From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
Received for publication, January 4, 2001, and in revised form, January 24, 2001
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ABSTRACT |
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In many cell types including myoblasts, growth
factors control proliferation and differentiation, in part, via the
mitogen-activated protein kinase (MAPK) pathway (also known as the
extracellular regulated kinase (Erk) pathway). In C2C12 myoblast cells,
insulin-like growth factor-1 and basic fibroblast growth factor
(bFGF) activate MAPK/Erk, and both growth factors promote myoblast
proliferation. However, these factors have opposing roles with respect
to differentiation; insulin-like growth factor-1 enhances muscle cell
differentiation, whereas bFGF inhibits the expression of the
muscle-specific transcription factors MyoD and myogenin. Cells treated
with bFGF and PD98059, a specific inhibitor of the MAPK pathway, show
enhanced expression of the muscle-specific transcription factors MyoD
and myogenin as compared with cells not exposed to this inhibitor.
Inhibiting MAPK activity also enhances myoblast fusion and the
expression of the late differentiation marker myosin heavy chain. Basic
FGF mediated repression of muscle-specific genes does not result from continued cell proliferation, since bFGF-treated cells progress through
only one round of cell division. We have identified a critical boundary
16 to 20 h after plating during which bFGF induced MAPK activity
is able to repress myogenic gene expression and differentiation. Thus,
the targets of MAPK that regulate myogenesis are functional at this
time and their identification is in progress.
The mitogen-activated protein kinase
(MAPK)1 signaling cascade has
been implicated in the regulation of numerous cellular processes including cell growth and differentiation. The activation of the MAPK
pathway occurs through ligand-induced activation of receptor tyrosine
kinases that results in receptor dimerization and autophosphorylation (1). The resulting phosphotyrosine residues of the activated receptors
associate with various downstream targets. For MAPK or Erk activation,
in general, receptor phosphorylation leads to the binding of Grb2/SOS
and activation of Ras, which in turn activates the kinase cascade
consisting of Raf, its target MEK1/2 (MAP/Erk kinase), followed by MAPK
activation. Activated MEK1/2 phosphorylates
Thr183 and Tyr185 of p42/p44
MAPK rendering the kinase active such that it phosphorylates cytoplasmic and nuclear substrates (reviewed in Refs. 2-4). MAPK is
strongly activated by many mitogens, suggesting these kinases may play
an important role in cell proliferation, and by inference, a negative
role in differentiation for appropriate cell types. Somewhat
paradoxically, however, the sustained activation of MAPK's appears to
be necessary for the differentiation and neurite outgrowth process in
the PC12 neuronal cell line (5-9). Microinjection of constitutively
active MAPK into PC12 cells results in their differentiation (8). Also,
co-injection of MAPK with the dual-specificity phosphatase MAP kinase
phosphatase-1, or the expression of dominant negative MAPK inhibits
neuronal cell differentiation (8). On the other hand, MAPK activity
antagonizes the ability of 3T3-L1 preadipocytes to differentiate.
Treatment of these cells with a MEK1 inhibitor (PD98059) enhances their
differentiation, whereas overexpression of MAPK inhibits this process
(10). Moreover, tumor necrosis factor Members of the MRF (muscle regulatory factor) family of proteins,
including Myf-5, MyoD, myogenin, and MRF4, regulate the differentiation
of pluripotent stem cells into multinucleated myotubes (reviewed in
Refs. 11-13). These proteins are transcription factors of the basic
helix-loop-helix type that can transactivate the promoters of many
muscle-specific genes when associated with ubiquitously expressed E box
proteins (12). Although the temporal pattern of MRF gene expression
that controls the progress of muscle cell differentiation has been
characterized, the signaling events that dictate whether myoblasts
either proliferate or exit the cell cycle and express muscle-specific
genes have not been fully described. The positive regulation of
myogenic progression may be controlled by several signal transduction
cascades, including phosphatidylinositol 3-kinase (PI 3-kinase)
(14-16) and the stress-activated protein kinase p38 (17, 18).
Insulin-like growth factor-1 promotes myogenesis in L6E9 cells by way
of downstream signaling through PI 3-kinase since ectopic expression of
a dominant negative p85 subunit of PI 3-kinase inhibits cell
differentiation (16). The addition of the PI 3-kinase inhibitors,
wortmannin or LY294002, to muscle cells inhibits their differentiation
as marked by lack of myogenin expression and creatine kinase activity
(14, 15). The stress-activated protein kinase, p38, plays a positive
role in the differentiation of muscle cells, and the expression of muscle-specific genes and fusion to myotubes is inhibited by the p38
inhibitor, SB203580 (17, 18). Both insulin-like growth factor-1 (IGF-1)
and basic fibroblast growth factor (bFGF) utilize the MAPK pathway, in
part, to promote C2C12 myoblast cell growth (19), but still
it is not clear what role p42/p44 MAPK plays in controlling the
differentiation of skeletal muscle cells. Thus the focus of this study
is to examine the growth factor activated MAPK signaling cascade in the
regulation of muscle cell differentiation.
The differentiation of C2C12 myoblasts seems to be a default pathway
for cells which are deprived of normal growth conditions (serum
deprivation). In vivo, the differentiation process is
tightly regulated by serum growth factors and by autocrine and
paracrine factors. The C2C12 cell line is sensitive to bFGF, a growth
factor known to regulate satellite cell function (20, 21). We show that
bFGF is a more potent activator of p42/p44 MAPK compared with IGF-1 and
this activation represses myogenesis. We use the specific inhibitor of
MEK1 (PD98059) to show that p42/p44 MAPK activity is important for
repression of muscle-specific gene expression and myoblast fusion. In
addition, we determined that bFGF does not repress differentiation by
maintaining the cells in a proliferative state even though it promotes
one round of cell division and maintains the expression of cyclin D1.
MAPK activity is critically important for the suppression of myogenesis
during the 16-20 h after plating. At this time the cells are in late
G1 phase and the early targets of MAPK appear to be
expressed during this period.
Cell Culture
Mouse myoblast cells (C2C12) (22, 23) were maintained as
subconfluent monolayers in Dulbecco's modified Eagle's medium (DMEM)
containing 4.5 g/liter glucose and L-glutamine supplemented with 20% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were rendered quiescent by suspension in
methylcellulose (24, 25). Subconfluent cells were trypsinized and
~2 × 107 cells were suspended in 100 ml of 4%
methylcellulose (Sigma) in DMEM supplemented with 20% FBS and
penicillin/streptomycin. After 72 h the cells were recovered by
dilution with 4 volumes of phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM dibasic
sodium phosphate, 1.8 mM monobasic potassium phosphate, pH
7.4), followed by centrifugation at 2000 × g at
4 °C. Cells were replated in DMEM supplemented with
penicillin/streptomycin and 0.4% calf serum for 1 h in the
presence of the MEK1 specific inhibitor, PD98059 (New England Biolabs,
Beverly, MA) or dimethyl sulfoxide carrier prior to addition of the
indicated concentration of bFGF or IGF-1 (Peprotech, Rocky Hill, NJ).
Approximately 2 × 106 cells were plated for analysis
by Western blotting and 5 × 106 cells were plated for
analysis by Northern blotting. Cells were supplemented every 24 h
with bFGF, IGF-1, and PD98059 for the duration of the experiment.
Phase-contrast Microscopy
Phase-contrast images of the cells were taken 96 h after
treatment using an Olympus IX70 Inverted Research Microscope in
conjunction with a Kodak EOS DCS5A digital camera (Eastman Kodak, New
Haven, CT).
Thymidine Incorporation Assay
Quiescent cells were plated into 24-well culture dishes at
3 × 105 cells per well.
[methyl-3H]thymidine (PerkinElmer Life
Sciences) was added to a final concentration of 1 µCi/ml and
incubated at 37 °C for 24 h. Cells were rinsed twice with PBS
and then fixed in 100% methanol for 10 min at room temperature.
Following fixation, cells were treated with 5% trichloroacetic acid
for 10 min on ice and then samples were collected in 0.3 M
sodium hydroxide and counted using a scintillation counter.
Flow Cytometric Analysis
Myoblasts and myotubes were trypsinized and resuspended in
ice-cold PBS. The cells were fixed by mixing with an equal volume of
100% ice-cold ethanol. Debris was removed by underlaying the fixed
cells with calf serum followed by centrifugation. The cell pellet was
washed once with PBS and then treated with 50 µg/ml RNase (Roche
Molecular Biochemicals, Indianapolis, IN) for 15 min at 37 °C.
Propidium iodide (Sigma) was added to a final concentration of 100 µg/ml and incubated for at least 30 min at room temperature before
analyzing in a Beckman Coulter Epics XL flow cytometer (Beckman
Coulter, Inc., Fullerton, CA).
Western Blotting
Cells were rinsed three times with PBS and then lysed in buffer
containing 25 mM Tris, pH 7.4, 50 mM NaCl,
0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS, 10 µM leupeptin, 1 µM pepstatin, 1 µM aprotinin, 0.1 µM phenylmethylsulfonyl
fluoride, 20 mM sodium fluoride, and 1 mM
sodium orthovanadate. Cell lysates were cleared of insoluble material
by centrifugation at 12,000 × g for 10 min at 4 °C.
Equal amounts of protein were loaded on a 10% or 6-12% continuous
gradient SDS-polyacrylamide gel (26). Separated proteins were
transferred electrophoretically to a 0.2-µm nitrocellulose membrane
(Bio-Rad) using Towbins transfer buffer (390 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol) (27). Membranes
were incubated in 10% nonfat milk in PBS-T (PBS plus 0.1% Tween 20) and then with the following primary antibodies in PBS-T and 2% bovine
serum albumin for 1 h at room temperature; F5D monoclonal antiserum (Developmental Studies Hybridoma Bank, University of Iowa)
specific for myogenin, MF20 monoclonal antiserum (Developmental Studies
Hybridoma Bank) specific for myosin heavy chain, anti-ACTIVE MAPK
antibody (Promega Corp., Madison, WI), that recognizes only the
activated p42/p44 MAPK species, pan-ERK antibody (Transduction Laboratories, Lexington, KY), PC10 (PCNA) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p27KIP1 antibody
(Transduction Laboratories), cyclin D1 antibody (PharMingen, San Diego,
CA). Horseradish peroxidase-conjugated secondary antibodies (Sigma)
were diluted 1:2000 in PBS-T and incubated for 1 h at room
temperature. Proteins were detected using Enhanced Chemiluminescence (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and quantitated using a Molecular Dynamics Densitometer (Amersham Pharmacia Biotech, Inc.) with MD ImageQuant Software version 3.2.
Northern Blotting
RNA was obtained as described by Chomczynski and Sacchi (28).
Cells were rinsed three times with PBS, lysed in Solution D (4 M guanidinium isothiocyanate, 25 mM sodium
citrate, pH 7.0, 0.5% Sarkosyl, 0.1 M
Quantitative Analysis
Determination of Cell Number--
Cells were detached from the
plate with 0.05% trypsin-EDTA and then resuspended in PBS with 0.4%
trypan blue. Viable cells as determined by trypan blue exclusion were
counted using a hemocytometer.
Determination of Protein Concentration--
The amount of
protein was determined using the bicinchoninic acid protein assay
reagent (Pierce, Rockfork, IL). Bovine serum albumin was used as a
protein standard.
Activation of the p42 and p44 MAP Kinase Isoforms Represses
Differentiation in C2C12 Cells--
Various signal transduction
pathways, such as the MAPK cascade, have been implicated in controlling
proliferation, growth arrest, and differentiation. The data concerning
the role of the p42/p44 MAPK pathway in myogenesis is conflicting as
both positive and negative regulatory roles have been suggested
(32-35). The present study addresses the role of bFGF induced p42/p44
MAPK activation in C2C12 differentiation. A cell suspension method was
used (24, 25) that permits growth arrest of C2C12 cells in Go
without subsequent induction of the myogenic program. This technique
allows us to characterize early signaling events induced by growth
factors in a synchronous way and to determine the effect of this
signaling on muscle cell differentiation.
Quiescent C2C12 cells were stimulated with the indicated concentrations
of bFGF or IGF-1 and MAPK phosphorylation was measured after 5 min by
Western blotting with a MAPK antibody that recognizes the activated
form of MAPK (Fig. 1). Activation of MAPK
by bFGF or IGF-1 requires nanomolar concentrations of growth factor,
with bFGF being a significantly more potent upstream activator of MAPK phosphorylation. Compared with IGF-1-treated cells, the addition of
bFGF (0.1-1 nM) to quiescent cells results in MAPK
phosphorylation that is ~13-fold higher than the activation following
IGF-1 treatment. Cells treated with the same growth factor
concentrations were examined for myogenin expression by Western blot
analysis. In control cells (no growth factor treatment) and in cells
treated with nanomolar concentrations of IGF-1, myogenin is expressed at 48 h, while bFGF-treated cells express less than 4% the amount of myogenin observed in control or IGF-1-treated cells. One interesting point is that cells treated with 10 nM IGF-1 exhibit a
slight decrease in myogenin expression due to the proliferative effects of IGF-1 at this concentration
(36).2
The results in Fig. 1 indicate a correlation between the level of bFGF
induced MAPK activity and myogenin expression. The MEK1 inhibitor,
PD98059, was used to block MAPK activation in response to growth factor
stimulation. Quiescent C2C12 cells were incubated with the indicated
concentrations of PD98059 and then stimulated with bFGF or IGF-1. The
dose dependence of the inhibitor on MAPK activity and myogenin protein
expression is shown in Fig. 2. In
agreement with the data presented in Fig. 1, IGF-1 and bFGF activate
the p42 and p44 isoforms of MAPK, although to different extents. Cells
treated with 3 nM IGF-1 and 20 µM PD98059
have no detectable MAPK activity, while cells treated with 1 nM bFGF and increasing concentrations of PD98059 still
retain some of their kinase activity. Even at the highest concentration
(100 µM) of PD98059 tested, the bFGF induced activation
of MAPK is inhibited by ~70%. Most likely, the inability of PD98059
to completely inhibit MAPK phosphorylation is due to the limited
solubility of the inhibitor (19, 37). Alternatively, since PD98059 is specific for MEK1, it may not block signaling that is MEK2 dependent. The addition of PD98059 to cells correlates with a loss of MAPK activity, but not to a loss of MAPK protein expression as determined by
probing the blot with pan-ERK which recognizes the 42- and 44-kDa
isoforms of MAPK (data not shown).
Myogenin protein expression was analyzed under these same conditions to
determine whether the level of MAPK activity correlates with the amount
of muscle-specific marker expression. C2C12 cells stimulated with IGF-1
express myogenin at 48 h after addition of growth factor, in a
manner similar to cells deprived of serum (Fig. 2 compare control and
IGF-1-treated cells). Similar results were observed with control and
IGF-1-treated cells since C2C12 cells express endogenous IGFs as they
differentiate (38-42), and the addition of 3 nM IGF-1 does
not have dramatic effect on differentiation. Meanwhile, the addition of
bFGF inhibits the expression of myogenin (Fig. 2). Interestingly, cells
treated with PD98059 in addition to either IGF-1 or bFGF exhibit
enhanced expression of myogenin protein 48 h after exposure to
growth factor (Fig. 2, lanes labeled FGF+PD and
IGF+PD). At the higher end of the concentration range for
PD98059, a 4.5-fold increase in myogenin expression is observed for
cells incubated with bFGF and PD98059, while a 1.5-fold increase occurs
in cells treated with IGF-1 and PD98059, compared with their
counterparts that saw growth factor alone. These data suggest that
inactivation of MAPK relieves a repression of myogenin expression and
that MAPK activity may play a role in suppressing differentiation in
C2C12 cells.
p42/p44 MAPK Activity Represses the Transcription of
Muscle-specific Genes and Blocking Kinase Activity Leads to Enhanced
Myotube Formation--
To determine whether the
MAPK-dependent reduction of myogenin protein is a result of
decreased mRNA expression, Northern blot analysis was performed.
Total mRNA was isolated at the indicated time during the
differentiation time course and the expression of myoD and
myogenin were examined (Fig.
3A). Up-regulation of both
myoD and myogenin mRNA is observed in control
(no growth factor stimulation) and IGF-1-treated cells by 24 h
post-stimulation. Cells treated with the PD98059 inhibitor, in addition
to IGF-1, undergo a more rapid induction of these mRNAs (Fig.
3A, compare IGF to IGF+PD at 24 h). Alternatively, C2C12 cells stimulated with bFGF exhibit minimal
expression of myoD and myogenin. Cells treated
with both bFGF and PD98059 express both myoD and
myogenin although to a lesser extent and in a delayed time
course compared with control cells. Thus, MRF expression
appears to be transcriptionally regulated by bFGF. To confirm that the
lack of muscle-specific gene expression is reflective of changes in
C2C12 phenotype, the expression of muscle-specific proteins and
myoblast fusion was examined. Quiescent C2C12 cells were treated as in
Fig. 3A and then at the indicated times the cells were
harvested and analyzed by Western blotting for myosin heavy chain (MHC)
and myogenin (Fig. 3B). The expression of muscle marker
proteins is induced in control (no growth factor stimulation) and
IGF-1-treated cells. Myogenin protein expression is evident 24-48 h
after stimulation, along with subsequent induction of MHC protein by
48-72 h. On the other hand, cells treated with bFGF lack expression of
myogenin and MHC, and fail to differentiate into myotubes (Fig.
3C). Cells treated with the MEK1 inhibitor exhibit enhanced
expression of differentiation markers (Fig. 3B, lanes
labeled FGF+PD and IGF+PD). The combined
treatment of IGF-1 and PD98059 results in an earlier induction of both
myogenin and MHC proteins (Fig. 3B, compare IGF
to IGF+PD at 24 h). Moreover, the treatment of cells
with bFGF and PD98059 also leads to fusion of cells into multinucleated myotubes (Fig. 3C) and the induction of myogenin and MHC
proteins (Fig. 3B, compare FGF to
FGF+PD). Cells treated with both bFGF and the inhibitor
demonstrate a 6-8-fold increase in myogenin over cells treated with
bFGF alone. The differentiation of bFGF-treated C2C12 cells cannot be
completely restored by the addition of PD98059, perhaps because of the
limited solubility of the inhibitor or because of its specificity for
MEK1 as mentioned above.
Basic FGF Promotes One Round of Cell Division, but Cells Fail to
Growth Arrest in Go--
The inhibition of differentiation
by MAPK activity may result from the ability of bFGF to maintain the
cells in a proliferative state. The possibility that bFGF is causing
the cells to proliferate throughout the "differentiation" time
course was tested by assaying the incorporation of tritiated thymidine
into DNA as a marker of cell cycle progression (S phase). Quiescent
cells treated with fetal bovine serum were used as an indicator of
continuous cell cycling and the amount of radiolabeled thymidine
detected in these cells steadily increases over the 96-h time course
(Fig. 4A). Otherwise,
quiescent cells were exposed to bFGF or IGF-1 in the presence or
absence of PD98059 and these cells incorporate radiolabeled thymidine
primarily during the 24-48-h time period after plating (Fig.
4A). Although cells treated with both bFGF and PD98059
undergo DNA synthesis, they have a profile of thymidine incorporation that is more similar to control cells than to bFGF-treated cells. The
bFGF-treated cells incorporate 20% of the labeled thymidine compared
with proliferating cells at times after 48 h, but they incorporate
150% of the radiolabel compared with differentiated (control) cells,
indicating that a small percentage of the cells may be maintained in
the cell cycle. To confirm that the synthesis of DNA and therefore
progression through S phase correlates with cell doubling, cells
treated under the same conditions were counted at each 24 h period to
determine changes in cell number. As shown in Fig. 4B, bFGF-
and IGF-1-treated cells (± PD) undergo only one round of cell
doubling, while cells treated with fetal bovine serum go through at
least three doublings over the 72 h time course. These data indicate
that both bFGF and IGF-1 initiate C2C12 cell cycle progression, but
after one round of division, the cells growth arrest.
The proliferative state of the cells over the 72 h time course was
analyzed by examining several markers of cell cycle in quiescent C2C12
cells treated with or without PD98059 and bFGF or IGF-1 (Fig.
5). Proliferating cell nuclear antigen
(PCNA) is a protein that associates with DNA polymerase subunits whose
expression is a marker of cycling cells. The expression of PCNA is
increased in growth factor-treated cells during the first 48 h
(Fig. 5, compare control with IGF-1 and bFGF
lanes) and after this time drops to below basal (0 h) levels.
These data are consistent with the thymidine incorporation results (see
Fig. 4), and suggests that the cells are not proliferating at later
times (72 h). The expression of PCNA in bFGF + PD-treated cells mirrors
the expression in cells treated with bFGF alone, which is not
surprising as their time course for DNA synthesis is similar.
Cyclin D1, which is expressed during G1 and S phase, was
examined to determine whether the expression of cell cycle regulatory proteins may be altered in cells exposed to bFGF. Cyclin D1 expression is barely detectable in cells not exposed to growth factor (Fig. 5,
lanes labeled control cells). On the other hand, bFGF (± PD)-treated cells express cyclin D1 through 30 h during which time
the cells are proliferating (see Fig. 4) and then its expression drops
to basal levels at 48 h. Cyclin D1 is also expressed in IGF-1 (± PD)-treated cells, although to a lesser extent and for a shorter time
than cells exposed to FGF. One striking difference in cyclin D1
expression is apparent at 72 h when bFGF-treated cells regain expression of cyclin D1 compared with their counterparts that were
exposed to IGF-1 or to both bFGF and PD98059. Thus, the maintained expression of cell cycle regulatory proteins in bFGF-treated cells may
prevent the progress of differentiation.
Cells exposed to bFGF are no longer cycling after 48 h, therefore,
we determined whether or not the cells were growth arrested in
Go by examining the expression of the
cyclin-dependent kinase inhibitor p27KIP1. At
time 0, the p27KIP1 expression is high since the cells were
made quiescent by suspension (Fig. 5, lane labeled 0 h). As
the cells differentiate, they maintain their level of
p27KIP1 expression, which decreases only when a small
population of the cells enter the cell cycle between 24 and 30 h
(Fig. 5, lanes labeled control). Also, expression of
p27KIP1 is diminished in IGF (± PD)-treated cells as they
proliferate, but it is regained at later times as the cells
differentiate. Similarly, bFGF + PD-treated cells expressed
p27KIP1, although to a lesser extent than control and
IGF-1-treated cells. On the other hand, the low level of
p27KIP1 expression in bFGF-treated cells leads us to
conclude that these cells are not growth arrested in Go. These
results give rise to an alternative hypothesis; bFGF-treated cells
undergo one round of cell division and then are "stuck" at some
point in the cell cycle.
To determine at what phase of the cell cycle C2C12 cells are in as a
function of the differentiation time course, we performed flow
cytometric analysis on differentiating, proliferating, and bFGF-treated
cells (Fig. 6). Quiescent C2C12 cells
were incubated with or without PD98059 and then stimulated or not with
FBS or bFGF. At the indicated time, the cells were fixed, stained with propidium iodide, and analyzed for DNA content using a flow cytometer. At time 0 about 90-95% of the cells are in
G0/G1. By 20 h of treatment, cells exposed
to FBS and bFGF enter S phase, while control cells (no serum) and bFGF + PD-treated cells are still maintained in G0/G1. Throughout the differentiation time
course, similar profiles of cell cycle progress are observed between
control and bFGF + PD-treated cells, and between bFGF- and FBS-treated
cells, although cells exposed to bFGF alone appear to accumulate in
G2/M phase at later times. These results suggest that after
16-20 h, the cells either proliferate or remain in
G0/G1 and later differentiate, and this
decision is dependent on the activation of MAPK (see Fig.
7).
p42/p44 MAPK Activity Is Required between 16 and 20 h After
Plating to Inhibit Myogenesis--
The fact that differentiating
(control) cells and bFGF-treated cells exhibit similar profiles for
their phases of the cell cycle up to 20 h, but differ thereafter,
suggests that bFGF may be regulating events that are occurring in the
20-h period after cell plating that block the pathway to
differentiation. To determine whether the continual presence of the
growth factor during this period was necessary to inhibit myogenin
expression, hence differentiation, bFGF was added at time 0 and then
withdrawn at the various times indicated (Fig. 7A). Progress
toward differentiation was monitored by myogenin expression 48 h
after plating. PD98059 was added upon withdrawal to prevent any MAPK
activation that may be occurring due to the presence of internalized
bFGF receptor (43) that might escape the washing procedure and
addition of PD98059 did not have a significant effect on myogenin
expression (not shown). Exposure of cells to bFGF for 12 h and
then withdrawal resulted in no inhibition of myogenin expression, but
after 16 h, a 40% decrease in this MRF was observed.
Interestingly, bFGF-dependent MAPK activity was somewhat
diminished at early time points, possibly due to the induction of MAP
kinase phosphatase-1 upon growth factor exposure (see Ref. 44, and
reviewed in Refs. 45 and 46). Next, cells were plated in DMEM and bFGF
was added at various times thereafter (Fig. 7B). Myogenin
expression at 48 h and MAPK activation immediately following bFGF
addition were monitored as in the previous figure. Addition of bFGF up
to 20 h after plating effectively inhibited myogenin expression
and MHC expression (data not shown), but this treatment was ineffective
when added 24 h after plating. However, bFGF added at 24 h
activated MAPK to the same extent as it does when added at any other
time, indicating that the ability of bFGF to activate MAPK is not lost
between 20 and 24 h of incubation and therefore is not the reason
for the ineffectiveness of bFGF at this time. Taken together, the data
indicate that the ability of bFGF to inhibit myogenesis is mediated by
MAPK activity at a time between 16 and 20 h after cell plating.
The growth and regeneration of postnatal and adult skeletal muscle
is mediated by satellite cells that reside between the basement
membrane and plasma membrane of myofibers. Satellite cells are usually
mitotically quiescent in mature muscle, but the cells become active
following injury, exercise, denervation, or in diseased states. Various
extracellular stimuli result in the proliferation, differentiation, and
fusion of satellite cells with nearby muscle fibers. Growth factors,
such as bFGF, IGF-1, and transforming growth factor- Our results also show that bFGF and IGF-1 are mitogenic in C2C12 cells
and cause one round of cell proliferation, even though these growth
factors differ in their level of MAPK activation and consequently,
their role in differentiation. Interestingly, IGF-1, which is a
positive mediator of muscle differentiation, activates MAPK to a slight
extent in G0 arrested C2C12 cells compared with bFGF.
Obstruction of the IGF-1 induced MAPK signal by PD98059 results in an
earlier induction of myogenesis. The slight activation of MAPK by IGF-1
is substantial enough to slightly delay the progression of
differentiation. We suggest that in growth factor-stimulated C2C12
cells, the ability of cells to differentiate is limited by the level of
activated MAPK. Since bFGF stimulates p42/p44 MAPK activity to a much
greater extent than IGF-1, only the former is able to inhibit the
myogenic program. In similar studies, PD98059 treatment of
IGF-1-stimulated L6A1 myoblasts results in markedly enhanced myogenin
expression and creatine kinase activity (15). These data suggests that
the activation of MAPK is a key regulator of the myogenic process.
Cells may have a sensing mechanism which distinguishes between the
intensity of cellular signals that allows for fine tuned regulation of differentiation.
One striking feature of bFGF-mediated repression is the limited
requirement for MAPK activity following stimulation. In fact, a MAPK
signal from the time of replating is not necessary, since bFGF can be
added to the cells 16-20 h after plating and the cells remain
undifferentiated. The addition of bFGF 24 h after replating is not
effective in repressing differentiation, even though MAPK is fully
activated under these conditions. These results indicate that there is
a critical time frame during which MAPK can inhibit differentiation,
probably because the MAPK substrates and/or their molecular interactors
are available during this window. After 24 h, the cells enter a
commitment phase where they are no longer affected by bFGF. Along
these lines, we have observed in serum-deprived C2C12 cells that
p42/p44 MAPK activity decreases by 24 h after withdrawal.3 It is not
surprising that the MAPK activity decreases as differentiation progresses, considering its strong "anti-myogenic" effects.
Previous studies (32, 34) suggest that p42/p44 MAPK activity increases during myoblast differentiation. It is possible that distinctions in
the cell culture methods may account for these differences from our
results, and indeed the cell suspension system used in our studies are
preferred to examine the regulation by signaling pathways. In any case,
all of our data suggests that p42/p44 MAPK activity inhibits
differentiation. Interestingly, it has been suggested that the
sustained p42 MAPK activity observed in the cells is due to an IGFII
autocrine loop (32). It is possible that early in differentiation an
insulin/IGF stimuli coordinates differentiation via activation of PI
3-kinase, as demonstrated in various myoblast cell lines, including
L6A1, L6E9 (14, 15), and in chicken embryo myoblasts (51) and that MAPK
activity may be needed at later times.
Recent studies which examine the role of MAPK in adipocyte
differentiation have suggested that one possible target of MAPK in
preadipocytes is peroxisome proliferator-activated receptor- It is known that bFGF acts as a mitogen and could be altering the cell
cycle proteins. Studies indicate that D-type cyclins are both
positively and negatively regulated by bFGF. We have shown that cyclin
D1 expression is up-regulated in bFGF-treated C2C12 cells and is
dependent on MAPK activation since cells treated with bFGF and PD98059
do not maintain cyclin D1 expression at later times. These results are
consistent with reports that in C2C12 myoblasts, bFGF induces cyclin D1
and inhibits cyclin D3 expression whereas in the absence of mitogens
cyclin D1 levels decrease (53). In addition, overexpression of cyclin
D1 in C2C12 (53) and 10T1/2 (54) cells inhibits differentiation, while overexpression of other cyclin D isoforms did not have any effect on
differentiation. In the latter study, it was shown that cyclin D1 acts
by inhibiting MyoD from transactivating muscle-specific promoters and
this inhibition was due to the phosphorylation of MyoD. The MRF's are
not expressed in bFGF-treated C2C12 cells and therefore cyclin
D1/cyclin-dependent kinase complexes are not regulating
myogenesis by altering their transactivation function. Since the effect
of bFGF on cyclin D1 expression is not apparent until 72 h, this
response may or may not be directly mediated by MAPK.
Finding the targets of signaling molecules that regulate the
differentiation process have been difficult since the signaling induced
by extracellular mediators usually occurs within minutes, but the
resulting effect on differentiation is not apparent for many hours or
days. We have identified the 16-20-h period that bFGF induced MAPK
activity is essential to inhibit myoblast differentiation. During this
time the cells are probably in late G1 phase considering that they do not incorporate thymidine until 24 h and they have the DNA content of cells in G1 (Figs. 4 and 6). These data
is consistent with the earlier results that bFGF inhibits MM14 myoblast differentiation at the G1 phase (55). Therefore, the MAPK
substrates at early times in the differentiation time course may be
proteins expressed during G0/G1 phase, and our
efforts will now focus on finding the targets of bFGF induced MAPK that
are responsible for inhibiting C2C12 differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, an inhibitor of
adipogenesis, activates MAPK and combined treatment of 3T3-L1 cells
with tumor necrosis factor
and PD98059 restores their
differentiation (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol), followed by acid phenol (pH 4.2):chloroform
extraction. Total RNA was precipitated in isopropyl alcohol at
20 °C, followed by centrifugation at 10,000 × g
at 4 °C for 20 min. Equal amounts (20 µg) of RNA were separated on
a 1% agarose, 6% formaldehyde gel and transferred to
GeneScreen nylon membrane (PerkinElmer Life Sciences) by capillary
action using 10 × SSC (1.5 M NaCl, 0.15 M
sodium citrate, pH 7.0) as the liquid phase. The RNA was cross-linked
to the membrane using the UV Stratalinker (Stratagene, La Jolla, CA).
RNA was checked for equal loading and transfer by UV visualization of
ethidium bromide rRNA staining. Prehybridization of nylon membranes was for 4-6 h at 42 °C in 50% formamide, 4 × SSC, 5 × Denhardts (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% bovine
serum albumin), 0.05 M sodium phosphate, pH 7.0, 0.5 mg/ml
sodium pyrophosphate, 1% SDS, 0.1 mg/ml tRNA carrier. Hybridization of
the blot was for 16-20 h at 42 °C in 50% formamide, 4 × SSC,
1 × Denhardts, 0.05 M sodium phosphate (pH 7.0), 0.5 mg/ml sodium pyrophosphate, 1% SDS, 0.1 mg/ml tRNA carrier, and the
labeled probe. The cDNA inserts used for probes were MyoD (29) and
myogenin (30). The cDNA probes were labeled with
[32P]dATP by random priming using the method of Feinberg
and Vogelstein (31) and purified with a NucTrap column (Stratagene).
The specific activity of the probes were at least 108 cpm.
Blots were washed in a stepwise gradient at moderate stringency (0.2 × SSC, 0.1% SDS at 42 °C) and exposed to film at
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
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Fig. 1.
Basic FGF is a much more potent
activator of p42/p44 MAPK than IGF-1. Methylcellulose-suspended
C2C12 cells were replated for 1 h as described under
"Experimental Procedures" and then treated with the indicated
concentration of bFGF or IGF-1. A, total protein was
harvested either 5 min (activated MAPK**) or 48 h (myogenin
expression) following growth factor addition and equal amounts (100 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis.
Proteins were transferred to a nitrocellulose membrane and analyzed for
activated MAPK (MAPK**) or myogenin by Western blot. B,
densitometry was performed on blots in part A using a
Molecular Dynamics densitometer with MD ImageQuant Software version
3.2: open bar, IGF; striped bar, FGF;
dotted bar, control. Minus ( ) indicates control
cells that were maintained in DMEM + 0.4% calf serum. The results are
representative of three independent experiments with similar
results.
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Fig. 2.
The inhibition of MAPK activity by the
MEK1 inhibitor, PD98059, is dose dependent and allows myogenin
expression. Suspended C2C12 cells were replated for 1 h with
the indicated concentration of PD98059, stimulated with 1 nM bFGF or 3 nM IGF-1 and then harvested 5 min
(activated MAPK**) or 48 h (myogenin expression) after growth
factor addition. A, total protein (100 µg) was separated
by 10% SDS-polyacrylamide gel electrophoresis, blotted to a
nitrocellulose membrane, and examined for activated MAPK isoforms
(MAPK**) or myogenin by Western blotting techniques. B,
densitometry was performed with blots from part A using a
Molecular Dynamics densitometer with MD ImageQuant Software version
3.2: open bar, IGF; striped bar, FGF;
dotted bar, control. Minus ( ) indicates control
cells that were maintained in DMEM + 0.4% calf serum. The results are
representative of three independent experiments with similar
results.
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[in a new window]
Fig. 3.
Inhibition of MAPK signaling results in
enhanced myogenin and MHC expression, and fusion to multinucleated
myotubes. Suspended C2C12 cells were replated for 1 h in the
presence of 40 µM PD98059 or dimethyl sulfoxide and then
treated with 1 nM bFGF or 3 nM IGF-1.
A, total RNA was isolated at the indicated times
and 20 µg from each sample was separated on an agarose/formaldehyde
gel followed by transfer to a nylon membrane. Blots were probed with at
least 107 cpm/ml of purified 32P-labeled
myogenin or MyoD cDNA. Ethidium bromide staining was used to
establish equal loading. B, total protein was harvested at
the indicated times, and equal amounts (100 µg) of protein were
separated by SDS-PAGE (6-12%), blotted to a nitrocellulose membrane,
and probed with F5D (anti-myogenin) and MF20 (anti-MHC) antibodies.
C, phase-contrast images of the cells were taken 96 h
after treatment using an Olympus IX70 Inverted Research Microscope.
Magnification equals ×15. Arrows indicate multinucleated
myotubes. Control cells were cultured in only DMEM + 0.4% calf serum.
The data presented is representative of three independent
experiments.
View larger version (25K):
[in a new window]
Fig. 4.
Growth factors alone do not maintain C2C12
cells in a continuous proliferative state. A, suspended
C2C12 cells were replated for 1 h in the presence of 40 µM PD98059 or dimethyl sulfoxide. Cells were then treated
with or without 1 nM bFGF, 3 nM IGF-1, or 20%
FBS. At 0, 24, 48, and 72 h, 1 µCi/ml
[methyl-3H]thymidine was added to the cells
and incubated for a total of 24 h. Immediately following the 24-h
incubation, cells were fixed in ice-cold methanol and the DNA was
collected as described under "Experimental Procedures." The total
amount of [methyl-3H]thymidine incorporated
was detected by scintillation counting. Control cells were cultured in
DMEM + 0.4% calf serum. At each time point, four wells/condition were
assayed. The data presented is representative of two separate
experiments with similar results. B, cells treated as in
part A were used to determine cell number at 24, 48, and
72 h. At each time point, cells were detached from the cell
culture plate with trypsin/EDTA, resuspended in PBS containing trypan
blue, and counted using a hemocytometer. The percentage of cell death
(<5%) as determined by trypan blue exclusion was similar for cells at
each condition. At each time point at least four individual
plates/condition were counted. The data presented is representative of
two separate experiments with similar results.
View larger version (30K):
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Fig. 5.
Basic FGF-treated cells do not growth arrest
in Go after one round of cell division. Suspended
C2C12 cells were replated for 1 h in the presence of 40 µM PD98059 or dimethyl sulfoxide and then treated with or
without 1 nM bFGF or 3 nM IGF-1. Total protein
was harvested at the indicated times. Equal amounts (100 µg) of
protein were separated by 10% SDS-polyacrylamide gel electrophoresis,
transferred to a nitrocellulose membrane, and probed with PCNA, cyclin
D1, p27KIP1, and MF20 (anti-MHC) antibodies. Control cells
were cultured in only DMEM + 0.4% calf serum. The data is
representative of three individual experiments with similar results,
except for the cyclin D1 data which is representative of two
independent experiments.
View larger version (21K):
[in a new window]
Fig. 6.
Characterization of cell cycle progress in
proliferating and differentiating C2C12 cells by flow cytometry.
Suspended C2C12 cells were replated for 1 h in the presence of 40 µM PD98059 or dimethyl sulfoxide and then treated with or
without 1 nM bFGF or 20% FBS. At the indicated time, cells
were collected and fixed in ice-cold ethanol as indicated under
"Experimental Procedures." On the day of analysis, cells were
treated with RNase, and then stained with 100 µg/ml propidium iodide
at room temperature. DNA content was analyzed by flow cytometry using a
Beckman-Coulter Epics XL Flow Cytometer with Expo software. The
experiment was performed twice with similar results.
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[in a new window]
Fig. 7.
Basic FGF acts to inhibit myoblast
differentiation between 16 and 20 h following plating and is
ineffective if added after 24 h. A, suspended
C2C12 cells were replated and then stimulated at time 0 with 1 nM bFGF. At the hour indicated, the cells were washed three
times with PBS, and replated in DMEM. Total protein was harvested after
washing (activated MAPK**) or at 48 h (myogenin expression).
B, suspended C2C12 cells were replated and then were
stimulated at the indicated hour with 1 nM bFGF. Total
protein was harvested 5 min after growth factor addition (activated
MAPK**) or at 48 h (myogenin expression). For both A
and B, equal amounts (100 µg) of protein were separated by
SDS-polyacrylamide gel electrophoresis (10%) and blotted to a
nitrocellulose membrane. Western blot analyses were performed with F5D
(anti-myogenin) and anti-activated MAPK antibody (MAPK**) antibodies,
as described under "Experimental Procedures." Minus ( )
cells without bFGF treatment (control cells). Plus
(+) cells treated with bFGF for 48 h. Densitometry was
performed with myogenin blots using a Molecular Dynamics densitometer
with MD ImageQuant Software version 3.2. These data is representative
of three separate experiments with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
have been
implicated in the regulation of satellite cell proliferation and
differentiation (reviewed in Refs. 20 and 21). Basic FGF is mitogenic,
but can also act as a negative regulator of myogenesis by inhibiting
the expression and/or activity of the MRFs (47-50). The signaling
events that mediate repression of myogenesis by bFGF have not been
clearly identified. The regulation of myogenesis has mainly been
studied using conditions where differentiation is achieved following
withdrawal of serum components. In the present study, a cell suspension
system was used that arrests the cells in G0 without the
subsequent induction of myogenic markers (25), allowing us to examine
the growth factor effects on a synchronized system and enabling us to
detect changes in signaling that may be crucial for the control of
differentiation. Using this system, p42/p44 MAPKs were identified as
potential regulators of bFGF-mediated myogenic inhibition. A robust
activation of p42/p44 MAPK occurs upon bFGF stimulation of
G0 arrested C2C12 cells. A chemical inhibitor of p42/p44
MAPK signaling, PD98059, was used to show that MAPK activity represses
the expression of several muscle-specific transcription factors,
including myogenin and MyoD, and the subsequent expression of
differentiation markers, such as myosin heavy chain. These results are
consistent with reports that overexpression of MAP kinase phosphatase-1
could inhibit mitogen-induced p42 MAPK activity, resulting in the
expression of MyoD, myogenin, and MHC (33). A potent bFGF induced
signal is sufficient for complete inhibition of muscle-specific marker expression (our results and Ref. 36). In addition, bFGF is
capable of overriding positive regulatory effects normally imposed by IGF-1 in C2C12 cells (36).
, a key regulator of adipogenesis that gets phosphorylated and this modification represses this transcription factors activity and thus
suppresses differentiation (52). As peroxisome proliferator-activated receptor-
is a key modulator of adipogenesis, one could
hypothesize that the primary transcriptional regulators of myogenesis
may also be regulated in this fashion. Of the four MRF family members, only MyoD and myf5 contain at least one potential p42/p44 MAPK phosphorylation site with the consensus Pro-X-(Ser/Thr)-Pro,
while myogenin and MRF4 contain the minimal phosphorylation site(s) (Ser/Thr)-Pro. In the quiescent C2C12 culture system used in these studies, it is unlikely that bFGF induced MAPK activity is altering the
activity of the MRFs, in particular MyoD and myogenin, since these genes are not expressed significantly in bFGF-treated cells. Basic FGF has been implicated in controlling the mRNA expression of
the muscle-specific transcription factors, MyoD and myogenin (Refs. 47
and 48, and our results). On the other hand, in myoblasts which express
these transcription factors post-translational modification, such as
phosphorylation of the MRF's, may be the underlying regulatory
mechanism for altering their transcriptional activity and/or their
association with important transcriptional cofactors. For instance,
PKC-dependent phosphorylation of myogenin has been shown to
inhibit its transcriptional activity (49). The possibility that bFGF
alters the interaction of MRF's with necessary cofactors has also been
addressed (50).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Andrew Lassar, Harvard Medical School, for the MyoD1 plasmid, Suzan Lazro, Dana Farber Cancer Institute, for assistance with the flow cytometer, Dr. Stephen Farmer, Boston University School of Medicine, for helpful discussions, reagents, and critical reading of the manuscript, and Drs. Ron Morrison and Matthew Nugent, Boston University School of Medicine, for helpful discussions and reagents.
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FOOTNOTES |
---|
* This work was supported by National Institute of Health Grants DK-30425 (to P. F. P.) and DK-49147 (to N. B. Ruderman).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.
Supported by National Institutes of Health Training Grant AG00115
from the NIA (to P. Polgar).
§ To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118. Tel.: 617-638-4044; Fax: 617-638-5339; E-mail: pilch@biochem.bumc.bu.edu.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M100091200
2 L. Tortorella and P. Pilch unpublished data.
3 L. Tortorella and P. Pilch, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; Erk, extracellular regulated kinase; MEK, MAPK/Erk kinase; SOS, son of sevenless; Grb2, growth factor receptor-bound protein; MRF, muscle regulatory factor; PI 3-kinase, phosphatidylinositol 3-kinase; IGF-1, insulin-like growth factor-1; bFGF, basic fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MHC, myosin heavy chain; PCNA, proliferating cell nuclear antigen; PBS, phosphate-buffered saline.
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