help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-1541
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
147/9/4363    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Strle, K.
Right arrow Articles by Kelley, K. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Strle, K.
Right arrow Articles by Kelley, K. W.
Endocrinology Vol. 147, No. 9 4363-4373
Copyright © 2006 by The Endocrine Society

C-Jun N-Terminal Kinase Mediates Tumor Necrosis Factor-{alpha} Suppression of Differentiation in Myoblasts

Klemen Strle, Suzanne R. Broussard, Robert H. McCusker, Wen-Hong Shen, Julie M. LeCleir, Rodney W. Johnson, Gregory G. Freund, Robert Dantzer and Keith W. Kelley

Laboratories of Immunophysiology (K.S., S.R.B., R.H.M., W.-H.S., J.M.L., K.W.K.) and Integrative Biology (R.W.J.), Department of Animal Sciences, and Department of Pathology, College of Medicine (G.G.F.), University of Illinois, Urbana, Illinois 61801; and Neurobiologie Intégrative (R.D.), Unité Mixte de Recherche Institut National de la Recherche Agronomique-Université de Bordeaux 2, 33077 Bordeaux Cedex, France

Address all correspondence and requests for reprints to: Keith W. Kelley, University of Illinois, Laboratory of Immunophysiology, Department of Animal Sciences, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, Illinois 61801. E-mail: kwkelley{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The stress kinase c-jun N-terminal kinase (JNK) was recently shown to be involved in the pathophysiology of major inflammatory conditions, including Alzheimer’s disease, stroke, obesity, and type II diabetes. However, the role of JNK in regulating inflammatory events in skeletal muscle is only beginning to be explored. IGF-I is the major hormone that promotes muscle growth and development. Here we used a novel, JNK interacting protein (JIP)-derived JNK peptide inhibitor to establish that JNK suppresses the biological activity of IGF-I in skeletal muscle progenitor cells. In these myoblasts, TNF{alpha} and its downstream receptor substrates, neutral-sphingomyelinase (N-SMase) and N-acetyl-D-sphingosine (C2-ceramide), induce JNK kinase activity in a time-dependent manner. Consistent with these results, TNF{alpha} induces JNK binding to insulin receptor substrate 1 (IRS-1) but is unable to inhibit IGF-I-induced IRS-1 tyrosine phosphorylation in myoblasts that are treated with the JNK peptide inhibitor. More importantly, JNK activation induced by TNF{alpha}, C2-ceramide, and N-SMase is associated with reduced expression of the critical muscle transcription factor myogenin as well as the differentiation marker myosin heavy chain (MHC). The JNK peptide inhibitor, but not the control peptide, completely reverses this inhibition of both myogenin and MHC. In the absence of IGF-I, TNF{alpha}, C2-ceramide, N-SMase and the JNK inhibitor are inactive, as shown by their inability to affect IRS tyrosine phosphorylation and protein expression of myogenin and MHC. These results establish that the resistance of muscle progenitor cells to IGF-I, which is caused by inflammatory stimuli, is mediated by the JNK stress kinase pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FAMILY OF stress-activated protein kinases (SAPK) consists of three c-jun N-terminal kinase (JNK) isoforms and at least 10 splice variants. All three JNK isoforms are activated by dual phosphorylation on tyrosine and threonine residues in response to a plethora of stress stimuli, including proinflammatory cytokines, ceramide, UV irradiation, DNA damage, and growth factor deprivation (1, 2, 3). JNK1 and -2 are ubiquitously expressed, whereas JNK3 is localized to the central nervous system. A novel peptide inhibitor of JNK was recently used in several elegant studies to asses the role of JNK in animal models of diabetes (4), Alzheimer’s disease (5), and stroke (6). The functional domain of this blocker is a sequence of approximately 20 amino acids that corresponds to the JNK binding domain (JBD) of JNK interacting protein (JIP), an endogenous regulator of JNK (7, 8, 9). JIP is a scaffold protein that normally assembles and activates the SAPK pathway complex. The therapeutic potential of JNK regulation has recently gained significant clinical attention because JNK is implicated in the pathophysiology of inflammatory, neurodegenerative, and metabolic disorders. For example, novel JIP-derived peptide inhibitors that block all three JNK isoforms (7) suppress pancreatic ß-cell loss and improve insulin sensitivity in obese db/db mice (4). Similarly, these inhibitors block neuronal death, decrease lesion size, and maintain normal motor skills after middle cerebral artery occlusion (6). In a model of Alzheimer’s disease, JNK peptide inhibitors also prevent ß-amyloid-induced apoptosis of cortical neurons and suppress the inhibition of long-term potentiation, which is an in vitro measure of memory (5). These data indicate that JNK is a convergence point for a variety of stress stimuli and that JNK inhibitory peptides may serve as valuable tools for therapeutic interventions in a variety of inflammatory conditions.

Despite recent advances in understanding proinflammatory cytokine-dependent regulation of myogenesis, the role of the SAPK JNK in regulating this process remains largely obscure. Muscle wasting that occurs in the sarcopenia of aging (10, 11, 12) and more prominently in cachectic AIDS and cancer patients (13, 14, 15, 16) is associated with a decline in IGF-I anabolic activity. IGF-I is a critical growth factor that promotes muscle differentiation, regeneration, and hypertrophy. However, even very low, physiological concentrations (0.01–1 ng/ml) of the proinflammatory cytokine TNF{alpha} inhibit the ability of IGF-I to induce global protein synthesis (17, 18) and promote expression of critical muscle-specific transcription factors, including myogenin (17, 18) and MyoD (18). These inhibitory actions of TNF{alpha} are associated with activation of ceramide-generating pathways, acid-sphingomyelinase (A-SMase), neutral (N)-SMase, and de novo ceramide synthesis, because blockers of each of these three pathways suppress the inhibition caused by TNF{alpha} and IL-1ß (18). Unfortunately, it is not yet clear whether ceramide induces IGF-I resistance directly or through other key downstream mediators such as the SAPK JNK. Recent results have attributed the catabolic actions of JNK in muscle tissue to its ability to suppress IGF-I expression (19). Consistent with these findings, a pharmacological inhibitor of JNK, SP600125, completely blocks the ability of TNF{alpha} to suppress GH-dependent mRNA expression of IGF-I (20). However, this mechanism does not explain the findings from cachectic AIDS patients in which administration of GH increases IGF-I serum concentrations to the same level as control subjects but does not ameliorate protein loss (15, 21). The possibility that JNK directly mediates proinflammatory cytokine inhibition of IGF-I-induced anabolic activity has not yet been explored.

Here we used a JIP-derived cell-permeable peptide inhibitor of JNK, I-JNK, which was previously established to specifically inhibit all three isoforms of JNK (7). We demonstrate that JNK is a key regulatory protein that mediates TNF{alpha}- and C2-ceramide (N-acetyl-D-sphingosine)-induced IGF-I resistance in murine myoblasts. Specifically, I-JNK prevents the ability of TNF{alpha} to inhibit IGF-I-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). More importantly, the suppressive actions of TNF{alpha} and C2-ceramide on IGF-I-dependent myogenin expression are inhibited by I-JNK, indicating that JNK is likely responsible for cytokine-mediated inhibition of differentiation. This hypothesis was tested directly by showing that I-JNK completely blocks the ability of TNF{alpha} to inhibit IGF-I-induced expression of embryonic myosin heavy chain (MHC), a well-defined marker of differentiation. Collectively, these results establish that JNK is a key downstream protein by which TNF{alpha} and ceramide induce IGF-I resistance and inhibit differentiation in muscle progenitors. These are some of the first findings to determine that JNK inhibits myogenesis by directly suppressing IGF-I anabolic activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant murine TNF{alpha}, murine IL-1ß, and human IGF-I were purchased from Intergen (Purchase, NY). N-SMase from Staphylococcus aureus and C2-ceramide, a cell-permeable analog of ceramide, were purchased from Sigma Aldrich Corp. (St. Louis, MO). D-JNK1 (I-JNK), a JNK peptide inhibitor 1, D-stereoisomer and control peptide (C-JNK) were purchased from Alexis Biochemicals (San Diego, CA). The nonradioactive SAPK/JNK assay kits were purchased from Cell Signaling Technology (Beverly, MA), and radioactive JNK assay kits were purchased from Upstate (Lake Placid, NY). DMEM (with 4.5 g/liter glucose, 0.584 g/liter glutamine) and penicillin/streptomycin were obtained from Bio Whittaker Cambrex (Walkersville, MD) and fetal bovine serum (<0.25 EU/ml of endotoxin) was purchased from HyClone (Logan, UT). Protein G Sepharose 4 Fast Flow beads were purchased from Amersham Biosciences (Uppsala, Sweden). Mouse monoclonal antibodies were obtained as follows: an IgG1 antibody to myogenin (F5D) was from Santa Cruz Biotechnology (Santa Cruz, CA), an IgG1 antibody to {alpha}-tubulin (B-5-1-2) was from Sigma Aldrich, an IgG1 antibody to embryonic MHC (F1.652) was from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA), and the phosphotyrosine-specific PY-20 antibody was obtained from Transduction Laboratories (Lexington, KY). The rabbit IgG anti-IRS-1 antibody was from Upstate. All other reagents were from Sigma Aldrich.

Optimal concentrations for C2-ceramide (1 µM) and N-SMase (25 mU) were determined as previously described and are considerably lower than reported in other cells (18). Preliminary dose-response experiments established that effective concentrations of I-JNK in inhibiting JNK activity ranged from 1–5 µM, which is consistent with results of others in various cell types (6, 7, 8, 9). No cell death was detected in the presence of I-JNK (up to 3 µM) over a 36-h period, as assessed by trypan blue staining (data not shown). Appropriate diluents for the reagents were added to the medium of all corresponding treatments. These diluents were as follows: PBS (final concentration, 0.1%) for I-JNK, dimethylsulfoxide (final concentration, 0.01%) for C2-ceramide, and glycerol (final concentration, 0.1%) for N-SMase.

Cell culture
Murine C2C12 myoblasts were obtained from American Type Culture Collection (Manassas, VA). Myoblasts were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C, 7% CO2, and 95% humidity. Cells were grown to 70% confluence. Before initiation of experiments, cells were washed three times in DMEM to remove growth factors and deprived of serum for 5 h.

In all experiments using I-JNK, myoblasts were pretreated with the inhibitor (I-JNK) or TAT peptide control (C-JNK) for 1 h before addition of TNF{alpha}, C2-ceramide, or N-SMase with or without IGF-I. In experiments measuring tyrosine phosphorylation of IRS-1, myoblasts were pretreated with TNF{alpha}, C2-ceramide, or anisomycin, the positive control for JNK activation, for 1 h before addition of IGF-I for an another 3 min. JNK association with IRS-1 was evaluated after a 15-min treatment with IGF-I or TNF{alpha}. In experiments measuring the inhibition of JNK kinase activity, myoblasts were pretreated with varying doses of I-JNK (1–3 µM) or the control peptide C-JNK (3 µM) for 1 h and then treated with TNF{alpha} for an additional 15 min. JNK kinase activity was measured by incubating myoblasts with TNF{alpha}, IGF-I, C2-ceramide, or N-SMase for 0, 5, 10, 15, 30, and 60 min. For the experiments measuring protein expression of myogenin and embryonic MHC, myoblasts were treated with TNF{alpha}, IL-1ß, C2-ceramide, or N-SMase for 1 h before addition of IGF-I for an additional 24 h.

Western blotting for IRS-1, c-Jun, myogenin, and MHC
After treatment, myoblasts were homogenized in buffer at pH 7.4 containing 50 mM HEPES, 150 mM NaCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, 1% glycerol, 1.5 mM MgCl2, 1% sodium deoxycholate, 100 mM NaF, 0.1% SDS, 1% Triton X-100, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 40 nM leupeptin, and 2 µg/ml pepstatin. To ensure that equal amounts of protein were added to all wells, protein concentrations of each sample were determined using the low-concentration protocol with a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). TNF{alpha}, C2-ceramide, N-SMase, and I-JNK did not alter protein concentrations in whole-cell lysates compared with untreated myoblasts. Proteins from whole-cell lysates were separated using SDS-PAGE. For experiments measuring IRS-1 (molecular mass = 165 kDa), proteins were separated on gels containing 7.5% polyacrylamide; in experiments where c-Jun (molecular mass = 35 kDa) was measured, proteins were separated on 10% polyacrylamide gels; for those with myogenin (molecular mass = 35 kDa), proteins were separated on 12.5% polyacrylamide gels. Proteins were transferred to Trans-Blot polyvinylidene fluoride membranes (Bio-Rad Laboratories) using a Bio-Rad Laboratories Mini Protean 3 system. Nonspecific antibody binding interactions were blocked by incubating membranes for 1 h in Tris-buffered saline with Tween 20 (TBS-T) [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] containing 1 or 5% BSA, as recommended by the manufacturer. Membranes were then rinsed once with TBS-T and incubated overnight at 4 C with antibodies specific to myogenin, MHC, IRS-1, PY-20, phosphorylated c-Jun, or {alpha}-tubulin. These antibodies were diluted 1/1000 or 1/2000 (for {alpha}-tubulin) in TBS-T containing 1% BSA or 5% BSA (phospho-c-Jun). Membranes were then washed four times for 5 min in TBS-T and incubated with antimouse or antirabbit horseradish-peroxidase-labeled secondary IgG antibodies diluted 1/2000 in TBS-T containing 1% BSA. Membranes were washed as before and developed with enhanced chemiluminescence ECL Western Blot Detection Reagent from Amersham Biosciences (Piscataway, NY). Blots were exposed to autoradiographic B-Plus x-ray film obtained from Central Illinois X-Ray (Bloomington, IL). Films were developed with a Fischer model K-Plus, automatic x-ray film processor from Fischer Industries Inc. (Geneva, IL). Protein band intensity was quantified by scanning autoradiograms with Canon CanoScan N1220U scanner (Lake Success, NY) followed by densitometric analysis with IMAGEJ software from the National Institutes of Health (Bethesda, MD). Densitometric summaries are presented as ratios of the densitometric values of tyrosine-phosphorylated IRS-1 protein to total IRS-1 protein or as densitometric values of the target proteins myogenin and MHC to {alpha}-tubulin protein-loading control. Data were standardized by dividing the individual sample ratios by the mean of the entire experiment.

Immunoprecipitation of IRS-1
The protocol for immunoprecipitations of IRS-1 has been previously described in detail by our laboratory (17, 22). In brief, approximately 1 x 106 myoblasts were treated as described in Results and lysed in 1 ml homogenization buffer. Proteins bound to IRS-1 were immunoprecipitated with 2 µg/ml anti-IRS-1-specific antibody and 20 µl protein G Sepharose beads overnight at 4 C. The protein and bead complexes were washed four times, resuspended in 100 µl Laemmli buffer, and heated for 10 min at 100 C. Proteins were separated on SDS-PAGE, transferred to polyvinylidene fluoride membranes, blotted with antiphosphotyrosine-specific PY-20 antibody, and exposed to film. The same membranes were then stripped, reblotted with an anti-IRS-1-specific antibody, and developed as described above for Western blotting.

Association of JNK with IRS-1 and JNK activity assay
The radioactive JNK kinase assay (Upstate) protocol was modified to measure the ability of JNK to associate with IRS-1 in addition to measuring JNK kinase activity. Approximately 2 x 106 myoblasts were treated as described in Results and lysed in homogenization buffer. Proteins that associated with IRS-1 were then immunoprecipitated for 5 h at 4 C with 4 µg/ml anti-IRS-1-specific antibody and 30 µl protein G Sepharose beads. After the wash, immunoprecipitates were incubated with 20 µl of ATP dilution buffer, 10 µl (1 µg) recombinant glutathione-S-transferase-conjugated c-Jun, and 20 µl stock [{gamma}-32P]ATP (20 µCi). The complexes were then mixed and incubated for 30 min at 30 C with constant shaking. This reaction was terminated by addition of 100 µl Laemmli buffer followed by heating for 5 min at 100 C. Proteins were separated on 10% SDS-PAGE gels. Subsequently, these gels were placed on filter paper, covered with plastic wrap, and vacuum dried. JNK activity and binding to IRS-1 were visualized on a Typhoon 9600 imaging system and on x-ray films exposed for 24 h at –80 C. Films were developed as described for Western blotting.

JNK kinase assay
After treatment, myoblasts were lysed and proteins immunoprecipitated overnight at 4 C with 20 µl (2 µg) c-Jun fusion protein attached to agarose beads. Protein and c-Jun/bead complexes were washed, resuspended in 100 µl kinase buffer containing 200 µM ATP, and incubated for 30 min at 30 C, as described by the manufacturer. The reaction was terminated with 100 µl Laemmli buffer, which was followed by heating for 5 min at 100 C. Proteins were separated on 10% SDS-PAGE gels and blotted with an anti-phosphoserine 63 c-Jun-specific antibody provided by the manufacturer. The films were exposed and quantified as described for Western blotting. Because c-Jun can be singly or doubly phosphorylated on S63 and S73, duplicate bands sometimes appear on the blots because of an increase in molecular mass from 35 kDa of singly phosphorylated c-Jun to 37 kDa of doubly phosphorylated protein.

Statistical analysis
Experiments were conducted as a completely randomized design. All data were analyzed using Statistical Analysis System (SAS version 8) (23) with a general linear model. Differences between treatments were detected using F-protected Duncan’s multiple-range tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An activator of JNK, anisomycin, inhibits the IGF-I-induced tyrosine phosphorylation of IRS-1
Genetic mutations in the IGF-I system are rarely implicated in muscle wasting. Consequently, defects in IGF-I signaling are generally attributed to environmental factors such as proinflammatory cytokines, although the details of this inhibition are not well defined. Recently we reported that TNF{alpha} significantly impairs downstream IGF-I signaling in both human breast cancer epithelial cells (22) and murine myoblasts (17) but does not affect the ability of IGF-I to bind to or activate the tyrosine kinase IGF-I receptor (IGF-IR). Instead, TNF{alpha} (17, 18) and the downstream phosphosphingolipid C2-ceramide (18) inhibit tyrosine phosphorylation of a key docking protein in the IGF-I signaling pathway, IRS-1. Here we extend these results to JNK by showing that a potent activator of this kinase, anisomycin, mimics TNF{alpha} and C2-ceramide inhibition of IRS-1 tyrosine phosphorylation (n ≥ 5; Fig. 1Go). C2C12 myoblasts were pretreated with TNF{alpha}, C2-ceramide, or anisomycin for 1 h before a 3-min incubation with or without IGF-I. As expected, IGF-I increased tyrosine phosphorylation of IRS-1 (P < 0.01; Fig. 1Go). This 10-fold increase in IRS-1 tyrosine phosphorylation was completely inhibited by TNF{alpha}, C2-ceramide, and anisomycin (P < 0.01; Fig. 1Go). In the absence of IGF-I, TNF{alpha}, C2-ceramide, and anisomycin did not affect IRS-1 tyrosine phosphorylation. These results indicate that activation of the SAPK JNK inhibits IGF-I signaling events in muscle progenitor cells. In addition, these data are consistent with the idea that JNK may serve as a key downstream component in a cytokine-induced signaling pathway that leads to IGF-I resistance in muscle.


Figure 1
View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1. Anisomycin inhibits IGF-I-induced IRS-1 tyrosine phosphorylation. C2C12 myoblasts were treated with TNF{alpha} (1 ng/ml), C2-ceramide (1 µM), or anisomycin (0.1 µM) for 1 h before a 3-min stimulation with or without IGF-I (50 ng/ml). Alternatively, C2C12 myoblasts were treated with I-JNK in the presence or absence of TNF{alpha} and IGF-I (A, last four lanes). IRS-1 proteins were immunoprecipitated using an IRS-1-specific antibody and separated on 7.5% SDS-PAGE gels. The membranes were probed with antibodies specific to phosphotyrosine residues, stripped, and reblotted with antibodies against IRS-1. A representative gel (A, first eight lanes), and a densitometric summary of at least five independent experiments (B), show that IGF-I increased tyrosine phosphorylation of IRS-1 by 7-fold, an effect that was inhibited by TNF{alpha}, C2-ceramide, and anisomycin (n ≥ 5). **, P < 0.01.

 
JNK is required for TNF{alpha}-induced inhibition of IGF-IR signaling
The ability of JNK to inhibit insulin-induced IRS-1 tyrosine phosphorylation is strongly implicated in insulin resistance and the pathophysiology of chronic disorders such as obesity and diabetes (4, 24, 25, 26). Similar inhibitory actions of JNK on insulin signaling have been reported in muscle (27, 28). The majority of these earlier studies examined the role of JNK in inducing insulin resistance and in regulation of glucose uptake and metabolism. However, despite the extensive similarities between insulin and IGF-IR signaling pathways and the critical role of IGF-I in muscle development, the possibility that JNK mediates cytokine-dependent regulation of IGF-I signaling in muscle cells has not been explored. To test this possibility, we first examined whether JNK could bind to IRS-1, because such an association was previously shown to lead to a reduction in insulin-induced IRS-1 tyrosine phosphorylation (29). Consequently, murine C2C12 myoblasts were treated for 15 min with IGF-I and TNF{alpha}. IRS-1 was then immunoprecipitated from whole-cell lysates, and a radioactive assay for JNK was performed using recombinant c-Jun as a substrate. These data show that JNK associates with IRS-1 in myoblasts stimulated with TNF{alpha} but not with IGF-I (n = 3; Fig. 2AGo). JNK association with IRS-1 was confirmed by an alternate approach using a phosphorylated JNK-specific antibody to immunoprecipitate P-JNK and then probing with an antibody against IRS-1. IRS-1 bands were detected in immunoprecipitates from C2C12 myoblasts treated for 15 min with an activator of JNK, anisomycin, as well as with TNF{alpha} (data not shown). To further explore the mechanism by which TNF{alpha}-activated JNK may inhibit tyrosine phosphorylation of IRS-1, we tested the ability of TNF{alpha} to induce serine 307 phosphorylation of IRS-1. For this experiment, C2C12 myoblasts were pretreated with I-JNK (2 µM) for 1 h before treatment with TNF{alpha} for an additional 15 or 60 min. A representative gel shows that TNF{alpha} potently induces IRS-1 S307 phosphorylation and, more importantly, that the inhibitor of JNK, I-JNK, suppresses this increase in IRS-1 serine phosphorylation (Fig. 2BGo). These data provide a likely mechanism by which TNF{alpha} inhibits IGF-I-induced tyrosine phosphorylation of IRS-1 in muscle cells. We directly explored this hypothesis by pretreating myoblasts with an inhibitor of JNK, I-JNK (2 µM), or an inactive inhibitor control peptide that lacks the JNK binding domain sequence, C-JNK (2 µM), for 1 h before treatment with TNF{alpha}. Cells were then stimulated for 3 min with or without IGF-I. TNF{alpha} completely inhibited IGF-I-induced tyrosine phosphorylation of IRS-1 (n = 7, P < 0.01; Fig. 1AGo, blot, and Fig. 2CGo, summary). More importantly, I-JNK (n = 5, P < 0.01; Fig. 1AGo, blot, and Fig. 2CGo, summary), but not an inactive inhibitor control, C-JNK (n = 2, P < 0.01; Fig. 2CGo), reversed the TNF{alpha}-dependent inhibition of IRS-1. Identical results were observed with a pharmacological inhibitor of JNK, SP600125 (10 µM), which totally reversed the ability of TNF{alpha} to suppress tyrosine phosphorylation of IRS-1 (data not shown). These results confirm that JNK is required for TNF{alpha} inhibition of IRS-1 tyrosine phosphorylation in muscle progenitor cells. More importantly, these data establish a direct role for JNK in inhibiting IGF-I receptor signaling events in muscle progenitor cells.


Figure 2
View larger version (41K):
[in this window]
[in a new window]
  		FIG. 2. JNK mediates TNF{alpha}-induced inhibition of IRS-1 tyrosine phosphorylation. A, C2C12 myoblasts were treated for 15 min with TNF{alpha} (1 ng/ml) or IGF-I (50 ng/ml). IRS-1-associated JNK kinase activity was determined by incubating IRS-1 immunoprecipitates with recombinant glutathione-S-transferase-conjugated c-Jun in the presence of [{gamma}-32P]ATP (20 µCi). TNF{alpha}, but not IGF-I, induced JNK association with IRS-1. This autoradiograph is representative of three independent experiments. Recombinant JNK (R-JNK) protein served as control. B, To examine the mechanism by which TNF{alpha} inhibits tyrosine phosphorylation of IRS-1, we pretreated C2C12 myoblasts with I-JNK (2 µM) for 1 h before treatment with TNF{alpha} for an additional 15 and 60 min. IRS-1 proteins were immunoprecipitated (IP) and separated on SDS-PAGE, and membranes were probed with an antibody specific for phosphorylated serine 307 (PS307) residues of IRS-1. Membranes were then stripped and reblotted with an IRS-1-specific antibody. The autoradiograph shows that TNF{alpha} potently induced serine 307 phosphorylation of IRS-1. More importantly, an inhibitor of JNK, I-JNK, completely blocked the TNF{alpha}-induced serine phosphorylation. C, To confirm the possibility that JNK binding to IRS-1 mediates TNF{alpha}-induced inhibition of IRS-1 tyrosine phosphorylation, C2C12 myoblasts were pretreated with I-JNK (2 µM) for 1 h before treatment with TNF{alpha} for an additional 1 h. Cells were then treated for 3 min with or without IGF-I. As expected, TNF{alpha} suppressed the ability of IGF-I to tyrosine phosphorylate IRS-1 (n = 7). An inhibitor of JNK, I-JNK (n = 5), but not an I-JNK control peptide (C-JNK; n = 2), completely reversed the inhibition by TNF{alpha}. Densitometric summaries are presented as a ratio of the mass of IRS-1 phosphotyrosine (PY) residues to that of the IRS-1 protein loading control. **, P < 0.01.

 
JNK enzymatic activity is induced by TNF{alpha} but not IGF-I
The SAPK JNK, is a member of the MAPK family and is induced in a variety of cells in response to stress signals, including UV light, DNA damage, proinflammatory cytokines, and ceramide (1, 2, 3). The mechanism of TNF{alpha}-dependent activation of JNK requires the ligation and trimerization of TNF receptor 1, binding of TNF receptor-associated factor 2 (TRAF2) with the receptor, and initiation of a kinase cascade that results in phosphorylation and activation of downstream MAPK-activated kinase kinases (MKK)-4 and -7. These two kinases activate JNK by dual phosphorylation on tyrosine and threonine residues (1, 2). However, the details of JNK activation in skeletal muscle are not fully understood. Here we show that JNK enzymatic activity in myoblasts is induced by TNF{alpha} (Fig. 3Go, A and B), but not by IGF-I (Fig. 3CGo). C2C12 myoblasts were stimulated with TNF{alpha} or IGF-I for 0, 5, 10, 15, 30, and 60 min. Cells were then lysed and subjected to a nonradioactive kinase assay using c-Jun as a substrate. A representative Western blot is shown in Fig. 3AGo, and a densitometric summary of three independent experiments is presented in Fig. 3BGo (n = 3). Maximal stimulation of JNK was 3-fold, as indicated by serine 63 phosphorylation of c-Jun, and occurred at 10 min after treatment with TNF{alpha} (P < 0.01). JNK phosphorylation remained elevated at 15 min (P < 0.05; Fig. 3BGo) and then declined to baseline. Despite results showing that JNK can be activated in muscle by the insulin signaling pathway (30, 31), which shares close homology with the IGF-I signaling pathway, IGF-I was unable to stimulate JNK phosphorylation at any time point (n = 2, P < 0.01; Fig. 3CGo). Similarly, JNK activity in myoblasts was not changed in response to growth factor deprivation (data not shown). These data indicate that JNK is activated in a time frame that is consistent with its role in cytokine-mediated regulation of IGF-IR signaling but that it is not involved in IGF-I-induced anabolic actions in muscle progenitor cells.


Figure 3
View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3. TNF{alpha}, but not IGF-I, induces JNK kinase activity. C2C12 myoblasts were treated with TNF{alpha} (1 ng/ml) or IGF-I (50 ng/ml) for 0, 5, 10, 15, 30, and 60 min. JNK kinase activity was determined by incubating proteins bound to c-Jun (2 µg) conjugated beads with 200 µM ATP for 30 min as described in the Materials and Methods. A and B, A representative gel (A) and a densitometric summary of three independent experiments (B) show that TNF{alpha} induces a 3-fold increase in JNK enzymatic activity after 10 min of stimulation. JNK activity remains elevated at 15 min and decreases at the later time points (n = 3). C, Conversely, IGF-I does not increase JNK enzymatic activity at any of the time points tested (n = 2). *, P < 0.05; **, P < 0.01.

 
N-SMase and C2-ceramide induce JNK enzymatic activity in a time-dependent manner
Ceramide is a sphingosine-based lipid that can be newly synthesized (32, 33) or generated by degradation of membrane sphingomyelin that is catalyzed by A-SMase and N-SMase (34, 35). In a recent study, ceramide was shown to activate JNK by directly binding to MAPK kinase kinase, Mek1. Subsequently, Mek1 phosphorylates and activates MKK4/Sek1, a MAPK kinase that lies just upstream of JNK (3). However, to our knowledge, the ability of ceramide and N-SMase to induce JNK kinase activity in skeletal muscle has not yet been established. Here we show that N-SMase (n = 2; Fig. 4AGo) and C2-ceramide (n = 3; Fig. 4BGo) induce JNK kinase activity in a time-dependent manner. Maximal activation of JNK by N-SMase occurred at 5 min (P < 0.01), declined slightly at 10 min (P < 0.05), and returned to basal levels at the later time points (Fig. 4AGo). Similarly, C2-ceramide induced a 4-fold increase in JNK kinase activity, but this increase did not occur until 10 min of stimulation (P < 0.01; Fig. 4BGo). JNK remained elevated at 15 min (P < 0.01) and then returned to baseline (Fig. 4BGo). In light of these results, it is tempting to conclude that one mechanism by which TNF{alpha} induces JNK kinase activity is by activation of N-SMase that catalyzes the generation of ceramide, although this hypothesis has not been tested directly. Furthermore, these data also indicate that JNK may serve as an important regulatory protein in skeletal muscle by transducing signals from several upstream catabolic factors such as TNF{alpha} and ceramide.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		FIG. 4. N-SMase and C2-ceramide mimic TNF{alpha}-induced activation of JNK enzymatic activity. C2C12 myoblasts were treated with N-SMase (25 mU) or C2-ceramide (1 µM) for 0, 5, 10, 15, 30, and 60 min. JNK activity was measured as described in Fig. 3Go. A, JNK enzymatic activity was induced 3-fold after 5 min stimulation with N-SMase, and remained elevated at 10 min (n = 2); B, similarly, a 10-min stimulation with C2-ceramide (C2-cer) induced a 4-fold increase in JNK enzymatic activity, which remained active at 15 min and then returned to basal levels (n = 3). *, P < 0.05; **, P < 0.01.

 
An inhibitor of JNK, I-JNK, suppresses the ability of TNF{alpha} to induce JNK enzymatic activity
Novel, JIP-derived cell-permeable peptide inhibitors of JNK have been suggested to ameliorate insulin resistance in type II diabetes (4, 7, 8, 9), ß-amyloid deposition and neurodegeneration in Alzheimer’s disease (5, 8), and neuronal death after ischemia (6, 8). The functional domain of these inhibitors is derived from a sequence of approximately 20 amino acids corresponding to the JBD in JIP that acts by blocking JNK association and activation of downstream substrates (7, 8). This 20-amino-acid sequence is then linked to a 10-amino-acid peptide that corresponds to a trans-activating domain protein (TAT) of the HIV. TAT can cross cell plasma membranes and has recently been reported to also traverse the blood-brain barrier (36). When linked to the 20-amino-acid JBD, TAT peptide allows for efficient transport of the inhibitor into cells (7, 8). Although the peptide inhibitors have been successfully used both in vitro and in vivo for extended periods of time, their ability to inhibit JNK in muscle progenitor cells has not been explored. To test this possibility, we treated C2C12 myoblasts with various concentrations (1, 2, or 3 µM) of I-JNK for 1 h before treatment with TNF{alpha} for an additional 10 min. As expected, TNF{alpha} potently induced JNK enzymatic activity, as demonstrated on a representative Western blot (Fig. 5AGo) and quantified by a densitometric summary of several independent experiments [control, TNF{alpha}, and I-JNK (1 µM), n = 4; I-JNK (2 µM), n = 3; I-JNK (3 µM), n = 2; P < 0.01; Fig. 5BGo]. The inhibitor of JNK, I-JNK, but not the I-JNK control peptide, C-JNK, suppressed the ability of TNF{alpha} to induce JNK kinase activity at all concentrations tested (P < 0.01; Fig. 5Go, A and B). These data indicate that I-JNK is a potent inhibitor of JNK in muscle cells and can serve as a valuable tool for elucidating the role of JNK in myogenesis.


Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 5. I-JNK is a potent cell-permeable peptide inhibitor of JNK. After a 1-h pretreatment with I-JNK or an inactive control, C-JNK (1, 2, and 3 µM), C2C12 myoblasts were treated with TNF{alpha} (1 ng/ml) for an additional 10 min. A, A representative gel shows that I-JNK, but not C-JNK, potently suppresses the TNF{alpha}-induced increase in JNK enzymatic activity at all doses tested; B, densitometric summary of several independent experiments indicates that the ability of TNF{alpha} to induce JNK enzymatic activity is abolished by I-JNK at all doses (n = 4, 1 µM; n = 3, 2 µM; n = 2, 3 µM). **, P < 0.01.

 
JNK mediates cytokine suppression of myogenesis by blocking IGF-I-induced myogenin expression
TNF{alpha} and ceramide inhibit the ability of IGF-I to induce protein synthesis and expression of the muscle-specific transcription factors myogenin and MyoD, which are required for myoblast differentiation (17, 18, 37). However, it remains unknown whether ceramide inhibits myogenesis directly or indirectly via other downstream substrates such as JNK. Furthermore, it is not yet known whether JNK mediates proinflammatory cytokine- and ceramide-induced inhibition of IGF-I in myoblasts. Here we confirm and significantly extend previous results by showing that the inhibitor of JNK, I-JNK, suppresses the ability of TNF{alpha} to inhibit IGF-I-induced myogenin expression (n = 4; Fig. 6Go). To examine this concept, C2C12 myoblasts were pretreated with I-JNK or C-JNK for 1 h before addition of TNF{alpha}. Cells were then cultured in the presence or absence of IGF-I for an additional 24 h. Myogenin expression was assessed by blotting membranes with a murine monoclonal antibody specific for myogenin. As previously reported, IGF-I promoted expression of myogenin (P < 0.01), and this effect was inhibited by TNF{alpha} (P < 0.01; Fig. 6Go). The control peptide, C-JNK, had no effect on TNF{alpha}-induced inhibition of myogenin (Fig. 6AGo, upper gel). However, when cells were pretreated with I-JNK, the ability of TNF{alpha} to inhibit IGF-I-induced myogenin expression was completely abolished (P < 0.01; Fig. 6AGo, lower gel, and B). Conversely, expression of an intracellular matrix protein, {alpha}-tubulin, did not change in response to any of the treatments, confirming that equal amounts of total protein were present in each lane (Fig. 6AGo). It is important to note that no effects of either the JNK inhibitor I-JNK or the control peptide C-JNK were observed in absence of TNF{alpha}. A densitometric summary of four independent experiments is shown in Fig. 6BGo. These data directly establish that JNK regulates myogenesis by causing TNF{alpha} inhibition of IGF-I-induced expression of muscle-specific transcription factors.


Figure 6
View larger version (35K):
[in this window]
[in a new window]
  		FIG. 6. JNK mediates the ability of TNF{alpha} to inhibit IGF-I-induced myogenin expression. To examine the role of JNK in cytokine-mediated suppression of myogenesis, C2C12 myoblasts were treated with I-JNK (2 µM) or C-JNK (2 µM) for 1 h before treatment with TNF{alpha} (1 ng/ml) for an additional 1 h. Cells were then incubated with IGF-I (50 ng/ml) for another 24 h. Proteins were separated on SDS-PAGE as described in the Materials and Methods. Expression of myogenin was determined by blotting membranes with a myogenin-specific antibody. An intracellular cytoplasmic protein, {alpha}-tubulin, served as a control to assure that equal amounts of protein were added to each lane. A and B, Representative gels with the control (C-JNK) and inhibitor (I-JNK) peptides (A) and a densitometric summary of independent experiments (B) show that TNF{alpha} completely blocks the ability of IGF-I to induce myogenin expression (n = 4). This inhibition is completely reversed by I-JNK (n = 3) but not C-JNK (n = 2). Densitometric summaries are presented as a ratio of the mass of myogenin to that of {alpha}-tubulin protein loading control. **, P < 0.01.

 
N-SMase- and C2-ceramide-induced inhibition of myogenin expression is mediated by JNK
Ceramide and N-SMase have recently been shown to mediate cytokine inhibition of myogenesis by blocking IGF-I-induced anabolic activity (18). Both C2-ceramide and N-SMase inhibit IGF-I-induced protein synthesis as well as expression of myogenin and MyoD. Here we used a peptide inhibitor of JNK to test the idea that this inhibition is mediated by JNK. Myoblasts were pretreated with I-JNK for 1 h before treatment with N-SMase and C2-ceramide. Cells were then incubated for an additional 24 h with IGF-I. Consistent with previous results and those in Fig. 6Go, IGF-I induced the expression of myogenin (P < 0.01; Fig. 7Go), and this induction was prevented by both N-SMase (n = 3, P < 0.01; Fig. 7AGo) and C2-ceramide (n = 3, P < 0.01; Fig. 7BGo). However, when myoblasts were pretreated with an inhibitor of JNK, the ability of N-SMase and C2-ceramide to inhibit myogenin expression was abolished (P < 0.01; Fig. 7Go). These data demonstrated that JNK is a required component by which TNF{alpha} and its downstream messengers N-SMase and ceramide inhibit IGF-I-induced myogenesis.


Figure 7
View larger version (16K):
[in this window]
[in a new window]
  		FIG. 7. N-SMase- and C2-ceramide-dependent inhibition of IGF-I-induced myogenin expression is mediated by JNK. C2C12 myoblasts were pretreated with I-JNK (2 µM) for 1 h before treatment with C2-ceramide (1 µM) or N-SMase (25 mU/ml) for another 1 h. These myoblasts were then incubated for an additional 24 h in the presence or absence of IGF-I (50 ng/ml). Myogenin expression was determined as in Fig. 6Go. Again, {alpha}-tubulin served as a protein loading control to ensure that equal amounts of protein were added to each lane. A, N-SMase blocks the IGF-I-induced increase in myogenin expression. JNK is required for this inhibition because an inhibitor of JNK, I-JNK, completely suppressed N-SMase-induced inhibition of myogenin expression (n = 3); B, a densitometric summary of three independent experiments confirms that like N-SMase, C2-ceramide (C2-cer) completely blocks the ability of IGF-I to induce the protein expression of myogenin. This inhibition is completely reversed by I-JNK (n = 3). Densitometric summaries for both A and B are presented as a ratio of the mass of myogenin to that of {alpha}-tubulin protein loading control. **, P < 0.01.

 
JNK is required for inhibition of IGF-I-induced myoblast differentiation by TNF{alpha}
Muscle differentiation is induced by sequential expression of key muscle transcription factors, including myogenin and MyoD, that belong to the family of myogenic regulatory factors (38, 39). As differentiation proceeds, myoblasts develop into myotubes and initiate expression of contractile proteins such as troponin, actin, tropomyosin, and MHC. IGF-I is a potent promoter of differentiation, as indicated by its ability to induce not only myogenin and MyoD but also the expression of myogenic differentiation markers such as MHC. Therefore, we hypothesized that TNF{alpha} antagonizes the ability of IGF-I to promote myogenesis by activating JNK. To test this possibility, C2C12 myoblasts were pretreated with I-JNK for 1 h before treatment with TNF{alpha} and IGF-I for an additional 24 h. A representative gel and densitometric summary are shown (n = 4; Fig. 8Go, A and B, respectively). As hypothesized, IGF-I induced expression of MHC, and this increase was blocked by TNF{alpha} (P < 0.01; Fig. 8Go, A and B). More importantly, I-JNK suppressed the ability of TNF{alpha} to block the IGF-I-induced increase in MHC (P < 0.01; Fig. 8Go), indicating that JNK is required for inhibition of MHC expression. These results confirm the myogenin data from Figs. 6Go and 7Go and extend them by showing that JNK is a key regulatory factor downstream of the cytokine receptors that is required for inhibition of muscle differentiation induced by IGF-I. Consequently, it is likely that JNK regulates the expression of other myogenic factors, such as MyoD, that are also controlled by proinflammatory cytokines and ceramide (18).


Figure 8
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 8. JNK enzymatic activity is required for cytokine-dependent suppression of IGF-I-induced differentiation. To examine the possibility that JNK is involved in cytokine-mediated inhibition of differentiation, C2C12 myoblasts were pretreated with I-JNK (2 µM) for 1 h before treatment with TNF{alpha} (1 ng/ml) and IGF-I (50 ng/ml) for an additional 24 h. Expression of a marker of differentiation, MHC, was determined by blotting membranes with an antibody specific to embryonic MHC. A and B, Representative gel (A) and a densitometric summary of independent experiments (B) show that the IGF-I-induced increase in MHC expression is completely abolished by TNF{alpha}. In the presence of I-JNK, TNF{alpha} does not inhibit IGF-I-induced MHC expression (n = 4). **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of JNK is closely associated with the pathophysiology of diabetes, Alzheimer’s disease, ischemia, and many other neurodegenerative and inflammatory disorders (40). Such profound actions of JNK can be in part attributed to its ability to mediate stress-induced resistance to several hormones, including GH (19), insulin (41), and glucocorticoids (42). These data, along with findings that macrophages from psychologically stressed mice develop glucocorticoid resistance (43, 44), indicate that JNK-induced hormone resistance may serve as a common mechanistic response to stress. Data in this report significantly extend this concept by demonstrating that the inflammatory mediator JNK is a key protein by which TNF{alpha} and C2-ceramide induce IGF-I resistance and inhibit myogenesis.

To decipher the function of JNK in multiple in vitro and in vivo systems, pharmacological inhibitors and, more recently, peptide blockers of JNK have been used extensively. Chemical inhibitors, primarily SP600125 and AS601245, competitively target the ATP binding site of JNK1, JNK2, and JNK3. However, despite their widespread use, the lack of specificity remains a significant concern with the use of these chemical inhibitors. For example, SP600125 inhibits more than 10 other kinases with equal or greater efficiency (45). Furthermore, in our hands, the concentration of SP600125 (10 µM) required to inhibit TNF{alpha}-induced phosphorylation of JNK at 10 min is toxic to the cells at the later time-points (24 h) that were used to test the role of JNK in myogenic factor expression (data not shown). Although a shorter 6-h treatment with SP600125 is less toxic and suppresses TNF{alpha} inhibition of myogenin expression, this approach greatly reduces the amount of time that myoblasts are treated with IGF-I (4 h). Consequently, the increase in IGF-I-induced myogenin expression is quite small compared with the increase observed in cells treated with IGF-I for a full 24 h (data not shown). Recently, novel membrane-permeable peptide inhibitors that interrupt all three JNK isoforms, but not other MAPKs, have been developed and successfully used in both animal and cell culture studies (7, 8, 9). Unlike chemical inhibitors that compete for the ATP site, the advantage of these peptide inhibitors is that they directly bind to JNK, forming a phenotype similar to JNK knockouts (7, 8, 9). In addition to their specificity, the D-stereoisomer of I-JNK is very stable and has been used successfully in vivo and in vitro for up to 2 wk (7). Here we used this novel peptide inhibitor in murine myoblasts to show that JNK is a key regulatory protein that mediates TNF{alpha}- and ceramide-induced IGF-I resistance (Fig. 5Go). Furthermore the concentration of I-JNK necessary to block TNF{alpha}-induced kinase activity of JNK is not toxic to the cells, even after 36 h in culture (data not shown). Consequently, these data indicate that peptide inhibitors of JNK provide important tools to decipher the role of JNK in skeletal muscle formation and wasting and possibly provide a means for development of more efficient and specific treatments for wasting conditions.

The role of JNK in regulating myogenesis is not well established. Although the majority of the data indicate that JNK is primarily involved in promoting catabolic, atrophic events in muscle cells, results of some studies suggest otherwise. Cotransfection of L6 rat skeletal myoblasts with JNK and MKK7 increases JNK kinase activity and leads to a reduction in myogenin expression in myoblasts that are allowed to undergo differentiation for 36 h (46). Conversely, two reports from another laboratory indicate that JNK activity is positively correlated with increased myogenin expression, although these effects were measured at 72 h after the initiation of differentiation and used a pharmacological blocker, SP600125, to inhibit JNK activity (47, 48). One possible explanation for these divergent results stems from the fact that cells were allowed to differentiate for different lengths of time before expression of myogenin was determined. Another possibility is that an endogenous transient elevation in JNK is required for myogenesis, whereas overexpression or induction of JNK via external stimuli such as TNF{alpha} promotes a more pronounced and inhibitory enzymatic activity of this kinase. This hypothesis is also consistent with the finding that JNK phosphorylation is transiently elevated in muscles that undergo mechanical stress (31, 49, 50), but this JNK activity is reduced before induction of hypertrophic signals such as Akt and p70S6K (51). The delay in a hypertrophic response could be attributed to the ability of JNK to inhibit Akt and p70S6K phosphorylation in mouse skeletal muscle (49). Consequently, these data are not inconsistent with our findings that show that JNK is a key regulatory factor downstream of TNF{alpha} and ceramide and is necessary for the inhibition of IGF-I-induced myogenin expression (Figs. 6Go and 7Go) as well as suppression of muscle differentiation as measured by expression of embryonic MHC (Fig. 8Go).

Receptor-associated docking molecules, such as the IRS proteins, link the tyrosine kinases of IGF-I and insulin receptors to downstream Src homology-2 domain-containing proteins and their intracellular signaling cascades. Although these docking proteins, particularly IRS-1 and IRS-2, exemplify significant functional redundancy, IRS-1 is primarily involved in regulating postnatal growth and metabolic processes in muscle (26). IRS-1 is activated by phosphorylation of tyrosine residues by receptor tyrosine kinases. Phosphorylated IRS-1 then recruits a plethora of Src homology-2 domain-containing proteins (26). Conversely, proinflammatory cytokines such as TNF{alpha} inhibit tyrosine phosphorylation of IRS-1 and suppress its ability to transduce receptor-induced mitogenic signals (26). One mechanism by which TNF{alpha} inhibits IRS-1 function is by promoting the association of the stress protein kinase JNK with the corresponding protein tyrosine binding (PTB) domains on IRS-1. JNK binding induces phosphorylation of Ser307 (Ser307 in mice and Ser312 in humans) (Fig. 2BGo), a residue located on the C terminus of the IRS-1 PTB domain (29). Phosphorylation of this serine residue disrupts the PTB domain and leads to dissociation of IRS-1 from the receptor and inhibition of the tyrosine phosphorylation of IRS-1 (52), thereby causing a rift in the link between the receptor and downstream signaling cascades. This mechanism of action is thought to contribute to the insulin resistance that is observed in diabetic states and obesity, although it is not yet clear whether the same mechanism induces IGF-I resistance in muscle cells.

The clinical relevance of IRS-1 inhibition is well recognized in diabetes but is much more speculative in muscle-wasting disorders. Perhaps the most important clues to determine IRS-1 function come from animal models engineered to lack these docking proteins. IRS-1-deficient mice are resistant to both IGF-I and insulin (26, 53). Consequently, these mice are 40% smaller and display impaired glucose uptake induced by insulin and IGF-I (26, 53, 54, 55). This is not surprising considering that IGF-I and GH are responsible for over 80% of postnatal growth (56), whereas insulin maintains glucose metabolic balance. Because IGF-I directly stimulates skeletal muscle growth and regeneration, inhibition of IGF-IR signaling by targeting key intermediates such as the IRS-1 docking proteins can significantly interrupt the anabolic actions of IGF-I in myogenesis. Data from the experiments reported here support this general hypothesis. Other results in both muscle (17, 37) and human breast cancer cells (22) have demonstrated that proinflammatory cytokines, including TNF{alpha}, do not alter tyrosine kinase activity of the IGF-IR. Instead, TNF{alpha} potently suppresses the ability of IGF-I to induce tyrosine phosphorylation of IRS-1 (17, 18, 37). We extended these results to show that ceramide acts like TNF{alpha} to induce IGF-I resistance by inhibiting tyrosine phosphorylation of IRS-1 (Fig. 1Go). Furthermore, we now show that TNF{alpha}, but not IGF-I, promotes JNK association with IRS-1 (Fig. 2AGo) and that inhibition of JNK with a specific cell-permeable peptide inhibitor completely suppresses (Fig. 2CGo) the ability of TNF{alpha} to inhibit IGF-I-induced tyrosine phosphorylation of IRS-1. Given the critical role of IRS-1 in IGF-IR signaling, it is likely that this inhibition of IRS-1 tyrosine phosphorylation is involved in the ability of TNF{alpha} to inhibit myogenesis.

Cachexia is a complex wasting disorder that results in a striking loss of fat and lean body mass (13, 57) and directly contributes to almost a third of cancer deaths that are not related to tumor burden itself (13). Yet, no viable therapy to prevent and reverse muscle loss has been developed. The most obvious reason is that the specific intracellular targets that promote wasting have not yet been identified. Although proinflammatory cytokines are closely implicated in the loss of muscle mass (13), it is often difficult to determine which cytokine is involved in eliciting wasting because of their overlapping and often synergistic functions. Data in this report, along with promising results from combination therapy (58), reinforce the idea that identification of a common key downstream mediator of several cytokines is an important step toward development of more effective treatments. The inhibition of JNK therefore shows promise for treatment for several disorders, including type II diabetes, Alzheimer’s disease, and stroke. Based upon data in this report, JNK inhibitors may also be useful for ameliorating IGF-I resistance and muscle loss in wasting disorders.


   Footnotes
 
This work was supported by National Institutes of Health to K.W.K. (AI50442 and MH51569), R.W.J. (AG16710 and AG023580), G.G.F. (DK064862), and R.D. (MH071349), and USDA to R.H.M. (AG2004-35206-14144).

Disclosure statement: The authors have read the Disclosure of Potential Conflicts of Interest and have nothing to declare.

First Published Online June 15, 2006

Abbreviations: A-SMase, Acid-sphingomyelinase; C2-ceramide, N-acetyl-D-sphingosine; C-JNK, inactive control for I-JNK; IGF-IR, IGF-I receptor; I-JNK, peptide inhibitor of JNK; IRS-1, insulin receptor substrate 1; JBD, JNK binding domain; JIP, JNK interacting protein; JNK, c-jun N-terminal kinase; MHC, myosin heavy chain; MKK, MAPK-activated kinase kinases; N-SMase, neutral-sphingomyelinase; PTB, protein tyrosine binding; SAPK, stress-activated protein kinase; SMase, sphingomyelinase; TBS-T, Tris-buffered saline with Tween 20.

Received December 5, 2005.

Accepted for publication June 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Roux PP, Blenis J 2004 ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68:320–344[Abstract/Free Full Text]
  2. Kyriakis JM, Avruch J 2001 Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807–869[Abstract/Free Full Text]
  3. Huwiler A, Xin C, Brust AK, Briner VA, Pfeilschifter J 2004 Differential binding of ceramide to MEKK1 in glomerular endothelial and mesangial cells. Biochim Biophys Acta 1636:159–168
  4. Kaneto H, Nakatani Y, Miyatsuka T, Kawamori D, Matsuoka TA, Matsuhisa M, Kajimoto Y, Ichijo H, Yamasaki Y, Hori M 2004 Possible novel therapy for diabetes with cell-permeable JNK-inhibitory peptide. Nat Med 10:1128–1132[CrossRef][Medline]
  5. Minogue AM, Schmid AW, Fogarty MP, Moore AC, Campbell VA, Herron CE, Lynch MA 2003 Activation of the c-Jun N-terminal kinase signaling cascade mediates the effect of amyloid-ß on long term potentiation and cell death in hippocampus: a role for interleukin-1ß? J Biol Chem 278:27971–27980[Abstract/Free Full Text]
  6. Borsello T, Clarke PG, Hirt L, Vercelli A, Repici M, Schorderet DF, Bogousslavsky J, Bonny C 2003 A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med 9:1180–1186[CrossRef][Medline]
  7. Bonny C, Oberson A, Negri S, Sauser C, Schorderet DF 2001 Cell-permeable peptide inhibitors of JNK: novel blockers of ß-cell death. Diabetes 50:77–82[Abstract/Free Full Text]
  8. Bogoyevitch MA 2005 Therapeutic promise of JNK ATP-noncompetitive inhibitors. Trends Mol Med 11:232–239[CrossRef][Medline]
  9. Bogoyevitch MA, Boehm I, Oakley A, Ketterman AJ, Barr RK 2004 Targeting the JNK MAPK cascade for inhibition: basic science and therapeutic potential. Biochim Biophys Acta 1697:89–101[Medline]
  10. Lamberts SW, van den Beld AW, van der Lely AJ 1997 The endocrinology of aging. Science 278:419–424[Abstract/Free Full Text]
  11. Grounds MD 2002 Reasons for the degeneration of ageing skeletal muscle: a central role for IGF-1 signalling. Biogerontology 3:19–24[CrossRef][Medline]
  12. Roubenoff R, Parise H, Payette HA, Abad LW, D’Agostino R, Jacques PF, Wilson PW, Dinarello CA, Harris TB 2003 Cytokines, insulin-like growth factor 1, sarcopenia, and mortality in very old community-dwelling men and women: the Framingham Heart Study. Am J Med 115:429–435[CrossRef][Medline]
  13. Argiles JM, Busquets S, Lopez-Soriano FJ 2005 The pivotal role of cytokines in muscle wasting during cancer. Int J Biochem Cell Biol 37:1609–1619[CrossRef][Medline]
  14. Crown AL, Cottle K, Lightman SL, Falk S, Mohamed-Ali V, Armstrong L, Millar AB, Holly JM 2002 What is the role of the insulin-like growth factor system in the pathophysiology of cancer cachexia, and how is it regulated? Clin Endocrinol (Oxf) 56:723–733[CrossRef][Medline]
  15. Kelley KW 2004 From hormones to immunity: the physiology of immunology. Brain Behav Immun 18:95–113[CrossRef][Medline]
  16. Mulligan K, Grunfeld C, Hellerstein MK, Neese RA, Schambelan M 1993 Anabolic effects of recombinant human growth hormone in patients with wasting associated with human immunodeficiency virus infection. J Clin Endocrinol Metab 77:956–962[Abstract]
  17. Broussard SR, McCusker RH, Novakofski JE, Strle K, Shen WH, Johnson RW, Freund GG, Dantzer R, Kelley KW 2003 Cytokine-hormone interactions: tumor necrosis factor {alpha} impairs biologic activity and downstream activation signals of the insulin-like growth factor I receptor in myoblasts. Endocrinology 144:2988–2996[Abstract/Free Full Text]
  18. Strle K, Broussard SR, McCusker RH, Shen WH, Johnson RW, Freund GG, Dantzer R, Kelley KW 2004 Proinflammatory cytokine impairment of insulin-like growth factor I-induced protein synthesis in skeletal muscle myoblasts requires ceramide. Endocrinology 145:4592–4602[Abstract/Free Full Text]
  19. Lang CH, Hong-Brown L, Frost RA 2005 Cytokine inhibition of JAK-STAT signaling: a new mechanism of growth hormone resistance. Pediatr Nephrol 20:306–312[CrossRef][Medline]
  20. Frost RA, Lang CH 2003 Regulation of insulin-like growth factor-I in skeletal muscle and muscle cells. Minerva Endocrinol 28:53–73[Medline]
  21. McNurlan MA, Garlick PJ, Steigbigel RT, DeCristofaro KA, Frost RA, Lang CH, Johnson RW, Santasier AM, Cabahug CJ, Fuhrer J, Gelato MC 1997 Responsiveness of muscle protein synthesis to growth hormone administration in HIV-infected individuals declines with severity of disease. J Clin Invest 100:2125–2132[Medline]
  22. Shen WH, Zhou JH, Broussard SR, Freund GG, Dantzer R, Kelley KW 2002 Proinflammatory cytokines block growth of breast cancer cells by impairing signals from a growth factor receptor. Cancer Res 62:4746–4756[Abstract/Free Full Text]
  23. SAS 2003 User’s guide, version 8.2. Cary, NC: SAS Institute
  24. Hotamisligil GS 2003 Inflammatory pathways and insulin action. Int J Obes Relat Metab Disord 27(Suppl 3):S53–S55
  25. Bennett BL, Satoh Y, Lewis AJ 2003 JNK: a new therapeutic target for diabetes. Curr Opin Pharmacol 3:420–425[CrossRef][Medline]
  26. White MF 2002 IRS proteins and the common path to diabetes. Am J Physiol Endocrinol Metab 283:E413–E422
  27. Hilder TL, Tou JC, Grindeland RE, Wade CE, Graves LM 2003 Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle. FEBS Lett 553:63–67[CrossRef][Medline]
  28. Prada PO, Zecchin HG, Gasparetti AL, Torsoni MA, Ueno M, Hirata AE, Corezola do Amaral ME, Hoer NF, Boschero AC, Saad MJ 2005 Western diet modulates insulin signaling, c-Jun N-terminal kinase activity, and insulin receptor substrate-1ser307 phosphorylation in a tissue-specific fashion. Endocrinology 146:1576–1587[Abstract/Free Full Text]
  29. Aguirre V, Uchida T, Yenush L, Davis R, White MF 2000 The c-Jun NH2-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser307. J Biol Chem 275:9047–9054[Abstract/Free Full Text]
  30. Kumar N, Dey CS 2004 Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones. Mol Cell Biochem 260:55–64[CrossRef][Medline]
  31. Leng Y, Steiler TL, Zierath JR 2004 Effects of insulin, contraction, and phorbol esters on mitogen-activated protein kinase signaling in skeletal muscle from lean and ob/ob mice. Diabetes 53:1436–1444[Abstract/Free Full Text]
  32. Xu J, Yeh CH, Chen S, He L, Sensi SL, Canzoniero LM, Choi DW, Hsu CY 1998 Involvement of de novo ceramide biosynthesis in tumor necrosis factor-{alpha}/cycloheximide-induced cerebral endothelial cell death. J Biol Chem 273:16521–16526[Abstract/Free Full Text]
  33. Merrill Jr AH, Jones DD 1990 An update of the enzymology and regulation of sphingomyelin metabolism. Biochim Biophys Acta 1044:1–12[Medline]
  34. Adam-Klages S, Adam D, Wiegmann K, Struve S, Kolanus W, Schneider-Mergener J, Kronke M 1996 FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 86:937–947[CrossRef][Medline]
  35. Wiegmann K, Schutze S, Machleidt T, Witte D, Kronke M 1994 Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005–1015[CrossRef][Medline]
  36. Banks WA, Robinson SM, Nath A 2005 Permeability of the blood-brain barrier to HIV-1 Tat. Exp Neurol 193:218–227[CrossRef][Medline]
  37. Broussard SR, McCusker RH, Novakofski JE, Strle K, Shen WH, Johnson RW, Dantzer R, Kelley KW 2004 IL-1ß impairs insulin-like growth factor I-induced differentiation and downstream activation signals of the insulin-like growth factor I receptor in myoblasts. J Immunol 172:7713–7720[Abstract/Free Full Text]
  38. Buckingham M 2001 Skeletal muscle formation in vertebrates. Curr Opin Genet Dev 11:440–448[CrossRef][Medline]
  39. Sabourin LA, Rudnicki MA 2000 The molecular regulation of myogenesis. Clin Genet 57:16–25[CrossRef][Medline]
  40. Manning AM, Davis RJ 2003 Targeting JNK for therapeutic benefit: from junk to gold? Nat Rev Drug Discov 2:554–565[CrossRef][Medline]
  41. Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS 2002 A central role for JNK in obesity and insulin resistance. Nature 420:333–336[CrossRef][Medline]
  42. Wang X, Wu H, Lakdawala VS, Hu F, Hanson ND, Miller AH 2005 Inhibition of Jun N-terminal kinase (JNK) enhances glucocorticoid receptor-mediated function in mouse hippocampal HT22 cells. Neuropsychopharmacology 30:242–249[CrossRef][Medline]
  43. Stark JL, Avitsur R, Padgett DA, Campbell KA, Beck FM, Sheridan JF 2001 Social stress induces glucocorticoid resistance in macrophages. Am J Physiol Regul Integr Comp Physiol 280:R1799–R1805
  44. Avitsur R, Stark JL, Dhabhar FS,