By itself, MEF2C-
retained minimal transcriptional
activity compared with the wild type protein (Fig.
7A, compare lanes 2 and 6). When co-expressed with wild type MEF2C, the mutant
protein was able to repress the transcriptional activity of the former
protein (Fig. 7A, lanes 2-5). However, the
mutant MEF2C protein did not repress activation by MyoD (lanes 8 and 9) or GAL4-VP16 (lanes 11 and
12) and therefore was specific to wild type MEF2C. To find
out whether the mutant protein could block the effect of p38 on the MCK
regulatory sequences, we transfected the different expression plasmids
with the MCK reporter gene (pe+AT-CAT). The
transcriptionally inactive MEF2C protein inhibited the induced
expression of MCK reporter mediated by MyoD (Fig. 7B,
lanes 1-4). Moreover, it also abolished the additional
activity contributed by MKK6 (Fig. 7B, lanes
5-7). The inhibition was specific to the MCK enhancer that
contained the MEF2-binding site, because the inactive MEF2C protein did
not inhibit MyoD activation of reporter gene containing only
MyoD-binding sites (4R-tk-CAT)(Fig. 7A, lanes 8 and 9). We conclude that MKK6 induced the transcription of
MCK via MEF2 protein(s) and that this induction was blocked by
competition of the transcriptionally inactive MEF2C protein.
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Fig. 7.
A transcriptionally inactive MEF2C protein
abrogates the effects of MKK6 on the MCK enhancer. A,
MEF2C- represses the transcriptional activity of wild type MEF2C
protein. 10T1/2 fibroblasts were transfected with different expression
vectors and reporter genes as indicated. Reporter genes:
Pe+AT-CAT contains enhancer and promoter elements of MCK,
4R-tk-CAT contains four MyoD-binding sites and the basal tk promoter,
and pGAL-CAT contains five GAL4-binding sites and a basal tk promoter.
MEF2C-
expression vector was transfected at 2 µg (×1),
4 µg (×2), and 8 µg (×4). Cells were lyzed
48 h after transfection, and protein extracts of the transfected
cells were used in a CAT assay according to the transfection efficiency
that was measured as described under "Experimental Procedures." The
chloramphenicol products were separated by thin layer chromatography
and quantified by phosphor imaging (Fuji). For each reporter gene, the
maximal CAT activity was set to 100 units. Average results from two
independent experiments are presented. B, MEF2C-
represses the induction of MCK transcription mediated by MyoD and MKK6.
10T1/2 fibroblasts were transfected with different plasmid DNA as
indicated. Reporter gene and expression vectors are described in
A. MEF2C-
expression vector was transfected at 2 µg
(×1) and 4 µg (×2). Transfection, CAT assay
and processing of the results were done as described for A.
Average results from three independent experiments are presented.
Error bars represent standard errors.
HLH MyoD) and another that contained
a substituted DNA-binding domain from the Drosophila
acheate-scute protein (GAL-T4basic MyoD). The transcription
mediated by GAL4-MyoD proteins was not affected by co-expression of
MKK6 (Fig. 8, lanes 7-12).
However, MKK6 augmented the activity of a chimeric GAL4-MEF2C
transcription factor (Fig. 8, lanes 1 and 2). The
activity of two MEF2C mutants that did not contain their p38
phosphorylation sites (293, 300A and 387A) was not significantly
increased by the expression of MKK6 (Fig. 8, lanes 3-6).
Therefore, we conclude that the transcriptional activity of GAL4-MyoD
was not affected, whereas the activity of GAL4-MEF2C was affected by
MKK6.
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Fig. 8.
Transcriptional activity of GAL4-MEF2C
protein is induced, whereas activities of GAL4-MyoD proteins are not
affected by MKK6. 10T1/2 fibroblasts were transfected with
different expression constructs as indicated and the pGAL4-CAT reporter
gene. Cells were lyzed 48 h after transfection, and protein
extracts of the transfected cells were used in a CAT assay according to
the transfection efficiency that was measured as described under
"Experimental Procedures." The chloramphenicol products were
separated by thin layer chromatography and quantified by phosphor
imaging (Fuji). Average results from three independent experiments are
presented. Error bars represent standard errors.
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Fig. 9.
Phosphorylation of MEF2 proteins in
cells. A, 293 cells transfected with GAL4 (lanes
8 and 9) or GAL4-MEF2C (lanes 1-7)
expression vector alone or with MKK6 were labeled with
[35S]methionine. As indicated, some transfected cells
were treated with SB 203580 (20 µM) for 36 h before
proteins were extracted. GAL4 proteins were immunoprecipitated from
protein extracts and separated using SDS-PAGE. Lanes 5-7,
immunoprecipitates were treated with alkaline phosphatase. The
asterisks represent truncated forms of GAL4-MEF2C.
B, dividing myoblasts (0 h in DM) and differentiating
myotubes (48 h in DM) were metabolically labeled with
[35S]methionine (lanes 1-4) or with
[32P]orthophosphate (lanes 5-9). MEF2A and
MEF2C proteins were immunoprecipitated and separated over SDS-PAGE. In
lanes 3 and 7, a competitive peptide against
which the MEF2 antibody was made was added to extracts prior to the
immunoprecipitation procedure. Lanes 4 and 9 are
control lanes; MEF2 antibody was not added to the immunoprecipitation
reaction.
DISCUSSION
,
,
, and
). We suggest
that p38
and
are the major isoforms involved in differentiation of L8 cells because 1) The inhibitor, SB 203580, that is specific to
the
and
isoforms (38-40) inhibited the differentiation of L8
cells (Figs. 2 and 3), and 2) immunodepletion of the
and
but
not of
and
isoforms from L8 extracts significantly inhibited the phosphorylation of GST-MEF2C (Fig. 1C). Interestingly,
our preliminary studies suggest that
and
isoforms are more
efficient than
and
isoforms in the induction of muscle
differentiation when transfected with MyoD. Farther studies will
elucidate the role of p38 isoforms in muscle differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Uri Nudel and Dr. David Yaffe for L8 cells. We thank Dr. Stephen J. Tapscott for the 10T1/2 MyoD-ER cell line and muscle reporter genes. We thank Dr. Jiahuai Han for MKK6 and GAL4-MEF2C constructs and the antibodies to different isoforms of p38. We thank Dr. Ami Aronheim and Dr. Michael Karin for offering us the pGEX-ATF2 plasmid. We thank Dr. Eric Olson for the MEF2C expression vector. We thank Dr. Bianca Raikhlin-Eisenkraft for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a United States-Israel Binational Foundation grant, by an Israel Cancer Association grant, and by funds from the Rappaport Foundation for Medical Research and the Foundation for Promotion of Research in the Technion, Israel Institute of Technology.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.
These authors contributed equally to the work.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Medicine, Technion-Israel Inst. of Technology, P.O. Box 9649, Haifa 31096, Israel. Tel.: 972-4-8295-287; Fax: 972-4-8535-773; E-mail: Bengal{at}tx.technion.ac.il.
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; MCK, muscle creatine kinase; ER, estrogen receptor; ERK, extracellular signal-regulated protein kinase; DM, differentiation medium; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MHC, myosin heavy chain.
2 J. McDermott, personal communication.
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REFERENCES |
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