NF-κB-Induced Loss of MyoD Messenger RNA: Possible Role in Muscle Decay and Cachexia

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Science  29 Sep 2000:
Vol. 289, Issue 5488, pp. 2363-2366
DOI: 10.1126/science.289.5488.2363


MyoD regulates skeletal muscle differentiation (SMD) and is essential for repair of damaged tissue. The transcription factor nuclear factor kappa B (NF-κB) is activated by the cytokine tumor necrosis factor (TNF), a mediator of skeletal muscle wasting in cachexia. Here, the role of NF-κB in cytokine-induced muscle degeneration was explored. In differentiating C2C12 myocytes, TNF-induced activation of NF-κB inhibited SMD by suppressing MyoD mRNA at the posttranscriptional level. In contrast, in differentiated myotubes, TNF plus interferon-γ (IFN-γ) signaling was required for NF-κB–dependent down-regulation of MyoD and dysfunction of skeletal myofibers. MyoD mRNA was also down-regulated by TNF and IFN-γ expression in mouse muscle in vivo. These data elucidate a possible mechanism that may underlie the skeletal muscle decay in cachexia.

The vertebrate skeletal muscle differentiation (SMD) program is under the strict control of the myogenic bHLH transcription factor family (MyoD, Myf5, myogenin, and MRF4) and of a second class of transcription factors termed myocyte enhancer factor–2 (MEF2A through MEF2D) (1, 2). MyoD and Myf5 are expressed in proliferating undifferentiated myoblasts and, upon growth factor withdrawal, are activated to initiate SMD that ultimately leads to the fusion of myoblasts into multinucleated myotubes (1, 2). Although mice lacking MyoD develop normally (3), MyoD-deficient skeletal muscle is severely impaired in its ability to regenerate after tissue injury, which suggests a specific role for MyoD in the replenishment of lost muscle (4).

NF-κB is a transcription factor expressed in a variety of cell types, including mature muscle (5), and it inhibits SMD by regulating the expression of cyclin D1 (6). The cytokine tumor necrosis factor (TNF) is a potent activator of NF-κB, causing it to translocate to the nucleus and bind to promoters and enhancers of genes involved in inflammatory and proliferative responses (7–9). TNF is an important mediator of skeletal muscle degeneration associated with cachexia, a debilitating syndrome characterized by extreme weight loss and whole-body wasting (10–12). Cachexia is frequently seen in patients afflicted with chronic diseases such as cancer and acquired immunodeficiency syndrome. It is striking that about one-third of cancer mortalities result from cachexia rather than tumor burden (13). Little is known about the molecular etiology of cachexia, and hence few targets have been identified for therapy. Here we investigate the potential role of NF-κB as a downstream effector of TNF-mediated skeletal muscle dysfunction.

TNF was recently shown to inhibit skeletal myogenesis in vitro (6, 14). We therefore investigated the potential role of NF-κB in this regulatory process. The addition of TNF to mouse C2C12 myocytes completely blocked their SMD program, as evidenced by the reduced expression of the differentiation markers myogenin and the cyclin-dependent kinase inhibitor p21, and by the complete absence of the late-stage differentiation marker myosin heavy chain (MHC) (Fig. 1A). TNF also caused a severe reduction in MyoD protein levels (Fig. 1B) but had no effect on the transcription factors Myf5 or MEF2D, which are also expressed but are inactive in undifferentiated myoblasts. Stable heterologous expression of MyoD restored the ability of C2C12 cells to maintain SMD (Fig. 1C) and form myotubes (15) in the presence of TNF, indicating that TNF inhibits myogenesis by down-regulating MyoD. The rapid loss of MyoD protein was preceded by an equally rapid loss ofMyoD mRNA (Fig. 1D). In addition, cells devoid of NF-κB activity [through stable expression of the NF-κB inhibitor IκBα (IκBαSR)] retained MyoD mRNA in the presence of TNF (Fig. 1D), indicating that NF-κB is a downstream TNF effector that regulates MyoD expression.

Figure 1

TNF-induced activation of NF-κB inhibits SMD through suppression of MyoD synthesis. (A) C2C12 cells were switched from growth medium [GM: Dulbecco's modified Eagle's medium–H (DMEM-H) and 20% fetal bovine serum] to differentiation medium [DM: DMEM-H, 2% horse serum, and insulin (5 μg/ml)] and treated with or without TNF (Promega) at 20 ng/ml for a 72-hour period (to sustain NF-κB activity, TNF was also added at 6, 12, and 24 hours after the initial treatment). At indicated times, whole-cell extracts were prepared and probed for SMD markers myogenin and the cyclin-dependent kinase inhibitor p21 (M-225 and C-19, Santa Cruz Biotechnology), MHC (MY-32, Sigma), or α-tubulin (Sigma). (B) TNF regulation is specific to MyoD. Extracts from C2C12 cells in DM treated with or without TNF were probed for MyoD, Myf5 (M-318 and C-20, respectively, Santa Cruz Biotechnology), and MEF2D (Transduction Laboratories). (C) Overexpression of MyoD restores SMD in the presence of TNF. C2C12 cells were infected with pBabepuro virus containing MyoD cDNA, and selected with puromycin (1 μg). Clones were screened for their expression of MyoD and p21 (15). Vector control cells and three independent clones—pBabeMyoD1, -4, and -7—were treated with TNF in DM, and extracts were probed for p21 (after 48 hours) and MHC (after 72 hours). (D) TNF regulation of MyoD is dependent on NF-κB activity. Vector control or cells lacking NF-κB activity (IκBαSR) were differentiated and treated with TNF. At the indicated times,MyoD and GAPDH mRNA expression was analyzed.

To examine which NF-κB subunits were involved in the regulation ofMyoD, reporter assays were performed in mouse 10T1/2 fibroblasts. Coexpression of p50 and p65 subunits, or of the p65 subunit alone, strongly blocked MyoD activity, and this effect was reversed with equivalent expression of the IκBαSR plasmid (16). Examination of whole-cell lysates from these transfections demonstrated that p65 alone was sufficient to dramatically reduce MyoD protein levels (Fig. 2A), and this inhibition was again lost after the expression of IκBαSR. In addition, similar to what was observed with TNF treatment of C2C12 cells, overexpression of p65 reduced MyoD mRNA levels (Fig. 2B). These results demonstrate that TNF regulation of MyoD in C2C12 cells is dependent on NF-κB activity and that the p65 subunit is sufficient to induce the down-regulation of MyoD mRNA.

Figure 2

MyoD is regulated by the p65 subunit of NF-κB. (A) p65 suppresses MyoD expression. 10T1/2 fibroblasts were transfected with MyoD, NF-κB, and IκBαSR expression plasmids (23). Whole-cell lysates were prepared and probed for MyoD and α-tubulin. (B) p65 suppresses MyoD mRNA expression. Transfections were repeated in 100-mm culture dishes with 0.5 μg of vector or of MyoD expression plasmid, with or without 1.0 μg of p65 expression plasmid. Cells were either kept in GM or differentiated in DM, and at indicated times after transfection, RNA was prepared and probed for MyoD and GAPDH mRNA expression.

These transfection results showed that p65 regulated MyoDtranscripts produced from a heterologous promoter. This suggested that NF-κB was ultimately targeting the MyoD mRNA itself for down-regulation. We therefore attempted to map the MyoDsequences that rendered the mRNA susceptible to p65-mediated down-regulation. Deletion analysis revealed that nucleotides 539 through 914 in the MyoD mRNA were required for this response (Fig. 3A). Introduction of thisMyoD fragment into the coding region of the GAPDHgene resulted in a p65-dependent reduction in GAPDH mRNA levels (Fig. 3B), confirming that p65 targets this MyoDsequence. We next examined whether NF-κB inhibition ofMyoD mRNA was direct or indirect (that is, requiring NF-κB–dependent transcription). MyoD mRNA decay rates, determined in experiments in which transcription was blocked with actinomycin D, were similar whether C2C12 myoctyes were treated or not treated with TNF (16), which suggests that regulation of MyoD is dependent on an NF-κB–responsive gene. To test the requirement for the transcription function of p65 in the regulation of MyoD, we performed reporter assays in mouse 10T1/2 fibroblasts with deletion mutants lacking regions of the p65 transactivation domain. Deletion of the entire transactivation domain of p65 [Fp65(313)] restored MyoD function (Fig. 3C). These data are consistent with those in Fig. 2A, where the p50 subunit of NF-κB, which lacks a transactivation domain, was also unable to inhibit MyoD expression. Taken together, these data indicate that MyoD mRNA is inhibited at the posttranscriptional level in a manner requiring NF-κB–dependent transcription.

Figure 3

Identification of the MyoD sequences regulated by p65-dependent transcription. (A) p65 inhibits MyoD in a region encompassing nucleotides 539 through 914. The MyoD cDNA was removed from plasmid pEMC11s, containing a long terminal repeat element, and ligated into pCDNA3 (Invitrogen), generating the plasmid pCMV-MyoD. 5′ and 3′ deletions were made in MyoD sequences (16). Numerical values present in the names of each plasmid denote the location of the deletions generated with respect to the 5′ and 3′ ends of the MyoD cDNA. Wild-type MyoD (lanes 1 and 2) or deletion plasmids (lanes 3 through 10), each at 0.5 μg, were individually transfected in 10T1/2 fibroblasts with or without a p65 expression plasmid (1.0 μg). RNA was extracted 24 hours after transfection, and MyoD was probed. Numerical values listed with arrows indicate the predicted sizes of exogenously expressed MyoD mRNA products. (B) p65 inhibits GAPDH expression containing MyoD sequences. Nucleotides 313 through 914 from MyoD were ligated into pCMVGAPDH, generating the plasmid pCMVGAPDH-MyoD. Transfections and RNA analysis were repeated as described in (A). The arrow indicates endogenous GAPDH expression in 10T1/2 fibroblasts. (C) MyoD regulation requires p65-dependent transcription. The upper portion shows transfections performed in 10T1/2 fibroblasts with a MyoD-responsive reporter (4RTK-Luc, containing four E-box sites fused to a minimal thymidine kinase promoter), the pCMV-MyoD expression plasmid, and epitope-tagged p65 expression plasmids containing either wild-type [Fp65(551)] or COOH-terminal deletions in the transactivation domain. Cell extracts were prepared with M-Per Extraction Buffer (Pierce) and tested in luciferase assays. The lower portion shows cell extracts from transfections that were probed with an antibody to FLAG (M2, Sigma) to verify the presence of equal p65 expression.

TNF is an important mediator in the skeletal muscle degeneration associated with cancer-induced cachexia (10–12). Direct inhibition of NF-κB also blocks cachexia in an animal model (17), suggesting a link between NF-κB and TNF in this disease. Although delivery of TNF is sufficient to induce weight loss in animals, in vitro studies showing that skeletal muscle explants incubated with TNF do not undergo degeneration (18) support the notion that at least one other factor is required to induce muscle wasting. Consistent with these findings, we found that in contrast to the effect of TNF on differentiating myoblasts, differentiated myotubes were completely refractory to this cytokine with respect to MyoD and MHC expression (Fig. 4A). Because other inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and IFN-γ also induce cachexia in animal models (19), we investigated the possibility that one of these cytokines in combination with TNF could affect skeletal muscle cellular function. Treatments with IL-1β or IL-6 alone or in combination with TNF had no effect on skeletal muscle–specific gene expression (Fig. 4A) (15). In addition, IFN-γ treatment alone had no effect (15). However, differentiated myotubes treated with TNF plus IFN-γ exhibited significant reductions in both MyoD and MHC protein expression (Fig. 4A). Consistent with these results, immunostaining analysis displayed dramatic diminishment of MHC after treatment with TNF plus IFN-γ (Fig. 4C) (16), which correlated with the inability of myofibers to maintain their contractile activities in culture (15). Apoptosis was observed in cultures treated with TNF plus IFN-γ, but only in those myocytes unable to complete their differentiation program (16), indicating that the loss of muscle cell structure was not a result of cell death. Thus, IFN-γ and TNF together regulate myotube degeneration. When C2C12 myotubes devoid of NF-κB activity were treated with TNF plus IFN-γ, they maintained both MyoD and MHC expression (Fig. 4, B and C), indicating that repression of MyoD and impairment of skeletal muscle function are NF-κB–dependent processes.

Figure 4

IFN-γ is required in addition to TNF to regulate skeletal muscle dysfunction. (A) MyoD and MHC were reduced in myotubes by treatment with TNF plus IFN-γ. C2C12 myocytes were differentiated for a 3-day period. DM was replaced with either no additive (lane 1); TNF at concentrations of 20 or 100 ng/ml (lanes 2 and 3, respectively); TNF (20 ng/ml) plus IL-1β (20 ng/ml; Promega) (lane 4); TNF (20 ng/ml) plus IL-6 (20 ng/ml; Promega) (lane 5); or TNF (20 ng/ml) plus IFN-γ (100 U/ml; Life Technologies) (lane 6). Treatments were repeated at 6, 12, and 24 hours. At 48 hours, whole-cell lysates were prepared and probed for MyoD, MHC, and α-tubulin. (B through D) Regulation of MyoD and myosin by TNF and IFN-γ is NF-κB–dependent. Vector control or IκBαSR-expressing myoblasts were differentiated as described in (A) and then treated with TNF and/or IFN-γ for 48 hours. Lysates were prepared and probed for MyoD and MHC (B), or cells were immunostained (24) for MHC (C). Scale bar, 15 μm. (D) MyoD was down-regulated by cytokines in vivo. Upper panels: BALB/c and B6/129 mice were given saline alone (vehicle) or a combination of 2 μg of TNF and 5000 U of IFN-γ in a volume of 0.1 ml in the right gastrocnemius muscle. Injections were repeated every 4 hours. At 12 hours, gastrocnemius muscles were dissected and quick-frozen in liquid nitrogen. At a later time, RNA was prepared with the use of TRIZOL reagent (Life Technologies), and Northern analyses were performed to detect MyoD and GAPDH expression. Lower panels: 1 × 107 vector control CHO cells or a mixture of CHO cells expressing human TNF (12) and mouse IFN-γ, clones 1 and 15 (20), were injected into right gastrocnemius muscles of nude mice. At day 12 after injection, gastrocnemius muscles were isolated, RNA was prepared, and Northern analysis was performed, probing for MyoD and GAPDH.

To test whether TNF and IFN-γ regulate MyoD expression in vivo, we injected cytokines directly into mouse skeletal muscle.MyoD mRNA levels were substantially reduced after injection of the cytokines (Fig. 4D). A mixture of tumor-forming Chinese hamster ovary (CHO) cells expressing TNF (12) and IFN-γ (20) was also injected into the skeletal muscle of nude mice to more accurately mimic a cachetic state. Again, these cytokines caused a pronounced decline in MyoD mRNA levels but had no effect on GAPDH mRNA (Fig. 4D). These results suggest thatMyoD is an in vivo target of cytokine signaling.

In adult skeletal muscle, MyoD is expressed at relatively low levels (21, 22). However, in response to injury, MyoD expression is induced from satellite cells, and genetic evidence clearly demonstrates that MyoD is required for these cells to proliferate and to reinitiate SMD necessary for the repair process (4, 21). Our data demonstrate that TNF, along with a second proinflammatory cytokine, IFN-γ, functions through NF-κB to suppress MyoD synthesis by repressing the accumulation ofMyoD mRNA. With respect to an injury model such as cachexia, we propose that the cytokines TNF and IFN-γ are likely to affect skeletal muscle regulation at two phases: first, when TNF alone would be sufficient to suppress MyoD expression, thereby inhibiting the formation of new myofibers; and second, when combined signaling between TNF and IFN-γ would be required to inhibit MyoD and cause the degeneration of newly formed myotubes. The combined cytokine effects, working through NF-κB, would lead to the inability to repair damaged skeletal muscle, thereby contributing to the overall wasting process associated with cachexia. Thus, direct inhibition of NF-κB may prove beneficial in reducing the muscle wasting associated with cachexia in cancer patients as well as with other disorders.

  • * Present address: Laboratory of Molecular Signaling, Department of Biologic and Material Science, University of Michigan, Ann Arbor, MI 48109, USA.

  • To whom correspondence should be addressed. E-mail: jhall{at}


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