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MUSCLE CELL BIOLOGY AND CELL MOTILITY
B
, but not c-Rel, in skeletal muscle atrophy
1Department of Health Sciences, Boston University, Boston; 2Genzyme, Waltham, Massachusetts; and 3Division of Immunology, Department of Medicine, Weill School of Medicine, Cornell University, New York, New York
Submitted 26 May 2006 ; accepted in final form 11 August 2006
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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B (NF-B) activity. Previous work showed that p50 is one of the NF-B family members required for this activation and for muscle atrophy. In this work, we tested whether another NF-B family member, c-Rel, is required for atrophy. Because endogenous inhibitory factor B
(IB) was activated (i.e., decreased) at 3 and 7 days of muscle disuse (i.e., hindlimb unloading), we also tested if IB, which binds and retains Rel proteins in the cytosol, is required for atrophy and intermediates of the atrophy process. To do this, we electrotransferred a dominant negative IB (IB
N) in soleus muscles, which were either unloaded or weight bearing. IBN expression abolished the unloading-induced increase in both NF-B activation and total ubiquitinated protein. IBN inhibited unloading-induced fiber atrophy by 40%. The expression of certain genes known to be upregulated with atrophy were significantly inhibited by IBN expression during unloading, including MAFbx/atrogin-1, Nedd4, IEX, 4E-BP1, FOXO3a, and cathepsin L, suggesting these genes may be targets of NF-B transcription factors. In contrast, c-Rel was not required for atrophy because the unloading-induced markers of atrophy were the same in c-rel–/– and wild-type mice. Thus IB degradation is required for the unloading-induced decrease in fiber size, the increase in protein ubiquitination, activation of NF-B signaling, and the expression of specific atrophy genes, but c-Rel is not. These data represent a significant advance in our understanding of the role of NF-B/IB family members in skeletal muscle atrophy, and they provide new candidate NF-B target genes for further study.
disuse; nuclear factor-B signaling; unloading; muscle wasting; ubiquitination; atrophy genes; inhibitory factor B
B (NF-B) transcription factors (12, 27, 34, 56).
The classical NF-B pathway is activated in muscle wasting because of disease (12, 21, 22). Our work shows that the removal of weight bearing leads to sustained NF-B activation in muscle as reflected by NF-B reporter activity and gel shift assays (26), more recently confirmed by others (6, 18). Biochemical data suggested that the NF-B Rel transcription factors p50 and c-Rel, and the inhibitor of B (IB) family member B cell lymphoma 3 (Bcl-3) were candidates for the NF-B activation resulting from muscle disuse. Indeed, unloading-induced NF-B activation and fiber atrophy were inhibited in soleus and plantaris muscles from mice null for either Nfkb1 (encodes p105/p50) or Bcl3, indicating that both proteins are required for atrophy (26). A role for c-Rel in muscle atrophy, however, remains to be tested. Because p50 is required for atrophy, the additional requirement of c-Rel could point toward involvement of c-Rel-p50 dimers, which are known to have strong transactivating properties (8, 57).
In addition, we recently showed a decrease in IB levels in muscle after 7 days of unloading (27), suggesting that IB could be a component of NF-B activation in disuse atrophy. Consistent with this are recent data showing that transgenic mice expressing a dominant negative IB in muscle had a 45% inhibition of denervation atrophy (12).
We have previously identified that the Nfkb1 gene and the Bcl3 gene are required for soleus and plantaris muscle disuse muscle atrophy, and others have shown genetic evidence for the involvement of IB in physiological models of atrophy. However, given the complexity of NF-B signaling, the role of the necessary components of this pathway in muscle atrophy are just beginning to be defined. The questions we addressed in this study were: 1) is c-Rel required for unloading atrophy or its biochemical intermediates, 2) is IB required for unloading atrophy, 3) is IB activation required for the normal increase in total ubiquitinated protein seen during unloading atrophy, and 4) what are candidate target genes of NF-B-dependent transcription during unloading atrophy? To answer the first question, we used c-rel–/– mice and found that muscle atrophy and its biochemical markers were changed to the same extent as wild-type mice, obviating a role for c-Rel in unloading atrophy. This is important, since it reduces the number of NF-B family members that could be involved in the atrophy process. To address the last three questions, rat soleus muscles were transduced with a dominant negative IB plasmid (IBN). By using electrotransfer of IBN in adult muscle, we had the advantage of avoiding any confounding effects of its overexpression during development. Expression of dominant negative IB inhibited atrophy by 40%. It also abolished the unloading-induced increase in NF-B activity and total protein ubiquitination. In addition, 6 out of 10 genes that are normally induced with unloading atrophy were significantly inhibited by expression of dominant negative IB. These data represent a significant advance in our understanding of the role of NF-B/IB family members in skeletal muscle atrophy, and they provide new candidate NF-B target genes for further study.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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BN cDNA. For the c-Rel experiments, 8-wk-old C57BL/6 mice were used as the wild-type strain (The Jackson Laboratory, Bar Harbor, ME) and c-rel–/– mice (background strain C57BL/6) were created as described previously (35). The use of animals in this study was approved by the Boston University Institutional Animal Care and Use Committee.
Expression and reporter plasmids.
The NF-B-GL3 reporter plasmid was used as previously described (27). An expression vector containing IBN cDNA (IB with amino acids 1–36 deleted), originally obtained from Dr. D. Ballard (11), was subcloned in the COOH terminus of an enhanced green fluorescence protein (EGFP) expression vector (EGFP-c1 from Clontech) to construct an IBN-EGFP fusion protein. Plasmid DNA was prepared using an Endotoxin-Free Mega Prep Kit (Qiagen).
DNA injection, electroporation, and hindlimb unloading.
The method for plasmid DNA injections in skeletal muscle has been previously detailed (38). Briefly, the DNA was ethanol precipitated and resuspended at 4°C overnight in 1x PBS before injection. The solei of some rats were coinjected with 45 µg NF-B-GL3 reporter plasmid plus 45 µg of either IBN-EGFP or EGFP-c1 in a volume of 50 µl. In this case, we take advantage of the fact that electroporation of mixes of two vectors in vivo typically produces cotransduction of a given fiber nearly 100% of the time (3, 41). Other rats had just the IBN-EGFP or the EGFP-c1 plasmid injected in the soleus muscle. The solei of mice were injected with 10 µg of the NF-B reporter plasmid in a volume of 5 µl. Following injection, electric pulses were delivered, using an electric pulse generator (Electro Square porator ECM 830; BTX), by placing two paddle-like electrodes on each side of the muscle. Five electric pulses were delivered at 125 V/cm, duration of 20 ms, and interpulse interval of 200 ms. This electroporation protocol did not induce damage in our hands (unpublished data) nor by Rossini et al. (15, 42). The efficiency of plasmid DNA uptake in muscle is the same in weight-bearing muscle and unloaded muscle (38).
Animals were randomly assigned to control or hindlimb-unloaded (HU) groups, and an elastic tail cast was applied to the HU group while still anesthetized. Later (24 h), rat and mouse hindlimbs were suspended off the cage floor by 1 mm, as previously described (26, 40). Muscles were extracted 7 days later (8 days after plasmid injection) except in the case where NF-B activity and IB expression were measured at 3 days of unloading (4 days after plasmid injection).
Muscle preparation and analysis. Soleus, plantaris, and gastrocnemius muscles were removed, rapidly weighed, and either processed immediately for protein or RNA assays or embedded in tissue-freezing medium and quick-frozen for sectioning and subsequent immunohistochemical analysis.
NF-B reporter activity.
Muscle was homogenized in passive lysis buffer (Promega), and luciferase activity was assessed as described previously (26).
Western blot and coimmunoprecipitation.
Protein concentration was determined on muscle homogenates in passive lysis buffer using a detergent-compatible assay (Bio-Rad). For immunoblotting, protein was denatured and separated on SDS-polyacrylamide gels (Bio-Rad). Proteins were transferred to an immobilon-FL polyvinylidene fluoride membrane (Millipore), blocked, and immunoblotted with the appropriate antibody diluted according to the manufacturer's instructions. Alexa Fluor 680 (Invitrogen) or IRDye800 (Rockland Immunochemicals) fluorescent dye-conjugated secondary antibodies were used for visualization. Immunoprecipitation was performed using a Catch and Release kit (Upstate) according to the manufacturer's instructions. Briefly, 1 mg of total protein from control samples were used, and either anti-p50 antibody (ab7971; abcam) or anti-green fluorescent protein (GFP) antibody (ab290; abcam) was used for immunoprecipitation. Samples were eluted with denaturing buffer, boiled, and separated by electrophoresis, as described above. Anti-p65 (ab7970) was from abcam, anti-IB (sc-371) and anti-phospho-IB (sc-21869) were from Santa Cruz Biotechnology, and anti-ubiquitin (U-5379) was from Sigma-Aldrich. Blots were imaged using an Odyssey system (Li-Cor Biosciences), which uses direct infrared fluorescence detection, with a wide linear dynamic range. For quantification, the sum of the total fluorescence in each lane was used to quantify total ubiquitinated protein in a sample, as shown in RESULTS. For IB blots, the fluorescence of the specific band was quantified in each lane.
Immunohistochemistry. Muscles were sectioned (10 µm) from the midbelly of the soleus and fixed in 4% paraformaldehyde. Mouse muscle cross sections were incubated with wheat germ agglutinin Texas Red-X conjugate (W21405 [GenBank] ; Molecular Probes) to visualize muscle fiber borders, and rat muscle cross sections were incubated with anti-laminin (L9393; Sigma-Aldrich) followed by Alexa Fluor 488 (Invitrogen) fluorescent dye conjugated secondary antibody. Fluorescence microscopy was used for visualization, and images were captured with a SPOT RT camera (Diagnostic Instruments). Fiber cross-sectional area measurements were calculated using the MetaMorph Imaging System (Universal Imaging).
RNA isolation and RT-PCR. Total RNA was isolated from the soleus muscle using a TriZOL-based protocol previously described (1). A subsequent "clean-up" step was used to improve the quality of the RNA using RNeasy columns (Qiagen). cDNA was synthesized using 1 µg total RNA in a 20-µl reaction using a RETROscript First Strand Synthesis Kit (Ambion) according to the manufacturer's instructions. The cDNA (2 µl) was then used as a template in a PCR reaction using specific primers for atrogin-1 (GenBank accession no. AF441120), Nedd4 (GenBank accession no. XM_486230), immediate-early response gene (IEX; GenBank accession no. X96437), translation initiation factor 4E binding protein 1 (4E-BP1; GenBank accession no. NM_007918), FOXO3a (GenBank accession no. AF114259), cathepsin L (GenBank accession no. X06086), sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1; GenBank accession no. M99223), myosin heavy chain IIb (MyHCIIb; GenBank accession no. AJ278733), activin IIb receptor (GenBank accession no. NM_007397), glutamine synthetase (GenBank accession no. NM_008131), p105 (GenBank accession no. XM_342346), Bcl-3 (GenBank accession no. NM_033601), and 18S (GenBank accession no. X01117) that were obtained from PrimerBank, where available (http://pga.mgh.harvard.edu/primerbank/index.html), or using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primers for PCR amplification were as follows: atrogin-1, forward, 5'-CAGCTTCGTGAGCGACCTC-3', and reverse, 5'-GGCAGTCGAGAAGTCCAGTC-3'; Nedd4, forward, 5'-GTGGGAAGAGAGGCAGGATGTC-3', and reverse, 5'-GCGAATTCACAGGAAGTGTAGGC; IEX, forward, 5'-ctaccagcgccatatcacct, and reverse, 5'-AGGTACCCCTGGTGCTCTTT-3'; 4E-BP1, forward, 5'-GGGGACTACAGCACCACTC-3', and reverse, 5'-CTCATCGCTGGTAGGGCTA-3'; FOXO3a, forward, 5'-GTCATGGGCCACGATAAGTT-3', and reverse, 5'-GGGCTGCTAACAGTCTCTGC-3'; cathepsin L, forward, 5'-CCCCAAGACTGTGGACTGGAGAGA-3', and reverse, 5'-TTTACAAGATCCATCCTTTGCTTC-3'; SERCA1, forward, 5'-CTGTCCATGTCCCTCCACTT-3', and reverse, 5'-GGGTGGTTATCCCTCCAGAT-3'; MyHCIIb, forward, 5'-TTGAAAAGACGAAGCAGCGAC-3', and reverse, 5'-AGAGAGCGGGACTCCTTCTG-3'; activin IIb receptor, forward, 5'-ACCCCCAGGTGTACTTCTG-3', and reverse, 5'-CATGGCCGTAGGGAGGTTTC-3'; glutamine synthetase, forward, 5'-TGAACAAAGGCATCAAGCAAATG-3', and reverse, 5'-CAGTCCAGGGTACGGGTCTT-3'; p105, forward, 5'-GCTACTCCCAGAGCACAAGG-3', and reverse, 5'-TCTCGCTGTGTGTGTTCCTC-3'; Bcl-3, forward, 5'-CCGGAGGCCCTTTACTACCA-3', and reverse, 5'-GGAGTAGGGGTGAGTAGGCAG-3'; and 18S, forward, 5'-CGCGGTTCTATTTTGTTGGT-3', and reverse, 5'-AGTCGGCATCGTTTATGGTC-3'. Once the conditions were optimized for each primer, PCR was performed, and the amplified samples were run on a 10% acrylamide gel, stained with Syto 60 red fluorescent nucleic acid stain (Molecular Probes), and visualized and quantified using an Odyssey infrared detection system (Li-Cor Biosciences).
Statistical analysis.
A one-tailed, Student's t-test was used for statistical analysis of IB protein expression, and a two-way ANOVA (loading condition vs. plasmid type) was used for analysis of all other variables (GraphPad Software, San Diego, CA). All data are expressed as means ± SE, and significance was established at the P < 0.05 level.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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B expression during muscle unloading.
Three and 7 days of hindlimb unloading caused a 32 and 27% decrease in soleus muscle IB protein levels, respectively, and a 32% decrease in the plantaris muscle at 7 days (Fig. 1). This early decrease in protein levels suggests that IB degradation may be involved in NF-B activation with unloading.
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B is initiated by its phosphorylation, we measured phospho-IB protein levels in unloaded and control muscle. There were no differences between control and unloaded muscle (data not shown), in agreement with our previous findings (27). It is possible that, in 7-day unloaded muscle, the phospho-IB may be at a new steady state, having the same amount of phosphorylation as in control muscle to maintain a lower level of IB. At later time points, the amount of phosphorylation needed may only depend on the amount of newly formed IB.
Effect of dominant negative IB on unloading-induced NF-B reporter activity.
To determine whether the observed IB degradation is necessary for unloading-induced NF-B activity, we transduced whole rat soleus muscles with a mutant IB plasmid (IBN, also known as the super repressor) that lacks amino acids 1–36 and therefore the phosphorylation sites necessary for subsequent proteasomal degradation. Overexpression of an IBN fusion protein (IBN-EGFP) in soleus muscles using electrotransfer was confirmed by Western blot analysis (Fig. 2). To confirm that IBN-EGFP functions the same as IBN, we found that tumor necrosis factor (TNF)--induced (10 ng/ml for 3 h) activation of the NF-B reporter in C2C12 myotubes was inhibited to the same extent when the cells were transfected with the IBN compared with the IBN-EGFP plasmid (data not shown).
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B reporter plasmid, 3 days of hindlimb unloading caused a twofold increase in NF-B reporter activity, whereas 7 days of unloading showed a fivefold increase compared with weight-bearing control muscles (Fig. 3). However, reporter activity was completely abolished in weight-bearing and unloaded soleus muscles that were coinjected with IBN-EGFP plus the NF-B reporter at both 3 and 7 days, suggesting that IB degradation is necessary for NF-B activation with hindlimb unloading. In these cotransfection experiments, all fibers that receive one plasmid alsoreceive the second plasmid (3, 41) so that the effectiveness of the test plasmid on reporter activity is independent of transfection efficiency. As mentioned, unloaded soleus muscles and weight-bearing soleus muscles show no difference in plasmid transfection efficiency, as assessed using Southern blotting (38).
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BN inhibited NF-B activity, we next determined whether it also inhibited muscle fiber atrophy with unloading. An anti-laminin antibody was used to visualize the borders of fibers in cross sections from weight-bearing and unloaded rats. The cross-sectional area of fibers expressing IBN-EGFP (fluorescing fibers) was compared with fibers not expressing IBN-EGFP (nonfluorescing fibers; Fig. 4). Unloading for 7 days caused a 40% decrease in fiber cross-sectional area in fibers not expressing IBN-EGFP compared with normal weight-bearing fibers. However, muscle fiber atrophy with unloading was inhibited by 40% in fibers expressing the super repressor, indicating that IB signaling is important in the atrophy process (Fig. 4). The frequency distribution of fiber area (Fig. 4D) illustrates the increase in the percentage of small fibers not expressing IBN-EGFP with unloading. This leftward shift in the distribution curve is attenuated in fibers expressing IBN-EGFP with unloading. In weight-bearing muscle, there were no differences in fiber cross-sectional area between fibers expressing vs. not expressing IBN-EGFP. In addition, we carried out experiments comparing fiber size from four different groups (2 weight bearing, 2 unloaded) of rats where only EGFP vs. IBN-EGFP expressing fibers were compared, and the same results were found (not shown). The transfection efficiency was between 40 and 75% of fibers based on EGFP expression.
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B on unloading-induced increase in ubiquitinated protein.
Because muscle atrophy involves significant activation of the ATP-dependent ubiquitin-proteasome pathway, we determined if IBN affected the commonly reported (53, 54, 58) increase in the amount of total ubiquitinated protein in atrophying muscle. Thus the increase in total protein ubiquitination was used as a marker of increased protein degradation. Because many proteins are ubiquitinated, the fluorescent signal from the entire lane of an anti-ubiquitin immunoblot was measured using the highly sensitive Odyssey Li-Cor system, as shown (Fig. 5). There was a 38% increase in the amount of ubiquitinated protein in unloaded muscles compared with weight-bearing control muscles. However, the dominant negative IB abolished this increase, suggesting that the Rel proteins bound by IB have a role in unloading-induced muscle proteolysis (Fig. 5). Besides the soleus, another muscle, the plantaris, also showed a 40% increase in the amount of ubiquitinated protein resulting from unloading (data not shown).
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B on the unloading-induced increases in mRNA expression of selected genes.
RT-PCR was used to study the effect of the dominant negative IB on the expression of selected genes known to be upregulated with atrophy based on our microarray study (46). The hindlimb unloading-induced increases in mRNAs encoding atrogin-1/MAFbx, Nedd4, IEX, 4E-BP1, FOXO3a, and cathepsin L (all of which have a role in regulating muscle protein content or apoptosis) were inhibited by 21–50% in muscles injected with the dominant negative IB. This suggests that these genes may be targets of NF-B transcription factors that are regulated by IB (Fig. 6). In-depth in vivo analysis of the promoter regions of this set of IB-responsive genes would be required to determine if they are bona fide targets of NF-B in unloaded muscle. The increase in mRNA expression of SERCA1, MyHCIIb, activin IIb receptor, and glutamine synthetase was unaffected by the super repressor, suggesting that not all atrophy-upregulated genes are NF-B targets.
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BN-EGFP on p50 and Bcl-3 expression.
Because the Bcl3 and Nfkb1 (encodes p105/p50) genes are regulated by NF-B (10, 48), and both are necessary for disuse atrophy (26), we determined if inhibition of IB by expression of IBN would inhibit mRNA and protein expression of these NF-B and IB family members in control and unloaded muscle. Bcl-3 mRNA and protein expression (Fig. 7, A and B) were significantly increased after 7 days of unloading; however, the super repressor significantly attenuated these increases. Although there were no unloading effects on p50 mRNA or protein expression [though nuclear protein levels are increased (27); Fig. 7, C and D], the super repressor significantly reduced p50 mRNA and protein expression at 7 days in both control and unloaded muscles.
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BN-EGFP.
Coimmunoprecipitation was used to help determine if p50 or p65 are bound by ectopically expressed IBN-EGFP, thereby retaining their cytosolic localization. We were successful in coimmunoprecipitating p65 by using an antibody to the GFP moiety of IBN-EGFP (Fig. 8). However, because the heavy-chain antibody IgG is 50 kDa, the possible identification of p50 binding to IBN-EGFP could not be done using this strategy. Therefore, immunoprecipitation was performed with anti-p50, and the immunoprecipitates were immunoblotted for IB. Thus the fusion protein showed association with p65 and p50 (Fig. 8).
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B reporter activity in wild-type vs. c-Rel knockout mice.
To determine if c-Rel is necessary for unloading-induced NF-B activation, an NF-B-dependent reporter was injected in soleus muscles of control and unloaded wild-type and c-Rel knockout mice. NF-B activity was increased with unloading to the same extent in wild-type and c-rel–/– mice (Fig. 9A), thus eliminating c-Rel as a contributing transcription factor in NF-B activation with unloading.
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Ubiquitinated protein in wild-type vs. c-Rel knockout mice. Because the majority of proteins that are degraded during muscle atrophy ultimately are handled by the ubiquitin proteasome pathway, we determined whether c-Rel is necessary for the unloading-induced increase in total ubiquitinated protein. Unloading for 7 days caused a similar increase in total ubiquitinated protein in wild-type (72%) and c-rel–/– (63%) mice compared with their respective weight-bearing controls (Fig. 9C).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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B signaling has emerged as one pathway that is involved in this process. Not only is NF-B activated early, as shown here at 3 days, but it is sustained in atrophying muscles because of 10 days of unloading (26). Moreover, mice null for either the Nfkb1 gene or the Bcl3 gene show profound inhibition of the unloading-induced NF-B activation, muscle fiber atrophy, and the slow to fast shift in myosin expression (26). However, because of the complexity of NF-B signaling, additional molecules involved in this signaling need investigation to understand the details of its regulation. It is possible that NF-B signaling of both IB-dependent and IB-independent pathways are involved (12, 26, 27). In addition, the genes targeted by NF-B need to be identified to understand how NF-B regulates muscle protein loss. In the present study, we show that, although NF-B activity is markedly increased in unloaded soleus muscles at 3 and 7 days, injection of a dominant negative IB plasmid (IBN) abolishes the unloading induced activation of NF-B. This super repressor also completely blocks the increase in ubiquitinated protein, and it inhibits skeletal muscle atrophy by 40%. The super repressor attenuated the unloading-induced increased mRNA expression of MAFbx/atrogin-1, Nedd4, IEX, 4E-BP1, FOXO3a, and cathepsin L, suggesting that these are candidate NF-B target genes. These findings show that IB is required, in part, for atrophy and markers of the underlying biochemical events. In contrast, we show that c-Rel is not involved in the regulation of muscle atrophy, thereby narrowing the number of NF-B transcription factors that could be involved with mediating atrophy.
Dominant negative IB.
We show that IB is decreased in unloaded muscle. Therefore, we experimentally blocked this loss during unloading atrophy to test which functions IB is regulating. We injected muscles with a plasmid encoding an NH2-terminally truncated version of IB that has a super repressing effect on the NF-B components that are normally regulated by native IB. Upon expression of this super repressor, we then explored the consequences on 1) NF-B activation; 2) muscle atrophy; 3) ubiquitination of total muscle protein; and 4) the expression genes upregulated during atrophy.
NF-B activity and muscle fiber atrophy.
The presence of the dominant negative IB downregulated NF-B signaling with muscle unloading. In fact, it eliminated baseline activity of the reporter even in weight-bearing muscles. We also found that the dominant negative IB inhibited unloading atrophy by 40%. Our finding that the super repressor attenuates muscle atrophy is in agreement with a recent study that used transgenic mice expressing a muscle-specific, constitutively active, IKK
(MIKK) which, in the classic NF-B pathway, causes phosphorylation of IB at serines 32 and 36, resulting in its ubiquitination and subsequent proteasomal degradation (12). The muscles of MIKK mice showed an increase in NF-B activity that was sufficient to induce significant muscle atrophy. However, when MIKK mice were crossed with transgenic mice expressing a muscle-specific IB super repressor (MISR), NF-B activity was abolished, and the atrophy reversed. Furthermore, muscle denervation and cancer cachexia caused a 53 and 33% reduction in fiber cross-sectional area, respectively, in wild-type mice, and this atrophy was attenuated by 45 and 55%, respectively, in MISR mice. Our data are in close agreement with these, since, here, the 40% unloading-induced fiber atrophy was inhibited by 40% in fibers expressing the super repressor. Our work on the role of IB shown here extends previous work by focusing on the role of IB in ubiquitination and atrophy gene expression in a physiological model of atrophy and by the use of a conditional expression system.
IB and ubiquitination in unloading atrophy.
Although there are several proteases that have been implicated in the initial steps of proteolysis in skeletal muscle atrophy (17, 24, 39), it is generally accepted that one of the last steps of protein degradation for the majority of intracellular proteins is via the ubiquitin proteasome pathway. In fact, most muscle atrophies lead to an increase in the amount of ubiquitinated protein (54), and it has previously been shown that 4, 8, and 14 days of unloading (53, 58) cause increased ubiquitin-protein conjugation. In agreement with this, we observed increased ubiquitinated protein resulting from 7 days of unloading. However, the dominant negative IB blocked this increase, suggesting that the cytosolic retention of Rel dimers by IB regulates the increased protein ubiquitination associated with atrophy. This is supported by the work of Li et al. (32) who demonstrated, using IBN in a stable C2C12 cell line, that NF-B is required for upregulation of UbcH2 mRNA (an E2 enzyme) and TNF--dependent ubiquitin-conjugating activity in differentiated myotubes. Furthermore, the increased proteolysis in MIKK mice was blocked by the proteasome inhibitor MG-132, providing further evidence that NF-B activation can stimulate proteasome-dependent proteolysis (12).
NF-B target genes.
So far, there is evidence that UbcH2 and the C3 proteasome subunit may be NF-B target genes during atrophy in cell culture (16, 32). Unloading atrophy upregulates other genes involved with ubiquitin-proteasomal degradation such as the ubiquitin protein ligases atrogin-1 and Nedd4 (46). We therefore determined the effect of dominant negative IB on the expression of these genes during unloading. IBN significantly attenuated the unloading-induced increase in both atrogin-1 and Nedd4, suggesting these genes may be targets of NF-B regulated by IB. Although the identification of bona fide NF-B target during atrophy requires more detailed investigation, preliminary analysis of the 5'-flanking region of these genes, using Promoser, Patser, TESS, and Transfac databases, indicates that atrogin-1 and Nedd4 have multiple putative NF-B binding sites.
Dominant negative IB also had a significant inhibitory effect on the expression of other genes that we previously found to be upregulated with atrophy (46). IBN expression attenuated the unloading-induced increase in IEX, 4E-BP-1, FOXO3a, and cathepsin L mRNA expression, whereas SERCA1, MyHCIIb, activin IIb receptor, and glutamine synthetase expression were not affected. Therefore, these latter genes would not be candidates for further study as NF-B targets. It is worth noting that all the genes studied here that were inhibited by the dominant negative IB play a role in regulating muscle protein content, whereas those that were unaffected do not. This could provide an explanation for the attenuation of muscle atrophy with NF-B inhibition.
The attenuated expression of IEX, which is involved in the regulation of cellular growth and survival, was not unexpected, since this gene has previously been confirmed as an NF-B target gene (55). In addition, IEX is known to be involved in apoptosis, and muscle atrophy is associated with myonuclear loss (2). Furthermore, cathepsin L, a lysosomal enzyme whose role appears to be the degradation of membrane proteins (37), has been shown to contain an NF-B binding motif (44), perhaps explaining the attenuation of the unloading-induced increase in cathepsin L mRNA by dominant negative IB. However, to our knowledge, this is the first study to suggest 4E-BP1 and FOXO3a as potential NF-B target genes.
Because not all fibers were transduced with IBN (40–75% efficiency), in some experiments where muscle homogenates were used, the results could have underestimated the effect of blocking endogenous IB. In the case of the mRNA expression data, it is not clear whether transduction of 100% of the fibers would show a greater effect on the inhibition of gene expression, but the effect was at least not overestimated. The complete inhibition of increased ubiquitinated protein, by expression of IBN, could be because of the transgene reducing the ubiquitin-protein conjugates in the transfected cells to levels below that in weight-bearing controls, thus causing the average values to be the same as that of the controls.
Regulation of p50 and Bcl-3 by IB in unloading.
Because the super repressor was found to have effects on muscle atrophy, we performed experiments to study whether it affected p50 and Bcl-3 expression, since they are both required for unloading atrophy (26) and since the genes encoding these proteins can be activated by NF-B transcription factors (10, 48). Our results suggest that the Rel proteins bound by IBN are inducers of p50 and Bcl-3 gene expression. IBN caused a reduction in p50 mRNA and protein expression, and it attenuated the unloading-induced increase in Bcl-3 mRNA and protein expression after 7 days of unloading. Promoter analysis of Bcl-3 has identified two NF-B-binding sites, both of which are targets for p65-mediated transactivation (10), whereas the p105/p50 promoter contains NF-B-binding sequences that can be activated by p50 or p65 alone or together (9). These results make a strong case for there being a transcriptional component to the p50 and Bcl-3 upregulation that is required for unloading atrophy.
Rel proteins released by IB in unloading.
It is generally accepted that IB binds to Rel dimers and retains their cytosolic residence because of the presence of ankyrin repeats that block the Rel protein's from being recognized by the nuclear transport machinery (45). However, when not bound to Rel proteins, IB has been shown to enter the nucleus via its nuclear localization sequence and remove DNA-bound Rel proteins (52), exporting them to the cytosol through its NH2-terminal nuclear export sequence (25). Therefore, the complete inhibition of NF-B activity because of expression of the dominant negative IB suggests it is binding Rel proteins in the cytosol that are involved in NF-B activation during unloading. We investigated the binding of IB to Rel proteins using immunoprecipitation. We found complexes containing IB together with p50 and p65. These data could be interpreted as indicating IB binding to any p65 complex (p65-p50 or p65-c-Rel) or any p50 complex (p50 homodimers, p65-p50, c-Rel-p50, or RelB-p50). However, although c-Rel binds IB with equal affinity as p65, c-Rel protein is expressed at low levels in skeletal muscle and, as discovered in this study, is not required for atrophy. Furthermore, IB does not associate with dimers containing RelB (7). Therefore, the most logical dimer candidates for super repressor binding appear to be p65-p50 and p50-p50 dimers. Although IB binds the p65-p50 heterodimer with much higher affinity than p50 homodimers, DNA binding of both can be inhibited by IB (36). In addition, the inhibition of NF-B activity by the dominant negative IB in both unloaded and weight-bearing muscle, below baseline levels, may be because of its sustained binding to Rel proteins in the cytosol (23).
The dominant negative IB has previously been used to inhibit the classic pathway of NF-B activity in skeletal muscle, which is activated during disease-induced muscle atrophy and involves p65-p50 heterodimers (12, 33). However, during unloading, although nuclear translocation of p50 is increased, nuclear localization of p65 was unchanged (27). Because p50 does not contain a transactivation domain, p50 homodimers only induce transcriptional activation when bound to Bcl-3, and we have previously shown that both p50 and Bcl-3 are necessary for NF-B activation and muscle atrophy (26). Therefore, the finding that the dominant negative IB inhibits NF-B activation during unloading could be because of its sequestration of p50. The finding of a complex containing both IBN and p65 could be a reflection of baseline p65-IB binding that is not related to unloading-induced NF-B activation. Additional experiments using a dominant negative form of p65 are needed to define the participation of p65 in this system.
Role of c-Rel in unloading atrophy.
Previously, we found that c-Rel was upregulated during unloading atrophy, and, although not to the same degree as p50 and Bcl3, the strong requirement for each of these latter factors inspired us to look at the contribution of c-Rel. We obtained a knockout for c-Rel, but, as this paper makes clear, the lack of this particular Rel protein does not disturb the progress of disuse atrophy. Therefore, these findings eliminate a role for c-Rel in NF-B activation and muscle atrophy during unloading.
In summary, the work presented here describes an important role for IB in unloading atrophy but not for the NF-B family member c-Rel. Transduction of soleus muscles with a dominant negative IB abolished unloading-induced NF-B activity and protein ubiquitination, and it significantly inhibited muscle atrophy and the expression of certain genes known to be upregulated with atrophy. This explains a good deal of the mechanism underlying the requirement for p50 and Bcl-3 in unloading atrophy and opens the way to the very important work of pursuing the atrophy genes whose regulation is mediated by NF-B. In addition, work is needed to determine the importance of the known kinases of IB during unloading and to determine if the genes identified here as being affected by the dominant negative IB are bona fide targets of NF-B.
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64 kDa).
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