Extracellular Signal-Regulated Kinase-1 and -2 Respond Differently to Mitogenic and Differentiative Signaling Pathways in Myoblasts
Dos D. Sarbassov,
Linda G. Jones and
Charlotte A. Peterson
Departments of Medicine and Biochemistry and Molecular Biology
University of Arkansas for Medical Sciences and the Geriatric
Research, Education, and Clinical Center McClellan Veterans
Hospital Little Rock, Arkansas 72205
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ABSTRACT
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In this report we show that extracellular
signal-regulated kinase-1 and -2 (ERK-1 and -2) respond differently to
signals that elicit proliferation and/or differentiation of myoblasts
using the C2C12 cell line and nondifferentiating mutant NFB4 cells
derived from them. Induction of differentiation by withdrawal of serum
rendered ERKs in C2C12 myoblasts relatively insensitive to
restimulation by serum. Instead, myogenic differentiation of C2C12
cells was associated with sustained activation of ERK-2 dependent on
the insulin-like growth factor II (IGF-II) autocrine loop. By contrast,
mutant NFB4 cells cultured under the same conditions remained
proliferative and demonstrated robust activation of ERKs in response to
serum. Similarly, a Gi-dependent signaling
pathway induced activation of ERKs in NFB4 cells, but not in C2C12
cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells
partially rescued by prolonged IGF-I treatment, ERK activity remained
responsive to Gi-dependent LPA stimulation,
whereas rescue of NFB4 cells by constitutive expression of myogenin or
MyoD, associated with activation of the IGF-II autocrine loop, rendered
the Gi-signaling pathway refractory to LPA
stimulation. Relatively high levels of G
i2
were detected in NFB4 cells and IGF-I treated NFB4 cells, which
correlated with responsive Gi signaling.
Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or
treatment with IGF-II was associated with loss of
G
i2 and inhibition of
Gi-dependent signaling. Thus, IGF-I and IGF-II
activate distinct signaling cascades, with IGF-II eliciting a stronger
differentiation effect correlated with down-regulation of
G
i2 protein. Short-term stimulation of NFB4
cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1
and -2 activation. Transient stimulation of NFB4 cells with IGF-I while
blocking activation of Gi-proteins is with
pertussis toxin resulted in preferential activation of ERK-2
characteristic of differentiated C2C12 cells, suggesting that
proliferation induced by IGF-I is Gi-dependent
and separable from the IGF-I-signaling pathway that leads to
differentiation.
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INTRODUCTION
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Many growth factor tyrosine kinase receptors transduce signals to
the cytoplasm through a common pathway involving the GTP-binding
protein p21ras, thereby leading to activation of sequential
protein kinase reactions. This pathway diverges upon activation of
extracellular signal-regulated kinases (ERKs), also referred to as
mitogen-activated protein kinases (MAPKs) (1, 2). GTP-bound
p21ras activates raf kinase by its recruitment to the
plasma membrane. Raf phosphorylates and thereby activates MAPK/ERK
kinase (MEK), which in turn phosphorylates ERKs on threonine and
tyrosine residues that are separated by one amino acid. This dual
phosphorylation of ERKs is required for their activation (3, 4, 5). The
biological role of ERKs, a typical family of serine/threonine kinases,
has been the subject of intensive study. Two members of this family,
ERK-1 and -2, are widely expressed and well characterized. Activation
of these ERKs is involved in cellular proliferation and
differentiation, indicating that downstream cellular responses
initiated by ERKs may vary and trigger mutually exclusive events
(6, 7, 8). For instance, stimulation of neuronal cells by a variety of
growth factors resulted in ERK activation leading to different cellular
responses. Insulin-like growth factor-I (IGF-I) and epidermal growth
factor appeared to be mitogenic for these cells, whereas neuronal
differentiation was induced by basic fibroblast growth factor and nerve
growth factor. Cellular proliferation correlated with a transient peak
of ERK activity, and sustained activation of ERKs was observed during
differentiation (9, 10, 11). These observations indicated that the duration
of ERK activity is critical for cell-signaling decisions.
Seven membrane-spanning receptors that interact with Bordella pertussis
toxin (PT)-sensitive heterotrimeric G proteins also are able to mediate
p21ras-dependent activation of ERKs (12, 13).
Receptor activation stimulates nucleotide exchange and
dissociation of the G protein, releasing the G
i subunit
in its GTP-bound state from the Gß
complex.
Gß
complex mediates src-dependent phosphorylation of
the epidermal growth factor receptor and thereby activation of the
p21ras pathway (14). Employing this mechanism, G
protein-coupled receptors can also contribute to
p21ras-dependent cellular response.
The role of ERK activity in myogenic growth and differentiation is not
well understood. The formation of skeletal muscle in embryogenesis
proceeds through commitment of mesodermal progenitors to the myogenic
lineage and subsequent differentiation of skeletal myoblasts into
terminally differentiated myotubes. Growth factors, such as basic
fibroblast growth factor and transforming growth factor-ß, play a
central role in maintenance of myoblasts in the proliferative state
that is nonpermissive for the expression of muscle-specific genes (15, 16). Basic fibroblast growth factor-stimulated proliferation is
accompanied by robust and transient ERK activation (17). Exit from the
cell cycle that can be forced by serum withdrawal from the medium
induces terminal differentiation. Transition of myoblasts to myotubes
is accompanied by activation of the IGF-II autocrine loop and appears
absolutely required (Ref. 18; reviewed in Ref.19). The IGF-I receptor
is the main mediator of both IGF-I and IGF-II signaling in myoblasts
(20), where IGF-I promoted proliferation followed by differentiation,
whereas IGF-II demonstrated less mitogenic effect (21). Activation of
the IGF-I receptor by binding of IGFs induces tyrosine phosphorylation
of insulin receptor substrate-1 (IRS-1), the major substrate for
insulin and type I IGF tyrosine kinase receptors, and Shc proteins. The
Grb-2 adaptor protein links IRS-1, and alternatively Shc proteins, to
the p21ras-signaling pathway, resulting in activation of
ERK-1 and -2 (22, 23). Recently it was shown that the ERK pathway
mediates primarily the proliferative effects of IGF-I on myoblasts
(24). In our previous study, we characterized the IGF signal
transduction pathway during myogenic differentiation of C2C12 myoblasts
and nondifferentiating mutant NFB4 cells that fail to activate the
IGF-II autocrine loop and require exogenous IGFs to induce
differentiation (25). Exogenous IGF-I partially rescued the mutant
phenotype, making these cells useful tools for manipulating the IGF
pathway and analyzing downstream signaling components.
In this study we analyzed ERK activity in C2C12 cells during myogenic
differentiation and also in nondifferentiating mutant NFB4 cells
derived from them by chemical mutagenesis. After exposure of cells to
low serum, ERK-1 and -2 in NFB4 cells remained responsive to
stimulation by mitogens whereas they were much less responsive in C2C12
cells. Reactivation of ERKs occurred in response to different mitogenic
signals and appeared dependent on signaling through Gi
proteins. Furthermore, induction of differentiation in both cell types
was correlated with preferential and sustained activation of ERK-2.
Activation of ERK-1 and -2 was an early response to IGF-I, which could
be blocked by PT leading to sustained activation of ERK-2. Thus, the
biphasic response of myoblasts to IGF-I, proliferation followed by
differentiation, was correlated with specific ERK activity.
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RESULTS
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ERK Reactivation in Response to Serum Stimulation Is Characteristic
of the Mutant NFB4 Phenotype (Fig. 1
)
To determine whether differences in ERK activity are associated
with the nondifferentiated phenotype of NFB4 cells, functional in-gel
kinase assays were performed using myelin basic protein as a substrate.
As myelin basic protein is a substrate for a variety of
serine/threonine kinases, several protein bands, which possessed kinase
activity that was not altered after serum stimulation in C2C12 and NFB4
cells, were visualized (Fig. 1A
). Both
C2C12 and NFB4 cell lines demonstrated low kinase activity in the mol
wt range of ERK-1 and -2, indicated as p44 and p42, in differentiation
medium (DM) low in serum (Fig. 1A
, lanes 1 and 3). After exposure to DM
for 24 h, stimulation of NFB4 cells by medium containing high
serum (growth medium, GM) induced activation of the p44 and p42, as
well as a 97-kDa kinase (Fig. 1A
, lane 4; p42, p44, and p97) that might
represent a novel kinase of this family, ERK-5 (26). By contrast, ERK
activation in response to serum was significantly reduced in C2C12
cells (Fig. 1A
, lane 2). Similar to NFB4 cells, C2C12 cells blocked
from differentiation by stable expression of constitutively active Ras
also demonstrated highly inducible ERK activity after stimulation by GM
(data not shown).

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Figure 1. ERK Activity, Phosphorylation, and Abundance in
C2C12 and NFB4 Cells after Serum Stimulation
Cell extracts from C2C12 (lanes 1 and 2) and NFB4 cells (lanes 3 and 4)
incubated in DM for 48 h with (lanes 2 and 4) or without (lanes 1
and 3) stimulation by GM for 10 min were analyzed by in-gel kinase
assay (panel A) and Western blot with ERK-1 and 2 (panel B) and
phospho-MAPK (panel C) antibodies.
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To confirm that p44 and p42 were, in fact, ERK-1 and -2, Western blots
with an antibody recognizing phosphorylated and nonphosphorylated ERK-1
and -2 were performed. This analysis showed that in C2C12 cells, ERK-2
was approximately 4 times more abundant than ERK-1 (Fig. 1B
, lanes 1
and 2). A small proportion of ERK-2 appeared to be phosphorylated as
detected by a shift in mobility, and this was relatively unaffected by
serum stimulation, consistent with in-gel kinase results. ERK-2
abundance in NFB4 cells was comparable to C2C12s (1.5-fold), whereas
ERK-1 was 4 times more abundant in NFB4 cells, as abundant as ERK-2
(Fig. 1B
, lanes 3 and 4). After stimulation by GM, both kinases became
phosphorylated in NFB4 cells but to different extents. ERK-1 became
completely phosphorylated whereas approximately half of the ERK-2
demonstrated altered mobility.
Phosphorylation of ERK-1 and -2 in C2C12 compared with NFB4 cells was
quantitated using a phospho-MAPK antibody specifically selected to
recognize only activated (phosphorylated) forms of ERK-1 and -2 (Fig. 1C
). This antibody reacted strongly with ERK-1 and -2 only in NFB4
cells stimulated by GM (Fig. 1C
, lane 4). Much less reactivity
(
20-fold less) was detectable in unstimulated cells (Fig. 1C
, lane
3). ERKs were weakly recognized in C2C12 cells, which increased
slightly after stimulation by GM (Fig. 1C
, lanes 1 and 2). This was
most apparent for ERK-2 due to its abundance (see Fig. 1B
). Overall,
phosphorylation of ERK-1 and -2 was increased 5-fold in NFB4 compared
with C2C12 cells in response to serum stimulation. These data indicated
that kinase activity of ERK-1 and -2 was correlated with their
phosphorylation state, and that NFB4 myoblasts that continued to
proliferate in low serum had ERK activity that was strongly serum
inducible.
Sustained Activation of ERKs during Myogenic
Differentiation Is Dependent on Activation of the IGF-II Autocrine Loop
(
Figs. 24

)
It was shown above that NFB4 cells that do not differentiate upon
serum withdrawal retained highly inducible ERK-1 and -2. Moreover,
activated ERK-2 was detectable in differentiated C2C12 cells (Fig. 1
, B
and C, lane 1), suggesting that sustained ERK activation may be
involved in myogenic differentiation. This was examined by performing a
time course of ERK phosphorylation during differentiation of C2C12
cells by phospho-MAPK Western blot (Fig. 2
). C2C12 cells have been well
characterized, and IGF-II is secreted into the medium within 24 h
of exposure to DM, and fully formed myotubes are present within 72
h (19, 20, 25). In GM and early during differentiation of C2C12 cells,
low level ERK phosphorylation was observed (Fig. 2
, top
panel, lanes 1 and 2). Phosphorylation of ERK-2 was increased
slightly (1.5-fold) by 12 h and within 36 h of incubation in
DM, ERK-2 became preferentially phosphorylated (4-fold over GM levels)
that was sustained (Fig. 2
, top panel, lanes 57),
indicating that activation of ERK-2 was associated with myogenic
differentiation of C2C12 cells. ERK-1 phosphorylation also increased
slightly with differentiation, although this was less apparent due to
low protein abundance. Neither ERK-1 nor ERK-2 abundance changed during
differentiation demonstrated with a p44/42 MAPK antibody recognizing
phosphorylated and nonphosphorylated ERKs (Fig. 2
, bottom
panel). ERK-2 phosphorylation correlated with tyrosine
phosphorylation of IRS-1 in C2C12 cells after 36 h of incubation
in DM, dependent on the IGF-II autocrine loop (D. D. Sarbassov and C.
A. Peterson, unpublished observations), implying involvement of the IGF
signal transduction pathway in sustained activation of ERK-2. To test
this idea, ERK activation in C2C12 cells in which the IGF-II autocrine
loop was inhibited by IGF-II antisense expression was analyzed (Fig. 3
). It has been shown that expression of
IGF-II antisense oligonucleotides in C2C12 cells blocked myogenic
differentiation and induced apoptosis (18). In our study, expression of
IGF-II antisense oligonucleotides in C2C12 cells resulted in loss of
accumulation of IGF-II precursor protein (Fig. 3A
, lane 4) compared
with control (Fig. 3A
, lane 3) and vector transfected (Fig. 3A
, lane 1)
cells, similar to the NFB4 phenotype (Fig. 3A
, lane 2). Furthermore,
IGF-II antisense-expressing cells differentiated poorly as indicated by
inhibition of myogenin and myosin heavy chain (MyHC) accumulation after
exposure to DM (Fig. 3B
, lane 4), again, similar to the NFB4 phenotype
(Fig. 3B
, lane 2). Treatment of IGF-II antisense-expressing cells with
exogenous IGFs restored the differentiated phenotype (Fig. 3B
, lane 5),
as in control C2C12 cells (Fig. 3B
, lane 3) and vector-transfected
cells (Fig. 3B
, lane 1). Moreover, C2C12 cells expressing IGF-II
antisense oligonucleotides were not able to activate ERK-2 in DM (Fig. 3C
, lanes 1 and 3). Exogenous IGF-I induced a 3-fold increase in
phosphorylation of ERK-2 (Fig. 3C
, lane 4) that required greater than
24 h incubation with the growth factor (Fig. 3C
, lane 2). Thus,
the IGF signal transduction pathway was necessary for activation of
ERK-2 during myogenic differentiation of C2C12 cells.

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Figure 2. Sustained Activation of ERKs Occurs during Myogenic
Differentiation
Cell extracts from C2C12 cells in GM (lane 1) and after exposure for
the indicated times to DM (lanes 27) were analyzed by Western blot
with phospho-MAPK (top panel) or ERK-1 and -2
(bottom panel) antibodies.
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Figure 3. Sustained Activation of ERKs Is Dependent on the
IGF-II Autocrine Loop
A, Cell extracts from control C2C12 cells (C, lane 3), C2C12 cells
transfected with empty vector (V, lane 1), C2C12 cells expressing
IGF-II antisense oligonucleotide (AS, lane 4), and NFB4 cells (lane 2)
were analyzed by Western blot with an antibody recognizing IGF-II
precursor protein. B, Cell extracts from C2C12 cells (C, lane 3), C2C12
cells transfected with empty vector (V, lane 1), C2C12 cells expressing
IGF-II antisense oligonucleotide (AS, lane 4), NFB4 cells (lane 2), and
antisense-expressing C2C12 cells treated with IGF-I (15 ng/ml, lane 5),
were analyzed by Western blot with MyHC and myogenin antibodies
simultaneously. All cells in panels A and B were cultured 48 h in
DM. C, Cell extracts from C2C12 cells expressing IGF-II antisense
oligonucleotide incubated for the indicated times in DM ± IGF-I
(15 ng/ml) were analyzed by Western blot with phospho-MAPK antibody.
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Figure 4. Blocking Sustained ERK Phosphorylation by
Inhibition of MEK1 Activity Interferes with Myogenic Differentiation
A, Cell extracts from C2C12 cells incubated for 36 h in DM with
(+) or without (-) the MEK1 inhibitor PD98059 were analyzed by Western
blot with phospho-MAPK (top panel) or myogenin
(bottom panel) antibodies. B, Cells treated as in panel
A were analyzed immunocytochemically for MyHC accumulation.
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To determine whether ERK phosphorylation in response to autocrine
IGF-II was required for differentiation, the inhibitor PD98059 that
prevents activation of MEK, thereby inhibiting phosphorylation and
activation of ERKs, was used (Fig. 4
).
After 36 h exposure to DM + PD98059, ERK phosphorylation was
significantly inhibited in C2C12 cells (Fig. 4A
, top panel),
whereas ERK accumulation was unaffected by the inhibitor (data not
shown). PD98059 treatment appeared to be detrimental to the
differentiation process. Although the expression of myogenin was
unaffected (Fig. 4A
, bottom panel) and myotubes did form,
they did not survive (Fig. 4B
). In the presence of the inhibitor, the
cells rounded up and detached from the plate compared with untreated
cells, suggesting that sustained ERK phosphorylation is required for
cell survival during differentiation.
Differential ERK Activation in Response to
Gi-Dependent Signaling Pathways (
Figs. 57

)
ERK activation in NFB4 cells in response to serum was also
produced by specific signaling molecules. Lysophosphatidic acid (LPA)
acts through its cognate receptor that interacts with PT-sensitive
heterotrimeric G proteins (13, 28, 29, 30). LPA activated ERKs in NFB4
cells but not in C2C12 cells assayed by the in-gel kinase assay (Fig. 5
, compare lanes 1 and 2). Under these
conditions activation of ERK-1 (p44) was more pronounced. Blocking the
Gi-dependent pathway by PT in NFB4 cells inhibited
activation of ERKs by LPA (Fig. 5
, lane 3). These data suggested that
activation of ERKs in NFB4 cells occurred through a
Gi-dependent pathway. Taken together with the results
described above, it appears that increased phosphorylation of ERK-1
relative to ERK-2 is associated with proliferation, whereas high levels
of phosphorylated ERK-2 relative to ERK-1 is characteristic of
differentiation.

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Figure 5. ERK Activity Is Responsive to
Gi-Dependent Signaling in NFB4 Cells
Cell extracts from C2C12 (lane 1) and NFB4 (lanes 2 and 3) cells in DM
with (lane 3) and without (lanes 1 and 2) PT preincubation were
stimulated by LPA for 10 min and analyzed by in-gel kinase assay.
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Figure 6. Distinct Responses to Gi-Dependent
Pathways in Partially Rescued NFB4 Cells
In-gel kinase assays of cell extracts from IGF-I treated NFB4 cells
(lanes 13) and cells expressing myogenin (lanes 46) or MyoD (lanes
79) stimulated for 10 min by GM (lanes 2, 5, and 8) or LPA (lanes 3,
6, and 9).
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Figure 7. Abundance of G i2 Protein and Its
Correlation with Myogenic Differentiation
A, Western blot with G i1-2 antibody of cell extracts
from C2C12 cells (lane 1), untreated NFB4 (lane 2), IGF-I-treated (lane
3), or IGF-II-treated (lane 6) NFB4 cells, and NFB4 cells expressing
myogenin (lane 4) or MyoD (lane 5). All cells were incubated in DM for
24 h. IGF-I was used at a concentration of 50 ng/ml and IGF-II at
100 ng/ml, because of differences in their affinity to the IGF-I
receptor (21, 39). B, Cellular extracts from NFB4 (lane 1) and
IGF-I-treated (lane 2) or IGF-II-treated (lane 3) NFB4 cells mentioned
in panel A were analyzed by Western blot with myogenin antibody.
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In parallel, we analyzed activation of ERKs in partially rescued NFB4
cells by IGF-I treatment or by overexpressing myogenin (NFB4/myogenin)
or MyoD (NFB4/MyoD). Mutant NFB4 cells fail to accumulate significant
levels of either transcription factor or activate IGF-II expression
(25). Prolonged treatment of NFB4 cells with IGF-I resulted in
activation of the myogenin gene in the absence of significant
endogenous IGF-II expression, whereas overexpression of myogenin and
MyoD in these cells resulted in activation of the IGF-II autocrine loop
(25). IGF-I treated NFB4, NFB4/myogenin, and NFB4/MyoD cells incubated
in DM demonstrated low ERK activity (Fig. 6
, lanes 1, 4, and 7). Stimulation of
IGF-I treated NFB4 cells by either GM or LPA strongly induced
activation of ERKs (Fig. 6
, lanes 2 and 3), whereas ERKs in
NFB4/myogenin and NFB4/MyoD cells were responsive only to GM (Fig. 6
, lanes 5 and 8) but not to LPA (Fig. 6
, lanes 6 and 9). Thus, NFB4 cells
rescued by direct IGF-I treatment and those rescued by myogenin or MyoD
expression differed in their response to LPA. In IGF-I-rescued NFB4
cells, ERKs, in particular ERK-1, were still responsive to the
Gi-dependent pathway, whereas in myogenin or
MyoD-transfected cells this pathway was inhibited.
The mitogenic effect of LPA involving activation of ERKs is mediated
through its receptor coupled to G
i2 protein. It might be
that activation of ERKs through a Gi-dependent pathway was
dependent on the abundance of G
i2 protein in C2C12 and
NFB4 cells. Similar levels of G
i1 protein were detected
in C2C12, NFB4, and rescued NFB4 cells, whereas the levels of
G
i2 were different (Fig. 7A
). A low level of G
i2
was found in C2C12 cells (Fig. 7A
, lane 1), and a 10-fold higher level
of this protein was detected in NFB4 cells relative to
G
i1 (Fig. 7A
, lane 2). Northern analysis also revealed
overexpression of G
i2 mRNA in NFB4 cells compared with
C2C12 cells (data not shown). IGF-I-rescued NFB4 cells (Fig. 7A
, lane
3) continued to accumulate G
i2 whereas the protein was 3
times less abundant in NFB4/myogenin and NFB4/MyoD cells relative to
G
i1 (Fig. 7A
, lanes 4 and 5). Thus, the abundance of
G
i2 correlated with the ERK response to LPA
stimulation.
We and others showed that IGF-I treatment inhibits activation of the
IGF-II autocrine loop in myoblasts (25, 31). Activation of the IGF-II
autocrine loop is normally associated with myogenic differentiation of
C2C12 cells and was also detected in NFB4/myogenin and NFB4/MyoD cells
(25), implying that the IGF-II-signaling pathway may lead to
down-regulation of G
i2. This appears to be the case as
incubation of NFB4 cells with exogenous IGF-II induced down-regulation
of G
i2 by 4-fold relative to G
i1 (Fig. 7A
, lane 6). These results suggest that NFB4 cells respond differently
to long-term exposure to IGF-I vs. IGF-II.
To determine whether the different effects of IGF-I and IGF-II on the
level of G
i2 protein correlated with induction of
myogenic differentiation in NFB4 cells, we analyzed expression of
myogenin. Myogenin accumulated to very low levels in NFB4 cells (Fig. 7
, lane 1). Incubation of NFB4 cells with IGF-I induced expression of
myogenin (Fig. 7B
, lane 2), whereas a higher level of myogenin was
detected in NFB4 cells incubated with IGF-II (Fig. 7B
, lane 3). Thus,
IGF-II appeared to be a stronger myogenic factor, compared with IGF-I,
consistent with previous reports (21, 25).
PT Alters ERK Activity in Response to IGF-I (Fig. 8
)
Rapid phosphorylation of ERKs in response to serum stimulation and
LPA treatment that was preferential for ERK-1 was characteristic of the
proliferative phenotype of the mutant NFB4 cells, whereas sustained
activation of ERK-2 was associated with IGF-dependent myogenic
differentiation. These observations suggest that ERK activity in NFB4
cells may be an indicator of the biphasic effects of IGF-I on
myoblasts: proliferation is the early response followed by
differentiation (21). This idea was tested by analyzing ERK activity in
NFB4 cells incubated in DM and stimulated by IGF-I for 10 min. Low
level phosphorylation of ERKs was detected in DM by the phospho-MAPK
antibody (Fig. 8
, lane 1). Short-term
IGF-I stimulation induced activation of both ERK-1 and -2, whereas LPA
stimulation resulted in preferential activation of ERK-1 (Fig. 8
, compare lanes 2 and 4; see also Fig. 5
). This effect was abrogated by
preincubation with PT (Fig. 8
, lane 5). Surprisingly, in cells
preincubated with PT, IGF-I induced preferential phosphorylation of
ERK-2 relative to ERK-1 (Fig. 8
, lane 3). Thus, in response to IGF-I,
high levels of G
i2 protein in NFB4 cells were linked
with activation of ERK-1 and -2. Blocking Gi signaling by
PT resulted in preferential activation of ERK-2, similar to the pattern
of ERK phosphorylation in differentiated C2C12 cells treated with IGF-I
(Fig. 8
, lane 6). These results suggest that early IGF-I signaling
events associated with proliferation can be altered by blocking
activation of G
i proteins, thereby mimicking signaling
events occurring during myogenic differentiation dependent on the
IGF-II autocrine loop.

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Figure 8. Alteration of the IGF-Signal Transduction Pathway
by PT in NFB4 Cells
Cell extracts from C2C12 (lane 6) and NFB4 cells (lanes 15) incubated
in DM for 24 h with (lanes 3 and 5) or without (lanes 1, 2, 4, and
6) PT preincubation stimulated by IGF-I (lanes 2, 3, and 6) for 10 min
were analyzed by Western blot with phospho-MAPK antibody.
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DISCUSSION
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In this study we showed an interplay of IGF-signaling pathways
with Gi proteins that appeared to be relevant in the
activation of ERKs and in the control of myoblast proliferation and
differentiation. We characterized activation of ERKs by high serum in
NFB4 cells that remained proliferative in DM, and in C2C12 cells that
withdraw from the cell cycle and differentiated under similar
conditions. Dramatic activation of ERKs was demonstrated for NFB4 cells
after stimulation, consistent with reports that these kinases remain
responsive in other cell types with a proliferative phenotype (6, 7, 11). A modest activation of ERK-2 was shown in differentiated C2C12
cells stimulated by high serum. In both C2C12 and NFB4 cells, ERK
activation by serum correlated with the relative abundance of the
proteins: ERK-1 and -2 accumulated to high, comparable levels in NFB4
cells, whereas C2C12 cells demonstrated a higher abundance of ERK-2
compared with ERK-1. Phosphorylation was also correlated with abundance
in the sustained activation of ERKs associated with myogenic
differentiation in C2C12 cells. Sustained activation of ERKs has
previously been observed during differentiation of neuronal cells (9, 11). Inhibition of MEK activation during differentiation interfered
with sustained ERK phosphorylation and differentiation consistent with
the conclusion that ERKs play a positive role during differentiation of
C2C12 cells (32). However, as myogenin gene expression was unaffected
by inhibition of ERK phosphorylation, it appears that sustained
activation of ERKs is necessary to maintain cells in a viable state,
allowing differentiation to proceed, rather than initiating the
process. In any case, these results are in apparent disagreement with
those of Coolican et al. (24), who recently reported that
PD98059 accelerates myogenic differentiation in L6A1 cells in response
to exogenous IGF-I. The most likely explanation is that the signaling
pathways activated by autocrine IGF-II in C2C12s myoblasts in the
experiments described here are not identical to those activated in
response to exogenous IGF-I in L6A1 myoblasts (Ref. 21; also discussed
below) resulting in a different cellular response to inhibition of ERK
phosphorylation during differentiation.
Activation of the IGF-II autocrine loop is required for myogenic
differentiation of C2C12 cells, and IGFs are survival factors for
myoblasts (18, 19). NFB4 cells fail to activate IGF-II gene expression
upon serum withdrawal, are unable to differentiate, and do not
demonstrate activation of ERK-2 in DM. Inhibition of IGF-II
accumulation in C2C12 cells also resulted in a nondifferentiated
phenotype and no ERK-2 activation. Exogenous long-term IGF-I treatment
was able to induce differentiation and concomitant activation of ERK-2.
By contrast, a Gi-signaling pathway was activated in NFB4
cells but not in C2C12 cells in response to LPA leading to preferential
ERK-1 activation. LPA acts through its cognate receptor that is coupled
with G
i2 protein, and the level of G
i2
protein correlated with PT-sensitive activation of ERK-1. A low
abundance of this protein was demonstrated in C2C12 and a high
abundance in NFB4 cells, suggesting that G
i2-signaling
contributes to the proliferative phenotype of NFB4 cells. Thus, a high
ratio of phosphorylated ERK-2/ERK-1 is associated with myogenic
differentiation, whereas a high ratio of phosphorylated ERK-1/ERK-2 is
associated with proliferation. These results suggest that ERK-1 and -2
may be functionally distinct. The mechanism whereby these kinases may
participate in different cellular processes is unknown but may involve
different substrates or different subcellular localization.
Although the IGF-I receptor is the main mediator of IGF-I and IGF-II,
the effects of these factors on myogenic differentiation were not
identical. It has been reported previously that IGF-I has a greater
mitogenic effect and IGF-II is more myogenic (21). IGF-I-treated NFB4
cells remained responsive to LPA stimulation, whereas NFB4/myogenin and
NFB4/MyoD cells, able to activate the IGF-II autocrine loop,
demonstrated nonresponsiveness to stimulation by LPA. Thus, rescue of
the mutant phenotype by IGF-I treatment or by indirect activation of
the IGF-II autocrine loop by myogenin or MyoD expression were distinct.
This is confirmed by the fact that in NFB4/myogenin, NFB4/MyoD, and
NFB4 cells directly treated with IGF-II (but not IGF-I),
G
i2 protein was down-regulated. Specific coupling of the
IGF-II receptor with G
i2 protein has been reported (33).
Long-term activation of G
i2 signaling by the IGF-II
receptor may result in down-regulation of G
i2 protein.
IGF-I was not able to down-regulate G
i2 protein possibly
due to its low affinity to the IGF-II receptor. This effect of IGF-II
may also be linked to the putative atypical IGF-I receptor associated
only with myogenic differentiation of myoblasts that preferentially
binds IGF-II (34). Thus, activation of the IGF-II autocrine loop
associated with myogenic differentiation of C2C12 cells and NFB4 cells
by expression of myogenin or MyoD demonstrated low-level
G
i2 protein accumulation correlated with
nonresponsiveness of these cells to PT-sensitive activation of ERKs by
LPA. The stronger myogenic effect of IGF-II vs. IGF-I might
be linked with the ability of IGF-II to down-regulate one of the
proliferative pathways associated with
G
i2-signaling.
Signaling through the IGF-I receptor was altered by PT, suggesting that
Gi-proteins are involved in this process. It has been shown
in other cell types that blocking Gi-dependent signaling
interfered with activation of ERKs by the IGF-I signal transduction
pathway (33, 35). Stimulation of NFB4 cells that overexpress
G
i2 protein by IGF-I induced activation of ERK-1 and -2.
Similar stimulation of NFB4 cells while blocking the activity of
Gi proteins with PT altered IGF signaling, resulting in
preferential activation of ERK-2 that mimicked ERK-2 activation during
normal differentiation of C2C12 cells. Thus, the dual effects of IGF-I
on myoblasts, induction of proliferation and differentiation, appear to
be mediated by different signaling pathways that result in distinct
patterns of ERK activation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfection
NFB4 is a subclone of the nondifferentiating NFB cell line
originally derived from the C2C12 mouse muscle cell line (25, 36). Both
cell lines were grown in serum-rich growth medium (GM): DMEM (GIBCO
BRL, Gaithersburg, MD) supplemented with 10% FBS and 10% defined
bovine serum (all serum from Hyclone, Logan, UT) at 37 C in a
humidified 10% CO2, 90% air atmosphere. Confluent cells
were washed with serum-free DMEM and maintained in DM: DMEM plus 2%
horse serum. For IGF-I (provided by Elena Moerman, University of
Arkansas for Medical Sciences, Little Rock, AR) or IGF-II (obtained
from PeproTech, Rocky Hill, NJ) treatment, cells were maintained in
DMEM containing 0.4% horse serum plus IGF-I or IGF-II at the indicated
concentrations added fresh daily. For short-term IGF-I stimulation,
IGF-I was added at 150 ng/ml for 10 min. LPA (Sigma Chemical Co., St.
Louis, MO) was added in serum-free DMEM at a concentration of 20
µM for 10 min with crystalline BSA (Calbiochem, San
Diego, CA) at 0.5 mg/ml. PT (List Biological Laboratories, Campbell,
CA) was applied in the medium at a concentration of 100 ng/ml and
preincubated for 2 h before each experiment. The MEK1 inhibitor
PD98059 (New England Biolabs, Inc., Beverly, MA) was applied in DM at a
concentration of 50 µM and was added fresh every 24
h as recommended by the manufacturer.
Introduction of a pEMSVscribe
2/IGF-II antisense expression plasmid
(18) or empty vector into C2C12 cells was performed by the calcium
phosphate coprecipitation method as described (36). C2C12 cells were
plated at 1.5 x 105 cells/100-mm tissue culture plate
and 24 h later, cells were washed and cotransfected with 10 µg
of a pEMSVscribe
2/IGF-II antisense expression plasmid together with
1 µg pSV2neo. The DNA was removed 24 h later with the addition
of fresh GM. After an additional 24 h, cells were split 1:4 and
refed with medium to which 400 µg/ml of G418 (Geneticin, GIBCO/BRL,
Gaithersburg, MD) had been added. Selection proceeded for 14 days and
G418-resistant clones were picked randomly. Pooled clones of NFB4 cells
constitutively expressing MyoD or myogenin have been described
previously (25).
Western Blot Analysis
After incubation and stimulation of cells, 100-mm dishes were
washed twice with cold PBS and lysed in 0.5 ml cold lysis buffer (50
mM HEPES, pH 7.4, 150 mM NaCl, 1.5
mM MgCl2, 1 mM EGTA, 100
mM NaF, 10 mM sodium pyrophosphate, 1
mM phenylmethylsulfonyl fluoride, 3 µg/ml of each
leupeptin and aprotinin, 6 µg/ml of antipain, 30 µg/ml of
benzamidine, 1 mM Na3VO4, 1%
Triton X 100) for 10 min by shaking. All manipulations of cell lysates
were at 4 C. Lysates were scraped into microcentrifuge tubes and
cleared of nuclei and detergent-insoluble material by centrifugation
for 10 min at 14,000 rpm. Samples (35 µg of protein) were resolved by
discontinuous electrophoresis through 7.5% SDS polyacrylamide gels and
electrophoretically transferred to PVDF membrane (Immobilon P,
Millipore, Bedford, MA). Blots were blocked for 1 h in 5% milk in
PBS plus 0.5% Tween-20 (PBST). Phospho-MAPK and ERK-1 and -2
antibodies (New England Biolabs) were applied at a 1:1000 dilution in
PBST containing 3% BSA (heat shock treated, Fisher, Pittsburgh, PA)
overnight at 4 C. G
i1 and G
i2
(Calbiochem, San Diego, CA), myogenin (hybridoma F5D, Dr. Woodring
Wright, University of Texas Southwestern Medical Center, Dallas, TX),
MyHC (A4.1025, 25), and IGF-II (sc-1417, Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) antibodies were applied at 1:1000 dilution in
PBST containing 3% milk and incubated for 1 h. Blots were washed
5 times with PBST for a total of 30 min, before and after incubating
with horseradish peroxidase-conjugated secondary antibody (Pierce,
Rockford, IL) diluted 1:4000 in PBST containing 4% milk for 1 h.
Renaissance Chemiluminescence Reagent (DuPont/NEN, Wilmington, DE) was
used as the detection system. Signals were quantitated using a
computing densitometer with LasarQuant software (Molecular Dynamics,
Sunnyvale, CA). Blots were stripped and reprobed as described (37).
Immunocytochemistry
Immunocytochemical analyses were performed essentially as
described (25). Briefly, cells were washed in PBS, fixed with 1%
paraformaldehyde, and then treated with ice-cold methanol. Cells were
incubated with undiluted MyHC A4.1025 hybridoma tissue culture
supernatant for 1 h at room temperature followed by incubations
with horseradish peroxidase-conjugated anti-mouse IgG. Peroxidase
reactivity was visualized using the DAB substrate kit (Vector
Laboratories, Burlingame, CA).
In-Gel Kinase Assay
The in-gel kinase assay was performed essentially as described
(38). Cell lysates were boiled in Laemmli sample buffer for 2 min and
resolved by discontinuous electrophoresis through 7.5% SDS
polyacrylamide gel containing 0.5 mg/ml myelin basic protein (Sigma).
The gel was fixed by four washes with 20% 2-propanol in buffer A (50
mM Tris-HCl buffer, pH 8.0, containing 2 mM
dithiothreitol) for 2 h, and SDS was removed by washing the gel
for 2 h in several volumes of buffer A, changing the solution
every 15 min. Proteins in the gel were denatured in 6 M
guanidine HCl for 2 h in buffer A and then renatured overnight at
4 C in buffer A containing 0.04% Tween 40 (Sigma). After preincubation
of the gel for 1 h in 5 ml of 40 mM HEPES, pH 8.0,
containing 2 mM dithiothreitol and 10 mM
MgCl2, the kinase reaction was carried out by incubation of
the gel for 1 h at 25 C in 5 ml of 40 mM HEPES, pH
8.0, containing 25 µCi of [
-32P]ATP, 40
µM ATP, 0.5 mM EGTA, 2 mM
dithiothreitol, and 10 mM MgCl2. After the
kinase reaction the gel was washed several times in 5% (wt/vol)
trichloroacetic acid containing 1% pyrophosphate until the
radioactivity reached background levels. After washes, the gel was
dried and exposed to x-ray film.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Peter Rotwein for providing pEMSVscribe
2/IGF-II
antisense expression plasmid, Dr. Woodring Wright for the myogenin
hybridoma, Elena Moerman for IGF-I, and Jane Taylor-Jones for
assistance with artwork.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Charlotte A. Peterson, Veterans Affairs Hospital, Research 151, 4300 West 7th Street, Little Rock, Arkansas 72205.
This work was supported by grants from the National Institutes of
Health-National Institute on Aging (to C.A.P).
Received for publication May 27, 1997.
Revision received September 8, 1997.
Accepted for publication September 23, 1997.
 |
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