Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-κB activation in response to tumor necrosis factor α

Abstract

Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.

Myeloid cell‐derived tumor necrosis factor‐alpha promotes sarcopenia and regulates muscle cell fusion with aging muscle fibers

“…For example, TNF‐α affects muscle mass through its catabolic role in regulating muscle protein content. TNF‐α treatment of differentiated myotubes activates NFκB, which can lead to reductions in protein content (Li, Schwartz, Waddell, Holloway, & Reid, 1998). TNF‐α also induces the ubiquitin‐proteasome system in cachexia, which can be attenuated by blocking the activation of NFκB signaling (Reid & Li, 2001).…”

“…TNF‐α can also decrease muscle protein content by inhibiting protein synthesis through the induction of IL‐6 or inhibition of insulin‐like growth factor‐I signaling (Alvarez et al, 2002; Frost, Lang, & Gelato, 1997). Increased TNF‐α levels can induce apoptosis in disease models and during muscle aging through the increase in cell death‐inducing receptor, Fas (CD95), and the interaction of the TNF‐α receptor complex and the Fas‐associated protein with death domain (Lees, Zwetsloot, & Booth, 2009; Li et al, 1998). Thus, multiple mechanisms may underlie the reduction in sarcopenia in TNF‐α‐mutant mice, in addition to the effects we report concerning influences on satellite cell function and muscle cell fusion during aging.…”

Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin–proteasome pathway

“…Watchorn et al (2001) have shown PIF to activate both the transcription factors NF-kB and STAT3 in hepatic cells. Li et al (1998) showed that TNF-a stimulated protein loss in C 2 C 12 myotubes involved the ubiquitin -proteasome proteolytic pathway and was accompanied by nuclear translocation of NF-kB to its targeted DNA sequence by stimulating phosphorylation, ubiquitin conjugation and proteolysis of I-kBa. Total protein content and myosin heavy chain levels in C 2 C 12 myotubes were found to be unaltered in cells transfected with viral plasmid constructs that overexpress mutant I-kBa proteins that are insensitive to degradation by the ubiquitin -proteasome pathway (Li and Reid, 2000).…”

NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin–proteasome system in skeletal muscle

“…In this process, protein substrates are conjugated with a polyubiquitin chain, which enables them to be recognised for degradation by the proteasome, a multi-subunit complex containing a range of proteolytic enzymes. The main mediators known to influence expression of polyubiquitin genes and proteasomal subunits are glucocorticoids (Wang et al, 1998), cytokines such as tumour necrosis factor-a (TNF-a) (Li et al, 1998) and proteolysis-inducing factor (PIF) (Lorite et al, 2001), a sulphated glycoprotein produced by cachexia-inducing murine and human tumours (Todorov et al, 1996), which specifically induces degradation of skeletal muscle (Lorite et al, 1998).…”

“…Glucocorticoids also induce gene transcription and protein synthesis of the NF-kB inhibitor, IkB, and inhibit the expression of cytokines (Almawi and Melemedjian, 2002). In contrast, induction of protein degradation by TNF-a, which is also mediated through the ubiquitin -proteasome proteolytic pathway (Li et al, 1998), appears to be mediated through proteasomal degradation of IkBa and translocation of NF-kB to the nucleus (Li and Reid, 2000). We have also recently shown that both PIF and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), an intracellular signal for the increased protein degradation induced by PIF , cause degradation of IkBa and nuclear accumulation of NF-kB, associated with an increased proteasome expression.…”