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Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Address all correspondence and requests for reprints to: Crystal M. Weyman, Cleveland Clinic Foundation, Department of Cell Biology, Cleveland, Ohio 44195. E-mail: weymanc{at}cesmtp.ccf.org
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Abstract |
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Introduction |
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Signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK plays a key role in many biological processes (6, 7, 8, 9). Both these biological processes and the signal from MEK to MAPK are initiated by the binding of certain extracellular growth factors to their cognate transmembrane receptors. Ligand-induced activation of the receptor’s intrinsic tyrosine kinase activity creates docking sites for adapter proteins such as SHC or GRB2, thus facilitating the localization of mSOS (the guanine nucleotide exchange factor for Ras) near Ras (10). Activation of Ras via mSOS enhanced exchange of GDP for GTP leads to the activation of the Raf family of serine kinases, which in turn phosphorylate and activate MEK. Activated MEK then phosphorylates and activates MAPK (11). Transmission of the extracellular signal through the cytoplasm via the MAPK phosphorylation cascade culminates with the translocation of activated MAPK to the nucleus and the subsequent phosphorylation and regulation of transcription factors (12), which presumably are ultimately responsible for eliciting the appropriate biological response.
Treatment of C2C12 myoblasts with concentrations of IGF-1 reported to enhance differentiation results in no appreciable increase in MAPK activity (13). The activation of MAPK by concentrations of IGF-1 that would inhibit C2C12 myoblast differentiation was not examined. Treatment of C2C12 myoblasts with FGF-2 activates MAPK, but the significance of the FGF-2-induced MAPK activation to the inhibition of differentiation was not determined (13). Treatment of MM14 myoblasts with FGF-2 activates MEK, but transmission of the signal to MAPK is prevented by a dual specificity phosphatase (14, 15). The significance of the FGF-2 induced MEK activation to the FGF-2 induced inhibition of differentiation was not determined.
An essential role for signaling by MEK has been demonstrated in many systems through the use of the selective chemical inhibitor PD 098059 (8, 16, 17). This inhibitor blocks the activation of MEK and, consequently, the activation of MAPK (17, 18). In this study, we show that either IGF-1 or FGF-2 inhibits the differentiation of 23A2 myoblasts and that both potently stimulate signaling through MEK to MAPK. We also demonstrate the significance of signaling through MEK by using PD 098059 to abrogate the inhibition of differentiation by either IGF-1 or FGF-2.
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Materials and Methods |
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Cells and cell culture
Undifferentiated, proliferating 23A2 myoblasts were maintained
in growth medium (BME supplemented with 10% FBS and 1% P/S) at low
density on gelatin coated dishes. Differentiation was induced by
treating the cells with differentiation medium (BME, 1% P/S). PD
098059 was dissolved in dimethyl sulfoxide to give a final
concentration of 37 mM and stored at -80 C. Dimethyl
sulfoxide alone (<0.15% vol/vol) was added to control cultures.
MAP kinase activity assay
Cell extracts were prepared in p21 buffer (20 mM
MOPS pH 7.4, 4 mM magnesium chloride, 200 mM
sucrose, 0.1 mM EDTA) supplemented with 1% CHAPS,
protease, and phosphatase inhibitors. TR10 antibody precoupled to
protein A sepharose was added to extracts followed by rotation for
1 h at 4 C. After sedimentation, pellets were washed three times
with Tris-buffered saline (TBS)/0.1% NP40, once with p21 buffer and
then incubated for 20 min at 37 C with 25 µM myelin basic
protein (MBP), 10 µM [
-32P]ATP (10
µCi, >4, 500 Ci/mmol), and 10 µM phosphatase
inhibitors in a total volume of 50 µl (19). Assays were performed
within the linear range of 32P incorporation and were
terminated by the addition of 2 x Laemmli buffer. Reaction
products were separated by discontinuous SDS-PAGE (15%). The gel was
dried and phosphorylated bands visualized and quantitated by
Phosphorimager analysis. MBP sometimes appears as a phosphorylated
doublet. This doublet has nothing to do with the treatment but rather
the batch of purchased MBP. Please refer to Fig. 2A
where MBP is a
single band regardless of whether the cells are treated with FGF-2 or
IGF-1 and to (see Fig. 5) where MBP is a doublet regardless of whether
the cells are treated with FGF-2 or IGF-1. The results presented in
Fig. 2B were simply obtained at different times with different batches
of MBP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) by the ability to elongate and fuse
into multinucleated myotubes (see Fig. 4B). To compare the effect of
IGF-1 signaling to FGF-2 signaling on the differentiation of 23A2
myoblasts, these cells were switched from GM to DM with increasing
concentrations of FGF-2 or IGF-1. Both IGF-1 at 7 ng/ml (1
nM) and FGF-2 at 10 ng/ml (0.6 nM) inhibit the
differentiation of 23A2 myoblasts as monitored by the expression of MHC
(Fig. 1) or by the ability to elongate and fuse into multinucleated
myotubes (data not shown, d.n.s., and Fig. 4B).
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). Treatment of 23A2 myoblasts cultured
in DM for either 3 or 18 h with either IGF-1 or FGF-2 for 10 min
induced a 7- to 8-fold stimulation of signaling through MEK to MAPK
(Fig. 2A). These results were confirmed by Western analysis with an
antibody specific for activated phosphoMAPK (Fig. 2B). Furthermore, the
signaling through MEK to MAPK induced by either IGF-1 or FGF-2 is
readily detected if IGF-1 or FGF-2 is added to GM or if added
simultaneously when cells are switched from GM to DM (d.n.s.).
Activated MAPK could be detected after stimulation with as little as 1
ng/ml of either growth factor with maximal activation in response to 5
ng/ml (Fig. 2C).
PD 098059 blocks the IGF-1 or FGF-2-induced signal from MEK to MAPK
and restores the myogenic potential to IGF-1 or FGF-2 treated
myoblasts
PD 098059 selectively blocks the activation of MEK, and
consequently, the activation of MAPK (17, 18). Cells were incubated for
10 min with increasing concentrations of PD 098059 before the addition
of IGF-1 or FGF-2 to determine the concentration of PD 098059 required
for maximum abrogation of the growth factor-induced signal from MEK to
MAPK. After 10 min of growth factor stimulation, the MAPK activity
immunoprecipitated from cells preincubated with 50 µM PD
098059 (Fig. 3A) was comparable to the
MAPK activity immunoprecipitated from unstimulated control cells. PD
098059 must be stable over the 48-h time period used to monitor the
induction of MHC to be a useful reagent to assess the significance of
the signal from MEK to MAPK to the inhibition of differentiation by
IGF-1 or FGF-2. Cells were treated with 50 µM PD 098059
(or vehicle) for 10 min or 48 h before the addition of either
IGF-1 or FGF-2. Transmission of the signal from MEK to MAPK was
assessed after 10 min of growth factor stimulation. The PD 098059
mediated inhibition of MAPK activation after 48 h in culture
medium was essentially identical to that obtained after only 10 min in
culture medium (Fig. 3B). These results are consistent with previous
reports from our lab (8) and others (16, 18) on the stability and
efficacy of PD 098059.
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)] and similar
fusion into multinucleated myotubes [(8 and Fig. 4B)]. Including
either 10 ng/ml IGF-1 or FGF-2 in the DM inhibits their differentiation
as monitored by the expression of MHC (Figs. 1 and 4A) and their
ability to elongate and fuse (Fig. 4B). A 10-min preincubation with PD
098059 before the addition of IGF-1 or FGF-2 (a treatment sufficient to
block the IGF-1 or FGF-2 induced signal from MEK to MAPK) also
abrogates the ability of IGF-1 or FGF-2 to effectively inhibit the
differentiation of 23A2 myoblasts restoring their ability to elongate
and fuse (Fig. 4B) and to express MHC at levels 70–80% of that
detected from 23A2 myoblasts cultured identically but without IGF-1 or
FGF-2 (Fig. 4A). We did note in roughly 10% of our experiments that PD
098059 alone appeared to enhance the expression of MHC by 15–20% when
compared with MHC expression from control cultures (Fig. 4A, top
panel). This result was not only irreproducible, but could not
possibly explain the levels of MHC obtained (70–80% of that detected
in control cultures) when the FGF-2 or IGF-1 induced signals through
MEK were abrogated by PD 098059. Furthermore, this slight increase in
MHC expression detected by Western analysis was not observed when cell
cultures were fixed and immunostained for MHC (d.n.s and Ref.8).
Persistent signaling through MEK is required for the inhibition of
differentiation by IGF-1 or FGF-2
The presence of FGF-2 in the culture medium is continually
required to prevent differentiation (22). In C2C12 myoblasts,
FGF-2-induced activation of MAPK was observed at 2 min and returned to
nearly basal levels by 10 min. A second but smaller increase in MAPK
activity could be seen at 15 min, which returned to basal by 60 min
(13). In 23A2 myoblasts, the peak of signaling through MEK to MAPK in
response to either IGF-1 or FGF-2 occurred within 60 min with maximal
activation observed after 10 min. MAPK activity remained roughly 70%
above basal, however, even after 48 h (Fig. 5, A and C), an increase similar to that
seen in 23A2 myoblasts transformed by oncogenic Ras (8). Western
analysis was performed to demonstrate that MAPK protein levels remained
unchanged after 3, 18, or 48 h in DM or after treatment for
48 h with 10 ng/ml of either FGF-2 or IGF-1 (Fig. 5B).
To determine if continuous signaling through MEK to MAPK was required
to inhibit differentiation, PD 098059 was added after the initial burst
of MAPK activity. The restoration of myogenic potential seen when cells
were preincubated with PD 098059 was similar to that seen when PD
098059 was added to cultures after the initial peak of signaling from
MEK to MAPK (Fig. 6), indicating that
persistent signaling through MEK is required for the inhibition of
differentiation by either IGF-1 or FGF-2.
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), 5 ng/ml was sufficient for
maximal signaling through MEK to MAPK (Fig. 2B). Having demonstrated
that persistent signaling through MEK by these growth factors is
required to inhibit differentiation, we speculated that it is not the
concentration of growth factor required to maximally stimulate
signaling (which occurs after 10 min) which is significant but rather
that the critical size of the initial dose of FGF-2 is directly related
to growth factor stability so that a persistent signal is transduced
over the time period necessary for the expression of MHC. To test this
hypothesis, media containing FGF-2 at 5 ng/ml and at 10 ng/ml was
incubated for 24 or 48 h in the tissue culture incubator before
transfer to naive 23A2 myoblasts. After 10 min, activation of the
MEK/MAPK pathway was determined. Media containing 10 ng/ml FGF-2 for
24 h before transfer for 10 min to naive 23A2 myoblasts stimulated
the signal from MEK to MAPK by 5-fold, whereas media containing 5 ng/ml
FGF-2 for 24 h before transfer did not stimulate this signal (Fig. 7). Media containing 10 ng/ml FGF-2 for
48 h before transfer for 10 min to naive 23A2 myoblasts stimulated
the signal from MEK to MAPK to MBP by 80% (d.n.s). This stimulation is
similar to the persistent activation seen when cells are treated with
10 ng/ml FGF-2 for 24 or 48 h (Fig. 5, A and C) and further
substantiates the observation in Fig. 6 that persistent signaling, and
not just the initial burst of signal observed within the first hour of
treatment (Fig. 5, A and C) is required to prevent differentiation from
occurring. Furthermore, the persistent signaling through MEK to MAPK
seen when cells are treated with 10 ng/ml FGF-2 for 48 h is not
found when cells are treated with 5 ng/ml for 48 h (d.n.s).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is important to make a distinction between a requirement for MEK activation and a requirement for MAPK activation. Although MEK is the immediate upstream activator of MAPK (11), activation of MEK does not always result in MAPK activity. In the MM14 skeletal myoblast cell line, FGF-2 is a potent activator of MEK1, but this signal only leads to the activation of MAPK after the cells have been in differentiation medium for at least 10 h, a time point at which FGF-2 can no longer inhibit their differentiation (14). Although we have found that either FGF-2 or IGF-1 is capable of signaling through MEK to MAPK 1) if added to growth medium, 2) if added simultaneously when cells are switched from growth medium to differentiation medium, or 3) if added after switching the cells to differentiation medium for either 3 or 18 h, a reagent that would selectively eliminate only MAPK signaling would be required to attribute any significance to this MAPK activation. It is also important to make a distinction between signals that are necessary and signals that are sufficient for the inhibition of skeletal myoblast differentiation. Although we have clearly demonstrated that signaling through MEK is required by FGF-2 and IGF-1 to inhibit differentiation, others have shown that signaling by constitutively active MEK is not sufficient to inhibit MyoD-induced differentiation in 10T1/2 fibroblasts (23).
We report here a distinction between the signaling pathways used by oncogenic Ras and both IGF-1 and FGF-2 in that we have previously used PD 098059 to show that the differentiation-defective phenotype established by oncogenic Ras in 23A2 myoblasts is MEK independent (8). Although it might seem likely that IGF-1 and FGF-2 signal through endogenous Ras, this has not been formally proven. It is possible, therefore, that the IGF-1 and FGF-2-induced activation of MEK, and subsequent activation of MAPK, is Ras independent. If IGF-1 and FGF-2 do signal through endogenous Ras, it remains formally possible that endogenous Ras and oncogenic Ras signal through distinct, as well as common, effectors. Although signaling through MEK was not required by oncogenic Ras to inhibit 23A2 myoblast differentiation, we used PD 098059 to demonstrate that oncogenic-Ras relies on signaling through MEK to permit serum-independent proliferation (8). PD 098059 also blocked the mitogenic effect of IGF-1 in L6 myoblasts (6) and the mitogenic effect of FGF-2 or IGF-1 in C2C12 myoblasts (13). FGF-2 and, to a lesser extent, IGF-1 induce serum-independent thymidine incorporation in 23A2 myoblasts, which is abrogated by the inclusion of PD 098059 (d.n.s.). This does not contribute to the restoration of the myogenic phenotype, however, because it is well documented that the ability of FGF-2, IGF-1 or oncogenic Ras to inhibit differentiation is independent of their ability to induce proliferation (1, 24, 25). Furthermore, we have previously shown that, although PD 098059 effectively abrogates the oncogenic Ras-induced serum independent proliferation of 23A2 myoblasts, PD 098059 is not able to overcome the oncogenic Ras-induced differentiation block (8).
Skeletal myoblast differentiation is positively regulated by activation of the muscle regulatory factor (MRF) family of basic helix-loop helix transcription factors. To positively regulate the expression of genes necessary for differentiation, the MRFs (MyoD, myogenin, Myf-5, and MRF4) heterodimerize with ubiquitously expressed E-proteins and bind to target E-box sequences located within specific promoters or enhancers. The signaling pathways used by inhibitors of differentiation ultimately impinge on the ability of the MRFs to induce transcription (26). Oncogenic Ras inhibits the ability of the muscle regulatory transcription factors, MyoD or MRF4, to induce skeletal myoblast differentiation without altering their dimerization, DNA binding, or inherent transcriptional activities (27). FGF-2 also inhibits the ability of these MRFs to induce differentiation without altering their dimerization or DNA binding. FGF-2 does, however, inhibit their inherent transcriptional activity (27). Thus, distinctions between the signaling pathways used by oncogenic Ras and FGF-2 that we have shown here are evident in the cytoplasm also exist at the level of MRF regulation. Unfortunately, the effect of IGF-1 signaling on MRF regulation has not been explored. The challenge now is to understand how the signal initiated by IGF-1 and FGF-2 and transmitted through MEK impinges on the transcription factors responsible for skeletal muscle specific gene expression.
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Acknowledgments |
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Footnotes |
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Received August 26, 1997.
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