We have a longstanding interest in respiratory muscle physiology and the effects of free radicals on muscle function. The current project examines an intracellular signaling mechanism in which free radicals may function as essential components. Tumor necrosis factor-alpha (TNF- alpha) mediates skeletal muscle atrophy and weakness in a variety of chronic diseases; however, little is known about the direct effects of TNFalpha on muscle or the signaling mechanism(s) involved. In nonmuscle cell types, TNFalpha stimulates production of reactive oxygen species (ROS) which can activate nuclear factor-kB (NF-kB). This TNF alpha/NF- kB pathway is not ubiquitous, however. Post-receptor signaling mechanisms for TNFalpha are cell type-specific and have not been investigated in muscle. The current project addresses this issue by testing the direct effects of TNFalpha on diaphragm and other skeletal muscle cells and by evaluating the TNF-alpha/NF-kB pathway as a putative signaling mechanism. Experiments will be conducted on fiber bundles isolated from mouse diaphragm and on cultured muscle cells to address four specific aims:
Aim 1. To determine whether TNF-alpha directly stimulates protein loss in muscle. Other investigators have concluded that TNFalpha does not stimulate muscle catabolism directly. We will test this assertion by exposing cultured muscle cells to TNFalpha for 24-96 hrs; catabolism will be assessed by measuring myocyte size, total protein content, and myosin levels.
Aim 2. Evaluate NF-kB activation as an early response of muscle to TNFalpha. We will evaluate NF-kB activation in TNF-alpha-stimulated muscle by testing for depletion of I-kB (a NF-kB inhibitory protein) and nuclear translocation of NF-kB.
Aim 3. Determine whether ROS mediate NF-kB activation in muscle. We will test the capacities of TNFalpha to stimulate ROS production, of exogenous ROS to activate NF-kB, and of antioxidant enzymes to inhibit NF-kB activation by TNFalpha.
Aim 4. Test the importance of ubiquitin in NF-kB activation by TNFalpha. In other cell types, TNFalpha stimulates I-kB conjugation with ubiquitin; this leads to I-kB proteolysis via the 26S proteasome complex thereby activating NF-kB. We will evaluate this mechanism in TNF-alpha-stimulated muscle by testing for increased ubiquitin conjugation and by testing the capacity of proteasome inhibitors to block NF-kB activation.
| Li, Wei; Moylan, Jennifer S; Chambers, Melissa A et al. (2009) Interleukin-1 stimulates catabolism in C2C12 myotubes. Am J Physiol Cell Physiol 297:C706-14 |
| Moylan, Jennifer S; Smith, Jeffrey D; Chambers, Melissa A et al. (2008) TNF induction of atrogin-1/MAFbx mRNA depends on Foxo4 expression but not AKT-Foxo1/3 signaling. Am J Physiol Cell Physiol 295:C986-93 |
| Hardin, Brian J; Campbell, Kenneth S; Smith, Jeffrey D et al. (2008) TNF-alpha acts via TNFR1 and muscle-derived oxidants to depress myofibrillar force in murine skeletal muscle. J Appl Physiol 104:694-9 |
| Smith, Melissa A; Moylan, Jennifer S; Smith, Jeffrey D et al. (2007) IFN-gamma does not mimic the catabolic effects of TNF-alpha. Am J Physiol Cell Physiol 293:C1947-52 |
| Moylan, Jennifer S; Reid, Michael B (2007) Oxidative stress, chronic disease, and muscle wasting. Muscle Nerve 35:411-29 |
| Durham, William J; Arbogast, Sandrine; Gerken, Eric et al. (2006) Progressive nuclear factor-kappaB activation resistant to inhibition by contraction and curcumin in mdx mice. Muscle Nerve 34:298-303 |
| Smith, Melissa A; Reid, Michael B (2006) Redox modulation of contractile function in respiratory and limb skeletal muscle. Respir Physiol Neurobiol 151:229-41 |
| Gong, Ming C; Arbogast, Sandrine; Guo, Zhenheng et al. (2006) Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells. J Appl Physiol 100:399-405 |
| Chen, Shuen-Ei; Gerken, Eric; Zhang, Yingmin et al. (2005) Role of TNF-{alpha} signaling in regeneration of cardiotoxin-injured muscle. Am J Physiol Cell Physiol 289:C1179-87 |
| Li, Yi-Ping; Chen, Yuling; John, Joseph et al. (2005) TNF-alpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle. FASEB J 19:362-70 |
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