Mammalian Target of Rapamycin (mTOR) Signaling Network in Skeletal Myogenesis*

Mammalian (or mechanistic) target of rapamycin (mTOR) regulates a wide range of cellular and developmental processes by coordinating signaling responses to mitogens, nutrients, and various stresses. Over the last decade, mTOR has emerged as a master regulator of skeletal myogenesis, controlling multiple stages of the myofiber formation process. In this minireview, we present an emerging view of the signaling network underlying mTOR regulation of myogenesis, which contrasts with the well established mechanisms in the regulation of cell and muscle growth. Current questions for future studies are also highlighted.

Among the myriad of cellular and developmental processes governed by mTOR, the development and physiology of skeletal muscle involve extensive regulation by mTOR signaling. The topics of mTOR signaling in insulin sensitivity and glucose metabolism in skeletal muscles and in the regulation of muscle mass have been discussed in excellent recent reviews (10 -12). In this minireview, we focus on the function and regulatory mechanisms of mTOR in skeletal myogenesis, the formation of skeletal muscle. During embryonic development, cells in somites commit to myogenic lineage and differentiate into myoblasts, which then fuse to form multinucleated myofibers (13). This is a highly coordinated process in which various environmental cues and signaling pathways integrate to activate a muscle-specific gene expression program (14,15). Embryonic myogenesis is largely recapitulated during adult skeletal muscle regeneration upon injury, which involves satellite cell (or other types of muscle stem cell) activation, proliferation, and differentiation to form new myofibers or to repair damaged myofibers (16 -18). Over the last decade, mTOR has emerged as a master regulator of skeletal myogenesis, exerting control at multiple levels via distinct signaling mechanisms. Here, we will recount the early observations that established mTOR as a key myogenic regulator, discuss recent progress in the field, and summarize our current understanding of the molecular pathways involving mTOR in myogenesis.

mTOR as a Key Regulator of Skeletal Myogenesis
The function of mTOR signaling in muscle growth and hypertrophy is relatively well understood, and the mechanisms are largely similar to those in cell growth regulation (10,11). However, a role for mTOR in myogenesis (formation of myofibers involving terminal differentiation and fusion) cannot be simply deduced from its requirement for muscle growth and hypertrophy (increase in protein content that may or may not involve fusion).
Pharmacological and Genetic Evidence for Role of mTOR in Myogenesis-Early clues for mTOR involvement in myogenesis came from the effect of rapamycin on myoblast differentiation in culture. Coolican et al. (19) first reported rapamycin inhibition of differentiation of rat L6 myoblasts. Reports from other laboratories followed, demonstrating that rapamycin inhibited the differentiation of mouse C2C12 cells (e.g. Refs. 20 -22). Rapamycin also inhibited skeletal muscle regeneration in rodents (23,24). However, an earlier report showed that rapamycin had no effect on IGF2 (insulin-like growth factor 2)-induced differentiation of L6, Sol8, and human myoblasts (25). The high concentration (300 ng/ml) of exogenous IGF2 used in that study prompted us to speculate that rapamycin might inhibit a myogenic step involving autocrine production of IGF2. This led to the discovery of IGF2 as an important target of mTOR regulation (discussed below) (26). The pharmacological evidence of mTOR involvement in myogenesis was validated by the ability of a rapamycin-resistant (RR) mutant of mTOR to support C2C12 cell differentiation and mouse muscle regeneration in the presence of rapamycin (22,24,27).
Kinase-independent Functions of mTOR in Myogenesis-Unexpectedly, RR-mTOR carrying a kinase-inactive mutation (RR/KI) also rescued differentiation from rapamycin inhibition (22), suggesting that the rapamycin-sensitive myogenic function of mTOR was independent of mTOR kinase activity, contrary to the well characterized mTOR function in cell growth. However, another kinase-inactive mutant of mTOR was reported to be incapable of supporting differentiation (27). The distinct mutations used to inactivate the mTOR kinase in the two studies, D2357E (22) and D2338A (27), are unlikely to explain the inconsistent results because both mutations abolished all measurable mTOR kinase activity and signaling to S6K1 (28). Although the two studies were carried out in the same cell line (C2C12), clonal variation could have led to the discrepancy. Recently, genetic evidence provided strong support for a kinase-independent role of mTOR in myogenesis in vivo: muscle-specific transgenic expression of RR/KI-mTOR, like RR-mTOR, rescued muscle regeneration in mice from rapamycin administration (24).
mTOR Regulation of Two Distinct Stages of Myogenesis-Myoblast differentiation is an ordered multistep process, involving cell cycle withdrawal, myogenic protein expression, and myocyte fusion. The fusion process can be divided into at least two stages that are molecularly separable: initial fusion to form nascent myotubes/myofibers and second-stage fusion to form mature myotubes/myofibers (29). Interestingly, myoblasts relying on KI-mTOR differentiated robustly, but myotubes were arrested at a smaller size (30), suggesting that the kinase activity of mTOR is dispensable for nascent myotube formation but is required for myotube maturation. In vivo studies fully corroborated this observation, as expression of RR/KI-mTOR in mouse muscles rescued new myofiber formation during regeneration, but not myofiber maturation, from rapamycin inhibition, whereas expression of RR-mTOR rescued both (24). Hence, rapamycin-sensitive mTOR signaling governs at least two different stages of myogenesis, nascent myotube/myofiber formation and myotube/myofiber maturation, by distinct mechanisms (Fig. 1).

IGF2 as a Critical Target of mTOR Signaling
A key mediator of kinase-independent myogenic mTOR signaling is IGF2. At the initiation of myoblast differentiation, mTOR controls Igf2 transcription via a muscle-specific enhancer independently of its kinase activity, and IGF2 in turn regulates differentiation through PI3K/Akt (26), an essential pathway for myogenesis ( Fig. 2) (31,32). Consistent with the in vitro findings, mTOR kinase-independent regulation of Igf2 expression is observed during the early phase of muscle regeneration in mice (24). Interestingly, mechanisms underlying the mTOR/IGF2 axis have turned out to be more complicated than previously expected. Our recent findings revealed that mTOR also up-regulates IGF2 production by suppressing a microRNA (miR-125b) that targets the Igf2 3Ј-UTR (33). This function of mTOR is again independent of its kinase activity ( Fig. 2) (33). A recent report identified yet another connection between mTOR and IGF2 in which mTOR directly phosphorylates the Igf2 mRNA-binding protein IMP2 (thus mTOR kinase-dependent), resulting in the activation of IGF2 translation in human rhabdomyosarcoma cells and mouse embryos (34).
It has yet to be tested whether this mechanism also underlies myogenesis. The multilayered control of the mTOR/IGF2 axis attests to the central importance of both proteins in myogenesis.
The mechanism by which mTOR regulates Igf2 transcription remains unknown. Recently, the transcriptional regulator YY1 (Yin Yang 1) was placed downstream of mTOR in skeletal muscle in the regulation of glucose metabolism (35). YY1 suppresses the expression of numerous IGF2/Akt signaling components (including Igf2), and mTOR removes this suppression, possibly through a physical interaction with YY1 (35). This mTOR regulation is apparently a nuclear event, distinct from the canonical mTOR signaling to the protein synthesis machinery in the cytoplasm. mTOR regulation of transcription by all three types of RNA polymerases has been reported (36,37). A nuclear presence of mTOR has long been observed (38). Indeed, mTOR has been found to associate with RNA polymerase I and III-transcribed genes (39,40), and raptor (mTORC1) binds to promoters of mitochondrial oxidative genes (41).
Because YY1 is known to be a negative regulator of skeletal myogenesis (42,43), it is tempting to speculate that YY1 may also be a target of mTOR in myogenesis. Although phosphorylation of YY1 has been shown to be critical for its suppression by FIGURE 1. Rapamycin-sensitive mTOR signaling controls distinct stages of skeletal myogenesis. Formation of nascent myotubes in vitro and myofibers in vivo is regulated by mTOR in a kinase-independent manner, whereas maturation of myotubes/myofibers requires mTOR kinase activity.

FIGURE 2. Two rapamycin-sensitive myogenic mTOR signaling pathways.
A kinase-independent mTOR pathway controls IGF2 expression through transcriptional regulation at a muscle-specific enhancer, as well as through suppression of miR-125b, which targets Igf2. IGF2 regulates myogenesis by activating the IGF1 receptor (IGF-IR) and PI3K/Akt signaling. A kinase-dependent mTOR pathway regulates myocyte fusion and myotube/myofiber maturation by controlling follistatin expression via a MyoD/miR-1/HDAC4 pathway. Amino acid signals are most likely upstream of the IGF2 pathway, but their role in the follistatin pathway is not clear. mTOR (35), there is no published evidence indicating that mTOR is the direct kinase of YY1 or that the kinase activity of mTOR is necessary for this function. Hence, mTOR suppression of YY1 has not yet been ruled out as a possible mechanism mediating mTOR kinase-independent regulation of Igf2 expression.
What may control the activation of mTOR upstream of IGF2? As a nutrient sensor, mTOR may transduce amino acid availability signals to instruct the expression of IGF2. Indeed, the role of amino acids has been demonstrated by their requirement in the activation of the Igf2 muscle-specific enhancer upon myogenic differentiation (26). Although it is not clear whether the same amino acid-sensing pathways upstream of mTOR in cell growth regulation (44,45) are at work in myogenesis, at least one of the components in those pathways, phospholipase D1 (PLD1), has a clear myogenic role. PLD1 is an enzyme that produces the lipid second messenger phosphatidic acid, which is critical for mitogenic activation of mTORC1 (45,46) and which was recently also shown to mediate amino acid signaling (47,48). Mechanical load-induced muscle growth through mTORC1 activation has been shown to involve PLD and phosphatidic acid (49). The requirement of PLD1 for myogenic differentiation has been demonstrated by RNAi experiments in C2C12 (50) and L6 (51) cells. Overexpression of PLD1 ameliorated rapamycin inhibition of L6 cell differentiation (52), consistent with a competition between phosphatidic acid and rapamycin for binding to mTOR (53) and suggesting that PLD1 acts through mTOR. In C2C12 cells, the activity of PLD1 can be stimulated by amino acids (48), and evidence has placed PLD1 upstream of the mTOR/IGF2 pathway (Fig. 2) (50). However, whether other components of the amino acid-sensing mTORC1 pathway, including human VPS34 and Rag (44,45), play any role in myogenesis awaits investigation.

mTOR Regulation of Myocyte Fusion
Earlier observations suggested that a myocyte-secreted factor(s) under the control of mTOR in a kinase-dependent manner is necessary for a second-stage fusion leading to maturation of myotubes (30). Follistatin was recently identified to be one such factor (54). Follistatin is a potent inducer of muscle growth and myogenesis (55,56), acting through antagonizing myostatin, GDF11, and activins in the TGF␤ superfamily (57,58). Elevated follistatin expression is also the underlying mechanism for enhanced muscle fusion elicited by histone deacetylase (HDAC) inhibitors or activation of a nitric oxide/cGMP pathway (59 -61). Rapamycin-sensitive and kinase-active mTOR controls follistatin expression both in culture and in vivo, and recombinant follistatin rescues myotube maturation, as well as regenerating myofiber growth, from rapamycin inhibition (54). Furthermore, mTOR controls the expression of miR-1, which suppresses HDAC4 and subsequently activates follistatin expression (54). Evidence also suggests that mTOR regulates the transcription of pri-miR-1 by stabilizing the master myogenic transcription factor MyoD (54), although the exact mechanism by which mTOR controls MyoD stability remains elusive, and whether this regulation occurs in the nucleus or cytoplasm is not known. Hence, an mTOR 3 MyoD 3 miR-1 3 HDAC4 3 follistatin pathway regulates a late-stage fusion that leads to muscle maturation (Fig. 2).
The regulation of MyoD by mTOR may not be limited to a specific stage or target during myogenesis. It is reasonable to speculate that mTOR might control a subset of (or many) MyoD-regulated myogenic programs. Similarly, the connection between mTOR and microRNAs may also be widespread. Our expression profiling study has revealed a group of microR-NAs differentially expressed during myoblast differentiation in a rapamycin-sensitive fashion (54). Given the central roles of both mTOR and microRNAs in myogenesis (62,63), it would not be surprising if there turned out to be a network of mTOR/ microRNA connections, through MyoD or otherwise, to control or fine-tune various steps of myogenesis.

Regulation by mTORC1
Examining the roles of canonical mTORC1 signaling components in myogenesis has led to some unexpected findings. Knockdown of raptor, the defining component of mTORC1, was found to enhance, rather than inhibit, myoblast differentiation (52,64), and overexpression of raptor suppressed myogenic differentiation (64). Evidence suggests that the negative function of raptor in differentiation of C2C12 cells is through serine phosphorylation and destabilization of IRS1 by mTORC1 and subsequent inhibition of PI3K/Akt signaling (Fig. 3) (64). This is analogous to the well established negative feedback loop between mTORC1 and IRS/PI3K/ Akt in insulin signaling (65-67). Jaafar et al. (52) have proposed an alternative mechanism in which S6K1 phosphorylation and suppression of rictor (mTORC2) (68, 69) mediate the negative effect of raptor on L6 cell differentiation (Fig. 3). In any event, it is clear that mTOR has a role in maintaining homeostasis of myogenesis. mTOR ablation in mouse muscles leads to severe myopathy, impaired oxidative capacity, increased glycogen stores, and decreased mitochondrial biogenesis, resulting in premature death (70). Interestingly, mice with muscle-specific deletion of raptor display dystrophy and metabolic changes similar to those observed in mTOR-deficient muscles (71), suggesting that mTORC1 is responsible for maintaining those functions. Although these observations may seem at odds with the in vitro results of raptor knockdown (64), hyperactive Akt was also found in the raptor knock-out mice as in the raptor knockdown cells and is believed to contribute to an increase in slow myofibers (71). It is also important to note that the muscle-specific ablation of raptor relies on a Cre recombinase driven by the human skeletal actin promoter (71), which is active only in differentiated myocytes (72). Revealing a role of raptor in early muscle development or muscle regeneration would require deletion of the gene prior to myogenic differentiation (e.g. using Pax7 or MyoD promoter-driven or -inducible Cre or in vivo delivery of RNAi prior to muscle degeneration).
The activator of mTORC1, Rheb, is also an inhibitor of myogenesis, as knockdown of Rheb enhances and overexpression of Rheb suppresses differentiation of C2C12 myoblasts (64). Rheb most likely exerts this negative regulation through raptor/ mTOR and suppression of IRS1 (64). This is in contrast to a positive role of Rheb in stimulating muscle hypertrophy (73). Although potential off-target effects of RNAi could complicate the interpretation of knockdown outcomes, consistent phenotypic observations from two independent shRNAs for each gene, corroborated by the effect of overexpression of that gene (64), lend confidence to the conclusions drawn from the RNAi studies. Nevertheless, in light of the discrepancy between in vitro RNAi and in vivo knock-out results, it would be prudent to further validate the RNAi studies by gene rescue and by in vivo gene deletion during early stages of muscle development.
The best characterized mTORC1 substrate, S6K1, does not appear to play a role in muscle differentiation despite its reported function in muscle hypertrophy, growth, and maintenance (74 -76). Although a correlation between S6K1 activity and muscle differentiation has been widely observed (19,22,77,78), S6K1 is dispensable for myoblast differentiation in vitro and formation of regenerating myofibers in vivo (24,26,64,75,76). A role of 4E-BP, another well characterized substrate of mTORC1, has not been examined directly in myogenesis. The lack of a positive contribution of the canonical mTORC1 signaling to myogenic differentiation suggests the existence of novel mTOR complexes and/or mechanisms responsible for rapamycin-sensitive functions of mTOR in myogenesis.

Regulation by mTORC2
Akt is an important regulator of skeletal myogenesis and muscle maintenance (23,32,79). mTORC2, with rictor as its defining component, activates Akt via phosphorylation at Ser-473 in most cellular contexts (80). Several groups employing RNAi reported rictor as being essential for myogenic differentiation (52,81,82). However, the mechanism of rictor action appears to vary depending on the in vitro culture systems. Shu and Houghton (81) have shown that expression of constitutively active Akt rescues C2C12 cell differentiation from rictor knockdown, supporting the idea that Akt mediates the myogenic function of rictor/mTORC2. These authors have also suggested that a relevant target of Akt is ROCK1 (81), the down-regulation of which is necessary for myoblast differentiation (83). In arginine vasopressin-induced L6 cell differentia-tion, however, Akt does not appear to play a major role (52). Instead, Némoz and co-workers (52) propose PKC␣, another known substrate of mTORC2 (84), as the mediator of mTORC2 function in myogenesis. Within this context, PLD1 may act upstream of mTORC2 (52), and this would be consistent with a reported role of PLD/phosphatidic acid in mTORC2 assembly (85). In addition, PLD1 regulation of mTORC2 may be linked to actin cytoskeleton rearrangement (51), as it is well established that mTORC2 regulates the actin cytoskeleton in various cell types (86,87).
Although mTORC1 is the canonical rapamycin-sensitive mTOR complex, prolonged rapamycin treatment disrupts mTORC2 assembly in many types of cells (88). Therefore, the inhibitory effect of rapamycin on myoblast differentiation and muscle regeneration could potentially be attributed to suppression of mTORC2 function. Indeed, Akt phosphorylation in C2C12 cells and PKC␣ phosphorylation in L6 cells have both been shown to be sensitive to rapamycin treatment (52,81). However, definitive evidence for mTORC2 being the relevant target of rapamycin in myogenesis is lacking. Although constitutively active Akt can rescue C2C12 cell differentiation from rapamycin treatment (26,81), this observation alone does not prove a role of mTORC2 because an equally plausible explanation is that rapamycin inhibition of mTOR-dependent IGF2 production can be overcome by constitutive activation of Akt downstream of IGF2 signaling (see Fig. 2

) (26).
Further confounding is the lack of evidence for mTORC2 being an essential kinase for Ser-473 Akt in skeletal muscle. Although knockdown or knock-out of rictor decreases Akt phosphorylation at Ser-473 (71,81,89), depletion of mTOR itself, surprisingly, does not impair this phosphorylation event in either muscle or myoblasts (64,70). In light of the inhibitory effect of rapamycin on phosphorylation of Akt Ser-473 (81), which is best explained by long-term inhibition of mTORC2 (88), one may speculate that mTORC2 is the kinase for Akt under physiological conditions and that, in the absence of mTOR, another kinase takes over. A candidate for this kinase could be integrin-linked kinase, which has been proposed to complex with rictor and phosphorylate Akt at Ser-473 in cancer cells (90).
Skeletal muscle-specific deletion of rictor in mice has revealed no obvious phenotype in muscle development or structure (71,89). Again, the gene deletion in those studies was dependent on the human skeletal actin and muscle creatine kinase promoters, which are active only in differentiated myocytes, precluding assessment of the role of mTORC2 in myogenesis. The strategies outlined above for examining raptor would also be applicable to rictor.
Functional divergence of mTOR and mTORC components is also observed in other physiological contexts in skeletal muscle. One example is the distinct effects of muscle-specific deletion of mTOR, raptor, or rictor on glucose metabolism. mTOR deletion increased basal glucose uptake in the slow-twitch soleus muscle (70), whereas rictor deletion decreased insulin-stimulated (but not basal) glucose uptake in the fast-twitch extensor digitorum longus muscle (89). Nevertheless, whole body glucose tolerance was not affected by mTOR deletion (70) but was impaired by rictor or raptor deletion (71,89). Another example is autophagy. Nutrient deprivation through mTORC1 inhibition is a well established pathway for activation of autophagy (91). However, in skeletal muscle, autophagy is induced by rictor knockdown but is minimally affected by rapamycin or mTOR knockdown; an Akt/FoxO pathway appears to regulate autophagy independently of the canonical nutrient-sensing mTORC1 pathway (92,93).

Other Pathways That Intersect with Myogenic mTOR Signaling
NET39 Interaction with mTOR-NET39 (nuclear envelope transmembrane protein 39) has been reported by Gerace and co-workers (94) to interact with mTOR in myotubes. NET39 knockdown enhanced myoblast differentiation, whereas NET39 overexpression suppressed it, at the same time diminishing mTORC1 activity and IGF2 expression. Furthermore, the positive effect of NET39 knockdown on differentiation is dependent on IGF2 (94). Hence, it appears that NET39 negatively regulates myogenesis, at least partially through inhibiting the mTOR/IGF2 axis (94). The exact mechanism by which NET39 inhibits mTOR is not clear.
FoxO1 as a Negative Regulator of mTOR/IGF2 Signaling-FoxO1 (forkhead box protein O1), a transcription factor directly inhibited by Akt, is a negative regulator of skeletal myocyte differentiation and a key effector of Akt in myogenesis (95,96). Overexpression of FoxO1 induces proteasome-dependent degradation of a subset of mTOR signaling components, including mTOR itself, resulting in suppression of Igf2 transcription (97). Exogenous IGF2 fully rescues myocyte differentiation from FoxO1 inhibition (97), suggesting that the mTOR/ IGF2 pathway is a major mediator of FoxO1 inhibitory function in skeletal myogenesis, thus forming a feedback loop. It should be noted that mTOR signaling is not the sole target of FoxO in myogenesis. FoxO1 also interacts with Notch and activates Notch target genes, leading to suppression of differentiation (96).
Potential Connection between Myostatin and mTOR-Myostatin, a member of the TGF␤ superfamily, is known to inhibit myogenesis through the anaplastic lymphoma kinase receptor and Smad2/3 phosphorylation (98). Glass and colleagues (82) reported that Akt phosphorylation is suppressed by myostatin in a Smad2/3-dependent manner in human skeletal myoblasts and that IGF1 rescues phospho-Akt and myogenic differentiation from myostatin inhibition without affecting Smad2/3. Hence, the effect of myostatin/Smad on Akt appears to be mediated by IGF1 and IGF1 receptor signaling. Interestingly, depletion of either raptor or rictor synergizes with myostatin to inhibit myogenic differentiation (82). Whereas rictor knockdown impairing Akt phosphorylation was expected, the observation that raptor knockdown enhanced Smad2/3 phosphorylation and facilitated suppression of phospho-Akt in the presence of myostatin (82) would require further mechanistic probing.

Conclusion and Perspective
Fifteen years after the initial observation of the rapamycin effect on myoblast differentiation, we now have great appreciation of the role of mTOR as a master regulator of skeletal myogenesis and considerable insights into the regulatory mechanisms. Rapamycin-sensitive mTOR signaling controls distinct stages of myogenic differentiation via both kinaseindependent and kinase-dependent pathways. Interestingly, skeletal muscle is the only system in which kinase-independent function of mTOR has been reported so far. Dissection of the roles of previously characterized mTOR signaling components in other biological contexts has also led to some unexpected revelations, including a negative function of canonical mTORC1 signaling and dispensability of S6K1 in myogenesis. Many other canonical components of mTOR signaling, such as PRAS40, mSin1, Protor, Deptor, mLST8, etc., have not been examined in myogenesis, and it is not possible to predict their functions given what we have learned so far. Novel targets of myogenic mTOR signaling are also expected. For instance, several microRNAs whose levels are modulated during myogenic differentiation in a rapamycin-sensitive manner are intriguing candidates for future investigation. Another unexplored question is whether amino acid signals are involved in other myogenic mTOR functions, including the fusion pathway (Fig. 2), and whether the amino acid-sensing mechanisms identified in cell growth regulation also operate in myogenesis. The current state of our knowledge raises a tantalizing possibility that novel mechanisms of mTOR regulation, such as yet-tobe-identified associating proteins, post-translational modifications, or subcellular localization of mTOR, may exist in myogenesis.
As an allosteric inhibitor of mTOR, rapamycin potentially spares many mTORC2 and mTORC1 substrates. Kinase inhibitors of mTOR (99) can be useful tools to capture additional kinase-dependent mTOR targets and processes in myogenesis. As rapamycin analogs and mTOR kinase inhibitors continue to be explored for their clinical applications in combating various human diseases, including cancer, our mechanistic understanding of mTOR-regulated skeletal myogenesis raises the possibility of potential adverse effects of these drugs on muscle regeneration but may also shed light on new drug targets in treating aging-or disease-related muscular dystrophies.