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Originally published In Press as doi:10.1074/jbc.M005815200 on July 13, 2000

J. Biol. Chem., Vol. 275, Issue 44, 34424-34432, November 3, 2000
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Phosphoinositide 3-Kinase Induces the Transcriptional Activity of MEF2 Proteins during Muscle Differentiation*

Yael Tamir and Eyal BengalDagger

From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, Haifa 31096, Israel

Received for publication, July 3, 2000, and in revised form, July 12, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The activity of phosphoinositide 3-kinase (PI3-K) is essential for the differentiation of skeletal muscle cells by largely unknown mechanisms. Here we show that inhibition of PI3-K activity by the pharmacological agent LY294002 affects early processes of myoblast differentiation including the transcriptional activation of myogenin. Previous studies indicated that transcription of myogenin was dependent on MyoD and MEF2 proteins. We find that expression of a dominant negative form of PI3-K or growth in the presence of LY294002 inhibits cellular activity of MEF2 but not of MyoD. Evidence reveals that whereas MEF2 transcriptional activity is inhibited, its DNA binding activity remains unaffected. Recent studies demonstrated that phosphorylation by p38 mitogen-activated protein kinase (MAPK) induced transcriptional activity of MEF2 proteins. We show that the phosphorylation of MEF2 occurring during muscle differentiation is prevented if the activity of PI3-K is inhibited. However, our results also indicate that p38 MAPK is not affected by PI3-K in muscle cells. Nevertheless, p38 MAPK can substitute for PI3-K in the induction of MEF2 and muscle transcription. Together, these findings indicate that PI3-K affects skeletal muscle differentiation by inducing phosphorylation and transcriptional activity of MEF2 proteins in a parallel but distinct route from p38 MAPK.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The differentiation of skeletal muscle cells involves two major stages: the withdrawal of myoblasts from the cell cycle and subsequent expression of myotube-specific genes. Proliferating myoblasts express two myogenic transcription factors from the basic helix-loop-helix family, MyoD and Myf5, prior to the onset of muscle differentiation (1-3). 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, as well as transcription factors from the MEF2 family. Together, myogenin and MEF2 family members cooperate in the activation of many muscle structural genes (4, 5).

Insulin-like growth factors (IGFs)1 have been implicated in the control of skeletal muscle growth and differentiation both in embryonic development and in muscle regeneration (6-9). Unlike other growth factors, IGF-I stimulates myoblast proliferation as well as differentiation (10-12). Studies have indicated that the effects of IGF-I on proliferation and differentiation are temporally separated. In proliferating myoblasts, IGFs increase the expression of factors involved in cell cycle progression (10, 13). After myoblasts are withdrawn from the cell cycle, IGFs promote muscle differentiation by inducing the expression or activity of myogenic regulatory factors (14). It was recently suggested that IGFs induce these two functions by activating separate signal transduction pathways (15, 16). Proliferation is mediated by the mitogen-activated protein (MAP) kinase pathway, whereas differentiation is mediated by phosphoinositide 3-kinase (PI3-K). Several studies proved the involvement of PI3-K in differentiation by inhibition of its activity. Two approaches were used: treatment of cells with chemical inhibitors like LY294002 and wortmannin or expression of a mutated form of the regulatory subunit of PI3-K, p85, functioning as a dominant negative (15, 17-19). These studies indicated that PI3-K activity was involved in the induction of muscle gene expression.

PI3-K activates downstream molecules like protein kinase B (PKB). The involvement of PKB in muscle differentiation was recently demonstrated. PKB induces muscle-specific transcription (17) and functions as a positive mediator of muscle cell survival (20). No other molecules that function downstream to PI3-K and PKB have so far been identified as being involved in myogenesis.

Some earlier studies suggested that IGF-I induced the expression of myogenin in L6 myoblasts (21). The induction of myogenin expression did not occur immediately following administration of IGF-I, suggesting that the effect was not direct and involved intermediary molecules.

The present study aimed at understanding how PI3-K affects muscle-specific transcription. We suggest that PI3-K affects skeletal muscle differentiation by inducing the transcriptional activity of MEF2 proteins. Our results explain why PI3-K is necessary for the expression of myogenin and of late muscle differentiation genes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

LY294002 and SB203580 were supplied by Calbiochem. PKB (catalog no. 9272) and phosphospecific PKB (Ser473) (catalog no. 9271) antibodies were supplied by New England BioLabs. An antibody to MEF2A and MEF2C proteins (SC-313) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). MyoD polyclonal antibody (SC-760) was from Santa Cruz Biotechnology, and a monoclonal antibody to myosin heavy chain (MF-20) was a gift from Dr. S. Tapscott. p38 MAPK antibody was from Santa Cruz Biotechnology (SC-535). Protein A-Sepharose was supplied by Sigma.

Plasmids

pEMSV-MyoD was described by Tapscott et al. (22). The 4R-tk-CAT reporter gene was described by Weintraub et al. (23). pe+AT-CAT was described by Buskin and Hauschka (24). MEF2-80MCK-CAT reporter was described by Zetser et al. (25). The myogenin 1565-LacZ was described by Cheng et al. (26). The activated allele of MKK6 (MKK6b(E)) was described by Han et al. (27). MEF2C (pCDNAI-MEF2C) and pCDNAI-MEF2(1-117)-VP16 expression vectors were a generous gift of Dr. E. Olson (28). GAL4-MEF2C and GAL4-MEF2A constructs were described by Han et al. (29), and GAL4-MyoD constructs were described by Weintraub et al. (30). Expression vector of p85Delta iSH2-N was described by Rodriguez-Viciana et al. (31).

Cell Culture

L8 cells were a gift of Dr. David Yaffe (32). 10T1/2 cells were obtained from ATCC (Manassas, VA). 10T1/2 cells that expressed the MyoD-estrogen receptor (ER) chimera protein were described by Hollenberg et al. (33). Cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 15% calf serum (Hyclone), penicillin, and streptomycin (growth medium (GM)). To induce differentiation, we used Dulbecco's modified Eagle's medium supplemented with 10 µg/ml insulin and 10 µg/ml transferrin (differentiation medium (DM)). Differentiation of 10T1/2 cells that expressed MyoD-ER protein was induced by the addition of DM that contained 10-7 M estradiol.

Growth of Cells in the Presence of LY294002 or SB203580

LY294002 and SB203580 were dissolved in dimethyl sulfoxide to a concentration of 10 mM that was added directly to the differentiation medium to a final concentration of 10-20 µM, as indicated. Control cells were incubated with the same volumes of dimethyl sulfoxide without the inhibitors. The medium was replaced every 24 h by medium containing fresh inhibitor.

Transfections

Transfections were performed by calcium phosphate precipitation as described (34) or using the TransFast reagent of Promega according to the recommended protocol. Cells in 6-cm TC dishes (Corning) were transfected with a total amount of 10 µg (or 5 µg, using TransFast) of the following plasmid DNA: 1 µg of pCMV-LacZ or pMSV-LacZ, 3 µg of CAT reporter gene, 3 µg of the various expression vectors. "Empty vectors" of pCDNA3 or EMSV containing promoters of cytomegalovirus and murine sarcoma virus long terminal repeat, respectively, were added to equalize the promoter content in each transfection experiment. Following transfection, the medium was replaced with either GM or DM for another 24-48 h. Transfection efficiencies were tested in soluble X-gal assays, and amounts of extracts used for the CAT assays were adjusted accordingly (35).

In Situ beta -Galactosidase Staining of Cells

Transfected cells were washed three times in phosphate-buffered saline and then fixed in 0.5% gluteraldehyde and washed again in phosphate-buffered saline. Cells were then incubated for 3 h at 37 °C in the dark with the following staining solution: 1 mg/ml X-gal, 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2. Stained cells were photographed.

Immunohistochemical Staining

Cells were fixed and immunostained as described (36). The primary antibodies used were polyclonal anti-MyoD (Santa Cruz Biotechnology) and monoclonal anti-MHC (MF-20). The immunochemically stained cells were viewed at × 200 magnification in a fluorescence microscope (Olympus, model BX50) and photographed.

RNA Analysis

RNA was extracted using TRI REAGENTTM (MRC Inc.) and analyzed by Northern blotting as described (36). Blots were hybridized with probes for MEF2A (pCDNA-MEF2A), myogenin (pEMSV-myogenin), myosin light chain 2 (MLC2) (PVZLC2), p21 (pCDNA-Waf1), and glyceraldehyde-3-phosphate dehydrogenase (pMGAP).

Western Analysis

Cells were lysed as described for the kinase assays, and equal amounts of extracted proteins (30-100 µg) were loaded and separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Membrane was incubated in blocking buffer (1× TBS, 0.1% Tween 20, 5% (w/v) nonfat dry milk) and then with the same buffer with the first and secondary antibodies. Between the second and third incubations, membranes were washed three times in 0.1% Tween 20 and 1× TBS. Immunoblotting was conducted with the following antibodies: anti-PKB, anti-phospho-PKB (New England BioLabs), anti-p38 MAPK (Santa Cruz Biotechnology), and anti-phospho-p38 MAPK (Thr180/Tyr182) (New England BioLabs) (1:1000). Proteins were visualized using the enhanced chemiluminescence kit of Pierce.

Metabolic Labeling of Cells and Immunoprecipitation of MEF2 Proteins

Labeling with [35S]Methionine-- Cells were incubated in methionine-free Dulbecco's modified Eagle's medium and dialyzed calf serum for 40 min and then incubated with 100 µCi/ml [35S]methionine for 3 h before proteins were extracted in radioimmune precipitation buffer (50 mM Tris, pH 7.9, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5 mM dithiothreitol, 0.1 mM Na3VO4, 2 µg/ml leupeptin, 20 mM p-nitrophenyl phosphate, and 100 µg/ml phenylmethylsulfonyl fluoride).

Labeling with [32P]Orthophosphate-- L8 cells were incubated in Dulbecco's modified Eagle's medium without sodium phosphate, to which [32P]orthophosphate was added at 0.75 mCi/ml for 3 h before proteins were extracted in radioimmune precipitation buffer.

Immunoprecipitation of MEF2-- Equal amounts of labeled proteins were incubated with antibody to MEF2 for 2 h and an additional 1 h with protein A-Sepharose beads. Beads were washed four times with radioimmune precipitation buffer containing 0.5 M NaCl and once in radioimmune precipitation buffer. Beads were resuspended in SDS sample buffer, and proteins were separated over 10% SDS-polyacrylamide gel electrophoresis.

Gel Shift Analysis of MEF2

The double-stranded DNA probe contained a binding site of MEF2 from the MCK enhancer region. The sequence of the sense strand was 5'-AGCTCGCTCTAAAAATAACCCTGGATCC-3'.

Proteins were extracted from 10T1/2 MyoD-ER cells that were induced to differentiate in DM and estradiol (10-7 M) during 24 h in the absence or presence of 20 µM of LY294002. 20 µg of proteins in binding buffer (10% glycerol, 2 mM spermidine, 0.1 mg/ml bovine serum albumin, 2 mM MgCl2, 0.02% Nonidet P-40, 0.1 mM EDTA, 50 mM KCl, 10 mM HEPES, pH 7.9) were added to 500 ng of poly(dI-dC)-poly(dI-dC) and labeled probe (50,000 cpm, ~1 ng). Proteins and DNA were incubated for 30 min at room temperature. Unlabeled competitor DNA or a specific antibody to MEF2 was incubated with proteins on ice prior to the addition of labeled probe. DNA-protein complexes were separated over 4% native polyacrylamide gel (0.25× TBE).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PI3-K is involved in early stages of myoblast cell differentiation. The involvement of PI3-K in skeletal muscle differentiation was previously demonstrated using different approaches to inhibit this kinase (15, 17-19). In order to study the involvement of PI3-K in the differentiation of L8 myoblasts, we added insulin to these cells at concentrations known to bind the IGF-I receptor (37). To find out whether the PI3-K pathway was activated, we monitored the phosphorylation state of PKB, which is downstream to PI3-K in the pathway. We found that phosphorylation of PKB was induced immediately after the addition of insulin to cells and gradually declined to its basal levels after 24 h (Fig. 1A). Phosphorylation of PKB was prevented in cells treated with LY294002, an inhibitor of PI3-K, suggesting that PKB phosphorylation was dependent on the activity of PI3-K (Fig. 1B, lane 3). Phosphorylation of PKB was only marginally reduced in cells treated with SB203580, an inhibitor of p38 MAPK (Fig. 1B, lane 4). Like insulin, treatment of myoblasts with IGF-1 induced the phosphorylation of PKB (data not shown). The immediate transient phosphorylation of PKB suggests that the pathway may be involved in the differentiation process during this time window. To test this possibility, L8 cells were induced to differentiate for 48 h, during which LY294002 was added for different periods of time. An inhibitor that was added during the first 24 h and then removed prevented differentiation as reflected by the low expression of MLC2 (Fig. 1C, lane 4). An inhibitor added for 24 h after a delay of 24 h did not affect differentiation (Fig. 1C, lane 5). These results suggest that PI3-K is involved during early stages of the differentiation process.


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  		Fig. 1.   PI3-K and PKB are active early in muscle differentiation. A, L8 cells were grown in DM for different periods of time as indicated, and proteins were extracted and separated over SDS-polyacrylamide gel electrophoresis. Western blotting identified the phosphorylated form of PKB (Ser473) and total PKB as described under "Experimental Procedures." P-PKB, phosphorylated PKB. B, L8 cells were grown in DM for 7 h in the absence or presence of 20 µM LY294002 or 20 µM SB203580. Total and phosphorylated PKB were identified by Western blotting. The asterisk represents a nonspecific band. C, L8 cells were grown in DM in the absence or presence of 20 µM LY294002 for different periods of time as indicated. At the indicated time points, total RNA was extracted and analyzed using the Northern technique. +/-, cells were grown for 24 h in DM and in the presence of LY294002, and then for an additional 24 h in DM only. -/+, Cells were grown in DM in the absence of an inhibitor and then for additional 24 h in DM in the presence of an inhibitor. The membrane was hybridized with probes for MLC2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Inhibition of PI3-K Affects the Expression of the Myogenin Gene-- The involvement of PI3-K during the initial 24 h of muscle differentiation prompted us to analyze the expression of "early induced genes" that are turned on during that period of time. We chose to study a 10T1/2 cell line expressing a chimera protein of MyoD and the hormone-binding domain of estrogen receptor (MyoD-ER) (33). The chimera protein resides in an inactive form in the cytoplasm of cells. The addition of estradiol induces its translocation to the nucleus, where it activates the expression of muscle genes. Induction of myogenesis in this cell line follows a very precise timetable, since it depends on the activation of the MyoD-ER protein. Cells were induced to differentiate in the absence or presence of LY294002, and the expression of several genes was analyzed (Fig. 2A). The expression of myogenin and myosin light chain 2 was significantly repressed in cells grown in the presence of the PI3-K inhibitor compared with cells that were induced to differentiate in the absence of the inhibitor (compare lanes 2-4 with lanes 5-7). However, the expression of p21-WAF1 and MEF2A was not affected in cells treated with the inhibitor. It was previously suggested that the expression of these two genes is dependent on the MyoD protein (38, 39). Therefore, the results in Fig. 2A indicate that the activity of the MyoD-ER protein was not affected, while other transcription factor(s) needed for the expression of myogenin and MLC2 genes were inhibited. To test this possibility, cells were induced to differentiate in the presence of the protein synthesis inhibitor cycloheximide (Fig. 2B, lane 4). Under these conditions, genes that are directly induced by MyoD independently of newly synthesized transcriptional mediators can be identified. The expression of p21-WAF1 and MEF2A was not affected, while that of myogenin and MLC2 was totally prevented in the presence of cycloheximide (compare lane 3 with lane 4). These results indicate that the activity of MyoD is not sufficient to induce the transcription of myogenin. The transcription of myogenin is affected by an additional transcription factor(s) that may be inhibited in cells grown in the presence of LY294002. To test whether the inhibitor of PI3-K affected the promoter activity of myogenin, we studied a reporter gene that contained the myogenin promoter driving the expression of the lacZ gene. The promoter was active in 10T1/2 cells expressing the MyoD-ER protein after their treatment with estradiol, as visualized by beta -galactosidase staining (Fig. 3). The expression of this reporter gene was inhibited in cells grown in the presence of LY294002, as observed by the lesser number of beta -galactosidase-stained cells (Fig. 3). The addition of LY294002 to cells transfected with another reporter gene driven by the cytomegalovirus promoter did not affect the activity of this promoter. Hence, LY294002 specifically inhibited the expression of the myogenin promoter, suggesting that PI3-K activity was involved in the transcription of myogenin.


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  		Fig. 2.   Treatment of 10T1/2 MyoD-ER cells with an inhibitor of PI3-K (LY294002) reduces the expression of the myogenin gene. A, 10T1/2 cells expressing a fusion protein of MyoD and the hormone-binding domain of the estrogen receptor were induced to differentiate for the indicated time periods by the addition of DM and estradiol. Some cells were also treated with 20 µM of LY294002 that was added with estradiol and DM. Total RNA was extracted and analyzed by Northern blotting. The membrane was sequentially hybridized with probes of the indicated cDNA molecules. B, 10T1/2 MyoD-ER cells were induced to differentiate in the presence of DM and estradiol for 24 h. Some cells were also treated with cycloheximide (50 µg/ml) or with cycloheximide and LY294002 (20 µM). Total RNA was extracted after 24 h in DM for Northern blotting. The membrane was hybridized with probes of the indicated cDNA molecules. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


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  		Fig. 3.   Treatment of 10T1/2 MyoD-ER cells with LY294002 inhibits the expression of a myogenin promoter-reporter gene. 10T1/2 cells expressing the MyoD-ER protein were transfected with a lacZ reporter gene driven by the promoter of myogenin, or with a lacZ reporter gene driven by the cytomegalovirus (CMV) promoter. The activity of MyoD-ER protein was induced by adding DM and estradiol to cells in the absence or presence of LY294002. The activity of the promoter was analyzed by a beta -galactosidase in situ staining assay (see "Experimental Procedures"). Similar results were obtained in a second independent experiment.

Inhibition of PI3-K Represses the Activity of MEF2 Transcription Factors-- The myogenin promoter contains binding sites for MyoD and MEF2 family members (40). In transgenic mice models, it was shown that both binding sites were necessary and sufficient for muscle-specific transcription of this gene (26). To analyze which of these transcription factors was affected by the PI3-K pathway, the activities of MyoD and MEF2 transcription factors were independently analyzed. 10T1/2 cells were transfected with an expression vector of MyoD and a specific reporter gene containing a minimal promoter and MyoD binding sites (4R-tk-CAT) or an expression vector of MEF2C and a reporter gene containing MEF2 binding sites (MEF2-MCK-CAT). Each transcription factor induced the expression of its respective reporter gene (Fig. 4, lanes 2 and 6). Treatment of the transfected cells with the inhibitor of PI3-K, LY294002, or transfection of a dominant negative form of PI3-K (p85Delta iSH2-N) significantly inhibited the activity of MEF2 (lanes 7 and 8) while only marginally affecting the activity of MyoD (lanes 3 and 4). These findings indicate that PI3-K is involved in the activity of the MEF2C transcription factor. The inhibition of myogenin expression by the PI3-K inhibitor observed in Figs. 2 and 3 might be mediated by suppression of MEF2 activity.


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  		Fig. 4.   The transcriptional activity of MEF2C protein is suppressed in cells grown in the presence of PI3-K inhibitors. 10T1/2 fibroblasts were transiently transfected with an expression vector of MyoD and a CAT reporter gene containing MyoD-binding sites upstream to a minimal promoter (4R-tk-CAT) or with an expression vector of MEF2C and a CAT reporter gene containing MEF2 binding sites upstream to a minimal promoter (MEF-MCK-CAT). Some cells were transfected with an expression vector of Delta p85 (p85Delta iSH2-N) as indicated. The Delta p85 plasmid is encoding for a mutant protein of the regulatory subunit of PI3-K functioning as a dominant negative of PI3-K. Cells were grown for 48 h in DM in the absence or presence of LY294002 (20 µM). Protein extracts were used in a CAT assay according to the transfection efficiencies (see "Experimental Procedures"). The maximal CAT activity for each reporter gene was adjusted to 100 units. Values are means from three independent experiments. Error bars represent S.E.

The Transactivation Potential of MEF2 Proteins Is Repressed by the PI3-K Inhibitor-- Classical transcriptional activators contain two separate activities: DNA binding and transactivation. Using a gel shift assay, we examined whether inhibition of PI3-K affected the DNA binding activity of MEF2 in the 10T1/2 cells expressing the chimera MyoD-ER protein. MEF2 binding activity was significantly induced in extracts of cells treated with estradiol (Fig. 5A, lane 2 versus lane 3), confirming previous results and those shown in Fig. 2 according to which MEF2 expression was induced during muscle differentiation (38, 41). Treatment of the cells with LY294002 during the period at which they were induced to differentiate did not affect the DNA binding activity of MEF2 proteins (Fig. 5A, lane 4). The binding was specific, as demonstrated by homologous competition and supershift of the complex with a specific MEF2 antibody (Fig. 5A, lanes 5 and 6). Therefore, the DNA binding activity of MEF2 is not affected when PI3-K is inhibited.


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  		Fig. 5.   DNA binding of MEF2 is not affected while its transactivation potential is repressed in cells grown in the presence of LY294002. A, 10T1/2 MyoD-ER cells were induced to differentiate for 24 h in DM with estradiol, in the absence or presence of 20 µM LY294002. Proteins were extracted, and DNA binding of MEF2 was analyzed by gel shift analysis. A DNA fragment containing the MEF2 binding site from the MCK enhancer was used as a probe. A 100-fold excess of unlabeled homologous fragment was added to one reaction, and a specific MEF2 antibody was added to another. B, 10T1/2 cells were transfected with different expression vectors as indicated and with a reporter gene containing the GAL4 binding sites in the promoter (GAL4-CAT). Transfected cells were grown in DM in the absence or in the presence of LY294002 (20 µM). Protein extracts were used in a CAT assay according to the transfection efficiencies (see "Experimental Procedures"). The maximal CAT activity for each expression vector was adjusted to 100 units. Values are means from three independent experiments. Error bars represent S.E.

The transactivation potential of MEF2 was studied in a GAL4 activator/reporter system. The transcriptional activators were chimeric proteins consisting of the transactivation domain (TAD) of MEF2 and the DNA-binding domain of the yeast GAL4 protein. The reporter gene contained GAL4 binding sites in the promoter. Using this system, we analyzed the activity of MEF2 TAD independently of its DNA binding activity. The transcriptional activities of several chimera activators were analyzed, all of which activated the transcription from the GAL4 reporter gene (Fig. 5B). Treatment of the transfected cells with LY294002 inhibited the activity of GAL4-MEF2 proteins (Fig. 5B, lanes 2 and 4) but did not affect the activities of GAL4-MyoD or GAL4-VP16 (lanes 6 and 8). We suggest therefore that the effect of PI3-K inhibitor is specific to the TAD of MEF2 proteins.

Expression of a MEF2C-VP16 Protein Partially Substitutes for the Function of PI3-K in Muscle-specific Transcription-- Given that the TAD of MEF2 protein is a target for PI3-K, we assumed that artificial activation of MEF2 could bypass the requirement for PI3-K activity. We tested a chimera protein containing the MEF2 DNA-binding domain (amino acids 1-117) linked to the VP16 TAD (MEF2C-VP16) in the activation of MEF2-responsive genes (28). Both MEF2C and MEF2C-VP16 activated transcription from a muscle creatine kinase reporter gene (pe+AT-CAT) (Fig. 6A, lanes 2 and 6). Treatment of cells with LY294002 or the expression of a mutated form of the regulatory subunit of PI3-K (p85Delta iSH2-N) significantly inhibited MCK transcription in cells transfected with MEF2C but did not affect transcription in cells transfected with MEF2C-VP16 expression vector (compare lanes 3 and 5 with lanes 7 and 9). Similar results were obtained when we analyzed the transcription of a minimal promoter containing MEF2 binding sites (data not shown).


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  		Fig. 6.   MEF2C-VP16 can substitute for PI3-K activity necessary for the transcription of muscle-specific genes. A, 10T1/2 cells were transiently transfected with expression vectors as indicated and a reporter gene containing the MCK enhancer and promoter (pe+AT-CAT). Cells were grown in DM for 48 h in the absence or in the presence of 20 µM LY294002 or 20 µM SB203580. Protein extracts were used in the CAT assay according to the transfection efficiencies (see "Experimental Procedures"). The maximal CAT activity in lanes 2 and 6 was adjusted to 100 units. Average results from three independent experiments are presented. Error bars represent S.E. B, 10T1/2 cells were transiently transfected as indicated. Cells were grown in DM for 36 h in the absence or in the presence of 20 µM LY294002, after which they were fixed and double-stained for MHC (rhodamine) and MyoD (fluorescein). Representative fields of fluorescently stained cells are shown. Right panels show 4,6-diamidino-2-phenylindole staining of the same fields. C, results were quantified and presented as a histogram. The percentage of differentiation was calculated by dividing the number of double-stained cells (MyoD and MHC) by the total number of MyoD-stained cells (single- and double-stained). Each bar in the histogram represents the results of counting about 200 transfected cells. The results are averages of two separate experiments.

To analyze whether MEF2C-VP16 can rescue the expression of endogenous muscle genes, we transfected expression vectors of MyoD and MEF2C, or alternatively MEF2C-VP16, to 10T1/2 cells that were induced to differentiate in the absence or presence of LY294002. We followed the expression of endogenous myosin heavy chain in the transfected cells by immunostaining with specific antibodies to MyoD and MHC (Fig. 6B). Treatment of cells transfected with expression vectors of MyoD and MEF2C with LY294002 reduced the percentage of MHC-expressing cells (Fig. 6C, lanes 1 and 2). Expression of MHC in cells that were transfected with expression vectors of MyoD and MEF2C-VP16 and treated with LY294002 was also inhibited, but to a lesser extent (Fig. 6C, lanes 3 and 4). Therefore, expression of MEF2C-VP16 could partially substitute for the activity of PI3-K needed for the expression of endogenous MHC.

Activity of PI3-K Affects the Phosphorylation State of MEF2 Proteins in L8 Muscle Cells-- Several kinases phosphorylate MEF2 proteins at their transactivation domain and induce their activity (29, 42-44). Since the results presented here suggest that PI3-K affects the transactivation potential of MEF2, we decided to examine whether this activity was also involved in the phosphorylation of MEF2. L8 cells at different stages of differentiation, grown in the presence or absence of LY294002, were incubated with orthophosphate. MEF2 proteins were immunoprecipitated from extracts of these cells and analyzed (Fig. 7). MEF2 proteins were not phosphorylated in myoblasts (lane 1) but were phosphorylated in cells grown for 24 h in differentiation medium (lane 2) (25). Treatment of cells with PI3-K inhibitor (LY294002) or p38 MAPK inhibitor (SB203580), while grown in differentiation medium, blocked the phosphorylation of MEF2 proteins (lanes 3 and 4). The similar steady state levels of MEF2 proteins that were metabolically labeled with methionine suggest that the differences in phosphate labeling were not due to differences in the amount of the proteins (Fig. 7, lower panel). These findings indicate that phosphorylation of MEF2 during muscle differentiation is affected by PI3-K as well as by p38 MAPK.


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  		Fig. 7.   Phosphorylation of MEF2 proteins in L8 cells is blocked by inhibitors of p38 MAPK (SB203580) and PI3-K (LY294002). L8 dividing myoblasts (0 h in DM) or differentiating cells growing for 24 h in DM in the absence or in the presence of the inhibitors LY294002 (20 µM) or SB203580 (20 µM) were incubated with [32P]orthophosphate (upper panel) or with [35S]methionine (lower panel). Cells were lysed, and MEF2A and MEF2C proteins were immunoprecipitated with MEF2-specific antibody and separated over SDS-polyacrylamide gel electrophoresis.

p38 MAPK Is Not Affected by PI3-K, but It Can Replace PI3-K Activity in Muscle-specific Transcription-- Previous studies indicated that p38 MAPK activity was induced in differentiating L8 cells and activated MEF2 proteins (25). The similarities in the effects of p38 MAPK and PI3-K on MEF2 activity suggest that these kinases share the same pathway. To test this possibility, L8 cells were grown in the presence of PI3-K inhibitor, LY294002, and the phosphorylation state of p38 MAPK was analyzed using an antibody recognizing the dually phosphorylated p38 MAPK. As was demonstrated before (25) phosphorylation of p38 MAPK was induced after 24 h in DM (Fig. 8A, lanes 1 and 2). The amount of phosphorylated p38 MAPK was even higher in cells grown for 24 h in DM and in the presence of PI3-K inhibitor (lane 3). Therefore, p38 MAPK is not a target of PI3-K in muscle cells. Phosphorylation of p38 MAPK was unaffected in cells growing in the presence of its own inhibitor, SB203580, as expected for a compound that inhibits activity but not the activation of p38 MAPK (45). Although these results suggest that PI3-K and p38 MAPK are not functioning in the same pathway, similarities in their activities (see Figs. 6 and 7) prompted us to analyze whether p38 MAPK can substitute for PI3-K in the induction of muscle-specific transcription. For that purpose, expression vectors of MyoD and an activated form of MKK6 were transfected to 10T1/2 cells induced to differentiate in the absence or presence of LY294002 (Fig. 8B). The expression of endogenous myosin heavy chain in the transfected cells was analyzed by immunostaining with specific antibodies to MyoD and MHC. MyoD induced the expression of endogenous MHC in 65% of the transfected cells grown in the absence of LY204992 (lane 1) and in 30% of cells grown in the presence of LY294002 (lane 2). Expression of activated MKK6 with MyoD induced the expression of MHC in 100% of the transfected cells, and the number of MHC-expressing cells was only minimally reduced (to 95%) if cells were grown in the presence of LY294002 (lanes 3 and 4). These data suggest that p38 MAPK activity can replace PI3-K in the induction of endogenous MHC expression.


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  		Fig. 8.   p38 MAPK is not activated by PI3-K, yet an activated form of MKK6 rescues the transcriptional activity of MEF2 when PI3-K activity is blocked. A, proteins were extracted from L8 dividing myoblasts (0 h in DM) or differentiating cells growing for 24 h in DM in the absence or in the presence of the inhibitors LY294002 (20 µM) or SB203580 (20 µM) and analyzed by Western blotting, using an antibody to p38 MAPK. An anti-phospho-p38 MAPK (Thr180/Tyr182) antibody was used and presented in the upper panel. An anti-total p38 MAPK antibody was used and presented in the lower panel. B, 10T1/2 cells were transiently transfected as indicated. Cells were grown in DM for 36 h in the absence or in the presence of 20 µM LY294002, after which they were fixed and immunostained using antibodies to MHC and MyoD. Results were quantified and presented as a histogram. The percentage of differentiation was calculated by dividing the number of double-stained cells (MyoD and MHC) by the total number of MyoD-stained cells (single- and double- stained). Each bar in the histogram represents the results of counting about 200 transfected cells. C, 10T1/2 cells were transiently transfected as indicated. Transfected cells were allowed to grow in DM in the absence or presence of LY294002 (20 µM) or SB203580 (20 µM) for 36 h. MKK6b indicates an expression vector of a constitutively active form of MKK6b. Delta p85 (p85Delta iSH2-N) indicates an expression vector of a mutated form of the p85 regulatory subunit that inhibits PI3-K activity. Protein extracts were used in a CAT assay according to the transfection efficiency measured as described under "Experimental Procedures." CAT activities of the experiments shown in lanes 2 and 6 were adjusted to 100 units, and other activities were calculated accordingly. Values are means ± S.E. from three independent experiments.

Next, we analyzed the transcriptional activity of a MCK reporter gene containing two MEF2 binding sites in the enhancer region (Fig. 8C). The transcription of MCK was induced by the expression of MyoD (Fig. 8B, lane 2). MCK transcription was repressed if cells were also treated with LY294002 or SB203580 or transfected with an expression vector of p85 functioning as a dominant negative of PI3-K (Delta p85) (lanes 3-5). Inhibition of PI3-K in transfected cells did not affect MCK transcription if the activated form of MKK6 was expressed (lanes 7 and 9). However, inhibition of p38 MAPK with SB203580 inhibited MCK transcription (lane 8), probably because this inhibitor blocks the activity of p38 MAPK downstream to the ectopically expressed MKK6 protein. Thus, the results presented in Fig. 8 indicate that although PI3-K does not affect the activity of p38 MAPK, these two kinases function in a similar way in the activation of muscle-specific transcription.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factors have been shown to induce muscle transcription via PI3-K (7), but how PI3-K affects muscle transcription has not been shown. In this study, we suggest that PI3-K affects muscle-specific transcription by regulating the activity of MEF2 proteins.

First, we have shown that the PI3-K pathway is active for 24 h after myoblasts are induced to differentiate. This is demonstrated by the immediate and transient phosphorylation of PKB, a kinase in the PI3-K pathway (Fig. 1A). Inhibition of PI3-K during the same period is sufficient to block the transcription of such muscle structural genes as MLC2 (Fig. 1C). The transcription of several "early genes" is induced within this time window. Analysis of the expression of three "early induced genes," p21-WAF1, MEF2A, and myogenin, reveals that only myogenin is affected by the activity of PI3-K (Fig. 2A). Inhibition of PI3-K activity does not affect the expression of p21-WAF1 and MEF2A. p21-WAF1 is involved in the withdrawal of myoblasts from the cell cycle. This raises the possibility that, although functioning during an early stage, PI3-K may not be involved in the exit of myoblasts from the cell cycle. Musaro and Rosenthal elegantly demonstrated that PI3-K affector, IGF-I, expressed postmitotically from the MLC promoter-induced myogenic differentiation (14). However, PI3-K may also control the exit of myoblasts from the cell cycle. Inhibition of PI3-K in chicken myoblasts blocked the expression of MyoD involved in the withdrawal of myoblasts from the cell cycle (18). In this work, the effects of PI3-K on differentiation were studied in a cell line expressing the MyoD protein under a constitutive promoter (10T1/2 MyoD-ER). Therefore, this cell line enabled us to study effects of PI3-K in stages subsequent to the expression of MyoD. Therefore, PI3-K may affect processes occurring during and after the exit of myoblasts from the cell cycle.

Previous studies indicated that IGFs induce the expression of myogenin (3). Inhibition of myogenin expression by antisense oligonucleotides blocked the myogenic effect of IGF-I in myoblasts (46). Our work indicates a correlation between the expression of myogenin and the activity of PI3-K (Figs. 2 and 3). Hence, it appears that IGFs affect the expression of myogenin via the activation of PI3-K. Members of two families of transcription factors, MyoD and MEF2, regulate transcription of myogenin. We aimed to define which of the transcription factors was affected by PI3-K. Studies with the inducible MyoD-ER cell line suggested that the activity of the MyoD-ER protein was not affected by PI3-K. In these cells, the expression of p21-WAF1 and MEF2A was still induced in the presence of the inhibitor of PI3-K (Fig. 2). MyoD-ER also induced the expression of these genes in the presence of cycloheximide, suggesting that these genes were activated directly by MyoD. The promoter of the waf1 gene contains E boxes necessary for the induction of transcription by the MyoD protein (39). MyoD-ER protein alone was not sufficient to induce the expression of myogenin if protein synthesis or PI3-K activity was inhibited. This raised the possibility that the PI3-K pathway could affect the newly synthesized MEF2 proteins (see Fig. 2). This assumption was substantiated by further studies. In a reporter gene assay, the transcriptional activity of MEF2C protein was significantly inhibited, while that of MyoD was marginally affected when cells were grown in the presence of PI3-K inhibitor or transfected with a dominant negative form of PI3-K (Fig. 4). It is possible that, although MyoD is not the prime target of PI3-K, its activity may have been affected as a result of physical and functional interactions with MEF2 proteins (28).

DNA binding activity was not affected (Fig. 5A), but the transactivation potential of MEF2 proteins was inhibited in cells grown in the presence of LY294002 (Fig. 5B). The effect of PI3-K on the TAD of MEF2 proteins was demonstrated using chimeras of MEF2. A protein containing the TAD of MEF2A or MEF2C and the DNA-binding domain of the yeast GAL4 was affected by PI3-K (Fig. 5B), while a protein containing the TAD of VP16 and the DNA-binding domain of MEF2C was not affected by PI3-K (Fig. 6A). Therefore, it seems likely that the TAD of MEF2 proteins is the main target of PI3-K. Moreover, the fact that MEF2C-VP16 protein can partly restore muscle-specific transcription when PI3-K activity is crippled strengthens the suggestion presented above that MEF2 proteins are involved in the PI3-K pathway.

Residues at the TAD of MEF2 are phosphorylated by p38 MAPK and big MAP kinase 1, also known as ERK5 (29, 43, 47). This phosphorylation leads to the transcriptional activation of the protein. In a previous study, we found that MEF2 proteins were phosphorylated during muscle differentiation and suggested that p38 MAPK was involved in their phosphorylation and subsequent activation (25). In this study, we found that inhibition of PI3-K as well as of p38 MAPK in L8 cells prevented the phosphorylation of MEF2 (Fig. 7). This observation raises the possibility that both p38 MAPK and PI3-K are involved in the phosphorylation of MEF2 proteins. The membrane-localized PI3-K is probably not directly phosphorylating nuclear MEF2 proteins. Therefore, PI3-K may cause MEF2 phosphorylation via another kinase. Results presented in Fig. 8A indicate that p38 MAPK is not the kinase mediating PI3-K activity. Two candidates that may function as mediators of PI3-K in the phosphorylation of MEF2 proteins are PKB and big MAP kinase 1. PKB mediates PI3-K activity in phosphorylating many substrates, while big MAP kinase 1 phosphorylates MEF2 proteins but is not related yet to one of the well established signaling pathways. Although p38 MAPK and PI3-K do not share the same signaling pathway, our data strongly indicate that both regulate the transcriptional activity of MEF2 proteins. This is shown by the ability of activated MKK6 to substitute PI3-K in the induction of MEF2 and muscle transcription (see Fig. 8). Interestingly, we observed that SB203580 was consistently more potent than LY294002 in the inhibition of MEF2 (see Figs. 6A and 8C). This activity of SB203580 may be explained by the recent observation that at micromolar concentrations this inhibitor blocks not only p38 MAPK but also the phosphorylation of PKB by phosphoinositide-dependent protein kinase 1 (48). Therefore, at the concentrations used in this work, SB203580 blocked both pathways affecting MEF2, while LY294002 blocked only one.

Two recent studies indicated that IGF-1 induced hypertrophy of differentiated myotubes through the calcium-activated phosphatase, calcineurin (49, 50). In another work, two independent pathways of calcium signaling, one of which was calcineurin, were shown to activate MEF2 transcription factors (51). These data and our results raise the possibility that IGFs affect MEF2 activity by inducing several signaling pathways.

In addition to their role in muscle differentiation, members of the MyoD family are also involved in controlling cell cycle progression (39, 52). The activity of the pRb protein is also required for the withdrawal of myoblasts from the cell cycle (53-55). Cells that do not express the pRb protein (Rb-/-) cannot undergo late events of muscle differentiation. A recent study by Lassar and colleagues (56) demonstrated that MEF2C was functionally inactive in Rb-/- cells. The DNA binding activity of MEF2 was normal, but its TAD was not functional, and the requirement for pRb was partially bypassed by fusing MEF2C to the VP16 TAD. In their study, the authors showed that MEF2 requires both MyoD and pRb for its full activity. Taken together, the above studies and our results suggest that pRb and PI3-K affect the transactivation potential of MEF2 proteins. We propose a model in which the PI3-K and/or p38 MAPK pathways are activated only in the presence of MyoD and pRb, which induce cell cycle withdrawal. Consequently, the IGF signal is transmitted and induces muscle differentiation only after myoblasts withdraw from the cell cycle. If proven correct, this is the first model that explains why withdrawal of myoblasts from the cell cycle is a prerequisite for activation of the differentiation program.

    ACKNOWLEDGEMENTS

We thank Uri Nudel and David Yaffe for the L8 cells. We thank Jiahuai Han, Eric Olson, Stephen Tapscott, and Julian Downward for the generous gifts of reagents. We thank Bianca-Raikhlin-Eisenkraft and Abbie Rosner for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a grant from the Israel Science Foundation (to E. B.) and by funds from the Rappaport Foundation for Medical Research and the Foundation for the Promotion of Research in the Technion, Israel Institute of Technology.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.

Dagger To whom correspondence should be addressed. Tel.: 972-4-8295-287; Fax: 972-4-8535-773; E-mail: bengal@tx.technion.ac.il.

Published, JBC Papers in Press, July 13, 2000, DOI 10.1074/jbc.M005815200

    ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; MCK, muscle creatine kinase; MAP, mitogen-activated protein; PI3-K, phosphoinositide 3-kinase; PKB, protein kinase B; ER, estrogen receptor; GM, growth medium; DM, differentiation medium; CAT, chloramphenicol acetyltransferase; X-gal, 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside; TAD, transactivation domain.

    REFERENCES
TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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