FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Y.-p.
Right arrow Articles by Reid, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Y.-p.
Right arrow Articles by Reid, M. B.
(The FASEB Journal. 1998;12:871-880.)
© 1998 FASEB

RESEARCH COMMUNICATION

Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-{kappa}B activation in response to tumor necrosis factor {alpha}

Yi-ping Lia, Robert J. Schwartzb, Ian D. Waddellc, Brian R. Hollowayc, and Michael B. Reida,1

a Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA
b Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA
c Zeneca Pharmaceuticals, Alderley Park, Cheshire, England, SK10 4TG


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
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 {kappa}B (NF-{kappa}B) to its targeted DNA sequence and stimulated degradation of I-{kappa}B{alpha}, an NF-{kappa}B inhibitory protein. TNF-{alpha} stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10–40 µM) blocked TNF-{alpha} activation of NF-{kappa}B. Catalase 1 kU/ml inhibited NF-{kappa}B activation by TNF-{alpha}; exogenous hydrogen peroxide 200 µM activated NF-{kappa}B and stimulated I-{kappa}B{alpha} degradation. These data demonstrate that TNF-{alpha} directly induces skeletal muscle protein loss, that NF-{kappa}B is rapidly activated by TNF-{alpha} in differentiated skeletal muscle cells, and that TNF-{alpha}/NF-{kappa}B signaling in skeletal muscle is regulated by endogenous reactive oxygen species.—Li, Y.-P., Schwartz, R. J., Waddell, I. D., Holloway, B. R., Reid, M. B. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-{kappa}B activation in response to tumor necrosis factor {alpha}. FASEB J. 12, 871–880 (1998)


Key Words: cachexia • myosin • cytokines • inflammatory disease • tumor necrosis factor • ubiquitin • free radicals


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
SKELETAL MUSCLE WASTING and negative nitrogen balance occur in a variety of chronic diseases that include sepsis, cancer, congestive heart failure, and AIDS (1). The ensuing loss of muscle mass and contractile dysfunction contribute to weakness, fatigue, and loss of mobility in affected individuals. A primary factor responsible for these changes is an increase in circulating tumor necrosis factor {alpha} (TNF-{alpha}),2 a polypeptide cytokine that promotes antitumor and immune responses. In inflammatory disease, circulating TNF-{alpha} levels can increase markedly, with serum values as high as 2.8 ng/ml reported for patients with rheumatoid arthritis (2) and 6 ng/ml for some cancer patients (3). Muscle loss has been documented in animals treated with exogenous TNF-{alpha} (4, 5), in animals that express a transgene (6), and in diseases that elevate endogenous TNF-{alpha}, i.e., experimental sepsis (7) or tumor implantation (8). Muscle wasting primarily reflects loss of structural protein; accordingly, TNF-{alpha}-induced catabolism depletes animals of muscle-specific proteins such as myosin heavy chains (4, 7). These data clearly identify TNF-{alpha} as a mediator of muscle pathology, but the underlying mechanism remains enigmatic. To increase our understanding of TNF-{alpha} effects, we used differentiated myotubes derived from skeletal muscles of mice (C2C12 cell line) and rats (primary cell cultures) to test three hypotheses.

Hypothesis 1. Prolonged exposure to TNF-{alpha} directly stimulates protein loss in skeletal muscle cells
The catabolic changes observed in vivo are strongly influenced by indirect effects of TNF-{alpha} on hormones that regulate muscle growth (8–11), on the expression of other cytokines (12, 13), and on TNF-induced anorexia (14, 15). To test for direct effects, previous investigators have incubated excised skeletal muscles with TNF-{alpha} for up to 3 h (5, 16–18). Neither amino acid release nor protein breakdown was altered under these conditions, leading others to conclude that TNF-{alpha} does not stimulate muscle catabolism directly. However, unlike earlier protocols, muscle wasting occurs in humans and experimental animals over a period of days to weeks. The possibility remains that TNF-{alpha} acts directly on muscle but that protein loss is detectable only over longer time spans (19).

Hypothesis 2. TNF-{alpha} activates NF-{kappa}B in differentiated muscle
TNF-{alpha} alters gene expression by activating nuclear transcription factors that bind to DNA promoter regions and influence transcription (20). In nonmuscle cell types, a principal transcription factor induced by TNF-{alpha} is nuclear factor {kappa}B (NF-{kappa}B). NF-{kappa}B exists in the cytoplasm as an inactive complex bound by the inhibitory protein I-{kappa}B{alpha}. TNF-{alpha} stimulates ubiquitin conjugation of I-{kappa}B{alpha} and subsequent degradation of I-{kappa}B{alpha} by the 26S proteasome. NF-{kappa}B is thereby activated and translocates to the nucleus (21, 22). Postreceptor signaling mechanisms vary among cell types, accounting for the pleiotropic nature of TNF-{alpha} (20). TNF-{alpha} signal transduction in skeletal muscle is largely unstudied and NF-{kappa}B activation has not been evaluated in differentiated muscle.

Hypothesis 3. Reactive oxygen species (ROS) are endogenous mediators of TNF-{alpha}/NF-{kappa}B signaling in muscle
TNF-{alpha} has been shown to stimulate ROS production by mitochondria (23, 24); in selected cell types, endogenous ROS mediate NF-{kappa}B activation (25). This component of TNF-{alpha}/NF-{kappa}B signaling is not universal; it is important in some cell types but not in others (26). The role of endogenous ROS in NF-{kappa}B activation has not been tested in skeletal muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Cultured myogenic cell line
Myoblasts derived from mouse skeletal muscle (C2C12 cells; American Type Culture Collection, Rockville, Md.) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% newborn calf serum and gentamicin at 37°C in the presence of 5% CO2. Myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% heat-inactivated horse serum. The muscle cells were examined for evidence of myotube formation and growth by using a light microscope (Labophot; Nikon Corp., Tokyo, Japan). Myotubes were treated with murine TNF-{alpha} (Boehringer-Mannheim, Indianapolis, Ind.) by adding 1–10 ng/ml TNF-{alpha} to the differentiation medium every 24 h.

Primary rat myocyte cultures
Neonatal rats (2 to 4 days old) were killed by cervical dislocation using methods approved by the Animal Research Committee of Baylor College of Medicine. Limb skeletal muscles were excised, minced with razor blades in a minimal volume of phosphate-bufferd saline (PBS), and enzymatically dissociated in dissociation buffer (0.1% trypsin, 0.1% collagenase type 2, and 0.025% DNAse in PBS) at 37°C for 5 min. The slurry was centrifuged in 20 ml DF20 (20% fetal bovine serum in DMEM) at 150 g for 1 min at 4°C and the supernatant was discarded. The dissociation process was repeated three times. The pelleted cells were incubated each time in dissociation buffer for 15 min and the supernatant was collected by centrifuging at 300 g for 7 min at 4°C. The dissociated cells were pelleted and resuspended in 1.082 gm/ml Percoll (Pharmacia, Piscataway, N.J.) for purification through a density gradient (1.050, 1.060, and 1.082 gm/ml) by centrifugation at 2000 g for 25 min at room temperature. The Percoll gradient was made in a buffer containing 6.8 gm/l NaCl, 0.4 gm/l KCl, 0.1 gm/l MgSO4, 1.5 gm/l NaH2PO4, 1.0 gm/l dextrose, and 4.76 gm/l HEPES (pH 7.3). The band containing myocytes at the interface between 1.060 and 1.082 gm/ml Percoll layers was collected and washed twice in the gradient buffer plus 0.02 gm/l phenol red. The cells were resuspended in Ham's F-10 nutrient mixture (GIBCO BRL, Gaithersburg, Md.) supplemented with 20% fetal calf serum, then plated in primaria dishes and grown at 37°C in the presence of 5% CO2. When the cells were 80% confluent, the medium was replaced by differentiation medium (DMEM supplemented with 2% heat inactivated horse serum) and the cells were allowed to differentiate for 96 h before experimentation.

Protein and DNA determinations
Myotubes were collected by trypsinization and divided into two parts for protein and DNA assays. Cells used for protein determinations were boiled in Laemmli buffer for 5 min and the lysates were assayed by the method of Lowry (27). DNA was extracted using the DANAzol reagent (GIBCO BRL) and measured by the DyNAquant 200 fluorometer (Pharmacia).

Preparations of cell extracts
Cytoplasmic extracts were prepared by treating the cells with three cycles of freezing-thawing in a buffer solution containing 10 mM HEPES-KOH (pH 7.9), 5% glycerol, 100 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF (phenylmethylsulfonyl fluoride), 1 µg/ml leupeptin, and 2 µg/ml aprotinin. Supernatants were collected after centrifuging samples for 10 s in a microcentrifuge. Nuclear extracts were prepared by resuspending the pellets in a solution of 20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 1 µg/ml leupeptin, and 2 µg/ml aprotinin. The resuspended pellets were incubated at 4°C for 20 min and centrifuged for 10 min in a microcentrifuge at 4°C; supernatants then were collected.

Electrophoresis mobility shift assay (EMSA)
EMSA assays were carried out as described previously (28). The sequence of the NF-{kappa}B binding DNA probe was 5'-AGTTGAGGGGACTTTCCCAGGC-3'. The mutant probe used for competition had six mutated nucleotides (shown in italics) within the binding sequence GGAGTCAACC. For supershift analyses, 300 µg of polyclonal antibody to the p65 subunit of NF-{kappa}B (anti-p65; Santa Cruz Biotechnologies, Santa Cruz, Calif.) was added to the EMSA incubation medium after nuclear proteins had bound to the DNA probe; incubation was continued for an additional 16 h at 4°C.

Analysis of MHCf synthesis
C2C12 cells were cultured, differentiated, and treated with 1, 3, or 6 ng/ml TNF-{alpha} for 72 h as described above. After 68 h incubation with TNF-{alpha}, myotubes were washed twice with methionine-free DMEM and incubated in methionine-free DMEM supplemented with 0.1 mCi/ml [35S]methionine (>1000 Ci/mmol, Amersham Life Science, Arlington Heights, Ill.) for 4 h. Cells were washed twice with PBS and collected by scraping; cytoplasmic extracts were prepared as described above. Approximately 100 µg of each extract was precleared and then tumbled with the MHCf antibody (Novocastra Laboratories, Newcastle, England) in 200 ml of buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA (pH 8.0), 0.25% gelatin, and 0.02% sodium azide at 4°C for 1 h. The immune complex was collected with protein G PLUS/protein A agarose beads (Oncogene Science, Cambridge, Mass.) by incubating the beads with the reaction mixture at 4°C for 1 h with tumbling. After three washes with buffer, the pellets were boiled in Laemmli buffer and the immunoprecipitate was separated on 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The labeled MHCf was visualized by exposing the dried gel to X-ray film. MHCf synthesis was quantified by analyzing autoradiographs with the use of ImageMaster densitometry software (Pharmacia).

Western blot analysis
Protein samples were separated with SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated in the presence of appropriate primary antibodies to MHCf (Novocastra) or I-{kappa}B{alpha} (Santa Cruz). Horseradish peroxidase was conjugated to the primary antibodies, using secondary antibodies. Antibodies were visualized by the enhanced chemiluminescence method (Amersham). The bands detected on the X-ray films were quantified using densitometry software (Pharmacia).

Ubiquitin conjugation assay
Immunoabsorbent microtiter plates (Nunc, Inc., Naperville, Ill.) were pretreated with 100 µl of 0.1% polyethylenimine for 6 h. The plates then were washed in PBS containing 0.05% Tween 20 (wash buffer). Lysed cells and standards were diluted in PBS; 100 µl of each dilution was transferred to the microtiter plate and incubated at 4°C for 16 h. The plates then were washed, blocked in 100 µl 1% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) for 2 h at 37°C, and rewashed. Rabbit anti-ubiquitin (Boehringer-Mannheim) was added to dilution buffer to achieve 4 µl/ml; 100 µl anti-ubiquitin solution was added to each well and incubated at 4°C for 16 h. Plates were rewashed; peroxidase-conjugated anti-rabbit immunoglobulin G (Amersham) was diluted 1500-fold in PBS and 100 µl was added to each well; plates were incubated at room temperature for 2 h. Plates were rewashed with PBS and each well was loaded with 100 µl substrate solution containing 0.15 ml/ml 3,3',5,5'-tetramethylbenzidine and 0.0025% hydrogen peroxide in 100 mM sodium acetate at pH 5.5. Color development was allowed to proceed for 20 min at room temperature, after which 50 µl 0.6 M H2SO4 was added to stop the reaction. The absorbance at 450 nm was determined for each well by using a Labsystems iEMS plate reader (Life Sciences International, Basingstoke, U.K.).

Statistics
Data were entered into computer files and evaluated for differences among groups by using commercial software (SigmaStat; Jandel Scientific, Corte Madera, Calif.). Time-dependent decrements in total protein content and MHCf levels were assessed using linear regression analysis (29). Changes in total conjugated ubiquitin content were assessed using one-way analysis of variance; differences at single time points were evaluated post hoc with a Tukey's test (29). Catalase effects on NF-{kappa}B activation were evaluated by using the Student's paired t test (29). Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Effects of TNF-{alpha} on myotube growth
Muscle cells underwent differentiation after serum restriction. Myoblasts fused to form myotubes that were visible by light microscopy within 24 h and that subsequently increased in size. Total protein content was measured as an index of myotube growth. Figure 1 illustrates data from a representative experiment. Protein content of control myotubes increased progressively for approximately 96 h and then remained constant for an additional 72 h, i.e., until 168 h postdifferentiation. During this phase, culture composition remained constant (85–90% differentiated myotubes, 10–15% residual myoblasts) and total DNA content was unchanged (data not shown). Relative to time-matched control cells, initiation of TNF-{alpha} exposure at either 48 or 96 h postdifferentiation caused a progressive loss in total protein over the ensuing 72 h. A standardized protocol was used in all subsequent experiments. TNF-{alpha} treatment was initiated 96 h after differentiation, when myotube protein content is relatively stable, and was continued for 72 h.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Time dependence of TNF-{alpha} effects on total protein in muscle cells. Addition of TNF-{alpha} 10 ng/ml to C2C12 myotubes either 48 or 96 h after serum restriction (denoted by arrows) caused progressive loss of total protein (filled circles) relative to time-matched control cells (open circles). Myotubes cultured in multiple plates were serum restricted at time 0 h; plates were removed at the times indicated for analysis of total protein; each data point denotes the protein content of an individual plate. Results are from one of five experiments in which TNF-{alpha} (10 ng/ml) was shown to decrease C2C12 myotube protein content.

Myosin is a major functional protein of adult skeletal muscle and loss of skeletal muscle myosin is typical in TNF-{alpha}-treated animals (4, 7). Figure 2 shows that TNF-{alpha} had similar effects on our cultured muscle cells; Western analysis revealed that MHCf content decreased in myotubes exposed to 1–6 ng/ml TNF-{alpha}. Figure 3 shows that decrements in total protein content and MHCf were concentration dependent; the fall in MHCf was greater than the fall in total protein, illustrating sensitivity of MHCf to TNF-{alpha}. The lower TNF-{alpha} concentrations of 1 and 3 ng/ml decreased MHCf content without significant loss of DNA, a pattern analogous to chronic muscle wasting. We assessed TNF-{alpha} effects on MHCf synthesis by measuring changes in [35S]methionine-labeled MHCf. No significant changes were observed after 1 ng/ml TNF-{alpha} (80% control ± 30 SE), 3 ng/ml (69±36), or 6 ng/ml (112±49) when compared with time-matched control myotubes (n = three/group).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. TNF-{alpha} decreases MHCf content. Western blot compares MHCf from control C2C12 myotube lysates with MHCf of myotubes exposed to 1, 3, or 6 ng/ml TNF-{alpha} for 72 h. Equal amounts of protein were loaded in each lane. Anti-MHCf bound to MHCf was detected by the enhanced chemiluminescence method and quantified by densitometry software (see Materials and Methods).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Concentration dependence of TNF-{alpha} effects on total protein and MHCf. Incubation with TNF-{alpha} (1–6 ng/ml) caused concentration-dependent losses of total protein (open circles) and MHCf (filled circles) in C2C12 myotubes; means shown ± SE; n = 4; P values depict differences of linear regression slopes from zero.

TNF-{alpha}/NF-{kappa}B signaling
We tested the capacity of TNF-{alpha} to stimulate translocation of NF-{kappa}B to the myotube nucleus and depletion of I-{kappa}B{alpha} from the cytosol. As shown by EMSA in Fig. 4, NF-{kappa}B binding to its consensus DNA binding sequence increased after TNF-{alpha} treatment; NF-{kappa}B binding was elevated at 15 min and peaked at 30 min. Specificity was further confirmed by supershift analysis in which a supershifted anti-p65/NF-{kappa}B complex was clearly detectable (data not shown). After 30 min, NF-{kappa}B binding decreased but still remained above control levels 120 min after TNF-{alpha} challenge (data not shown). This response did not differ appreciably between C2C12 myotubes ( Fig. 4A) and primary myotubes ( Fig. 4B). TNF-{alpha} also induced rapid disappearance of I-{kappa}B{alpha} from the cytoplasm. Figure 5 shows Western analyses of cytoplasmic extracts from the same cells in which NF-{kappa}B translocation was measured (above). Rapid breakdown of I-{kappa}B{alpha} was evident 15 min after TNF-{alpha} treatment; this was followed by a period of slow recovery that remained incomplete at 120 min.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. Activation of NF-{kappa}B by TNF-{alpha} demonstrated by EMSA. A) Nuclear extracts of C2C12 myotubes treated with 3 ng/ml TNF-{alpha} for 15 or 30 min (lane 3 and 4) were compared with extract from control myotubes (lane 2) and with extract-free buffer (lane 1). Lanes 5 and 6 are competitions with 500-fold concentration of cold DNA probe or 500-fold concentration of cold DNA probe with mutated NF-{kappa}B binding sequence (see Materials and Methods), using the same extract as lane 4. These results are from one of five separate experiments in which TNF-{alpha} was shown to activate NF-{kappa}B in C2C12 myotubes. B) Nuclear extracts of rat primary myotubes treated with TNF-{alpha} (6 ng/ml) for 15 or 30 min (lanes 3 and 4) were compared with extract from control myotubes (lane 2) and with extract-free buffer (lane 1); results are representative of duplicate experiments in which TNF-{alpha} was shown to activate NF-{kappa}B in primary myotubes.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. I-{kappa}B{alpha} degradation induced by TNF-{alpha}. Western blot (inset) shows I-{kappa}B{alpha} levels in cytoplasmic extracts before TNF-{alpha} exposure (0 min) and 15, 30, 60, or 120 min thereafter; a polyclonal anti-I-{kappa}B (Santa Cruz) was used to detect I-{kappa}B{alpha}. Plot of optical density measurements from the same gel quantifies I-{kappa}B{alpha} levels. Results are from one of four separate experiments in which TNF-{alpha} was shown to induce I-{kappa}B{alpha} degradation in C2C12 myotubes; similar I-{kappa}B{alpha} degradation was also observed in primary myotubes exposed to TNF-{alpha} (data not shown).

We subsequently tested involvement of the ubiquitin/proteasome pathway. TNF-{alpha} (3 ng/ml) stimulated ubiquitin conjugation of muscle proteins ( Fig. 6). Enzyme-linked immunosorbent assay (ELISA) measurements detected an increase in total conjugated ubiquitin within 30 min of exposure to TNF-{alpha}; ubiquitin conjugation peaked at 60 min, reaching 330% of the control value. Pharmacologic inhibition of 26S proteasome activity inhibited NF-{kappa}B activation by TNF-{alpha} ( Fig. 7). Myotubes were pretreated for 1 h with MG132, a competitive inhibitor of 26S proteasome peptidase activity (30), and then were challenged with TNF-{alpha} (3 ng/ml). Pretreatment with MG132 inhibited NF-{kappa}B activation in a dose-dependent manner; partial inhibition was detectable after MG132 10 µM whereas MG132 40 µM produced nearly complete inhibition.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 6. Total cellular ubiquitin conjugation stimulated by TNF-{alpha}. C2C12 myotubes were treated with TNF-{alpha} (3 ng/ml) for the time periods indicated. Total conjugated ubiquitin in myotube lysates was measured by ELISA as described in Methods; means shown ± SE; n = 6; overall significance of time-dependent changes was P < 0.01; *value different from value at time 0 (P<0.05).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 7. The 26S proteasome inhibitor MG132 blocks TNF-{alpha} activation of NF-{kappa}B. EMSA demonstrates a concentration-dependent inhibition of TNF-{alpha}-stimulated NF-{kappa}B activity by MG132. MG132 dissolved in DMSO (lane 3, 4, and 5) or DMSO only (final concentration of DMSO 0.2%, lane 6) was added to the cell culture medium 1 h before a 30 min TNF-{alpha} treatment. Results are from one of two experiments on C2C12 myotubes that yielded similar results.

ROS effects on TNF-{alpha}/NF-{kappa}B signaling
We tested the role of endogenous ROS by pretreating muscle cells with catalase 1 kU/ml, an antioxidant enzyme that dehydrates hydrogen peroxide to water (31) and decreases cytosolic oxidant levels in skeletal muscle fibers (32). One hour after catalase treatment, muscle cells were challenged with TNF-{alpha} (3 ng/ml). Figure 8 shows results from experiments in C2C12 myotubes. Catalase pretreatment consistently inhibited NF-{kappa}B activation (three of three trials; P<0.025), which identifies endogenous hydrogen peroxide or its redox derivatives as modulators of the TNF-{alpha}/NF-{kappa}B pathway. As a positive control, we also tested the capacity of exogenous hydrogen peroxide to activate NF-{kappa}B. Hydrogen peroxide (200 µM) stimulated NF-{kappa}B binding to DNA in the absence of TNF-{alpha} ( Fig. 9A); this effect peaked at approximately 2 h and was still detectable after 4 h. Exogenous hydrogen peroxide also stimulated degradation of I-{kappa}B{alpha} ( Fig. 9B); protein levels in the cytosol were decreased at 2 h and had partially recovered after 3 h. Subsequent studies using primary myotubes confirmed the role of ROS in NF-{kappa}B activation. Pretreatment with catalase 1 kU/ml inhibited activation by TNF-{alpha} and hydrogen peroxide stimulated activation (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. Catalase inhibits NF-{kappa}B activation by TNF-{alpha}. EMSA demonstrates that 1 h pretreatment with catalase (1 kU/ml: lane 3) depresses subsequent NF-{kappa}B activation stimulated by 30 min incubation with TNF-{alpha} (3 ng/ml: lane 2); representative results from one of three separate experiments that used C2C12 myotubes; mean effect was a 39.3% ± 8.8 SE decrease in optical density of NF-{kappa}B band by catalase pretreatment (P<0.025). Similar inhibition was also seen in studies of rat primary myotubes (data not shown).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 9. Exogenous hydrogen peroxide stimulates NF-{kappa}B translocation and I-{kappa}B{alpha} degradation. A) Hydrogen peroxide added to the culture medium (200 µM) activated NF-{kappa}B as demonstrated by EMSA; representative results from one of three experiments using C2C12 myotubes; hydrogen peroxide also was shown to activate NF-{kappa}B in rat primary myotubes (data not shown). B) Hydrogen peroxide (200 µM) induced I-{kappa}B{alpha} degradation as demonstrated by Western blot; representative results are from one of two duplicate experiments using C2C12 myotubes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
TNF-{alpha} is a primary mediator of skeletal muscle pathology in inflammatory disease, but surprisingly little is known about the direct effects of TNF-{alpha} on muscle cells. Nor has the mechanism (or mechanisms) of postreceptor signaling been examined in differentiated skeletal muscle. The findings of this project establish that TNF-{alpha} directly stimulates protein loss in skeletal muscle, that a TNF-{alpha}/NF-{kappa}B pathway exists in differentiated muscle, and that endogenous ROS modulate this signaling mechanism. The physiologic relevance of these observations is bolstered by our use of differentiated myotubes rather than undifferentiated cells. Also, data on TNF-{alpha} signaling mechanisms in muscle-derived C2C12 myotubes were confirmed when using primary cultures from rat limb muscle. These strategies increased the likelihood that TNF-{alpha} actions observed in this study resemble those in mature skeletal muscle.

TNF-{alpha} stimulates protein loss
Loss of muscle mass is the hallmark of TNF-{alpha}-induced muscle pathology. TNF-{alpha} was previously designated `cachectin' in recognition of its catabolic action (33, 34), and is thought to cause muscle wasting and negative nitrogen balance in human diseases such as cancer and sepsis (1). Because myosin heavy chains undergo accelerated breakdown, myosin has been used as a muscle-specific structural protein to assess TNF-{alpha}-induced catabolism (4, 7). Experimental animals lose muscle mass when treated with exogenous TNF-{alpha} (4, 5) or subjected to pathologic processes that elevate endogenous TNF-{alpha}, e.g., sepsis (7) or tumor implantation (8).

The mechanism of TNF-{alpha} effects in vivo remains enigmatic. It has long been recognized that TNF-{alpha} may stimulate catabolism via indirect mechanisms. TNF-{alpha} alters circulating levels of hormones that regulate muscle growth, including insulin, glucagon, thyroid hormone, glucocorticoids, and catecholamines (8, 9, 11), and affects tissue sensitivity to such factors (10). TNF-{alpha} also stimulates production of catabolic cytokines (e.g., prostaglandin E2 and interleukin-1) (12, 13) and promotes anorexia (14, 15). Each of these effects indirectly promotes muscle wasting. In contrast, previous attempts to demonstrate a direct catabolic effect of TNF-{alpha} on skeletal muscle have been uniformly unsuccessful (5, 16–18). Excised rodent muscles have been exposed to supraphysiologic TNF-{alpha} concentrations of 20 ng/ml (18) to 26,000 ng/ml (5) for up to 3 h. No changes were observed in extracellular alanine release (18), tyrosine release (5), or net protein breakdown (16, 17), leading earlier investigators to conclude that TNF-{alpha} does not directly stimulate loss of muscle protein.

The current experiments provide the first evidence that TNF-{alpha} concentrations similar to those measured in patients (2, 3) can directly stimulate protein loss and decrease MHCf levels in skeletal muscle cells. We were able to resolve these direct effects by monitoring TNF-{alpha} actions on cultured myotubes for several days. The response we observed is similar to the chronic changes seen in cachexia: skeletal muscle atrophy without overt cell death. Our data indicate that loss of myosin in TNF-{alpha}-treated animals (4, 7) may reflect a direct effect of the cytokine on protein regulation by the myocyte. Our data further suggest that this is not an effect on synthesis. TNF-{alpha} consistently decreased MHCf content of the myotubes with no detectable effect on MHCf synthesis. In our system, therefore, MHCf depletion appears to be mediated by TNF-{alpha}-induced proteolysis.

TNF-{alpha} activates NF-{kappa}B in muscle
Cellular responses to TNF-{alpha} differ markedly among tissues. The pleiotropic response to TNF-{alpha} arises from a complex cascade of postreceptor signaling events that vary according to cell type (20). TNF-{alpha} receptor binding stimulates at least three distinct signal transduction pathways (35, 36). One pathway stimulates apoptosis via interaction of the TNF-{alpha}–receptor complex and the Fas-associated protein with death domain (FADD). A second pathway activates Jun-N-terminal kinases (JNK) and the transcription factor AP-1. The third pathway activates NF-{kappa}B, a primary mediator of transcriptional control by TNF-{alpha} (37) and the focus of our present study. NF-{kappa}B was characterized originally as part of an inactive heterotrimeric complex (38) that usually comprises the DNA binding proteins p50 and p65 plus the inhibitory protein I-{kappa}B{alpha} (39, 40). TNF-{alpha} activates NF-{kappa}B by stimulating phosphorylation, ubiquitin conjugation, and proteolysis of I-{kappa}B{alpha}, which leads to nuclear translocation of NF-{kappa}B (41).

The principal support for TNF-{alpha}/NF-{kappa}B signaling in differentiated muscle derives from intact animal studies in which TNF-{alpha} has been shown to activate the ubiquitin/proteasome pathway. Acute, intravenous injection of TNF-{alpha} causes time-dependent increases in free ubiquitin (42), conjugated ubiquitin (42), and ubiquitin mRNA (43) in the limb muscles of intact rats. Ubiquitin mRNA is increased in muscle by experimental sepsis (44) or tumor implantation (19), and proteasome inhibitors suppress protein breakdown in rodent muscle after experimental sepsis (46–48). Ubiquitin mRNA levels are also elevated in excised muscle after 3 h exposure to TNF-{alpha} in vitro (19), indicating that TNF-{alpha} can stimulate ubiquitin gene expression directly.

The current study provides new information about the regulation of this pathway in skeletal muscle. Most important, our data demonstrate that TNF-{alpha} activates NF-{kappa}B in differentiated muscle cells. This establishes the relevance of this pathway in skeletal muscle and validates previous observations in undifferentiated myoblasts (49). Second, we have shown that TNF-{alpha} rapidly stimulates ubiquitin conjugation to muscle proteins. This response occurred within 30 min and peaked at 60 min. The time course suggests rapid activation of an existing ubiquitin pool rather than synthesis of new protein. This early response appears to precede the rise in ubiquitin mRNA that may occur several hours later (19). Third, proteasome inhibitors completely prevented NF-{kappa}B translocation in our system. This indicates that ubiquitin/proteasome interactions are obligatory for TNF-{alpha}/NF-{kappa}B signaling in skeletal muscle, which agrees with observations in other cell types (21).

The role of ROS
We have long been interested in the physiological importance of ROS as modulators of respiratory and limb muscle function (32, 50–52). Recent data from nonmuscle cell types demonstrate that ROS mediate intracellular signal transduction (53–55). TNF-{alpha}/NF-{kappa}B signaling is the most widely recognized of the ROS-sensitive pathways. TNF-{alpha} stimulates mitochondrial production of superoxide anions (23, 24), which undergo enzymatic metabolism and electron transfer reactions to produce hydrogen peroxide and hydroperoxide derivatives. These may enhance NF-{kappa}B activation (25).

The importance of ROS in NF-{kappa}B activation appears to be tissue specific. ROS modulate NF-{kappa}B in human breast cell lines (56) and T cell lines (57–60) but not in monocytic cell lines (61) and epidermal cells (26). Previous reports suggest that ROS may influence TNF-{alpha} effects on skeletal muscle. Experimental endotoxemia increases TNF-{alpha} levels in muscle (62) and causes muscle dysfunction due to oxidative injury (63, 64). Data from undifferentiated myoblasts indicate that TNF-{alpha} signaling also may be redox sensitive since it is influenced by glutathione metabolism and exogenous hydrogen peroxide (49).

The present study demonstrates that ROS modulate TNF-{alpha}/NF-{kappa}B signaling in differentiated muscle. Catalase inhibited TNF-{alpha} activation of NF-{kappa}B by approximately 40%, identifying hydrogen peroxide or its redox derivatives as important components of the pathway. These values probably underestimate the magnitude of ROS effects because catalase (60 kDa) is not thought to cross the cell membrane (31). Instead, exogenous catalase is likely to function as a pericellular `sink' for hydrogen peroxide, facilitating diffusion from the cell and thereby lowering cytosolic ROS concentrations indirectly (32). It is unlikely that exogenous catalase metabolized all hydrogen peroxide synthesized within the cell; some amount of ROS-mediated NF-{kappa}B activation probably persisted. As a positive control, we also demonstrated that exogenous hydrogen peroxide stimulates NF-{kappa}B translocation and I-{kappa}B{alpha} degradation. These responses were induced when using 200 µM hydrogen peroxide, a concentration that is not overtly toxic to muscle (51), which measurably increases oxidant levels in the myocyte cytosol (unpublished observation) and alters other intracellular processes, i.e., excitation-contraction coupling (51).


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
We conclude that TNF-{alpha} directly stimulates protein loss in skeletal muscle. The TNF-{alpha} signal is transduced in part by activation of NF-{kappa}B, a process that involves ubiquitin conjugation and proteasomal degradation of I-{kappa}B{alpha}. NF-{kappa}B activation is accelerated by endogenous hydrogen peroxide or its derivatives, which act at an upstream site to enhance I-{kappa}B{alpha} degradation. The relative importance of this pathway in regulating protein loss is not known. NF-{kappa}B may be the sole effector, altering gene expression to increase expression of ubiquitin and regulated proteases. Alternatively, TNF-{alpha}-induced catabolism may be driven by the FADD-mediated pathway or by the MAP kinase/JNK/AP-1 pathway, and NF-{kappa}B may oppose the process, assuming a protective role analogous to that of NF-{kappa}B in apoptosis. No data at present allow us to discriminate between these alternatives.


   ACKNOWLEDGMENTS
 
This study was supported by the Baylor/Zeneca Research Alliance and by National Institutes of Health grant HL45721. We thank Jim Agan for technical assistance, Dr. Michael Schneider for providing skeletal muscles from neonatal rats, and Melanie Moody for assistance with graphics.


   FOOTNOTES
 
1 Correspondence: Pulmonary Medicine, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: reid{at}bcm.tmc.edu

2 Abbreviations: TNF-{alpha}, tumor necrosis factor {alpha}; NF-{kappa}B, nuclear factor {kappa}B; I-{kappa}B{alpha}, inhibitory protein {kappa}B{alpha}; ROS, reactive oxygen species; MHCf, adult fast-type myosin heavy chain; EMSA, electrophoresis mobility shift assay; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; JNK, Jun-N-terminal kinases; FADD, Fas-associated protein with death domain.

Received for publication November 18, 1997. Accepted for publication February 12, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

  1. Espat, N. J., Copeland, E. M., and Moldawer, L. L. (1994) Tumor necrosis factor and cachexia: a current perspective. Surg. Oncol. 3, 255–262[Medline]
  2. Vreugdenhil, G., Lowenberg, B., VanEijk, H. G., and Swaak, J. G. (1992) Tumor necrosis factor-{alpha} is associated with disease activity and the degree of anemia in patients with rheumatoid arthritis. Eur. J. Clin. Invest. 22, 488–493[Medline]
  3. Nakashima, J., Tachibana, M., Ueno, M., Baba, S., and Tazaki, H. (1995) Tumor necrosis factor and coagulapathy in patient with prostate cancer. Cancer Res. 55, 4881–4885[Abstract/Free Full Text]
  4. Buck, M., and Chojkier, M. (1996) Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. EMBO J. 15, 1753–1765[Medline]
  5. Garcia-Martinez, C., Lopez-Soriano, F. J., and Argiles, J. M. (1993) Acute treatment with tumour necrosis factor-{alpha} induces changes in protein metabolism in rat skeletal muscle. Mol. Cell. Biochem. 125, 11–18[Medline]
  6. Cheng, J., Turksen, K., Yu, Q., Schreiber, H., Teng, M., and Fuchs, E. (1992) Cachexia and graft-vs-host-disease-type skin changes in keratin promoter-driven TNF-{alpha} transgenic mice. Genes & Dev. 6, 1444–1456[Abstract/Free Full Text]
  7. Ahmad, S., Karlstad, M. D., Choudhry, M. A., and Sayeed, M. M. (1994) Sepsis-induced myofibrillar protein catabolism in rat skeletal muscle. Life Sci. 55, 1383–1391[Medline]
  8. Tessitore, L., Costelli, P., and Baccino, F. M. (1993) Humoral mediation for cachexia in tumour-bearing rats. Br. J. Cancer 67, 15–23[Medline]
  9. Lawson, D. H., Richmond, A., Nixon, D. W., and Rudman, D. (1982) Metabolic approach to cancer cachexia. Annu. Rev. Nutr. 2, 277–301[Medline]
  10. Morrison, S. D. (1989) Cancer cachexia. In Influence of Tumor Development on the Host (Liotta, A. L., ed) pp. 176–213, Kluwer Academic Publishers, Amsterdam
  11. Mealy, K., van Lanschott, J. J., Robinson, B. G., Rounds, J., and Wilmore, D. W. (1990) Are the catabolic effects of tumor necrosis factor mediated by glucocorticoids? Arch. Surg. 125, 42–48[Abstract]
  12. Dayer, J. M., Beutler, B., and Cerami, A. (1985) Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J. Exp. Med. 162, 2163–2168[Abstract/Free Full Text]
  13. Gelin, J., Moldawer, L. L., Lonnroth, C., Sherry, C., Chizzonite, T., and Lundholm, K. (1991) Role of endogenous tumor necrosis factor-{alpha} and interleukin-1 for experimental tumor growth and the development of cancer cachexia. Cancer Res. 51, 415–421[Abstract/Free Full Text]
  14. Michie, H. R., Sherman, M. L., Spriggs, D. R., Rounds, J., Christie, M., and Wilmore, D. W. (1989) Chronic TNF infusion causes anorexia but not accelerated nitrogen loss. Ann. Surg. 209, 19–24[Medline]
  15. Socher, S. H., Freidman, A., and Martinez, D. (1988) Recombinant tumor necrosis factor induces acute reductions in food intake and body weight in mice. J. Exp. Med. 167, 1957–1962[Abstract/Free Full Text]
  16. Goldberg, A. L., Kettelhut, I. C., Furuno, K., Fagen, J. M., and Barascos, V. (1988) Activation of protein breakdown and prostaglandin E2 production in rat skeletal muscle in fever is signaled by a macrophage product distinct from interleukin-1 or other known monokines. J. Clin. Invest. 81, 1378–1383
  17. Goodman, M. N. (1991) Tumor necrosis factor induces skeletal muscle protein breakdown in rats. Am. J. Physiol. 260, E727–E730[Abstract/Free Full Text]
  18. Rofe, A. M., Conyers, R. A. J., Bais, R., Gamble, J. R., and Vadas, M. A. (1987) The effects of recombinant tumour necrosis factor (cachectin) on metabolism in isolated rat adipocyte, hepatocyte and muscle preparations. Biochem. J. 247, 789–792[Medline]
  19. Llovera, M., Garcia-Martinez, C., Lopez-Soriano, F. J., and Argiles, J. M. (1997) TNF can directly induce the expression of ubiquitin-dependent proteolytic system in rat soleus muscles. Biochem. Biophys. Res. Commun. 230, 238–241[Medline]
  20. Schutze, S., Machleidt, T., and Kronke, M. (1992) Mechanisms of tumor necrosis factor action. Semin. Oncol. 2, 16–24
  21. Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) The ubiquitin-proteasome pathway is required for processing the NF-{kappa}B1 precursor protein and the activation of NF-{kappa}B. Cell 78, 773–785[Medline]
  22. Chen, Z., Hagler, J., Palombella, V. J., Melandri, F., Scherer, D., Ballard, D., and Maniatis, T. (1995) Signal-induced site-specific phosphorylation targets I-{kappa}B{alpha} to the ubiquitin-proteasome pathway. Genes & Dev. 9, 1586–1597[Abstract/Free Full Text]
  23. Schulze-Osthoff, K., Bakker, A. C., Vanhaesebroeck, B., Beyaert, R., Jacob, W. A., and Fiers, W. (1992) Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial function. Evidence for the involvement of mitochondrial radical generation. J. Biol. Chem. 267, 5317–5323[Abstract/Free Full Text]
  24. Hennet, T., Richter, C., and Peterhans, E. (1993) Tumour necrosis factor-{alpha} induces superoxide anion generation in mitochondria of L929 cells. Biochem. J. 289, 587–592
  25. Flohe, L., Brigelius-Flohe, R., Saliou, C., Traber, M. G., and Packer, L. (1997) Redox regulation of NF-{kappa}B activation. Free Radical Biol. Med. 22, 1115–1126[Medline]
  26. Brennan, P., and O'Neill, L. (1995) Effects of oxidants and antioxidants on nuclear factor {kappa}B activation in three different cell lines: Evidence against a universal hypothesis involving oxygen radicals. Biochem. Biophys. Acta 1260, 167–175[Medline]
  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275[Free Full Text]
  28. Li, Y.-P., Baskin, F., Davis, R., Wu, D., and Hersh, L. B. (1995) A cell type-specific silencer in the human choline acetyltransferase gene requiring two distinct and interactive E boxes. Mol. Brain Res. 30, 106–114[Medline]
  29. Zar, J. H. (1974) Biostatistical Analysis, Prentice-Hall, Inc., Engelwood Cliffs, N.J
  30. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L. (1994) Inhibitors of the proteasome block degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78, 761–771[Medline]
  31. Beckman, J. S., and Freeman, B. A. (1986) Antioxidant enzymes as mechanistic probes of oxygen-dependent toxicity. In Physiology of Oxygen Radicals (Taylor, A. E., Matalon, S., and Ward, P. A., eds) pp. 39–53, American Physiological Society, Bethesda, Maryland
  32. Reid, M. B., Haack, K. E., Franchek, K. M., Valberg, P. A., Kobzik, L., and West, M. S. (1992) Reactive oxygen in skeletal muscle: I. Intracellular oxidant kinetics and fatigue in vitro. J. Appl. Physiol. 73, 1797–1804[Abstract/Free Full Text]
  33. Beutler, B., Mahoney, J., LeTrang, N., Pekala, P., and Cerami, A. (1985) Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced Raw264.7 cells. J. Exp. Med. 161, 984–995[Abstract/Free Full Text]
  34. Beutler, B., and Cerami, A. (1986) Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature (London) 320, 584–588[Medline]
  35. Hsu, H., Shu, H.-B., Pan, M.-G., and Goeddel, D. V. (1996) TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299–308[Medline]
  36. Liu, Z.-G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-{kappa}B activation prevents cell death. Cell 87, 565–576[Medline]
  37. Barnes, P. J., and Karin, M. (1997) Nuclear factor-{kappa}B—a pivotal transcription factor in chronic inflammatory disease. New Engl. J. Med. 336, 1066–1071[Free Full Text]
  38. Sen, R., and Baltimore, D. (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705–716[Medline]
  39. Siebenlist, U., Franzoso, G., and Brown, K. (1994) Structure, regulation, and function of NF-{kappa}B. Annu. Rev. Cell Biol. 10, 405–455
  40. Baeuerle, P. A., and Baltimore, D. (1996) NF-{kappa}B: ten years after. Cell 87, 13–20[Medline]
  41. Beg, A. A., Finco, T. S., Nantermet, P. V., and Baldwin, A. S., Jr. (1993) Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I-{kappa}B{alpha}: a mechanism for NF-{kappa}B activation. Mol. Cell. Biol. 13, 3301–3310[Abstract/Free Full Text]
  42. Garcia-Martinez, C., Agell, N., Llovera, M., Lopez-Soriano, F. J., and Argiles, J. M. (1993) Tumour necrosis factor-{alpha} increases the ubiquitinization of rat skeletal muscle proteins. FEBS Lett. 323, 211–214[Medline]
  43. Garcia-Martinez, C., Llovera, M., Agell, N., Lopez-Soriano, F. J., and Argiles, J. M. (1994) Ubiquitin gene expression in skeletal muscle is increased by tumour necrosis factor-{alpha}. Biochem. Biophys. Res. Commun. 201, 682–686[Medline]
  44. Garcia-Martinez, C., Llovera, M., Agell, N., Lopez-Soriano, F. J., and Argiles, J. M. (1995) Ubiquitin gene expession in skeletal muscle is increased during sepsis: involvement of TNF-{alpha} but not IL-1. Biochem. Biophys. Res. Commun. 217, 839–844[Medline]
  45. Llovera, M., Carbo, N., Garcia-Martinez, C., Costelli, P., Tessitore, L., Baccino, F. M., Agell, N., Bagby, G. J., Lopez-Soriano, F. J., and Argiles, J. M. (1996) Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour-bearing rats. Biochem. Biophys. Res. Commun. 221, 653–655[Medline]
  46. Tawa, N. E., Odessey, R., and Goldberg, A. L. (1997) Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscle. J. Clin. Invest. 100, 197–203[Medline]
  47. Mitch, W. E., and Goldberg, A. L. (1996) Mechanisms of muscle wasting; the role of the ubiquitin-proteasome pathway. New Engl. J. Med. 335, 1897–1905[Free Full Text]
  48. Hochstrasser, M. (1995) Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr. Opin. Cell Biol. 7, 215–223[Medline]
  49. Sen, C. K., Khanna, S., Resznick, A. Z., Roy, S., and Packer, L. (1997) Glutathione regulation of tumor necrosis factor{alpha}-induced NF-{kappa}B activation in skeletal muscle-derived L6 cells. Biochem. Biophys. Res. Commun. 237, 645–649[Medline]
  50. Reid, M. B., Shoji, T., Moody, M. R., and Entman, M. L. (1992) Reactive oxygen in skeletal muscle: II. Extracellular release of free radicals. J. Appl. Physiol. 73, 1805–1809[Abstract/Free Full Text]
  51. Reid, M. B., Khawli, F. A., and Moody, M. R. (1993) Reactive oxygen in skeletal muscle: III. Contractility of unfatigued muscle. J. Appl. Physiol. 75, 1081–1087[Abstract/Free Full Text]
  52. Reid, M. B. (1996) Reactive oxygen and nitric oxide in skeletal muscle. News Physiol. Sci. 11, 114–119[Abstract/Free Full Text]
  53. Sen, C. K., and Packer, L. (1996) Antioxidant and redox regulation of gene transcription. FASEB J. 10, 709–720[Abstract]
  54. Demple, B., and Amabile-Cuevas, C. F. (1991) Redox redux: the control of oxidative stress responses. Cell 67, 837–839[Medline]
  55. Meyer, M., Schreck, R., Muller, J. M., and Baeuerle, P. A. (1994) Redox control of gene expression by eukaryotic transcription factors NF-{kappa}B, AP-1 and SRF/TCF. In Oxidative Stress, Cell Activation and Viral Infection (Pasquier, C., Olivier, R. Y., Auclair, C., and Packer, L., eds) pp. 217–235, Birkhauser Verlag, Basel, Switzerland
  56. Kretz-Remy, C., Mehlen, P., Mirault, M.-E., and Arrigo, A.-P. (1996) Inhibition of I-{kappa}B{alpha} phosphorylation and degradation and subsequent NF-{kappa}B activation by glutathione peroxidase overexpression. J. Cell Biol. 133, 1083–1093[Abstract/Free Full Text]
  57. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-{kappa}B transcription factor and HIV-1. EMBO J. 10, 2247–2258[Medline]
  58. Schmidt, K. N., Traenckner, E. B., Meier, B., and Baeuerle, P. A. (1995) Induction of oxidative stress by okadaic acid is required for activation of transcription factor NF-{kappa}B. J. Biol. Chem. 10, 27136–27142
  59. Sappey, C., Legrand-Poels, S., Best-Belpomme, M., Favier, A., Rentier, B., and Piette, J. (1994) Stimulation of glutathione peroxidase activity decreases HIV type activation after oxidative stress. AIDS Res. Hum. Retroviruses 10, 1451–1461[Medline]
  60. Makropoulos, V., Bruening, T., and Schultze-Osthoff, K. (1996) Selenium-mediated inhibition of transcription factor NF-{kappa}B and HIV-1 LTR promoter activity. Arch. Toxicol. 70, 277–283[Medline]
  61. Israel, N., Gougerot-Pocidalo, M. A., Aillet, F., and Virelizier, J. L. (1992) NF-{kappa}B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J. Immunol. 149, 3386–3393[Abstract]
  62. Shindoh, C., Hida, W., Ohkawara, Y., Yamuchi, K., Ohno, I., Takashima, T., and Shirato, K. (1995) TNF-{alpha} mRNA expression in diaphragm muscle after endotoxin administration. Am. J. Resp. Crit. Care Med. 152, 1690–1691[Abstract]
  63. Supinski, G., Nethery, D., and Dimarco, A. (1993) Effect of free radical scavengers on endotoxin-induced respiratory muscle dysfunction. Am. Rev. Resp. Dis. 148, 1318– 1324[Medline]
  64. Shindoh, C., Dimarco, A., Nethery, D., and Supinski, G. (1992) Effect of PEG-superoxide dismutase on the diaphragmatic response to endotoxin. Am. Rev. Resp. Dis. 145, 1350–1354[Medline]



This article has been cited by other articles:


Home page
 Eur Respir JHome page
P. D. Wagner
Possible mechanisms underlying the development of cachexia in COPD
Eur. Respir. J., March 1, 2008; 31(3): 492 - 501.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
T. Barker and M. G. Traber
Reply from Tyler Barker and Maret G. Traber
J. Physiol., January 1, 2008; 586(1): 309 - 310.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. A. Smith, J. S. Moylan, J. D. Smith, W. Li, and M. B. Reid
IFN-{gamma} does not mimic the catabolic effects of TNF-{alpha}
Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1947 - C1952.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
I. Vogiatzis, G. Stratakos, D. C M Simoes, G. Terzis, O. Georgiadou, C. Roussos, and S. Zakynthinos
Effects of rehabilitative exercise on peripheral muscle TNF{alpha}, IL-6, IGF-I and MyoD expression in patients with COPD
Thorax, November 1, 2007; 62(11): 950 - 956.
[Abstract] [Full Text] [PDF]

Home page
 ThoraxHome page
R A Stockley
Biomarkers in COPD: time for a deep breath
Thorax, August 1, 2007; 62(8): 657 - 660.
[Full Text] [PDF]

Home page
 FASEB J.Home page
C. Dogra, H. Changotra, N. Wedhas, X. Qin, J. E. Wergedal, and A. Kumar
TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine
FASEB J, June 1, 2007; 21(8): 1857 - 1869.
[Abstract] [Full Text] [PDF]