Regulation of adult skeletal muscle growth and regeneration by the IL-6 cytokine family
There are several approaches to studying the role of satellite cells and trophic factors in muscle function. Relatively simpler models such as denervation (atrophy) and overloading (compensatory hypertrophy), where the sciatic nerve is severed or where the soleus and plantaris muscles are forced to compensate for the loss of the surgically isolated gastrocnemius, respectively, are two examples that occur in the absence of inflammation, thereby minimizing the confounding contribution of inflammatory cells. For example, increasing the mechanical load on adult skeletal muscle by overloading constitutes one of the most extreme modes of inducing hypertrophic growth of the tissue. IL-6 and LIF are induced in overloaded muscles during the process of hypertrophy in rodents [20-22]. LIF (and IL-6) expression is also significantly induced by resistance exercise in human muscle and in electrically stimulated cultured human myotubes . Confirming the role of these cytokines in muscle hypertrophy, both LIF and IL-6 knockout mice were shown to have an impaired hypertrophic response to overloading [20, 21]. Myofiber hypertrophy in response to overloading requires not only increased net protein synthesis, but also accretion of new nuclei from the progeny of satellite cells. Notably, the impaired hypertrophic muscle growth in IL-6 null mice has been ascribed to blunted accretion of myonuclei, while protein synthesis pathways are preserved . This impaired myonuclei incorporation is a consequence of the defective proliferation and migration capacities of satellite cells in the absence of IL-6 , reinforcing the idea that muscle-produced IL-6 critically regulates satellite cell functions. Similarly, LIF has been shown to control the proliferation of satellite cells both in mice and humans . Indeed, exogenous LIF can induce human myoblast proliferation, an effect that likely occurs via induction of the cell proliferation-associated factors c-Myc and JunB. These effects can be abrogated by genetic interference with the LIF receptor. Treatment with IL-6 also promotes murine satellite cell proliferation via regulation of the cell-cycle-associated genes cyclin D1 and c-myc . Importantly, a complementary role to IL-6 in stimulating muscle growth may be attributed to IL-4 because it promotes myoblast fusion without affecting their proliferative capacity . Similar to IL-6, IL-4 is produced by exercising muscle [4, 24] and the expression of both cytokines has been shown to depend on the transcription factor serum response factor. Thus, serum response factor can be used by the myofibers to translate mechanical cues into paracrine growth-promoting signals that impact positively on satellite cell proliferation and fusion .
Functional studies in rodents have shown that IL-6 and LIF also contribute to muscle regeneration after injury and this may involve a similar stimulatory effect on satellite cell proliferation [25-28]. However, the cellular source of IL-6 and LIF in the damaged muscle tissue is less clear than in overloaded muscle. It is likely that distinct cell types contribute to increasing the local production of cytokines, which in turn impact on satellite cells to modify their reparative functions. In regenerating muscle, IL-6 is produced by infiltrating macrophages and neutrophils , by fibro-adipogenic progenitors , as well as by satellite cells [30, 31], thus implying potential paracrine and autocrine functions of IL-6 in satellite cell-dependent myogenesis. Early studies showed that cultured human myoblasts produced and secreted IL-6 in response to treatment with proinflammatory cytokines such as IL-1β or tumor necrosis factor (TNF)α . Thus, inflammatory cells infiltrating the injured muscle not only produce IL-6, but also may secrete other proinflammatory cytokines which might lead to further IL-6 expression by satellite cells, thus increasing the concentration of IL-6 in the local satellite cell microenvironment.
This local increase in IL-6 may lead not only to proliferation of satellite cells, but also to their differentiation and fusion, thus playing a dual role in myogenesis. For example, cultured myoblasts undergoing differentiation have been shown to be a source of IL-6 and, more importantly, ablation of IL-6 expression with specific siRNAs reduced the extent of myoblast differentiation and fusion. However, genetic overexpression or addition of exogenous IL-6 augmented the expression of muscle-specific genes, supporting its promyogenic function . The requirement of IL-6 for myogenic differentiation has been recently confirmed genetically, because myoblasts derived from IL-6 null mice displayed reduced differentiation and fusion capacities in vitro . Like IL-6, LIF has also been associated with myoblast differentiation . Thus, the IL-6 family of cytokines appears as a pivotal regulator of myogenesis, acting during both proliferation and differentiation. This dual mode of action is similar to IGF, a critical regulator of myogenesis, which can promote both proliferation and differentiation of satellite cells. Even more interestingly, it has been proposed that during myogenic differentiation, IGF1 can activate the signal transducer and activator of transcription/suppressor of cytokine signaling (STAT/SOCS) pathway , which has classically been associated with the intracellular transmission of IL-6 cytokine family signals.
Activation of distinct JAK/STAT signaling pathways by the IL-6 family of cytokines regulate satellite cell-dependent myogenesis
Many intracellular signaling pathways are known to regulate myogenesis. Among them, the p38 mitogen-activated protein kinase (MAPK), insulin-like growth factor/phosphatidylinositol 3-kinase/AKT, calcium/calmodulin-activated protein kinase, and calcineurin positively regulate myogenic differentiation [16, 37-40]. Alternatively, the extracellular signal-regulated kinase (ERK) pathway has dual roles: it inhibits differentiation at the early stage of differentiation, but promotes myocyte fusion at the late stages of differentiation [41-43]. Similarly, NF-ĸB has been shown to promote myoblast proliferation, while also favoring differentiation at later stages by acting as a downstream mediator of p38 MAPK signaling [33, 44]. Interestingly, IL-6 expression in differentiating myoblasts was shown to depend on p38 MAPK and NF-ĸB signaling pathways, and is an effector of their myogenic activities .
Consistent with the dual functions of IL-6 and LIF in myogenesis that have emerged in recent years, the JAK/STAT signaling pathway has also been associated with both promotion of myoblast proliferation and/or differentiation. The distinct myogenic actions appear to depend on the particular intracellular mediators of the JAK/STAT pathway engaged at every step. Early studies showed that proliferating satellite cells in regenerating muscle-expressed activated (phosphorylated) STAT3 , and cultured myoblasts showed expression of activated STAT3 when stimulated with LIF [45, 46]. In addition, STAT3 was shown to be capable of associating directly with MyoD and inhibiting its myogenic activities when overexpressed in C2C12 cells .
In recent years, much additional progress has been made in the understanding of the role of the JAK/STAT pathway as an essential intracellular mediator of the IL-6 family of cytokines in myogenesis, particularly from studies by Wu and colleagues [48-51]. In vivo analysis of JAK1, STAT1 and STAT3 molecules in regenerating muscles has revealed that they are activated at early times when satellite cells proliferate rapidly . Consistent with this, the JAK1/STAT1/STAT3 pathway was shown to be necessary for myoblast proliferation in vitro, based on its capacity to regulate the expression of cell-cycle-associated genes such as p27, p21 and Id1. Further analysis showed that stimulation of myoblast proliferation by LIF treatment requires formation of a STAT1/STAT3 complex, because genetic interference with these molecules abrogates LIF-mediated proliferation. This result is consistent with the delayed muscle regeneration of LIF−/− mice, which can be rescued by delivery of exogenous LIF . Thus, LIF is essential for the proliferation of myoblasts both in vivo and in vitro. Consistent with this, IL-6-dependent activation of STAT3 was also shown to be required for satellite cell proliferation in response to muscle overloading, and inhibition of this pathway in IL-6 null mice impaired myofiber hypertrophy in vivo as well as satellite cell proliferation in vitro [21, 22].
However, important studies by Wu and colleagues demonstrated that activation of the JAK1/STAT1/STAT3 pathway not only stimulates myoblast proliferation, but also prevents their premature differentiation by blocking the expression of genes critical for myoblast differentiation and fusion, such as MyoD, MEF2 and myogenin . In this way, the JAK1/STAT1/STAT3 pathway constitutes a differentiation checkpoint, ensuring that differentiation commences only when a sufficient number of myoblast cell progeny have been generated during the proliferative phase. Consistent with this, specific knockdown of JAK1 or STAT1 reduces myoblast proliferation and leads to premature differentiation. Thus, LIF, and to certain extent, IL-6, play dual roles in proliferating myoblasts by inducing the JAK1/STAT1/STAT3 pathway, which is able to promote their proliferation and also inhibit their precocious differentiation. Interestingly, exposure of myoblasts to LIF in differentiating conditions appeared to maintain the number of proliferating cells as differentiation proceeded . More precisely, LIF treatment reduced the percentage of cells positive for active caspase 3 through a MEK/ERK-dependent pathway. Because previous studies had shown that caspase 3 activity is required for myogenic differentiation , LIF might inhibit differentiation not only via modulation of the JAK1/STAT1/STAT3 pathway, but also through inhibition of caspase 3.
At variance with the JAK1-mediated actions, Wu's group demonstrated that JAK2 is required for myogenic differentiation in a pathway requiring STAT2 and STAT3, because pharmacological and genetic interference with JAK2, STAT2 or STAT3 activation prevented the differentiation process . Whereas the JAK1/STAT1/STAT3 pathway repressed the expression of MyoD and MEF2, the JAK2/STAT2/STAT3 pathway enhanced their expression, consistent with their opposite action on myogenesis. The JAK2/STAT2/STAT3 pathway also regulates the expression of hepatocyte growth factor and IGF2 in differentiating cells . The expression of hepatocyte growth factor was shown to be repressed by the JAK2/STAT2/STAT3 pathway at the initial stages of differentiation in agreement with the known role of this growth factor in promoting proliferation and inhibiting differentiation. Alternatively, IGF2 was induced by the same pathway as differentiation progressed, a result consistent with the capacity of IGF2 to stimulate myotube formation and growth [54, 55]. The role of STAT2 and STAT3 might not be fully redundant because STAT2 appeared to mediate principally the action of JAK2 on hepatocyte growth factor expression, whereas the expression of IGF2 was mediated by both STAT2 and STAT3 . Taken together, these studies indicate that various members of the JAK/STAT family are involved in the regulation of muscle cell proliferation and differentiation through different partners and effectors, thereby exerting distinct actions throughout myogenesis. It is not yet well known which ligands engage the JAK2/STAT2/STAT3 pathway during myogenic differentiation. Because IL-6 is expressed in differentiating myoblasts, and it promotes their differentiation and fusion [33, 34], IL-6 (as well as LIF)  may be a potential trigger of these actions.
A conclusion that can be deduced from the studies described above is that the JAK1/STAT1/STAT3 pathway needs to be tuned down for cessation of myoblast proliferation and commencement of differentiation. Consistent with this idea, the kinase activity of JAK1 is reduced upon differentiation . Three families of regulators of JAK/STAT signaling are known: the SOCS family of proteins, the protein inhibitor of activated STAT (PIAS) family of proteins and the SH2-containing phosphatase family of proteins . These proteins target distinct members of the JAK/STAT pathway in distinct cellular compartments. SOCS1 and SOCS3 target JAK1 and gp130, respectively, near the plasma membrane to prevent cytoplasmic STATs from being activated, whereas PIAS1 principally targets activated STAT1 in the cell nucleus and prevents it from binding to DNA. The inhibition at distinct levels and positions might function to ensure that the pathway can be effectively turned off thus allowing progression of myogenesis. The fact that STAT1 and STAT3 are capable of inducing the inhibition of the pathway by activating the expression of SOCS1 and SOCS2, but not PIAS1, in a feedback inhibitory mechanism constitutes a further level of specificity and regulation of this pathway during myogenesis . It is worth noting that in differentiating myoblasts, IGF was shown to induce SOCS gene transcription, suggesting that this protein could be involved in the differentiation process . In agreement with this notion, SOCS3 overexpression in human myoblasts resulted in an increased expression of genes associated with skeletal muscle growth, although the mechanism underlying this effect requires further investigation . Interestingly, SOCS3 signaling during aging appears dysregulated [57, 58], and therefore, the decline in the regenerative capacity of muscle with aging may be connected to STAT3/SOCS3 dysregulation. PIAS1 has been shown to modulate myogenesis also through the interaction with proteins that can regulate myoblast differentiation, such as SnoN or Msx1 [59-61]. PIAS1 interacts with and directly sumoylates SnoN, which increases the SnoN capacity to block myogenin gene expression and subsequent cell differentiation [59, 61]. In addition, PIAS1 was also shown to interact with and sumoylate Msx1, which is then capable of binding to the MyoD promoter thus repressing MyoD gene expression . However, the precise role of PIAS1 in myogenic differentiation remains debatable, because distinct phenotypes have been obtained after interfering with its expression [59, 60]. Therefore, additional studies are necessary to confirm the myogenic function of these molecules. Similarly, further investigation is necessary to decipher the function the SH2-containing phosphatase family inhibitors of JAK/STAT. Although SH2-containing phosphatase has been associated with myoblast differentiation , siRNA knockdown did not affect the activity of the JAK1/STAT1/STAT3 pathway during the differentiation process .
Finally, evidence that other IL-6 family members oncostatin M and cardiotrophin-1 are also active in myogenesis in vitro and in vivo was recently provided [51, 63]. Oncostatin M was shown to inhibit myoblast differentiation by activating the JAK1/STAT1/STAT3 pathway. STAT1 can interact with, and repress the transcriptional activity of, MEF2 in vitro. Furthermore, prolonged expression of oncostatin M in injured skeletal muscles resulted in defective regeneration. By contrast, treatment of myoblasts with cardiotrophin-1 inhibited their differentiation. However, this action was preferentially mediated through activation of MEK/ERK signaling [51, 63], which in turn might interfere with activation of critical myogenic regulatory factors. These findings suggest that cardiotrophin-1 and oncostatin M may be implicated in the maintenance of the undifferentiated state in muscle progenitor cells, in collaboration with the proliferative actions of IL-6 and LIF. Collectively, these data suggest that several members of the IL-6 family of cytokines contribute to myogenesis in vitro and muscle regeneration and growth in vivo, acting at distinct stages of these processes in a timely and regulated fashion, through distinct signaling pathways and effectors.