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(The FASEB Journal. 2002;16:529-538.)
© 2002 FASEB

Activation of an alternative NF-{kappa}B pathway in skeletal muscle during disuse atrophy

R. BRIDGE HUNTER, ERIC J. STEVENSON, ALAN KONCAREVIC, HEATHER MITCHELL-FELTON, DAVID A. ESSIG* and SUSAN C. KANDARIAN1

Boston University, Department of Health Sciences, Boston, Massachusetts, USA; and
* Geneva College, Department of Biology, Beaver Falls, Pennsylvania, USA

1Correspondence: Boston University, Department of Health Sciences, 635 Commonwealth Ave., Room 443, Boston, MA 02215, USA. E-mail: skandar{at}bu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although cytokine-induced nuclear factor {kappa}B (NF-{kappa}B) pathways are involved in muscle wasting subsequent to disease, their potential role in disuse muscle atrophy has not been characterized. Seven days of hind limb unloading led to a 10-fold activation of an NF-{kappa}B-dependent reporter in rat soleus muscle but not the atrophy-resistant extensor digitorum longus muscle. Nuclear levels of p50 were markedly up-regulated, c-Rel was moderately up-regulated, Rel B was down-regulated, and p52 and p65 were unchanged in unloaded solei. The nuclear I{kappa}B protein Bcl-3 was increased. There was increased binding to an NF-{kappa}B consensus oligonucleotide, and this complex bound antibodies to p50, c-Rel, and Bcl-3 but not other NF-{kappa}B family members. Tumor necrosis factor alpha (TNF-{alpha}) and TNF receptor-associated factor 2 protein were moderately down-regulated. There was no difference in p38, c-Jun NH2-terminal kinase or Akt activity, nor were activator protein 1 or nuclear factor of activated T cell-dependent reporters activated. Thus, whereas several NF-{kappa}B family members are up-regulated, the prototypical markers of cytokine-induced activation of NF-{kappa}B seen with disease-related wasting are not evident during disuse atrophy. Levels of an anti-apoptotic NF-{kappa}B target, Bcl-2, were increased fourfold whereas proapoptotic proteins Bax and Bak decreased. The evidence presented here suggests that disuse muscle atrophy is associated with activation of an alternative NF-{kappa}B pathway that involves the activation of p50 but not p65.—Hunter, R. B., Stevenson, E. J., Koncarevic, A., Mitchell-Felton, H., Essig, D. A., Kandarian, S. C. Activation of an alternative NF-{kappa}B pathway in skeletal muscle during disuse atrophy.


Key Words: unloading • cytokine • Bcl-2 protein • NF-kappaB p50 • NF-kappaB p65 • Bcl-3 protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SKELETAL MUSCLE WASTING is associated with disease states such as cancer, sepsis, HIV infection, and end-stage heart failure (1 , 2) . The rise in inflammatory cytokine production in these disorders is thought to trigger the loss of skeletal muscle mass (3 4 5) . Cytokines, particularly tumor necrosis factor {alpha} (TNF-{alpha}), are potent activators of the prototypical NF-{kappa}B transcription factor (p65/p50) in cultured muscle cells (4 , 6 7 8) . This NF-{kappa}B pathway is activated by phosphorylation, ubiquitination, and proteolysis of the inhibitory protein I{kappa}B, which otherwise binds and retains the NF-{kappa}B heterodimer in the cytosol (9) . TNF-{alpha}-induced NF-{kappa}B activation leads to muscle protein loss (4 , 8) and inhibition of NF-{kappa}B protects against TNF-{alpha}-induced protein degradation in cultured muscle cells (4 , 6) ; these relationships are more difficult to demonstrate in whole muscle. NF-{kappa}B regulates the transcription of a wide variety of genes including those involved in immune and inflammatory responses, cell survival, and cell adhesion (10) .

Skeletal muscle wasting is also a result of chronic disuse such as that due to bed rest, sedentary lifestyles, exposure to space flight, and muscle denervation (11 , 12) . An important similarity between the muscle loss seen in cachexia and that in disuse is that protein degradation is mediated primarily by the ubiquitin–proteasome pathway (1 , 12 , 13) . However, whether cytokine or NF-{kappa}B pathways are involved in the regulation of muscle atrophy due to disuse has not been explored. Other than the classic p65/p50/I{kappa}B pathway, there are no studies of the presence of other NF-{kappa}B/I{kappa}B family members in skeletal muscle such as c-Rel, Rel B, p52, or Bcl-3 and no studies of the potential involvement of any NF-{kappa}B/I{kappa}B family members with muscle disuse. Potential targets of NF-{kappa}B in skeletal muscle are anti-apoptotic genes. For instance, there is evidence that TNF receptor-associated factor 2 (TRAF2) is a target during myogenesis (14) and evidence from type 2A muscular dystrophy patients that NF-{kappa}B activation targets survival gene induction (15) . The aim of the present study was to determine whether cytokine or NF-{kappa}B/I{kappa}B pathways are involved in muscle disuse atrophy and, if so, to identify candidate target genes of NF-{kappa}B in rat soleus muscles 7 days after the removal of hind limb weight bearing (i.e., unloading).

In this work, we show a 10-fold increase in the activity of a transfected NF-{kappa}B-dependent reporter in unloaded soleus muscles compared with weight-bearing controls, but the reporter was not activated in unloaded, atrophy-resistant extensor digitorum longus (EDL) muscles. Nuclear extracts from unloaded solei showed increased binding to NF-{kappa}B oligonucleotides; this complex bound antibodies to p50, c-Rel, and Bcl-3 but not other NF-{kappa}B family members. This was accompanied by a marked increase in the nuclear level of the NF-{kappa}B protein p50 and moderate increases in c-Rel and Bcl-3. There was no evidence for the canonical NF-{kappa}B pathway involving activation of p65 or I{kappa}B, nor of TNF-{alpha} activation or inflammatory responses based on the lack of induction of p38, c-Jun NH2-terminal kinase (JNK), and Akt activity, activator protein 1-(AP-1) and nuclear factor of activated T cells (NFAT) -dependent transcription, or TNF-{alpha} and TRAF2 expression. The relative level of an anti-apoptotic NF-{kappa}B target, Bcl-2, was increased fourfold but unloading did not trans-activate a bcl-2 reporter plasmid. We have shown the activation of an alternative NF-{kappa}B pathway with skeletal muscle disuse, distinct from that seen in cachexia, involving activation of p50, Bcl-3, and possibly c-Rel.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Hind limb unloading
Female Wistar rats (8-wk-old) were randomly assigned to control or hind limb unloading groups. To induce muscle atrophy, the rat’s hind limbs were suspended by elastic tail casts, as described (16) . After 1 or 7 days, control and unloaded animals were anesthetized with pentobarbital sodium (50 mg/kg); soleus muscles from right and left hind limbs were removed, quickly weighed, and immediately processed for protein assays as described below.

Protein isolation procedures
Nuclear extracts were prepared from rat skeletal muscle according to Blough et al. (17) . The cytosolic extract was obtained from the first supernatant of the nuclear extract preparation. The supernatant was added to Millipore Ultrafree-4 centrifugal columns, pre-wet with 0.5 ml of dilution buffer (20 mM HEPES, 40 mM KCL, 10% glycerol, 0.2 mM EDTA, 1 mM DTT), and centrifuged (7500 g) at 4°C for 30 min; 0.8 ml of dilution buffer was added to the column and the 30 min spin was repeated. Protein concentration was determined using the Bio-Rad Bradford protein assay for the nuclear samples and the Detergent Compatible Assay for the cytosolic samples.

Whole-muscle lysates were isolated by homogenizing freshly excised muscle in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol phosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF), sonicating 4 x 5 s, shaking for 45 min at 4°C, centrifuging at 15,000 g for 10 min, and using the supernatant as lysate.

Microsomal isolations were prepared according to Cameron et al. (18) . Muscles were homogenized in a buffer containing 50 mM Tris (pH 7.4), 1 mM EDTA, 1 mM ß-mercaptoethanol, 0.1 mg/ml leupeptin, 1 mg/ml pepstatin, and 0.1 mg/ml PMSF. The homogenate was centrifuged at 45,000 g for 10 min. The pellet was resuspended in buffer without ß-ME, solubilized in 1% CHAPS for 20 min, and centrifuged at 45,000 g for 20 min. The supernatant was treated as microsomal protein. Determination of protein concentration was performed using a detergent compatible assay (Bio-Rad, Hercules, CA).

Reporter plasmids
The NFAT-GL3 (also called NFATx3) contains a trimerized distal NFAT site from the human interleukin 2 (IL-2) gene; the AP1-GL3 contains five copies of the AP1 site from the metallothionein promoter; NF-{kappa}ß-GL3 contains a trimerized NF-{kappa}ß site from the Ig{kappa} light chain enhancer. These sequences were each inserted into the IL-2 minimal promoter (19) driving expression of luciferase in pGL3-basic and were gifts from Dr. S. Ho. The bcl-2 construct contains 3.9 kb of the 5' flank and promoter sequence ligated upstream of the luciferase gene in pBluescript II and was a gift from Dr. L Boxer (20) . The bcl-2 sequence contains 7 NF-{kappa}B binding sites.

Somatic gene transfer using intramuscular DNA injection
Plasmid DNA injections into rat skeletal muscle have been detailed before (21) . Plasmid DNA was prepared using an Endotoxin-Free Mega Prep Kit (Qiagen, Chatsworth, CA). The DNA was ethanol precipitated and then resuspended at 4°C overnight in 25% sucrose-1 x PBS. Soleus and EDL muscles were injected with 75 µg plasmid DNA in a volume of 50 µl. Muscles were removed for analysis of luciferase activity 8 days after injection. Seven days of unloading do not influence the efficiency of plasmid DNA uptake compared with weight-bearing controls (21) . Firefly luciferase activity was determined using a luciferase assay kit (Promega, Madison, WI) and a Turner Designs Luminometer (model TD 20/20).

Western analysis
Protein (30 µg) from soleus muscle samples were denatured in SDS loading buffer, boiled for 3 min, centrifuged briefly to remove insoluble material, and separated on SDS-polyacrylamide gels. Protein was transferred onto Hybond ECL (Amersham, Little Chalfont, UK) nitrocellulose membrane. Membranes were blocked in 5% nonfat milk diluted in TBS-Tween for 1 h, then incubated for 1–2 h with the appropriate antibody diluted according to the manufacturer. Horseradish peroxidase (HRP) -conjugated secondary antibodies and a chemiluminescent detection system (Amersham) were used for visualization. Extracts used for positive controls were obtained from BD Transduction Laboratories (Lexington, KY: mouse macrophage and Jurkat whole-cell lysates), CST (Beverly, MA: untreated and TNF-{alpha} treated HeLa cell extracts), Geneka (Montreal, Canada: C2C12 nuclear extracts), and Santa Cruz (Santa Cruz, CA: serum-starved 3T3, Jurkat, HuT78, WEHI, A-431, and CTLL whole-cell lysates and KNRK nuclear extracts).

Antibodies
Monoclonal antibodies used for immunoblotting obtained from BD Transduction Laboratories included NF-{kappa}B p65 (N67620), Bcl-2 (B46620), Bcl-XL (B22620), and Mcl-1 (M54020). Polyclonal antibodies from CST included Bad (9290), I{kappa}B-{alpha} (9240), and phospho-specific (Ser 32) I{kappa}B-{alpha} (9240). Antibodies from Santa Cruz Biotechnology included polyclonals Rel B (sc-226), NF-{kappa}B p52 (sc-298), c-Rel (sc-70), Bcl-3 (sc-185), TRAF2 (sc-876), Bax (sc-493), and Bak (sc-832) and the monoclonal NF-{kappa}B p50 (sc-8414). Monoclonal anti-calcineurin (C-1956) was purchased from Sigma (St. Louis, MO) and polyclonal anti-TNF-{alpha} (AF-510-NA) from R&D Systems (Oxon, UK). Secondary antibodies were obtained from Vector Laboratories (Burlingame, CA: HRP-labeled horse anti-mouse) and Transduction Laboratories (HRP-labeled goat anti-rabbit).

Gel shift assay
The 1 x NF-{kappa}B probe (5'-ATCCTCGGAAAGTCCCCTCTG-3') was purchased from Genosys (Woodlands, TX) and the 148-mer 3 x NF-{kappa}B probe (5'-CTAGCCCCGATGTTTTCTGAGTTACTTTTGTATCCCCACCCCCCCTCGACATCCTC-GGAAAGTCCCCTCTGAGATCCTCGGAAAGTCCCCTCTG-AGATCTCAGAGGGGACTTTCCGAGGATTCGAGGACAT-TTTGACACCCCCATAAT-3') was as obtained by restriction digestion of the NF{kappa}B-GL3 using SspI and NheI. Gel shift assays were performed as described (22) . Probes were end labeled with [{gamma}-32P] dATP (New England Nuclear, Boston, MA) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Binding reactions were performed for 30 min at 20°C in 20 µl volume. The binding reaction contained buffer [10 mM HEPES (pH 7.9), 50 mM KCl, 0.2 mM EDTA, 2.5 mM DTT, 10% (w/v) glycerol, and 0.05% Nonidet P-40], 50,000 cpm (0.1–0.2 ng) of labeled DNA probe, 10 µg of nuclear extract (for 3xNF{kappa}B probe) or 15 µg (for 1xNF{kappa}B probe) of either control soleus or hind limb unloaded soleus nuclear extracts, and 10 ng (for 1xNF{kappa}B probe) or 20 ng (for 3xNF{kappa}B probe) of nonspecific DNA competitor poly (dI·dC). Specificity of DNA binding was determined by addition of 0- to 20-fold molar excess of unlabeled competitor DNA to the binding reactions. Supershifts were performed by preincubation with the antibodies indicated for 30 min before the addition of the labeled probe. The binding reactions were loaded onto 5% (1xNF-{kappa}B probe) or 4% (3xNF-{kappa}B probe) nondenaturing polyacrylamide gels and electrophoretically resolved in 0.5 x TBE buffer, pH 8.2, at 30 mA for 3.5 h.

In vitro kinase activity assays
In vitro kinase activity was measured in whole-tissue lysates according to the instructions from relevant kits (CST). To measure JNK activity, 250 µg protein was incubated with c-Jun fusion protein followed by addition of cold ATP. Phospho c-Jun substrate was detected by immunoblotting (Cat. #9810). Recombinant JNK used as positive control. To measure Akt and p38 activity, 200 µg protein was immunoprecipitated with either anti-p38 or anti-Akt immobilized antibodies, followed by addition of cold ATP and GSK-3{alpha}/ß (Akt) or ATF-2 (p38) fusion protein substrate, respectively. Phospho-GSK-3{alpha}/ß (Cat. #9840) or phospho-ATF-2 (Cat. #9820) was detected by immunoblotting.

Statistics
For immunoblotting and enzyme activity assays, Student’s t test for unpaired data was used to determine statistical significance between control and unloaded groups at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reporter activity in control and unloaded skeletal muscle
Soleus muscles lost 30% of their muscle mass after 7 days of unloading (not shown). TNF-{alpha} and other cytokines are known to activate AP-1-dependent genes via members of the MAP kinase pathway (23) , but there was no change in the activity of an AP-1-dependent reporter plasmid after 7 days of unloading (Fig. 1 a). An NFAT-dependent reporter known to be activated by immunoreceptors, G-protein receptors, or changes in intracellular calcium fluxes (24) was not activated with unloading (Fig. 1a ). However, there was a 10-fold increase in the activity of a NF-{kappa}B-dependent reporter. In comparison, the EDL muscle, which is not atrophied by 7 days of unloading, did not show increases in NFAT or NF-{kappa}B-dependent reporter activity, but there was a significant increase in the AP-1-dependent reporter (Fig. 1b ). Thus, the increase in NF-{kappa}B activity in the soleus is not likely due to inflammation from the injection. The injury caused by injection of endotoxin-free plasmid DNA is mild and brief; there was no evidence of injury or inflammation 5 days after plasmid injection (25) . Analysis of reporter activity measured here was 8 days after injection of the plasmid and 7 days after the initiation of muscle unloading. Injection of a different NF-{kappa}B-dependent reporter obtained from Stratagene containing only a TATA box downstream of 5 NF-{kappa}B consensus sites showed up-regulation (6-fold) at 7 days of unloading (not shown).



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  		Figure 1. In vivo injection of rat soleus and EDL muscles with different cis reporter plasmids analyzed after 7 days of unloading for firefly luciferase activity. a) Soleus muscle unloading led to a 30% loss of muscle mass and a 9-fold activation of a NF-{kappa}B-dependent reporter vs. normal weight-bearing controls (P<0.05). Unloading did not activate AP-1- or NFAT-dependent reporters. b) Unloaded EDL muscles did not atrophy after 7 days and did not activate NF-{kappa}B or NFAT reporters. The AP-1 reporter was activated 5.6-fold (P<0.05). Data are means ± SE of at least 8 muscles per group.

In earlier work using Southern blotting, we showed no difference in the efficiency of plasmid uptake in whole muscle due to unloading as long as rats were weight bearing for 1 day after the injection procedure (21) . However, soleus muscles take up injected plasmid more efficiently than EDL muscles, and so reporter activities between muscle types are not comparable (21) .

NF-{kappa}B family members in atrophied muscle
The increased NF-{kappa}B reporter activity led us to examine which family members may be involved. Nuclear levels of p65 (Rel A) were not different due to unloading (Fig. 2 a). Cytosolic levels of p65 were higher than nuclear levels but there were no differences in expression due to unloading (not shown). Nuclear Rel B (Fig. 2b ) decreased 47% whereas p52 expression was unchanged (Fig. 2c ). c-Rel expression increased by 1.7-fold (Fig. 2d ). Nuclear levels of p50 were increased 2.3-fold by unloading and there were additional faster migrating p50-reactive bands in nuclear extracts from unloaded muscles (Fig. 2e ). The appearance of these bands is consistent with the notion that multiple active p50 proteins exist and are either products of differential proteolytic processing or cotranslational products of the p105 gene (26 27 28 29) . Cytosolic levels of p50 were unchanged with unloading (Fig. 2f ). Cytosolic p105 could not be detected, but an anti-p50 immunoreactive band running at ~150 kDa was unchanged due to unloading (not shown). It is not clear whether partial or complete proteolytic processing contributed to the appearance of increased p50 in the nucleus.



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  		Figure 2. Expression of NF-{kappa}B family members in nuclear extracts from control (Con) and 7 day hind limb unloaded (HU) soleus muscles. Nuclear protein levels measured using antibodies specific to p65 (a), Rel b (b), p52 (c), c-Rel (d), p50 (e). Cytosolic p50 expression (f). HuT 78 (HuT) and CTLL cell lysates and Jurkat and KNRK nuclear extracts were used as positive controls. p50 and c-Rel increased expression 2.3- and 1.7-fold, respectively (P<0.05); additional p50 immunoreactive bands were evident only in unloaded extracts. Expression of Rel B decreased 47% (P<0.05). Blots quantified by densitometric analysis. Data are means ± SE of at least 4 muscles per group.

Expression of I{kappa}B proteins
Given the increased nuclear levels of p50, we next determined whether Bcl-3, an I{kappa}B transcription factor, was activated with unloading. Bcl-3 can bind p50 homodimers, particularly when phosphorylated (30 , 31) , and induce trans-activation (32 33 34) . Bcl-3 immunoreactive band intensity was increased by 2.2-fold in nuclei from unloaded muscle (Fig. 3 a). The faster migrating band may be the nonphosphorylated species as shown in other studies (29 , 35 , 36) .



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  		Figure 3. Expression of I{kappa}B family members in whole-muscle lysates from control and 7 day hind limb unloaded (HU) solei. Relative protein level measured using antibodies specific to Bcl-3 (a), I{kappa}B-{alpha} (b), and phospho-specific (Ser 32) I{kappa}B-{alpha} (c). WEHI whole-cell lysates, Untreated HeLa (UH) cell lysates and TNF-{alpha} treated HeLa (TH) cell lysates were used as positive controls. Bcl-3 protein level increased 2.2-fold (P<0.05). I{kappa}B-{alpha} expression was decreased by 20% (P<0.05) and phosphorylated I{kappa}B-{alpha} was variable. Blots quantified by densitometric analysis. Data are means ± SE of at least 4 muscles per group.

To determine whether I{kappa}B was involved, possibly in the release of cytosolic p50, we examined the expression level and phosphorylation state of I{kappa}B (phospho-Ser 32) in cytosolic and whole-muscle lysates from control and unloaded muscles. There was a 20% decrease in I{kappa}B levels and no consistent difference in I{kappa}B phosphorylation status with unloading (Fig. 3b, c ). In fact, I{kappa}B phosphorylation was quite variable, which may be due to potential trauma associated with muscle dissection.

Protein binding to NF-{kappa}B DNA consensus sequence
To determine whether members of the NF-{kappa}B family were differentially binding to the NF-{kappa}B promoter sequence in nuclei from unloaded muscle, gel shift assays were performed (Fig. 4 ).There was consistently more binding to an NF-{kappa}B oligonucleotide containing a single NF-{kappa}B consensus sequence in extracts from unloaded vs. control muscle (Fig. 4b ). The faster migration of recombinant p50 binding to the oligonucleotide suggests that the band in the nuclear extract lanes is not p50 alone. The binding activity from nuclear extracts was successfully competed by excess cold oligonucleotide (Fig. 4c ). The same sequence contained in the NF-{kappa}B reporter plasmid (‘3x NF-{kappa}B’) was used for DNA binding experiments (Fig. 4d ). Again, more binding activity was seen in extracts from unloaded muscle, although the distinct bands are difficult to visualize (Fig. 4e ). These experiments are difficult because of the long oligonucleotide (148-mer) and the use of nuclear extracts from whole muscle, which have relatively low transcriptional activity. Supershift assays using the 3x NF-{kappa}B oligo showed that anti-p50 and anti-Bcl-3 caused a supershift but p65 and Mcl-1 did not (Fig. 4f ). Anti-Mcl-1 was used as a control, irrelevant monoclonal antibody. Anti-p52 and Rel B did not supershift the bands but c-Rel caused a clear supershift, with a stronger complex in the unloaded extract (Fig. 4g ). These results were repeated several times.



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  		Figure 4. Specific binding of proteins to NF-{kappa}B sites on 1x (a) and 3x (d) NF-{kappa}B oligonucleotides. b) Gel shift assays performed with 15 µg soleus nuclear extract and 0.1 ng of 5' end-labeled 1x NF-{kappa}B oligonucleotide (21-mer) separated on 5% gel. Multiple lanes of control and unloaded represent extracts from separate rats. Unloaded extracts show increased binding compared with control. Recombinant (r) p50 used for comparison. c) Competition with 0-, 1-, 5-, and 20-fold molar excess of unlabeled NF-{kappa}B oligonucleotide with hind limb unloaded (HU) extracts demonstrates specificity of binding to the NF-{kappa}B site. e) Gel shift assays performed with 10 µg of soleus nuclear extract and 0.1 ng of 5' end-labeled 3x NF-{kappa}B 148-mer. Arrows indicate binding complexes. Unloaded extracts have more binding than controls. Cold competition with 10-fold molar excess 3x NF-{kappa}B demonstrates binding specificity. f) Incubation with antibodies specific for p50, p65, Mcl-1 (control), and Bcl-3 shows supershifts for p50 and Bcl-3 only. Arrows labeled with an asterisk represent supershifted bands. Recombinant p50 plus probe and anti-p50 demonstrates antibody fidelity. g) Anti-c-Rel shows a supershift in both control and HU, but the complex is much lighter in control. From the same gel, anti-Rel B and anti-p52 improved complex formation but did supershift the bands.

TNF-{alpha} pathways in atrophied muscle
The activity or expression of TNF-{alpha} and other proteins known to be involved in cytokine pathways were measured. Although the trigger for unloading-induced atrophy may be intracellular, the possibility of muscle cytokine production by an autocrine mechanism has been shown for TNF-{alpha} as well as other cytokines (37 , 38) . Though detected at only low levels, there was a moderate decrease in TNF-{alpha} protein levels in 7 day unloaded soleus muscles (Fig. 5 a). TRAF2, an adaptor protein required for TNF-{alpha}-activated JNK and NF-{kappa}B pathways (39 , 40) , was down-regulated in both whole-muscle lysates (Fig. 5b ) and cytosolic extracts (not shown) from 7 day unloaded muscles. TRAF2 is expressed at high levels in mature muscle fibers and is thought to have a role in muscle cell viability during development (14) .



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  		Figure 5. Expression and activity of TNF-{alpha} pathway components. Expression was measured using specific antibodies to TNF-{alpha} (a) and TRAF2 (b). Enzyme activity was measured using in vitro kinase assays for p38 (c), JNK (d), and Akt (e) in lysates from control and 7 day unloaded soleus muscles. Recombinant rat TNF-{alpha} (rTNF) and WEHI (W) cell lysates used as positive controls. No increase in expression or activity of proteins was found. TNF-{alpha} and TRAF2 expression decreased with atrophy (P<0.05). Blots quantified by densitometric analysis. Data are means ± SE from at least 4 muscles per group.

Whereas TNF-{alpha} and TRAF2 are upstream components of cytokine pathways, p38 and JNK are downstream MAP kinases activated by immune and inflammatory triggers such as TNF-{alpha} and other cytokines. There was no difference in p38 or JNK activity after either 1 (not shown) or 7 days of soleus muscle unloading (Fig. 5c, d ). This in conjunction with the lack of AP-1 or NFAT-dependent reporters with unloading suggests that cytokine-mediated pathways are not activated with disuse atrophy, and is consistent with a report indicating the lack of complement activation during hind limb muscle unloading (41) . The activity of protein kinase B (PKB/Akt), an I{kappa}B kinase known to be activated by cytokines (42) , was not different at either 1 or 7 days of unloading (Fig. 5e ). This observation is consistent with the lack of change in I{kappa}B phosphorylation status.

Expression of Bcl-2 family members
Members of the Bcl-2 family are targets of NF-{kappa}B (40) . We measured the expression of several proteins in this family in unloaded muscle. The strongly anti-apoptotic Bcl-2 protein was increased by fourfold in lysates from unloaded muscles compared with control (Fig. 6 a). The anti-apoptotic Bcl-XL was not changed due to unloading (Fig. 6b ). However, the proapoptotic Bax protein was down-regulated by 46% in both whole-cell (Fig. 6c ) and microsomal fractions (not shown). The proapoptotic protein Bad (Fig. 6d ) was unchanged, and Bak (Fig. 6e ) decreased by 38% due to unloading. Muscle unloading did not trans-activate a bcl-2 reporter gene containing 7 NF-{kappa}B sites (Fig. 6f ), suggesting post-rather than transcriptional regulation. The expression of calcineurin, a phosphatase involved in the regulation of both Bad and Bcl-2, was unchanged with unloading (not shown).



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  		Figure 6. Expression of Bcl-2 family members in control and 7 day unloaded soleus muscle whole-cell lysates. Relative protein level measured using antibodies specific to Bcl-2 (a), Bcl-XL (b), Bax (c), Bad (d), and Bak (e). Jurkat, mouse macrophage (M), CTLL, serum-starved 3T3 (SS), and A-431 whole-cell extracts used as positive controls. Expression of anti-apoptotic Bcl-2 increased 4-fold with unloading (P<0.05). Expression of proapoptotic Bax decreased ~50% with unloading (P<0.05). Bak was moderately lower (38%; P<0.05). Transfected bcl-2 reporter gene not activated by muscle unloading (f). Mcl-1 expression not detected in muscle extracts (not shown). Blots quantified by densitometric analysis. Data are means ± SE of at least 4 muscles per group. *Significance of at least P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Members of the NF-{kappa}B family of trans regulators are activated by a broad range of signals including cytokines, mitogens, free radicals, and stress (9 , 10) . During the muscle wasting associated with disease (i.e., cachexia), NF-{kappa}B induction appears to be triggered by cytokines (4 , 5) . The present work demonstrates that muscle disuse atrophy in healthy young rodents is also associated with the activation of NF-{kappa}B; however, the trigger does not appear to be of cytokine origin and the activated NF-{kappa}B pathway is distinct from that seen with cachexia or TNF-{alpha}. Although NF-{kappa}B activation is seen in different types of endoplasmic reticulum (ER) stress (43) , there is no evidence of prototypical ER stress markers in disuse atrophy (44) . Moreover, we show the lack of NF-{kappa}B p65 activation, a component of the ER stress response (45) .

The left panel of Fig. 7 summarizes key components of TNF-{alpha}-induced NF-{kappa}B pathways that were assessed in the present study. Known relationships in this pathway have recently been reviewed (46) , and some of these components have been shown to be involved in disease-related muscle wasting (5 , 6) . We did not show involvement of TNF-{alpha}-induced NF-{kappa}B activation in disuse atrophy but rather an alternative NF-{kappa}B pathway that does not appear to be associated with inflammation. Such a pathway has recently been uncovered and involves activation of IKK{alpha} (47) . IKK{alpha} activation is not required for inflammatory or cytokine activation of NF-{kappa}B (47 , 48) . IKK{alpha} is required for limb morphogenesis (48 , 49) and IGF-II-induced muscle differentiation (50) , but p65 activation is not involved. Whereas IKK{alpha} was not assessed here, immunoblotting indicated that nuclear levels of p50, c-Rel, and Bcl-3 are up-regulated with muscle disuse, but not p65, p52, or Rel B. Gel shift assays consistently showed more protein binding to NF-{kappa}B oligonucleotides in extracts from unloaded muscle; however, of the NF-{kappa}B/I{kappa}B family members, only p50, c-Rel, and Bcl-3 were identified as components of the binding complex. This is in contrast to cytokine-(TNF-{alpha}) or disease-dependent NF-{kappa}B activation, which involves p65:p50 heterodimers and is dependent on cytosolic I{kappa}B phosphorylation and degradation (4 , 6 , 8) . We did not find evidence of I{kappa}B activation or of cytokine signaling based on p38 or JNK activity assays or AP-1/NFAT-dependent reporter assays. Thus, we have shown that the activation of NF-{kappa}B during disuse atrophy involves a pathway distinct from that used by cytokines.



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  		Figure 7. Proposed model for roles of NF-{kappa}B pathways in cachexia/cytokine-induced muscle atrophy vs. disuse-induced muscle atrophy. Data from literature support involvement of the classic TNF-{alpha}/NF-{kappa}B pathway in disease-associated muscle atrophy (left panel). The data do not support involvement of classic NF-{kappa}B pathways in disuse atrophy but rather an alternative NF-{kappa}B pathway involving nuclear p50 and Bcl-3 binding activity (right panel). See text for details.

The right panel of Fig. 7 shows an alternative NF-{kappa}B pathway that could be involved during muscle atrophy. Even though the contribution of c-Rel is not included in this model, it should be noted that p50 and c-Rel can form trans-activating heterodimers (51) . There is evidence in the literature for at least two different mechanisms involving Bcl-3 and p50 nuclear translocation and trans-regulation. In one mechanism, Bcl-3-p50 complexes are activated by inducible phosphorylation of the p105 precursor via I{kappa}B kinase. The phosphorylated p105 from the p105–p50 complex is then proteolytically processed to form a p50 homodimer (52) . Bcl-3 has a strong affinity for p50 homodimers and its binding causes nuclear translocation of the complex, binding to NF-{kappa}B sites, and transcriptional regulation. An alternative mechanism is the interaction of Bcl-3 with two p105–p50 complexes. This leads to the release of Bcl-3-(p50)2, its nuclear translocation, and regulation of transcription (36) . However, the mechanism of activation of p50, Bcl-3, or c-Rel during muscle atrophy awaits further investigation.

There is precedent for physiological activation of NF-{kappa}B without induction of p65. Certain tumors show activation of NF-{kappa}B family members similar to that seen here in disuse atrophy. Breast tumors show activated c-Rel, p50, and p52 but not p65 (53 , 54) . Bcl-3, which can trans-activate by forming a complex with p50 or p52 homodimers (55 , 56) , has been shown to be activated in breast tumors (54) . Similarly, in mouse skin tumors, p50 homodimers are the major component of DNA binding complexes without activation of p65, Rel B, or c-Rel (57) . Nuclear Bcl-3 was overexpressed as well. The overexpression of Bcl-3 can induce nuclear p50 homodimer formation in other systems (33 , 36 , 58) . Although the significance of the up-regulation of NF-{kappa}B family members in these tumors is not known, anti-apoptotic targets have been proposed (53) .

Though the target genes of NF-{kappa}B during muscle wasting are unclear, this family of trans factors is known to regulate genes involved in cell survival (40) . There is evidence from type 2A muscular dystrophy patients that classic NF-{kappa}B activation targets survival gene induction in skeletal muscle (15) . TRAF2 is an anti-apoptotic target of TNF-{alpha}-induced NF-{kappa}B in cultured muscle cells (14) . Members of the anti-apoptotic Bcl-2 family are known targets of NF-{kappa}B in nonmuscle (59 60 61) . Here, Bcl-2 protein level was markedly increased in atrophied muscle. Consistent with an anti-apoptotic phenotype was the observation that expression of proapoptotic proteins Bak and Bax was decreased with muscle atrophy. However, bcl-2 was not a transcriptional target of NF-{kappa}B because a transfected reporter plasmid containing 3.9 kb of bcl-2 regulatory region was not up-regulated by unloading. Another survival gene, HO-1, has been shown to be a target of NF-{kappa}B in liver (62) , and its promoter region contains multiple NF-{kappa}B binding sites (63) . Since we showed HO-1 protein is up-regulated by unloading (44) , it could be a potential target of NF-{kappa}B in atrophying muscle. Although we do not know whether NF-{kappa}B activates survival genes in atrophying muscle, it seems a mechanism may be in place that protects atrophying fibers from cell death. Skeletal muscle fibers are postmitotic and multinucleated, and though there appears to be a loss of myonuclei during unloading atrophy (64) , whole-muscle cells are not lost. Thus, up-regulation of survival genes such as bcl-2 or HO-1 may temper the stimuli leading to myonuclear loss from destroying the entire cell.

In summary, the present work provides insight into a signaling pathway involved in unloading-induced muscle atrophy. An NF-{kappa}B pathway is activated that is distinct from disease-related atrophy involving the NF-{kappa}B family member p50, the I{kappa}B family member Bcl-3, and possibly c-Rel. Even though the target genes of NF-{kappa}B trans-activation during muscle atrophy are unclear, survival genes that are up-regulated with unloading are possibilities. NF-{kappa}B may also activate genes involved in post-transcriptional control of target genes such as bcl-2.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grant AR41705, an American College of Sports Medicine Foundation award, and a Dudley Allen Sargent award to R.B.H. During this work S.K. was the recipient of an Established Investigator Award from the American Heart Association.

Received for publication November 9, 2001. Revision received January 8, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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