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Akt1 and Akt2 Differently Regulate Muscle Creatine Kinase and Myogenin Gene Transcription in Insulin-Induced Differentiation of C2C12 Myoblasts

Department of Molecular Medicine, Osaka University Graduate School of Medicine (C-4), Osaka 565-0871, Japan; and Human Genetics Program, Fox Chase Cancer Center (J.R.T.), Philadelphia, Pennsylvania 19111

Address all correspondence and requests for reprints to: Satoru Sumitani, M.D., Department of Molecular Medicine, Osaka University Graduate School of Medicine (C-4), 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail: . sumitani{at}imed3.med.osaka-u.ac.jp

	Abstract

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Insulin and IGFs are potent inducers of skeletal muscle differentiation. Although PI3K is known to be involved in skeletal muscle differentiation, its downstream targets in this process are not clearly defined. We investigated the roles of Akt and mammalian target of rapamycin (mTOR) in skeletal muscle differentiation. LY294002, a pharmacological inhibitor of PI3K, and the immunosuppressant rapamycin inhibited insulin-induced differentiation of C2C12 myoblasts. LY294002 and rapamycin suppressed myosin heavy chain expression and myotube formation. Transient reporter assays showed that both inhibitors repress muscle creatine kinase (MCK) and myogenin gene transcription. Heterologous expression of Akt1/PKB potently suppressed MCK gene transcription without affecting myogenin gene transcription, whereas heterologous expression of Akt2 increased myogenin and MCK gene transcription. Finally, overexpression of myogenin rescued the inhibitory effect of rapamycin on MCK gene transcription, whereas it failed to rescue the inhibitory effect of LY294002 and Akt1. These results suggest that insulin regulates myogenic differentiation chiefly at the level of myogenin gene transcription via PI3K and mTOR. PI3K activity, but not mTOR, may regulate transcriptional activity of myogenin. Our data also suggest that Akt1 and Akt2 play distinct roles in myogenic differentiation.

	Introduction

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DIFFERENTIATION OF skeletal muscle cells involves three major steps, namely, withdrawal of myoblasts from the cell cycle, subsequent expression of myotube-specific genes, and formation of multinucleated myotubes (1, 2). Proliferating myoblasts express two muscle regulatory factors (MRFs) belonging to the basic helix-loop-helix family, MyoD and Myf5, before the initiation of myogenic differentiation. Once activated, MyoD and Myf5 induce the withdrawal of myoblasts from the cell cycle and the expression of another myogenic basic helix-loop-helix factor, myogenin. Insulin and IGFs have been implicated in the control of skeletal muscle growth and differentiation in both embryonic development and skeletal muscle regeneration (3, 4). Unlike other growth factors, insulin and IGFs stimulate myoblast proliferation as well as differentiation via the IGF-I receptor (3, 5). Previous studies have indicated that the effects of IGFs on myoblast proliferation and differentiation are temporally separated. In proliferating myoblasts, IGFs increase the expression of factors involved in cell cycle progression. After myoblasts are withdrawn from the cell cycle, IGFs promote skeletal muscle differentiation by inducing the expression or activity of MRFs and myocyte enhancer-binding factor 2 family transcription factors. It was recently suggested that IGFs induce these two functions by activating separate signal transduction pathways (6, 7, 8). Proliferation is mediated by the Raf/MEK/ERK pathway, whereas differentiation is mediated by the PI3K pathway.

Among downstream targets regulated by PI3K, the role of a rapamycin-sensitive pathway in myogenic differentiation has been addressed with conflicting results. It has recently been reported that the inhibition of mammalian target of rapamycin (mTOR) through the immunosuppressant rapamycin blocked myogenic differentiation of rat L6 myoblasts (6) and mouse C2C12 myoblasts (9), whereas another group reported that rapamycin did not inhibit myogenic differentiation of rat, mouse, and human skeletal muscle cells (10). The serine/threonine kinase Akt/PKB has been implicated in many biological processes regulated by PI3K. To date, three isoforms of Akt/PKB, i.e. Akt1/PKB, Akt2/PKBß, and Akt3/PKB, have been identified. The role of Akt/PKB in IGF-induced myogenic differentiation is not clearly known.

In the present study we employed an experimental condition, using mouse C2C12 myoblasts, in which the addition of exogenous insulin was required for promoting myogenic differentiation and addressed the roles of the PI3K pathway in insulin-induced myogenic differentiation. In addition, we focused on whether Akt1/PKB and Akt2/PKBß have distinct roles in skeletal muscle differentiation.

	Materials and Methods

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Materials
Culture media and FBS were obtained from Asahi Technoglasses, Inc. (Tokyo, Japan). BSA and porcine insulin were purchased from Sigma-Aldrich Corp. (Tokyo, Japan). The SuperScript II preamplification system was obtained from Life Technologies, Inc. (Gaithersburg, MD). LY294002 and rapamycin were purchased from BIOMOL Research Laboratories, Inc. (Plymouth, PA). Monoclonal antibody against hemagglutinin (HA) epitope (12CA5) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Monoclonal antibody against actin (clone C4) was obtained from ICN Biomedicals, Inc. (Aurora, OH). The monoclonal antibodies against sarcomeric MHC (clone MF20) and myogenin (clone 5FD) developed by Drs. Fischman and Wright, respectively, were obtained from the Developmental Studies Hybridoma Bank maintained by University of Iowa, Department of Biological Sciences (Iowa City, IA). Horseradish peroxidase-conjugated antimouse IgG, protein G-Sepharose, and ECL reagent were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Crosstide was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Cytomegalovirus-promoter driven eukaryotic expression vector pcDNA3.1 was purchased from Promega Corp. (Madison, WI). Superfect reagent and Plasmid Maxi kit were obtained from QIAGEN (Hilden, Germany). Other reagents were of the highest analytical grade.

Plasmid constructions
Murine Akt1 and Akt2 was cloned from cDNA of mouse 3T3-L1 adipocytes. First strand cDNA was generated with the Superscript II preamplification system according to the manufacturer’s instructions. The HA epitope-tagged construct of Akt1 was prepared by amplifying the murine Akt1 cDNA (GenBank accession no. X65087) in-frame with the HA epitope (YPYDVPDYA) instead of the first methionine with the following primers: sense, 5'-ACC ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT AAC GAC GTA GCC ATT GTG AA (sequences coding for HA epitope are underlined); and antisense, 5'-TCT AGA TCA GGC TGT GCC ACT GGC TG. HA epitope-tagged murine Akt2 (GenBank accession no. U22445) was amplified using the following primers: sense, 5'-ACC ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT AAT GAG GTA TCT GTC ATC; and antisense, 5'- CTC TCG GAT GCT GGC. Myristoylated/palmitylated (m/p)-HA-Akt1, which contains a 12-amino acid N-terminal sequence encoding myristoylation/palmitylation signals of nonreceptor tyrosine kinase LCK (MGCGCGCSSHPEDD) followed by HA epitope, was constructed with the primer 5'-ACC ATG GGC TGT GGC TGC AGC TCA CAC CCG GAA GAT GAC TAC CCA TAC GAT GTT CCA and the antisense primer 5'-TCT AGA TCA GGC TGT GCC ACT GGC TG with HA-Akt1 as a template. K179M-HA-Akt1 was constructed by substituting K179 (AAG) for M179 (ATG) by an overlapping PCR method using mutagenesis primers 5'-TAT GCC ATG ATG ATC CTC AAG AAG GAG GTC and 5'-CTT GAG GAT CAT CAT GGC ATA GTA GCG GCC with HA-Akt1 as a template. The authenticity of all Akt derivatives was confirmed by automated DNA sequencing, followed by subcloning into pcDNA3.1. pcDNA3-E299K-HA-Akt2 was described previously (11). pcDNA3.1-myogenin was constructed by subcloning the BamHI/HindIII fragment of mouse myogenin (12). pcDNA3.1-p85 was constructed by subcloning the EcoRI fragment containing p85 from pSG5-p85 (13). pCMV-p110* (14) was described previously. The MCK-Luc reporter gene contains sequences of the rabbit MCK gene from positions -650 to +45 relative to the start of transcription fused in front of the luciferase gene (15). The myogenin-Luc reporter gene contains sequences of the mouse myogenin gene from -133 to +3 relative to the start of transcription fused in front of the luciferase gene (16). All plasmids were prepared with the Plasmid Maxi kit.

Cell culture
C2C12 mouse myoblasts (American Type Culture Collection, Manassas, VA) were maintained in growth medium (DMEM supplemented with 10% FBS). To induce differentiation, cells at 60–80% confluence were washed once with serum-free DMEM, and the media were shifted to differentiation medium (DMEM supplemented with 0.1% FBS and 5 µg/ml insulin). The media were changed every 24 h. Stock solutions of inhibitors were prepared in dimethylsulfoxide and diluted into the media with the final concentration of dimethylsulfoxide adjusted to 0.1%. 10T1/2 fibroblasts were cultured in DMEM supplemented with 10% FBS.

Protein extraction
C2C12 cells grown on six-well plates were washed with ice-cold PBS and then scraped into Laemmli’s sample buffer containing proteinase inhibitors (1 µM pepstatin A, 1 µM leupeptin, 3 µg/ml aprotinin, and 200 µM phenylmethylsulfonylfluoride) and phosphatase inhibitors (2 mM sodium fluoride, 2 mM sodium orthovanadate, and 10 nM microcystin-LR). Lysates were boiled for 5 min and passed through 25-gauge syringe five times to shear DNA, then the lysates were cleared by centrifugation at 15,000 rpm for 10 min. Soluble proteins were quantitated using the bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL) with BSA as a standard.

Immunoblotting
Samples were electrophoresed on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Immunoblot analysis was carried out essentially as described previously (17). The primary antibodies were used at a 1:1000 dilution. The antigen-antibody interaction was visualized using ECL reagent and exposed to an x-ray film.

Immunofluorescence
C2C12 cells plated on glass coverslips were fixed with 2% formaldehyde for 15 min at room temperature, followed by incubation with 100 mM glycine/PBS for 15 min to quench excess formaldehyde. Then the cells were permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature. After blocking nonspecific binding sites in 5% goat serum/PBS for 1 h at room temperature, the cells were incubated with MF20 antibody at a 1:100 dilution in buffer A (PBS containing 0.05% Tween 20 and 1% BSA) for 30 min at 37 C. After washing with PBS, the cells were incubated with Cy3-conjugated goat antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:200 dilution in buffer A for 30 min at 37 C. Nuclei were counterstained with 1 µg/ml Hoechst 33342. Subsequently, the cells were extensively washed with buffer A and mounted onto glass slides. The specimens were examined with an Axiovert 135 fluorescent microscope equipped with LSM410 confocal microscopy (Carl Zeiss, Jena, Germany).

Transient transfections and reporter assay
Transient transfections were performed with Superfect reagent according to the manufacturer’s instructions. Briefly, C2C12 cells were plated on 24-well culture dishes at 80,000 cells/well the day before transfection. The cells were transfected with 0.5 µg reporter plasmids and 0.475 µg expression vectors. To normalize transfection efficiency, 0.025 µg control plasmid, which contains the Renilla reniformis luciferase gene under thymidine kinase promoter (pRL-TK-Luc), were cotransfected. After transfection, the cells were cultured in growth media for 24 h, then the media were switched to differentiation media. After 48 h, the cells were lysed, and dual luciferase reporter assay was performed according to the manufacturer’s instructions. Luciferase activity was measured by Lumat LB9501 luminometer (Berthold Detection Systems, Pforzheim, Germany). All experiments were performed in triplicate. Relative luciferase units were defined as the ratio of luciferase activity of reporter plasmids to that of control plasmids. All data for reporter analysis were presented as fold stimulation compared with the relative luciferase units obtained in myoblasts.

In vitro kinase assay of Akt
C2C12 cells plated on six-well plates were washed with ice-cold PBS and extracted with lysis buffer [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 20 mM ß-glycerophosphate, 10 nM microcystin-LR, and proteinase inhibitors] on ice for 15 min at 4 C. Lysates were centrifuged at 12,000 x g for 10 min at 4 C, and the HA-Akt proteins were immunoprecipitated from 400 µg cell-free extracts with the anti-HA epitope 12CA5 monoclonal antibody coupled to protein G-Sepharose. The immune complexes were washed three times with lysis buffer, followed by washing with wash buffer (HEPES-buffered saline solution and 0.1% Triton X-100) and finally with kinase buffer [20 mM MOPS (pH 7.2), 25 mM ß-glycerophosphate, 5 mM EDTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol]. Aliquots of immunoprecipitates were subjected to immunoblot analysis using anti-HA antibody. In vitro kinase assays were performed for 30 min at room temperature in a 60-µl reaction mixture containing 30 µl immunoprecipitates in kinase buffer, 30 µM Crosstide (GRPRTSSFAEG) as substrate, 22.5 mM MgCl₂, 150 µM ATP, 10 µM PKA inhibitor peptide, and 5 µCi [-³²P]ATP. All reactions were stopped by transferring the aliquots onto P81 phosphocellulose paper, followed by washing with 0.75% phosphoric acid five times.

Statistics
Data are expressed as the mean ± SE from multiple experiments. Differences between control values and experimental values were determined by ANOVA or Dunnett’s test as appropriate. Statistical calculations were performed using the StatView version 5.0 package (SAS Institute, Inc., Cary, NC). P < 0.05 was considered to be significant.

	Results

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Insulin induces MHC expression and MCK gene transcription via the PI3K pathway
To address the roles of the PI3K pathway in insulin-induced myogenic differentiation, we examined the effects of pharmacological inhibitors of distinct signaling pathways on myotube formation and MHC expression. Immunofluorescence analysis revealed that LY294002 (10 µM) blocked the formation of multinucleated myotubes stained by MF20 (Fig. 1A). Although some myocytes were MF20 positive, they never formed multinucleated myotubes. Rapamycin (2 ng/ml) inhibited both myotube formation and MHC expression comparable to LY294002. Thus, PI3K and mTOR may be required for insulin to induce myotube formation and MHC expression. Next, we investigated whether PI3K activity was required for insulin to induce MCK gene transcription. Consistent with the results in immunofluorescence analysis, both LY294002 and rapamycin significantly attenuated insulin-induced activation of MCK-Luc promoter activity (Fig. 1B). LY294002 or rapamycin alone did not affect MCK promoter activity in the absence of insulin (data not shown). Furthermore, a combination of LY294002 and rapamycin completely abrogated insulin-induced activation of MCK promoter activation.

View larger version (35K): [in this window] [in a new window]   Figure 1. Insulin induces MHC expression and MCK gene transcription via the PI3K pathway. A, C2C12 myoblasts on glass coverslips were allowed to differentiate in DMEM/0.1% FBS with insulin (5 µg/ml) in the presence or absence (None) of the inhibitors (LY294002, 10 µM; rapamycin, 2 ng/ml). After 48 h, cells were fixed and subjected to immunofluorescence analysis with anti-MHC antibody (A–C). The same fields stained with Hoechst 33342 are also shown to localize the nuclei (D–F). Representative results of three independent experiments are shown. B, C2C12 myoblasts were transiently transfected with MCK-Luc and grown in DMEM/10% FBS for 24 h. Cells were allowed to differentiate in DMEM/0.1% FBS with or without insulin (5 µg/ml) in the presence or absence of the inhibitors (LY294002, 10 µM; rapamycin, 2 ng/ml). After 48 h, cells were lysed and subjected to luciferase assay. Average results from three independent experiments are presented. Error bars represent SEs. *, P < 0.05; **, P < 0.01 (by Dunnett’s test, with insulin without inhibitor as a reference category). C, C2C12 myoblasts were transiently transfected with MCK-Luc and various expression vectors and allowed to differentiate in DMEM/0.1% FBS with insulin (5 µg/ml). The following expression vectors were used: constitutively active form of p110 (p110*) and dominant negative p85 (p85). *, P < 0.05 (by ANOVA, with insulin and mock transfected as a reference category).

Because insulin activates multiple intracellular signaling pathways in addition to the PI3K pathway (19), we questioned whether activation of the PI3K pathway is sufficient to induce MCK gene transcription. Therefore, we examined the effect of a constitutively active form of p110 (p110*) on MCK promoter activity. Cotransfection of p110* without insulin caused an increase in MCK promoter activity comparable to that with insulin (Fig. 1C).

The roles of Akt1 and Akt2 in MCK gene transcription induced by insulin
In subsequent experiments we addressed the role of Akt in insulin-induced MCK gene transcription. We also examined whether Akt1 and Akt2, both of which are expressed in C2C12 myoblasts (20, 21), have different effects on insulin-induced MCK gene transcription. When Akt1 and Akt2 expression vectors were introduced into C2C12 myoblasts, Akt1 and Akt2 proteins were expressed to the similar extent (Fig. 2A). In vitro kinase assay with Crosstide as a substrate revealed that insulin activated both wild-type Akt1 and Akt2 (Fig. 2B) and that LY294002 (10 µM) completely blocked insulin-induced activation of both wild-type Akt1 and Akt2 (data not shown). In addition, insulin activated neither kinase-dead Akt1 (K179M-Akt1) nor Akt2 (E299K-Akt2). The membrane-bound form of Akt1 (m/p-Akt1) was constitutively active.

View larger version (24K): [in this window] [in a new window]   Figure 2. Insulin activates heterologous Akt1 and Akt2 in C2C12 myoblasts. C2C12 cells were transiently transfected with vector alone (mock), wild-type Akt1 (Akt1), kinase-dead Akt1 (K179M), constitutively active Akt1 (m/p), wild-type Akt2 (Akt2), and kinase-dead Akt2 (E299K) and grown for 24 h. After serum starvation for 3 h in DMEM, cells were challenged with insulin, and in vitro kinase assay with Crosstide as a substrate was performed as described in Materials and Methods. A, Aliquots of immunoprecipitates were subjected to immunoblot analysis using anti-HA antibody. HC, Mouse IgG heavy chain. B, Cells were stimulated with () or without () insulin (5 µg/ml for 10 min), and an in vitro kinase assay was carried out. Data are the mean of two independent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 3. The roles of Akt1 and Akt2 in MCK gene transcription. A, C2C12 myoblasts were transiently transfected with MCK-Luc and various expression vectors and allowed to differentiate in DMEM/0.1% FBS with or without insulin (5 µg/ml). After 48 h, cells were lysed and subjected to luciferase assay. Average results from three independent experiments are presented. Error bars represent SEs. #, P < 0.05 (by Dunnett’s test, with mock without insulin as a reference category). **, P < 0.01 (by Dunnett’s test, with mock with insulin as a reference category). B, C2C12 myoblasts were transiently transfected with MCK-Luc and constitutively active Akt1(m/p-Akt1) and allowed to differentiate in DMEM/0.1% FBS with or without rapamycin (2 ng/ml). After 48 h, cells were lysed and subjected to luciferase assay. Average results from three independent experiments are presented. Error bars represent SEs. **, P < 0.01 (by ANOVA).

View larger version (13K): [in this window] [in a new window]   Figure 4. Insulin increases myogenin gene transcription via the PI3K pathway. A, C2C12 myoblasts were transiently transfected with myogenin-Luc and grown in DMEM/10% FBS for 24 h. pRL-TK-Luc was also transfected to normalize transfection efficiency. Cells were allowed to differentiate in DMEM/0.1% FBS with or without insulin (5 µg/ml) in the presence or absence of various inhibitors (LY294002, 10 µM; rapamycin, 2 ng/ml). After 48 h, cells were lysed and subjected to luciferase assay. Average results from three independent experiments are presented. Error bars represent SEs. **, P < 0.01 (by Dunnett’s test, with insulin alone as a reference category). B, C2C12 myoblasts were transiently transfected with myogenin-Luc with various expression vectors and allowed to differentiate in DMEM/0.1% FBS with or without insulin (5 µg/ml). The following expression vectors were used: constitutively active form of p110 (p110*) and dominant negative p85 (p85). After 48 h, cells were lysed and subjected to luciferase assay. Average results from three independent experiments are presented. **, P < 0.01 (by ANOVA, with insulin alone as a reference category).

View larger version (17K): [in this window] [in a new window]   Figure 7. Our proposed model for a role of PI3K in myogenin and MCK gene expression. PI3K increases myogenin gene expression via mTOR and Akt2. Akt1 activated by PI3K has an inhibitory effect on MCK gene expression via modulating the transcriptional activity of myogenin. Bold arrows indicate stimulatory effects. Dashed arrows indicate inhibitory effects.

View larger version (21K): [in this window] [in a new window]   Figure 5. The roles of Akt1 and Akt2 in myogenin gene transcription. C2C12 myoblasts were transiently transfected with myogenin-Luc and various expression vectors and allowed to differentiate in DMEM/0.1% FBS with or without insulin (5 µg/ml). After 48 h, cells were lysed and subjected to luciferase assay. Average results from eight independent experiments are presented. Error bars represent SEs. **, P < 0.01 (by Dunnett’s test, with mock without insulin as a reference category).

View larger version (19K): [in this window] [in a new window]   Figure 6. Overexpression of myogenin rescues the inhibitory effect of rapamycin on insulin-induced MCK gene transcription, but not that of LY294002 and Akt1. A, C2C12 cells were transiently transfected with pcDNA3.1 (mock) or pcDNA3.1-myogenin (myogenin) and grown for 24 h. Cell lysates were prepared and subjected to SDS-PAGE. Immunoblotting was performed with antimyogenin antibody. Equal loading was confirmed by reprobing with antiactin antibody. B, C2C12 myoblasts were transiently transfected with myogenin with or without cotransfection of Akt1. Cells were allowed to differentiate in DMEM/0.1% FBS and insulin (5 µg/ml) in the presence or absence of inhibitors (LY294002, 10 µM; rapamycin, 2 ng/ml). After 48 h, cells were lysed and subjected to luciferase assay. Average results from three independent experiments are presented. Error bars represent SEs. **, P < 0.01 (by Dunnett’s test, with no inhibitor as a reference category).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Myogenic differentiation involves at least three major distinct steps: 1) commitment in which proliferating myoblasts withdraw irreversibly from the cell cycle, 2) induction of muscle-specific genes, and 3) fusion of committed myoblasts into multinucleated myotubes (31). We showed that PI3K activity and mTOR were required for MHC expression, myotube formation, and transcriptional activation of myogenin and MCK genes. Our data are in agreement with the report by Xu et al. (25), who showed that PI3K and p70 S6K are crucial signaling molecules mediating the stimulatory effect of IGFs on myogenin expression. Importantly, we have shown that heterologous expression of myogenin does not rescue the inhibitory effect of LY294002 on MCK gene transcription, but it completely rescues that of rapamycin. These results suggest that PI3K activity, but not mTOR, may regulate the transcriptional activity of myogenin in addition to myogenin gene transcription. To corroborate this notion, Tamir and Bengal (32) demonstrated that PI3K enhances the transcriptional activity of muscle enhancer-binding factor 2 proteins that promote myogenic differentiation in cooperation with MRFs.

The role of Akt/PKB in myogenic differentiation have not been well described. The first clue to a role for Akt/PKB in cell differentiation was provided by the finding that myristoylated, constitutively active Akt1 promoted adipogenic differentiation of 3T3-L1 preadipocytes (22). Later, Jiang et al. (33) reported that constitutively active Akt1 retrovirally transfected into chicken embryo fibroblasts induced myogenic differentiation. Furthermore, they demonstrated that kinase-dead Akt1 attenuated myogenic differentiation, suggesting that activation of Akt1 is necessary and sufficient to induce skeletal muscle differentiation. In contrast, Hajduch et al. (34) reported that neither myristoylated Akt1 nor kinase-dead Akt1 affected myogenic differentiation of stably transfected L6 myoblasts. Neither study, however, addressed the role of Akt2 in myogenic differentiation. Although heterologous expression of Akt1 blocked MCK gene transcription induced by insulin, transfection of constitutively active Akt1 (m/p-Akt1) promoted MCK gene transcription without insulin. How can we reconcile these apparently contradictory results? As myristoylated, constitutively active Akt1 induces the activation of p70 S6K in 3T3-L1 adipocytes (22), and mTOR may be a direct target for Akt1 (35), it is possible that constitutively active Akt1 promotes MCK gene transcription via mTOR activation. In fact, inhibition of mTOR by rapamycin significantly attenuated MCK gene transcription induced by m/p-Akt1. Previous work has shown that Akt1 and Akt2 are transiently associated with the cellular membrane after growth factor stimulation and are then translocated to the nucleus (36). As LCK, whose myristoylation/palmitylation motif was placed in the N terminus of Akt1 in our construct, is tightly associated with the cell membrane (37), m/p-Akt1 may be precluded from translocating to the nucleus and, therefore, be unable to interact with the physiological target of Akt1 in the nucleus. In this regard, we observed that m/p-Akt1 did not repress insulin-induced MCK gene transcription (data not shown). Moreover, we speculate that there may be a subtle difference in substrate specificity between Akt1 and Akt2 toward nuclear proteins, because the in vitro kinase assays using Crosstide as a substrate showed that insulin activated heterologous Akt1 and Akt2, and that kinase-dead Akt1 did not suppress MCK gene transcription.

The exact mechanism by which Akt1 inhibited MCK gene transcription remains unknown. As transfection of Akt1 did not affect insulin-induced myogenin gene transcription, and overexpression of myogenin did not rescue the inhibitory effect of Akt1 on MCK gene transcription, we speculate that Akt1 may suppress the activity of myogenin to induce MCK gene by a posttranscriptional mechanism. In agreement with the posttranscriptional modulation of myogenin, the recent reports suggest that expression of myogenin is not sufficient for a complete myogenic program. Zhang et al. (38) showed that mice lacking both p21CIP1 and p57KIP2 cyclin-dependent kinase (CDK) inhibitors fail to form myotubes regardless of the proper expression of myogenin. As p21CIP1 is a predominant inhibitory subunit of the CDK complex in C2C12 myotubes (38), Akt1 may affect the expression or the activity of p21CIP1 CDK inhibitor. Alternatively, Akt1 may directly influence the function of the retinoblastoma protein, because myoblasts lacking the retinoblastoma protein induce an aberrant skeletal muscle cell differentiation program characterized by normal expression of myogenin and p21CIP1, but attenuated expression of late differentiation markers such as MHC (39).

Another important finding in the present study is that Akt2, but not Akt1, increased MCK gene and myogenin gene transcription, suggesting that Akt2, but not Akt1, promotes myogenic differentiation. In contrast to ubiquitous expression of Akt1, the expression of Akt2 is greater in insulin-responsive tissues, especially in skeletal muscle (20, 40). Furthermore, Akt2, but not Akt1, was up-regulated during myogenic differentiation of C2C12 myoblasts (20), Sol8 myoblasts (41), and 10T1/2-MyoD cells (40). These findings support the idea that Akt2 may play a major role in myogenic differentiation. At present, it is not known how heterologous expression of Akt2 increases myogenin gene transcription. As kinase-dead Akt2 did not induce myogenin promoter activation, serine/threonine kinase activity of Akt2 is essential for inducing myogenin gene transcription. One testable hypothesis may be that transfection of Akt2 induces IGF-II expression, as overexpression of the IGF-II gene enhances myogenic differentiation of C2C12 myoblasts (42). It has been shown that overexpression of human Akt2 can transform NIH-3T3 fibroblasts (43). It is presently unknown why overexpression of Akt2 can be associated with two different biological consequences, namely cell transformation and cell differentiation. It could be due to the presence of an accessory protein in skeletal muscle that modifies the activity of Akt2. In this regard, Mitsuuchi et al. (44) reported an adapter protein, APPL, that appears to interact preferentially with Akt2, and APPL is highly expressed in skeletal muscle.

In conclusion, we demonstrate that insulin regulates myogenic differentiation of C2C12 myoblasts chiefly via the PI3K pathway. Our proposed signaling pathways downstream of PI3K to myogenin and MCK gene expression are depicted in Fig. 7. PI3K activity, but not mTOR, may also regulate the transcriptional activity of myogenin. Among targets of PI3K, we suggest that Akt1 and Akt2 play distinct roles in myogenic differentiation. Akt1 may inhibit the transcriptional activity of myogenin, whereas Akt2 may promote myogenic differentiation via increasing myogenin gene transcription.

	Acknowledgments

 
We thank Drs. Michael D. Waterfield, Anke Klippel, Yo-ichi Nabeshima, Issei Komuro, and Kenneth Walsh for plasmids and reagents. We also thank Dr. Yasushi Fujio for helpful comments.

	Footnotes

 
This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan (to H.K. and S.K.).

Abbreviations: APPL, Adapter protein containing PH domain, PTB domain and leucine zipper motif; CDK, cyclin-dependent kinase; CMV, cytomegalovirus; HA, hemagglutinin; MCK, muscle creatine kinase; MEK, MAPK/ERK kinase; MHC, myosin heavy chain; MOPS, 3-[N-morpholino]propanesulfonic acid; m/p, myristoylated/palmitylated; MRF, muscle regulatory factor; mTOR, mammalian target of rapamycin.

Received July 24, 2001.

Accepted for publication November 6, 2001.

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