The ubiquitin–proteasome and autophagy–lysosome machinery are activated in atrophying muscles
Activation of the cell's proteolytic systems is transcriptionally regulated, and a subset of genes that are commonly up- or down-regulated has been identified in atrophying skeletal muscle, regardless of the catabolic condition [72-75]. These common genes are thought to regulate the loss of muscle components, and were thus designated atrophy-related genes or ‘atrogenes’ [75-77]. Among the up-regulated atrophy-related genes are transcripts belonging to the ubiquitin–proteasome and autophagy–lysosome systems. The up-regulation of several ubiquitin–proteasome and autophagy-related genes is normally blocked by Akt through negative regulation of Forkhead box O (FoxO) transcription factors [77-79].
In muscle, the ubiquitin–proteasome system is required to remove sarcomeric proteins in response to changes in muscle activity. The rate-limiting step of the ubiquitination process, which affects subsequent proteasome-dependent degradation, is catalysed by the E3 enzyme, which is a ubiquitin ligase. Among the known E3s, only a few are both muscle-specific and up-regulated during muscle loss. The first to be identified were atrogin-1/MAFbx (muscle atrophy F-box) and muscle RING finger 1 (MuRF1). Mice lacking atrogin-1/MAFbx and MuRF1 are resistant to muscle atrophy induced by denervation . Moreover, knockdown of atrogin-1 prevents muscle loss during fasting , whereas MuRF1 knockout mice (but not atrogin-1 knockout mice) are also resistant to dexamethasone-induced muscle atrophy . So far, very few muscle proteins have been identified as substrates for atrogin-1, and those that have been identified appear to be involved in growth-related processes or survival pathways. For example, atrogin-1 promotes degradation of MyoD, a key muscle transcription factor, and of eukaryotic translation initiation factor 3 subunit F (eIF3-f), an important activator of protein synthesis [82, 83]. In the heart, atrogin-1 ubiquitinates and reduces the levels of calcineurin A, an important factor triggering cardiac hypertrophy in response to pressure overload . Interestingly, immunoprecipitation experiments in C2C12 myoblasts and myotubes have found that atrogin-1 interacts with sarcomeric proteins, including myosins, desmin and vimentin, as well as transcription factors, components of the translational machinery, enzymes involved in glycolysis and gluconeogenesis, and mitochondrial proteins . Whether atrogin-1 ubiquitinates these proteins has yet to be proven. Conversely, MuRF1 was reported to interact with and control the half-life of many important muscle structural proteins, including troponin I , myosin heavy chains [87, 88], actin , myosin binding protein C and myosin light chains 1 and 2 . Presumably, additional E3s that have not yet been identified are also activated during atrophy to promote the clearance of soluble cellular proteins and to limit/regulate anabolic processes. A recent paper reported that Trim32 (tripartite motif-containing protein 32) is a crucial E3 ligase for the degradation of thin filaments (actin, tropomyosin and troponins), α-actinin and desmin . However, Trim32 knockout mice are not protected from atrophy, but instead show impaired recovery of muscle mass after atrophy . Another E3 ubiquitin ligase that has been found to play a critical role in atrophy is TRAF6 (TNF receptor-associated factor) , which mediates the conjugation of Lys63-linked polyubiquitin chains to target proteins. Lys48-linked polyubiquitin chains are a signal for proteasome-dependent degradation, but Lys63-linked polyubiquitin chains play other roles, such as regulating autophagy-dependent cargo recognition by interacting with the scaffold protein p62 (also known as SQSTM1) [94-96]. Muscle-specific TRAF6 knockout mice have a decreased amount of polyubiquitinated proteins, almost no Lys63-polyubiquitinated proteins in starved muscles , and are resistant to muscle loss induced by denervation, cancer or starvation [93, 97, 98]. The mechanism of this protection involves both direct and indirect effects of TRAF6 on protein breakdown. In fact, TRAF6-mediated ubiquitination is required for the optimal activation of c-Jun N-terminal kinase, AMPK, FoxO3 and NF-κB . All of these factors are crucial regulators of atrogin-1 and MuRF1 expression and of several autophagy-related genes. Inhibition of TRAF6 reduces the induction of atrogin-1 and MuRF1, thereby preserving muscle mass under catabolic conditions.
Specific ubiquitin ligases may be involved in different models of muscle wasting and at different stages of the atrophy process. For instance, the HECT domain ubiquitin ligase Nedd4-1 has been reported to be up-regulated mainly during muscle disuse. Indeed, deletion of the Nedd4-1 gene specifically in skeletal muscle results in partial protection from muscle atrophy in denervated type II fibers. However, Nedd4-1 knockout mice have smaller muscles, suggesting that this E3 may play additional roles during myogenesis or in the control of protein synthesis .
Mul1 is a mitochondrial ubiquitin ligase that plays an important role in mitochondrial network remodeling. Mul1 is up-regulated by the FoxO family of transcription factors under catabolic conditions, such as fasting or denervation, and causes mitochondrial fragmentation and removal via autophagy (mitophagy) . Importantly, knocking down Mul1 spares muscle mass during fasting. Mul1 ubiquitinates the mitochondrial pro-fusion protein mitofusin 2, causing its degradation via the proteasome system. The exact mechanism that triggers Mul1-dependent mitochondrial dysfunction and mitophagy is unclear, but it has been reported that mitofusin degradation is permissive for mitochondrial fission and mitophagy .
Carboxy terminus of Hsc70 interacting protein (CHIP) is another ubiquitin ligase, which regulates ubiquitination and lysosomal-dependent degradation of filamin C, a muscle protein found in the Z-line . Filamins undergo unfolding and refolding cycles during muscle contraction, and are therefore prone to irreversible damage . Alterations to filamin structure trigger binding of the co-chaperone BAG3, which is a complex comprising the chaperones Hsc70 and HspB8, as well as the ubiquitin ligase CHIP. CHIP ubiquitinates BAG3 and filamin, which are recognized and delivered to the autophagy system by p62 . Interestingly, filamin B half life is controlled, at least during myogenesis, by another ubiquitin ligase, ASB2β, which is mainly expressed in muscle cells. In this case, ubiquitination of filamin B by ASB2β leads to proteasome-dependent degradation .
In skeletal muscle, E3 ligases also have important regulatory functions in signaling pathways. For example, it was recently found that the ubiquitin ligase Fbxo40 (F-box only protein) regulates anabolic signals . Fbxo40 ubiquitinates and affects the degradation of insulin receptor substrate 1, a downstream effector of insulin receptor-mediated signaling. Inhibition of Fbxo40 by RNAi induces hypertrophy in myotubes, and Fbxo40 knockout mice display bigger muscle fibers .
Although some E3 ligases involved in muscle protein ubiquitination and breakdown have been identified, very little is known about how ubiquitinated proteins are recognized and delivered to the proteasome. ZNF216 has been identified as an important player in the recognition and delivery of ubiquitinated proteins to the proteasome during muscle atrophy. Interestingly, ZNF216 is up-regulated by FoxO transcription factors in atrophying muscles, and ZNF216-deficient mice are partially resistant to muscle loss during denervation. The absence of ZNF216 in muscle leads to accumulation of polyubiquitinated proteins .
Another important system for extraction and degradation of ubiquitinated proteins from larger structures is the p97/valosin containing protein (VCP) ATPase complex. p97/VCP is induced during denervation, and over-expression of a dominant-negative p97/VCP reduces overall proteolysis by the proteasome and lysosome pathways, and blocks the accelerated protein breakdown induced by FoxO3. Interestingly, p97 and its co-factors, Ufd1 and p47, have been found to be associated with specific myofibrillar proteins, suggesting a role for p97 in extracting ubiquitinated proteins from myofibrils .
Although a great body of research has focused on the ubiquitination process, little is known about the role of deubiquitination and its contribution to muscle atrophy. The largest class of deubiquitinating enzymes are ubiquitin-specific proteases. So far, only two ubiquitin-specific proteases (USP14 and USP19) have been found to be up-regulated in atrophying muscles [73, 107]. Knockdown of USP19 in myotubes results in decreased protein degradation and reverts dexamethasone-induced loss of myosin heavy chain .
Macroautophagy, hereafter referred to as autophagy, is the other proteolytic system that is activated in catabolic conditions and that is under FoxO regulation . The various types of autophagy, including their regulation and involvement in muscle homeostasis, have been reviewed recently . Briefly, autophagy is a highly conserved homeostatic mechanism that is used for the degradation and recycling, through the lysosomal machinery, of bulk cytoplasm, long-lived proteins and organelles . Although autophagy was initially considered a non-selective degradation pathway, the presence of more selective forms of autophagy is becoming increasingly evident. Indeed, autophagy may trigger the selective removal of specific organelles, such as mitochondria, via mitophagy. In mammals, parkin, PINK1 (phosphatase and tensin homolog-induced putative kinase 1), and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3 and Bnip3L) have been shown to regulate mitophagy, and inactivation of the genes encoding these proteins leads to mitochondrial abnormalities [112, 113]. PINK1 is normally absent in healthy mitochondria because it is constitutively degraded by mitochondrial proteases. However, once mitochondria are damaged, PINK1 is no longer degraded and accumulates. PINK1 induces parkin recruitment to mitochondria, promoting mitophagy through ubiquitination of outer mitochondrial membrane proteins that are recognized by p62, which then brings autophagic vesicles to ubiquitinated mitochondrial proteins [114, 115]. Bnip3 and Bnip3L are BH3-only proteins that are localized at the outer membrane of the mitochondria after cellular stress, and reportedly bind directly to LC3 (MAP1LC3A microtubule-associated protein 1 light chain 3 alpha), thereby recruiting the autophagosome to damaged mitochondria [116, 117]. In atrophying muscle, the mitochondrial network is dramatically remodeled following fasting or denervation, and autophagy via Bnip3 contributes to mitochondrial remodeling [101, 118-120]. Expression of the fission machinery is sufficient to cause muscle wasting in mice, whereas inhibition of mitochondrial fission prevents muscle loss during denervation, indicating that disruption of the mitochondrial network is a crucial amplificatory loop of the muscle atrophy program [101, 118]. Conversely, impairment of basal mitophagy is deleterious to muscle homeostasis, and leads to the accumulation of damaged and dysfunctional mitochondria . Accordingly, the phenotype of mice with muscle-specific inactivation of various genes coding for autophagy-related proteins, such as Atg7, Atg5 or nutrient-deprivation autophagy factor-1 (NAF-1), a Bcl-2-associated autophagy regulator, results in atrophy, weakness and various myopathic features [122-124]. In addition, altered regulation of autophagy-related genes leads to muscle dysfunction. Histone deacetylases 1 and 2 (HDACs) were found to regulate muscle autophagy by controlling the expression of autophagy genes. Muscle-specific ablation of both HDAC1 and HDAC2 results in partial perinatal lethality, and the HDAC1/2 knockout mice that do survive develop a progressive myopathy characterized by impaired autophagy [125, 126].
Several studies have shown that the IGF1 and/or insulin signaling suppress protein breakdown while promoting muscle growth [127-129]. Additional data supporting the role of the IGF1 pathway in regulating muscle atrophy have been obtained from studies of Akt. Electroporation of constitutively active Akt in adult myofibers completely blocks muscle atrophy induced by denervation . Akt transgenic mice display muscle hypertrophy and protection from denervation-induced atrophy [19, 20, 130], showing that the Akt pathway promotes muscle growth and simultaneously blocks protein degradation [20, 33]. In particular, Akt regulates both the ubiquitin–proteasome system and the autophagy–lysosome pathway, and this action is mediated by FoxO transcription factors. The FoxO family members that are important for skeletal muscle include three isoforms: FoxO1, FoxO3 and FoxO4. Akt phosphorylates all FoxOs, promoting their export from the nucleus to the cytoplasm. As predicted, the reduced activity of the Akt pathway observed in various models of muscle atrophy leads to decreased levels of phosphorylated FoxO in the cytoplasm and a marked increase in nuclear FoxO  (Fig. 3). The translocation and transcriptional activity of FoxO members is sufficient to promote atrogin-1 and MuRF1 expression, and muscle atrophy. Studies utilizing FoxO3 over-expression in adult muscle or muscle-specific FoxO1 transgenic mice showed markedly reduced muscle mass and fiber atrophy [77, 132, 133]. In contrast, FoxO knockdown by RNAi blocks the up-regulation of atrogin-1 expression during atrophy and prevents muscle loss [77, 134].
Figure 3. Protein degradation regulates protein synthesis. In the presence of growth factors, the PI3K–Akt/protein kinase B pathway sequesters FoxO1/3/4 transcription factors in the cytoplasm. In the absence of growth factors, Akt is inactive, and therefore, FoxOs are translocated into the nucleus and induce the transcription of target genes that regulate the ubiquitin–proteasome and autophagy–lysosome systems. mTOR senses the amino acids derived from the proteasome, or, when localized on lysosomes, the amino acid flux derived from lysosomal protein breakdown, and is therefore activated.
Download figure to PowerPoint
Cross-talk between protein breakdown and protein synthesis is not limited to Akt, but also involves FoxO. Activation of FoxO in Drosophila muscle up-regulates 4E-BP1  and represses mTOR via sestrin . Consistently, in mammals, FoxO3 reduces total protein synthesis in adult muscle . Thus, when Akt is active, protein breakdown is suppressed, and when FoxO is induced, protein synthesis is further suppressed. This is not trivial, as FoxO activity is regulated by several post-translational modifications, including phosphorylation, acetylation and mono- and polyubiquitination . Adding an additional level of complexity, the regulatory consequences of these changes appear to be specific for individual FoxO members. For example, recent evidence suggests that acetylation negatively regulates FoxO3 activity, but has no effect on FoxO1 . Mutants of FoxO3 that mimic the effect of acetylation have cytosolic localization and a reduced capacity to induce transcription of the gene encoding atrogin-1, and cause muscle atrophy . Most of these regulatory mechanisms are Akt-independent, and may play a role in muscle atrophy induced by oxidative or energy stress.
Other studies have revealed a connection between AMPK and FoxO3. AMPK phosphorylates several Akt-independent sites on FoxO3, thereby stimulating its transcriptional activity [141, 142]. Indeed, treatment of muscle cultures with 5-aminoimidazole-4-carboxamide riboside (AICAR), an activator of AMPK, increases protein breakdown and atrogin-1 expression via the FoxO family . It has recently been shown that FoxO3 is activated via AMPK in myofibers to induce expression of atrogin-1 and MuRF1 under conditions of energy stress [101, 144]. Activation of AMPK also leads to induction of some autophagy-related genes encoding proteins such as LC3 and Bnip3.
Increased oxidative stress occurs during denervation and hindlimb suspension. During these disuse conditions, nNOS moves from the sarcolemma, where it is bound to the dystrophin–glycoprotein complex, to the cytosol. Free cytosolic nNOS induces oxidative stress and enhances FoxO3-mediated transcription of atrogin-1 and MuRF1, thereby causing muscle loss . Interestingly, the NF-κB pathway is not involved in nNOS-mediated muscle atrophy . Similarly, when dihydropyridine receptor (DHPR) is reduced in adult muscle by RNAi, muscle atrophy is triggered via nNOS relocalization and FoxO3 activation . However, in this latter setting, the genes up-regulated by FoxO3 are those encoding the autophagy regulators LC3, vacuolar protein sorting 34 (VPS34) and Bnip3 as well as the lysosomal enzyme cathepsin L. In humans, the diaphragm of patients that are mechanically ventilated undergoes rapid atrophy caused by activation of proteolytic systems, including autophagy, through Akt inhibition and FoxO1 induction . Interestingly, oxidative stress is increased and therefore contributes to FoxO activation in this example of disuse-mediated atrophy.
FoxO activity is also modulated by direct or indirect actions of co-factors and by interaction with other transcription factors. FoxOs have been found to interact with PGC-1α, a critical co-factor for mitochondrial biogenesis [148, 149]. Maintaining high levels of PGC-1α under catabolic conditions (either in transgenic mice or by transfecting adult myofibers) spares muscle mass during denervation, fasting, heart failure, aging and sarcopenia – similar to the effect observed for expression of constitutively active FoxO3 [5, 150, 151]. Similar beneficial effects were recently obtained by over-expression of PGC-1β, a homolog of PGC-1α . The positive action on muscle mass of these co-factors is due to inhibition of autophagy–lysosome and ubiquitin–proteasome degradation. PGC-1α and PGC-1β reduce protein breakdown by inhibiting the transcriptional activity of FoxO3 and NF-κB, but do not affect protein synthesis. Thus, these co-factors prevent the excessive activation of proteolytic systems by inhibiting the action of the pro-atrophy transcription factors without perturbing the translational machinery.
We recently reported that the transcription factor JunB blocks atrophy and promotes hypertrophy in adult muscles . Indeed, JunB blocks myofiber atrophy of denervated tibialis anterior muscles and cultured myotubes induced by FoxO3 over-expression, dexamethasone treatment or starvation. Under these conditions, JunB prevents activation of atrogin-1 and partially prevents activation of MuRF1, thereby reducing the increase in overall protein degradation induced by activated FoxO3. Further analysis revealed that JunB does not inhibit FoxO3-mediated activation of the autophagy–lysosome system, but only ubiquitin–proteasome degradation, by inhibiting atrogin-1 and MuRF1 induction under catabolic conditions. In fact, JunB directly binds FoxO3, thereby preventing its recruitment to the promoters of key atrogenes. Moreover, JunB over-expression is sufficient to induce dramatic hypertrophy of myotubes and adult muscle. These hypertrophic changes depend on increased protein synthesis, without affecting the basal rate of protein degradation. The growth-promoting effects mediated by JunB in muscle resemble the effects of inhibiting the TGFβ pathway [33, 34]. Indeed, JunB over-expression markedly suppresses myostatin expression in transfected myotubes and decreases the phosphorylation of Smad3, the transcription factor downstream of the myostatin–TGFβ signaling pathway .
Inflammatory cytokines and NF-κB signaling
NF-κB transcription factors are expressed in skeletal muscle and are activated by inflammatory cytokines, particularly tumor necrosis factor α (TNFα). Indeed, inflammation is a potent trigger of muscle wasting and cachexia . NF-κB is maintained in the inactive state by binding of a family of inhibitory proteins called IκB. The increase in the TNFα level induces activation of an IκB kinase (IKKβ) complex that phosphorylates IκB, resulting in its ubiquitination and proteasomal degradation. This leads to nuclear translocation of NF-κB and activation of NF-κB-mediated gene transcription .
Transgenic mice that over-express IKKβ specifically in muscle show severe muscle wasting that is mediated, at least in part, by the ubiquitin ligase MuRF1, but not by atrogin-1 . In contrast, muscle-specific inhibition of NF-κB by transgenic expression of a constitutively active IκB mutant does not induce an overt phenotype, but denervation atrophy is substantially reduced . Mice deficient for the p105/p50 subunit of NF-κB are partially resistant to muscle atrophy induced by hindlimb unloading . However, one of the effects of TNFα and pro-inflammatory cytokines is to induce insulin resistance and suppression of the IGF1–Akt pathway [157-159]. Therefore, Akt phosphorylation should always be monitored when NF-κB signaling is altered, as Akt inhibition may substantially contribute to muscle wasting. Indeed IKKβ conditional knockout mice are resistant to muscle atrophy but show activation of Akt . The significance of decreased muscle atrophy following IKKβ ablation and the degree to which this effect is Akt-dependent remains unclear. Nevertheless, these findings highlight the relevance of the cross-talk between the two pathways, and future studies are required to elucidate the respective contributions of the IKKβ–NF-κB and Akt–FoxO pathways to muscle atrophy.
A recent study revealed an unexpected connection between TNFα signaling and myogenin on MuRF1 and atrogin-1 expression: TNFα treatment causes up-regulation of myogenin, MuRF1 and atrogin-1. Interestingly, a G protein-coupled receptor blocks TNFα-mediated myogenin up-regulation by activating Gαi2  and expression of muscle-specific ubiquitin ligases. However, the precise mechanisms of TNFα-mediated myogenin regulation, the interplay with Gαi2 and the implications for muscle wasting are still far from fully understood.
TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that was recently found to induce muscle atrophy [158, 162]. TWEAK acts on responsive cells by binding to fibroblast growth factor-inducible 14 (Fn14), a small cell-surface receptor. Fn14 is up-regulated in denervated muscle, allowing NF-κB activation and consequently MuRF1 (but not atrogin-1) expression . TWEAK knockout mice display reduced atrophy after denervation, as well as reduced NF-κB activation and MuRF1 expression. However, Fn14 does not increase under all conditions of muscle atrophy; for instance, it is not induced by dexamethasone treatment. Another important player in NF-κB signaling is the ubiquitin ligase TRAF6, which is required for Fn14 up-regulation during fasting . As noted earlier, TRAF6 is also required for activation of FoxO3 and AMPK in starved muscles and for induction of the ubiquitin–proteasome and autophagy–lysosome systems .
The pro-inflammatory cytokines TNFα, IL-6 and IL-1 also activate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Interestingly, sepsis and cancer induce STAT3 phosphorylation in muscles, and STAT3 inhibition spares muscle mass in tumor-bearing mice . Moreover, over-expression of Stat3 is sufficient to induce muscle atrophy and to up-regulate atrogin-1. However, another recent study identified an unexpected role of Stat3 in autophagy regulation. Stat3 has been reported to block VPS34 expression, resulting in alteration of assembly of the Vps34–Beclin1–Vps15–Atg14 complex, and therefore autophagy inhibition and muscle degeneration . This Stat3-dependent regulation of autophagy occurs downstream of Fyn tyrosine kinase.
Other signaling pathways
Myostatin inhibition and its role in muscle growth has been described above; however, the mechanism of myostatin activation and its role and capacity to trigger muscle atrophy remain unclear. Myostatin activation has been reported to induce massive , mild or no atrophy at all [166, 167]. However, in muscle cell cultures, myostatin was reported to up-regulate essential atrophy-related ubiquitin ligases. This regulation was found to be FoxO-dependent and NF-κB-independent . Importantly, myostatin expression is controlled by FoxO1, supporting the concept that the myostatin pathway synergizes with Akt–FoxO signaling . A recent study showed that inhibition of myostatin by soluble ACVR2B prevents and fully reverses skeletal muscle loss and atrophy of the heart in tumor-bearing animals . Such treatment dramatically prolongs the survival of these animals, suggesting potential therapeutic efficacy in patients with cancer cachexia. Reports attempting to dissect the downstream signaling have shown that Smad2 and Smad3 are the principle transcription factors that mediate myostatin's effects on muscle mass [33, 34, 36, 100]. However, as mentioned above, specific transcriptional targets of Smad2 and Smad3 are still unknown, and mechanisms of Smad-dependent atrophy remain to be established.
Glucocorticoid levels are increased in many pathological conditions associated with muscle loss. Glucocorticoid treatment induces atrogin-1 and MuRF1 expression and muscle wasting in cell culture and in vivo [16, 77, 88, 127, 170]. In contrast, adrenalectomy or treatment with a glucocorticoid receptor antagonist attenuate muscle loss in some diseases [170, 171]. The mechanisms of glucocorticoid-mediated muscle atrophy were recently unraveled. Once in the nucleus, the glucocorticoid receptor activates expression of two target genes, encoding REDD1 (regulated in development and DNA damage responses 1) and KLF15 (Krüppel-like factor 15) . REDD1 inhibits mTOR activity by sequestering 14-3-3 and increasing TSC1/2 activity. Inhibition of mTOR is permissive for activation of an atrophy program via KLF15. Indeed, mTOR activation attenuates glucocorticoid-induced muscle atrophy. KLF15 is a transcription factor that is involved in several metabolic processes in skeletal muscle, for instance up-regulation of branched-chain aminotransferase 2. KLF15 participates in muscle catabolism via transcriptional regulation of FoxO1, atrogin-1 and MuRF1. Moreover, KLF15 negatively affects mTOR through up-regulation of branched-chain aminotransferase 2, which in turn induces branched-chain amino acid degradation. Interestingly, FoxO1 and glucocorticoid receptor cooperate to up-regulate MuRF1 expression .