Tumor necrosis factor alpha (TNFalpha) has been implicated as a mediator of muscle wasting through nuclear factor kappa B (NF-kappaB) -dependent inhibition of myogenic differentiation. The aim of the present study was to identify the regulatory molecule(s) of myogenesis targeted by TNFalpha/NF-kappaB signaling. TNFalpha interfered with cell cycle exit and repressed the accumulation of transcripts encoding muscle-specific genes in differentiating C2C12 myoblasts. Overexpression of a p65 (RelA) mutant lacking the transcriptional activation domain attenuated the TNFalpha-mediated inhibition of muscle-specific gene transcription. The ability of muscle regulatory factor MyoD to induce muscle-specific transcription in 10T1/2 fibroblasts was also disrupted by wild-type p65, demonstrating that NF-kappaB transcriptional activity interferes with the function of MyoD. Inhibition of muscle-specific gene expression by TNFalpha was restored by overexpression of MyoD, whereas endogenous MyoD protein abundance and stability were reduced by TNFalpha through increased proteolysis of MyoD by the ubiquitin proteasome pathway. Last, the inhibitory effects of TNFalpha on myogenic differentiation were demonstrated in a mouse model of skeletal muscle regeneration, in which TNFalpha caused a delay in myoblast cell cycle exit. These results implicate that TNFalpha inhibits myogenic differentiation through destabilizing MyoD protein in a NF-kappaB-dependent manner, which interferes with skeletal muscle regeneration and may contribute to muscle wasting.

Transforming growth factor β1 (TGF-β1) is a cardinal cytokine in the pathogenesis of airway remodeling, and promotes epithelial-to-mesenchymal transition (EMT). As a molecular interaction between TGF-β1 and Jun N-terminal kinase (JNK) has been demonstrated, the goal of this study was to elucidate whether JNK plays a role in TGF-β1-induced EMT. Primary cultures of mouse tracheal epithelial cells (MTEC) from wild-type, JNK1–/– or JNK2–/– mice were comparatively evaluated for their ability to undergo EMT in response to TGF-β1. Wild-type MTEC exposed to TGF-β1 demonstrated a prominent induction of mesenchymal mediators and a loss of epithelial markers, in conjunction with a loss of trans-epithelial resistance (TER). Significantly, TGF-β1-mediated EMT was markedly blunted in epithelial cells lacking JNK1, while JNK2–/– MTEC underwent EMT in response to TGF-β1 in a similar way to wild-type cells. Although Smad2/3 phosphorylation and nuclear localization of Smad4 were similar in JNK1–/– MTEC in response to TGF-β1, Smad DNA-binding activity was diminished. Gene expression profiling demonstrated a global suppression of TGF-β1-modulated genes, including regulators of EMT in JNK1–/– MTEC, in comparison with wild-type cells. In aggregate, these results illuminate the novel role of airway epithelial-dependent JNK1 activation in EMT.

Muscle wasting and increased circulating levels of inflammatory cytokines, including TNF-alpha, are common features of chronic obstructive pulmonary disease. To investigate whether inflammation of the lung is responsible for systemic inflammation and muscle wasting, we adopted a mouse model of pulmonary inflammation resulting from directed overexpression of a TNF-alpha transgene controlled by the surfactant protein C (SP-C) promoter. Compared with wild-type mice, SP-C/TNF-alpha mice exhibited increased levels of TNF-alpha in the circulation and increased endogenous TNF-alpha expression in skeletal muscle, potentially reflecting an amplificatory response to circulating TNF-alpha. Decreased muscle and body weights observed in SP-C/TNF-alpha mice were indicative of muscle wasting. Further evaluation of the SP-C/TNF-alpha mouse musculature revealed a decreased muscle regenerative capacity, shown by attenuated myoblast proliferation and differentiation in response to reloading of disuse-atrophied muscle, which may contribute to skeletal muscle wasting. Importantly, incubation of cultured myoblasts with TNF-alpha also resulted in elevated TNF-alpha mRNA levels and inhibition of myoblast differentiation. Collectively, our results demonstrate that chronic pulmonary inflammation results in muscle wasting and impaired muscle regeneration in SP-C/TNF-alpha mice, possibly as a consequence of an amplificatory TNF-alpha expression circuit extending from the lung to skeletal muscle.

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) inhibits myogenic differentiation via the activation of nuclear factor-kappaB (NF-kappaB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C(2)C(12) cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha-treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha-induced activation of NF-kappaB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappaB. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.