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Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor
Y P Li1, R J Schwartz, I D Waddell
1Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
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.
Insights
Tumor necrosis factor alpha (TNF-alpha) directly causes skeletal muscle protein loss by activating nuclear factor kappaB (NF-kappaB) signaling. This process involves reactive oxygen species and leads to muscle atrophy in chronic diseases.
Area of Science:
- Cellular biology
- Molecular biology
- Physiology
Background:
- Skeletal muscle atrophy is linked to chronic diseases and tumor necrosis factor alpha (TNF-alpha).
- The direct impact of TNF-alpha on differentiated muscle cells and its signaling pathways remain unclear.
Purpose of the Study:
- To investigate the direct effects of TNF-alpha on differentiated skeletal muscle cells.
- To elucidate the signaling mechanisms underlying TNF-alpha-induced muscle atrophy.
Main Methods:
- Utilized mouse C2C12 myotubes and primary rat skeletal muscle cultures.
- Administered TNF-alpha and measured protein content, myosin heavy chain (MHCf) levels, DNA content, and MHCf synthesis.
- Assessed nuclear factor kappaB (NF-kappaB) activation, I-kappaBalpha degradation, and ubiquitin conjugation.
- Employed proteasome inhibitor (MG132) and catalase to explore signaling pathways and reactive oxygen species involvement.
Main Results:
- TNF-alpha induced dose- and time-dependent reductions in total protein and MHCf content in differentiated myotubes.
- These effects occurred at low TNF-alpha concentrations without altering DNA content or MHCf synthesis.
- TNF-alpha activated NF-kappaB binding and stimulated I-kappaBalpha degradation, indicating NF-kappaB pathway activation.
- Reactive oxygen species (ROS) mediate TNF-alpha's activation of NF-kappaB and I-kappaBalpha degradation, as shown by catalase inhibition and hydrogen peroxide's effects.
Conclusions:
- TNF-alpha directly causes skeletal muscle protein loss in differentiated cells.
- NF-kappaB signaling is rapidly activated by TNF-alpha in skeletal muscle.
- The TNF-alpha/NF-kappaB pathway in skeletal muscle is regulated by endogenous reactive oxygen species.