GSK-3beta negatively regulates skeletal myotube hypertrophy

Dharmesh R. Vyas, Espen E. Spangenburg, Tsghe W. Abraha, Thomas E. Childs, and Frank W. Booth

Departments of Veterinary Biomedical Sciences and Physiology, and the Dalton Cardiovascular Institute, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether changes in glycogen synthase kinase-3beta (GSK-3beta ) phosphorylation contribute to muscle hypertrophy, we delineated the effects of GSK-3beta activity on C2C12 myotube size. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible gene activity and possible modulation of NFAT activation by GSK-3beta . Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., both inhibit GSK-3beta activity) increased the area of C2C12 myotubes by 80 and 85%, respectively. The application of IGF-I (250 ng/ml) elevated GSK-3beta phosphorylation and reduced GSK-3beta kinase activity by ~800% and ~25%, respectively. LY-294002 (100 µM) and wortmannin (150 µM), specific inhibitors of phosphatidylinositol 3'-kinase, attenuated IGF-I-induced GSK-3beta phosphorylation by 67 and 92%, respectively. IGF-I suppressed the kinase activity of GSK-3beta . IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporter activity. Cotransfection of a constitutively active GSK-3beta (cGSK-3beta ) inhibited the induction by IGF-I of NFAT-inducible reporter activity. LiCl, which inhibits GSK-3beta , removed the block by cGSK-3beta on IGF-I-inducible NFAT-responsive reporter gene activity. These data suggest that the IGF-I-induced increase in skeletal myotube size is signaled, in part, through the inhibition of GSK-3beta .

skeletal muscle; signaling; glycogen synthase kinase-3beta ; insulin-like growth factor I; nuclear factors of activated T cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN RESPONSE TO AN INCREASE in functional demand, striated muscle undergoes a phenotypic adaptation characterized by a compensatory increase in mass with an alteration in contractile and metabolic properties of the cell (3). Studies on the molecular mechanisms underlying growth factor-induced skeletal muscle hypertrophy have elucidated various prohypertrophic signaling molecules. However, little is known regarding potential negative regulators of the adaptational circuitry in response to insulin-like growth factor I (IGF-I)-stimulated muscle hypertrophy.

IGF-I is capable of activating many signal transduction pathways; however, recent data suggest that specific activation by IGF-I of the phosphatidylinositol 3'-kinase (PI3'-kinase) pathway significantly contributes to muscle hypertrophy (2). IGF-I binds to the IGF-I receptor with high affinity, and this binding of IGF-I to its specific receptor ultimately leads to activation of the 85-kDa regulatory subunit of PI3'-kinase. Activation of PI3'-kinase induces the activation of protein kinase B (PKB or Akt) by way of phosphorylation of specific serine/threonine residues. Bodine et al. (2) suggested that activation of Akt is a major contributor of muscle hypertrophy process. Furthermore, a downstream target of Akt is a signaling protein termed glycogen synthase kinase-3beta (GSK-3beta ). Akt reduces GSK-3beta kinase activity through specific phosphorylation of Ser9 (7). Decreases in activity (2) and increases in phosphorylation (2; unpublished observations) of GSK-3beta occur in overloaded skeletal muscle in animals. Although the importance of the inhibition of GSK-3beta is not completely understood, it appears that the reduction in GSK-3beta kinase activity affects the activity of various transcription factors and global protein synthesis (11). Obviously, if GSK-3beta can impact both the regulation of transcription factor activation and protein synthesis, then it is very possible that GSK-3beta could play an important role in skeletal muscle hypertrophy.

Recent data have implicated a role for GSK-3beta as a negative regulator of cardiac muscle hypertrophy (1, 6, 10). For example, inactivation of GSK-3beta (phosphorylation of Ser9) appears to be requisite for transcriptional activation of the atrial natriuretic factor (ANF) gene, an established marker of cardiac hypertrophy (10). Furthermore, the regulation of cardiac hypertrophy-responsive gene expression is mediated via the modulation by GSK-3beta of the NFAT (nuclear factors of activated T cells) and GATA family of transcription factors (6). In addition, Rommel et al. (12) found that inactivation of GSK-3beta significantly contributed to skeletal muscle hypertrophy.

Recent evidence points to an important role of GSK-3beta in the IGF-I-stimulated skeletal muscle hypertrophy through the PI3'-kinase/Akt signaling cascades (12). In the current study, we undertook a more detailed examination of the role of GSK-3beta as a negative regulator of skeletal muscle hypertrophy. We hypothesized that inhibiting GSK-3beta activity through IGF-I exposure would increase the activity of an NFAT-inducible reporter gene.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Recombinant human IGF-I was purchased from Austral Biologicals (San Ramon, CA). The PI3'-kinase inhibitors LY-294002 and wortmannin were from Calbiochem (San Diego, CA) and Sigma (St. Louis, MO), respectively. All antibodies were purchased from Cell Signaling Technology (Beverly, MA) unless otherwise indicated: phospho-GSK-3beta (Ser9) rabbit polyclonal antibody raised against human phospho-GSK-3beta Ser9 peptide (1:1,000 dilution); GSK-3beta mouse monoclonal antibody raised against amino acids 1-160 of rat GSK-3beta (1:2,500 dilution; BD Transduction Laboratories, Lexington, KY); phospho-Akt (Ser473) rabbit polyclonal antibody raised against mouse phospho-Ser473 Akt peptide (1:1,000 dilution); and Akt rabbit polyclonal antibody raised against amino acids 466-479 of mouse Akt (1:300 dilution). pGSK-3beta A9 (cGSK-3beta ) was a gift from James R. Woodgett (University of Toronto, Canada) (15).

Cell culture. All cell culture experiments were performed by using C2C12 skeletal muscle myoblasts (ATCC), maintained at 37°C and 10% CO2, in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Rockville, MD) supplemented with 1% penicillin-streptomycin antibiotic. Myoblasts were maintained at a subconfluent seeding density in growth medium; DMEM was supplemented with 20% fetal bovine serum (FBS). Differentiation into myotubes was induced by transferring myoblasts into differentiation medium (DM) consisting of DMEM with 2% horse serum. IGF-I and/or LiCl was added to the medium at the same time as the medium was changed to DM according to the previously described techniques of Semsarian et al. (13). Medium was changed every 24 h to ensure maintenance of optimal concentrations of IGF-I and LiCl. Cultures were maintained in supplemented or nonsupplemented DM for various indicated time points between 1 min and 5 days.

Myotube staining and area calculation. Myotubes were stained after 5 days in DM by using a modified Wright stain with minor modifications to the manufacturer's recommendations (Sigma). Briefly, after aspiration of the DM, the culture plates were gently washed twice with ice-cold 1× PBS. Next, the cells were fixed in 10% methanol for 15 min and stained with Wright solution for 2 min. The myotubes were then visualized by using a microscope (Olympus BH-2) and a SPOT Insight imaging color camera (Diagnostic Instruments). The image system was calibrated by using a micrometer at each magnification. Myotube area was quantified according to the previously described techniques of Semsarian et al. (13, 14) at ×10 magnification. The area of the myotubes was quantified by using SPOT Imaging software (Diagnostic Instruments). The area of each myotube is expressed in square micrometers. The individual area of 100 total myotubes was quantified from multiple separate microscope fields on three separate culture plates. The myotubes chosen were only those for which the entire outline (i.e., sarcolemma) could be clearly visualized within the microscope field. Smaller myotubes were not ignored if it was possible to visualize the entire myotube.

Transient transfection and reporter gene assays. Transient transfections were performed by using the Lipofectamine Plus reagent (Life Technologies) in accordance with the manufacturer's specifications. Briefly, the transfections were carried out in 24-well tissue culture plates (3 × 104 cells/well) with a total of 0.4 µg of DNA per transfection in serum-free DMEM. All assays were performed by using equimolar ratios of DNA, and the total amount of DNA per transfection was adjusted by using the pUC19 empty vector. Three hours after the transfection, the culture medium was changed to DMEM supplemented with 10% FBS overnight. The myoblasts were then washed twice with sterile PBS and subsequently received 2% horse serum medium supplemented with IGF-I and/or LiCl for an additional 18 h. The percent FBS used in the transfection experiments was intentionally reduced to minimize background NFAT-luciferase (NFAT-Luc) expression vector stimulation by endogenous serum growth factors. In those cultures in which LiCl was in the medium, cells were preconditioned in LiCl (10 mM) for 2 h before the onset of the experiment, which then remained present during the entire experiment. At the end of the incubation period, the cells were lysed with Reporter Lysis Buffer (Promega, Madison, WI), and luciferase activities were measured with the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's recommendations. Each sample was cotransfected with 0.4 µg of pRL-null (Promega) control reporter vector expressing Renilla luciferase, which was found to be optimum (data not shown). The pRL-null Renilla luciferase values obtained ranged from 737 to 1,413 relative light units across all conditions. In addition, cells that were not transfected with any reporter constructs exhibited minimal luciferase values of <100 relative light units. NFAT reporter firefly luciferase values were divided by pRL Renilla luciferase values to normalize for differences in transfection efficiency.

Immunoblotting analysis. For cell culture experiments, myoblasts were grown on 100-mm plates and scraped with the use of 400 µl of lysis buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 10 mM Na4P2O7, 100 mM NaF, 25 mM beta -glycerophosphate, 1 mM Na4VO4, 1 mM dithiothreitol (DTT), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml tosyl-L-phenylalanine chloromethyl ketone, 10 µg/ml Nalpha -p-tosyl-L-lysine chloromethyl ketone, 0.1 µM okadaic acid, 1 mM benzamidine, and 2 mM Pefabloc SC Plus]. The scraped cells were lysed by rotation for 30 min at 4°C. Protein concentrations were determined by standard Bradford assay (Bio-Rad), and equal amounts (25 µg) were fractionated by SDS-PAGE on a 10% gel. The samples were electrophoretically transferred to a nitrocellulose membrane in transfer buffer containing 25 mM Tris · HCl (pH 8.3), 192 mM glycine, and 20% (vol/vol) methanol. Membranes were stained with Ponceau S (Sigma) to verify equal loading between lanes and subsequently blocked in 5% nonfat dry milk/0.05% (vol/vol) Tween 20 for 1 h at room temperature. The filter was then incubated in primary antibody for 12 h at 4°C and washed six times (5 min each) with TBS-T (25 mM Tris-base, pH 8.3, 150 mM NaCl, and 0.05% Tween 20). Next, the filter was incubated (21°C, 1 h) in 1:8,000 dilution of a 1 mg/ml donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Amersham) and washed with TBS-T. Immunocomplexes were treated with the enhanced chemiluminescence reagent (ECL; NEN Life Science Products) and visualized by autoradiography.

GSK-3beta immune complex kinase assay. GSK-3beta activity was measured by using 100 µg of total cellular protein prepared as described in Immunoblotting analysis. The extract was diluted to 400 µl with fresh cell lysis buffer and immunoprecipitated with 1 µg of the monoclonal anti-GSK-3beta antibody by rotation for 2 h at 4°C. The immune complexes were isolated by the addition of 25 µl of a 50% slurry of protein G-Sepharose and incubation for 1.5 h. Immunoprecipitates were washed four times in lysis buffer and twice in kinase reaction buffer (8 mM MOPS, pH 7.4, 0.2 mM EDTA, 10 mM magnesium acetate, 1 mM Na4VO4, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 µM okadaic acid, and 2 mM Pefabloc SC Plus). Kinase assays were performed in 40 µl of total reaction buffer containing 62.5 µM phospho-glycogen synthase peptide-2 (Upstate Biotechnology), 20 mM MgCl2, 125 µM ATP, and 10 µCi [gamma -32P]ATP. The reaction mixture was allowed to proceed for 30 min at 30°C with shaking, and 25 µl of the supernatant were spotted onto Whatmann P81 phosphocellulose paper. The filter squares were washed five times for 5 min each in 0.75% phosphoric acid. Next, the filters were briefly rinsed in acetone, dried at room temperature, and subjected to liquid scintillation counting. A sample including all assay components except the immunoprecipitate was included in each experiment as a background control.

Statistics. All values are means ± SE. All differences between groups were determined by using either two-tailed Student's t-test or one-way ANOVA. If ANOVA revealed significant effects, Tukey's post hoc test was applied. For all statistical tests, a 0.05 level was used as statistical significance.


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INTRODUCTION
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Inhibitors of active GSK-3beta produce hypertrophy of C2C12 myotubes. When LiCl, an inhibitor of GSK-3beta activity, was included in the culture medium, the area of C2C12 myotubes was 85% greater (Fig. 1). Inclusion of IGF-I (250 ng/ml), instead of LiCl, in the incubation medium resulted in a similar increase in myotube area (80%). The IGF-I-induced increase in myotube area confirmed the finding of Rommel et al. (12).


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Fig. 1.   Top: increases in C2C12 myotube area after insulin-like growth factor (IGF-I; 250 ng/ml) or LiCl (10 mM) exposure. *Statistical difference from control myotubes at the P < 0.05 level. Values are means ± SE; n = 3 independent culture dish experiments performed on 3 different days. Bottom: representative examples of myotubes depicted after 5 days of exposure to either IGF-I or LiCl in differentiation medium. Arrows indicate C2C12 myotubes.

IGF-I produces a transient phosphorylation of GSK-3beta in C2C12 myotubes. GSK-3beta phosphorylation was increased 732, 908, 914, and 1,312% after exposure to IGF-I (250 ng/ml) for 15 min, 30 min, 1 h, and 12 h, respectively, in C2C12 myotubes (Fig. 2A). However, after 24 h of IGF-I exposure, GSK-3beta phosphorylation returned to baseline levels. These observations extend the single time point (15 min) reported by Rommel et al. (12) and show that the GSK-3beta phosphorylation effect is transient.


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Fig. 2.   A: changes in glycogen synthase kinase-3beta (GSK-3beta ) phosphorylation in C2C12 myotubes after exposure to IGF-I (250 ng/ml) for various times, indicated in minute (m) or hours (h). Representative examples of GSK-3beta (Ser9 and total) immunoblots are depicted at bottom. B: decreases in GSK-3beta kinase activity after exposure to IGF-I (250 ng/ml) for various times. Control samples were normalized to 100% and the data are expressed as percent decreases in kinase activity after IGF-I exposure. *Statistical difference from control myotubes at the P < 0.05 level. Values are means ± SE; n = 3 independent culture dish experiments performed on 3 different days.

IGF-I suppresses GSK-3beta activity. GSK-3beta kinase activity in C2C12 myotubes was decreased by 12-33% at 1 min, 15 min, 30 min, 1 h, 12 h, and 24 h after IGF-I was added to the incubation medium (Fig. 2B).

IGF-I increases Akt phosphorylation in C2C12 myotubes. Two-, four-, three-, and onefold increases in Akt phosphorylation (Ser473) were detected after C2C12 myotubes were exposed to IGF-I (250 ng/ml) for 15 min, 30 min, 1 h, and 12 h, respectively (Fig. 3A). After 24 h of IGF-I exposure, Akt phosphorylation returned to baseline levels. These observations extend the single time point (15 min) reported by Rommel et al. (12) and show that the Akt phosphorylation effect is transient. In addition, PI3'-kinase inhibitors decreased Akt phosphorylation: LY-294002 (100 µM), an inhibitor of PI3'-kinase, suppressed IGF-I-induced Akt phosphorylation by 54% in myotubes (Fig. 3B), and wortmannin (150 µM), a different inhibitor of PI3'-kinase, inhibited Akt by 56% in the presence of IGF-I.


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Fig. 3.   A: increases in Akt phosphorylation in C2C12 myotubes after exposure to IGF-I (250 ng/ml) for various times. Phosphorylation status of Akt is expressed as the amount of phosphorylated Akt (Ser473) over the total amount of Akt. Representative examples of immunoblots for Akt (Ser473 and total) are depicted at bottom. Absence of the horizontal bar labeled "IGF-I" indicates that no IGF-I is present. *Statistical difference from control at the P < 0.05 level. B: inhibition of Akt phosphorylation with pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3'-kinase) activity in C2C12 myotubes after exposure to IGF-I (250 ng/ml). Akt expression was calculated in the same manner as described in A. Representative Akt (Ser473 and total) immunoblots are depicted at bottom. C, control (myotubes not receiving any inhibitors); LY, myotubes receiving LY-294002 (100 µM); W, myotubes receiving wortmannin (150 µM). *Statistical difference from control at the P < 0.05 level; #statistical difference from IGF-I at the P < 0.05 level. C: inhibition of GSK-3beta phosphorylation with pharmacological inhibitors of PI3'-kinase activity in C2C12 myotubes after exposure to IGF-I (250 ng/ml). GSK-3beta expression was calculated in the same manner as described in Fig. 2. Representative examples of GSK-3beta (Ser9 and total) immunoblots are also shown. *Statistical difference from all conditions at the P < 0.05 level; dagger statistical difference from control at the P < 0.05 level; #statistical difference from LY and control at the P < 0.05 level. In A-C, values are means ± SE; n = 3 independent culture dish experiments performed on 3 different days.

PI3'-kinase inhibitors block GSK-3beta phosphorylation. LY-294002 (100 µM), an inhibitor of PI3'-kinase, suppressed IGF-I-induced GSK-3beta phosphorylation by 67% in myotubes (Fig. 3C), and wortmannin (150 µM), a different inhibitor of PI3'-kinase, inhibited GSK-3beta phosphorylation by 92% in the presence of IGF-I. These results confirm the findings of Rommel et al. (12).

Constitutively active GSK-3beta decreases NFAT-inducible reporter gene activity. As a biological probe for the presence of nuclear NFAT, a promoter containing multiple NFAT binding sites driving a firefly luciferase reporter gene, was transfected into myoblasts. To normalize for transfection efficiencies, pRL-null Renilla luciferase was cotransfected (values ranged from 737 to 1,221 relative light units across all conditions). Whereas LiCl had no effect on NFAT-inducible luciferase activity, IGF-I increased luciferase activity by 145% in C2C12 myotubes (Fig. 4). However, the inclusion of a constitutively active GSK-3beta (cGSK-3beta A9; Ser9 mutated to an alanine) with IGF-I reversed the enhancement by IGF-I of NFAT-inducible luciferase activity. Furthermore, the addition of LiCl with IGF-I and cGSK-3beta rescued the reversal by cGSK-3beta of IGF-I's stimulation of NFAT-driven luciferase activity. There was no change in NFAT-driven luciferase activity when the medium was supplemented with LiCl compared with the NFAT-only condition. A 190% increase in luciferase activity occurred when myoblasts transfected with the constitutively active pGSK-3beta A9 were exposed to medium supplemented with both IGF-I and LiCl.


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Fig. 4.   Changes in NFAT (nuclear factors of activated T cells)-driven luciferase activity after exposure to IGF-I or LiCl in differential medium. A: all data are expressed as the ratio of NFAT firefly luciferase values to control plasmid Renilla luciferase values. +IGF-I, myotubes exposed to IGF-I (250 ng/ml); +cGSK, myoblasts transfected with constituently active mutant pGSK-3beta A9; +LiCl; myoblasts exposed to LiCl (100 µM). *Statistical difference from all groups except IGF-I + cGSK + LiCl at the P < 0.05 level. #Statistical difference from all groups except +IGF-I at the P < 0.05 level. Values are means ± SE; n = 6 independent culture dish experiments performed on 3 different days. B: schematic of results shown in A. During the experiments depicted in A, all conditions contained NFAT promoter-luciferase reporter construct (NFAT-Luc). Differential media conditions were as follows: control, 2% horse serum (HS) only; IGF-I, 2% HS and 250 ng/ml IGF-I; cGSK, constitutively active GSK-3beta ; LiCl, 10 mM LiCl. left-right-arrow, no change; up-arrow , increase.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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The purpose of this study was to determine whether GSK-3beta plays a role in skeletal myotube hypertrophy. Of key significance was the initial finding that noncompetitive inhibition of GSK-3beta by LiCl was associated with an 85% increase in myotube surface area in culture without an increase in NFAT reporter gene activity. The observation showing an increase in muscle cell size associated with GSK-3beta inhibition confirms the recent results of Rommel et al. (12), who used a dominant-negative GSK-3beta cDNA to produce profound C2C12 myotube hypertrophy. Our findings strongly suggest that GSK-3beta inhibition, or another pathway activated and/or inhibited by Li+, may be involved in increasing myotube size. LiCl has been widely used to assess the functional role of this enzyme in various contexts (16). Although the nonspecific effects of LiCl are limited, recent evidence does suggest that it may act as an inhibitor of casein kinase-2, p38 kinase, and mitogen-activated protein kinase kinase-2 (5). However, the LiCl-associated increase in myotube area and other corroborative reports in the literature (6, 12) favor a prohypertrophic mechanism for Li+ via inhibition of GSK-3beta in skeletal myotubes.

These skeletal muscle findings are similar to a recent finding for a critical role for GSK-3beta as a negative regulator of cardiac hypertrophy in culture and in whole animals (1, 6, 10). For example, Haq et al. (6) observed that exposure of neonatal cardiomyocytes to LiCl reversed the inhibitory effects of cGSK-3beta (GSK-3beta A9 mutant) on protein synthesis and sarcomere organization. Also, in cardiac tissue, expression of cGSK-3beta A9 attenuated the myocardial hypertrophy induced by the expression of a constitutively active calcineurin in transgenic mice (1). Therefore, GSK-3beta may have a role in hypertrophy of both cardiac and skeletal muscle.

An alternative approach to inhibit GSK-3beta activity is to apply IGF-I to muscle cells (12). IGF-I signals the phosphorylation of Akt, which in turn inactivates GSK-3beta by phosphorylation of Ser9 (10). IGF-I has been shown to produce myotube hypertrophy and increase GSK-3beta phosphorylation (12). In other cell types the phosphorylation of GSK-3beta by IGF-I decreases GSK-3beta activity (7, 11). We therefore pursued an experiment to test whether the IGF-I-induced reduction of GSK-3beta kinase activity would be associated with an increase in NFAT-responsive gene activity. The NFAT-luciferase reporter plasmid selected for these experiments contained four direct repeats of the NFAT binding sequence (-286 to -257) from the human interleukin-2 (IL-2) gene. As expected, IGF-I increased the NFAT-responsive reporter gene activity by 145% in C2C12 myotubes (Fig. 4). Moreover, the increase in NFAT-driven reporter gene was blocked by the simultaneous overexpression of cGSK-3beta A9. Unexpectedly though, LiCl, a noncompetitive inhibitor of GSK-3beta , did not enhance NFAT-inducible luciferase activity but did increase C2C12 myotube area. We interpreted the latter result to suggest that LiCl increases myotube area independently of an enhancement of NFAT-inducible transcriptional activity. Whereas cGSK-3beta blocked the stimulation by IGF-I of NFAT-responsive luciferase activity, addition of LiCl to this mixture to noncompetitively inhibit GSK-3beta rescued the stimulation by IGF-I of NFAT-reporter gene activity to a value seen with IGF-I alone. Because LiCl alone did not increase NFAT-responsive reporter gene activity, we infer that IGF-I in the presence of LiCl is able to enhance this NFAT-inducible marker by a GSK-3beta -independent mechanism (Fig. 5).


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Fig. 5.   Suggested mechanism for operation of GSK-3beta in IGF-I-induced skeletal muscle hypertrophy. Data from other reports (1, 6, 10, 12) were used to construct this drawing. Increased concentrations of IGF-I induced elevations in PI3'-kinase activity. A substrate for PI3'-kinase is Akt, which PI3'-kinase phosphorylates on Ser473, inducing an increase in Akt kinase activity. This elevation in Akt kinase activity can then actively phosphorylate GSK-3beta on Ser9, thereby inhibiting GSK-3beta activity. This reduction in GSK-3beta activity allows NFAT to remain in the nucleus for a longer period of time, thereby promoting increased activation of hypertrophic genes. Solid arrows indicate the proposed action of IGF-I signaling proteins, whereas dotted arrows indicate the directional change in response to IGF-I.

GSK-3beta has been shown to have multiple cellular targets. For example, Harwood (7) recently described GSK-3beta as a "vital regulatory kinase with a plethora of significant cellular targets, some of which include cytoskeletal and transcription factor proteins." GSK-3beta is also known to effect protein synthesis by altering the phosphorylation status of the alpha -subunit of the eukaryotic initiation factor eIF-2. More specifically, phosphorylation of Ser540 on eIF-2beta by GSK-3beta results in inhibition of the GDP/GTP exchange activity of eIF-2 and an overall reduction in protein synthesis (11). Therefore, the effects of inhibition of GSK-3beta kinase activity could most likely be multifactorial with regard to skeletal muscle hypertrophy.

Our current results regarding the effect of IGF-I on NFAT transcriptional activity differ from those recently published by Rommel et al. (12). They reported that the addition of 10 ng/ml IGF-I in the culture medium of C2C12 myotubes, 48 h postdifferentiation, resulted in a cytoplasmic localization of inactivated NFATc1. In contrast, we observed an increase in NFAT-induced transcriptional activity following the addition of 250 ng/ml IGF-I to C2C12 myoblasts, concurrent with cell transfer into DM. Our observation thus agrees with the findings of Semsarian et al. (14). While the reasons for this discrepancy between our report and that of Rommel et al. (12) remain unclear, plausible explanations point to the differences in IGF-I concentration and time point of growth factor exposure. Semsarian and colleagues (13, 14) observed C2C12 hypertrophy following the addition of 250 ng/ml IGF-I to the cell medium before or at the time of myoblast differentiation into postmitotic skeletal myotubes. Importantly, myotube hypertrophy was not noted if IGF-I was introduced at a concentration of 25 ng/ml or at additional time points beyond 24 h postdifferentiation (13). Therefore, although our methods mimicked those of Semsarian et al., who found myotube hypertrophy and NFATc1 accumulation in the nucleus at our chosen IGF-I concentration (250 ng/ml), the study of Rommel et al. (12) employed an IGF-I dosage (10 ng/ml) and time point of exposure previously shown by Semsarian et al. (13) to be ineffective in producing myotube hypertrophy. Furthermore, the NFAT-luciferase reporter assay system used in our study allows the direct measure of the transcriptional activity of various activated NFAT isoproteins. Crabtree (4) indicates that, because of the unusual NFAT-DNA binding domain, NFAT exhibits weak DNA binding and requires an unknown nuclear partner for tight association with DNA. Therefore, our utilization of an NFAT-driven reporter construct allows a direct biological measure of nuclear NFAT-inducible activity by IGF-I. In addition, Rommel et al. (12) reported the phosphorylation status and the cytoplasmic or nuclear location of only one isoform, NFATc1. There is recent evidence indicating that NFAT isoforms NFATc2 (8) and NFATc3 (9) play important roles in the control of skeletal muscle size and myogenesis, respectively, which strongly suggests that NFATc1 is not the only member of this family of transcription factors involved in the regulation of myotube hypertrophy. We therefore conclude that IGF-I increases NFAT-inducible activity.

To determine how IGF-I signals GSK-3beta phosphorylation in our cells, further experiments were performed in C2C12 myotubes. Rommel et al. (12) suggested that IGF-I-induced phosphorylation of GSK-3beta occurs due to activation of the PI3'-kinase/Akt pathway. Here, we confirm and extend their report of increased GSK-3beta phosphorylation by our observation that the kinase activity of GSK-3beta is also inhibited by IGF-I (Fig. 3B). Taken together, these observations strongly suggest that PI3'-kinase and Akt are upstream mediators of the IGF-I-mediated phosphorylation of GSK-3beta in skeletal myotubes.

In summary, our results indicate that phosphorylation-mediated inhibition of GSK-3beta via the PI3'-kinase/Akt cascade could play an important regulatory role in skeletal myotube hypertrophy. Noncompetitive deactivation of GSK-3beta by LiCl resulted in a marked increase in skeletal myotube area without activating NFAT-inducible luciferase activity. The IGF-I-increased NFAT-inducible reporter gene activity was prevented by constitutive activation of GSK-3beta . However, NFAT reporter gene activity was increased when IGF-I was placed together with LiCl and a constitutively active GSK-3beta . These data show that IGF-I can produce increases in NFAT-inducible transcriptional activity independently of GSK-3beta . Taken together, these data suggest that a dose of 250 ng/ml IGF-I does, in fact, induce NFAT transcriptional activity and that this increase in transcriptional activity can be attenuated by GSK-3beta . Finally, our data and that of Rommel et al. (12) clearly indicate that GSK-3beta can play an important regulatory role in modulating skeletal myotube size.


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-19393.


    FOOTNOTES

Address for reprint requests and other correspondence: F. W. Booth, Dept. of Veterinary Biomedical Sciences, E102 Vet. Med. Bldg., Univ. of Missouri, 1600 E. Rollins, Columbia, MO 65211 (E-mail: boothf{at}missouri.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 3, 2002;10.1152/ajpcell.00049.2002

Received 30 January 2002; accepted in final form 28 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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