Activation of the Ubiquitin Proteasome Pathway in a Mouse Model of Inflammatory Myopathy: A Potential Therapeutic Target

Abstract

Objective Myositis is characterized by severe muscle weakness. We and others have previously shown that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of myositis. The present study was undertaken to identify perturbed pathways and assess their contribution to muscle disease in a mouse myositis model. Methods Stable isotope labeling with amino acids in cell culture (SILAC) was used to identify alterations in the skeletal muscle proteome of myositic mice in vivo. Differentially altered protein levels identified in the initial comparisons were validated using a liquid chromatography tandem mass spectrometry spike-in strategy and further confirmed by immunoblotting. In addition, we evaluated the effect of a proteasome inhibitor, bortezomib, on the disease phenotype, using well-standardized functional, histologic, and biochemical assessments. Results With the SILAC technique we identified significant alterations in levels of proteins belonging to the ER stress response, ubiquitin proteasome pathway (UPP), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. We validated the myositis-related changes in the UPP and demonstrated a significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1) in myositis, but not in muscle affected by other dystrophies or normal muscle. Inhibition of the UPP with bortezomib significantly improved muscle function and also significantly reduced tumor necrosis factor α expression in the skeletal muscle of mice with myositis. Conclusion Our findings indicate that ER stress activates downstream UPPs and contributes to muscle degeneration and that UCHL-1 is a potential biomarker for disease progression. UPP inhibition offers a potential therapeutic strategy for myositis.

Cell death, clearance and immunity in the skeletal muscle

“…129 Upregulation of the expression of MHC class I molecules in skeletal muscle fibers is an early feature of IIM and ectopic expression of the molecule induced ER stress response and resulted in a disease that shares several features with human IIM. 130,131 Considering that muscle injury and the associated inflammatory response represent rather common event in vivo, 109 the relative rarity of IIM indicates that a number of events must simultaneously occur to set the stage for self-sustaining autoimmunity. We have recently observed that apoptotic myoblasts are immunogenic only if appropriate adjuvants are provided in the injured/regenerating muscle.…”

Bortezomib inhibits C2C12 growth by inducing cell cycle arrest and apoptosis

“…BTZ prolongs survival during lethal hemorrhagic shock in rats [11]. Rayavarapu et al showed that BTZ significantly improved muscle function and reduced tumor necrosis factor α expression in the skeletal muscle of mice with myositis [12]. Furthermore, the potential use of BTZ in treating cancer cachexia has also been attempted [13].…”

Idiopathic inflammatory myopathies: from immunopathogenesis to new therapeutic targets

“…In addition to making the cells vulnerable to cytotoxic T cell injury, overexpression of HLA class I antigens leads to protein accumulation and misfolding in the endoplasmic reticulum (ER). Such misfolding activates the ER stress pathway leading to activation of the NFκB pathway with enhanced synthesis of pro‐inflammatory cytokines and endogenous class I MHC up‐regulation, and ER death pathway through caspase 12, leading to muscle degeneration . In presence of necrotic cell materials, muscle autoantigens released from regenerating muscles may activate IFN .…”

“…Inhibition of the ubiquitin proteasome pathway with bortezomib improved muscle function in a mouse model of myositis …”

“…Such misfolding activates the ER stress pathway leading to activation of the NFjB pathway with enhanced synthesis of pro-inflammatory cytokines and endogenous class I MHC up-regulation, and ER death pathway through caspase 12, leading to muscle degeneration. 33,34 In presence of necrotic cell materials, muscle autoantigens released from regenerating muscles may activate IFN. 35 Further studies suggest that PAMPs or alarmins, notably high mobility group box 1 (HMB1), released from degenerated and necrotic muscle cells, activate myoblasts through the TLR3 pathway.…”