- Email: [email protected]
Cytokine Signaling in Skeletal Muscle Wasting
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Review
Cytokine Signaling in Skeletal Muscle Wasting Jin Zhou,1,6 Bin Liu,2,6 Chun Liang,3,6 Yangxin Li,4,* and Yao-Hua Song5,* Skeletal muscle wasting occurs in a variety of diseases including diabetes, cancer, Crohn's disease, chronic obstructive pulmonary disease (COPD), disuse, and denervation. Tumor necrosis factor a (TNF-a) is involved in mediating the wasting effect. To date, a causal relationship between TNF-a signaling and muscle wasting has been established in animal models. However, results from clinical trials are conflicting. This is partly due to the fact that other factors such as TNF-like weak inducer of apoptosis (TWEAK) and interleukin 6 (IL-6) are also involved in skeletal muscle wasting. Because muscle wasting is often associated with physical inactivity and reduced food intake, therapeutic interventions will be most effective when multiple approaches are used in conjunction with nutritional support and exercise. Inflammatory Cytokines and Cachexia Cachexia (see Glossary) is characterized by weakness, weight loss, and muscle atrophy, due to severe chronic illness, and is often fatal. Cachexia is seen in patients with a variety of serious illnesses, including cancer, chronic obstructive pulmonary disease (COPD), acquired immune deficiency syndrome (AIDs), multiple sclerosis, congestive heart failure, etc. Chronic inflammation is often seen in cachexia of various causes. TNF-/ is an inflammatory cytokine implicated in muscle wasting conditions associated with various diseases [1–4] and has been called cachexin, or cachectin, which refers to substances that cause severe body weight loss. TNF-/ is produced by different cell types including macrophages, lymphocytes, and skeletal muscle cells, and is involved in both local and systemic inflammation [5]. The cytokine exerts its effect through two receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 is believed to mediate the muscle wasting effect, whereas TNFR2 is protective [6–9] (see Box 1 for details). TWEAK is small pleiotropic cytokine and member of the TNF-/ superfamily with multiple biological functions, including stimulation of apoptosis, and induction of inflammatory cytokines. TWEAK is also involved in muscle injury and atrophy and has been shown to play a role in muscle wasting [10]. The cytokine signals through the fibroblast growth factor-inducible 14 (Fn14) receptor and activates nuclear factor (NF)-kB (see Box 2 for details). Finally, interleukins IL-6 and IL-1b have also been implicated in muscle wasting [2,11]. In this review, we will discuss the role of these cytokines in the development of cachexia, and highlight current and emerging treatment options to prevent muscle atrophy.
TNF-a in Conditions Promoting Muscle Wasting Inflammatory cytokines such as TNF-/, IL-1b, and IL-6 are involved in mediating the wasting effect in cachectic patients. With regard to TNF-/, systemic TNF-/ infusion in healthy volunteers
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Trends Muscle wasting is the result of an imbalance between anabolic and catabolic metabolism due to inflammation, physical inactivity, and inadequate nutrition. TNF-/, IL-6, and TWEAK shift the metabolism balance toward a catabolic process. However, current therapies such as neutralizing antibodies/decoy receptors against TNF-/, IL-6, and nonsteroidal anti-inflammatory drugs had limited success when used alone. These specific therapies will be more successful when combined with nutritional support, appetite stimulators, and exercise, because combinatorial approaches will not only inhibit protein degradation but also promote protein synthesis. Clinical trials are warranted and will yield more conclusive results by measuring cytokine levels from each patient prior to treatment.
1 Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, P.R. China 2 Cardiovascular Disease Center, The First Hospital of Ji Lin University, Changchun, Jilin, 130021, P.R. China 3 Department of Cardiology, ChangZheng Hospital, Second Military Medical University, Shanghai, 200003, P.R. China 4 [3_TD$IF]Department of Cardiovascular [4_TD$IF]Surgery and [5_TD$IF]Institute of Cardiovascular [6_TD$IF]Science, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215123, P.R. China 5 Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Collaborative Innovation Center of Hematology, Soochow University, Suzhou,[7_TD$IF] P.R. China [2_TD$IF]6 These authors contributed equally to this work
http://dx.doi.org/10.1016/j.tem.2016.03.002 © 2016 Elsevier Ltd. All rights reserved.
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Box 1. TNF-/ Receptor Signaling TNFR1 belongs to the death receptor family of proteins that contain death domains, which can induce apoptosis via the caspase pathway. Upon TNF-/ binding to TNFR1, TNFRSF1A-associated via death domain (TRADD) is recruited to TNFR1. TRADD then recruits Fas-associated protein with death domain (FADD) that triggers the caspase cascade to induce apoptosis. TNFR2 does not contain a death domain, and therefore is not able to induce apoptosis through caspase-dependent mechanisms. Instead, TNFR2 promotes survival via phosphorylation of Etk and subsequent activation of Akt (Figure I) [7,8]. TNFR1 and TNFR2 recruit separate downstream adaptor molecules to induce a cellular response, but converge on TNF receptor-associated factor 2 (TRAF2), to activate IKK and NF-kB [77]. TNFR1 recruits TRADD and RIP1 prior to engaging with TRAF2, whereas TNFR2 can interact with TRAF2 directly [7]. TNFR1-triggered NF-kB activation leads to increased expression of muscle RING-finger protein 1 (MuRF1) and subsequent protein degradation through the ubiquitin proteasome pathway. NF-kB activation also inhibits satellite cell activation and differentiation, by promoting DNA methylation of the Notch-1 promoter [78], and by inhibiting MyoD expression [79], respectively. TNFR2 can activate the alternative NF-kB pathway through NIK [77] and its impact on myogenesis remains to be identified. In addition to TRAF2, six other TRAFs have been identified so far to transmit signals via the TNFRs. From those, TRAF6 is the only one involved in protein degradation. TRAF6 is an E3 ubiquitin ligase and a critical autophagy regulator [80]. TRAF6 mRNA levels are significantly upregulated in skeletal muscles of mice subjected to denervation, cancer cachexia, or diabetes [27]. By contrast, TRAF6 knockout (TRAF6mko, muscle-specific) mice show reduced expression of MuRF1, and atrogin1, and are resistant to denervation and cancer-induced muscle wasting [27]. In a denervation model, activation of NF-kB in skeletal muscle is inhibited in TRAF6mko mice, compared with TRAF6f/f mice. Because NF-kB activation induces the expression of MuRF1 [27], TRAF6-induced muscle atrophy is likely mediated by the NF-kB/ubiquitin/proteasome pathway. However, the upstream signaling molecules that regulate TRAF6 remain unknown. Recent studies suggest that the CD40–TRAF6 interaction leads to obesity-associated insulin resistance [81], which is a condition that induces muscle wasting.
TNFR1
TNFR2
TRADD FADD
RIP-1 NIK
EtK
TRAF2 Caspase-8 p-Etk IKK Apoptosis NF-κB
MuRF1
Protein degradaon
Akt MyoD
Satellite cell differenaon
Survival and protein synthesis
Figure I. TNF-/ Signaling Pathway. TNF-/ can bind to TNFR1 and TNFR2. TNFR1 activation can induce apoptosis via the TRADD/FADD/Caspase 8 pathway, or activate NF-kB via RIP1/TRAF2/IKK. NF-kB activation results in inhibition of myogenic differentiation and protein degradation. Stimulation of TNFR2 can also lead to NF-kB activation via TFAF2 or NIK. Alternatively, TNFR2 signaling could promote survival and protein synthesis through the Etk/Akt pathway. Abbreviations: TNF-/, tumor necrosis factor /; TRADD, TNFRSF1A-associated via death domain; FADD, Fas-associated protein with death domain; RIP1, receptor-interacting protein 1; TRAF2, TNF receptor-associated factor 2; IKK, IkB kinase; NIK, NF-kB-inducing kinase; Etk, epithelial/endothelial tyrosine kinase.
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*Correspondence: [email protected] (Y. Li) and [email protected] (Y.-H. Song).
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Box 2. TNF-like Weak Inducer of Apoptosis (TWEAK) Signaling
Glossary
TWEAK is an emerging cytokine and member of the TNF-/ superfamily involved in muscle injury and atrophy. TWEAK binds to the fibroblast growth factor-inducible 14 (Fn14) receptor and activates NF-kB (Figure I). Fn14 is expressed at low levels in many tissues and cell types under physiological conditions but becomes upregulated during injury and in inflamed tissues. During acute ischemic injury, TWEAK is released from macrophages to induce satellite cell proliferation through activation of classical NF-kB and Notch signaling [82]. By contrast, TWEAK can suppress satellite cell selfrenewal through inversely modulating notch and NF-kB signaling pathways, as shown in a cardiotoxin injury model [20]. It is not clear how TWEAK/Fn14 activation lead to NF-kB activation, but according to Kumar et al., Fn14-induced NF-kB activation may be mediated by TRAF6, at least in a denervation model [27]. Enwere et al. [83] showed that TWEAK can either promote or inhibit myogenic differentiation depending on its concentration. At low concentrations (10–100 ng/ml), TWEAK activates the alternative pathway (NIK/IKK/) and promotes myoblast fusion [83]. At high concentrations (500 ng/ml), TWEAK activates the classical pathway (IKKb) to inhibit differentiation [83]. Thus, TWEAK can maintain muscle differentiation at physiological conditions; however, under pathological conditions TWEAK becomes overexpressed and it induces muscle atrophy [10]. A recent study suggests that TWEAK/Fn14 signaling could also activate nicotinamide adenine dinucleotide phosphate (NADPH) and release reactive oxygen species (ROS) in a Nox2-dependent manner [84]. Increased ROS production resulting from chronic inflammation has been implicated in muscle wasting [85]. Heart failure-induced skeletal muscle atrophy is associated with increased Nox2 expression, ROS production, and NFkB activation that lead to increased expression of MuRF1 and activation of the proteasome [86]. Therefore, both TNF-/ and TWEAK can induce muscle atrophy by the induction of E3 ubiquitin ligase MuRF1 via different signaling pathways.
Activin-A: a growth factor that stimulates synthesis and secretion of follicle stimulating hormone. Activin-A was recently found to be associated with muscle loss in cancer models. Amyotrophic lateral sclerosis (ALS): or Lou Gehrig's disease, a progressive neurodegenerative disease that affects neurons in the brain and spinal cord. Cachexia: or wasting syndrome, is a multifactorial syndrome characterized by weight loss and muscle atrophy, fatigue, weakness, and significantly reduced food intake due to loss of appetite, in people not actively trying to lose weight. Nutritional intervention to reverse the loss of body mass is unsuccessful even with an increase in calorie intake, indicating a primary pathology. Chronic obstructive pulmonary disease (COPD): is a chronic obstructive lung disease characterized by poor airflow and impaired exercise capacity due to skeletal muscle wasting. Denervation: is a loss of nerve supply due to injury or motor neuron diseases and peripheral neuropathies. Duchenne muscular dystrophy (DMD): a genetic disease caused by mutation of the dystrophin gene, which encodes the dystrophin protein that is critical in maintaining muscle cell integrity. Patients with dystrophin mutation develop muscle wasting. Muscle atrophy: a decrease of muscle mass. Myostatin: a member of the TGF-b family. It is a negative regulator of skeletal muscle growth. Myotonic dystrophy type 1 (DM1): is a multisystem disorder characterized by muscle wasting, cataracts, and heart conduction defects. The disease is caused by an abnormal expansion of CTG repeat in the dystrophia myotonica protein kinase (PMDK) gene. Sarcopenia: is a disease associated with loss of muscle mass and strength due to aging. Wasting: muscle loss and weakness.
TWEAK
Fn14
Alternave pathway
Classical pathway
NIK/IKKα
IKKβ
RelB/p52
p65/p50
NADPH
TRAF6
ROS
NF-κB
MuRF1
MyoD
Myogenesis ↑
Myogenesis ↑
Proteasome dependent degradaon Muscle Muscle atrophy
Figure I. The Role of TWEAK/Fn14 Signaling in Myogenesis and Muscle Atrophy. Depending on its concentration, TWEAK could either promote or inhibit myogenesis. At low concentrations, TWEAK activates the alternative pathway (NIK/IKK/) and promotes myoblast fusion. At high concentrations, TWEAK activates the classical pathway (IKKb) to inhibit differentiation. TWEAK/Fn14 signaling could also lead to protein degradation by activating NF-kB via TRAF6 or NADPH. For abbreviations, see text in Box 2 above.
was shown to increase protein breakdown and inhibit protein synthesis [12], and elevated TNF/ levels correlate with muscle loss in patients with cancer [1,2], COPD [3], and disuse [4]. The TNF chimeric monoclonal antibody infliximab, an agent that blocks TNF-/ action, can increase muscle strength and volume in patients with Crohn's disease [13], and long-term treatment with etanercept, another TNF-/ inhibitor, results in body weight gain in rheumatoid arthritis (RA) patients [14]. By contrast, a Phase II study that combined the chemotherapy drug gemcitabine and infliximab did not show a benefit in preserving lean body mass or survival in cachectic
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patients with pancreatic cancer [15]. A Phase I/II study compared etanercept with gemcitabine versus gemcitabine alone for treatment of patients with advanced pancreatic cancer and the results were also disappointing because the addition of etanercept did not alleviate cancerrelated cachexia [16]. One possible explanation for this discrepancy is that the patients that participated in these clinical trials might express varied cytokine levels [16,17]. Recently, TWEAK has been identified as a key mediator of wasting in denervation [18] and cancer [19]. TWEAK induces muscle RING-finger protein 1 (MuRF1) expression via NF-kB activation [10] (Box 2). TWEAK also reduces satellite cell pool by inhibiting Notch signaling and activating NF-kB [20]. In addition to the NF-kB pathway, TNF-/ and TWEAK also signal to p38 MAP kinase [21,22] and the Jak/Stat pathway, which in turn upregulates the expression of atrogin-1 and MuRF1 [23]. Therefore, interventions targeting these pathways should be considered when designing novel therapies for cachexia.
Cancer Cachexia Cancer cachexia, induced by both tumor and chemotherapy, is characterized by anorexia and weight loss [24]. While its pathogenesis remains largely unknown, recent data suggest that TWEAK and IL-6 might be involved in cancer-related wasting. TWEAK Under pathological conditions, TWEAK can become overexpressed and induce a cachectic phenotype in part by the induction of the E3 ligase MuRF1 [10]. Using the Lewis lung carcinoma (LLC) and colon-26 (C-26) tumor model, Johnston et al. [25] demonstrated that these two types of tumors induce cachexia in TWEAK or Fn14-deficient mice (germline deletions), suggesting that tumor-derived rather than host-derived TWEAK or Fn14 might play a role in these cancer cachexia models. Antibodies against tumor-derived Fn14, but not TWEAK, prevented tumorinduced inflammation and loss of fat and muscle mass [25], suggesting that an as-yet unidentified Fn14 ligand might exist that triggers its activation. Alternatively, Fn14 might be able to trigger downstream events independent of TWEAK. A shorter form of Fn14 (Fn14-DEC) was recently identified in human cancer cell lines [26] that lacks the extracellular domain and thus cannot bind TWEAK. Fn14-DEC monomers can self-assemble within cells into dimers and activate NF-kB. While it is unclear what Fn14 downstream molecules are activated in tumors that then act on muscle cells to induce atrophy, activin-A or myostatin have been suggested as potential candidates [25]. Some of the TWEAK/Fn14 signals might be transmitted through TNF receptor-associated factor 6 (TRAF6) [27], subsequently activating NF-kB. Indeed, it has been shown that expression of TRAF6 is increased in the LLC model and deletion of TRAF6 prevents muscle wasting in response to tumor growth [27]. The role of TRAF6 in animal models of cancer cachexia was recently confirmed in a clinical study. Both TRAF6 and ubiquitin were found to be significantly upregulated in muscle of gastric cancer patients compared with muscles from patients with benign diseases [28]. Thus, TRAF6 has become an important therapeutic target for treating cancer cachexia. IL-6 IL-6 is an important inflammatory marker that has been associated with increased mortality in cancer patients [2]. Clinical studies showed that plasma IL-6 levels increased 11-fold in the cancer cachectic group compared with weight-stable controls [29]. A study of 98 patients with advanced cancer showed that IL-6 but not TNF-/ predicted survival rates [30]. The role of IL-6 in cancer cachexia was confirmed in the APCMin+[1_TD$IF] mouse, which is an established model of colon cancer that develops cachexia. The progression of muscle atrophy in these mice was related to circulating IL-6 levels, and overexpression of IL-6 APCMin+ mice accelerated the development of
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cachexia [31]. In vitro studies showed that recombinant IL-6 could inhibit C2C12 myogenic differentiation when added to the differentiation medium [32].
Chemotherapy-Induced Cachexia Cancer-associated cachexia is not only caused by tumor but also by chemotherapy. The chemotherapy drug cisplatin induces muscle atrophy by activating myostatin, IL-6, TNF-/, and IL-1b, which can be prevented by the orexigenic peptide ghrelin in the LLC-induced cachexia model [33]. Cyclophosphamide-, doxorubicin-, and 5-fluorouracil-induced cachexia is associated with increased circulating IL-6, followed by decreased insulin-like growth factor 1 (IGF-1) levels, and can be prevented by germline deletion of IL-6 [34]. Therefore, different chemotherapy agents induce cachexia via distinct mechanisms and further research is needed to understand their transduction pathways.
COPD COPD is most often caused by cigarette smoking, and mice exposed to cigarette smoke daily for 8 weeks exhibited a 157% increase in serum TNF-/, accompanied by significant decrease of peroxisome proliferator-activated receptor g co-activator 1/ (PGC-1/) mRNA levels in soleus and extensor digitorum longus muscles [35]. Analysis of markers and regulators of skeletal muscle oxidative phenotype (oxphen) such as citrate synthase and PGC-1/ in vastus lateralis muscle biopsies of patients with advanced COPD and healthy smoking control participants showed elevated muscle TNF-/ mRNA, and reduced mRNA levels of oxidative markers/regulators in 23% of the COPD patients [3]. The authors were not able to detect TNF-/ protein in muscle biopsies. So the question is whether circulating or lung-derived TNF-/ would have an impact on muscle function. In a transgenic mouse model overexpressing TNF-/ in the lung, TNF-/ impaired peripheral skeletal muscle function due to excessive formation of superoxide in contracting skeletal muscle [36,37]. However, a study of 426 COPD patients over a 3-year period found that TNF-/ levels were associated with muscle loss only in patients who were cachectic at entry into the study; the authors concluded that inflammation is a consequence rather than a cause of initial loss of fat-free mass [38]. At this point, the role of TNF-/ in COPD-induced muscle wasting remains unclear, and a better way to clarify this issue would be to study the effect of cigarette smoke exposure in TNF-/ knockout mice.
Denervation and Neuron Muscular Diseases Motor neurons normally provide trophic factors that promote protein synthesis in muscle. Loss of nerve supply due to denervation results in molecular and cellular changes in skeletal muscle and atrophy [39]. Recent studies suggest that TWEAK and TRAF6 may be involved in this process. Transgenic expression of TWEAK specifically in skeletal muscle induces atrophy via activation of NF-kB and upregulation of MuRF1 [10]. Interestingly, TWEAK overexpression was associated with a transition from slow (type I) to fast (type II) fiber type, and TWEAK-induced muscle atrophy is predominantly restricted to fast-type fibers [10]. TWEAK/Fn14 also plays an important role in myotonic dystrophy type 1 (DM1). Expression of Fn14 correlated with severity of muscle pathology in skeletal muscle of mouse models of DM1 and in tissues from DM1 patients, whereas Fn14 germline deletion improved muscle pathology and function [40]. Blocking TWEAK, a ligand for Fn14, with an anti-TWEAK antibody improved muscle histopathology and functional outcomes in affected mice [40]. Therefore, unlike cancerinduced cachexia [25], TWEAK appears to signal through Fn14 to induce muscle atrophy in myotonic dystrophy.
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In the SOD1G93A mouse, a model of amyotrophic lateral sclerosis (ALS), TWEAK is upregulated in spinal cord astrocytes, which was believed to induce neuron death by releasing IL-6 [41]. Genetic ablation of TWEAK or treatment with anti-TWEAK antibody significantly reduced astrocyte activation and ameliorated muscle wasting in these mice. However, these approaches failed to improve motor function and motor neuron survival [41]. These results suggest that astrocyte activation and TWEAK upregulation may not be the primary cause of motor neuron death. Alternatively, as the authors suggested, TWEAK is a downstream effector of interferon g (IFNg) and reducing astrocyte activation by depleting TWEAK may not impact the deleterious effect of TWEAK-independent IFNg signaling on astrogliosis and motor neuron death. Thus, the role of TWEAK in the development of ALS remains unknown. TRAF6 is another factor involved in denervation-induced muscle atrophy and skeletal musclespecific depletion of TRAF6 prevented myofibril degradation upon denervation [27]. TRAF6 is activated by TWEAK/Fn14 and can subsequently activate NF-kB-dependent protein degradation through the ubiquitin proteasome pathway [27]. TRAF6 also regulates the autophagy system because the expression of the autophagy proteins Beclin1 and LC3B was upregulated in denervated muscle but reduced in TRAF6 knockout mice [27]. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), TRAF6 activity was increased in skeletal muscle, and muscle-specific deletion of TRAF6 reduced fiber necrosis and improved muscle function. Ablation of TRAF6 (muscle-specific deletion) also enhanced myofiber regeneration resulting from satellite cell proliferation in young mdx mice [42]. However, ablation of TRAF6 exacerbated muscle injury and increased fibrosis in 9-month-old mdx mice. The authors suggested that TRAF6 is an important autophagy regulator that removes damaged organelles. Old mice are more reliant on autophagy due to the progressive nature of the disease. Although animal studies have clearly demonstrated a role of TWEAK and TRAF6 in denervation and neuron muscular disease-induced muscle wasting, therapeutic interventions based on inhibition of either TWEAK or TRAF6 signaling should proceed with caution because both molecules play a physiological role in maintaining muscle mass and function.
Sarcopenia While the mechanisms underlying sarcopenia remain unknown, reduced physical activity may play a role [4]. In older adults, just 14 days of reduced steps induced reductions in myofibril protein synthesis and muscle mass, accompanied by impairments in insulin sensitivity and elevated TNF-/ levels (while IL-6 levels were not elevated) [4], suggesting that TNF-/ inhibitors may be beneficial for the elderly. Animal studies demonstrated that TWEAK/Fn14 is also involved in sarcopenia. Fn14 transcription was significantly increased in both tibial anterior (TA) and gastrocnemius (GA) muscles of 18-month-old mice, compared with 3-month-old mice [43]. Genetic ablation of Fn14 attenuated age-associated skeletal muscle atrophy in mice and reduced the expression of ubiquitinated proteins. However, the expression of atrogin-1 and MuRF1 was not altered by Fn14 ablation, suggesting that perhaps other as-yet unidentified ubiquitin ligases may be responsible for muscle protein ubiquitination and degradation. Alternatively, atrogin-1 and MuRF1 expression follows a clear time course and their quantitation could have been too late to detect meaningful differences in the Fn14 mutant mice. TWEAK affects skeletal muscle mitochondrial content, and age-related loss in skeletal muscle oxidative capacity was rescued in TWEAK global knockout mice [44]. The expression of several molecules involved in oxidative metabolism such as PGC1/ and succinate dehydrogenase (SDH) was significantly higher in skeletal muscle of TWEAK knockout mice. Thus, both TNF-/ and TWEAK/Fn14 are implicated in the pathogenesis of sarcopenia, although their signal transduction pathways may be different.
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Cardiac Cachexia Cardiac cachexia is a serious complication of congestive heart failure [45]. TNF-/ and angiotensin II (ang II) have been implicated in cardiac cachexia, but the association of TNF-/ signaling with ang II has not been clarified in atrophic conditions. Two recent studies provided new insight on how ang II might induce muscle loss. One study [46] showed that transcription factor EB (TFEB) is a mediator of ang II-induced muscle wasting through upregulation of MuRF1, and ang II-induced MuRF1 expression and atrophy could be prevented by targeting TFEB with siRNA. MuRF1 expression is also regulated by histone deacetylase 5 (HDAC5) that binds to TFEB and inhibits its interaction with the MuRF1 promoter [46]. HDAC5 is associated with protein [10_TD$IF]kinase [1_TD$IF]D1 (PKD), which associates with, phosphorylates, and mediates 14-3-3-dependent export of HDAC5 from the nucleus. Muscle-specific knockout of PKD1 prevented ang II-induced wasting. A second study [47] showed that mitochondrial oxidative enzymes were significantly decreased, while superoxide production was increased, in the skeletal muscle of ang II-treated mice. Because ang II plays a central role in cardiac cachexia, ang II receptor blockade should alleviate muscle wasting. Along this line, losartan, an ang II type I receptor blocker, improved muscle activity and reduced inflammation in older mice [48].
Muscle Injury and Regeneration TWEAK is involved in muscle regeneration postinjury. In the cardiotoxin (CTX) injury model, TWEAK negatively regulates muscle regeneration by inhibiting myogenic differentiation via activation of NF-kB [49]. TWEAK-induced activation of NF-kB is counterbalanced by Nrf2, a transcription factor that regulates antioxidative enzymes. Nrf2 global knockout mice showed increased TWEAK expression, enhanced fibrosis, and insufficient regeneration [50]. In contrast to its deteriorating roles in wasting conditions, TNF-/ signaling promotes muscle regeneration. When the proinflammatory capacity of M1 monocytes was assessed in men ex vivo by stimulating whole blood with lipopolysaccharide (LPS), IFNg, granulocyte macrophage colony-stimulating factor (GM-CSF), and TNF-/ cytokine production was positively associated with lean body mass and muscle strength [51]. When the role of TNF-/ signaling in CTX-injured soleus muscle in TNFR1 and TNFR2 global double-knockout mice was analyzed, it was shown that inflammation was resolved and the regeneration process was nearly complete at day 12 postinjury in wild-type mice; however, immune cells remained in the injured muscle with little signs of regeneration in the double-knockout mice [52]. At the molecular level, reduced levels of myogenin and p21 indicated that myogenesis was blocked in the injured knockout mice. Along this line, TNF-/ could promote bone repair by promoting the recruitment and differentiation of muscle-derived stromal cells to injury sites [53]. However, in a mouse model of hindlimb ischemia, the limb reperfusion after ischemia was significantly higher in TNFR1-deficient mice (germline deletion) after injury, accompanied by reduced apoptosis, when compared with wildtype controls, but the lack of TNFR1 signaling impaired angiogenesis at later stages of ischemic injury [54]. In light of the role that TNF-/ plays in promoting muscle regeneration, anti-TNF-/ agents such as etanercept may not be the best choice for treating inflammatory diseases or cachexia in patients who are also suffering from acute injury.
Pharmacotherapy of Muscle Wasting Exercise Exercise improves blood flow and releases growth factors that rejuvenate muscles and other tissues [55], as well as reducing inflammation. When the expression of E3 ligase, IGF-1, and TNF-/ was measured in 60 chronic heart failure (CHF) patients who underwent a 4-week endurance training program [55], it was found that the expression of MuRF1 and TNF-/ in
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skeletal muscle was reduced, while that of IGF-1 was increased, with exercise. Additionally, resistance training-induced increase of IGF-1 was mediated by PGC-1/4, a novel form of PGC1/ [56]. The effect of chronic exercise on TNF-/ and atrogin-1 expression was evaluated in a mouse hindlimb unloading model after the mice received a treadmill running exercise for 6 weeks after unloading [57]. Results showed that chronic exercise attenuated a decrease in muscle weight and inhibited expression of TNF-/, IL-6, and atrogin-1 in the atrophied gastrocnemius muscle. TNF-/ is increased in inflammatory bowel diseases (IBDs), which is often associated with muscle wasting. Mice subjected to 4 months of free wheel running (WR) exhibited reduced expression of TNF-/ and caspase-7 and -8 in intestinal lymphocytes [58]. When the effect of high-fat diet was tested in rats with colitis forced to a treadmill running exercise regimen for 6 weeks, exercise accelerated the healing of colitis, significantly decreased plasma IL-1b, TNF-/, TWEAK, and leptin levels, and increased IL-6 plasma levels [59]. Along these lines, it was previously suggested that the beneficial anti-inflammatory effects of exercise are mediated by muscle-derived IL-6, because recombinant human IL-6 infusion was shown to inhibit endotoxin-induced increases in circulating TNF-/ in healthy humans [60]. Furthermore, IL-6 level was increased during a wheelchair marathon in athletes with cervical spinal cord injuries, accompanied by decreased levels of TNF-/, suggesting an inhibitory role of IL-6 on TNF-/ release [61]. While IL-6 signaling in monocytes generates a proinflammatory response, IL-6 signaling in myocytes creates an anti-inflammatory environment [60]. Nonsteroidal anti-inflammatory drugs (NSAIDs) NSAIDs such as celecoxib and ibuprofen have been used to treat cachexia with some success. A Phase II trial examined the efficacy and safety of celecoxib (COX-2 inhibitor) on cancer cachexia. Patients with advanced cancers were treated with celecoxib daily (200–300 mg/day) for 4 months. The treatment led to a significant increase of lean body mass, grip strength, and a reduction of circulating TNF-/ levels [62]. Side effects such as grade 1/2 anemia and neuropenia were observed in only a few patients, and cardiovascular events were not observed in any of the patients at this moderate dose [62]. The effect of ibuprofen on cancer cachexia was evaluated in the C-26 tumor model; ibuprofen improved muscle mass and reduced the expression of atrogin-1 and MuRF1 in tumor-bearing mice [63]. Thus, results from both clinical and animal studies support the use of NSAIDs to treat cancer cachexia (Box 3).
Box 3. Microbiota and Cachexia Although inflammation and bacterial infections can lead to cachexia, certain bacteria strains could actually prevent muscle atrophy. Schieber et al. [87] made a ground-breaking discovery while working on the dextran sulfate sodium (DSS) intestinal injury model, trying to determine the cause of muscle wasting in mice. While C57Bl/6 mice obtained from Jackson Laboratories (Jax mice) exhibited muscle wasting, C57Bl/6 from the University of California, Berkeley, colony (CB mice) showed significantly less wasting, when treated with DSS, suggesting that differences in the microbiota composition between Jax and CB mice were responsible for the differences observed in muscle wasting. Subsequently, they were able to isolate an Escherichia coli (O21:H+) strain that was absent from Jax mice. Oral administration of E. coli O21:H+ resulted in colonization of the intestine of Jax mice and on DSS treatment, E. coli O21:H+ Jax mice showed significantly less wasting than did DSS-treated vehicle mice. E. coli O21:H+ also prevented muscle wasting induced by other microbes. This protection was associated with challenge-induced translocation of E. coli O21:H+ to white adipose tissue and activation of the NLRC4 inflammasome via IL-18, which prevented muscle atrophy by activating IGF-1 signaling pathway [87]. Although previous studies demonstrated some beneficial effect of NSAIDs in treating muscle wasting, the discovery that IL-18 is involved in muscle maintenance leads to the questioning of the validity of NSAIDs as a therapeutic intervention for cachexia because NSAIDs can inhibit the release of IL-18 [88] and could potentially impair IGF-1 signaling.
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Etanercept Etanercept is a recombinant fusion protein that functions as a decoy receptor to neutralize TNF/. The drug has been used to treat inflammatory disorders including RA, a chronic inflammatory disease associated with muscle loss. RA-induced muscle atrophy is likely the result of increased production of TNF-/ and IL-6 [64], which is associated with insulin resistance [65]. A meta-analysis of eight studies with 260 subjects revealed that TNF-/ inhibitor therapy reduces insulin resistance in RA [65]. In another study [14], significant weight gain in RA patients treated with etanercept, twice weekly for 12 consecutive months, was observed. The most common side effects associated with etanercept when used to treat inflammatory diseases include infusion site reaction, headache, and upper respiratory infections such as tuberculosis [66]. When used for treating cachexia, it was tolerated well without tuberculosis or any side effects that would cause patient withdrawal, although increased serum uric acid levels have been noted [14]. TWEAK Antibody Animal studies demonstrated that blocking antibodies against the TWEAK antibody can improve muscle function in mice affected by myotonic dystrophy [40] and ALS [41]. However, antibodies against tumor-derived Fn14, but not TWEAK, prevented tumor-induced cachexia [25]. The antiFn14 treatment protected both the oxidative type IIa and glycolytic type IIx/b fibers. These results suggest that neutralizing antibodies against both TWEAK and Fn14 should be explored further in various models of muscle atrophy, as well as in clinical trials. Myostatin Inhibitor The myokine myostatin can inhibit myogenesis in an autocrine manner. Upregulation of myostatin has been reported in several muscle wasting conditions [67,68]. Chronic treatment of mice with a human monoclonal antibody (REGN1033) that blocks myostatin increased muscle mass and force production, and prevented the loss of muscle mass in several muscle wasting models including immobilization and dexamethasone treatment [68]. In aged mice, REGN1033 improved muscle function during treadmill exercise [68]. Although untested, REGN1033 might be beneficial for the elderly, because, at least in old rats, exercise alone was not able to reduce TNF-/ level [69]. Activin Receptor Type IIB (ActRIIB) Inhibitor ActRIIB and type I belong to the transforming growth factor b (TGF-b) family. Increased ActRIIB expression has been implicated in cancer cachexia. In the C-26 tumor mouse model, recombinant soluble ActRIIB (sActRIIB) was able to inhibit both myostatin and activin-A-mediated Smad2/3 signal transduction and prevented muscle wasting [70]. Importantly, this treatment stimulated stem cell growth and prolonged animal survival. A recent study using the LLC cachexia model confirmed the beneficial effect of sActRIIB [71]. IL-6 Antibody A strong correlation exists between serum IL-6 level and cachexia in patients with chemotherapy-resistant lung cancer [72]. A humanized anti-IL-6 antibody was shown to reverse fatigue and prevent muscle loss in patients with non-small cell lung cancer [73]. Transplantation of lung cancer cells expressing IL-6 induces cachexia in mice, which can be ameliorated by the IL-6 antibody. In a Phase II study, selumetinib, a mitogen-activated protein kinase kinase (MEK) inhibitor, promoted muscle gain in patients with cholangiocarcinoma [74]. A limitation of this study was the lack of a placebo control group. Besides its role in tumor growth inhibition, selumetinib also inhibited the secretion of IL-6, TNF-/, and IL-1b, which might explain its beneficial effects on muscle gain. However, caution must be taken when using this drug as treatment for cachexia because MEK signaling is critical to myogenic differentiation.
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Etanercept
Exercise
TNF
IL-6
Inflammaon
Skeletal muscle IGF-1
IL-6 anbody
NSAID
ROS Atrogin-1
Weakness and atrophy MuRF1
sActRIIB
Myostan
Food intake
Ghrelin
Figure 1. Current Treatment Options for Cachexia. Chronic inflammation plays a central role in the development of cachexia. Therefore, both nonspecific anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and specific cytokine blockers such as etanercept and selumetinib can be used to inhibit inflammation. However, these treatments would be most useful when combined with ghrelin to stimulate food intake. Exercise and myostatin inhibitors are particularly useful to maintain muscle mass and function in the elderly.
Ghrelin Ghrelin is an orexigenic peptide that is produced by cells in the gastrointestinal tract and acts on the hypothalamus to stimulate food intake [75]. Thus, ghrelin has been proposed as a treatment for muscle wasting (Figure 1). Analysis of Phase II, randomized, placebo-controlled, double-blind trials showed that treatment with anamorelin (a ghrelin receptor agonist) for 12 weeks, increased lean body weight in patients with cancer cachexia [76]. Adverse events associated with this treatment include fatigue, asthenia, atrial fibrillation, and dyspnea.
Concluding Remarks Inflammatory cytokines such as TNF-/ and IL-6 play an important role in the development of cachexia. These cytokines induce muscle atrophy by activating the myostatin and ubiquitin proteasome pathway, or by inhibiting protein synthesis and mitochondrial biogenesis. Therapeutic interventions including etanercept (TNF-/ inhibitor) and neutralizing antibodies that block IL-6 or myostatin have shown promising results in animal studies. However, these approaches had limited success in clinical studies, partly due to the fact that cachexia is a syndrome that is multifactorial in nature and any single therapy is insufficient to stop or prevent muscle loss. Combinational therapies that simultaneously target inflammation and proteolysis while promoting protein synthesis and mitochondrial biogenesis will have a better outcome. Exercise will have additional benefit because it will not only stimulate protein synthesis but also inhibit inflammation. TWEAK/Fn14 and TRAF6 have emerged as potential new targets during the past 5 years. However, their mechanisms of action and transduction pathways remain largely unknown and their roles in cachexia need to be verified in clinical studies before any potential therapeutic intervention can be developed. The ubiquitin proteasome system has been an obvious target for intervention, but activation of this system is not responsible for all types of cachexia; and it also
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has an important physiological role by removing unneeded and misfolded proteins, and therefore inhibition of the proteasome may have unwanted side effects. It is likely that each type of cachexia has a unique signature in terms of the nature of cytokines that pull the trigger to initiate the catabolic process, their cellular origin, time course of expression, and the transduction pathways that they use to mediate their actions on proteolysis. However, our current knowledge of these areas remains limited, and the complex interaction among different cytokines and networks are far from being understood (see Outstanding Questions). Further research is necessary to define the contributing factors and underlying mechanisms leading to cachexia and to translate this knowledge into new therapies. [12_TD$IF]Acknowledgments This work was supported by the project for the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Review
Cytokine Signaling in Skeletal Muscle Wasting Jin Zhou,1,6 Bin Liu,2,6 Chun Liang,3,6 Yangxin Li,4,* and Yao-Hua Song5,* Skeletal muscle wasting occurs in a variety of diseases including diabetes, cancer, Crohn's disease, chronic obstructive pulmonary disease (COPD), disuse, and denervation. Tumor necrosis factor a (TNF-a) is involved in mediating the wasting effect. To date, a causal relationship between TNF-a signaling and muscle wasting has been established in animal models. However, results from clinical trials are conflicting. This is partly due to the fact that other factors such as TNF-like weak inducer of apoptosis (TWEAK) and interleukin 6 (IL-6) are also involved in skeletal muscle wasting. Because muscle wasting is often associated with physical inactivity and reduced food intake, therapeutic interventions will be most effective when multiple approaches are used in conjunction with nutritional support and exercise. Inflammatory Cytokines and Cachexia Cachexia (see Glossary) is characterized by weakness, weight loss, and muscle atrophy, due to severe chronic illness, and is often fatal. Cachexia is seen in patients with a variety of serious illnesses, including cancer, chronic obstructive pulmonary disease (COPD), acquired immune deficiency syndrome (AIDs), multiple sclerosis, congestive heart failure, etc. Chronic inflammation is often seen in cachexia of various causes. TNF-/ is an inflammatory cytokine implicated in muscle wasting conditions associated with various diseases [1–4] and has been called cachexin, or cachectin, which refers to substances that cause severe body weight loss. TNF-/ is produced by different cell types including macrophages, lymphocytes, and skeletal muscle cells, and is involved in both local and systemic inflammation [5]. The cytokine exerts its effect through two receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 is believed to mediate the muscle wasting effect, whereas TNFR2 is protective [6–9] (see Box 1 for details). TWEAK is small pleiotropic cytokine and member of the TNF-/ superfamily with multiple biological functions, including stimulation of apoptosis, and induction of inflammatory cytokines. TWEAK is also involved in muscle injury and atrophy and has been shown to play a role in muscle wasting [10]. The cytokine signals through the fibroblast growth factor-inducible 14 (Fn14) receptor and activates nuclear factor (NF)-kB (see Box 2 for details). Finally, interleukins IL-6 and IL-1b have also been implicated in muscle wasting [2,11]. In this review, we will discuss the role of these cytokines in the development of cachexia, and highlight current and emerging treatment options to prevent muscle atrophy.
TNF-a in Conditions Promoting Muscle Wasting Inflammatory cytokines such as TNF-/, IL-1b, and IL-6 are involved in mediating the wasting effect in cachectic patients. With regard to TNF-/, systemic TNF-/ infusion in healthy volunteers
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Trends Muscle wasting is the result of an imbalance between anabolic and catabolic metabolism due to inflammation, physical inactivity, and inadequate nutrition. TNF-/, IL-6, and TWEAK shift the metabolism balance toward a catabolic process. However, current therapies such as neutralizing antibodies/decoy receptors against TNF-/, IL-6, and nonsteroidal anti-inflammatory drugs had limited success when used alone. These specific therapies will be more successful when combined with nutritional support, appetite stimulators, and exercise, because combinatorial approaches will not only inhibit protein degradation but also promote protein synthesis. Clinical trials are warranted and will yield more conclusive results by measuring cytokine levels from each patient prior to treatment.
1 Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, P.R. China 2 Cardiovascular Disease Center, The First Hospital of Ji Lin University, Changchun, Jilin, 130021, P.R. China 3 Department of Cardiology, ChangZheng Hospital, Second Military Medical University, Shanghai, 200003, P.R. China 4 [3_TD$IF]Department of Cardiovascular [4_TD$IF]Surgery and [5_TD$IF]Institute of Cardiovascular [6_TD$IF]Science, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215123, P.R. China 5 Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Collaborative Innovation Center of Hematology, Soochow University, Suzhou,[7_TD$IF] P.R. China [2_TD$IF]6 These authors contributed equally to this work
http://dx.doi.org/10.1016/j.tem.2016.03.002 © 2016 Elsevier Ltd. All rights reserved.
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Box 1. TNF-/ Receptor Signaling TNFR1 belongs to the death receptor family of proteins that contain death domains, which can induce apoptosis via the caspase pathway. Upon TNF-/ binding to TNFR1, TNFRSF1A-associated via death domain (TRADD) is recruited to TNFR1. TRADD then recruits Fas-associated protein with death domain (FADD) that triggers the caspase cascade to induce apoptosis. TNFR2 does not contain a death domain, and therefore is not able to induce apoptosis through caspase-dependent mechanisms. Instead, TNFR2 promotes survival via phosphorylation of Etk and subsequent activation of Akt (Figure I) [7,8]. TNFR1 and TNFR2 recruit separate downstream adaptor molecules to induce a cellular response, but converge on TNF receptor-associated factor 2 (TRAF2), to activate IKK and NF-kB [77]. TNFR1 recruits TRADD and RIP1 prior to engaging with TRAF2, whereas TNFR2 can interact with TRAF2 directly [7]. TNFR1-triggered NF-kB activation leads to increased expression of muscle RING-finger protein 1 (MuRF1) and subsequent protein degradation through the ubiquitin proteasome pathway. NF-kB activation also inhibits satellite cell activation and differentiation, by promoting DNA methylation of the Notch-1 promoter [78], and by inhibiting MyoD expression [79], respectively. TNFR2 can activate the alternative NF-kB pathway through NIK [77] and its impact on myogenesis remains to be identified. In addition to TRAF2, six other TRAFs have been identified so far to transmit signals via the TNFRs. From those, TRAF6 is the only one involved in protein degradation. TRAF6 is an E3 ubiquitin ligase and a critical autophagy regulator [80]. TRAF6 mRNA levels are significantly upregulated in skeletal muscles of mice subjected to denervation, cancer cachexia, or diabetes [27]. By contrast, TRAF6 knockout (TRAF6mko, muscle-specific) mice show reduced expression of MuRF1, and atrogin1, and are resistant to denervation and cancer-induced muscle wasting [27]. In a denervation model, activation of NF-kB in skeletal muscle is inhibited in TRAF6mko mice, compared with TRAF6f/f mice. Because NF-kB activation induces the expression of MuRF1 [27], TRAF6-induced muscle atrophy is likely mediated by the NF-kB/ubiquitin/proteasome pathway. However, the upstream signaling molecules that regulate TRAF6 remain unknown. Recent studies suggest that the CD40–TRAF6 interaction leads to obesity-associated insulin resistance [81], which is a condition that induces muscle wasting.
TNFR1
TNFR2
TRADD FADD
RIP-1 NIK
EtK
TRAF2 Caspase-8 p-Etk IKK Apoptosis NF-κB
MuRF1
Protein degradaon
Akt MyoD
Satellite cell differenaon
Survival and protein synthesis
Figure I. TNF-/ Signaling Pathway. TNF-/ can bind to TNFR1 and TNFR2. TNFR1 activation can induce apoptosis via the TRADD/FADD/Caspase 8 pathway, or activate NF-kB via RIP1/TRAF2/IKK. NF-kB activation results in inhibition of myogenic differentiation and protein degradation. Stimulation of TNFR2 can also lead to NF-kB activation via TFAF2 or NIK. Alternatively, TNFR2 signaling could promote survival and protein synthesis through the Etk/Akt pathway. Abbreviations: TNF-/, tumor necrosis factor /; TRADD, TNFRSF1A-associated via death domain; FADD, Fas-associated protein with death domain; RIP1, receptor-interacting protein 1; TRAF2, TNF receptor-associated factor 2; IKK, IkB kinase; NIK, NF-kB-inducing kinase; Etk, epithelial/endothelial tyrosine kinase.
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*Correspondence: [email protected] (Y. Li) and [email protected] (Y.-H. Song).
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Box 2. TNF-like Weak Inducer of Apoptosis (TWEAK) Signaling
Glossary
TWEAK is an emerging cytokine and member of the TNF-/ superfamily involved in muscle injury and atrophy. TWEAK binds to the fibroblast growth factor-inducible 14 (Fn14) receptor and activates NF-kB (Figure I). Fn14 is expressed at low levels in many tissues and cell types under physiological conditions but becomes upregulated during injury and in inflamed tissues. During acute ischemic injury, TWEAK is released from macrophages to induce satellite cell proliferation through activation of classical NF-kB and Notch signaling [82]. By contrast, TWEAK can suppress satellite cell selfrenewal through inversely modulating notch and NF-kB signaling pathways, as shown in a cardiotoxin injury model [20]. It is not clear how TWEAK/Fn14 activation lead to NF-kB activation, but according to Kumar et al., Fn14-induced NF-kB activation may be mediated by TRAF6, at least in a denervation model [27]. Enwere et al. [83] showed that TWEAK can either promote or inhibit myogenic differentiation depending on its concentration. At low concentrations (10–100 ng/ml), TWEAK activates the alternative pathway (NIK/IKK/) and promotes myoblast fusion [83]. At high concentrations (500 ng/ml), TWEAK activates the classical pathway (IKKb) to inhibit differentiation [83]. Thus, TWEAK can maintain muscle differentiation at physiological conditions; however, under pathological conditions TWEAK becomes overexpressed and it induces muscle atrophy [10]. A recent study suggests that TWEAK/Fn14 signaling could also activate nicotinamide adenine dinucleotide phosphate (NADPH) and release reactive oxygen species (ROS) in a Nox2-dependent manner [84]. Increased ROS production resulting from chronic inflammation has been implicated in muscle wasting [85]. Heart failure-induced skeletal muscle atrophy is associated with increased Nox2 expression, ROS production, and NFkB activation that lead to increased expression of MuRF1 and activation of the proteasome [86]. Therefore, both TNF-/ and TWEAK can induce muscle atrophy by the induction of E3 ubiquitin ligase MuRF1 via different signaling pathways.
Activin-A: a growth factor that stimulates synthesis and secretion of follicle stimulating hormone. Activin-A was recently found to be associated with muscle loss in cancer models. Amyotrophic lateral sclerosis (ALS): or Lou Gehrig's disease, a progressive neurodegenerative disease that affects neurons in the brain and spinal cord. Cachexia: or wasting syndrome, is a multifactorial syndrome characterized by weight loss and muscle atrophy, fatigue, weakness, and significantly reduced food intake due to loss of appetite, in people not actively trying to lose weight. Nutritional intervention to reverse the loss of body mass is unsuccessful even with an increase in calorie intake, indicating a primary pathology. Chronic obstructive pulmonary disease (COPD): is a chronic obstructive lung disease characterized by poor airflow and impaired exercise capacity due to skeletal muscle wasting. Denervation: is a loss of nerve supply due to injury or motor neuron diseases and peripheral neuropathies. Duchenne muscular dystrophy (DMD): a genetic disease caused by mutation of the dystrophin gene, which encodes the dystrophin protein that is critical in maintaining muscle cell integrity. Patients with dystrophin mutation develop muscle wasting. Muscle atrophy: a decrease of muscle mass. Myostatin: a member of the TGF-b family. It is a negative regulator of skeletal muscle growth. Myotonic dystrophy type 1 (DM1): is a multisystem disorder characterized by muscle wasting, cataracts, and heart conduction defects. The disease is caused by an abnormal expansion of CTG repeat in the dystrophia myotonica protein kinase (PMDK) gene. Sarcopenia: is a disease associated with loss of muscle mass and strength due to aging. Wasting: muscle loss and weakness.
TWEAK
Fn14
Alternave pathway
Classical pathway
NIK/IKKα
IKKβ
RelB/p52
p65/p50
NADPH
TRAF6
ROS
NF-κB
MuRF1
MyoD
Myogenesis ↑
Myogenesis ↑
Proteasome dependent degradaon Muscle Muscle atrophy
Figure I. The Role of TWEAK/Fn14 Signaling in Myogenesis and Muscle Atrophy. Depending on its concentration, TWEAK could either promote or inhibit myogenesis. At low concentrations, TWEAK activates the alternative pathway (NIK/IKK/) and promotes myoblast fusion. At high concentrations, TWEAK activates the classical pathway (IKKb) to inhibit differentiation. TWEAK/Fn14 signaling could also lead to protein degradation by activating NF-kB via TRAF6 or NADPH. For abbreviations, see text in Box 2 above.
was shown to increase protein breakdown and inhibit protein synthesis [12], and elevated TNF/ levels correlate with muscle loss in patients with cancer [1,2], COPD [3], and disuse [4]. The TNF chimeric monoclonal antibody infliximab, an agent that blocks TNF-/ action, can increase muscle strength and volume in patients with Crohn's disease [13], and long-term treatment with etanercept, another TNF-/ inhibitor, results in body weight gain in rheumatoid arthritis (RA) patients [14]. By contrast, a Phase II study that combined the chemotherapy drug gemcitabine and infliximab did not show a benefit in preserving lean body mass or survival in cachectic
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patients with pancreatic cancer [15]. A Phase I/II study compared etanercept with gemcitabine versus gemcitabine alone for treatment of patients with advanced pancreatic cancer and the results were also disappointing because the addition of etanercept did not alleviate cancerrelated cachexia [16]. One possible explanation for this discrepancy is that the patients that participated in these clinical trials might express varied cytokine levels [16,17]. Recently, TWEAK has been identified as a key mediator of wasting in denervation [18] and cancer [19]. TWEAK induces muscle RING-finger protein 1 (MuRF1) expression via NF-kB activation [10] (Box 2). TWEAK also reduces satellite cell pool by inhibiting Notch signaling and activating NF-kB [20]. In addition to the NF-kB pathway, TNF-/ and TWEAK also signal to p38 MAP kinase [21,22] and the Jak/Stat pathway, which in turn upregulates the expression of atrogin-1 and MuRF1 [23]. Therefore, interventions targeting these pathways should be considered when designing novel therapies for cachexia.
Cancer Cachexia Cancer cachexia, induced by both tumor and chemotherapy, is characterized by anorexia and weight loss [24]. While its pathogenesis remains largely unknown, recent data suggest that TWEAK and IL-6 might be involved in cancer-related wasting. TWEAK Under pathological conditions, TWEAK can become overexpressed and induce a cachectic phenotype in part by the induction of the E3 ligase MuRF1 [10]. Using the Lewis lung carcinoma (LLC) and colon-26 (C-26) tumor model, Johnston et al. [25] demonstrated that these two types of tumors induce cachexia in TWEAK or Fn14-deficient mice (germline deletions), suggesting that tumor-derived rather than host-derived TWEAK or Fn14 might play a role in these cancer cachexia models. Antibodies against tumor-derived Fn14, but not TWEAK, prevented tumorinduced inflammation and loss of fat and muscle mass [25], suggesting that an as-yet unidentified Fn14 ligand might exist that triggers its activation. Alternatively, Fn14 might be able to trigger downstream events independent of TWEAK. A shorter form of Fn14 (Fn14-DEC) was recently identified in human cancer cell lines [26] that lacks the extracellular domain and thus cannot bind TWEAK. Fn14-DEC monomers can self-assemble within cells into dimers and activate NF-kB. While it is unclear what Fn14 downstream molecules are activated in tumors that then act on muscle cells to induce atrophy, activin-A or myostatin have been suggested as potential candidates [25]. Some of the TWEAK/Fn14 signals might be transmitted through TNF receptor-associated factor 6 (TRAF6) [27], subsequently activating NF-kB. Indeed, it has been shown that expression of TRAF6 is increased in the LLC model and deletion of TRAF6 prevents muscle wasting in response to tumor growth [27]. The role of TRAF6 in animal models of cancer cachexia was recently confirmed in a clinical study. Both TRAF6 and ubiquitin were found to be significantly upregulated in muscle of gastric cancer patients compared with muscles from patients with benign diseases [28]. Thus, TRAF6 has become an important therapeutic target for treating cancer cachexia. IL-6 IL-6 is an important inflammatory marker that has been associated with increased mortality in cancer patients [2]. Clinical studies showed that plasma IL-6 levels increased 11-fold in the cancer cachectic group compared with weight-stable controls [29]. A study of 98 patients with advanced cancer showed that IL-6 but not TNF-/ predicted survival rates [30]. The role of IL-6 in cancer cachexia was confirmed in the APCMin+[1_TD$IF] mouse, which is an established model of colon cancer that develops cachexia. The progression of muscle atrophy in these mice was related to circulating IL-6 levels, and overexpression of IL-6 APCMin+ mice accelerated the development of
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cachexia [31]. In vitro studies showed that recombinant IL-6 could inhibit C2C12 myogenic differentiation when added to the differentiation medium [32].
Chemotherapy-Induced Cachexia Cancer-associated cachexia is not only caused by tumor but also by chemotherapy. The chemotherapy drug cisplatin induces muscle atrophy by activating myostatin, IL-6, TNF-/, and IL-1b, which can be prevented by the orexigenic peptide ghrelin in the LLC-induced cachexia model [33]. Cyclophosphamide-, doxorubicin-, and 5-fluorouracil-induced cachexia is associated with increased circulating IL-6, followed by decreased insulin-like growth factor 1 (IGF-1) levels, and can be prevented by germline deletion of IL-6 [34]. Therefore, different chemotherapy agents induce cachexia via distinct mechanisms and further research is needed to understand their transduction pathways.
COPD COPD is most often caused by cigarette smoking, and mice exposed to cigarette smoke daily for 8 weeks exhibited a 157% increase in serum TNF-/, accompanied by significant decrease of peroxisome proliferator-activated receptor g co-activator 1/ (PGC-1/) mRNA levels in soleus and extensor digitorum longus muscles [35]. Analysis of markers and regulators of skeletal muscle oxidative phenotype (oxphen) such as citrate synthase and PGC-1/ in vastus lateralis muscle biopsies of patients with advanced COPD and healthy smoking control participants showed elevated muscle TNF-/ mRNA, and reduced mRNA levels of oxidative markers/regulators in 23% of the COPD patients [3]. The authors were not able to detect TNF-/ protein in muscle biopsies. So the question is whether circulating or lung-derived TNF-/ would have an impact on muscle function. In a transgenic mouse model overexpressing TNF-/ in the lung, TNF-/ impaired peripheral skeletal muscle function due to excessive formation of superoxide in contracting skeletal muscle [36,37]. However, a study of 426 COPD patients over a 3-year period found that TNF-/ levels were associated with muscle loss only in patients who were cachectic at entry into the study; the authors concluded that inflammation is a consequence rather than a cause of initial loss of fat-free mass [38]. At this point, the role of TNF-/ in COPD-induced muscle wasting remains unclear, and a better way to clarify this issue would be to study the effect of cigarette smoke exposure in TNF-/ knockout mice.
Denervation and Neuron Muscular Diseases Motor neurons normally provide trophic factors that promote protein synthesis in muscle. Loss of nerve supply due to denervation results in molecular and cellular changes in skeletal muscle and atrophy [39]. Recent studies suggest that TWEAK and TRAF6 may be involved in this process. Transgenic expression of TWEAK specifically in skeletal muscle induces atrophy via activation of NF-kB and upregulation of MuRF1 [10]. Interestingly, TWEAK overexpression was associated with a transition from slow (type I) to fast (type II) fiber type, and TWEAK-induced muscle atrophy is predominantly restricted to fast-type fibers [10]. TWEAK/Fn14 also plays an important role in myotonic dystrophy type 1 (DM1). Expression of Fn14 correlated with severity of muscle pathology in skeletal muscle of mouse models of DM1 and in tissues from DM1 patients, whereas Fn14 germline deletion improved muscle pathology and function [40]. Blocking TWEAK, a ligand for Fn14, with an anti-TWEAK antibody improved muscle histopathology and functional outcomes in affected mice [40]. Therefore, unlike cancerinduced cachexia [25], TWEAK appears to signal through Fn14 to induce muscle atrophy in myotonic dystrophy.
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In the SOD1G93A mouse, a model of amyotrophic lateral sclerosis (ALS), TWEAK is upregulated in spinal cord astrocytes, which was believed to induce neuron death by releasing IL-6 [41]. Genetic ablation of TWEAK or treatment with anti-TWEAK antibody significantly reduced astrocyte activation and ameliorated muscle wasting in these mice. However, these approaches failed to improve motor function and motor neuron survival [41]. These results suggest that astrocyte activation and TWEAK upregulation may not be the primary cause of motor neuron death. Alternatively, as the authors suggested, TWEAK is a downstream effector of interferon g (IFNg) and reducing astrocyte activation by depleting TWEAK may not impact the deleterious effect of TWEAK-independent IFNg signaling on astrogliosis and motor neuron death. Thus, the role of TWEAK in the development of ALS remains unknown. TRAF6 is another factor involved in denervation-induced muscle atrophy and skeletal musclespecific depletion of TRAF6 prevented myofibril degradation upon denervation [27]. TRAF6 is activated by TWEAK/Fn14 and can subsequently activate NF-kB-dependent protein degradation through the ubiquitin proteasome pathway [27]. TRAF6 also regulates the autophagy system because the expression of the autophagy proteins Beclin1 and LC3B was upregulated in denervated muscle but reduced in TRAF6 knockout mice [27]. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), TRAF6 activity was increased in skeletal muscle, and muscle-specific deletion of TRAF6 reduced fiber necrosis and improved muscle function. Ablation of TRAF6 (muscle-specific deletion) also enhanced myofiber regeneration resulting from satellite cell proliferation in young mdx mice [42]. However, ablation of TRAF6 exacerbated muscle injury and increased fibrosis in 9-month-old mdx mice. The authors suggested that TRAF6 is an important autophagy regulator that removes damaged organelles. Old mice are more reliant on autophagy due to the progressive nature of the disease. Although animal studies have clearly demonstrated a role of TWEAK and TRAF6 in denervation and neuron muscular disease-induced muscle wasting, therapeutic interventions based on inhibition of either TWEAK or TRAF6 signaling should proceed with caution because both molecules play a physiological role in maintaining muscle mass and function.
Sarcopenia While the mechanisms underlying sarcopenia remain unknown, reduced physical activity may play a role [4]. In older adults, just 14 days of reduced steps induced reductions in myofibril protein synthesis and muscle mass, accompanied by impairments in insulin sensitivity and elevated TNF-/ levels (while IL-6 levels were not elevated) [4], suggesting that TNF-/ inhibitors may be beneficial for the elderly. Animal studies demonstrated that TWEAK/Fn14 is also involved in sarcopenia. Fn14 transcription was significantly increased in both tibial anterior (TA) and gastrocnemius (GA) muscles of 18-month-old mice, compared with 3-month-old mice [43]. Genetic ablation of Fn14 attenuated age-associated skeletal muscle atrophy in mice and reduced the expression of ubiquitinated proteins. However, the expression of atrogin-1 and MuRF1 was not altered by Fn14 ablation, suggesting that perhaps other as-yet unidentified ubiquitin ligases may be responsible for muscle protein ubiquitination and degradation. Alternatively, atrogin-1 and MuRF1 expression follows a clear time course and their quantitation could have been too late to detect meaningful differences in the Fn14 mutant mice. TWEAK affects skeletal muscle mitochondrial content, and age-related loss in skeletal muscle oxidative capacity was rescued in TWEAK global knockout mice [44]. The expression of several molecules involved in oxidative metabolism such as PGC1/ and succinate dehydrogenase (SDH) was significantly higher in skeletal muscle of TWEAK knockout mice. Thus, both TNF-/ and TWEAK/Fn14 are implicated in the pathogenesis of sarcopenia, although their signal transduction pathways may be different.
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Cardiac Cachexia Cardiac cachexia is a serious complication of congestive heart failure [45]. TNF-/ and angiotensin II (ang II) have been implicated in cardiac cachexia, but the association of TNF-/ signaling with ang II has not been clarified in atrophic conditions. Two recent studies provided new insight on how ang II might induce muscle loss. One study [46] showed that transcription factor EB (TFEB) is a mediator of ang II-induced muscle wasting through upregulation of MuRF1, and ang II-induced MuRF1 expression and atrophy could be prevented by targeting TFEB with siRNA. MuRF1 expression is also regulated by histone deacetylase 5 (HDAC5) that binds to TFEB and inhibits its interaction with the MuRF1 promoter [46]. HDAC5 is associated with protein [10_TD$IF]kinase [1_TD$IF]D1 (PKD), which associates with, phosphorylates, and mediates 14-3-3-dependent export of HDAC5 from the nucleus. Muscle-specific knockout of PKD1 prevented ang II-induced wasting. A second study [47] showed that mitochondrial oxidative enzymes were significantly decreased, while superoxide production was increased, in the skeletal muscle of ang II-treated mice. Because ang II plays a central role in cardiac cachexia, ang II receptor blockade should alleviate muscle wasting. Along this line, losartan, an ang II type I receptor blocker, improved muscle activity and reduced inflammation in older mice [48].
Muscle Injury and Regeneration TWEAK is involved in muscle regeneration postinjury. In the cardiotoxin (CTX) injury model, TWEAK negatively regulates muscle regeneration by inhibiting myogenic differentiation via activation of NF-kB [49]. TWEAK-induced activation of NF-kB is counterbalanced by Nrf2, a transcription factor that regulates antioxidative enzymes. Nrf2 global knockout mice showed increased TWEAK expression, enhanced fibrosis, and insufficient regeneration [50]. In contrast to its deteriorating roles in wasting conditions, TNF-/ signaling promotes muscle regeneration. When the proinflammatory capacity of M1 monocytes was assessed in men ex vivo by stimulating whole blood with lipopolysaccharide (LPS), IFNg, granulocyte macrophage colony-stimulating factor (GM-CSF), and TNF-/ cytokine production was positively associated with lean body mass and muscle strength [51]. When the role of TNF-/ signaling in CTX-injured soleus muscle in TNFR1 and TNFR2 global double-knockout mice was analyzed, it was shown that inflammation was resolved and the regeneration process was nearly complete at day 12 postinjury in wild-type mice; however, immune cells remained in the injured muscle with little signs of regeneration in the double-knockout mice [52]. At the molecular level, reduced levels of myogenin and p21 indicated that myogenesis was blocked in the injured knockout mice. Along this line, TNF-/ could promote bone repair by promoting the recruitment and differentiation of muscle-derived stromal cells to injury sites [53]. However, in a mouse model of hindlimb ischemia, the limb reperfusion after ischemia was significantly higher in TNFR1-deficient mice (germline deletion) after injury, accompanied by reduced apoptosis, when compared with wildtype controls, but the lack of TNFR1 signaling impaired angiogenesis at later stages of ischemic injury [54]. In light of the role that TNF-/ plays in promoting muscle regeneration, anti-TNF-/ agents such as etanercept may not be the best choice for treating inflammatory diseases or cachexia in patients who are also suffering from acute injury.
Pharmacotherapy of Muscle Wasting Exercise Exercise improves blood flow and releases growth factors that rejuvenate muscles and other tissues [55], as well as reducing inflammation. When the expression of E3 ligase, IGF-1, and TNF-/ was measured in 60 chronic heart failure (CHF) patients who underwent a 4-week endurance training program [55], it was found that the expression of MuRF1 and TNF-/ in
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skeletal muscle was reduced, while that of IGF-1 was increased, with exercise. Additionally, resistance training-induced increase of IGF-1 was mediated by PGC-1/4, a novel form of PGC1/ [56]. The effect of chronic exercise on TNF-/ and atrogin-1 expression was evaluated in a mouse hindlimb unloading model after the mice received a treadmill running exercise for 6 weeks after unloading [57]. Results showed that chronic exercise attenuated a decrease in muscle weight and inhibited expression of TNF-/, IL-6, and atrogin-1 in the atrophied gastrocnemius muscle. TNF-/ is increased in inflammatory bowel diseases (IBDs), which is often associated with muscle wasting. Mice subjected to 4 months of free wheel running (WR) exhibited reduced expression of TNF-/ and caspase-7 and -8 in intestinal lymphocytes [58]. When the effect of high-fat diet was tested in rats with colitis forced to a treadmill running exercise regimen for 6 weeks, exercise accelerated the healing of colitis, significantly decreased plasma IL-1b, TNF-/, TWEAK, and leptin levels, and increased IL-6 plasma levels [59]. Along these lines, it was previously suggested that the beneficial anti-inflammatory effects of exercise are mediated by muscle-derived IL-6, because recombinant human IL-6 infusion was shown to inhibit endotoxin-induced increases in circulating TNF-/ in healthy humans [60]. Furthermore, IL-6 level was increased during a wheelchair marathon in athletes with cervical spinal cord injuries, accompanied by decreased levels of TNF-/, suggesting an inhibitory role of IL-6 on TNF-/ release [61]. While IL-6 signaling in monocytes generates a proinflammatory response, IL-6 signaling in myocytes creates an anti-inflammatory environment [60]. Nonsteroidal anti-inflammatory drugs (NSAIDs) NSAIDs such as celecoxib and ibuprofen have been used to treat cachexia with some success. A Phase II trial examined the efficacy and safety of celecoxib (COX-2 inhibitor) on cancer cachexia. Patients with advanced cancers were treated with celecoxib daily (200–300 mg/day) for 4 months. The treatment led to a significant increase of lean body mass, grip strength, and a reduction of circulating TNF-/ levels [62]. Side effects such as grade 1/2 anemia and neuropenia were observed in only a few patients, and cardiovascular events were not observed in any of the patients at this moderate dose [62]. The effect of ibuprofen on cancer cachexia was evaluated in the C-26 tumor model; ibuprofen improved muscle mass and reduced the expression of atrogin-1 and MuRF1 in tumor-bearing mice [63]. Thus, results from both clinical and animal studies support the use of NSAIDs to treat cancer cachexia (Box 3).
Box 3. Microbiota and Cachexia Although inflammation and bacterial infections can lead to cachexia, certain bacteria strains could actually prevent muscle atrophy. Schieber et al. [87] made a ground-breaking discovery while working on the dextran sulfate sodium (DSS) intestinal injury model, trying to determine the cause of muscle wasting in mice. While C57Bl/6 mice obtained from Jackson Laboratories (Jax mice) exhibited muscle wasting, C57Bl/6 from the University of California, Berkeley, colony (CB mice) showed significantly less wasting, when treated with DSS, suggesting that differences in the microbiota composition between Jax and CB mice were responsible for the differences observed in muscle wasting. Subsequently, they were able to isolate an Escherichia coli (O21:H+) strain that was absent from Jax mice. Oral administration of E. coli O21:H+ resulted in colonization of the intestine of Jax mice and on DSS treatment, E. coli O21:H+ Jax mice showed significantly less wasting than did DSS-treated vehicle mice. E. coli O21:H+ also prevented muscle wasting induced by other microbes. This protection was associated with challenge-induced translocation of E. coli O21:H+ to white adipose tissue and activation of the NLRC4 inflammasome via IL-18, which prevented muscle atrophy by activating IGF-1 signaling pathway [87]. Although previous studies demonstrated some beneficial effect of NSAIDs in treating muscle wasting, the discovery that IL-18 is involved in muscle maintenance leads to the questioning of the validity of NSAIDs as a therapeutic intervention for cachexia because NSAIDs can inhibit the release of IL-18 [88] and could potentially impair IGF-1 signaling.
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Etanercept Etanercept is a recombinant fusion protein that functions as a decoy receptor to neutralize TNF/. The drug has been used to treat inflammatory disorders including RA, a chronic inflammatory disease associated with muscle loss. RA-induced muscle atrophy is likely the result of increased production of TNF-/ and IL-6 [64], which is associated with insulin resistance [65]. A meta-analysis of eight studies with 260 subjects revealed that TNF-/ inhibitor therapy reduces insulin resistance in RA [65]. In another study [14], significant weight gain in RA patients treated with etanercept, twice weekly for 12 consecutive months, was observed. The most common side effects associated with etanercept when used to treat inflammatory diseases include infusion site reaction, headache, and upper respiratory infections such as tuberculosis [66]. When used for treating cachexia, it was tolerated well without tuberculosis or any side effects that would cause patient withdrawal, although increased serum uric acid levels have been noted [14]. TWEAK Antibody Animal studies demonstrated that blocking antibodies against the TWEAK antibody can improve muscle function in mice affected by myotonic dystrophy [40] and ALS [41]. However, antibodies against tumor-derived Fn14, but not TWEAK, prevented tumor-induced cachexia [25]. The antiFn14 treatment protected both the oxidative type IIa and glycolytic type IIx/b fibers. These results suggest that neutralizing antibodies against both TWEAK and Fn14 should be explored further in various models of muscle atrophy, as well as in clinical trials. Myostatin Inhibitor The myokine myostatin can inhibit myogenesis in an autocrine manner. Upregulation of myostatin has been reported in several muscle wasting conditions [67,68]. Chronic treatment of mice with a human monoclonal antibody (REGN1033) that blocks myostatin increased muscle mass and force production, and prevented the loss of muscle mass in several muscle wasting models including immobilization and dexamethasone treatment [68]. In aged mice, REGN1033 improved muscle function during treadmill exercise [68]. Although untested, REGN1033 might be beneficial for the elderly, because, at least in old rats, exercise alone was not able to reduce TNF-/ level [69]. Activin Receptor Type IIB (ActRIIB) Inhibitor ActRIIB and type I belong to the transforming growth factor b (TGF-b) family. Increased ActRIIB expression has been implicated in cancer cachexia. In the C-26 tumor mouse model, recombinant soluble ActRIIB (sActRIIB) was able to inhibit both myostatin and activin-A-mediated Smad2/3 signal transduction and prevented muscle wasting [70]. Importantly, this treatment stimulated stem cell growth and prolonged animal survival. A recent study using the LLC cachexia model confirmed the beneficial effect of sActRIIB [71]. IL-6 Antibody A strong correlation exists between serum IL-6 level and cachexia in patients with chemotherapy-resistant lung cancer [72]. A humanized anti-IL-6 antibody was shown to reverse fatigue and prevent muscle loss in patients with non-small cell lung cancer [73]. Transplantation of lung cancer cells expressing IL-6 induces cachexia in mice, which can be ameliorated by the IL-6 antibody. In a Phase II study, selumetinib, a mitogen-activated protein kinase kinase (MEK) inhibitor, promoted muscle gain in patients with cholangiocarcinoma [74]. A limitation of this study was the lack of a placebo control group. Besides its role in tumor growth inhibition, selumetinib also inhibited the secretion of IL-6, TNF-/, and IL-1b, which might explain its beneficial effects on muscle gain. However, caution must be taken when using this drug as treatment for cachexia because MEK signaling is critical to myogenic differentiation.
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Etanercept
Exercise
TNF
IL-6
Inflammaon
Skeletal muscle IGF-1
IL-6 anbody
NSAID
ROS Atrogin-1
Weakness and atrophy MuRF1
sActRIIB
Myostan
Food intake
Ghrelin
Figure 1. Current Treatment Options for Cachexia. Chronic inflammation plays a central role in the development of cachexia. Therefore, both nonspecific anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and specific cytokine blockers such as etanercept and selumetinib can be used to inhibit inflammation. However, these treatments would be most useful when combined with ghrelin to stimulate food intake. Exercise and myostatin inhibitors are particularly useful to maintain muscle mass and function in the elderly.
Ghrelin Ghrelin is an orexigenic peptide that is produced by cells in the gastrointestinal tract and acts on the hypothalamus to stimulate food intake [75]. Thus, ghrelin has been proposed as a treatment for muscle wasting (Figure 1). Analysis of Phase II, randomized, placebo-controlled, double-blind trials showed that treatment with anamorelin (a ghrelin receptor agonist) for 12 weeks, increased lean body weight in patients with cancer cachexia [76]. Adverse events associated with this treatment include fatigue, asthenia, atrial fibrillation, and dyspnea.
Concluding Remarks Inflammatory cytokines such as TNF-/ and IL-6 play an important role in the development of cachexia. These cytokines induce muscle atrophy by activating the myostatin and ubiquitin proteasome pathway, or by inhibiting protein synthesis and mitochondrial biogenesis. Therapeutic interventions including etanercept (TNF-/ inhibitor) and neutralizing antibodies that block IL-6 or myostatin have shown promising results in animal studies. However, these approaches had limited success in clinical studies, partly due to the fact that cachexia is a syndrome that is multifactorial in nature and any single therapy is insufficient to stop or prevent muscle loss. Combinational therapies that simultaneously target inflammation and proteolysis while promoting protein synthesis and mitochondrial biogenesis will have a better outcome. Exercise will have additional benefit because it will not only stimulate protein synthesis but also inhibit inflammation. TWEAK/Fn14 and TRAF6 have emerged as potential new targets during the past 5 years. However, their mechanisms of action and transduction pathways remain largely unknown and their roles in cachexia need to be verified in clinical studies before any potential therapeutic intervention can be developed. The ubiquitin proteasome system has been an obvious target for intervention, but activation of this system is not responsible for all types of cachexia; and it also
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has an important physiological role by removing unneeded and misfolded proteins, and therefore inhibition of the proteasome may have unwanted side effects. It is likely that each type of cachexia has a unique signature in terms of the nature of cytokines that pull the trigger to initiate the catabolic process, their cellular origin, time course of expression, and the transduction pathways that they use to mediate their actions on proteolysis. However, our current knowledge of these areas remains limited, and the complex interaction among different cytokines and networks are far from being understood (see Outstanding Questions). Further research is necessary to define the contributing factors and underlying mechanisms leading to cachexia and to translate this knowledge into new therapies. [12_TD$IF]Acknowledgments This work was supported by the project for the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Outstanding Questions What is the specific cytokine responsible for each cachectic condition? What are the cellular sources of cytokine production? How could we use this information to design a personalized treatment for each cachectic patient tailored to his/her own cytokine profile? Does TWEAK have a physiological role in muscle maintenance? What is the pathological concentration of TWEAK found in patients with cachexia? The answers to these questions hold the key for the development of future therapeutic interventions. What is the downstream signaling pathway mediating the muscle wasting effect of Fn14? How does Fn14 signaling interact with other negative regulators of muscle mass such as NF-kB and glycogen synthase kinase (GSK)3b?
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