Positive Feedback Regulation between Akt2 and MyoD during Muscle Differentiation

Akt2 is a member of the Akt/PKB family, which is involved in a variety of cellular events including cell survival, proliferation, and differentiation. During skeletal muscle differentiation, the Akt2 but not Akt1 expression was significantly increased. Microinjection of anti-Akt2 but not anti-Akt1 antibody efficiently abrogated myogenesis, indicating that Akt2 plays a specific role in muscle differentiation. It has been well documented that ectopic expression of MyoD is sufficient to induce muscle differentiation in myoblasts. However, the mechanism of induction of Akt2 during muscle differentiation and the significance of Akt2 protein in MyoD-induced myogenesis are largely unknown. In this study, we provide direct evidence that Akt2 is transcriptionally regulated by MyoD and activates MyoD-myocyte enhancer binding factor-2 (MEF2) transactivation activity. The Akt2 promoter was isolated and found to contain nine putative E-boxes (CANNTG), which are putative MyoD binding sites. Electrophoretic mobility shift analyses revealed that MyoD bound to eight of the sites. The expression of MyoD significantly enhanced Akt2 promoter activity and up-regulated Akt2 mRNA and protein levels. Moreover, Akt2 but not Akt1 was activated during differentiation. The expression of Akt2 activated MyoD-MEF2 transcriptional activity and induced myogenin expression. These data indicate that there is a positive feedback regulation loop between Akt2 and MyoD-MEF2 during muscle differentiation, which is essential for MyoD-induced myogenesis.

Skeletal muscle differentiation requires an ordered multiple step process in which myoblasts irreversibly exit from the cell cycle, elongate, and fuse into multinucleated myotubes. This program is driven by the expression of the MyoD family of transcription factors and the myocyte enhancer binding factor-2 (MEF2) 1 family members (1). The MyoD family (also called myogenic regulatory factors) of basic helix-loop-helix proteins includes MyoD, myogenin, Myf5, and myogenic regulatory factor-4. Forced expression of MyoD transcription factor can inhibit cell cycle progression and induce muscle differentiation. The transcription of many muscle-specific genes is activated by the binding of MyoD factors to a simple consensus sequence of CANNTG termed an E-box, present in regulatory regions of many muscle-specific genes. The MEF2 family of transcription factors includes MEF2A, MEF2B, MEF2C, and MEF2D, which belongs to the MADS (MCM1, agamous, deficiens, serum response factor) box transcription factors. Evidence indicates that the members of MyoD and MEF2 families interact with each other to synergistically induce muscle-restricted target genes (2). One of the targets is the gene encoding myogenin, which is one of the earliest molecular markers for myoblasts committed to differentiation. The up-regulation of myogenin in concomitant with the induction of the cyclin-dependent protein kinase inhibitor p21 Waf/Cip1 indicates that the cells have irreversibly exited from the cell cycle and entered the differentiation program (3).
Unlike most growth factors that stimulate myoblast proliferation and inhibit muscle differentiation, the insulin-like growth factors (IGF-I and IGF-II) are potent stimulators of muscle differentiation through induction of myogenin and MEF2 (4). However, the intracellular myogenic signaling process dependent on IGFs is poorly understood. Studies on signaling through IGF receptors have revealed two main pathways, MAPK and PI3K, by which these signals might be transmitted. Several reports showed that the PI3K inhibitors (LY294002 and wortmannin) and a dominant negative p85␣ (the PI3K regulatory subunit) block IGF-induced myogenesis, whereas the MAPK inhibitor PD098059 enhanced IGF-stimulated muscle differentiation (5)(6)(7). Moreover, recent studies demonstrate that constitutively activated PI3K enhanced the transcriptional activity of both MyoD and MEF2 (6,8). Taken collectively, these studies strongly indicate the essential role of PI3K in myogenesis.
The serine/threonine protein kinase Akt (also named PKB) is a major downstream target of PI3K and has been implicated in muscle differentiation (9). Three different isoforms of Akt have been identified including Akt1/PKB␣, Akt2/PKB␤, and Akt3/ PKB␥, all of which are activated by growth factors in a PI3Kdependent manner. The full activation of the Akt requires phosphorylation at Thr 308 (Akt1), Thr 309 (Akt2), or Thr 305 (Akt3) in the activation loop and Ser 473 (Akt1), Ser 474 (Akt2), or Ser 472 (Akt3) in the C-terminal activation domain (10). The most studied isoform is Akt1, which mediates IGF signaling to regulate cell survival, cell growth, GLUT4 translocation, and muscle differentiation. It has been shown that ectopic expression of constitutively activated Akt1 can promote extensive differentiation in different myoblast cell lines in the absence of IGF-I and can reverse the inhibitory effects of PI3K inhibitors LY294002 and wortmannin on myogenic differentiation (5,9,11). However, several studies including ours show that both the mRNA and protein levels of the endogenous Akt1 were not changed, whereas Akt2 was elevated during muscle differentiation, suggesting that Akt2 but not Akt1 plays a specific role in myogenesis under physiological condition (12)(13)(14). A recent study provides compelling supporting evidence by showing that microinjection of Akt2 antibody inhibited the differentiation of muscle cells, whereas anti-Akt1 antibody did not inhibit cell differentiation (15). However, the mechanism by which Akt2 is involved in myogenesis is currently unknown. In this study, we cloned the Akt2 promoter and demonstrated that MyoD transcriptionally regulates AKT2. During muscle differentiation, elevated Akt2 in turn activated MyoD-MEF2 transactivation activity resulting in myogenin expression.

EXPERIMENTAL PROCEDURES
Cell Culture, Plasmids, and Materials-Human epithelial kidney (HEK)293 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. C2C12 mouse myoblasts were grown in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum (growth medium). To induce differentiation, C2C12 cells were maintained in Dulbecco's modified Eagle's medium containing 10% horse serum (differentiation medium). pCSA-MyoD was kindly provided by Dr. Lassar (Harvard Medical School, Boston, MA). The FLAG-tagged MyoD was constructed by subcloning MyoD into p3X-FLAG-CMV10 (Sigma). MEF2 and myogenin-Luc plasmids are described elsewhere (6). The antibodies to Akt1 and Akt2 were purchased from Upstate Biotechnology, and anti-MyoD, myogenin, actin, and FLAG antibodies were from Santa Cruz Biotechnology.
Transcription Start Site Mapping of Human AKT2 Gene-For the analysis of the Akt2 transcription start site, human OVCAR3 mRNA was reverse-transcribed at 55°C using Superscript reverse transcriptase (Invitrogen) and an Akt2 exon 1-specific reverse complement oligonucleotide 5Ј-TTCTTTGATGACAGACACCTCATT-3Ј. Synthesized cDNAs were amplified by polymerase chain reaction using a series of forward primers specific for the DNA sequences within the 8,500 bp upstream of the translation start site and a reverse primer from the coding region of exon 1 (GenBank TM accession number AF452411), and the products of these reactions were resolved by agarose gel electrophoresis.
Cloning and Analysis of the Human Akt2 Promoter-For the reporter analysis of the Akt2 promoter, DNA fragments containing Akt2 genomic sequences were amplified from a cosmid clone (pWE9 -3), which was obtained by the screening of a human placenta genomic library (Stratagene) with 5Ј sequence of Akt2 cDNA using the polymerase chain reaction and primers derived from human genomic Akt2 (GenBank TM accession number NT011250). Amplified DNA fragments were subcloned into the luciferase reporter vector pGL3 (Promega). The integrity of all constructs was confirmed by DNA sequencing. Luciferase assays were performed using the luciferase assay system (Promega), and activities were normalized to ␤-galactosidase activity.
Northern and Western Blot Analysis-Northern blot analysis of total cellular RNA was performed according to standard procedures. Hybridized 32 P-labeled probes were visualized and quantified using Phosphor-Imager analysis (Molecular Dynamics). Western blot analysis was performed as described previously (12).
Competition and Supershift Controls for Electrophoretic Mobility Shift Analysis-For competition controls, nuclear extracts were incubated with radiolabeled probes in the presence of 100-fold molar excess unlabeled competitor probe prior to PAGE. For supershift assay, 1 l of anti-FLAG antibody was incubated with nuclear extracts for 20 min at room temperature prior to the addition of radiolabeled probe and PAGE.

Akt2 Promoter Contains Nine MyoD Binding Elements-To
analyze the transcriptional regulation of the serine/threonine protein kinase Akt2, we cloned the 5Ј-flanking region of Akt2 gene from a pWE-15 cosmid human placenta genomic library using the 5Ј-non-coding region of Akt2 cDNA as probe. Three overlapping cosmid clones were obtained. Sequence analyses revealed that the Akt2 gene consists of 14 exons. Exon 1 is an untranslated region, and the first intron is Ͼ7.6 kilobases in length. The translation initiation site, ATG, of Akt2 protein resides within the exon 2 (Fig. 1A). The transcription start site, which was determined by 5Ј-RACE PCR, lies 7,829 bp upstream of the translation start site. Transcription element analyses of the 2,000 bases of upstream of the transcription start site of the Akt2 gene, which is considered the putative Akt2 promoter, revealed multiple binding sites for MyoD, Oct1, and p300 and single sites for AP1, C/EBP␤, C/EBP, CREB, and SP1 (Fig. 1B). The transcription factor that has the most binding sites in Akt2 promoter is MyoD (nine putative MyoD binding sites: Ϫ1852/Ϫ1841, Ϫ1741/Ϫ1731, Ϫ1502/Ϫ1493, Ϫ981/ Ϫ972, Ϫ759/Ϫ736, Ϫ422/Ϫ413, Ϫ405/Ϫ396, Ϫ250/Ϫ242, and Ϫ41/Ϫ32). A MyoD binding site is also called an E-box and its consensus sequence is CANNTG (Fig. 1B).
Defining the MyoD Binding Site(s) in the Akt2 Promoter -To determine the MyoD binding elements, we carried out the electrophoretic mobility shift analysis. Nine double-stranded oligonucleotides, each containing an E-box from the Akt2 promoter, were labeled with 32 P and incubated with the nuclear extract from FLAG-tagged MyoD-transfected HEK293 cells. The quality of the nuclear extracts was examined with oligonucleotides derived from an E-box of MEF-2 (data not shown). Mobility shift was observed in Ϫ1861/Ϫ1832, Ϫ1750/Ϫ1722, Ϫ1512/Ϫ1483, Ϫ754/Ϫ726, Ϫ432/Ϫ403, and Ϫ415/Ϫ386, Ϫ260/Ϫ231, and Ϫ51/Ϫ22 fragments (Fig. 1C). The formation of the electrophoretically retarded complexes was inhibited when an excess of unlabeled oligonucleotides (competitor) were introduced (middle lane of each E-box). Moreover, an addition of an anti-FLAG antibody to the reaction mixtures induced the supershift of the protein-DNA complexes appearing in Ϫ1861/ Ϫ1832, Ϫ1750/Ϫ1722, Ϫ1512/Ϫ1483, Ϫ754/Ϫ726, Ϫ432/Ϫ403, Ϫ415/Ϫ386, Ϫ260/Ϫ231, and Ϫ51/Ϫ22 (Fig. 1C). These results indicate that eight of the Akt2 protomer-derived E-box oligonucleotides can specifically bind MyoD.
MyoD Transactivates the Akt2 Promoter-To investigate whether MyoD regulates the transcription of Akt2, a 3.1-kilobase genomic fragment corresponding to the region from bases Ϫ2898 to ϩ220 containing nine putative MyoD binding sites, was subcloned upstream of the luciferase gene in pGL3 basic vector (pGL3-AKT2/3.1). A co-transfection of pGL3-Akt2/3.1 with MyoD into HEK293 cells resulted in a significant increase in reporter activity compared with the control sample co-transfected with the reporter and an empty vector (pcDNA3). Moreover, Akt2 promoter is regulated by MyoD in a dose-dependent manner ( Fig. 2A). A similar level of induction of Akt2 reporter activity was also observed upon the transfection of 10T1/2 cells, which lack the endogenous MyoD (data not shown).
To define the MyoD-responsive regions in this promoter, we constructed a group of deletion reporters containing the Akt2 promoter serially deleted from the 5Ј end of the Ϫ2898 to ϩ220 fragment (Fig. 2B). The deletion of Ϫ2898 to Ϫ1531 significantly increased the MyoD responsiveness by ϳ40%, even though two potential myoD binding sites were eliminated, suggesting the presence of inhibitory elements for MyoD responsiveness within this region. The further deletion of E-box 7 reduced MyoD responsiveness by 15%. The deletion of the region from Ϫ642 to Ϫ86 (pGL3-AKT2-0.3), containing a cluster of three E-boxes, deceased the MyoD responsiveness by ϳ16%. Nevertheless, pGL3-AKT2-0.3, which contains only an E-box, was still induced by MyoD Ͼ4.5-fold (Fig. 2B), suggesting that the E-box 1 could be a major MyoD response site within the promoter.

Akt2 Is Induced by MyoD during the Muscle Differentiation-We next examined whether MyoD induces mRNA of
Akt2. Because 10T1/2 myoblast do not express MyoD and are unable to differentiate to myotubes, we have established a 10T1/2 cell line, which was stably transformed with a MyoD expression vector. These MyoD-transformed cells expressing myocyte-specific markers form multinucleated myotubes when exposed to mitogen-poor differentiation medium (17,18). The levels of Akt2 mRNA were evaluated in parental and MyoDtransfected 10T1/2 cells in both growth medium and differentiation medium. Akt2 mRNA was significantly increased in the 10T1/2-MyoD cells, but this induction did not occur in the parental 10T1/2 fibroblasts when exposed to the differentiation culture medium (Fig. 2C).
We further investigated the status of Akt1 and Akt2 in C2C12 cells, which express endogenous MyoD, during differentiation. Western immunoblot analysis revealed that Akt1 protein is stably expressed at a relative high level prior to and during differentiation (Fig. 2E). However, both mRNA and protein levels of Akt2 were very low in C2C12 myoblasts cultured in high mitogen growth medium but progressively increased following exposure of cultures to differentiation medium (Fig. 2, D and E). Moreover, Akt2 kinase activity was induced after switching the culture to differentiation medium . Total RNAs (20 g) were subjected to Northern blot analysis with [ 32 P]dCTP-labeled Akt2 probe. D and E, Akt2 is elevated at mRNA and protein levels and activated during C2C12 cell differentiation. The C2C12 cells were cultured in differentiation medium (growth medium) for the indicated times. RNA and cell lysates were analyzed by Northern and Western blots with indicated isotope-labeled probes and antibodies, respectively. Phosphorylated Akt2 migrated slower and was abrogated by PP2A phosphatase treatment (E, panel 2).
Akt2 Induces Myogenin and Activates MyoD-dependent Reporter Genes-The induction of mRNA/protein and kinase activity of Akt2 during muscle differentiation suggests that it regulates muscle-specific gene(s) that controls differentiation. In fact, a previous report shows that ectopical expression of Akt1 and Akt2 could induce muscle-specific gene muscle creatine kinase, and Akt2 was more effective than Akt1 (15). However, the mechanism of Akt induction of muscle-specific gene expression has not been well documented. To explore this hypothesis further, we tested the effects of Akt2 on myogenin expression and MyoD transcriptional activity. G133-Luc, which is a 133-bp myogenin proximal promoter containing MyoD and MEF2 binding sites or 4RE-Luc, which is a MyoDdependent reporter gene containing four MyoD binding sites, was co-transfected into C2C12 myoblasts with either wild type Akt2 or constitutively active Akt2. The expression of the wild type Akt2 induced G133-Luc and 4RE-Luc reporter activities at 1.6-and 1.5-folds, respectively, whereas the levels of G133-Luc and 4RE-Luc reporter activities were significantly increased (4.2-and 2.5-fold) in the cells transfected with the constitutively active Akt2 (Fig. 3A). Consistent with the reporter results, myogenin expression was induced by both wild type and constitutively active Akt2 in C2C12 cells after 16 h of exposure to differentiation medium (Fig. 3B). These data suggest that AKT2 can up-regulate the endogenous myogenin expression and promote the MyoD transcriptional activity during muscle differentiation. DISCUSSION Previous studies demonstrate that IGF1-induced muscle differentiation was primarily mediated by the PI3K/Akt pathway (5)(6)(7)(8)(9). Among the three isoforms of Akt family, Akt2 is highly expressed in skeletal muscle (12) and plays a specific role in muscle differentiation as demonstrated by up-regulation of Akt2 but not Akt1 and Akt3 during differentiation and abrogation of myotube formation by anti-Akt2 antibody (15). However, the mechanisms by which Akt2 is up-regulated during the muscle differentiation and stimulates myotube formation are currently unclear. In this report, we provide evidence showing that Akt2 promoter possesses multiple MyoD binding sites, that the expression of Akt2 was induced by MyoD through stimulation of its promoter activity, and that the elevated Akt2 activated MyoD transactivation and induced muscle-specific gene myogenin expression to trigger muscle cell differentiation. Our data indicate a positive feedback regulation loop between Akt2 and MyoD during skeletal muscle differentiation (Fig. 4).
In ectopic expression systems, three isoforms of Akt display very similar functions including muscle differentiation. In fact, previous studies have mostly focused on Akt1 and demonstrated that Akt1 is a critical intermediate in IGF1-induced muscle differentiation hypertrophy and muscle survival (5)(6)(7)(8)(9)19). A previous study shows that the expression of constitutively activated Akt1 induces the transactivation activity of MyoD and MEF-2 (6). However, under physiological conditions, Akt2 seems to play more important roles in myogenesis, because mRNA and protein levels of Akt2 but not Akt1 were up-regulated during muscle differentiation (Fig. 2). In addition, accumulated studies have shown clear differences between these three isoforms in terms of biological function. (a) Akt1 expression is relatively uniform in various normal organs, whereas high levels of Akt2 are detected in skeletal muscle and heart (12,20,21). (b) The inhibition of Akt2 but not Akt1 expression abrogates IGF1-induced muscle differentiation (15). (c) Akt2 and Akt3 but not Akt1 are amplified and/or up-regulated in certain types of human cancer (22). (d) NIH3T3 cells are transformed by wild type Akt2 but not Akt1 and Akt3 (23). (e) Akt2-and Akt1-deficient mice displayed different phenotypes. Akt2 knock-out mice exhibited a typical type 2 diabetic phenotype that cannot be compensated by the presence of Akt1 and Akt3 (24). In contrast, Akt1 Ϫ/Ϫ mice exhibited no diabetic phenotype (25, 26) but showed an impairment in organismal growth, i.e. smaller when compared with wild type littermates. Such relatively subtle phenotype of Akt1 Ϫ/Ϫ mice suggests that Akt2 and Akt3 may substitute to some extent for Akt1 (25). Nevertheless, these data indicate that there are non-redundant functions between three isoforms of Akt in certain tissue and/or cell types. In this study, we cloned Akt2 promoter and identified multiple MyoD binding sites in Akt2 (Fig. 1) but not in Akt1 promoter, 2 further indicating the different transcriptional regulation between Akt1 and Akt2.
Previous studies have shown that MyoD and MEF2 transcription factors interact with each other to synergistically induce muscle-restricted target gene expression resulting in muscle differentiation (2), and that class II histone deacetylase (HDAC) 4 and 5 bind to MEF2 and inhibit MEF2/MyoD transactivation activity and muscle differentiation (27). Calcium/ calmodulin-dependent protein kinase induces muscle differentiation by phosphorylation of HDACs 4 and 5 and shuttling of the phosphorylated HDACs from nuclear MEF2⅐HDAC complex to the cytoplasm (28). It has been also shown that class II HDACs-repressed muscle differentiation can be overcome by treating cells with IGF1, which induces HDACs export from the nucleus (29). However, the mechanism by which IGF1 regulates HDACs 4 and 5 has not been well characterized. In this report, we have shown that the protein level and kinase activity of Akt2 were elevated during muscle differentiation. The up-regulation of Akt2 was because of MyoD induction of Akt2 promoter activity, whereas activated Akt2 might result from autocrine production of IGFs by myoblasts under differentiation medium condition (4). Nevertheless, elevated Akt2 during muscle differentiation could mediate IGFs signals to regulate HDACs 4 and 5 functions, even though a previous study showed that Akt1 did not phosphorylate HDACs 4 and 5 (28). Additional studies are required to define the mechanism of Akt2 activation of MyoD-MEF2 transcriptional activity.