Pradyut K. Paul

Tumor Necrosis Factor-related Weak Inducer of Apoptosis Augments Matrix Metalloproteinase 9 (MMP-9) Production in Skeletal Muscle through the Activation of Nuclear Factor-κB-inducing Kinase and p38 Mitogen-activated Protein Kinase A POTENTIAL ROLE OF MMP-9 IN MYOPATHY

Destruction of skeletal muscle extracellular matrix is an important pathological consequence of many diseases involving muscle wasting. However, the underlying mechanisms leading to extracellular matrix breakdown in skeletal muscle tissues remain unknown. Using a microarray approach, we investigated the effect of tumor necrosis factor-related weak inducer of apoptosis (TWEAK), a recently identified muscle-wasting cytokine, on the expression of extracellular proteases in skeletal muscle. Among several other matrix metalloproteinases (MMPs), we found that the expression of MMP-9, a type IV collagenase, was drastically increased in myotubes in response to TWEAK. The level of MMP-9 was also higher in myofibers of TWEAK transgenic mice. TWEAK increased the activation of both classical and alternative nuclear factor-κB (NF-κB) signaling pathways. Inhibition of NF-κB activity blocked the TWEAK-induced production of MMP-9 in myotubes. TWEAK also increased the activation of AP-1, and its inhibition attenuated the TWEAK-induced MMP-9 production. Overexpression of a kinase-dead mutant of NF-κB-inducing kinase or IκB kinase-β but not IκB kinase-α significantly inhibited the TWEAK-induced activation of MMP-9 promoter. The activation of MMP-9 also involved upstream recruitment of TRAF2 and cIAP2 proteins. TWEAK increased the activity of ERK1/2, JNK1, and p38 MAPK. However, the inhibition of only p38 MAPK blocked the TWEAK-induced expression of MMP-9 in myotubes. Furthermore the loss of body and skeletal muscle weights, inflammation, fiber necrosis, and degradation of basement membrane around muscle fibers were significantly attenuated in Mmp9 knock-out mice on chronic administration of TWEAK protein. The study unveils a novel mechanism of skeletal muscle tissue destruction in pathological conditions.

The E3 ubiquitin ligase TRAF6 intercedes in starvation-induced skeletal muscle atrophy through multiple mechanisms.

ABSTRACT Starvation, like many other catabolic conditions, induces loss of skeletal muscle mass by promoting fiber atrophy. In addition to the canonical processes, the starvation-induced response employs many distinct pathways that make it a unique atrophic program. However, in the multiplex of the underlying mechanisms, several components of starvation-induced atrophy have yet to be fully understood and their roles and interplay remain to be elucidated. Here we unveiled the role of tumor necrosis factor receptor-associated factor 6 (TRAF6), a unique E3 ubiquitin ligase and adaptor protein, in starvation-induced muscle atrophy. Targeted ablation of TRAF6 suppresses the expression of key regulators of atrophy, including MAFBx, MuRF1, p62, LC3B, Beclin1, Atg12, and Fn14. Ablation of TRAF6 also improved the phosphorylation of Akt and FoxO3a and inhibited the activation of 5′ AMP-activated protein kinase in skeletal muscle in response to starvation. In addition, our study provides the first evidence of the involvement of endoplasmic reticulum stress and unfolding protein response pathways in starvation-induced muscle atrophy and its regulation through TRAF6. Finally, our results also identify lysine 63-linked autoubiquitination of TRAF6 as a process essential for its regulatory role in starvation-induced muscle atrophy.

TWEAK and TRAF6 regulate skeletal muscle atrophy.

Purpose of reviewTo discuss the roles and mechanisms of action of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in skeletal muscle atrophy. Recent findingsProinflammatory cytokines are known to mediate muscle atrophy in many chronic disease states. However, their role in the loss of skeletal muscle mass in disuse conditions has just begun to be elucidated. Further, the initial signaling events leading to the activation of various catabolic pathways in skeletal muscle under different atrophic conditions are also less well understood. The TWEAK–Fn14 system has now been identified as a novel inducer of skeletal muscle wasting. Adult skeletal muscles express minimal levels of Fn14, the bona fide TWEAK receptor. Specific conditions of atrophy such as denervation, immobilization, or unloading rapidly induce the expression of Fn14 leading to TWEAK-induced activation of various proteolytic pathways in skeletal muscle. Recent studies have also demonstrated that the expression and activity of TRAF6 are increased in distinct models of muscle atrophy. Muscle-specific ablation of TRAF6 inhibits the induction of atrophy program in response to starvation, denervation, or cancer cachexia. Moreover, TWEAK also appears to activate some catabolic signaling through TRAF6-dependent mechanisms. SummaryRecent findings have uncovered TWEAK and TRAF6 as novel regulators of skeletal muscle atrophy. These proteins should potentially be used as molecular targets for prevention and/or treatment of muscular atrophy in future therapies.

Genetic Ablation of TWEAK Augments Regeneration and Post-Injury Growth of Skeletal Muscle in Mice

Impairment in the regeneration process is a critical determinant for skeletal muscle wasting in chronic diseases and degenerative muscle disorders. Inflammatory cytokines are known to cause significant muscle wasting, however, their role in myofiber regeneration is less clear. In this study we have investigated the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in skeletal muscle regeneration in vivo. Our results show that expression levels of TWEAK and its receptor Fn14 are significantly increased in skeletal muscles of mice after injury. Genetic deletion of TWEAK increased the fiber cross-sectional area and levels of embryonic isoform of myosin heavy chain in regenerating tibial anterior muscle. Conversely, muscle-specific transgenic overexpression of TWEAK reduced the fiber cross-sectional area and levels of the embryonic myosin heavy chain in regenerating muscle. TWEAK induced the expression of several inflammatory molecules and increased interstitial fibrosis in regenerating muscle. Genetic ablation of TWEAK suppressed, whereas overexpression of TWEAK increased, the activation of nuclear factor-kappa B without affecting the activation of Akt or p38 kinase in regenerating myofibers. Primary myoblasts from TWEAK-null mice showed enhanced differentiation in vitro, whereas myoblasts from TWEAK-Tg mice showed reduced differentiation compared with wild-type mice. Collectively, our study suggests that TWEAK negatively regulates muscle regeneration and that TWEAK is a potential therapeutic target to enhance skeletal muscle regeneration in vivo.

Elevated levels of active matrix metalloproteinase-9 cause hypertrophy in skeletal muscle of normal and dystrophin-deficient mdx mice

Matrix metalloproteinases (MMPs) are a group of extracellular proteases involved in tissue remodeling in several physiological and pathophysiological conditions. While increased expression of MMPs (especially MMP-9) has been observed in skeletal muscle in numerous conditions, their physiological significance remains less-well understood. By generating novel skeletal muscle-specific transgenic (Tg) mice expressing constitutively active mutant of MMP-9 (i.e. MMP-9G100L), in this study, we have investigated the effects of elevated levels of MMP-9 on skeletal muscle structure and function in vivo. Tg expression of enzymatically active MMP-9 protein significantly increased skeletal muscle fiber cross-section area, levels of contractile proteins and force production in isometric contractions. MMP-9 stimulated the activation of the Akt signaling pathway in Tg mice. Moreover, expression of active MMP-9 increased the proportion of fast-type fiber in soleus muscle of mice. Overexpression of MMP-9 also considerably reduced the deposition of collagens I and IV in skeletal muscle in vivo. In one-year-old mdx mice (a model for Duchenne muscular dystrophy, DMD), deletion of the Mmp9 gene reduced fiber hypertrophy and phosphorylation of Akt and p38 mitogen-activated protein kinase. Collectively, our study suggests that elevated levels of active MMP-9 protein cause hypertrophy in skeletal muscle and that the modulation of MMP-9 levels may have therapeutic value in various muscular disorders including DMD.

TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle.

Skeletal muscle wasting is a major reason for morbidity and mortality in many chronic disease states, disuse conditions and aging. The ubiquitinproteasome and autophagy-lysosomal systems are the two major proteolytic pathways involved in regulation of both physiological and pathological muscle wasting. Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an important adaptor protein involved in receptor-mediated activation of various signaling pathways in response to cytokines and bacterial products. TRAF6 also possesses E3 ubiquitin ligase activity causing lysine-63-linked polyubiquitination of target proteins. We have uncovered a novel role of TRAF6 in regulation of skeletal muscle mass. Muscle-wasting stimuli upregulate the expression, as well as the auto-ubiquitination, of TRAF6 leading to downstream activation of major catabolic pathways in skeletal muscle. Muscle-specific depletion of TRAF6 preserves skeletal muscle mass in a mouse model of cancer cachexia or denervation. Inhibition of TRAF6 also blocks the expression of the components of the ubiquitin-proteasome system (UPS) and autophagosome formation in atrophying skeletal muscle. While more investigations are required to understand its mechanisms of action in skeletal muscle, our results indicate that blocking TRAF6 activity can be used as a therapeutic approach to preserve skeletal muscle mass and function in different disease states and conditions.

Reciprocal Interaction between TRAF6 and Notch Signaling Regulates Adult Myofiber Regeneration upon Injury

ABSTRACT Skeletal muscle is a postmitotic tissue that repairs and regenerates through activation of a population of stem-cell-like satellite cells. However, signaling mechanisms governing adult skeletal muscle regeneration remain less understood. In the present study, we have investigated the role of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein involved in receptor-mediated activation of multiple signaling pathways in regeneration of adult myofibers. Skeletal muscle-specific depletion of TRAF6 in mice (TRAF6mko) improved regeneration of myofibers upon injury with a concomitant increase in the number of satellite cells and activation of the Notch signaling pathway. Ex vivo cultures of TRAF6mko myofiber explants demonstrated an increase in the proliferative capacity of myofiber-associated satellite cells accompanied by an upregulation of Notch ligands. Deletion of TRAF6 also inhibited the activity of transcription factor NF-κB and the expression of inflammatory cytokines and augmented the M2c macrophage phenotype in injured muscle tissues. Collectively, our study demonstrates that specific inhibition of TRAF6 improves satellite cell activation and skeletal muscle regeneration through upregulation of Notch signaling and reducing the inflammatory repertoire.