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gp130 signaling is involved in both muscle hypertrophy and atrophy. gp130 cytokines, IL-6 and LIF, are known as ‘myokines’, which means they are produced and released from skeletal muscles during exercise and are involved muscle adaptation to exercise in autocrine/paracrine manner. Both IL-6 and LIF have demonstrated their role in overload induced muscle hypertrophy. Further research showed that IL-6 and LIF can stimulate satellite cell proliferation which contribute to muscle hypertrophy. However, chronic elevation of gp130 cytokines and/or activation of gp130 downstream signaling pathways

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in skeletal muscles, are key mediators of muscle wasting in cancer cachexia. In this section, we will review (1) major gp130 ligands and gp130 downstream signaling pathways, (2) role of gp130 cytokines and downstream signaling pathways in muscle hypertrophy and myogenesis and (3) role of gp130 cytokines and downstream signaling pathways in muscle wasting.

gp130 ligands and downstream pathways.

Glycoprotein 130 kDa (gp130) is a ubiquitously expressed trans-membrane protein. It serves as the signal transduction unit of a family of cytokines: the IL-6 family. The members of this family are IL-6, IL-11, IL-27, IL-30, IL-31, oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC) and ciliary neurotrophic factor (CNTF) (2). These cytokines utilize type I cytokine receptors which consist of a ligand binding α-receptor subunit and a signal transducing β- receptor subunit containing a cytoplasmic signaling domain. Each gp130 cytokine is characterized by a certain profile of receptor recruitment but all cases involve at least one molecule of gp130. IL-6, IL-11 and CNTF first bind specifically to their respective α- receptor subunits. Once bound to the α-receptor, the complex then binds to two β-receptors to form a dimer. The remaining gp130 cytokines do not bind to an α-receptor, but bind to their respective β-receptor directly and signal via heterodimers of either gp130 and the LIFR (LIF, CNTF, CT-1 and CLC) or gp130 and the OSMR (OSM). Regardless of different binding pattern, this ligand-receptor protein complex then activates constitutively bound Janus family kinases, JAK1, JAK2 or Tyk2, which then phosphorylates tyrosine residues in the distal cytoplasmic domains of gp130. Phosphorylated gp130 can act as a docking

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site for SH2 domains of Signal Transducer and Activator of Transcription (STAT) family of transcription factors (STAT1, 3 and 5). Subsequent phosphorylation of STATs by gp130 then induces STAT dimerization and translocation to the nucleus where they induce gene transcription. Thus, JAK/STAT activation is a classical hallmark of gp130 dependent cytokine signaling. Dimerization of gp130 cytokine receptors also leads to the induction of the MAPK cascade, including ERK1/2, JNK1/2 and p38 MAPK. gp130 activation of MAPKs is primary mediated through the recruitment of the protein tyrosine phosphatase SHP2 to the gp130 phosphorylation site. Phosphorylation of Tyr759 on gp130 results in the recruitment of SHP-2 (Src homology domain-containing protein tyrosine phosphatase- 2), allowing its phosphorylation by JAK. Phosphorylated SHP-2 then interacts with Grb2 (growth-factor receptor bound protein 2), which leads to the activation of the Ras-Raf- ERK1/2 cascade (2). gp130 cytokines can also activate the stress-activated members of the MAPK family: p38 and JNK (180, 181), but the signal transduction pathways resulting in their activation remain poorly understood.

gp130 cytokines can also lead to the activation signaling cascades involving PI3K. This enzyme modifies certain phosphatidylinositides, so that the serine/threonine Akt is recruited to the plasma membrane, where it becomes activated through phosphorylation by PDK1 (phosphoinositide-dependent kinase-1). can induce the activation of mTOR signaling IL-6-induced activation of the PI3K/Akt pathway is involved in protection against apoptosis, as well as in enhanced proliferation in multiple myeloma cells (182-184), cardiac myocytes (185, 186) and basal carcinoma cells (187). The molecular mechanism linking gp130 engagement to the activation of the PI3K/Akt pathway is not well understood.

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Some evidence exist suggesting that IRS-1 and its adaptor protein Gab1 are involved in gp130 activation of PI3K (188, 189). Notably, PI3K activation upon gp130 cytokines is observed in a cell-type specific manner. For example, no significant Akt activation could be observed in IL-6 treated HepG2 hepatoma cells (190). Whether gp130 cytokines can induce PI3K/Akt signaling activation in skeletal muscle cells has not been examined.

Activation of gp130/STAT3 signaling is transient in most biological systems because of the efficient inhibition mechanisms for STAT inactivation. This inhibition mechanisms of gp130/STAT signaling pathway can inhibit the constant activation of JAK1/STAT1/STAT3 pathway. 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 (191). These proteins target to distinct members of the gp130/STAT signaling pathway: SOCS1 and SOCS3 target JAK1 and gp130, respectively, near the plasma membrane to prevent cytoplasmic STATs from being activated, whereas PIAS1 principally targets activated STAT proteins in the cell nucleus and prevents it from binding to DNA (191). SOCS proteins transcription is induced by IL-6 and LIF and since they inhibit tyrosine phosphorylation of gp130, STAT1 and STAT3. SOCS proteins contain a SH2 domain and was shown to directly interact with the kinase domain of JAK2, resulting in a reduced tyrosine kinase activity of JAK2. Thus SOCS is a JAK inhibitor that is induced by the STAT3 activation and eventually leads to feedback regulation of gp130/STAT3

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IL-6 and LIF are induced in overloaded muscles during the process of hypertrophy in rodents (7). LIF and IL-6 expression is also significantly induced by resistance exercise in human muscle and in electrically stimulated cultured human myotubes (192). Both LIF and IL-6 knockout mice were shown to have an impaired hypertrophic response to overloading, which confirms the role of these cytokines in muscle hypertrophy (7, 8). Overload induced protein synthesis induction was not attenuated by IL-6 knock-out mice. It was believed that impaired hypertrophic muscle growth is ascribed to blunted accretion of myonuclei, which is caused by the defective proliferation and migration capacities of satellite cells in the absence of IL-6 (8). Indeed, IL-6 can activate murine satellite cell proliferation via regulation of cyclin D1 and c-myc (193). Similarly, exogenous LIF can induce human myoblast proliferation via induction of the cell proliferation associated factors c-Myc and JunB (192). The overload induced myofiber hypertrophy also requires IL-4, which promotes myoblast fusion without affecting their proliferative capacity. Similar to IL-6, IL- 4 is expressed in skeletal muscles in response to overload and exercise (8, 194). The expression of both cytokines has been shown to depend on the transcription factor serum response factor (8). 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. Collectively, these results demonstrate that gp130 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.

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JAK/STAT signaling pathway is also associated with muscle growth and hypertrophy through the promotion of myoblast proliferation. Proliferating satellite cells in regenerating muscle showed activated STAT3 (195). STAT3 signaling directly associates with MyoD expression and overexpression of d.n.STAT3 in C2C12 myoblast can inhibit its myogenic activities (196). JAK1, STAT1 and STAT3 signaling pathways are activated in early stage of muscle regeneration which is characterized by rapid proliferation of satellite cells (197). 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. STAT1/STAT3 complex is also necessary for LIF stimulation of myoblast proliferation (197), which is consistent with the delayed muscle regeneration of LIF knock- out mice, which can be rescued by delivery of exogenous LIF (7). However, activation of the JAK1/STAT1/STAT3 pathway can also prevent premature differentiation of myoblasts by blocking the expression of genes critical for myoblast differentiation and fusion, such as MyoD, MEF2 and myogenin, while knockdown of JAK1 or STAT1 reduces myoblast proliferation and leads to premature differentiation (197). These results suggest that the JAK1/STAT1/STAT3 pathway serves as a differentiation checkpoint, ensuring that differentiation commences only when a sufficient number of myoblast cell progeny have been generated during the proliferative phase. SOCS dependent negative feedback is important to inhibit JAK1/STAT signaling, which allows the cessation of myoblast proliferation and commencement of differentiation. Kinase activity of JAK1 is reduced upon differentiation, which is associated with increased expression of SOCS1 and SOCS2 (195). In conclusion, LIF and IL-6 induction of JAK1/STAT1/STAT3 pathway plays dual role in proliferating myoblasts, that is, promote their proliferation and also inhibit their

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In addition to JAK/STAT3 signaling pathway, several intracellular signaling pathways downstream gp130 are known to regulate myogenesis. The p38 MAPK, PI3K/AKT, calcium/calmodulin activated protein kinase, and calcineurin positively regulate myogenic differentiation (198), while ERK1/2 pathway activates myoblast proliferation and inhibits differentiation at the early stage of differentiation, but promotes myocyte fusion at the late stages of differentiation (199, 200). Similarly, NF-ĸB also promotes myoblast proliferation, while also favoring differentiation at later stages by acting as a downstream mediator of p38 MAPK signaling (201).

Role of gp130 cytokines in muscle atrophy

Since early 1990’s, it has been recognized that elevated circulating IL-6, as well as complex underlying cytokine network, could participate in cachexia development (118). IL-6/gp130 signaling has been demonstrated to play a key role in several cancer cachexia animal models. Inhibiting IL-6 signaling by neutralizing antibodies showed a protective effect on body weight loss in C26 tumor bearing mice (39). The IL-6 dependent muscle mass loss has also been demonstrated in ApcMin/+ and C26 tumor-implanted mice (125, 202, 203). Recently, LIF has also been demonstrated to play a key role in C26 induced muscle wasting (204). However, whether IL-6/gp130 signaling is sufficient and/or has a direct role in the induction of muscle atrophy remains controversial. Further research is still required to determine the mechanisms underlying IL-6 /gp130 signaling in cachexia induced muscle wasting.

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Whether IL-6 induces muscle protein degradation is still under debate. In fact, different in vivo and in vitro studies got controversial results. Several early studies showed protein degradation rate in isolated rat muscles exposed to recombinant IL-6 (205), or after injection of IL-6 into wild type mice (206, 207), was unaltered. Similarly, exogenous IL-6 administration showed no effect on the proteolytic rate of rat and murine myotubes (208). These results are also supported by the fact that infusion of a single dose of IL-6 to rats did not induce ubiquitin gene upregulation in muscle (209). In contrast to these negative results, increased muscle proteolysis was observed after high doses or long-term administration of IL-6 in rats or mice (210). IL-6 transgenic mice showed elevated circulating levels IL-6 and displayed severe muscle atrophy by the age of 10 weeks, together with the activation of the proteolysis in muscles (211). The inhibition of IL-6 signaling by sustained administration of mouse IL-6R antibody completely reversed the muscle wasting in IL-6 overexpressing mice (212) and C26 tumor bearing wild type mice (151). These results demonstrate that IL-6 at least has a permissive role in the development of skeletal muscle proteolysis and muscle wasting when other cachectic factors are also present. Some evidence suggest IL-6 may synergize with other cytokines to induce muscular atrophy in some disease states. For example, activation of the rennin-angiotensin system can cause severe muscle wasting, which is commonly found in congestive heart failure or chronic kidney disease (213).

Consistent with IL-6 and LIF role, the role of STAT3 signaling in cachexia induced muscle wasting has been well established. Increased muscle STAT3 phosphorylation has

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been observed in several cachexia models. Overexpression of constitutively active STAT3 in normal mouse muscle by plasmid transfection induced a significant reduction of myofiber cross sectional area (202, 214). Transfection of dominant negative STAT3 mutants, or STAT3 short hairpin, prevented the atrophy induced by injection of Chinese hamster ovary cells overexpressing IL-6 in athymic nude mice and in the C26 adenocarcinoma mouse model (202). A recent study further demonstrates STAT3 signaling activation contributes to muscle wasting in C26 and LLC cachexia model by inhibiting muscle protein synthesis, as well as stimulating caspase-3, myostatin, and the ubiquitin- proteasome system (215). The disruption of SOCS3 dependent negative feedback loop may contribute to constant muscle STAT3 signaling activation in cachexia. Indeed, augmented SOCS3 mRNA expression was reported in cachexia induced wasting muscle undergoing an exacerbated proteolysis. Interestingly, the SOCS3 protein level was unchanged or even reduced in this model, despite SOCS3 mRNA upregulation. This discrepancy can be interpreted as a JAK/STAT-driven increase in proteolytic degradation of SOCS3 protein (202). Taken together, these results strongly suggest STAT3 activation is the major mediator of IL-6 dependent muscle wasting in cachexia.

The role of STAT3 activation in cachexia induced muscle protein synthesis suppression still needs to be examined. Some evidence support IL-6 may indirectly suppress muscle protein synthesis though its interference with the growth hormone/IGF-1 axis. Suppressed serum IGF-1 and muscle IGF-1/Akt signaling was observed in ApcMin/+ mice, an IL-6 dependent cachexia model (18). Short term IL-6 administration to wild type mice and humans reduced circulating IGF-1 levels. Transgenic mouse models overexpressing IL-6

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showed reduced growth, which is accompanied with high serum IL-6 level, low serum IGF- 1 levels and enhanced muscle SOCS3 mRNA expression (216). Neutralization of IL-6 activities in IL-6 transgenic mice rescued the circulating IGF1 levels and fully restored growth, reinforcing the casual relationship between high IL-6 and low IGF1 levels in plasma (216). Some existing evidence also suggests that AMPK signaling activation is involved in IL-6 suppression of muscle protein synthesis. Late stage of cachectic ApcMin/+ showed increased muscle AMPK phosphorylation (18). Systemic IL-6 overexpression in ApcMin/+ mice produced a dose-dependent suppression of mTOR signaling which correspond to AMPK phosphorylation, and chronic IL-6 administration was enough to induce AMPK phosphorylation in cultured myotubes (20). IL-6 suppression of myotube mTOR activity was rescued by AMPK inhibition (20). However, induction of muscle and myotube AMPK phosphorylation by LLC, which is another gp130 dependent cachexia model, was not attenuated by IL-6 or gp130 signaling inhibition (19), which indicates IL- 6/gp130 signaling is not the only inducer of muscle AMPK signaling in cachexia.