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Mol Cell Biol. May 2000; 20(9): 3256–3265.
PMCID: PMC85619

Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways

Abstract

In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.

Peptide growth factors regulate cell fate by activating specific transmembrane receptors, leading to the stimulation of multiple intracellular signal transduction pathways (64). Insulin-like growth factors I and II (IGF-I and -II) are small, structurally related proteins of fundamental importance for normal somatic growth and for the survival, proliferation, and differentiation of different cell types (5, 32, 57). The actions of both IGFs are mediated by the IGF-I receptor, a ligand-activated tyrosine protein kinase that is related to the insulin receptor (32, 44), and are modulated by a family of specific IGF binding proteins (13, 32).

IGF action is critical for the normal development and maintenance of skeletal muscle. Mice engineered to lack the IGF-I receptor exhibit profound muscle hypoplasia and die in the neonatal period because of inadequate strength to inflate the lungs (46). Conversely, mice with overexpression of IGF-I in muscle develop increased muscle mass secondary to myofiber hypertrophy (4, 12). In cultured myoblasts, IGF action stimulates terminal differentiation through an autocrine pathway dependent on the expression and secretion of IGF-II (18, 20, 22, 45, 47, 56). IGF-II also plays a key role in maintaining cell survival during the transition from proliferating to terminally differentiating myoblasts (58). The signal transduction pathways involved in IGF-mediated muscle cell survival have not been identified. Preliminary studies have suggested that two classes of regulated intracellular enzymes, phosphatidylinositol 3-kinase (PI3-kinase) and extracellular regulated kinases (ERKs), are involved in different aspects of IGF-facilitated muscle differentiation (14, 33, 34, 49, 53, 54), although the mechanisms by which these signaling molecules collaborate with specific myogenic regulatory factors remain undefined.

In this work we addressed the signal transduction pathways involved in IGF-mediated muscle cell survival by studying both wild-type C2 myoblasts and a derived cell line that lacks endogenous expression of IGF-II (58). These cells undergo apoptotic death in low-serum differentiation medium (DM), which can be prevented by IGF analogs that activate the IGF-I receptor or by the unrelated growth factor platelet-derived growth factor BB (PDGF-BB). We find that IGF-I and PDGF-BB use distinct signaling pathways to maintain myoblast viability. Treatment with IGF-I leads to the sustained stimulation of PI3-kinase and its downstream kinase, Akt, but only transient activation of the Ras-Raf-Mek-ERK pathway. By contrast, PDGF caused sustained stimulation of ERK1 and -2, but only transient induction of Akt, even though it also activated PI3-kinase to the same extent and duration as IGF-I. Forced expression of a constitutively active PI3-kinase or a conditionally active Akt maintained myoblast survival in the absence of growth factors, as did a constitutively active Mek1. Blockade of Mek activity by a specific pharmacological inhibitor prevented PDGF-mediated but not IGF-stimulated muscle cell survival, while interference with PI3-kinase activity inhibited only IGF-mediated survival. Our results thus show that distinct and apparently independent signal transduction pathways promote muscle cell survival in response to different growth factors.

MATERIALS AND METHODS

Materials.

Tissue culture supplies, fetal calf serum (FCS), newborn calf serum, horse serum, Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), PDGF-BB, and G418 were purchased from Gibco-BRL Life Technologies (Grand Island, N.Y.). R3IGF-I was obtained from Gropep (Adelaide, Australia), and Effectene was from Qiagen (Chatsworth, Calif.). Restriction enzymes, ligases, and polymerases were purchased from New England Biolabs (Beverly, Mass.), American Allied Biochemical (Aurora, Colo.), or Promega Biotec (Madison, Wis.). Protease inhibitor tablets were obtained from Roche Molecular Biochemicals (Indianapolis, Ind.), and 4-hydroxytamoxifen (HT; dissolved in ethanol at a concentration of 50 mM) was from Sigma Chemical Co. (St. Louis, Mo.). The bicinchoninic acid (BCA) protein assay kit was from Pierce Chemical Co. (Rockford, Ill.). Nitrocellulose was obtained from Schleicher & Schuell (Keene, N.H.). Reagents for enhanced chemiluminescence (ECL) were purchased from Amersham Pharmacia Biotechnology (Piscataway, N.Y.). X-ray film was from Kodak (Rochester, N.Y.). LY294002 (Biomol Research Laboratories, Plymouth Meeting, Pa.) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 30 mM and stored at −20°C until use. UO126 (Promega Biotec) was diluted to a concentration of 20 mM in DMSO and stored at −20°C until use. Kinase assay kits for Akt and ERKs were purchased from New England Biolabs and used as specified by the manufacturer. The ApopTag fluorescein in situ apoptosis detection kit was obtained from Intergen (Purchase, N.Y.) and used as described by the manufacturer. Lab Tek-II chamber slides were supplied by Nalge Nunc International (Naperville, Ill.). All other chemicals were reagent grade and were obtained from commercial suppliers.

Antibodies were from the following sources. Anti-Akt, anti-ERK1 and -2, anti-phospho-Akt, and anti-phospho-ERK were from New England Biolabs; antiphosphotyrosine (PY20) coupled to agarose was purchased from Transduction Laboratories (Lexington, Ky.); anti-Myc epitope tag was a gift from William Skach, Oregon Health Sciences University, Portland. Conjugated secondary antibodies were from Sigma.

Recombinant plasmids were obtained from the following sources. pEGFP-N3 was purchased from Clontech (Palo Alto, Calif.); wild-type human Mek1 and constitutively active rabbit Mek1 were gifts from Philip Stork, of Oregon Health Sciences University; constitutively active and inert PI3-kinases were obtained from Anke Klippel, Chiron Corporation (Emeryville, Calif.); inducible Akt was a gift from Richard Roth, Stanford University School of Medicine (Palo Alto, Calif.).

Cell culture.

C2 myoblasts stably transfected with an IGF-II antisense cDNA (C2AS12 cells [58]) were grown on gelatin-coated tissue culture dishes in DMEM containing 10% heat-inactivated FCS, 10% heat inactivated newborn calf serum, l-glutamine (2 mM), and G418 (400 μg/ml) (growth medium) until ~100% confluent density was reach. Parental C2 cells were incubated in growth medium lacking G418. Differentiation was initiated following washing with PBS by incubating cells in DM containing DMEM plus 2% horse serum or in DM supplemented with PDGF-BB or the long-lasting IGF-I analogue R3IGF-I at the indicated concentrations. At different intervals, adherent, viable cells were trypsinized and counted either by hematocytometer or by Coulter particle counter. Nonadherent, dead cells in the culture medium were counted similarly. For studies using inhibitors, cells were first incubated in DM supplemented with PDGF-BB or R3IGF-I for 18 h. Subsequently, the medium was replaced with DM containing fresh growth factors plus either an inhibitor (LY294002 at 30 μM, UO126 at 10 μM) or an equal volume of DMSO, and myoblasts were incubated for a further 3 or 6 h before adherent and detached cells were counted. For kinase assays, confluent C2AS12 cells were preincubated in DM for 1 h before addition of growth factors. Cos7 cells were grown in DMEM supplemented with 10% heat inactivated FCS and l-glutamine.

Construction of a bicistronic expression plasmid for an inducible Akt.

A 0.7-kb fragment containing the internal ribosome entry site from mouse encephalomyocarditis virus (25) was subcloned using SalI and BglII sites into the polylinker of pEGFP-N3 to generate pEGFP-IRES. A modified human Akt-1 cDNA was added 5′ to the internal ribosome entry site as a 2.1-kb BamHI-SalI fragment. The Akt cDNA, described previously (40), contains a truncated amino terminus encoding a myristoylation sequence and a carboxyl terminus fused in frame to the modified ligand binding domain of mouse estrogen receptor α. The recombinant plasmid was tested initially in Cos7 cells. Cells were grown in six-well dishes and were transfected with 2 μg of DNA by the calcium phosphate precipitation method (51). Expression of enhanced green fluorescent protein (EGFP) was assessed by fluorescence microscopy (Nikon Eclipse TE 300) 48 to 72 h after transfection, and Akt expression was analyzed after incubation of cells with 1 μM HT by immunoblotting or kinase assay as described below.

Transfection of myoblasts.

C2AS12 myoblasts were plated at ~50,000 cells/ml onto 6- or 12-well dishes and cultured for 24 h as previously described (58). Transfections were performed using Effectene, and 2.0 μg of total plasmid DNA per well of a six-well dish, as specified by the manufacturer. For the Mek1 and PI3-kinase plasmids, cotransfections were performed with 0.5 μg of pEGFP-N3 and 1.5 μg of the plasmid of interest in each well of a 12-well dish. After incubation with DNA for 18 to 24 h, fresh growth medium was added to the cells for an additional 24 h, followed by incubation in DM without or with PDGF or R3IGF-I as described above.

Cell survival assays of transfected cells.

C2AS12 myoblasts were transfected in parallel as described. When cells were confluent at ~48 h after transfection (time zero [T0]), two wells were harvested and the total number of cells per well was counted by hemocytometer (T0total). Transfection efficiency was determined by averaging the fraction of cells expressing EGFP in 20 hematocytometer fields at a magnification of ×200 (T0transfected). The remaining wells were incubated in DM without or with growth factors or HT for 24 h. Cells were then harvested, and total and transfected myoblasts were counted. Percent survival of transfected cells was determined as (T24transfected/T0transfected) × (T24total/T0total) × 100.

Protein extraction and immunoblotting.

After the cells were washed twice with cold PBS containing 1 mM sodium orthovanadate, total cellular proteins were isolated by incubation for 30 min at 4°C in radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1.0% NP-40, 1.0% deoxycholate) containing protease inhibitors, 1 μM okadaic acid, and 1 mM sodium orthovanadate. After removal of insoluble material by centrifugation, the protein concentration of the supernatant was measured by BCA assay. Protein extracts (60 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) under denaturing and reducing conditions before transfer onto 0.2 μM nitrocellulose membranes at 18 V for 45 min using a semidry blotter. Membranes were blocked for 2 h at 25°C using Tris-buffered saline containing 5% nonfat dry milk before being incubated with primary antibody (anti-Akt or anti-phospho-Akt, 1:1,000; anti-ERK or anti-phospho-ERK, 1:1,000; anti-Myc tag, 1:1,000). After addition of horseradish peroxidase-conjugated secondary antibodies, proteins were detected by ECL and membranes were exposed to X-ray film. Results were quantitated by densitometry (Bio-Rad GS 700 densitometer).

Kinase assays.

For PI3-kinase assays, cells were first lysed in a buffer consisting of 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.4 mM sodium orthovanadate, 1 μg of leupeptin per ml, and 1 μg of aprotinin per ml. Protein concentrations were determined by BCA protein assay. Cell lysates (400 μg) were immunoprecipitated overnight at 4°C using the antiphosphotyrosine antibody coupled to agarose beads (35). Immune complexes were collected by centrifugation and then washed twice in PBS containing 1% Triton X-100, twice in 10 mM Tris-HCl (pH 7.5) containing 0.5 mM LiCl, and twice in 10 mM Tris-HCl (pH 7.5) containing 100 mM NaCl and 1 mM EDTA. Washed samples were suspended in 35 μl of kinase assay buffer (20 mM HEPES [pH 8.0], 0.4 mM EGTA, 10 mM MgCl2, 100 μM sodium orthovanadate), and 10 μl of phosphatidylinositol at 1 mg/ml was added in the same buffer. Following a 20-min incubation at 25°C, 10 μM [γ-32P]ATP was added, and the reaction was continued for an additional 15 min before being stopped by addition of 80 μl of 1 M HCl and 160 μl of a 1:1 solution of chloroform-methanol. The organic phase was reextracted after centrifugation with 160 μl of chloroform-methanol solution, and the organic phase was separated by thin-layer chromatography (35) before exposure of the plates to X-ray film. Results were quantitated by densitometry.

Immune complex-kinase assays for ERKs or Akt were performed following the protocols described in the kits purchased from New England Biolabs. Cell lysates were incubated with immobilized antibodies overnight at 4°C. Immune complexes were washed twice in cell lysis buffer and twice in kinase buffer followed by addition of kinase assay buffer containing either Elk-1, the substrate for ERKs, or glycogen synthase kinase 3α (GSK-3α), substrate for Akt. After a 30-min incubation at 30°C the reaction was stopped by addition of 6× SDS loading buffer. Samples were then separated by SDS-PAGE and transferred to nitrocellulose membranes as described above. Immunoblotting was performed with primary antibodies to either phospho-Elk-1 or phospho-GSK-3α. After addition of conjugated secondary antibodies, detection by ECL, and exposure to X-ray film, results were quantitated by densitometry.

TUNEL assay.

C2AS12 cells were grown on gelatin-coated four-chamber Lab Tek-II slides. Confluent cells were incubated in DM without or with either PDGF-BB (0.4 nM) or R3IGF-I (2 nM) for 24 h before analysis of DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay using the protocol supplied by the manufacturer. Fluorescence and phase-contrast photomicrographs were taken at 400× using a Nikon Eclipse TE 300 fluorescence microscope.

Statistical analysis.

Data are presented as the mean ± standard error of the mean (SE). Statistical significance was determined using an independent Student t test. Results were considered statistically significant when P < 0.05.

RESULTS

Prevention of muscle cell death by IGF-I or PDGF.

We previously showed that C2 myoblasts engineered to lack IGF-II underwent rapid apoptotic death when incubated in DM (58). Addition of exogenous IGF-I, IGF-II, or other IGF-I analogues prevented cell death, as did other growth factors such as PDGF (58). As pictured in Fig. Fig.1A,1A, only ~50% of muscle cells remained adherent and alive after a 24-h incubation in DM. By contrast, treatment with R3IGF-I (2 nM) or PDGF (0.4 nM) maintained complete survival. Under these conditions, neither growth factor stimulated cell proliferation, since the total number of adherent plus detached and dead cells remained constant throughout the 24-h study interval (Fig. (Fig.1A).1A). Additionally, no proliferation was seen in myoblasts incubated in DM alone. Further cell death occurred upon longer incubations in DM. Only 30% of cells were viable at 48 h, compared with nearly 100% survival after IGF-I or PDGF treatment. Treatment with growth factors also prevented DNA fragmentation, as assessed by TUNEL assay. As shown in Fig. Fig.1B,1B, the majority of still-adherent cells are TUNEL positive after incubation in DM, compared with very few of the myoblasts incubated with IGF-I or PDGF.

FIG. 1FIG. 1
IGF-I and PDGF promote myoblast survival. (A) Cell counts of living (left) or dead (right) C2AS12 myoblasts after a 24-h incubation in DM without or with R3IGF-I (2 nM) or PDGF-BB (0.4 nM). The asterisk at the left indicates that significantly fewer cells ...

Parental C2 cells also underwent apoptotic death in DM, although to a more limited extent than observed with IGF-II-deficient cells. As shown in Fig. Fig.1C,1C, ~65% of C2 cells survived after a 24-h incubation in DM, compared to nearly 100% of myoblasts incubated with IGF-I (0.4 nM) or PDGF (0.3 nM). Under these conditions, little cell proliferation was seen, as total cell number remained constant. Unlike IGF-II-deficient myoblasts, little additional death occurred in parental C2 cells during longer incubations in DM alone (<2% between 24 and 48 h or between 48 and 72 h [data not shown]), potentially reflecting the effects on survival of endogenously expressed IGF-II, which increases markedly in abundance after 24 h in DM (60). Thus, growth factor treatment prevents apoptotic death of both parental and IGF-II-deficient skeletal myoblasts.

PDGF-stimulated MAP kinase activity promotes myoblast survival.

The results from Fig. Fig.11 prompted us to evaluate the signal transduction cascades involved in growth factor-regulated muscle cell survival. We first focused on the Ras-Raf-Mek-Erk pathway, which has been implicated previously in muscle differentiation (6, 27, 53). Figure Figure22 shows results of time course studies examining ERK phosphorylation and enzymatic activity as a function of treatment with PDGF or IGF-I. Figure Figure2A2A shows that PDGF stimulates the rapid and sustained phosphorylation of ERK1 and -2, as assessed with an antibody specific for phosphorylation sites at threonine 183 and tyrosine 185 (11, 55). ERK phosphorylation was detected at 5 min, the earliest time point examined, and persisted for up to 240 min after PDGF treatment. Similar results were obtained for ERK enzymatic activity, as evaluated by an in vitro kinase assay. After PDGF treatment, ERK activity was induced nearly 27-fold above baseline by 5 min and remained at 65% of maximal values at 240 min (Fig. (Fig.2B).2B). By contrast, incubation of confluent myoblasts with IGF-I resulted in transient ERK phosphorylation and enzymatic activation, which quickly declined to basal levels after 15 min.

FIG. 2
PDGF treatment leads to sustained stimulation of ERK1 and -2. (A) Representative immunoblot using a phospho-specific ERK1 and -2 antibody and whole cell protein extracts from C2AS12 cells treated with either PDGF (0.4 nM) or R3IGF-I (2 nM) for the indicated ...

To evaluate the role of ERK activation in growth factor-stimulated muscle cell survival, confluent myoblasts were treated with PDGF or IGF-I for 18 h, followed by a 6-h incubation with either growth factor plus the Mek1 inhibitor UO126 (21). Under these conditions, treatment of C2AS12 cells with IGF-I maintained complete myoblast survival (Fig. (Fig.3A),3A), even in the presence of UO126 at the lowest dose that fully blocked activation of ERKs (data not shown). By contrast, addition of UO126 led to the death of ~50% of PDGF-treated cells within 6 h (Fig. (Fig.3A),3A), with most of the myoblasts dying by 3 h (data not shown). Addition of UO126 also caused a small decline in survival in cells incubated in DM alone.

FIG. 3
Treatment with an inhibitor of Mek1 attenuates PDGF-mediated muscle cell survival. (A) Confluent C2AS12 myoblasts were treated with PDGF (0.4 nM) or R3IGF-I (2 nM) for 18 h, followed by a 6-h incubation with either growth factor plus UO126 (10 μM). ...

UO126 also decreased PDGF-stimulated survival of parental C2 myoblasts. Addition of the Mek inhibitor led to the rapid death of cells incubated with PDGF but had little effect on IGF-treated myoblasts (Fig. (Fig.3B).3B). A small decrease in viability also was seen in cells incubated in DM without added growth factors.

Based on these results, we next asked if Mek1 was able to function as a myoblast survival factor. C2AS12 cells were cotransfected with expression plasmids for the marker protein EGFP plus either a constitutively active or wild-type Mek1. The latter protein requires activation by phosphorylation (11, 55). Viability of transfected cells was assessed after a 24-h incubation in DM or in DM supplemented with PDGF (0.4 nM). Transfection with active Mek1 (Mek1*) significantly enhanced survival compared with cells transfected with wild-type Mek1 (Fig. (Fig.4A,4A, P < 0.0005), which had a survival rate similar to that seen in untransfected myoblasts (compare with Fig. Fig.1).1). Incubation with PDGF maintained complete viability of cells transfected with either plasmid, indicating that the process of transfection was not toxic to the cells. An enzymatic assay showed that as expected there was significantly more ERK activity in cells transfected with Mek1* than in myoblasts transfected with wild-type Mek1 (Fig. (Fig.4B).4B). In addition, incubation of cells with the Mek inhibitor UO126 completely abolished the ability of Mek1* to maintain myoblast survival, even in the presence of PDGF (Fig. (Fig.5).5). Taken together, the results in Fig. Fig.33 to to55 demonstrate that the Mek-ERK pathway mediates myoblast survival in response to PDGF and indicate that continuous activation of this pathway is required for PDGF-stimulated cell survival, since its inhibition resulted in rapid cell death.

FIG. 4
Forced expression of active Mek1 maintains myoblast survival. (A) C2AS12 myoblasts were transiently transfected with recombinant expression plasmids encoding either a constitutively active Mek1 (Mek1*) or wild-type enzyme (Mek1WT). Cell counts of transfected ...
FIG. 5
Reversal of muscle cell survival by the Mek1 inhibitor UO126. Transfected myoblasts were incubated in DM or in DM supplemented with PDGF (0.4 nM) for 18 h, followed by addition of UO126 (10 μM) for 6 h. Cell counts of viable myoblasts were performed ...

Activation of PI3-kinase by IGF-I and PDGF.

Several studies have shown that PI3-kinase is activated by treatment of cells with either IGF-I or PDGF (8, 26, 50) and have implicated this enzyme as a key intermediate in cell survival regulated by both growth factors (19, 36, 37, 61, 66). Incubation of myoblasts with either growth factor led to the acute and sustained induction of enzymatic activity, as assessed by in vitro kinase assay, with PDGF appearing to be more effective than IGF-I (Fig. (Fig.6).6). As expected, PI3-kinase activity was blocked by the specific inhibitor LY294002 (63).

FIG. 6
Both IGF-I and PDGF stimulate PI3-kinase activity. (A) Autoradiograph showing results of an in vitro PI3-kinase assay using cell extracts from confluent myoblasts incubated in DM without or with R3IGF-I (2 nM) or PDGF-BB (0.4 nM) for 5 min, performed ...

PI3-kinase activity is necessary for IGF-regulated muscle cell survival.

To evaluate the role of the PI3-kinase pathway in growth factor-stimulated muscle cell survival, confluent myoblasts were treated with PDGF or IGF-I for 18 h, followed by a 6-h incubation with either growth factor plus the PI3-kinase inhibitor LY294002 (30 μM). Incubation with PDGF maintained nearly complete cell survival (Fig. (Fig.7A),7A), even in the presence of LY294002 at the lowest dose that fully blocked enzymatic activity in vitro (data not shown). By contrast, LY294002 caused the death of ~50% of IGF-treated myoblasts within 6 h of addition (Fig. (Fig.7A),7A), with most of the cells dying within 3 h (data not shown). Addition of LY294002 also caused a small decrease in survival of myoblasts incubated in DM alone. LY294002 also decreased IGF-mediated survival in parental C2 cells. Addition of the PI3-kinase inhibitor caused rapid death of myoblasts incubated with IGF-I but did not block PDGF-stimulated cell survival (Fig. (Fig.7B).7B). A small inhibitory effect also was observed in myoblasts incubated in DM without added growth factors.

FIG. 7
A PI3-kinase inhibitor blocks IGF-mediated myoblast survival. (A) C2AS12 cells were incubated in DM or in DM supplemented with PDGF (0.4 nM) or R3IGF-I (2 nM) for 18 h, followed by addition of LY294002 (30 μM) for 6 h. Cell counts of viable myoblasts ...

Based on these results, we next asked if PI3-kinase could function as a muscle cell survival factor. C2AS12 myoblasts were cotransfected with expression plasmids for the marker protein EGFP plus either a constitutively active or inert PI3-kinase (p110* and p110Δkin, respectively [31]). Viability of transfected cells was assessed after incubation for 24 h in DM or in DM supplemented with IGF-I. In the absence of growth factors, transfection of p110* significantly enhanced survival compared with cells transfected with the inactive enzyme, p110Δkin (Fig. (Fig.8,8, P < 0.004), which had a survival rate similar to that seen in untransfected myoblasts (Fig. (Fig.1).1). Treatment with IGF-I maintained complete viability of cells transfected with either plasmid, indicating that transfection was not harmful to the cells. Incubation of transfected myoblasts with the PI3-kinase inhibitor LY294002 for 6 h overcame the ability of p110* to sustain myoblast survival, even in the presence of IGF-I (Fig. (Fig.9).9). Taken together, these results demonstrate that PI3-kinase mediates myoblast survival in response to IGF-I and indicate that sustained activation of this pathway is required for IGF-stimulated cell survival, since its disruption caused rapid myoblast death.

FIG. 8
Forced expression of active PI3-kinase maintains myoblast survival. (A) C2AS12 myoblasts were transiently transfected with recombinant expression plasmids encoding either a constitutively active PI3-kinase (p110*) or an inactive enzyme (p110Δkin). ...
FIG. 9
Reversal of muscle cell survival by the PI3-kinase inhibitor LY294002. Transfected C2AS12 myoblasts were incubated in DM or in DM supplemented with R3IGF-I (2 nM) for 18 h, followed by addition of LY294002 (30 μM) for 6 h. Cell counts of viable ...

IGF-stimulated Akt kinase activity promotes myoblast survival.

The serine threonine kinase Akt occupies a critical role in growth factor-regulated cell survival as an intermediate between PI3-kinase and inhibition of death-promoting factors (7, 9, 10, 15, 17, 23, 24, 48). Figure Figure1010 shows results of time course studies examining Akt phosphorylation and enzymatic activity as a function of treatment of C2AS12 myoblasts with IGF-I or PDGF. Figure Figure10A10A demonstrates that IGF-I stimulates the rapid and sustained phosphorylation of Akt, as assessed with an antibody specific for the phosphorylation site at serine 473 (1). Akt phosphorylation was seen at 5 min, the earliest time point examined, and remained readily detectable for up to 240 min after IGF treatment. Similar results were obtained for Akt enzymatic activity, as evaluated by in vitro kinase assay. Enzymatic activity was induced by 5 min and remained elevated at 240 min (Fig. (Fig.10B).10B). By contrast, incubation of cells with PDGF resulted in transient increases in Akt phosphorylation and enzymatic activity, with the latter returning to baseline values after 15 min.

FIG. 10
IGF treatment leads to sustained activation of Akt. (A) Representative immunoblot using a phospho-specific Akt antibody and whole cell protein extracts from C2AS12 cells treated with either R3IGF-I (2 nM) or PDGF (0.4 nM) for the indicated times (top) ...

To assess the role of Akt in promoting myoblast survival, we next asked if an inducible Akt fusion protein was able to maintain viability in the absence of growth factors. C2AS12 cells were transfected with an expression plasmid containing a modified human Akt-1 with a truncated and myristoylated amino terminus and a carboxyl terminus fused in frame to the modified ligand binding domain of mouse estrogen receptor α (40). Survival of transfected cells was assessed after incubation for 24 h in DM or in DM supplemented with IGF-I, with the inducer HT, or with both compounds. As seen in Fig. Fig.11A,11A, activation of Akt by HT significantly enhanced survival compared with untreated cells (P < 0.003), which had a survival rate similar to that observed in untransfected myoblasts (compare with Fig. Fig.1).1). Incubation with IGF-I also maintained complete survival, indicating that neither transfection nor HT was toxic to the cells. Both phosphorylation and kinase activity of the Akt fusion protein were stimulated by HT, but levels of protein expression were not regulated (Fig. (Fig.11B11B and C). Taken together, these results demonstrate that the PI3-kinase–Akt pathway mediates myoblast survival in response to IGF-I.

FIG. 11
Akt promotes muscle cell survival. (A) C2AS12 myoblasts were transiently transfected with a recombinant expression plasmid encoding an inducible Akt (iAkt). Cell counts of transfected myoblasts were performed 24 h after incubation in DM or in DM supplemented ...

DISCUSSION

In previous studies, we established that cultured myoblasts engineered to lack IGF-II underwent rapid apoptotic death when incubated in low-serum DM and demonstrated that survival could be maintained by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as PDGF-BB (58). These prior results showed that IGF-II functioned as an autocrine survival factor for skeletal myoblasts and also suggested that different growth factors might use common mechanisms to maintain muscle cell viability. Surprisingly, we now find that IGF-I and PDGF-BB use distinct signaling pathways to keep myoblasts alive. Treatment with IGF-I leads to sustained stimulation of PI3-kinase and its downstream kinase, Akt, but causes only transient activation of the Ras-Raf-Mek-ERK pathway. In conjunction with these findings, forced expression of a constitutively active PI3-kinase or a conditionally active Akt maintained myoblast survival in the absence of growth factors, while blockade of induction of Mek1 and -2 by the specific inhibitor UO126 did not prevent IGF-mediated myoblast survival. By contrast, treatment with PDGF caused sustained stimulation of ERK1 and -2 but only transient activation of Akt, even though PDGF also induced PI3-kinase activity to at least the same extent and duration as IGF-I. In both parental C2 cells and IGF-II-deficient myoblasts, PDGF-mediated myoblast survival was blocked by UO126 but was not diminished by LY294002, a specific inhibitor of PI3-kinase that prevented IGF-regulated cell survival. In further support of the key role of the Mek-ERK pathway in PDGF-mediated muscle cell survival, forced expression of a constitutively active Mek1 could maintain myoblast viability in the absence of growth factors. Since inhibition of ectopic Mek1 by UO126 led to cell death that could not be prevented by PDGF, these results in aggregate show that distinct and apparently independent signal transduction pathways promote muscle cell survival in response to different growth factors. These observations also indicate that continuous stimulation of these signaling pathways is required for growth factor-regulated myoblast viability, since their inhibition resulted in rapid cell death.

The role of PI3-kinase to activate Akt and the participation of Akt in cell survival mediated by growth factors, including IGF-I and PDGF, have been well documented (17, 23, 36, 37, 42, 43). In several cell types interference with this pathway blocked growth factor regulated viability (17, 19, 36, 37, 42, 43), although this has not been shown previously in skeletal muscle. The central role of Akt in preventing cell death also has been affirmed through the demonstration of its ability to directly inhibit proapoptotic molecules (9, 10, 15, 48). Phosphorylation by Akt of pro-caspase 9, one of the effectors of apoptosis (52, 62), blocks its proteolytic processing and activation (10), thereby inhibiting the enzymatic machinery of cell death. Phosphorylation by Akt of BAD, a proapoptotic member of the Bcl-2 family, impairs its ability to inhibit the activity of antiapoptotic members of this family and prevents cell death (15). Phosphorylation of the forkhead transcription factor FKHR-L1 by Akt results in its exit from the nucleus and sequestration in the cytoplasm by 14-3-3 proteins, thus blocking its ability to induce transcription of genes whose protein products promote cell death (9). Similar mechanisms are probably involved in the inactivation by Akt of Afx and FKHR, other members of forkhead family which are regulated by growth factors (7, 41, 59). Another substrate of Akt, GSK-3, also may participate in promoting apoptosis when not inactivated by phosphorylation by Akt (48). It is not yet known whether any of these molecules are important mediators of cell death in myoblasts.

In contrast to the PI3-kinase–Akt pathway, which appears to play a key role in preventing cell death in many cell types, the function of different mitogen-activated protein (MAP) kinases in modulating cell survival is not well characterized. Distinct components of the Ras-Raf-Mek-ERK pathway have been implicated in both apoptosis and cell survival under different circumstances (16). Cell death induced by an activated Myc in fibroblasts may be blocked by PI3-kinase and Akt (36) but appears to be enhanced by activated Ras or Raf1 (36). By contrast, in other cell types, Ras mediates survival by activating PI3-kinase (38, 39). Similarly, forced expression of an activated Mek1 is sufficient to maintain viability in the absence of other survival factors in PC12 cells (65), and sustained stimulation of the Mek-ERK pathway by neurotrophins may counteract the cell death induced by neurotoxic drugs (2, 30). These latter observations are potentially similar to our results for IGF-II-deficient muscle cells, although in each case the mechanism of survival is unknown. In contrast to these findings, other MAP kinases, including p38 and c-Jun N-terminal kinase/stress-activated protein kinase, appear to promote cell death by as yet uncharacterized mechanisms (16, 65).

One of the more surprising observations in this study was the transient induction of Akt by PDGF, even though growth factor treatment led to the sustained stimulation of PI3-kinase to levels that were at least equivalent to those induced by IGF-I. These results may reflect selectivity in the isoforms of PI3-kinase that are activated by the PDGF or IGF-I receptors, since several PI3-kinase regulatory subunits have been detected in muscle cells (3, 33). Alternatively, they may indicate interference by other signaling pathways with PI3-kinase activity or subcellular localization, or they may be a consequence of growth factor-selective differences in the Akt isoforms induced, since both Akt1 and Akt2 are expressed in our cells (data not shown). None of these possibilities have been assessed yet. In at least one other model system, interference of one signaling pathway by two others helps define cell fate (29). In cultured chicken smooth muscle cells, IGF action maintains differentiation through a pathway dependent on PI3-kinase and Akt (28, 29). PDGF also induces these enzymes but promotes dedifferentiation through activation of two distinct MAP kinases, ERK1 and -2, and p38 (29). Inhibition of these enzymes by pharmacological agents resulted in induction of smooth muscle differentiation by PDGF through the PI3-kinase–Akt pathway (29), thus indicating that the actions of one signal transduction pathway were impeded by the concerted effects of two others. In contrast to results observed in smooth muscle cells, where selective use of inhibitors could redirect cellular fate, in skeletal myoblasts inhibition of Mek1 by UO126 did not uncover a second survival pathway activated by PDGF. We do not know yet if the p38 pathway is regulated by PDGF in our system. Therefore, in our muscle cell model, the mechanisms used by IGF-I or PDGF to promote survival appear to be distinct and not interchangeable.

In summary, we have established that two different growth factors maintain muscle cell viability by specific nonoverlapping mechanisms. IGF-I stimulates PI3-kinase and Akt activity to promote survival, while PDGF-mediated survival requires the Mek-ERK pathway. Although the physiological relevance of IGF-mediated inhibition of cell death in skeletal muscle remains to be established, such regulation may provide a means for fine-tuning muscle mass during embryonic or adult life. Understanding the ways in which different growth factors promote muscle cell viability will provide an opportunity for controlling myogenesis through selective manipulation of specific signal transduction pathways.

ACKNOWLEDGMENTS

We thank the following individuals for gifts of plasmids: Anke Klippel, Chiron Corporation, for constitutively active and inert PI3-kinases; Richard Roth, Stanford University School of Medicine, for inducible Akt; and Philip Stork, Oregon Health Sciences University, for wild-type human Mek1 and constitutively active rabbit Mek1. The antibody to the Myc epitope tag was a gift from William Skach, Oregon Health Sciences University. We appreciate the technical assistance of Barb Rainish.

This study was supported by research grant 5RO1-DK42748 from the National Institutes of Health to P.R.

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