Tumor Necrosis Factor-regulated Biphasic Activation of NF-kappa B Is Required for Cytokine-induced Loss of Skeletal Muscle Gene Products*

Katherine J. Ladner, Michael A. CaligiuriDagger §, and Denis C. GuttridgeDagger ||

From the Division of Human Cancer Genetics, the Dagger  Department of Molecular Virology, Immunology, and Medical Genetics, the § Division of Hematology and Oncology, and the  Department of Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

Received for publication, July 16, 2002, and in revised form, November 12, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NF-kappa B activation is classically defined as a transient response initiated by the degradation of Ikappa B inhibitor proteins leading to nuclear import of NF-kappa B and culminating with the resynthesis of Ikappa Balpha and subsequent inactivation of the transcription factor. Although this type of regulation is considered the paradigm for NF-kappa B activation, other regulatory profiles are known to exist. By far the most common of these is chronic or persistent activation of NF-kappa B. In comparison, regulation of NF-kappa B in a biphasic manner represents a profile that is scarcely documented and whose biological significance remains poorly understood. Here we show using differentiated skeletal muscle cells, that tumor necrosis factor (TNF) induces NF-kappa B activation in a biphasic manner. Unlike the first transient phase, which is terminated within 1 h of cytokine addition, the second phase persists for an additional 24-36 h. Biphasic activation is mediated at both the levels of NF-kappa B DNA binding and transactivation function, and both phases are dependent on the IKK/26 S proteasome pathway. We find that regulation of the first transient phase is mediated by the degradation and subsequent resynthesis of Ikappa Balpha , as well as by a TNF-induced expression of A20. Second phase activity correlates with persistent down-regulation of both Ikappa Balpha and Ikappa Bbeta proteins, derived from a continuous TNF signal. Finally, we demonstrate that inhibition of NF-kappa B prior to initiation of the second phase of activity inhibits cytokine-mediated loss of muscle proteins. We propose that the biphasic activation of NF-kappa B in response to TNF may play a key regulatory role in skeletal muscle wasting associated with cachexia.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Rel/NF-kappa B (NF-kappa B)1 is a dynamic transcription factor family involved in the regulation of innate immune response, cellular proliferation and differentiation, and cell survival (1-3). In mammalian cells this family consist of RelA/p65, c-Rel, RelB, p50 (p105 precursor) and p52 (p100 precursor). These proteins are distinguished by a REL homology domain contained in the amino terminus, which specify protein dimerization, DNA binding, and the nuclear localization signal (NLS) (1-3). This family can be further subdivided into proteins that contain a transactivation domain at their carboxyl terminus (p65, c-Rel, and RelB) and those that do not (p50 and p52). Although in vitro, each of these proteins possesses the ability to homo- or heterodimerize, the prototypic form of NF-kappa B consists of the p50/p65 heterodimer. In most cells, the majority of NF-kappa B resides in the cytoplasm, bound to the Ikappa B inhibitory protein family, which include Ikappa Balpha , Ikappa Bbeta , Ikappa Bepsilon , Bcl-3, p100, and p105. These proteins function as inhibitors through ankyrin repeats, which bind to the REL domain of NF-kappa B and mask the NLS site thus preventing NF-kappa B nuclear translocation (1-3).

Activation of NF-kappa B is mediated through the Ikappa B kinase complex (IKK), which functions to phosphorylate two serine residues on Ikappa B proteins (4). Phosphorylation of these residues causes the ubiquitination and subsequent degradation of Ikappa B proteins by the 26 S proteasome complex (4). Upon loss of Ikappa B, NF-kappa B is free to translocate to the nucleus where it binds to its cognate DNA sequence and interacts with the basal transcription machinery and transcriptional co-activators to stimulate gene expression (5). One of the numerous genes induced by NF-kappa B is its own inhibitor, Ikappa Balpha (6-8). Once resynthesized, usually within 1 h of NF-kappa B activation depending on the activating signal and cell type, Ikappa Balpha is transported to the nucleus where it binds and inhibits NF-kappa B DNA binding (9). NF-kappa B is sequestered back to the cytoplasm through a nuclear export signal located in the amino terminus of Ikappa Balpha (10, 11). In this fashion, the rapid resynthesis of Ikappa Balpha ensures the equally rapid turnover of NF-kappa B activity.

NF-kappa B activation is stimulated by a wide variety of both intra- and extracellular stimuli, including inflammatory cytokines, viral and bacterial products, growth factors, and pro-oncogenic signals (12). Among the proinflammatory cytokines, tumor necrosis factor alpha  (TNFalpha ) is one of the most potent activators of NF-kappa B in cell types possessing TNF receptors (13). TNF is produced primarily from macrophages and functions in the early phases of infection by activating and recruiting other immune cells through the production of chemokines and other proinflammatory cytokines (14). Overproduction of TNF is also thought to contribute to the pathophysiology of several diseases including septic shock, cancer, AIDS, diabetes, and rheumatoid arthritis (14). In cancer and AIDS, chronic production of TNF has been further linked to the degeneration of skeletal muscle associated with tissue wasting or cachexia (15, 16).

Skeletal muscle wasting involves the proteolytic degradation of myofibrillar proteins as well as the inability to synthesize new muscle gene products (15, 16). In vitro, TNF has been shown to function as a potent inhibitor of skeletal myogenesis (17, 18). This activity is mediated through NF-kappa B (17), which promotes the down-regulation of the myogenic bHLH transcription factor, MyoD (19). Absence of MyoD is known to impair skeletal myogenesis (20), and compromise the efficiency of muscle regeneration in response to injury (21). In contrast, TNF activity alone is not sufficient to cause full degradation of mature muscle (22), which has prompted the notion that TNF functions in concert with other proinflammatory cytokines, such as interleukin-1beta (IL-1), interleukin-6 (IL-6), and interferon gamma  (IFNgamma ) to induce wasting (16, 23). Consistent with this thinking, recent evidence showed that the combined treatment of skeletal muscle with TNF and IFNgamma induced strong down-regulation of muscle-specific gene products (17). Importantly, cytokine-mediated muscle loss was prevented in myotubes lacking NF-kappa B activity, suggesting that this transcription factor may function as a critical regulator of skeletal muscle integrity.

To gain greater insight on the role of NF-kappa B in cytokine-induced muscle wasting, the present study was undertaken to elucidate the regulation of NF-kappa B in response to TNF and IFNgamma signaling in differentiated skeletal muscle. Since IFNgamma has not been demonstrated on its own to activate NF-kappa B, our initial goal was to determine whether IFNgamma signaling could synergize with TNF to activate NF-kappa B to a threshold level required for muscle decay. Utilizing differentiated C2C12 muscle cultures, results showed that IFNgamma did not potentiate TNF-induced activation of NF-kappa B, suggesting that the synergistic action of TNF and IFNgamma to induce muscle loss functions downstream from the initial point of NF-kappa B activation. However, in the course of this analysis, we observed that treatment of C2C12 myotubes with TNF alone caused a clear and pronounced biphasic activity of NF-kappa B. In contrast to the first phase, which was potent but transient, the activity of the second phase was equally potent but persisted, lasting an additional 24-36 h following TNF treatment. This type of NF-kappa B activity profile is unlike the classical scheme represented by a single transient phase. It also differs from the more commonly described chronic or persistent activity of NF-kappa B. Thus, in this study differentiated skeletal muscle cells were utilized to characterize the biphasic activation pattern of NF-kappa B in response to TNF, and to determine its biological significance with respect to cytokine-induced muscle wasting.

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Reagents-- Human and murine TNFalpha were purchased from Promega and Roche Molecular Biochemicals, respectively. Recombinant IL-1 and IL-6 were purchased from Promega, IFNgamma from Invitrogen and LPS from Alexis. Antibodies p50 (NLS), Ikappa Balpha (C-21), Ikappa Bbeta (C-20), Ikappa Bepsilon (G-4), and Myf-5 (C-20) were from Santa Cruz Biotechnology. Antibody p65 was obtained from Rockland, anti-phospho-Ikappa Balpha (Ser-32) from Cell Signaling, anti-alpha -tubulin (B-5), and anti-myosin heavy chain (MY-32) from Sigma. A Cdk4 antibody was generously provided by Y. Xiong (University of North Carolina, Chapel Hill). Secondary anti-rabbit and anti-mouse IgG antibodies conjugated to horseradish peroxidase were purchased from Promega. Oregon Red goat anti-mouse IgG was purchased from Molecular Probes. The proteasome inhibitor MG-132 and p38 MAP kinase inhibitor SB203580 were obtained from Calbiochem. IKK inhibitors Bay 11-7085 was obtained from Biomol and PS-1145 was generously provided by J. Adams (Millennium Pharmaceuticals). Protease and phosphatase inhibitor cocktails were purchased from Sigma.

Cell Culture-- Murine C2C12 myoblasts were obtained from ATCC and primary murine myoblasts were provided by J. Samulski (University of North Carolina, Chapel Hill). Cells were grown in Dulbecco's modified Eagle's medium with high glucose (DMEM-H), supplemented with 20% fetal bovine serum and antibiotics (Invitrogen). Cells were passaged every 2-3 days. For the induction of differentiation, cells were trypsinized and replated at 60-70% confluence in growth medium. The following day, cells were washed once in phosphate-buffered saline (PBS), and then switched to DMEM-H supplemented with 2% horse serum and 10 µg/ml insulin.

EMSAs and Western Blot Procedures-- Nuclear extracts in EMSAs were performed as previously described (24). Briefly, 5 µg of nuclear extract was incubated with 1 mM phenylmethylsulfonyl fluoride and 1 µg of poly(dI-dC)-poly(dI-dC) (Amersham Biosciences) for 10 min at room temperature. To this mixture 2 × 104 cpm of a 32P-labeled oligonucleotide probe corresponding to the promoter of the class I major histocompatibility complex gene (5'-CAGGGC TGGGGATTCCCCATCTCCACAGTTTCACTTC- 3') was added (NF-kappa B binding site is underlined) in a buffer consisting of 10 mM Tris-HCl, pH 7.7, 50 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 10% glycerol. Complexes were resolved on a non-denaturing 5% polyacrylamide gel, and then subsequently exposed for 4-8 h on X-OMAT film (Kodak). For supershifts, antibodies raised against specific subunits of NF-kappa B were preincubated with nuclear extracts for 10 min at room temperature before the addition of phenylmethlsulfonyl fluoride and poly(dI-dC)-poly(dI-dC).

For Western blot analysis, extracts were prepared as previously described in the presence of protease inhibitors (24). For detection of phospho-Ikappa Balpha , phosphatase inhibitors were also included in extract buffers. Following protein fractionation on SDS-polyacrylamide gels and protein transfer to nitrocellulose membranes (Gelman Corp.), blocking was performed for overnight in 5% nonfat dry milk in a buffer consisting of 25 mM Tris-HCl, pH 8.0, 125 mM NaCl, and 0.1% Tween 20 (TBST). Primary and secondary antibodies were diluted in 0.5% nonfat dry milk in TBST, and incubated from 1-2 h room temperature. The only exception was the anti-phospho-Ikappa Balpha antibody, which was diluted in 5% milk and incubated overnight at 4 °C. Membranes were treated with enhanced chemiluminescence (PerkinElmer Life Sciences), and proteins were visualized either by exposing to X-OMAT film or by using the ChemiDoc gel documentation system (Bio-Rad Laboratories, CA).

Northern and RT-PCR Analyses-- Total RNA was isolated with TRIzol (Invitrogen), fractionated on a 1.4% agarose gel and subsequently transferred overnight to a charged nylon membrane (Biodyne, Gelman Corporation). RNA was cross-linked with a UV crosslinker (Stratagene), and prehybridized with QuickHyb (Stratagene). Random prime probes were generated with Rediprime II (Amersham Biosciences) and [alpha -32P]dCTP. Probes were boiled with 100 µg/ml salmon sperm DNA and hybridized for 1 h at 68 °C. Membranes were washed two times in 2× SSC (1× is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.1% SDS at room temperature for 10 min. followed by one wash in 0.1× SSC, 0.1% SDS at 65 °C for 30 min. For semiquantitative RT-PCR analysis, 2 µg of total RNA from differentiated C2C12 myotubes were used in reactions with Access RT-PCR according to the manufacturer (Promega). Forward (CGGCATGGATCTCAAAGACAACC) and reverse (GAAGACTCCTCCCAGGTATATGG) primers were used to amplify a 252-bp fragment from the murine TNFalpha mRNA. Forward (GCTGTGAAGATACGAGAGAGAACC) and reverse (CACATGTACTGACAAGCTGCATGC) primers were used to amplify a 290-bp fragment from the murine A20 gene.

Generation of Cell Lines-- C2C12 myoblasts stably expressing a wild type (3xkappa B-Luc) or mutated (3xkappa B-Mut-Luc) NF-kappa B-responsive luciferase reporter, and cells expressing the Ikappa Balpha -SR transgene were previously described (24). The stable expression of the A20 gene in C2C12 cells was performed by transiently transfecting 2 µg of a human A20 expression plasmid in myoblasts grown on a 60-mm culture dishes. 48 h post-transfection, cells were passaged at 1:50 and 1:100 of their density in the presence of 1 mg of Geneticin. Resistant clones were expanded and pooled and tested for their expression of A20 by semiquantitative RT-PCR analysis using forward (GTGAAGATACGGGAGAGAACTCC) and reverse (GTACCAAGTCTGTGTCCTGAACG) primers, which amplifies a 310-bp product. The generation of MyoD-expressing fibroblasts was accomplished by infecting Ikappa Balpha +/+ and -/- fibroblasts with a MyoD-expressing retrovirus as previously described (17).

Immunofluorescence-- Staining experiments were performed as previously described (24). Briefly, following incubations cells were gently rinsed 3× with PBS. C2C12 myotubes were fixed in 2% formaldehyde/1× PBS solution for 30 min. Cells were permeabilized with 0.5% Nonidet P-40, PBS for 5 min at room temperature and then blocked with horse serum 1:100 dilution in PBS. Cells were incubated for 1 h with anti-skeletal myosin heavy chain diluted at 1:500 in 3% bovine serum albumin, PBS followed by a 1-h incubation in the dark with an anti-mouse Oregon Red IgG (Molecular Probes) diluted at 1:250 in 3% bovine serum albumin, PBS. Following additional washes with PBS and water, cells were mounted with coverslips and examined on a Nikon fluorescent microscope equipped with a SPOT digital camera.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Loss of Muscle-specific Gene Products by TNFalpha and IFNgamma Is Dependent on NF-kappa B Activity-- TNF is strongly linked with muscle wasting associated with cancer-induced cachexia (15). Studies indicate, however, that activities of other proinflammatory cytokines, including IL-1, IL-6, or IFNgamma , are required along with TNF to induce loss of skeletal muscle proteins (25-27). Specifically, combinatorial treatment of mature muscle with TNF and IFNgamma was recently shown to promote pronounced decreases in the levels of the muscle-specific transcription factor MyoD (17). Consistent with these previous findings, we again found that treatments of C2C12 myotube cultures with TNF + IFNgamma led to the dramatic down-regulation of MyoD protein expression (Fig. 1A). In contrast, treatment of differentiated C2C12 cells with TNF and IL-1 or TNF and IL-6 produced no changes in MyoD expression. These effects by TNF + IFNgamma treatment were specific, since similar decreases in protein levels were not observed with the closely related muscle transcription factor, Myf-5 (19). In addition, treatments with both TNF + IFNgamma led to a significant decrease in the expression of the myofibrillar protein, myosin heavy chain (MHC) (Fig. 1, B and C). Importantly, loss of MyoD and MHC was prevented in myotubes stably expressing the NF-kappa B transdominant inhibitor, Ikappa Balpha -SR (non-degradable form of Ikappa Balpha ), supporting the notion that NF-kappa B activity is required for cytokine-induced muscle loss.


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Fig. 1.   NF-kappa B is required for cytokine-induced muscle protein loss. A, C2C12 vector or Ikappa Balpha SR stably expressed myoblasts were differentiated in DM for a period of 3 days. At this time, cultures were refed with fresh DM supplemented with either no addition (untreated) or treated for two 24-h periods with TNF (20 ng/ml) with either IL-1 (20 ng/ml), IL-6 (20 ng/ml), or IFNgamma (100 units/ml). Whole cell extracts were prepared, and 50 µg of total protein was used in Western blot analysis to probe for Ikappa Balpha and Ikappa Balpha SR (which is epitope-tagged and therefore is a slower migrating protein), MyoD, Myf-5, and alpha -tubulin used as a loading control. B, vector control or Ikappa Balpha SR-expressing myotubes were treated as described above, and subsequently stained by immunofluorescence to detect for MHC expression. Images are shown at 20× magnification digitally captured by a SPOT camera. C, from immunostained myotubes as described in B, MHC expression was quantitated using BioRad Quantity One software. The data were calculated from the area of MHC staining from a minimum of 20 randomly chosen fields of cells, under different treatment conditions, visualized at 4× magnification.

TNF Induces a Biphasic Activation of NF-kappa B in Skeletal Muscle-- Although IFNgamma is not known to induce NF-kappa B activity directly, it has been demonstrated to potentiate the activation of NF-kappa B in response to TNF (28). We therefore questioned whether the synergy observed between TNF and IFNgamma signaling that led to the loss of MyoD and MHC resulted from such a potentiation in NF-kappa B activity. To address this question, we treated C2C12 myotube cultures with either cytokine alone or in combination and monitored for NF-kappa B activity. Treatment with TNF alone caused the expected rapid but transient induction of NF-kappa B (Fig. 2A). Activation levels peaked at 30 min post-TNF treatment and returned near basal levels by 1 h. In contrast, IFNgamma treatment alone did not induce NF-kappa B activity, nor did IFNgamma potentiate the activity of NF-kappa B in the presence of TNF (Fig. 2A). Similar results were obtained when treatments were repeated for longer time periods (Fig. 2B). These results suggest that the ability of IFNgamma to synergize with TNF to induce muscle loss is not associated with an enhanced NF-kappa B activity.


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Fig. 2.   TNF induces a biphasic activation of NF-kappa B in C2C12 myofibers. A and B, IFNgamma does not potentiate TNF-induced activation of NF-kappa B. C2C12 myotubes were treated with either TNF (20 ng/ml) or IFNgamma (100 units/ml) or a combination of both cytokines, and at indicated times, nuclear extracts were prepared and NF-kappa B was monitored by EMSA. C, EMSA was performed, monitoring NF-kappa B binding activity from myotubes treated with TNF for up to 48 h. D, extracts obtained from treated samples, as described in C, were used in supershift EMSA by incubating with antibodies raised against either the p50 or p65 subunit of NF-kappa B. Arrowheads denote supershifted complexes. E, NF-kappa B biphasic activation correlates with p65 nuclear translocation. Nuclear extracts (30 µg) prepared from myotubes treated with TNF, as described in C were used in Western blot analyses probing for p65, cdk4, or Myf-5.

During the course of this analysis we made the observation that following the initial activation phase of NF-kappa B in response to TNF, a second NF-kappa B activation phase developed. This second phase of activity was nearly as potent as the first, but unlike the first phase whose signal terminated within 1 h, second phase activity persisted for an additional 24-36 h (Fig. 2C). To identify NF-kappa B subunits contributing to DNA binding activities, supershift EMSAs were performed. Myotube nuclei were observed to contain predominantly p50 and p65 subunits, and as previously determined (24), no binding activity was detected with other NF-kappa B subunits, p52, c-Rel, or RelB (data not shown). Furthermore, equivalent binding of p50/p65 subunits were observed in both phases of NF-kappa B activity in response to TNF, demonstrating that no apparent changes in binding complexes occurred during the biphasic activation (Fig. 2D). To address whether the biphasic profile identified by EMSA analysis reflected simply changes in NF-kappa B DNA binding activity, or rather represented changes in nuclear translocation events, nuclear Western analyses were performed. As expected, a clear increase in nuclear p65 was observed within 30 min of TNF treatment, reflecting NF-kappa B nuclear translocation during the first phase of activation (Fig. 2E). Levels of nuclear p65 decreased within 1 h, but then increased a second time beginning at 6 h out to 48 h post-TNF treatment, suggestive of a second nuclear translocation event. This effect was specific to p65 since no change in nuclear protein expression was detected for the cyclin-dependent kinase, cdk4, or the muscle-specific transcription factor, Myf-5.

Next we addressed the specificity of this biphasic activation profile with respect to TNF. Since IL-1 and LPS are also potent inducers of NF-kappa B (1, 2), we used these agents to treat differentiated C2C12 muscle cultures. Results showed that similar to TNF, both IL-1 and LPS could induce the first phase of NF-kappa B activity with comparable kinetics, but neither agent promoted the second activation phase (Fig. 3), suggestive that, in differentiated skeletal muscle, the biphasic activation of NF-kappa B may be specific to TNF-mediated signaling.


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Fig. 3.   NF-kappa B biphasic activation may be specific to TNF signaling. C2C12 myotubes were treated with TNF (20 ng/ml), IL-1 (20 ng/ml), or LPS (10 µg/ml) for various times, and NF-kappa B activity was monitored by EMSA.

One of the caveats in using the C2C12 culture system is that these cells are immortalized, and thus under differentiation conditions one typically observes a substantial number of satellite cells that fail to fuse into multinucleated myotubes (Fig. 4A). Given this limitation, the possibility existed that the biphasic activation profile of NF-kappa B that we had observed was occurring in the undifferentiated satellite cells rather than in differentiated myotubes. To address this point, we utilized primary muscle cells, which in contrast to C2C12 cells are capable of full myotube conversion when induced to differentiate (Fig. 4A). Results showed that in TNF-treated primary myotube cultures, NF-kappa B was also activated in a biphasic manner similar to that seen in C2C12 differentiated cells (Fig. 4B). Supershift EMSA analysis was also consistent with results obtained in C2C12 cells, demonstrating the presence of both p50 and p65 subunits of NF-kappa B during both phases of DNA binding activity (Fig. 4C). These data thus demonstrate that the biphasic activation of NF-kappa B occurs in differentiated skeletal muscle.


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Fig. 4.   TNF induces the biphasic activation of NF-kappa B in primary myotubes. A, C2C12 (upper panel) or primary myoblasts (lower panel) were differentiated for 2 or 3 days, respectively in DM. In C2C12 cultures, arrowheads denote cells that were unable to complete their differentiation and that remain as myoblasts. B, primary myotube cultures were treated with TNF for indicated times and NF-kappa B activity was monitored by EMSA. C, supershift EMSA was performed with nuclear extracts prepared for EMSA in B and incubated with antibodies raised against NF-kappa B subunits p50 or p65.

The Biphasic Activity of NF-kappa B in Response to TNF Is Transcriptionally Competent-- Next we addressed whether NF-kappa B DNA binding activity correlated with transcriptional competency. To make this determination, reporter assays were performed using a C2C12 cell line containing a stably integrated NF-kappa B-responsive heterologous promoter (3xkappa B-Luc). These cells were induced to differentiate into myotubes and subsequently treated with TNF. Results showed that NF-kappa B transcriptional activity was regulated in a similar biphasic fashion (Fig. 5A). In contrast, no activation was observed by TNF when the assay was repeated with C2C12 cells containing a mutant version of the NF-kappa B promoter (3xkappa B-Mut-Luc), indicating that this biphasic regulation of transcriptional activity is specific to NF-kappa B. To further assess NF-kappa B transcriptional activity, we monitored the expression of a known NF-kappa B-regulated gene. Consistent with reporter data, results showed that Ikappa Balpha gene expression was maximally induced after 1 h of TNF treatment, which then decreased over the next few hours, but increased again steadily over a 24-h period (Fig. 5B). Taken together, these results demonstrate that NF-kappa B is transcriptionally competent during each activation phase in skeletal muscle in response to TNF.


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Fig. 5.   TNF induces NF-kappa B biphasic transcriptional activity. A, C2C12 myoblasts containing a stable NF-kappa B-responsive promoter (3xkappa B-Luc), or a mutated NF-kappa B promoter (3xkappa B-Mut-Luc) were differentiated into myotubes, and treated with TNF. At indicated times cell extracts were prepared, and luciferase assays were performed. B, RNA prepared from TNF-treated myotubes was used in Northern blot analysis probing for Ikappa Balpha . The blot was stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase to control for RNA loading.

The Biphasic Activity of NF-kappa B Is Regulated by Ikappa B Proteins-- In response to most inducing stimuli, classical activation of NF-kappa B occurs from the stimulation of the IKK complex, leading to the direct phosphorylation of two serine residues contained on each of Ikappa Balpha , Ikappa Bbeta , or Ikappa Bepsilon proteins (4). In response to TNF and other pro-inflammatory stimuli, this activity is mediated specifically by the beta -subunit of IKK. The phosphorylation of Ikappa B proteins leads to their ubiquitination and subsequent degradation via the 26 S proteasome. Degradation of Ikappa B proteins unmasks the nuclear localization signal of NF-kappa B allowing for nuclear translocation and NF-kappa B-dependent transcription (4). Termination of NF-kappa B DNA binding and transcriptional activity is largely mediated by NF-kappa B-dependent resynthesis of Ikappa Balpha , which binds and exports NF-kappa B from the nucleus back to the cytosol (10, 11).

Having partially characterized the biphasic activation profile of NF-kappa B in skeletal muscle, we next set out to address its regulatory mechanism. To initiate this analysis we first asked whether the biphasic regulation was dependent on proteasome activity. Pre-incubation of C2C12 myotube cultures with the proteasome inhibitor, MG-132 completely blocked TNF-mediated activation of NF-kappa B (Fig. 6A), indicative that the first phase of activity is proteasome-dependent. To assess the requirement of proteasome activity in the second phase of NF-kappa B activation, the proteasome inhibitor was added to cells 1 h post-TNF treatment, a time shown to precedes the initiation of the second phase, as demonstrated by EMSA and reporter data. In comparison to vehicle-treated myotubes, MG-132 strongly inhibited NF-kappa B activity in the second activation phase (Fig. 6B), demonstrating that the proteasome is required throughout the biphasic regulation of NF-kappa B. Next, we assessed IKK activity during biphasic regulation by monitoring the phosphorylated state of Ikappa Balpha . TNF treatment of C2C12 myotubes caused the rapid phosphorylation of Ikappa Balpha , which decreased within 30 min in conjunction with the degradation of the inhibitor protein (Fig. 6C). At 1 h post-TNF treatment, resynthesized Ikappa Balpha was again highly phosphorylated, suggesting that IKK activity is maintained throughout the first transient activation phase of NF-kappa B. To examine the status of IKK during the second activation phase, C2C12 myotubes were treated with TNF for 1 h, and subsequently supplemented with proteasome inhibitor to inhibit the degradation of Ikappa Balpha . Results showed that in MG132-treated cells, Ikappa Balpha phosphorylation was highly maintained during the second phase of NF-kappa B activity (Fig. 6D), indicating that biphasic activation of NF-kappa B is mediated through a IKK/proteasome-dependent pathway.


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Fig. 6.   Biphasic activation of NF-kappa B activation by TNF is regulated by the IKK/proteasome pathway and Ikappa B proteins. A and B, both phases of NF-kappa B activity in response to TNF is proteasome-dependent. C2C12 myotubes were incubated with either vehicle alone (DMSO, 0.1%) or the proteasome inhibitor, MG-132 (50 µM) for 1 h prior to the addition of TNF (A), or added at 1 h post-TNF treatment (B). At indicated times, nuclear extracts were prepared, and NF-kappa B activity was determined by EMSA. C and D, IKK is active during both phases of NF-kappa B activity. Myotubes were treated with TNF throughout (C) or at 1 h supplemented with either vehicle or MG-132 (D). At indicated times, cytoplasmic extracts were prepared, and Western blot analyses were performed probing for the phosphorylation of Ser-32 on Ikappa Balpha or total Ikappa Balpha expression. E, TNF induces the down-regulation of Ikappa B proteins. Myotubes were treated with TNF, and at indicated times cytoplasmic extracts were prepared. Western blot analyses were subsequently performed with 30 µg of extracts probing for Ikappa Balpha , Ikappa Bbeta , and Ikappa Bepsilon .

In light of the above results, we next examined levels of cytoplasmic Ikappa B proteins during TNF treatment. With respect to Ikappa Balpha , initial rapid degradation and resynthesis was followed by a second degradative regulation starting at 2 h (Fig. 6C), and levels remained down-regulated throughout a 48-h treatment period (Fig. 6E). With Ikappa Bbeta , expression also decreased at 30 min in response to cytokine treatment, but unlike Ikappa Balpha , was not resynthesized and instead was persistently down-regulated. In contrast to alpha  and beta  forms, we repeatedly observed that Ikappa Bepsilon was not degraded by TNF treatment, but rather levels of this protein increased over time. Thus, biphasic activation of NF-kappa B correlates with loss of both Ikappa Balpha and Ikappa Bbeta proteins.

To more closely examine the role of Ikappa Balpha in the regulation of NF-kappa B biphasic activity, TNF treatments were performed on myotubes stably expressing the NF-kappa B transdominant inhibitor, Ikappa Balpha -SR, which is incapable of degradation in response to an NF-kappa B stimuli. Results showed that in contrast to vector control myotubes, which displayed a pronounced biphasic activation pattern of NF-kappa B, Ikappa Balpha -SR-expressing myotubes lacked both first and second phases (Fig. 7A). Consistent with this finding, levels of Ikappa Balpha -SR protein was unchanged during TNF treatment, in comparison to Ikappa Balpha in vector control cells (Fig. 7B). These results not only demonstrate that the first activation phase is a prerequisite for the second, but they also confirm that Ikappa Balpha degradation is essential for the induction of the first transient phase. Interestingly, under these experimental conditions, Ikappa Bbeta down-regulation was observed in both vector control and Ikappa Balpha -SR-expressing myotubes (Fig. 7B), suggestive that regulation of the first activation phase is specific to Ikappa Balpha .


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Fig. 7.   Ikappa Balpha regulates the first transient phase of NF-kappa B activity. A, degradation of Ikappa Balpha is required for the induction of NF-kappa B. Vector control or Ikappa Balpha -SR-expressing myotubes were treated with TNF, and at indicated times nuclear extracts were prepared to analyze NF-kappa B activity by EMSA (A), and cytoplasmic extracts were prepared to probe for levels of Ikappa Balpha and Ikappa Bbeta (B). C-E, Ikappa Balpha resynthesis is required for the termination of the first activation phase. C, Ikappa Balpha +/+ and -/- fibroblasts were infected with retrovirus expressing the MyoD transgene (pBabeMyoD). Cells were selected by puromycin treatment, clones were pooled, and MyoD expression was verified by Western blotting. D, MyoD-expressing Ikappa Balpha +/+ and -/- fibroblasts were grown to near confluency and either maintained in growth medium (GM) or induced to differentiate (DM). Myotube phenotype was identified by morphology and by immunofluorescence staining for MHC. E, Ikappa Balpha +/+ and -/- myotube cultures were treated with TNF, and at selected times, nuclear and cytoplasmic extracts were prepared to analyze NF-kappa B activity (upper panel) or levels of Ikappa Balpha (lower panel).

Based on the above results, we next examined the requirement of Ikappa Balpha resynthesis in the termination of this first activation phase. To make this determination, Ikappa Balpha +/+ and -/- fibroblasts (29) were converted to skeletal muscle cells by expression of the MyoD transgene (Fig. 7C). Under differentiation conditions, both Ikappa Balpha wild type and null cells were readily converted to myosin-expressing myotubes (Fig. 7D), although consistently fewer and smaller myotubes were detected in null cells. TNF treatment of Ikappa Balpha +/+ skeletal muscle cultures induced typical NF-kappa B transient activity, correlating with the degradation and resynthesis of Ikappa Balpha (Fig. 7E). In contrast, induced NF-kappa B activity was sustained in Ikappa Balpha -/--treated muscle cultures, indicating that Ikappa Balpha resynthesis is required for the inactivation of NF-kappa B within the first phase.

Having identified that Ikappa Balpha is a required factor regulating both the activation and termination of NF-kappa B activity within the transient first phase in response to TNF, we next set out to gain insight on the role of Ikappa Balpha and Ikappa Bbeta in the second activation phase of NF-kappa B. Examination of cytoplasmic extracts had showed that both Ikappa Balpha and Ikappa Bbeta were persistently down-regulated during the second activation phase (Fig. 6E), indicating that Ikappa B proteins may also be involved in the regulation of this latter phase. In support of this notion, we observed that removal of TNF following the first activation phase abolished the induction of the second phase, both at the level of NF-kappa B DNA binding and transactivation function (Fig. 8, A and B, compare wash versus TNF lanes). Importantly, inhibition of the second phase correlated with restored levels of both Ikappa Balpha and Ikappa Bbeta proteins (Fig. 8C), suggesting that chronic TNF signaling may be required for the persistent down-regulation of Ikappa B proteins in order to maintain second activation phase of NF-kappa B.


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Fig. 8.   Second activation phase of NF-kappa B correlates with down-regulation of Ikappa B proteins, dependent on continuous TNF signaling. A, the second phase of NF-kappa B activity is dependent on exogenous TNF. A and B, C2C12 myotubes were treated with TNF for 1 h and either left alone (TNF) or washed extensively with PBS and subsequently refed with fresh differentiation medium (wash). At indicated times, nuclear extracts were preparedm and NF-kappa B DNA binding activity was determined (A), or total RNA was prepared from cytoplasmic extracts and NF-kappa B transactivation function was assessed by Northern analysis probing for Ikappa Balpha (B). C, second phase activation correlates with down-regulation of Ikappa B proteins. C2C12 myotubes were treated as in A, and at indicated times cytoplasmic extracts were prepared and probed for Ikappa B proteins by Western analyses.

A20 Contributes to the Regulation of NF-kappa B Biphasic Activity in Response to TNF-- In addition to the role of Ikappa Balpha in the regulation of NF-kappa B in differentiated muscle cells, we sought to expand our analysis of this regulatory mechanism by examining other potential factors that function upstream of this inhibitory protein. One such candidate is the zinc finger protein A20, whose expression is induced by TNF in an NF-kappa B-dependent manner (30). A20 functions as an inhibitor of NF-kappa B activity by interfering with TNF receptor signaling at multiple points in the transduction pathway (31-33). To analyze the potential regulatory role of A20 in differentiated muscle cells, we first examined A20 expression in response to TNF. Consistent with findings in other cell types (34), TNF treatment of myotubes induced maximal expression of A20 gene expression within 30 min to 1 h, and expression returned to near basal levels by 2 h (Fig. 9A). This was in sharp contrast to the biphasic expression profile of both an NF-kappa B-responsive reporter and the Ikappa Balpha gene, as seen in Fig. 5. To examine whether induction of A20 contributes to the regulation of NF-kappa B biphasic activity, skeletal muscle cells were generated to overexpress the A20 gene (Fig. 9B). Vector control and A20 expressing C2C12 cells were differentiated and subsequently treated with TNF. In comparison to vector control cells, overexpression of A20 in myotubes caused a substantial reduction of NF-kappa B activation (Fig. 9C). Consistent with the inhibitory function of A20 on IKK activity, the phosphorylation state of Ikappa Balpha was also strongly down-regulated in A20-expressing myotubes, which correlated with a reduction in Ikappa Balpha degradation. These results therefore demonstrate that, in addition to the resynthesis Ikappa Balpha , TNF-mediated induction of A20 leads to the regulation of NF-kappa B biphasic activity.


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Fig. 9.   A20 regulates biphasic activation of NF-kappa B. A, C2C12 myotubes were treated with TNF. At indicated times, total RNA was prepared and Northern analysis was performed probing for A20 (upper panel). These results were confirmed by repeating the experimental conditions outlined in A and by performing semiquantitative RT-PCR for A20 gene expression (lower panel). B, C2C12 myoblasts were transfected with a vector control plasmid or an expression plasmid for the human A20 gene. Drug-resistant clones were pooled and A20 expression was verified by RT-PCR. C, A20-expressing myoblasts were induced to differentiate into myotube cultures, and subsequently treated with TNF. At indicated times, nuclear extracts were prepared to analyze NF-kappa B activity (upper panel), and cytoplasmic extracts were prepared to probe for the phosphorylated and unphosphorylated states of Ikappa Balpha (lower panel)

Biphasic Activity of NF-kappa B Is Required for Cytokine-induced Skeletal Muscle Protein Loss-- Having characterized the biphasic activity of NF-kappa B in skeletal muscle, and analyzed the regulatory mechanism, we concluded this study by addressing its physiological significance. Specifically, we wanted to investigate whether both phases of NF-kappa B activity were required for cytokine-mediated damage of skeletal muscle. As shown in Fig. 1, inhibition of NF-kappa B activity through the stable expression of the Ikappa Balpha -SR transgene in C2C12 myotubes strongly blocked TNF/IFN-dependent muscle protein loss. Stable expression of the Ikappa Balpha -SR in these cells was also shown to inhibit NF-kappa B activation in response to TNF (Fig. 7A), confirming that at least the first phase of NF-kappa B activity is required for cytokine-mediated loss of muscle specific proteins. To address whether the second phase also plays a role in this regulation, we sought to selectively inhibit NF-kappa B activity during this activation phase. Our initial attempt to transiently express the Ikappa Balpha -SR transgene in myotubes by use of an adenoviral gene delivery system was unsuccessful (data not shown). As an alternative approach, NF-kappa B inhibition was selectively inhibited by the use of IKK inhibitor compounds, Bay 11-7085 and PS-1145. Consistent with previous findings (35), IKK compounds inhibited NF-kappa B activation in C2C12 myotube cultures in a dose-dependent manner (Fig. 10A). These inhibitors were then used to treat myotubes 1 h following the addition of TNF + IFNgamma (thereby allowing the first phase of NF-kappa B activity to occur), and the levels of muscle-specific gene products were subsequently analyzed. Our analysis showed that both NF-kappa B inhibitor compounds significantly blocked cytokine-mediated loss of MHC and MyoD expression (Fig. 10, B and C). The fact that these inhibitors were not able to completely restore MyoD expression most likely results from the gradual inactivation of these compounds in conditioned medium, as well as from the regulatory events that occurred during the first phase of NF-kappa B activity. To address the specificity of these inhibitors, C2C12 myotubes were treated with increasing doses of SB203580, an inhibitor of the p38 stress-activated mitogen-activated protein kinase. In contrast to the IKK inhibitors, p38 inhibition did not abrogate the ability of cytokines to decrease MyoD expression (Fig. 10C). Taken together, these results indicate that cytokine-mediated loss of muscle-specific proteins is dependent on both the first and second phases of NF-kappa B activity in response to TNF signaling.


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Fig. 10.   Biphasic activity of NF-kappa B is required for cytokine-induced loss of muscle proteins. A, Bay 11-7085 and PS-1145 block TNF-induced NF-kappa B activation in C2C12 myotubes. Myotube cultures were preincubated with increasing concentrations of IKK inhibitor compounds for 90 min prior to a 15-min treatment of TNF (20 ng/ml). Nuclear and cytoplasmic extracts were prepared and either assayed for NF-kappa B activity by EMSA (upper panel) or probed for Ikappa Balpha expression by Western blotting (IB, lower panel). IKK inhibitors block cytokine-induce loss of skeletal muscle proteins. B, C2C12 myotubes were treated with TNF + IFNgamma for 1 h and subsequently supplemented with either no additional treatment (untreated) or treated with dimethyl sulfoxide, Bay 11-7085 (10 µM), or PS-1145 (20 µM) for two 24-h periods. At this time, cells were fixed, and immunofluorescence was performed probing for MHC as described under "Experimental Procedures." C, myotube cultures were treated with TNF + IFNgamma as in B and subsequently supplemented with increasing doses of Bay 11-7085 (5 and 10 µM), PS-1145 (10 and 20 µM), or the p38 inhibitor compound SB203580 (1 and 5 µM). Cell lysates were prepared, and Western analyses were performed probing for MyoD and alpha -tubulin, used as a loading control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The basis of this study was founded on previous results showing that skeletal muscle protein loss depended on the combined signaling activities of inflammatory cytokines TNF and IFNgamma (17). Results also demonstrated that NF-kappa B activity was required for cytokines to induce muscle damage (17). To gain further insight into how NF-kappa B potentially functions in cytokine-induced muscle wasting, we sought to first examine how these cytokines signal to NF-kappa B in differentiated muscle. Unlike the earlier described synergistic actions of TNF and IFNgamma on NF-kappa B signaling in endothelial and neuronal cell types (28), our current results demonstrate that addition of IFNgamma does not potentiate TNF-induced activity of NF-kappa B. However, in the course of this analysis we discovered that in skeletal muscle, NF-kappa B activity was regulated in a biphasic manner by TNF. In contrast to the first phase of NF-kappa B activity, which was potent but transient, the second identified phase was nearly as potent, and it persisted out to nearly 2 days following initial TNF treatment. Importantly, two other known inducers of NF-kappa B, IL-1 or LPS, did not induce this biphasic activity, suggesting that this regulation on NF-kappa B may be specific to certain NF-kappa B inducing signals.

Biochemical and genetic analyses have soundly established that NF-kappa B is an inducible transcription factor, where the majority is maintained as an inactive complex through the cytoplasmic retention by Ikappa B inhibitors (1, 2, 36). Stimulation of cells by a wide variety of signals leads to proteolytic degradation of Ikappa B proteins, and subsequent nuclear translocation of NF-kappa B. It has also been well accepted that activation of NF-kappa B, by most physiological inducing signals, is a transient response, reaching its peak usually within 30 min followed by a return to basal levels within 1-4 h. The transient nature of NF-kappa B activity is in part due to the resynthesis of Ikappa Balpha (6-8), which binds and exports NF-kappa B from the nucleus back to the cytoplasm (10, 11). Depending on the inducer and/or cell type, however, other activation profiles of NF-kappa B have been described. By far the most common of these profiles is the described persistent or constitutive activity of NF-kappa B that occurs when NF-kappa B activity does not return back to its basal state following its initial induction phase. Ghosh and co-workers (37) had earlier described such a phenomenon in B cells treated with LPS. In these cells, the persistent activity of NF-kappa B was derived from its binding to a hypophosphorylated form of Ikappa Bbeta , which protects NF-kappa B from Ikappa Balpha binding and inactivation, thereby facilitating NF-kappa B nuclear import. A similar mechanism of protection by Ikappa Bbeta was attributed to the persistent activity of NF-kappa B observed in myeloid cells infected with the HIV virus (38). Other mechanisms have been presented to account for such a persistent activity. For example, the human T-cell leukemia virus type 1 protein, Tax, has been shown to bind and stimulate the IKK complex resulting in chronic NF-kappa B activity (39, 40). In mature B cells, evidence suggests that a calcium-mediated signaling pathway accounts for the chronic degradation of Ikappa Balpha , leading to constitutively high levels of c-Rel activity (41). In addition, continual loss of Ikappa Bbeta has been attributed to the prolonged activation state of NF-kappa B in glial cells treated with IL-1 (42). Furthermore, persistent activity of NF-kappa B is often associated with pathophysiological states such as cancer (12).

In contrast to these types of activation profiles, biphasic activity of NF-kappa B, as described in this report, is a type of regulatory profile less well described, and whose regulatory mechanisms and biological relevance remain poorly understood. With respect to TNF signaling, Han and Brasier (43) described such a phenomenon in cultured hepatocytes, in which the duration of the second phase of NF-kappa B activity was measured for 6 h correlating with the loss of Ikappa Bbeta . Kemler and Fontana (44) also detected biphasic activity of NF-kappa B in glial and neuroblastoma cell lines for up to 4 h post-TNF treatment, and in this case activation was shown to result from the combined losses of both Ikappa Balpha and Ikappa Bbeta proteins. Similar biphasic activity has been described in response to other stimuli including viral infection (45), and most recently, LPS signaling (46). Given these results, in conjunction with our current data acquired in skeletal muscle, it would appear that the ability of NF-kappa B to undergo biphasic activation is not cell type- or stimuli-specific, nor is it restricted to a single regulatory mechanism. In addition, the recent citing that NF-kappa B undergoes biphasic activation in rat skeletal muscle in response to ischemia/reperfusion, suggest that biphasic regulation of NF-kappa B is relevant in this tissue in vivo (47).

Unclear to this point however is the significance of this regulatory profile. Are the changes in NF-kappa B DNA binding activity reflected in its transcriptional activity, and what is the biological relevance of the second phase? At least in skeletal muscle, we have revealed that NF-kappa B is transcriptionally competent in both phases of its DNA binding activation profile. This was demonstrated by a reporter-based assay and also confirmed by analyzing the gene expression profiles of NF-kappa B responsive genes, Ikappa Balpha and TNFalpha (Fig. 5 and data not shown, respectively). In regard to the biological relevance of the second phase, our data demonstrate that this may be associated with the strength and duration of activation. In skeletal muscle, the strength of activity of this second phase was seen to nearly match that of the first, and in sharp contrast to other cell types (43, 44), remained active for nearly 48 h following cytokine treatment. Interestingly, this time coincides with the same period at which significant decreases of muscle specific proteins were detected in cytokine treated myotubes (Fig. 1). Data showing that the inhibition of NF-kappa B activity during this second phase blocked cytokine-mediated decreases in both MyoD and MHC would suggest that NF-kappa B biphasic activity is required for skeletal muscle decay. Although it has not yet been elucidated, it is likely that the second phase of NF-kappa B activity will be found to regulate multiple biological functions in various cell types.

Use of the differentiated skeletal muscle cell system has revealed much regarding this biphasic activity of NF-kappa B in response to TNF signaling. Our results demonstrate that both activation phases are derived from independent nuclear translocation events, which contain NF-kappa B complexes that, as stated above, are transcriptionally competent. The results obtained in both C2C12 and primary skeletal myotubes also reveal that the subunit composition of NF-kappa B is seemingly identical during each activation phase. This indicates that following the termination of the first phase, where the p65 subunit is exported from the nucleus, a similar transcription factor complex is poised once again to be activated and undergo a second round of nuclear translocation. The data also argue that both activation phases were induced through the IKK/26 S proteasome signaling pathway. Collectively, these results portray a profile of NF-kappa B activity, which would appear to be defined by two identical regulatory mechanisms.

Despite these similarities, our results revealed distinguishing features between each activation phase. The most obvious is the duration of NF-kappa B activity during each phase. While the first phase resembled a typical transient activation profile, where NF-kappa B activity is quickly terminated, the second phase persisted for nearly an additional 48 h. Our results using the Ikappa Balpha -SR showed that the induction of the first phase is dependent on the rapid degradation of Ikappa Balpha . The additional use of Ikappa Balpha -null muscle cultures demonstrated that the termination of this first phase is also dependent on the resynthesis of Ikappa Balpha within the first hour of TNF treatment. Although a similar analysis was not conducted with A20-null myotubes, our results support the notion that the observed rapid induction of A20 in response to TNF treatment functions in conjunction with Ikappa Balpha to terminate the first phase of NF-kappa B. Termination of this phase most likely occurs both through the nuclear export of NF-kappa B by Ikappa Balpha , as well as through the inhibition of TNF receptor signaling and IKK activation by A20. Results further demonstrated that induction of the first phase is a prerequisite for the second more persistent phase of NF-kappa B. In the presence of TNF, both Ikappa Balpha and Ikappa Bbeta proteins are persistently down-regulated. In addition, removal of TNF from myotube cultures (as shown in Fig. 8) caused the termination of this second phase and correlatively restored levels of Ikappa B proteins. These results support the conclusion that regulation of both Ikappa Balpha and Ikappa Bbeta proteins define the duration of the second phase of NF-kappa B activity. However, results also indicated that down-regulation of Ikappa Bbeta occurred even in the absence of the second phase of NF-kappa B activity (Fig. 7), bringing into question the essentialness of beta  degradation during this regulatory process. The development of an NF-kappa B transdominant inhibitor form of Ikappa Bbeta (analogous to Ikappa Balpha -SR) will be required to formally address the role of this Ikappa B protein in the first and second phases of NF-kappa B. Another contributing factor regulating the second activation phase may be related to the expression of A20. Interestingly, in contrast to the biphasic expression pattern observed with an NF-kappa B responsive reporter and the Ikappa Balpha gene, A20 expression was not found to be biphasic. In light of previous findings that absence of A20 in fibroblasts leads to chronic down-regulation of Ikappa Balpha and persistent activity of NF-kappa B (48), it is tempting to speculate that the persistent second phase of NF-kappa B activity results from the absence of A20 resynthesis.

Despite these current findings, it remains unknown at this point how inflammatory cytokines IFNgamma and TNF signal in skeletal myotubes to promote muscle loss. With respect to TNF signal transduction, we have elucidated that the biphasic activation of NF-kappa B is required in order for both TNF and IFNgamma to mediate the loss of muscle specific gene products. Importantly, biphasic activation of NF-kappa B required that myotube cultures be continuously treated with exogenous TNF (Fig. 8). Elevated levels of TNF are often associated with chronic inflammatory conditions (14). This suggest that the biphasic regulation of NF-kappa B is likely to reflect a pathophysiological condition where TNF levels remain high in the circulation, as is often the case in cancer cachexia (15). Our results also showed that IFNgamma did not potentiate the ability of TNF to activate NF-kappa B (Fig. 2). This would imply that the synergism exhibited by these cytokines occurs as a result of independent activation of their respective downstream effectors such as NF-kappa B for TNF and STATs/IRFs for IFNgamma (49). As seen in other instances (50), these signaling molecules may converge further downstream, possibly on a common promoter to regulate the expression of a yet unidentified gene that may potentially be a critical regulator of skeletal muscle wasting associated in cancer-induced cachexia.

    ACKNOWLEDGEMENTS

We thank J. Adams for generously providing PS-1145, C. Y. Wang for the A20 expression plasmid, and A. S. Baldwin for his support and Ikappa Balpha -null fibroblasts (with permission from A. A. Beg). We also thank M. W. Mayo and members of the Guttridge laboratory, especially S. Acharyya, for critical review of this manuscript and insightful discussions.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health Grants K01 CA97953 (to D. C. G.) and P30 CA16058.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Division of Human Cancer Genetics, Dept. of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, 420 W. 12th Avenue, Columbus, OH 43210. Tel.: 614-688-3137; Fax: 614-247-6842; E-mail: Guttridge-1@medctr.osu.edu.

Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M207129200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; TNFalpha , tumor necrosis factor alpha ; IL, interleukin; LPS, lipopolysaccharide; IFNgamma , interferon gamma ; NLS, nuclear localization sequence; Ikappa Balpha -SR, IkBalpha super repressor; IKK, Ikappa B kinase; MHC, myosin heavy chain; DM, differentiation medium; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcription polymerase chain reaction; PBS, phosphate-buffered saline.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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