Muscle wasting is a debilitating complication of cancer, AIDS, and other chronic inflammatory diseases. Loss of muscle mass results primarily from accelerated protein degradation via the ubiquitin-proteasome pathway (1
). Proteins degraded by this mechanism are first covalently linked to a chain of ubiquitin molecules, which marks them for rapid breakdown by the 26S proteasome (2
). The selectivity of ubiquitin targeting to protein substrates is primarily a function of ubiquitin ligases (E3 proteins), a family of enzymes that catalyze the transfer of activated ubiquitin from a specific ubiquitin carrier (E2) protein to a lysine residue on the substrate.
In skeletal muscle, three E3 proteins are known to regulate ubiquitin conjugation in catabolic states: E3α, atrogin1/MAFbx, and MuRF1. E3α interacts with E214k
to attach ubiquitin to protein substrates according to the “N-end rule” (3
). Atrogin1/MAFbx and MuRF1 are up-regulated and appear to be essential for accelerated muscle protein loss in a variety of experimental models of catabolism. These include fasting, diabetes, cancer, renal failure, hindlimb suspension, immobilization, denervation, sepsis, and lipopolysaccharide administration (4
). Because the atrogin1/MAFbx and MuRF1 genes respond to such diverse catabolic conditions, it is important to determine the cellular mechanisms that regulate expression of these genes.
In studies of atrogin1/MAFbx regulation, we recently found that mRNA levels are increased by exposing muscle cells to exogenous H2
). Skeletal muscle myocytes continuously generate H2
and other reactive oxygen species (ROS) that function as intracellular signaling molecules (10
). ROS production (11
) and ROS-mediated signaling (13
) are stimulated by tumor necrosis factor-α (TNF-α), a catabolic cytokine (15
) that increases general activity of the ubiquitin conjugating pathway (16
). Therefore, as observed for H2
, we hypothesized that TNF-α would up-regulate atrogin1/MAFbx expression in skeletal muscle. The present study tested this hypothesis, measuring changes in mRNA levels and ubiquitin conjugating activity in muscle cells stimulated by TNF-α. Our approach incorporated several experimental preparations including differentiated myotubes, excised muscles, and TNF-α-treated mice. Responses to TNF-α were compared with those elicited by H2
, a positive control.
We evaluated mitogen-activated protein kinases (MAPKs) as potential second messengers for TNF-α. Data from nonmuscle cell types indicate that TNF-α and H2
activate MAPKs, including p38, ERK1/2, and JNK (13
). p38 MAPK has been identified as a potential regulator of muscle catabolism (19
). Recent studies show that p38 activity in skeletal muscle is elevated by procatabolic states, which include limb immobilization (20
), acute quadriplegic myopathy (21
), Type 2 diabetes (22
), neurogenic atrophy (21
), and aging (23
). To test p38 MAPK involvement in atrogin1/MAFbx regulation, we measured TNF-α effects on total and phosphorylated enzyme levels in muscle and tested the effects of p38 MAPK inhibitors on TNF-α-stimulated atrogin1/MAFbx gene expression. Control studies screened ERK1/2 and JNK for similar involvement.
Our results suggest the atrogin1/MAFbx gene product is up-regulated in response to TNF-α, an event that precedes the rise in general ubiquitin conjugating activity. Atrogin1/MAFbx up-regulation in response to either TNF-α or H2O2 is associated with activation of p38 MAPK, ERK1/2, and JNK. The former appears to be essential for atrogin1/MAFbx up-regulation since p38 inhibitors block increases in gene expression and ubiquitin conjugating activity.