GCN2 deficiency protects mice from denervation-induced skeletal muscle atrophy via inhibiting FoxO3a nuclear translocation
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Several recent clinical studies have indicated that dietary supplementation with branched-chain amino acids (BCAA), particularly with leucine, is an effective anti-atrophic therapy (Bauer et al., 2015; Tsien et al., 2015; English et al., 2016). In animal models, BCAA can prevent denervation (Ribeiro et al., 2015), hindlimb suspension (Maki et al., 2012; Jang et al., 2015) or dexamethasone-induced (Yamamoto et al., 2010) muscle atrophy. General control nonderepressible 2 kinase (GCN2) is a well-known amino-acid sensor. Under conditions of amino-acid deprivation, the increased level of uncharged transfer RNA (tRNA) activates GCN2 through binding to the histadyl-tRNA synthetase-like domain (Wek et al., 1995). Upon activation, GCN2 phosphorylates eukaryotic initiation factor 2 alpha at Ser51, which leads to translational arrest and restoration of amino acid homeostasis (Wek et al., 1995; Sood et al., 2000). As amino acids are potent modulators of protein turnover in skeletal muscle, we proposed that GCN2 may affect denervation-induced muscle atrophy, but the detail mechanism remains unclear.
To investigate whether GCN2 activation directly causes FoxO3a nuclear translocation in muscle atrophy, we generated a stable C2C12 cell line (mGCN2-C2C12) with doxycycline (Dox)-controlled expression of flag-tagged mouse GCN2 (Fig. 2C), and found that GCN2 overexpression induced by Dox (Dox+) significantly increased the ratio of FoxO3a in the nucleus (Fig. 2D). Furthermore, co-immunoprecipitation experiments using lysates from FoxO3a plasmid-transfected mGCN2-C2C12 cells with Dox demonstrated that GCN2-Flag and FoxO3a-EGFP were specifically co-precipitated with anti-GFP and anti-Flag antibodies, respectively (Fig. 2E), demonstrating that GCN2 and FoxO3a can physically interact with each other in cells. Using the differentiated mGCN2-C2C12 cells, we also found that overexpression of GCN2 exacerbated dexamethasone-induced upregulation of Atrogin-1 and LC3-II in differentiated C2C12 cells (Fig. S4).
It has been demonstrated dietary deprivation of essential amino acids, which activates GCN2 via increasing uncharged tRNA levels (Wek et al., 1995), caused diffuse atrophy in the rectus femoris muscles (Kamata et al., 2014). In contrast, leucine or other BCAA supplementation, which attenuates GCN2 activity (Wek et al., 1995), has been regarded as a potential pharmaconutrient for the treatment of numerous muscle wasting conditions (Bauer et al., 2015; Tsien et al., 2015; English et al., 2016). In agreement with those findings, we demonstrated that GCN2 deletion attenuates, whereas GCN2 overexpression exacerbates denervation-induced muscle atrophy. Furthermore, the detrimental effect of GCN2 in denervation-induced atrophy was related to FoxO3a activation, which upregulates genes involved in both the ubiquitin-proteasome pathway and autophagy in muscle atrophy (Sandri et al., 2004; Bertaggia et al., 2012; Wei et al., 2013; Guo et al., 2016). Thus, reducing GCN2 activity may be a potential therapeutic approach for the clinical treatment of muscle atrophy.
This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81470520, 91643206, 91743104, 31371430 to HW and 31300976 to BW) and Chinese Academy of Sciences (KJRH2015-005 and Hundred Talents Program).
Yuting Guo, Huiwen Wang, Yinglong Tang, Yue Wang, Mengqi Zhang, Zhiguang Yang, Eric Nyirimigabo, Bin Wei, Zhongbing Lu, and Guangju Ji declare that they have no conflicts of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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