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Endocrinology Vol. 143, No. 2 511-516
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Early Stimulation and Late Inhibition of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation by IGF-I: A Potential Mechanism Mediating the Switch in IGF-I Action on Skeletal Muscle Cell Differentiation

Saleh Adi, Bassam Bin-Abbas, Nan-Yan Wu and Stephen M. Rosenthal

Department of Pediatrics, University of California, San Francisco, California 94143-0434

Address all correspondence and requests for reprints to: Stephen M. Rosenthal, M.D., Department of Pediatrics, P.O. Box 0434, University of California, San Francisco, California 94143-0434. E-mail: smr{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I has a unique biphasic effect on skeletal muscle cell differentiation. Initially, IGF-I inhibits differentiation and promotes proliferation of skeletal myoblasts. Subsequently, IGF-I switches to stimulating differentiation of these cells. The mechanisms responsible for this switch in IGF action remain unknown. We have examined the role of extracellular signal-regulated kinase (Erk)1/2 signaling in mediating the early inhibitory and late stimulatory effects of IGF-I on the gene expression of myogenin, a skeletal muscle-specific transcription factor essential for myogenic differentiation. We find that, concurrent with its early inhibitory and late stimulatory effects on myogenin mRNA, IGF-I has a biphasic but opposite effect on phosphorylation of Erk1/2: initially, IGF-I increases and subsequently decreases the phosphorylation of Erk1/2 in comparison to untreated cells. Cotreatment with an inhibitor of Erk1/2 activation prevents the early IGF-I-stimulation of Erk1/2 phosphorylation and partially reverses IGF-I-inhibition of myogenin mRNA. Conversely, preventing the late IGF-I-induced decrease in Erk1/2 phosphorylation blocks IGF-I-stimulation of myogenin mRNA. Our data indicate that the time-dependent, opposing effects of IGF-I on skeletal muscle cell differentiation are mediated, at least in part, by biphasic but opposite effects on activation of the Erk1/2 MAPK signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE HAVE PREVIOUSLY demonstrated that IGF-I has opposing, time-dependent actions in skeletal myoblasts: initially, IGF-I induces proliferation and inhibits differentiation; subsequently, IGF-I promotes cell cycle exit and stimulates differentiation (1, 2). These opposing effects of IGF-I on myogenic differentiation are mediated, at least in part, by early inhibition and subsequent stimulation of the gene expression of myogenin (1, 2, 3), a skeletal muscle-specific transcription factor essential for myogenic differentiation (4, 5, 6). The mechanisms responsible for this switch in IGF action are not well understood but may include alterations in IGF-I receptor signaling.

Upon ligand binding, the IGF-I receptor activates a number of downstream signaling cascades including the Raf-1/MAPK kinase (MEK)1/2/extracellular signal-regulated kinase (Erk)1/2 pathway, which has been shown to play a major role in regulating cell growth and differentiation in a variety of cell types (7, 8, 9, 10, 11, 12, 13, 14, 15). In skeletal muscle cells, previous studies have demonstrated that activation of the Ras/MAPK(Erk1/2) signaling pathway inhibits myogenic differentiation induced by serum withdrawal (9, 10) while blocking this pathway augments this process (9, 11, 14, 15). Furthermore, with respect to IGF effects in skeletal muscle cells, signaling through the MAPK/Erk1/2 pathway has been shown to mediate the initial proliferative effect of IGF-I (12) and to mediate IGF-I inhibition of differentiation in skeletal myoblasts (14). However, the role of MAPK/Erk1/2 signaling in mediating the late stimulatory effect of IGF-I on muscle cell differentiation has not been clearly defined. In view of studies that demonstrate that inhibiting the activity of the Ras/MAPK(Erk1/2) signaling pathway augments the process of muscle differentiation, we hypothesized that this pathway not only mediates the early inhibitory effect of IGF-I, but also the late stimulatory effect of IGF-I on muscle cell differentiation. Concurrent with its early inhibitory and late stimulatory effects on myogenin mRNA, we find that IGF-I has a biphasic but opposite effect on phosphorylation of Erk1/2: initially, IGF-I increases and subsequently decreases the phosphorylation of Erk1/2 in skeletal myoblasts. Preventing the early increase in Erk1/2 phosphorylation partially reverses IGF-I inhibition of myogenin mRNA. Furthermore, preventing the time-dependent decrease in Erk1/2 phosphorylation blocks the ability of IGF-I to stimulate myogenin gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The following materials were purchased: [{alpha}-32P]deoxy-CTP (3000 Ci/mmol) from Amersham Pharmacia Biotech (Piscataway, NJ), tissue culture medium components and FBS (HyClone Laboratories, Inc., Logan, UT) from the Cell Culture Facility (University of California, San Francisco), BSA from Sigma (St. Louis, MO), Super Signal Substrate from Pierce Chemical Co. (Rockford, IL), the MEK inhibitors PD098059 from New England Biolabs, Inc. (Beverly, MA) and UO126 from Cell Signaling Technology (Beverly, MA), and sodium (Na)-orthovanadate from Calbiochem (La Jolla, CA). Antibodies to the following peptides were purchased: Erk1/2 from Zymed Laboratories, Inc. (South San Francisco, CA), phospho-Erk1/2, MEK1/2, and phospho-MEK1/2 from Cell Signaling Technology. Antirabbit IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Des (1, 2, 3)IGF-I was a gift from Genentech, Inc. (South San Francisco, CA). A plasmid containing a myogenin cDNA was provided by E. N. Olson (University of Texas Southwestern Medical Center, Dallas, TX).

Cell culture
Rat L6E9 skeletal myoblasts [provided by B. Nadal-Ginard (16)] were maintained in DMEM/1% glutamine and antibiotics supplemented with 20% FBS. For IGF treatment studies, cells were placed in serum-free/1% BSA differentiation medium (DM) with vehicle (0.1 M acetic acid) or des (1, 2, 3)IGF-I (20 ng/ml) for the times indicated. The des (1, 2, 3)IGF-I analog of IGF-I was used to minimize any potential effects of IGF binding proteins (IGFBPs) (17, 18, 19) because des ( 1, 2, 3)IGF-I has a much reduced affinity for IGFBPs but an affinity for binding to the IGF-I receptor which is comparable to that of native IGF-I (19). In studies with IGF-I in the presence of an inhibitor, PD098059 or UO126 was added 1 h before the IGF peptide, whereas sodium orthovanadate was added immediately before IGF-I.

Western blotting
At the indicated times, cells were washed with cold PBS and harvested immediately in lysis buffer (1% NP-40, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl [pH 7.4], 20 mM NaF, 2 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The lysates were cleared by centrifugation at 15,000 x g for 10 min. Aliquots containing 30–50 µg protein were resolved by SDS-PAGE (7.5%) and transferred to nitrocellulose membranes in 20 mM Tris-HCl, pH 8.0, 150 mM glycine, and 20% (vol/vol) methanol at 4 C. Membranes were blocked with 3% nonfat dry milk in PBS/0.05% Tween 20 buffer and incubated with a phospho-Erk1/2 antibody. Immunoreactive bands were detected by incubation with horseradish peroxidase-conjugated secondary antibodies followed by an enhanced chemiluminescence reagent and autoradiography using Hyperfilm (Amersham Pharmacia Biotech) as previously described (20). Membranes were then stripped and reincubated with an antibody which detects total (phosphorylated and unphosphorylated) Erk1/2. Immunoreactive bands were detected by enhanced chemiluminescence, as above. In some experiments, membranes were stripped again and reprobed sequentially with antibodies against phospho-MEK and total MEK.

RNA isolation and Northern blotting
At the indicated times, cells were harvested and total RNA isolated by extraction in Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s recommendations. RNA was quantitated by spectrophotometric determination at 260 nm, and 25 µg of RNA per sample were denatured in formaldehyde, subjected to electrophoresis in 1% agarose gels, transferred to nylon membranes (Amersham Pharmacia Biotech), and fixed by UV cross-linking. Myogenin cDNA was labeled using random primers to 109 cpm/µg. Nylon membranes were prehybridized, hybridized, and washed as previously described (1, 2), and exposed to a PhosphorImager screen for detection and quantitation of myogenin mRNA using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Early stimulation of Erk1/2 phosphorylation by IGF-I is inhibited by PD098059 or UO126
We have previously demonstrated that treatment of skeletal myoblasts with IGF-I initially maintains a proliferative state associated with inhibition of differentiation (1). We have also shown that the inhibitory effect of IGF-I on differentiation is independent of its mitogenic effect because inhibition of proliferation does not prevent IGF-I-suppression of differentiation (1). The MAPK/Erk1/2 signaling pathway has been shown to play an important role in mediating the mitogenic effect of IGF-I in skeletal myoblasts (12), whereas its role in mediating the concomitant IGF-I-inhibition of differentiation remains incompletely defined.

To examine the role of the MAPK/Erk1/2 pathway in mediating the inhibitory effect of IGF-I on skeletal muscle cell differentiation, we used the MEK1/2 inhibitors PD098059 and UO126 to block IGF-I activation of Erk1/2. As expected, Erk1/2 are phosphorylated in myoblasts maintained in growth medium (GM) (Fig. 1Go, lane 1), and their phosphorylation decreases upon switching into differentiation medium (DM) (Fig. 1Go, lanes 2 and 6) (9, 11, 12, 14). Treatment with the IGF-I analog, des (1, 2, 3)IGF-I (20 ng/ml) maintains a high level of phosphorylation of Erk1/2 (Fig. 1Go, lanes 4 and 8). Pretreatment with PD098059 (30 µM) (or 10 µM UO126, not shown) prevents IGF-I-induced phosphorylation of Erk1/2 (Fig. 1Go, lanes 5 and 9).



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  		Figure 1. Stimulation of Erk1/2 phosphorylation by des (1 2 3 )IGF-I and its inhibition by PD098059. L6E9 myoblasts maintained in 20% FBS-supplemented medium were either harvested (GM) or placed in serum-free DM in the absence or presence of des (1 2 3 )IGF-I (20 ng/ml) for 30 min or 6 h alone or in combination with PD098059 (30 µM), then harvested for Western blot analysis. Phospho-Erk1/2 (upper panel) was detected by an antibody that recognizes only the Thr202/Tyr204-phosphorylated forms of Erk1/2, and total Erk/1/2 (lower panel) was detected by an antibody that recognizes both phosphorylated and unphosphorylated forms. A representative of five independent experiments is shown.

 
Blocking Erk1/2 phosphorylation partially prevents IGF-I inhibition of myogenin mRNA
The induction of myogenin expression is one of the earliest and essential events in skeletal muscle cell differentiation (6, 21). While late treatment with IGF-I (>24 h) stimulates muscle differentiation, early treatment with IGF-I (<24 h) inhibits this process, associated with inhibition of myogenin mRNA (1). To evaluate the role of the MAPK/Erk1/2 signaling pathway in IGF-I-inhibition of myogenin gene expression, myoblasts were transferred from GM to DM in the absence or presence of des (1, 2, 3)IGF-I (20 ng/ml) and/or PD098059 (30–50 µM) or UO126 (10 µM). In undifferentiated myoblasts (Fig. 2Go, lane 1), myogenin mRNA is nondetectable and is increased after 7 h in DM (Fig. 2Go, lane 2). As expected (1), addition of des ( 1, 2, 3) IGF-I, 20 ng/ml, virtually completely inhibits the induction of myogenin mRNA (Fig. 2Go, lane 3). Treatment with PD098059 or UO126 alone increases myogenin mRNA in comparison to control cells (Fig. 2Go, lanes 4, 6, and 8 compared with lane 2) while the combination of either MEK inhibitor with IGF-I partially reverses IGF-I-inhibition of myogenin mRNA (Fig. 2Go, lanes 5, 7, and 9). These data indicate that the early inhibitory effect of IGF-I on skeletal muscle cell differentiation is partially mediated by the MAPK/Erk1/2 signaling pathway.



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  		Figure 2. Effect of MEK inhibitors PD098059 and UO126 on the early inhibition of myogenin mRNA by IGF-I. L6E9 cells maintained in GM were either harvested or placed in serum-free DM in the absence or presence of des (1 2 3 )IGF-I (20 ng/ml) for 7 h alone or in combination with PD098059 or UO126, then harvested for Northern blot analysis and detection of myogenin mRNA (upper panel). Ethidium bromide (EtBr) staining of the gel was used to verify equal loading and quality of RNA samples (middle panel). The bar graph represents a quantitation of myogenin mRNA by PhosphorImager analysis. A representative of three independent experiments is shown.

 
IGF-I switches from stimulating to inhibiting the MAPK/Erk1/2 pathway
Our data in Figs. 1Go and 2Go demonstrate an inverse relationship between Erk1/2 phosphorylation and the expression of myogenin in skeletal myoblasts. A relatively high level of Erk1/2 phosphorylation in GM (or in the presence of IGF-I) is associated with absence of myogenin expression (Fig. 3Go, lane 1). Induction of myogenin mRNA in DM is associated with a decrease in Erk1/2 phosphorylation (Fig. 3Go, lane 2), and further inhibition of Erk1/2 phosphorylation by PD098059 (30 µM) augments myogenin gene expression (Fig. 3Go, lane 3). In view of these findings, we hypothesized that the late stimulatory effect of IGF-I on muscle cell differentiation (1, 3) may be mediated by inhibition of the MAPK/Erk1/2 pathway by IGF-I. To test this hypothesis, we first examined the phosphorylation of Erk1/2 following IGF-I treatment for up to 48 h. As seen in Fig. 4Go, treatment with des (1, 2, 3)IGF-I, 20 ng/ml, initially stimulates Erk1/2 phosphorylation (Fig. 4Go, lane 3 vs. lane 2) but subsequently switches to inhibiting the phosphorylation of Erk1/2 at greater than or equal to 24 h (Fig. 4Go, lanes 5 and 7 vs. lanes 4 and 6).



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  		Figure 3. The relationship between the level of Erk1/2 phosphorylation and myogenin gene expression in differentiating myoblasts. Parallel dishes of L6E9 cells maintained in GM were either harvested or placed in serum-free DM in the absence or presence of 30 µM PD098059 for 7 h, then harvested for either Western or Northern blot analysis and detection of Erk1/2 proteins (as in Fig. 1Go) or myogenin mRNA (as in Fig. 2Go), respectively. A representative of four independent experiments is shown.

 


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  		Figure 4. A biphasic effect of IGF-I on Erk1/2 phosphorylation in skeletal myoblasts. L6E9 cells maintained in GM were either harvested or placed in DM in the absence or presence of des (1 2 3 )IGF-I (20 ng/ml) for the times indicated, then harvested for analysis of Erk1/2 phosphorylation as described in Fig. 1Go. Total Erk1/2 proteins are shown in the lower panel. A representative of four independent experiments is shown.

 
To address the question of whether the time-dependent effects of IGF-I on Erk1/2 phosphorylation are specific for these kinases or represent a general effect on the MAPK/Erk1/2 signaling cascade, we also examined the phosphorylation of MEK under the same conditions. Similar to Erk1/2, MEK phosphorylation decreases upon switching from GM to DM (Fig. 5Go, lanes 2 and 4 vs. lane 1), and treatment with IGF-I initially stimulates the phosphorylation of MEK (Fig. 5Go, lane 3 vs. lane 2) but subsequently decreases MEK phosphorylation (Fig. 5Go, lane 5 vs. lane 4).



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  		Figure 5. A biphasic effect of IGF-I on MEK phosphorylation in skeletal myoblasts. L6E9 cells maintained in GM were either harvested or placed in DM in the absence or presence of des (1 2 3 )IGF-I (20 ng/ml) for the times indicated, then harvested for analysis of MEK phosphorylation. Phospho-MEK (upper panel) was detected by an antibody that recognizes only the Ser217/221-phosphorylated forms of MEK1/2, and total MEK (lower panel) was detected by an antibody that recognizes both the phosphorylated and unphosphorylated forms of MEK1/2. A representative of three independent experiments is shown.

 
Sodium orthovanadate prevents the late inhibition of Erk1/2 phosphorylation and stimulation of myogenin mRNA by IGF-I
It has been recently demonstrated that inhibition of Erk1/2 activation by cell-to-cell contact is critical for initiating cell cycle exit in confluent vascular endothelial cells (22, 23). This inhibition was associated with an increase in phosphatase activity which was sensitive to treatment with Na- orthovanadate (22, 23). We hypothesized that the initial mitogenic effect of IGF-I in skeletal myoblasts results in up- regulation of phosphatase activity and inhibition of the MAPK/Erk1/2 signaling pathway. Thus, we examined the effects of Na-orthovanadate on IGF-I-induced inhibition of Erk1/2 phosphorylation in skeletal myoblasts. As seen in Fig. 6Go, addition of Na-orthovanadate (1–5 µM) at time zero completely prevents the decrease in Erk1/2 phosphorylation observed after 48 h of IGF treatment (Fig. 6Go). Next we examined the effect of Na-orthovanadate on IGF-I-stimulation of myogenin gene expression. As expected, myogenin mRNA is nondetectable in myoblasts maintained in GM and is induced after 48 h in serum-free DM (Fig. 7Go, lanes 1 and 2). Treatment with des (1, 2, 3)IGF-I (20 ng/ml) causes an approximate 2-fold increase in myogenin mRNA [Fig. 7Go, lane 3 vs. lane 2 (1, 3)]. Addition of 5 µM Na-orthovanadate alone has no effect on myogenin gene expression (Fig. 7Go, lane 4) but completely inhibits IGF-I-stimulation of myogenin mRNA (Fig. 7Go, lane 5 vs. lane 4).



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  		Figure 6. Effect of Na-orthovanadate (SOV) on the late inhibition of Erk1/2 phosphorylation by IGF-I. L6E9 myoblasts were either harvested at time 0 (GM) or placed in serum-free medium in the absence or presence of des (1 2 3 )IGF-I (20 ng/ml) with or without Na- orthovanadate for 48 h. Cells were then harvested for analysis of Erk1/2 phosphorylation as described in Fig. 1Go. A representative of three independent experiments is shown.

 


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  		Figure 7. Effect of Na-orthovanadate (SOV) on the late stimulation of myogenin mRNA by IGF-I. L6E9 myoblasts were either harvested at time 0 (GM) or placed in DM in the absence or presence of des (1 2 3 )IGF-I (20 ng/ml) with or without 5 µM Na-orthovanadate for 48 h. Cells were then harvested for Northern analysis (A) and quantitation of myogenin mRNA (B) as described in Fig. 2Go. Ethidium bromide staining of the gel is shown in the middle panel. A representative of three independent experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGF-I has a unique, biphasic effect in skeletal myoblasts: an initial mitogenic effect associated with inhibition of differentiation, followed by cessation of proliferation and stimulation of differentiation (1). All of these effects are thought to be mediated by the IGF-I receptor (24, 25) (reviewed in Refs. 7, 26), but the mechanisms responsible for the switch in IGF action from early inhibition to late stimulation of differentiation remain unknown. The MAPK/Erk1/2 signaling pathway is well recognized to mediate a variety of IGF-I actions (7, 8) and to influence skeletal muscle differentiation (9, 10, 11, 12, 13, 14, 15). Myogenic differentiation is inhibited by constitutive activation of this pathway (9, 10), whereas inhibition of MAPK/Erk1/2 signaling augments the differentiation process (9, 11, 14, 15). We find that the early inhibitory effect of IGF-I on induction of myogenin expression is mediated, in part, through an increase in activation of the MEK/Erk1/2 pathway. Furthermore, we find that the opposing late stimulatory effect of IGF-I on myogenin gene expression is associated with a decrease in activation of MEK/Erk1/2. Preventing this time-dependent decrease in Erk1/2 activation completely blocks IGF-I stimulation of myogenin expression. We also find that the onset of myogenic differentiation induced by serum withdrawal, demonstrated by induction of myogenin gene expression, is associated with a rapid but incomplete deactivation of MAPK indicated by a partial decrease in Erk1/2 phosphorylation. Further suppression of MAPK/Erk1/2 activation by treatment with the MEK inhibitor PD098059 enhances the expression of myogenin, demonstrating an inverse relationship between MAPK/Erk1/2 activation and the level of myogenin expression in differentiating myoblasts.

While previous studies have suggested that some degree of MAPK/Erk1/2 activity is required for maintenance of the terminally differentiated phenotype but not for initiating the process of skeletal muscle differentiation (27), our findings demonstrate that the residual MAPK activity in differentiating myoblasts actually plays a role in restraining the induction of differentiation. In fact, our data indicate that decreasing the activation of the MEK/Erk1/2 pathway may be required for induction of differentiation. This is supported by the observation of Bennet and Tonks that inactivation of MAPK/Erk1/2 by ectopic expression of a selective MAPK phosphatase (MKP-1) is sufficient to induce the expression of myogenin even in the presence of serum (11). The mechanisms for this residual MAPK activity in differentiating L6E9 myoblasts are unclear, though unlikely to be a consequence of endogenous IGF-I or IGF-II expression during the 6–7 h time frame of the experiments shown in Figs. 1Go and 2Go. While a variety of skeletal muscle cell lines express high levels of IGF peptides (in particular, IGF-II) following initiation of differentiation induced by serum withdrawal (28, 29, 30, 31, 32), L6E9 cells are known to express little or no IGF-I and express relatively low levels of IGF-II after 24 h of differentiation (31, 33). However, Florini et al. (31) reported that those muscle cell lines which express the least IGF peptides are the most responsive to exogenous IGFs. Thus, L6E9 cells have been widely used as a model of exogenous IGF-induced skeletal muscle differentiation (1, 2, 16, 17, 26, 31, 34). The residual MAPK activity in L6E9 muscle cells during the initial 7 h of serum-free treatment is thus unlikely to be a consequence of IGF peptide secretion but more likely related to other, as yet unidentified, factors.

With respect to the early inhibitory effect of IGF-I on myogenin expression, our finding that PD098059, at a concentration which completely inhibits IGF-I-stimulated phosphorylation of Erk1/2, only partially reverses IGF-I inhibition of myogenin mRNA is consistent with the findings of Weyman and Wolfman in 23A2 myoblasts (14). These data indicate that the inhibitory effect of IGF-I on differentiation of skeletal myoblasts is mediated, in part, through MEK/Erk1/2 signaling and, in part, through other mechanisms. In view of the essential role of PI3K signaling in myogenic differentiation (35, 36), we have examined whether PI3K activity is suppressed in skeletal myoblasts at the time when IGF-I inhibits myogenin expression. Not surprisingly, we found that IGF-I strongly activates, rather than inhibits PI3K signaling, and that partially or completely blocking this activation has no effect on IGF-I inhibition of myogenin mRNA (data not shown).

Several reports have previously concluded that the late stimulatory effect of IGF-I on muscle cell differentiation is mediated through the PI3K signaling pathway (12, 34, 37). This was based on studies in which signaling through PI3K was interrupted by expression of a dominant negative PI3K or by treatment with an inhibitor of PI3K (12, 34, 37). The presence of these inhibitors prevents IGF-stimulated differentiation (12, 34, 37). However, even in the absence of IGF treatment, blocking PI3K signaling alone can completely prevent myogenic differentiation induced by serum withdrawal (34, 37). Therefore, it is not surprising to find that IGF-I can no longer stimulate differentiation in the absence of an intact PI3K signaling pathway. In contrast, in the current study, we hypothesized that the late stimulatory effect of IGF-I on myogenic differentiation is mediated by the concurrent decrease in MEK/Erk1/2 phosphorylation. To prevent this decrease in phosphorylation of Erk1/2, we used Na-orthovanadate, a nonspecific inhibitor of phosphatase activity. At a concentration that effectively prevents IGF-I induced suppression of Erk1/2 phosphorylation, Na-orthovanadate alone has no effect on myogenin expression, but virtually completely prevents the increase in myogenin mRNA by IGF-I. These findings suggest that a Na-orthovanadate- sensitive phosphatase activity may be up-regulated in cells treated with IGF-I for 48 h. Recently, it has been shown in vascular endothelial cells that cell cycle exit induced by contact inhibition is mediated by up-regulation of phosphatase activity in confluent cells, which leads to suppression of Erk1/2 activation (22, 23). Treatment with Na-orthovanadate increased Erk1/2 activation and induced reentry into the cell cycle of contact-inhibited vascular endothelial cells (22). It is therefore reasonable to speculate that, in skeletal muscle cells, the initial mitogenic response to IGF-I leads to increased cell number and cell-to-cell contact, resulting in up-regulation of phosphatase activity, which inhibits Erk1/2 activation. This decrease in Erk1/2 activity could then lead to cessation of proliferation and, by relieving some of the restraining effects of Erk1/2 signaling on myogenic differentiation, to the acceleration of the differentiation process.

While phosphatases may play a role in the time-dependent decrease in both MEK and Erk1/2 phosphorylation by IGF-I, it is possible that other mechanisms, including IGF-I receptor down-regulation, also contribute to this process in skeletal muscle cells. We have previously reported that IGF-I receptor down-regulation (at the levels of mRNA, receptor biosynthesis, receptor degradation, and receptor binding) occurred after 18 h of exogenous IGF peptide treatment in BC3H-1 muscle cells (30, 38). Thus, it is possible that the IGF-induced decrease in MAPK activity after 24 and 48 h in the present study is, in part, a consequence of IGF-I-induced down-regulation of the IGF-I receptor. The parallel decrease in phosphorylation of Erk1/2 with the upstream MEK1/2 (see Fig. 5Go) is consistent with decreased IGF-I receptor signaling via the MEK/MAPK pathway, which may be a consequence of IGF-I receptor down-regulation. Characterization of IGF-I receptor expression following IGF-I treatment in L6E9 cells will be an important avenue for further investigation of the mechanisms responsible for the time-dependent, opposing effects of exogenous IGF peptides on MAPK phosphorylation.

To our knowledge, the current study is the first demonstration that IGFs cause a time-dependent, biphasic effect on MEK/Erk1/2 phosphorylation. While an early stimulatory effect of IGF-I on Erk1/2 phosphorylation in skeletal myoblasts is expected (12), we find that after 24–48 h, IGF-I decreases the phosphorylation of MEK and Erk1/2. Furthermore, our data indicate that this switch in IGF effect on activation of the MAPK/Erk1/2 pathway mediates the switch in IGF-I action from inhibition to stimulation of skeletal muscle cell differentiation.


   Footnotes
 
This work was supported by grants from the National Institutes of Health [K08 DK-02412 (to S.A.) and R01 DK-44181 (to S.M.R.)], from the Endocrine Fellows Foundation (to B.B.A.), and the March of Dimes Birth Defects Foundation [FY98-0129 (to S.M.R.)].

Abbreviations: DM, Differentiation medium; Erk, extracellular signal-regulated kinase; GM, growth medium; IGFBP, IGF binding protein; MEK, MAPK kinase.

Received July 26, 2001.

Accepted for publication October 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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