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Endocrinology Vol. 145, No. 11 4991-4996
Copyright © 2004 by The Endocrine Society

Akt Phosphorylation Is Not Sufficient for Insulin-Like Growth Factor-Stimulated Myogenin Expression but Must Be Accompanied by Down-Regulation of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Phosphorylation

Nicki Tiffin, Saleh Adi, David Stokoe, Nan-Yan Wu and Stephen M. Rosenthal

Department of Pediatrics (N.T., S.A., N.-Y.W., S.M.R.), University of California San Francisco, San Francisco, California 94143; and Cancer Research Institute (D.S.), University of California San Francisco, San Francisco, California 94115

Address all correspondence and requests for reprints to: Stephen M. Rosenthal, Department of Pediatrics, Box 0434, University of California San Francisco, 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 differentiation. Initially, IGF-I inhibits expression of myogenin, a skeletal muscle-specific regulatory factor essential for myogenesis. Subsequently, IGF-I switches to stimulating expression of myogenin. The mechanisms that mediate this switch in IGF action are incompletely understood. Several laboratories have demonstrated that the phosphatidylinositol-3-kinase/Akt signaling pathway is essential for myogenic differentiation and have suggested that this pathway mediates IGF-I stimulation of myogenin mRNA expression, an early critical step in the differentiation process. These studies, however, did not address concurrent Akt and MAPK/ERK1/2 phosphorylation, the latter of which is also known to regulate myogenic differentiation. In the present study in rat L6E9 muscle cells, we have manipulated ERK1/2 phosphorylation with either an upstream inhibitor or activator and examined concurrent levels of Akt and ERK1/2 phosphorylation and of myogenin mRNA expression in response to treatment with IGF-I. We find that even in the presence of phosphorylated Akt, it is only when ERK1/2 phosphorylation is inhibited that IGF-I can stimulate myogenin mRNA expression. Thus, although Akt phosphorylation may be necessary, it is not sufficient for induction of myogenic differentiation by IGF-I and must be accompanied by a decrease in ERK1/2 phosphorylation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I ACTION IS critical for normal skeletal muscle development: Knockout mice lacking the IGF-I receptor have pronounced muscle hypoplasia and die at birth (1). Overexpression of IGF-I in muscle causes increased muscle mass and myofiber hypertrophy (2, 3). Unlike most growth factors, which are believed to stimulate muscle cell proliferation and inhibit differentiation, IGF-I is able to stimulate both proliferation and differentiation of muscle cells in culture (4, 5, 6, 7). These two processes are temporally separated in myoblasts. The early proliferative response to IGF-I is typified by inhibited expression of myogenin, a skeletal muscle-specific regulatory factor essential for myogenic differentiation (8, 9, 10), and the subsequent stimulation of differentiation by IGF-I is accompanied by substantially increased myogenin expression (5, 11).

The mechanisms that mediate the switch in IGF action from inhibition to stimulation of muscle differentiation are incompletely understood. Upon ligand binding, the IGF-I receptor activates a number of downstream signaling cascades, including the Raf-1/MAPK kinase (MEK)1/2/ERK1/2, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways (12). Several laboratories have demonstrated that the PI3K/Akt signaling pathway is essential for myogenic differentiation that occurs in response to serum withdrawal and have suggested that this pathway mediates IGF-I stimulation of myogenin expression (13, 14, 15, 16, 17). However, the MAPK/ERK1/2 pathway, known to regulate myogenic differentiation (18, 19, 20, 21, 22, 23, 24, 25), has also been implicated in IGF-stimulated muscle differentiation (14, 20, 26). In particular, MAPK/ERK1/2 inhibition was shown to augment muscle differentiation stimulated by IGF-I (14, 20). In addition, we have reported that IGF-I stimulation of myogenin mRNA expression is associated with a time-dependent decrease in MEK and ERK1/2 phosphorylation and that blocking this decrease prevented the ability of IGF-I to switch from inhibiting to stimulating myogenin mRNA (26). However, studies addressing the relative importance of stimulated PI3K/Akt signaling vs. inhibition of the MAPK/ERK1/2 pathway in the ability of IGF-I to induce myogenic differentiation have not been previously reported.

In the present study, we have manipulated ERK1/2 phosphorylation with either an upstream inhibitor or activator and examined concurrent ERK and Akt signaling and expression of myogenin mRNA in response to treatment with IGF-I. We find that even in the presence of phosphorylated Akt, it is only when ERK1/2 phosphorylation is inhibited that IGF-I can stimulate expression of myogenin mRNA, an early critical step in the muscle differentiation process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
L6E9 rat skeletal myoblasts (27) were grown to 30% confluence in DMEM (Cell Culture Facility, University of California San Francisco (UCSF)/10% fetal bovine serum (growth medium, GM). For time = 0 h, cells in GM were harvested. Differentiation medium (DM) was DMEM/1% BSA and 1% horse serum. For all IGF-I treatment studies, des(1–3)IGF-I (gift from Genentech, South San Francisco, CA) was used at 20 ng/ml. Des(1–3)IGF-I is an analog of IGF-I with greatly reduced affinity for IGF-binding proteins but unaltered affinity for the IGF-I receptor, and was used to minimize any effects of IGF-binding proteins (28). PD98059 (Cell Signaling Technology, Beverly, MA), a specific inhibitor of MEK1, was used at 30 µM. Sodium orthovanadate was purchased from Calbiochem (La Jolla, CA). A plasmid containing a myogenin cDNA was provided by E. N. Olson (University of Texas Southwestern Medical Center, Dallas, TX).

Plasmids
The pBabe-puro retroviral vector was used (29), containing an estradiol-inducible mutant Raf-1 construct with enhanced ability to activate downstream MEK1/2 and ERK1/2 due to two activating point mutations, Y340D and Y341D, called {Delta}Raf-1:ER(DD) (30, 31). As a negative control, the pBabe-puro vector containing {Delta}Raf301:ER, a kinase-inactive Raf construct, was used. pBabe-puro/Raf constructs were kindly provided by Dr. Martin McMahon, Cancer Research Institute, UCSF.

Cell infections
Transfection of packaging cells.
Phoenix cells in GM were transfected with pBabe-puro constructs containing {Delta}Raf-1:ER(DD), {Delta}Raf301:ER, or no insert. Thirty microliters of fuGENE6 (Roche Diagnostics, Indianapolis, IN) were added to 170 µl of OptiMEM medium (Cell Culture Facility, UCSF). Ten micrograms of plasmid DNA were added to the fuGENE6/OptiMEM mix and incubated for 30 min at room temperature. The mixture was added drop-wise to the cells. The cells were incubated for 24 h at 37 C/5% CO2, and then the medium replaced with fresh GM. Forty-eight hours after transfection, the supernatant was harvested in 1-ml aliquots and 10 ml fresh GM added to the cells. Additional aliquots of supernatant were harvested 72 h after transfection. Aliquots were immediately frozen at –80 C.

Infection of L6E9 cells.
Cells were grown to approximately 30% confluence. One hundred microliters of each viral supernatant were combined with 24 µl polybrene (1 mg/ml) (Specialty Media, Phillipsburg, NJ) and 2876 µl GM. The medium on cells was replaced with the viral mix and grown for 3 h at 37 C/5% CO2. Then 9 ml of GM was added and the cells grown for an additional 48 h. The cells were split 1:10 into GM containing 1.2 µg/ml puromycin (Sigma Chemical Co., St. Louis, MO). Surviving colonies were pooled and tested by Western blotting for constitutive phosphorylation of ERK1/2 in response to treatment with 1 µM estradiol (Sigma).

Western blot analysis
Cells were harvested after a prescribed time in DM by scraping into lysis buffer (20 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA; 0.5–1% Triton X-100; 140 mM NaCl; 25 mM NaF; 1 mM dithiothreitol, 1 mM Na3VO4) containing Complete protease inhibitor (Roche). Proteins were heated to 95 C for 3 min, DNA sheared in a 21-gauge needle, and spun for 10 min at 10,000 rpm. Total protein concentration was determined using the Bio-Rad DC protein assay, according to manufacturer’s instructions. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked overnight in TBS, 0.1% Tween, and 15% milk. Primary antibodies were diluted in TBS, 0.1% Tween, and 2% BSA, and the membrane was incubated with the primary antibody for 2 h at room temperature. The membrane was washed three times for 15 min with TBS and 0.1% Tween, and the secondary antibody was applied in TBS, 0.1% Tween, and 2% BSA for 0.5 h. The membrane was washed three times for 20 min with TBS and 0.1% Tween, and the image was developed using the ECL chemiluminescent system (Amersham Biosciences, Piscataway, NJ). Antibodies used were anti-phospho-ERK1/2 (Cell Signaling Technology) and anti-total ERK1/2 (Cell Signaling Technology) at 1:1000, anti-phospho-Akt (ser473) and anti-total Akt (Cell Signaling Technology) at 1:1000, and horseradish peroxidase-antimouse IgG and horseradish peroxidase-antirabbit IgG (Zymed Laboratories, South San Francisco, CA) at 1:10,000. Autoradiograms were scanned for band densitometry analysis using a Macintosh computer and the public domain NIH Image program, version 1.62 (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/H). Statistical analysis was performed using one-tail Student’s t test, two-sample assuming equal variance.

Northern blot analysis
Total RNA was isolated from L6E9, L6E9/pBabe-puro, L6E9/{Delta}Raf-1:ER(DD), and L6E9/{Delta}Raf301:ER cells in culture using Trizol (Invitrogen Life Technologies, Carlsbad, CA) according to manufacturer’s instructions. Twenty-five micrograms of total RNA were electrophoretically separated on a 0.95% agarose gel containing 2.2 M formaldehyde, transferred to a nylon membrane (Amersham Biosciences), and cross-linked to the membrane by UV cross-linking. Total RNA was shown by ethidium bromide staining. A 32P-radiolabeled probe was synthesized using a random primer method to incorporate [32P]dCTP into complementary strands of myogenin cDNA (Prime-a-Gene, Promega, Madison, WI). After 5 h of incubation at 42 C in a standard prehybridization solution supplemented with 100 µg/ml denatured salmon testes DNA, the membrane was exposed overnight to 2 x 106 counts/min·ml radiolabeled probe in a 50% formamide standard hybridization solution at 42 C. The membrane was rinsed and washed twice for 20 min in 2x standard saline citrate and 0.1% SDS at 42 C before undergoing autoradiography. Band densitometry was assessed as described above.

In vitro kinase assays
Monolayers of L6E9 cells were washed with cold PBS and lysed by scraping into lysis buffer [1% (vol/vol) Triton X-100, 10 mM Tris-HCl (pH 7.4 at 4 C), 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 500 µM Na3VO4, and 1x Roche Complete EDTA-free protease inhibitor cocktail] at 4 C. After centrifugation (10,000 x g for 10 min at 4 C) to remove insoluble components, endogenous Akt was immunoprecipitated using the anti-Akt antibody and protein A-Sepharose at 4 C for 1 h. After washing the immunoprecipitated protein/bead complex, kinase activity was assayed using the synthetic peptide GRPRTSSFAEG (Crosstide) as a substrate in a reaction mixture containing 20 mM Tris-HCl (pH 7.5), 75 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 20 µM ATP, 50 µM Crosstide, and 5 µCi [{gamma}-32P]ATP in a volume of 20 µl per assay. The reaction was allowed to proceed for 15 min at 30 C and then was stopped by the addition of Tricine sample buffer. The phosphopeptide was separated on a 16% Tricine gel, and the amount of 32P radioactivity was assessed using a STORM phosphorImager (Amersham Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF stimulation of myogenin expression occurs when ERK1/2 phosphorylation is down-regulated, despite Akt phosphorylation
We have previously demonstrated that IGF-I treatment of L6E9 skeletal myoblasts [using the des(1–3)IGF-I analog] results in time-dependent opposing effects on ERK1/2 phosphorylation and expression of myogenin mRNA. Initially, IGF-I stimulates ERK1/2 phosphorylation with concomitant inhibition of myogenin expression; subsequently, IGF-I inhibits ERK1/2 phosphorylation in association with increased expression of the myogenin gene (5, 26, 32). To examine concurrent effects of IGF-I treatment on Akt phosphorylation and on myogenin expression, we treated L6E9 cells for up to 48 h in the absence or presence of the des(1–3)IGF-I analog. As seen in Fig. 1AGo, IGF-I significantly up-regulates Akt phosphorylation after 6, 24, and 48 h of treatment. Despite this continuous stimulation of phospho-Akt, IGF-I has a biphasic effect on myogenin mRNA similar to our previously published results (26), where IGF-I initially inhibits myogenin mRNA and subsequently switches to stimulating myogenin mRNA (Fig. 1BGo).



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  		FIG. 1. Continuous stimulation of Akt phosphorylation by IGF-I despite biphasic effect on myogenin mRNA. L6E9 myoblasts were studied for up to 48 h in DM in the absence or presence of the des(1–3)IGF-I analog (IGF). A, Western blot of phosphorylated and total Akt. Shown also are mean ± SD of scanning densitometry analysis for two independent experiments. *, P < 0.05 for IGF- vs. non-IGF-treated cells at each time point. B, Northern blot of myogenin mRNA. Ethidium bromide staining of the gel shows 28S RNA. Shown also are mean ± SD of scanning densitometry analysis for three independent experiments. *, P < 0.05 for IGF- vs. non-IGF-treated cells at each time point.

 
We examined further the relationship between Akt phosphorylation, ERK1/2 phosphorylation, and myogenin expression in response to IGF-I by treating cells in the absence or presence of the MEK1 inhibitor PD98059 (33). After 6 h in DM containing the des(1–3)IGF-I analog, ERK1/2 phosphorylation is present and myogenin mRNA is virtually not detectable despite up-regulation of Akt phosphorylation and Akt kinase activity at this time (Fig. 2Go; see also Fig. 1AGo, 6Go h ± IGF-I). However, when ERK1/2 phosphorylation is inhibited using PD98059 in this context (6 h in DM with IGF-I treatment), myogenin mRNA expression is significantly induced (Fig. 2DGo). As expected, Akt phosphorylation and kinase activity in response to IGF-I treatment were not affected by the MEK1 inhibitor (Fig. 2Go, A and B). Thus, even in the presence of phosphorylated Akt, it is only when ERK1/2 phosphorylation is inhibited that myogenin mRNA expression is induced in response to treatment with IGF-I.



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  		FIG. 2. Despite phospho-Akt, IGF-I stimulates myogenin mRNA only when phospho-ERK1/2 is inhibited. L6E9 myoblasts were studied in DM with the des(1–3)IGF-I analog for 6 h in the absence or presence of the MEK inhibitor, PD98059 (PD). A, Western blot of phosphorylated and total Akt. Shown also are mean ± SD of scanning densitometry analysis for three independent experiments. B, Akt kinase activity. An average of two experiments (mean ± SD) is shown. C, Western blot of phosphorylated and total ERK1/2. Shown also are mean ± SD of scanning densitometry analysis for two independent experiments. Black bars represent phospho-ERK1; white bars represent phospho-ERK2. *, P < 0.05 for PD- vs. non-PD-treated cells. D, Northern blot of myogenin mRNA with ethidium bromide staining of the gel showing 28S RNA. Bar graph represents mean ± SD of scanning densitometry analysis for two independent experiments. *, P < 0.05 for PD- vs. non-PD-treated cells.

 


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  		FIG. 6. Na-orthovanadate (SOV) does not prevent the late inhibitory effect of IGF-I on phosphorylation of ERK1/2 and MEK. Western blot is of phosphorylated and total ERK1/2 and MEK from L6E9 cells in DM for 48 h in the absence or presence of the des(1–3)IGF-I analog without and with increasing doses of SOV. A representative of four independent experiments is shown.

 
Up-regulated ERK1/2 phosphorylation in differentiating L6E9/{Delta}Raf-1:ER(DD) cells prevents IGF-I-stimulated myogenin expression
We sought to explore further the relationship between inhibited ERK1/2 phosphorylation and stimulated myogenin expression in response to IGF-I. Previously, we demonstrated that treatment of L6E9 myoblasts with Na-orthovanadate prevented the late, time-dependent decrease in ERK1/2 phosphorylation by IGF-I and prevented the ability of IGF-I to switch from inhibiting to stimulating myogenin mRNA (26). A limitation of these earlier studies is that the effects of Na-orthovanadate are not limited to phospho-ERK1/2. Accordingly, we sought to specifically up-regulate phospho-ERK1/2 by infecting L6E9 myoblasts with pBabe-puro/{Delta}Raf-1:ER(DD), containing an estradiol-inducible mutant Raf-1construct with enhanced ability to activate downstream MEK1/2 and ERK1/2. Estradiol treatment of L6E9/{Delta}Raf-1:ER(DD) cells in DM resulted in up-regulated ERK1/2 phosphorylation at 24 and 48 h (Fig. 3Go, lanes 2 and 5). This up-regulation of ERK1/2 phosphorylation was not affected by treatment with IGF-I (Fig. 3Go, lanes 3 and 6). In L6E9 parent cells, IGF-I switches from inhibiting to stimulating myogenin expression after 24–48 h (Fig. 1BGo). In contrast, myogenin expression was undetectable for up to 48 h when L6E9/{Delta}Raf-1:ER(DD) cells in DM were treated with estradiol (Fig. 4Go, lanes 4–6), even after 48 h of treatment with IGF-I (Fig. 4Go, lanes 7–9). However, the level of myogenin expression was also very low in these cells even without induction of {Delta}Raf-1:ER(DD) by the addition of estradiol (Fig. 4Go, lanes 1–3). We therefore treated L6E9/{Delta}Raf-1:ER(DD) cells with PD98059 and measured expression of myogenin mRNA. We found that addition of PD98059 increased expression of myogenin mRNA in this cell line (Fig. 5Go, lane 2), and estradiol treatment completely abolished expression of myogenin mRNA (Fig. 5Go, lane 3). We have also shown that estradiol treatment of parent L6E9 cells does not inhibit myogenin expression (Fig. 4Go, lanes 8–10). Similarly, estradiol did not inhibit myogenin expression in L6E9 cells infected with the empty pBabe-puro vector and in L6E9 cells infected with the kinase-inactive {Delta}Raf301:ER construct (data not shown). Cells expressing kinase-inactive {Delta}Raf301:ER, which does not affect endogenous Raf activity, were used as a control to confirm that it is the kinase activity of the Raf protein that has an effect, rather than the presence of the protein itself.



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  		FIG. 3. Estradiol (E) up-regulates phospho-ERK1/2 in L6E9/{Delta}Raf-1:ER(DD) cells in the absence or presence of the des(1–3)IGF-I analog. Cells were placed in DM for up to 48 h in the absence or presence of E and IGF. Western blot is of phosphorylated and total ERK1/2. Shown also are mean ± SD of scanning densitometry analysis for two independent experiments.

 


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  		FIG. 4. Myogenin mRNA is inhibited in L6E9/{Delta}Raf-1:ER(DD) cells treated with estradiol (E) without and with the des(1–3)IGF-I analog. Northern blot is from cells treated for up to 48 h in DM in the absence or presence of estradiol and IGF. Shown for comparison are parent L6E9 cells. Ethidium bromide staining of the gel shows 28S RNA. A representative of two independent experiments is shown.

 


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  		FIG. 5. Myogenin mRNA is augmented by PD98059 (PD) and inhibited by estradiol (E) in L6E9/{Delta}Raf-1:ER(DD) cells. Northern blot is of cells in DM for 24 h in the absence or presence of PD or estradiol. Ethidium bromide staining of the gel shows 28S RNA. A representative of three independent experiments is shown.

 
Na-orthovanadate does not prevent the late inhibitory effect of IGF-I on phosphorylation of ERK1/2 and MEK
In view of the importance of down-regulated ERK1/2 phosphorylation in IGF-stimulated expression of myogenin, we examined the potential role of MAPK phosphatases on the previously reported (26) time-dependent inhibitory effect of IGF-I on ERK1/2 phosphorylation. As seen in Fig. 6Go (lanes 1 and 2), treatment of myoblasts with the des(1–3)IGF-I analog for 48 h markedly inhibits ERK1/2 phosphorylation. In the absence of IGF-I treatment, increasing doses of the phosphatase inhibitor Na-orthovanadate, alone, result in a dose-dependent increase in phospho-ERK1/2, implying the presence of MAPK phosphatases in the untreated state. However, even in the presence of up to 10 µM Na-orthovanadate, IGF-I is still capable of inhibiting ERK1/2 phosphorylation (Fig. 6Go, lanes 3–8). Similar to the effects of IGF-I on ERK1/2 phosphorylation, IGF-I treatment for 48 h inhibits phospho-MEK at doses of Na-orthovanadate up to 10 µM (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although it is well established that both the PI3K/Akt and MAPK/ERK1/2 pathways regulate muscle differentiation (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26), studies to address the relative importance of stimulated PI3K/Akt signaling vs. inhibition of the MAPK/ERK1/2 pathway in the ability of IGF-I to switch from inhibition to stimulation of muscle differentiation have not been previously reported. Here, we find that Akt phosphorylation in response to IGF-I treatment is not alone sufficient to drive expression of myogenin, a skeletal muscle-specific transcription factor essential for differentiation. Despite IGF-induced Akt phosphorylation, it is only when ERK1/2 phosphorylation is inhibited that IGF-I can stimulate expression of myogenin mRNA.

Earlier studies have concluded that the stimulatory effect of IGF-I on muscle cell differentiation is mediated through the PI3K signaling pathway (14, 15, 16). This was based in part on studies in which signaling through PI3K was interrupted by expression of a dominant negative PI3K or by treatment with an inhibitor of PI3K (14, 15, 16). The presence of these inhibitors prevents IGF-stimulated differentiation (14, 15, 16). However, even in the absence of IGF treatment, blocking PI3K signaling alone can completely prevent myogenic differentiation induced by serum withdrawal (13, 15, 16). Therefore, it is not surprising to find that IGF-I can no longer stimulate differentiation in the absence of an intact PI3K signaling pathway.

A more recent study using an inducible Akt in mouse C2 myoblasts demonstrated that Akt expression could promote muscle differentiation even in the presence of the chemical inhibitor of PI3K, LY294002 (17). The results of this study further support the importance of PI3K and its downstream effector Akt in the muscle differentiation process (17). Although this study did not address MAPK/ERK1/2 signaling, down-regulated phospho-ERK1/2 may have been a factor allowing differentiation to proceed as these studies were carried out under conditions of confluent cell density (17). Contact inhibition between cells is known to cause down-regulation of ERK1/2 phosphorylation in various cell types (34, 35, 36, 37), and myoblasts in culture will begin spontaneous myogenic differentiation at confluence despite the presence of exogenous growth factors. In the present study, we have transferred L6E9 cells to differentiation medium and started IGF-I treatment at approximately 30% density to avoid effects of cell confluence. Cells remained subconfluent throughout the experiments (data not shown).

The initial decrease in ERK1/2 phosphorylation is necessary for the initiation of IGF-stimulated myogenin expression. However, we previously reported that after the initial decrease, ERK1/2 phosphorylation levels slowly increase as differentiation of myoblasts proceeds (26). This is consistent with similar observations of the requirement for ERK1/2 phosphorylation in the later stages of myogenesis, particularly myoblast fusion to form multinucleate myofibers (22, 24, 25, 38). Thus, a decrease in ERK1/2 phosphorylation may be required for initiation and early commitment to myogenesis, whereas terminal differentiation and fusion in the final stages of myogenesis require some degree of ERK1/2 phosphorylation to proceed.

In view of the importance of down-regulated ERK1/2 phosphorylation in IGF-induced myogenin expression, we explored potential mechanisms responsible for the late time-dependent inhibitory effect of IGF-I on phospho-ERK1/2. Although a Na-orthovanadate-sensitive phosphatase activity is present in untreated myoblasts in differentiation medium, we find that IGF-I is capable of inhibiting both phospho-ERK1/2 and phospho-MEK even in the presence of doses of Na-orthovanadate up to 10 µM. Thus, it would appear that Na-orthovanadate-sensitive phosphatase activity does not play a principal role in the late inhibitory effect of IGF-I on both phospho-ERK1/2 and phospho-MEK. The decrease in both phospho-ERK1/2 and phospho-MEK is consistent with decreased IGF-I receptor signaling in the MEK/MAPK pathway, which may be a consequence of IGF-I receptor down-regulation.

We believe that our observations of a biphasic and opposite effect of IGF-I on ERK1/2 phosphorylation and myogenin expression, on a background of persistent Akt phosphorylation, could provide a unifying explanation for the various observed roles of the PI3K and MAPK pathways in IGF-I-induced myogenesis. We have shown that initiation of differentiation in the early response of subconfluent L6E9 cells to IGF-I treatment requires a decrease in ERK1/2 phosphorylation and that although Akt phosphorylation may be required for myogenesis to proceed, it is not able to drive initiation of myogenin expression unless accompanied by this decrease in ERK1/2 phosphorylation. This model does not exclude a later requirement for phosphorylation of ERK1/2 for myoblasts already committed to myogenic differentiation to undergo fusion and form multinucleate myofibers.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (NIH) (R01 DK44181) and Pfizer, Inc., to S.M.R. and from the NIH (K08 DK-02412) to S.A.

Present address for N.T.: SANBI, University of Western Cape, Bellville 7535, South Africa.

Present address for S.A.: California Pacific Medical Center Research Institute, San Francisco, California 94115.

Present address for N.-Y.W.: Lung Biology Center, University of California San Francisco, San Francisco, California 94110.

Abbreviations: DM, Differentiation medium; GM, growth medium; MEK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase.

Received January 28, 2004.

Accepted for publication July 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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