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IL-6 and LIF are members of the IL-6 family of cytokines that have recognized paradoxical regulatory roles in skeletal muscle mass. Short term muscle production of IL- 6 and LIF are necessary for overload induced muscle hypertrophy, but the cellular mechanisms involved in this regulation have not been completely identified. mTOR signaling is a key controller of muscle protein synthesis, which is a driver of muscle mass regulation. IL-6 family of cytokines regulates upstream activators of mTORC1 including PI3K/Akt and ERK1/2, but the muscle mTOR signaling regulation of by the IL-6 family of cytokines remains poorly understood. Therefore, we investigated the short term effects of IL-6 and LIF induced cellular signaling on the regulation of C2C12 myotube mTORC1 signaling and protein synthesis. C2C12 myotubes were subjected to short term IL-6 or LIF adminsitration. Signaling through the gp130 receptor and downstream effectors including PI3K/Akt, STAT3 and ERK1/2 were investigated by administration of specific siRNA or pharmaceutical inhibitors. Short term IL-6 or LIF administration increased myotube protein synthesis and activated STAT3 and Akt-mTOR signaling pathways. The induction of mTOR signaling and protein synthesis by IL-6 or LIF was attenuated by either gp130 siRNA knock-down or Akt signaling inhibition. Inhibition of STAT3 and mTOR signaling did not block the IL-6/LIF induction of myotube protein synthesis. These results demonstrate that short term IL-6 and LIF exposure can induce protein synthesis in culture myotubes through the activation of gp130-Akt signaling pathway, and IL-6/LIF induced Akt signaling can activate protein synthesis via mTOR independent mechanisms.

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3.2 Introduction

Skeletal muscle mass has a well-documented role in health and quality of life. To this end, our knowledge of processes governing skeletal muscle mass, which involves the balance between protein synthesis and degradation, have been rapidly expanding (1). However, significant gaps remain in our understanding of skeletal muscle regulation of protein turnover by systemic changes related to nutritional status, physical activity level, inflammation and underlying disease. The effectors of these systemic conditions targeting skeletal muscle protein turnover include mechanical stimuli, cytokines, growth factors, and hormones (1, 217). The Interleukin-6 (IL-6) family of cytokines regulate many physiological and pathological processes including immune system regulation, inflammation, wound healing, and cell survival (2). Skeletal muscle serves as both a biological target and source of the IL-6 family of cytokines. Two members of IL-6 family of cytokines, IL-6 and Leukemia Inhibitory Factor (LIF), have recognized roles in both skeletal muscle hypertrophy and atrophy (5, 218). Under physiological conditions, repeated contractions during exercise, overload or muscle injury can induce plasma elevation and muscle production of IL-6 and LIF (192, 219), which is thought to initiate important autocrine and paracrine signaling that contributes to muscle hypertrophy by stimulate myocyte proliferation and differentiation (7, 8, 192). Notably, the systematic IL-6 release in response to exercise is in a temporal manner, increasing rapidly and remaining elevated for a few hours (36, 220). Unlike the beneficial effects of short term actions of IL-6 and LIF under physiological conditions, long term, pathological elevation of circulating IL-6 and LIF are tightly associated with muscle wasting (9, 118, 204, 221, 222). Currently, the duration of IL-6 family of cytokines exposure is the plausible explanation of their

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differential effects on muscle mass regulation (5), but further research is still needed to understand this complex dichotomy.

IL-6 and LIF can induce multiple intracellular signaling pathways via type I cytokine receptors. These receptors contain a ligand binding α-receptor subunit and two signal transducing β-receptor subunits, which contain a cytoplasmic signaling transducing domain (2, 223). Glycoprotein 130 (gp130), a transmembrane protein, functions as the β- receptor subunits for the IL-6 and LIF. The ligand-receptor protein complex activates constitutively bound Janus family kinases 2 (JAK2), which phosphorylates tyrosine residues in the cytoplasmic domains of gp130 (2, 223). Phosphorylated gp130 can phosphorylate and activate STAT3, leading to STAT3 dimerization and nuclear translocation to induce target gene transcription. Notably, activated STAT3 signaling induces SOCS3 expression. SOCS3 can suppress gp130 dependent signaling through direct interaction with the kinase domain of JAK2 (2, 224), which forms a feedback inhibition mechanisms that prevents constant induction of gp130 dependent signaling pathways under physiological conditions (224). Phosphorylated gp130 also induces ERK1/2 signaling through the recruitment of protein tyrosine phosphatase SHP-2 (Src homology domain- containing protein tyrosine phosphatase 2), allowing SHP2 phosphorylation by JAK2. Phosphorylated SHP2 then interacts with Grb2 (growth-factor receptor bound protein 2), leading to the activation of the Ras-Raf-ERK1/2 cascade (2). Finally, IL-6 family of cytokines can induce the phospho-inositide-3-kinase (PI3K)/Akt signaling axis (2), but the molecular events mediating this regulation are not well understood. PI3K-Akt and ERK1/2 signaling induction by the IL-6 also demonstrates cell-type specific regulation. For

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example, PI3K/Akt activation could be observed in IL-6 treated multiple myeloma cells (182-184), cardiac myocytes (185, 186) and basal carcinoma cells (187), but not HepG2 hepatoma cells (190). Despite a growing body of evidence for cytokine induced gp130 signaling in cells, additional work is needed to better define the specific regulatory role of this signaling pathway in skeletal muscle fibers.

The induction of protein synthesis has a well-documented role in skeletal muscle hypertrophy (1). The mammalian target of rapamycin (mTOR), which functions in the multi-protein complex, mTORC1, plays an essential role in the regulation of protein synthesis and skeletal muscle mass (14). Activated mTORC1 can induce muscle protein synthesis through the phosphorylation of the p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein (4E-BP1), leading to increased ribosomal biogenesis and cap-dependent protein translation initiation (13). Many muscle hypertrophic factors, such as growth factors like insulin-like growth factor 1 (IGF-1) and mechanical stimuli, can stimulate muscle mTOR signaling and protein synthesis through signaling pathways which converge on the phosphorylation of mTORC1 suppressor, tuberous sclerosis 1/2 (TSC1/2). IGF-1 can activate PI3K-Akt signaling, which further relieves TSC1/2 suppression on Ras homolog enriched in brain (Rheb) by phosphorylation of TSC2, allowing Rheb to stimulate mTORC1 (14, 15). Induction of Extracellular signal- Regulated Kinases 1/2 (ERK1/2) signaling by mechanical overload also phosphorylates and inhibits TSC2, which contributes to early activation of muscle mTORC1 (16). Muscle produced of IL-6 and LIF also plays a key role in muscle overload induced muscle hypertrophy. However, whether they can stimulate mTOR signaling and subsequent protein

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gp130 signaling induction by IL-6 family of cytokines have the potential to induce upstream stimulators of mTOR, including Akt and ERK1/2 signaling pathways, leading to the increased protein synthesis. In cardiomyocytes, induction of gp130 by IL-6 or LIF leads to PI3K/Akt signaling activation and subsequent protein synthesis increase, which is a key mechanism of IL-6 and LIF induction of cardiac hypertrophy (186, 225). IL-6 induction of ERK1/2 signaling in cardiomyocytes has also been demonstrated to be necessary and sufficient for its cardioprotection effect (186). However, the specific interaction between gp130 and the PI3K/Akt-mTORC1 or ERK1/2-mTORC1 signaling axis within muscle cells requires further investigation. The purpose of this study is to investigate if short term administration of IL-6 family of cytokines, IL-6 or LIF will regulate myotube protein synthesis by activating gp130 dependent signaling pathways. We hypothesized that short term IL-6 or LIF will induce mTOR signaling and protein synthesis in cultured myotubes by activation the gp130-Akt-mTOR signaling axis. The role of gp130 and its potential downstream effectors, including PI3K-Akt-mTOR, STAT3, ERK1/2 signaling pathways were investigated with the siRNA knock-down or the administration of their specific pharmaceutical inhibitors.

3.3 Methods