Departments of Veterinary Biomedical Sciences and Physiology, and the Dalton Cardiovascular Institute, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
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To
determine whether changes in glycogen synthase kinase-3 (GSK-3
)
phosphorylation contribute to muscle hypertrophy, we delineated the
effects of GSK-3
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-3
. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., both
inhibit GSK-3
activity) increased the area of
C2C12 myotubes by 80 and 85%, respectively.
The application of IGF-I (250 ng/ml) elevated GSK-3
phosphorylation
and reduced GSK-3
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-3
phosphorylation by 67 and 92%, respectively. IGF-I suppressed
the kinase activity of GSK-3
. 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-3
(cGSK-3
) inhibited the induction by IGF-I of NFAT-inducible reporter
activity. LiCl, which inhibits GSK-3
, removed the block by cGSK-3
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-3
.
skeletal muscle; signaling; glycogen synthase kinase-3; insulin-like growth factor I; nuclear factors of activated T
cells
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INTRODUCTION |
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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-3 (GSK-3
). Akt reduces GSK-3
kinase activity through specific phosphorylation of Ser9 (7).
Decreases in activity (2) and increases in phosphorylation (2; unpublished observations) of GSK-3
occur in overloaded
skeletal muscle in animals. Although the importance of the inhibition
of GSK-3
is not completely understood, it appears that the reduction in GSK-3
kinase activity affects the activity of various
transcription factors and global protein synthesis (11).
Obviously, if GSK-3
can impact both the regulation of transcription
factor activation and protein synthesis, then it is very possible that
GSK-3
could play an important role in skeletal muscle hypertrophy.
Recent data have implicated a role for GSK-3 as a negative regulator
of cardiac muscle hypertrophy (1, 6, 10). For example,
inactivation of GSK-3
(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-3
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-3
significantly contributed to skeletal muscle hypertrophy.
Recent evidence points to an important role of GSK-3 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-3
as a negative regulator of skeletal muscle hypertrophy. We hypothesized
that inhibiting GSK-3
activity through IGF-I exposure would increase the activity of an NFAT-inducible reporter gene.
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METHODS |
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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-3 (Ser9) rabbit polyclonal antibody raised against human
phospho-GSK-3
Ser9 peptide (1:1,000 dilution); GSK-3
mouse monoclonal antibody raised against amino acids 1-160 of rat
GSK-3
(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-3
A9
(cGSK-3
) 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 -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
N
-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-3 immune complex kinase assay.
GSK-3
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-3
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 [
-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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RESULTS |
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Inhibitors of active GSK-3 produce hypertrophy of
C2C12 myotubes.
When LiCl, an inhibitor of GSK-3
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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IGF-I produces a transient phosphorylation of GSK-3 in
C2C12 myotubes.
GSK-3
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-3
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-3
phosphorylation effect is transient.
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IGF-I suppresses GSK-3 activity.
GSK-3
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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PI3'-kinase inhibitors block GSK-3 phosphorylation.
LY-294002 (100 µM), an inhibitor of PI3'-kinase, suppressed
IGF-I-induced GSK-3
phosphorylation by 67% in myotubes (Fig. 3C), and wortmannin (150 µM), a different inhibitor of
PI3'-kinase, inhibited GSK-3
phosphorylation by 92% in the presence
of IGF-I. These results confirm the findings of Rommel et al.
(12).
Constitutively active GSK-3 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-3
(cGSK-3
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-3
rescued the reversal by cGSK-3
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-3
A9 were exposed to medium supplemented
with both IGF-I and LiCl.
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DISCUSSION |
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The purpose of this study was to determine whether GSK-3 plays
a role in skeletal myotube hypertrophy. Of key significance was the
initial finding that noncompetitive inhibition of GSK-3
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-3
inhibition confirms the recent results of Rommel et al.
(12), who used a dominant-negative GSK-3
cDNA to
produce profound C2C12 myotube hypertrophy. Our
findings strongly suggest that GSK-3
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-3
in skeletal myotubes.
These skeletal muscle findings are similar to a recent finding for a
critical role for GSK-3 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-3
(GSK-3
A9 mutant) on protein synthesis and sarcomere
organization. Also, in cardiac tissue, expression of cGSK-3
A9
attenuated the myocardial hypertrophy induced by the expression of a
constitutively active calcineurin in transgenic mice (1).
Therefore, GSK-3
may have a role in hypertrophy of both
cardiac and skeletal muscle.
An alternative approach to inhibit GSK-3 activity is to apply IGF-I
to muscle cells (12). IGF-I signals the phosphorylation of
Akt, which in turn inactivates GSK-3
by phosphorylation of Ser9 (10). IGF-I has been shown to produce
myotube hypertrophy and increase GSK-3
phosphorylation
(12). In other cell types the phosphorylation of GSK-3
by IGF-I decreases GSK-3
activity (7, 11). We therefore
pursued an experiment to test whether the IGF-I-induced reduction of
GSK-3
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-3
A9. Unexpectedly though,
LiCl, a noncompetitive inhibitor of GSK-3
, 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-3
blocked the stimulation by IGF-I of NFAT-responsive
luciferase activity, addition of LiCl to this mixture to
noncompetitively inhibit GSK-3
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-3
-independent mechanism (Fig.
5).
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GSK-3 has been shown to have multiple cellular targets. For example,
Harwood (7) recently described GSK-3
as a "vital regulatory kinase with a plethora of significant cellular targets, some
of which include cytoskeletal and transcription factor proteins." GSK-3
is also known to effect protein synthesis by altering the phosphorylation status of the
-subunit of the eukaryotic initiation factor eIF-2. More specifically, phosphorylation of Ser540
on eIF-2
by GSK-3
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-3
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-3 phosphorylation in our cells,
further experiments were performed in C2C12
myotubes. Rommel et al. (12) suggested that IGF-I-induced
phosphorylation of GSK-3
occurs due to activation of the
PI3'-kinase/Akt pathway. Here, we confirm and extend their report of
increased GSK-3
phosphorylation by our observation that the kinase
activity of GSK-3
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-3
in skeletal myotubes.
In summary, our results indicate that phosphorylation-mediated
inhibition of GSK-3 via the PI3'-kinase/Akt cascade could play an
important regulatory role in skeletal myotube hypertrophy. Noncompetitive deactivation of GSK-3
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-3
. However,
NFAT reporter gene activity was increased when IGF-I was placed
together with LiCl and a constitutively active GSK-3
. These data
show that IGF-I can produce increases in NFAT-inducible transcriptional
activity independently of GSK-3
. 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-3
. Finally, our data and that of
Rommel et al. (12) clearly indicate that GSK-3
can play
an important regulatory role in modulating skeletal myotube size.
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ACKNOWLEDGEMENTS |
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This research was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-19393.
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FOOTNOTES |
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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.
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