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Endocrinology Vol. 144, No. 7 2988-2996
Copyright © 2003 by The Endocrine Society

Cytokine-Hormone Interactions: Tumor Necrosis Factor {alpha} Impairs Biologic Activity and Downstream Activation Signals of the Insulin-Like Growth Factor I Receptor in Myoblasts

Suzanne R. Broussard, Robert H. MCCusker, Jan E. Novakofski, Klemen Strle, Wen Hong Shen, Rodney W. Johnson, Gregory G. Freund, Robert Dantzer and Keith W. Kelley

Laboratories of Immunophysiology (S.R.B., R.H.M., K.S., W.H.S., K.W.K.), Developmental Endocrinology (J.E.N.) and Integrative Biology (R.W.J.), Departments of Animal Sciences and Pathology and College of Medicine (G.G.F.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Institut National de la Recherche Agronomique-Institut National de la Santé et de la Recherche Médicale, Unité 394, Unité de Recherches de Neurobiologie Integrative (R.D.), 33077 Bordeaux, France

Address all correspondence and requests for reprints to: Suzanne Broussard, University of Illinois, Laboratory of Immunophysiology, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, Illinois 61801. E-mail: broussar{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF{alpha} is elevated following damage to skeletal muscle. Here we provide evidence that TNF{alpha} acts on muscle cells to induce a state of IGF-I receptor resistance. We establish that TNF{alpha} inhibits IGF-I-stimulated protein synthesis in primary porcine myoblasts. Similar results were observed in C2C12 murine myoblasts, where as little as 0.01 ng/ml TNF{alpha} significantly inhibits protein synthesis induced by IGF-I. TNF{alpha} also impairs the ability of IGF-I to induce expression of a key myogenic transcription factor, myogenin. The inhibition by TNF{alpha} of IGF-I-induced protein synthesis and expression of myogenin is not due to direct killing of myoblasts by TNF{alpha}. Although IGF-I induces an approximately 19-fold induction in tyrosine phosphorylation of the ß-chains of its receptor, TNF{alpha} does not inhibit this autophosphorylation. Instead, TNF{alpha} significantly reduces by approximately 50% IGF-I-stimulated tyrosine phosphorylation of two of the major downstream receptor docking molecules, insulin receptor substrate (IRS)-1 and IRS-2. These results establish that low picogram concentrations of TNF{alpha} acts on both porcine and murine myoblasts to impair tyrosine phosphorylation of both IRS-1 and IRS-2, but not the receptor itself. These data are consistent with the notion that very low physiological concentrations of TNF{alpha} interfere with both protein synthesis and muscle cell development by inducing a state of IGF-I receptor resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOKINES FROM THE immune system are now known to control many aspects of endocrine function (1). Hormones such as GH (2) and neurotransmitters like norepinephrine (3) regulate the activity of leukocytes. The possibility has only recently been considered, however, that hormones and cytokines might regulate the function of each other by using common intracellular substrates. For example, there is now accumulating evidence that receptors for some cytokines, such as IL-2, IL-4, IL-9, and interferon-{gamma}, use intracellular docking molecules that were first identified for the insulin receptor (4). Conversely, prolactin was first shown to activate mammary gland factor [now known as signal transducers and activators of transcription (Stat)-5] (5), and Janus kinase (JAK)-Stat family members are now well accepted to be activated by cytokines such as IL-6 (6) as well as other hormones like GH (7). Simultaneous stimulation of both hormone and cytokine receptors can antagonize each other’s functions, as evidenced by the recent finding that TNF{alpha} impairs activation of signaling elements of the insulin receptor (8). The emerging idea is that cytokines from the immune system use intracellular substrates that are also regulated by hormone receptors, and this regulation is likely to control cytokine and hormone specificity and redundancy. This concept is consistent with the original ideas of Blalock (9), in which both hormones and cytokines serve important messenger roles in immune-endocrine communication.

Growth and regeneration of skeletal muscle requires fusion of progenitor mononucleated myoblasts into multinucleated terminally differentiated myofibers (reviewed in Ref. 10). Myoblasts fuse with myofibers, leading to incorporation of myoblast nuclei into myofibers. This fusion process is essential for maintaining a constant DNA to protein ratio and for increasing expression of proteins to promote skeletal muscle hypertrophy (reviewed in Ref. 11). The proinflammatory cytokine TNF{alpha} increases significantly during inflammatory muscle myopathies (12, 13) and in both AIDS (14) and cancer (15) cachexia. One approach that has been employed to increase muscle mass in AIDS patients (14) and in the frail elderly (16) is a regimen of GH therapy to increase plasma IGF-I concentrations. IGF-I has long been recognized to control muscle mass by enhancing myofiber hypertrophy and by stimulating myogenesis (17). However, AIDS patients with elevated levels of proinflammatory cytokines do not increase muscle mass to the same degree as their healthy controls receiving GH (14). Similarly, a rise in plasma concentrations of proinflammatory cytokines in the aged is negatively related to both muscle mass and strength in elderly individuals (16). These studies suggest that proinflammatory cytokines somehow interfere with the IGF-I promotion of muscle growth.

It is becoming increasingly evident that some cytokines can interfere with the actions of hormones (reviewed in Ref. 18). We have reported that proinflammatory cytokines reduce the ability of IGF-I to promote survival of primary cerebellar granule neurons (19) and to induce proliferation of epithelial cells (20). Here we sought to extend this concept to skeletal muscle myoblasts by exploring the possibility that the proinflammatory cytokine, TNF{alpha}, acts at the post-receptor level to impair the ability of IGF-I to promote the synthesis of protein. We confirm earlier findings with human myoblasts to show that picogram concentrations of TNF{alpha} impair IGF-I-induced protein synthesis in both primary porcine and murine C2C12 myoblasts. Surprisingly, tyrosine autophosphorylation of the ß-chains of the IGF-I receptor is not affected by TNF{alpha}. Instead, TNF{alpha} inhibits the ability of IGF-I to tyrosine phosphorylate its two major docking proteins in myoblasts, insulin receptor substrate-1 (IRS-1) and IRS-2. This inhibition by TNF{alpha} leads to diminished expression of the key myogenic transcription factor myogenin. These data establish that stimulation of TNF receptors by low physiological concentrations of TNF{alpha} impairs activity of the IGF-I receptor, thereby inhibiting synthesis of protein in skeletal muscle myoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of primary myoblasts
Primary porcine skeletal muscle myoblasts were isolated from 1-d-old piglets. Crossbred piglets were obtained from the Swine Research Center at the University of Illinois at Urbana. One-day-old piglets were euthanized immediately before removal of the longissimus muscle. The University of Illinois Institutional Laboratory Animal Care Advisory Committee approved all procedures involving piglets. A single cell suspension was generated by mincing the longissimus muscle and washing it twice with Hanks’ balanced salt solution to remove red blood cells. The muscle was then digested for 2 h with 1 mg/ml collagenase, 0.2 mg/ml elastase, and 0.5 mg/ml hyaluronidase. Cells were cultured in DMEM supplemented with 4.5 g/liter glucose, 3.7 g/liter sodium bicarbonate, 100 U/ml of penicillin G, and 100 µg/ml of streptomycin (all from Sigma, St. Louis, MO). The DMEM was supplemented with 20% low endotoxin fetal bovine serum (FBS, HyClone Laboratories, Inc., Logan, UT). Primary skeletal muscle myoblasts were enriched by preincubating cells in Petri dishes (3 h) to remove adherent fibroblasts and transferring the myoblasts to gelatin-coated 24-well tissue culture plates. After 5–7 d in culture, the cells were more than 70% confluent and consisted of more than 90% myoblasts as determined with a myoblast-specific monoclonal antibody (5–1H11; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and analysis with a Mo Flo Cytometer (Cytomation, Fort Collins, CO) (21).

Culture of myoblasts
C2C12 murine myoblast cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). C2C12 myoblasts were cultured in DMEM supplemented with 10% FBS (HyClone Laboratories, Inc.). The FBS culture medium contained nondetectable concentrations of endotoxin, as determined by the Limulus amebocyte lysate assay (22). Cells were incubated at 37 C in a modified atmosphere of 7% CO2.

De novo protein synthesis
A constant number of myoblasts (5 x 105 cells) was seeded into each well of a 24-well polystyrene plates (Costar, Corning, NY) and grown to 70–80% confluence. Cells were then washed three times in DMEM to remove growth factors and then cultured in serum-free DMEM for 5 h. Fresh serum-free medium (0.5 ml) was added, and then cells were pretreated for 1 h with specified concentrations of recombinant TNF{alpha}. Primary porcine myoblasts were treated with human recombinant TNF{alpha} (0.1, 1, or 10 ng/ml) and murine C2C12 myoblasts were treated with murine recombinant TNF{alpha} (0.01, 0.1, 1 or 10 ng/ml) purchased from the Intergen Co. (Purchase, NY). Myoblasts were then treated with IGF-I (50 ng/ml) and pulsed with 1.5 µCi of 3H-phenylalanine (L-[2,3,4,5,6-3H]phenylalanine; Amersham Pharmacia Biotech, Piscataway, NJ) for an additional 5 h. Phenylalanine incorporation was determined by harvesting both adherent and nonadherent cells with a PHD cell harvester (Cambridge Technology, Inc., Cambridge, MA), collecting protein on glass fiber filters (Whatman International Ltd., Maidstone, UK) and washing four times to remove unincorporated 3H-phenylalanine. Incorporation of 3H-phenylalanine into protein was measured using a LS 6000IC Beckman Scintillation Counter (Beckman, Fullerton, CA; LS6000IC). The amount of 3H-phenylalanine incorporation is presented as protein synthesis.

Live and apoptotic cell populations
Myoblasts (70–80% confluent) were treated with TNF{alpha} and IGF-I as described for the de novo protein synthesis experiments. C2C12 myoblasts were washed three times and cultured in DMEM for 2 h. Fresh medium was added, and cells were then pretreated with or without TNF{alpha} (0.1, 1, or 10 ng/ml) for 1 h and subsequently incubated an additional 5 h in the presence or absence of IGF-I. The entire myoblast population was analyzed by pooling cells in the culture supernatant with adherent cells collected by trypsinization (0.25%). Hoechst 33342 (7 ng/ml, Sigma) was added for 2 min at room temperature and the myoblasts were placed on ice before addition of propidium iodide (2 µg/ml), as we have previously described (22). A Cytomation MoFlo flow cytometer was used to analyze 5 x 104 cells, which distinguished dying cells with permeable surface membranes (propidium iodide positive) from the apoptotic (Hoechst positive propidium iodide, negative) population.

Immunoprecipitation
C2C12 myoblasts were washed three times in DMEM and cultured without FBS for 5 h. The cells were then pretreated either with or without recombinant TNF{alpha} (0.1, 1, or 10 ng/ml) for 1 h and stimulated with 50 ng/ml IGF-I for 3 min. Following treatment, cells were lysed in ice-cold buffer (1% Nonidet P-40; 100 mM NaCl; 25 mM benzamidine; 1 mM phenylmethylsulfonyl fluoride; 2 µg/ml aprotinin; 40 nM leupeptin; 2 µg/ml pepstatin; 50 mM NaF; 1 mM dithiothreitol; 2 mM sodium orthovanadate; and 50 mM Tris, pH 7.4). Insoluble cellular debris was removed by centrifugation at 16,000 x g for 10 min. The resultant supernatant was incubated with an excess of anti-IGF-I receptor ß-chain, anti-IRS-1, or anti-IRS-2 antibodies (1 µg; all purchased from Upstate Biotechnology, Inc., Lake Placid, NY) and 25 µl of protein G Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) by rotating at 4 C overnight, as previously described (19). Nonspecific proteins were removed by washing the immunocomplexes three times in cold lysis buffer. The samples were then heated in Laemmli buffer at 100 C for 7 min, followed by separation of proteins in 7% SDS-PAGE gels. The proteins were then immobilized on PVDF (polyvinylidene difluoride; Bio-Rad Laboratories, Inc., Hercules, CA) membranes and blotted with the appropriate antibody. Tyrosine phosphorylation was detected using a phosphotyrosine antibody (PY20, Transduction Laboratories, Inc., Lexington, KY). Immunoblots were then stripped by heating in the presence of 0.1 M ß-mercaptoethanol, as described by the manufacturer (Bio-Rad Laboratories, Inc.). The mass of protein in each lane was then determined by blotting with either IGF-I receptor, IRS-1 or IRS-2 antibodies. The membranes were subsequently incubated with a secondary sheep antimouse or donkey antirabbit IgG horseradish peroxidase-linked antibodies (both from Pharmacia Biotech), developed with enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL) and exposed to autoradiographic film (Eastman Kodak Co., Rochester, NY). Autoradiograms were quantified using a Duoscan T1200 densitometer equipped with AGFA Fotolook 3.00 software (NucleoTech, San Mateo, CA), as we have previously described (23). These results were then expressed as a ratio of the densitometric density of the mass of tyrosine phosphorylated protein divided by the mass of total protein in that lane, and this ratio was termed "Fold Induction."

Myogenin expression
Myoblasts that were approximately 70–80% confluent were washed three times in DMEM and then treated with or without 0.01, 0.1, or 1 ng/ml TNF{alpha} for 1 h. The C2C12 myoblasts were cultured either in the presence or absence of IGF-I for 24–30 h. Adherent cells were lysed in RIPA buffer (1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 1x PBS, 2 µg/ml aprotinin, 40 nM leupeptin, and 2 µg/ml pepstatin) and centrifuged at 16,000 x g at 4 C for 15 min. Total protein was measured using the Bio-Rad Laboratories, Inc. DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Seventy-five micrograms of protein were loaded onto 12% SDS-PAGE gels. Following resolution of proteins by size, they were transferred to Trans-Blot PVDF membranes (Bio-Rad Laboratories, Inc.). Myogenin was measured using a M-225 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein loading was determined by subsequent blotting with the monoclonal anti-{alpha}-tubulin clone B-5-1-2 antibody (Sigma). Myogenin expression is presented as the ratio of the densitometric value of myogenin relative to the {alpha}-tubulin loading control.

Statistical analysis
All experiments were independently replicated at least three times, and data were summarized as mean ± SEM. Densitometric intensities of autoradiograms were standardized by dividing intensity of the band of interest by that of its loading control. These data, as well as those for phenylalanine incorporation, were standardized and presented as "Fold Induction." All data were analyzed as a randomized, complete block design using standard ANOVA procedures with the Statistical Analysis System for Windows (24). Treatment differences were detected using Duncan’s new multiple range test (24). Two-sided P values of P < 0.05 (*) or P < 0.01 (**) were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proinflammatory cytokines inhibit IGF-I-induced de novo protein synthesis
IGF-I is a well-recognized stimulator of protein synthesis in skeletal muscle cells. High concentrations (~100 ng/ml) of TNF{alpha} inhibit protein synthesis in human myoblasts (25), but the mechanisms that are responsible for this inhibition are unknown. To begin to elucidate the cellular events responsible for inhibition of IGF-I actions by proinflammatory cytokines in myoblasts, we first established a model using protein synthesis as a biological marker. Primary porcine myoblasts were enriched to greater than 90%, as determined by the 5–1H11 myoblast-specific monoclonal antibody (21). All growth factors were then removed from myoblasts by incubating cells for 5 h in DMEM. Primary porcine myoblasts were subsequently pretreated with or without TNF{alpha} for 1 h and then pulsed with 3H-phenylalanine in the presence or absence of IGF-I for an additional 5 h. IGF-I increased (P < 0.01) de novo protein synthesis in primary porcine myoblasts by 1.5 ± 0.1-fold (Fig. 1AGo). TNF{alpha} decreased IGF-I-stimulated de novo protein synthesis in a dose-dependent manner. At a concentration as low as 0.1 ng/ml, TNF{alpha} inhibited (P < 0.05) IGF-I-stimulated protein synthesis by 42%. At a concentration of 1 ng/ml, TNF{alpha} completely inhibited de novo IGF-I-induced protein synthesis. The highest concentration of TNF{alpha} (10 ng/ml) did not further inhibit the ability of IGF-I to promote protein synthesis. No reduction in endogenous protein synthesis was detected in primary porcine myoblasts treated with TNF{alpha} in the absence of IGF-I.



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  		Figure 1. TNF{alpha} inhibits IGF-I stimulation of phenylalanine incorporation in primary porcine (A) and murine (B) myoblasts. Cells were treated with increasing concentrations of cytokine for 1 h and then pulsed with 3H-phenylalanine in the presence or absence of 50 ng/ml IGF-I for 5 h. Protein synthesis is presented as the amount of 3H-phenylalanine incorporation relative to the experimental average. The increase in protein synthesis stimulated by IGF-I was significantly inhibited by TNF{alpha} in a dose-dependent manner. A, IGF-I stimulated (P < 0.01) de novo protein synthesis by 1.5-fold in primary porcine myoblasts. As little as 0.01 ng/ml TNF{alpha} significantly inhibited IGF-I-induced protein synthesis (*, P < 0.05; n = 3). Increasing the dose of TNF{alpha} to 1 or 10 ng/ml resulted in complete inhibition (**, P < 0.01) of protein synthesis stimulated by IGF-I. B, TNF{alpha} concentration of 0.01 ng/ml significantly reduced IGF-I-induced protein synthesis in C2C12 myoblasts (*, P < 0.05) and, identical to the porcine myoblasts, 1 and 10 ng/ml TNF{alpha} completely blocked (**, P < 0.01) the induction by IGF-I. Only the highest concentration of TNF{alpha} (10 ng/ml) significantly ({delta}; P < 0.05; n = 5) reduced endogenous protein synthesis in C2C12 myoblasts.

 
Because the inhibitory effect of TNF{alpha} could be detected only in the presence of IGF-I, we next explored the concept of receptor cross talk in murine C2C12 myoblasts. These cells responded in a fashion similar to porcine myoblasts when treated with the combination of cytokine and growth factor. IGF-I induced a 1.6 ± 0.1-fold increase (Fig. 1BGo; P < 0.01) in protein synthesis in 70–80% confluent cultures of C2C12 myoblasts. A significant reduction in IGF-I-stimulated protein synthesis was detected at the lowest concentration of TNF{alpha} (0.01 ng/ml) that was tested. Similar to primary porcine myoblasts, 0.1 ng/ml TNF{alpha} reduced (69%; P < 0.01) de novo protein synthesis. At a concentration of 1 ng/ml, TNF{alpha} acted as in primary porcine myoblasts by completely blocking (P < 0.01) IGF-I-stimulated protein synthesis. In the absence of IGF-I, neither 0.1 nor 1 ng/ml TNF{alpha} affected de novo protein synthesis. A small but significant reduction (-22%, P < 0.05) in protein synthesis occurred in C2C12 myoblasts treated with 10 ng/ml TNF{alpha} alone. This small effect is likely due to inhibition of endogenous IGF-II-induced protein synthesis by myoblasts (26). These data establish that TNF{alpha} significantly impairs de novo protein synthesis in both primary porcine myoblasts and in C2C12 cells at concentrations as low as 0.1 ng/ml. In the absence of IGF-I, low concentrations of TNF{alpha} do not affect protein synthesis.

TNF{alpha} does not induce cell death in myoblasts
One possible explanation for the ability of TNF{alpha} to inhibit IGF-I-stimulated protein synthesis is that TNF{alpha} simply kills myoblasts. Indeed, there are conflicting reports as to whether TNF{alpha} induces apoptosis in myoblasts (27, 28) or has no affect on cell survival (29). We first measured cytotoxicity by determining the proportion of live cells with intact surface membranes as assessed by impermeability to propidium iodide. Nearly all (95 ± 0.4%) C2C12 myoblasts cultured in DMEM for 6 h excluded propidium iodide (Fig. 2AGo). IGF-I slightly improved survival of myoblasts to 98 ± 0.4%, and this increase was significant (P < 0.01). TNF{alpha} alone was not cytotoxic to myoblasts, even at the highest concentration of 10 ng/ml. Furthermore, TNF{alpha} did not impair IGF-I from promoting survival of murine myoblasts.



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  		Figure 2. Inhibition of IGF-I-stimulated protein synthesis by TNF{alpha} is not due to cell death. A, Identical to the protein synthesis experiments, C2C12 myoblasts were pretreated with TNF{alpha} for 1 h followed by treatment with or without IGF-I for 5 h. Cells were stained with propidium iodide and Hoechst 33342 before analysis by flow cytometry. Propidium iodide-negative cells were considered to be alive. TNF{alpha} alone did not increase cell death compared with myoblasts cultured in DMEM (P > 0.10). IGF-I significantly (**, P < 0.01; n = 3) protected myoblasts from cell death in either the absence or presence of TNF{alpha}. B, Apoptotic cells were detected as those negative for propidium iodide and positive for Hoechst 33342. TNF{alpha} did not increase (P > 0.10) the proportion of apoptotic cells, nor did it interfere with the ability of IGF-I to save myoblasts (P > 0.10; n = 3).

 
As expected, a greater proportion of cells could be detected as dying when Hoechst 33342 was used to measure the apo-ptotic population. However, it still was not possible to detect cell death caused by any concentration of TNF{alpha}. In the absence of exogenous growth factors, only 11 ± 1% of murine myoblasts underwent early apoptotic cell death (Fig. 2BGo). IGF-I significantly (P < 0.01) reduced this apoptotic population of myoblasts to 2 ± 1%. Addition of TNF{alpha}, in either the presence or absence of IGF-I, did not affect the proportion of apoptotic myoblasts. These data show that TNF{alpha} alone does not cause apoptotic death in murine C2C12 cells, which establishes that TNF{alpha}-induced cellular death is not the mechanism by which TNF{alpha} inhibits IGF-I-stimulated protein synthesis. We next explored the possibility that TNF{alpha} acts by targeting intracellular pathways that are activated by the IGF-I receptor.

Autophosphorylation of the IGF-I receptor is not affected by TNF{alpha}
The IGF-I ligand imparts biological functions by binding and activating the type I IGF receptor (30). After ruling out the possibility that TNF{alpha} inhibited protein synthesis induced by IGF-I in a cytotoxic-dependent manner, we next examined whether TNF{alpha} directly diminished the ability of IGF-I to autophosphorylate ß-chains of the IGF-I receptor. Peak tyrosine phosphorylation of the IGF-I receptor occurs 3 min following IGF-I stimulation and is significantly diminished by 10 min (data not shown). A 3-min stimulation with 50 ng/ml IGF-I caused a 19 ± 4-fold increase in tyrosine phosphorylation of the ß-chain of the IGF-I receptor (Fig. 3Go, A and B). Addition of TNF{alpha}, even the highest concentration of 10 ng/ml, did not affect the ability of IGF-I to cause autophosphorylation of the IGF-I receptor ß-chain. These data indicate that TNF{alpha} inhibition of IGF-I-stimulated protein synthesis occurs by a mechanism independent of autophosphorylation of the IGF-I receptor.



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  		Figure 3. TNF{alpha} does not reduce autophosphorylation of the IGF-I receptor. A, C2C12 cells were treated with TNF{alpha} for 1 h and then stimulated for 3 min with IGF-I. Cell lysates were then prepared and were immunoprecipitated with an antibody to the ß-chains of the IGF-I receptor. Following SDS-PAGE and transfer to PVDF membranes, the proteins were detected by Western blotting using antibodies to phosphotyrosine (PY) or the immunoprecipitating antibody. TNF{alpha} did not affect tyrosine phosphorylation of the IGF-I receptor, as shown in this representative Western blot. B, A densitometric summary of four independent experiments showed that IGF-I (3 min) induced a 19-fold increase (P < 0.01) in tyrosine phosphorylation of the IGF-I receptor. TNF{alpha} did not affect (P > 0.10) tyrosine phosphorylation of the IGF-I receptor in either the absence or presence of IGF-I.

 
TNF{alpha} inhibits IGF-I-stimulated tyrosine phosphorylation of IRS docking proteins
We previously established that TNF{alpha} interferes with the ability of IGF-I to promote cell survival (19) and proliferation (20) by impairing activation of the docking molecules, IRS-1 and IRS-2, that directly interact with the IGF-I receptor. As expected, IGF-I (50 ng/ml) caused a 10 ± 1 and 9 ± 2-fold increase in tyrosine phosphorylation of IRS-1 (Fig. 4Go, A and B) and IRS-2 (Fig. 4Go, C and D), respectively. Addition of TNF{alpha} at only 0.1 ng/ml significantly reduced (P < 0.01) IGF-I-stimulated tyrosine phosphorylation of IRS-1 by 61%. Higher concentrations of 1 and 10 ng/ml TNF{alpha} caused a similar inhibition of 66% and 70%, respectively. IGF-I-stimulated IRS-2 tyrosine phosphorylation declined (P < 0.05) by 40, 47, and 66%, respectively, with 0.1, 1, and 10 ng/ml TNF{alpha}. In the absence of IGF-I, TNF{alpha} had no affect on tyrosine phosphorylation or mass of either IRS-1 or IRS-2. Taken together, these data demonstrate that TNF{alpha} induces a state of IGF-I receptor resistance by directly targeting IRS docking proteins.



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  		Figure 4. TNF{alpha} inhibits the ability of IGF-I to stimulate tyrosine phosphorylation of IRS-1 and IRS-2. A, IRS-1 was immunoprecipitated from whole cell lysates of C2C12 myoblasts that were pretreated with TNF{alpha} for 1 h before a 3-min stimulation with IGF-I. Following separation on 7% SDS-PAGE gels, the proteins were transferred to PVDF membranes and blotted with antibodies specific for either phosphotyrosine or IRS-1. A representative autoradiogram showed that IGF-I induced substantial tyrosine phosphorylation of IRS-1, and this effect was reduced by pretreatment with increasing concentrations of TNF{alpha}. B, A summary of three independent experiments established that IGF-I caused a 10-fold increase (P < 0.01) in tyrosine phosphorylation of IRS-1, and as little as 0.1 ng/ml TNF{alpha} inhibited this event. C, C2C12 myoblasts were treated exactly as described in (A) and IRS-2 was precipitated from whole cell lysates. TNF{alpha} alone did not induce tyrosine phosphorylation of IRS-2, but it did inhibit the ability of IGF-I to phosphorylate IRS-2. D, A densitometric summary confirmed that in the absence of IGF-I, TNF{alpha} did not affect (P > 0.10) tyrosine phosphorylation of IRS-2. However, TNF{alpha} impaired (P < 0.01) the ability of IGF-I to cause tyrosine phosphorylation of IRS-2.

 
IGF-I-induced myogenin expression is blocked by TNF{alpha}
Myogenin is a critical transcription factor that initiates myoblast differentiation (31, 32). One of the major properties of IGF-I is to induce expression of proteins required for myogenesis (33). Therefore, we explored the possibility that the reduction of IGF-I-induced protein synthesis by TNF{alpha} would inhibit expression of this key myogenic transcription factor. Identical to the previous experiments, growth factors were removed from 70–80% confluent C2C12 myoblasts to facilitate differentiation of myoblasts into myotubes. Stimulation with IGF-I for 24–30 h induced a 2.2 ± 0.2-fold increase in the mass of myogenin, and this property of IGF-I was impaired by TNF{alpha} (Fig. 5Go, A and B). For example, IGF-I-induced expression of myogenin was significantly (P < 0.05) inhibited by 55% with as little as 0.01 ng/ml of TNF{alpha}. Pretreatment with 0.1 ng/ml TNF{alpha} suppressed the IGF-I-stimulated induction of myogenin by 76%, and a complete block occurred with 1 ng/ml TNF{alpha} (P < 0.01). Importantly, there was no reduction in basal expression of myogenin at 24–30 h by any concentration of TNF{alpha} in the absence of IGF-I. Collectively, these data provide strong evidence that TNF{alpha} inhibits myogenesis by blocking activation of docking proteins used by the IGF-I receptor. This results in reduced de novo protein synthesis, including expression of a transcription factor that is required to promote myoblast differentiation, myogenin.



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  		Figure 5. TNF{alpha} decreases IGF-I-induced expression of the myogenic transcription factor, myogenin. A, C2C12 myoblasts were pretreated with TNF{alpha} for 1 h before addition of 50 ng/ml IGF-I for 24–30 h. Whole cell lysates (75 µg) were separated on 12% SDS-PAGE gels, transferred to PVDF membranes and blotted with antibodies to both myogenin and {alpha}-tubulin. In the absence of IGF-I, there was little apparent effect of increasing concentrations of TNF{alpha} on the mass of either myogenin or {alpha}-tubulin. However, in the presence of IGF-I, the mass of myogenin doubled (P < 0.01), and this increase in IGF-I-induced expression of myogenin was inhibited in a dose-responsive manner by TNF{alpha}. B, A quantitative summary of three independent experiments established that IGF-I induced (P < 0.01) a greater than 2-fold increase in the mass of myogenin. As little as 0.01 ng/ml TNF{alpha} (*, P < 0.05) significantly reduced IGF-I stimulated expression of myogenin, and both 0.1 and 1 ng/ml TNF{alpha} completely blocked (**, P < 0.01) myogenin induction by IGF-I. TNF{alpha} alone did not affect the mass of either myogenin or {alpha}-tubulin (P > 0.10).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proinflammatory cytokines are tightly linked to muscle wasting and reduced regeneration capacity (34). Current dogma supports that TNF{alpha} acts directly on skeletal muscle cells to reduce fusion of progenitor myoblasts into multinucleated myofibers and cause atrophy of existing myofibers. Here we significantly extend this concept by establishing that very low concentrations of TNF{alpha} act by interfering with the action of a critical muscle growth factor, IGF-I. We first show that, at concentrations of TNF{alpha} at 1 ng/ml or less, its ability to inhibit de novo protein synthesis in both porcine (Fig. 1AGo) and murine (Fig. 1BGo) myoblasts can be detected only in the presence of exogenous IGF-I. This inhibitory effect of TNF{alpha} is not due to cytotoxicity (Fig. 2Go). We next considered the possibility that TNF{alpha} directly inhibits activation of the IGF-1 receptor, but we found TNF{alpha} does not impair the tyrosine kinase activity of the ß-chains of the IGF-1 receptor, as assessed by their tyrosine phosphorylation (Fig. 3Go). Instead, the IGF-I-stimulated tyrosine phosphorylation of both IRS-1 (Fig. 4Go, A and B) and IRS-2 (Fig. 4Go, C and D) is significantly blunted by TNF{alpha}, at a concentration of only 0.1 ng/ml TNF{alpha}. Furthermore, TNF{alpha} inhibits the ability of IGF-I to induce expression of a specific protein that is required for myoblast differentiation, myogenin (Fig. 5Go). These data establish that TNF{alpha} significantly reduces expression of myogenin and impairs the ability of IGF-I to activate early receptor signaling events that lead to synthesis of new protein by myoblasts.

IGF is a strong inducer of protein synthesis in myoblasts (25, 33). IGF-I-induced protein synthesis is important for controlling the expression of myogenic transcription factors required for myoblasts to differentiate (33) and to promote the maturation of myotubes (35). Conversely, the proinflammatory cytokine TNF{alpha} inhibits accumulation of myogenic transcription factors and subsequent differentiation of primary human myoblasts (35A ) and murine C2 myoblasts (36, 37, 38). What is not known is the mechanism by which TNF{alpha} impairs the expression of these transcription factors. Indeed, one of the complications with these reports of TNF{alpha} inhibiting myogenesis is the presence of growth factors due to supplementation of culture medium with either fetal bovine or horse serum (36, 37, 38). To circumvent this problem, we used cells that express receptors for both TNF{alpha} and IGF-I and cultured them in serum-free medium to explore the mechanisms by which TNF{alpha} might impair a biological activity induced by IGF-I, de novo protein synthesis. We found that the molecular mechanism is likely to be caused by TNF{alpha} inducing a state of IGF-I receptor resistance.

Physiological concentrations of TNF{alpha} significantly inhibit IGF-I-stimulated protein synthesis in both primary porcine myoblasts (0.1 ng/ml; Fig. 1AGo) as well as in the well-characterized C2C12 myoblasts (0.01 and 0.1 ng/ml; Fig. 1BGo). Importantly, in the absence of IGF-I, TNF{alpha} at 1 ng/ml or less did not reduce endogenous protein synthesis. However, higher concentrations of TNF{alpha} reduced basal protein synthesis in C2C12 myoblasts. These results are similar to those of Frost et al. (25), who demonstrated that TNF{alpha} (100 ng/ml) blocked IGF-I-stimulated protein synthesis in human myoblasts. However, this high concentration of TNF{alpha} alone also decreased basal protein synthesis to an equal extent, making it difficult to establish that proinflammatory cytokines specifically interfere with the actions of IGF-I rather than being caused by a direct effect of TNF{alpha}, such as inducing cell death.

A hallmark of TNF{alpha} is its cytotoxicity in a variety of cell types, whereas IGF-I is a well-recognized survival factor (39). A potential explanation for the TNF{alpha} inhibition of IGF-I-stimulated de novo protein synthesis was that TNF{alpha} simply killed myoblasts, resulting in a decreased number of cells available for IGF-I to induce protein synthesis. There are conflicting reports in the literature as to whether TNF{alpha} induces apoptosis in myoblasts. For example, there are several reports that TNF{alpha} promotes apoptosis in myoblasts (27, 28, 36). Experiments in all of these papers used high concentrations of TNF{alpha} (10–20 ng/ml) in cells cultured in the presence of either 2% horse serum or 0.5% fetal bovine serum (36). Lower concentrations of TNF{alpha} (1 ng/ml) did not induce apoptosis (28, 36) but significantly inhibited differentiation. In contrast, we removed fetal bovine serum before treating myoblasts with TNF{alpha}. Under these conditions, concentrations of TNF{alpha} ranging from 0.1–10 ng/ml did not affect cell survival. Therefore, induction of apoptosis by TNF{alpha} may be due to cytokine interactions with factors present in serum, similar to our results in epithelial cells (20).

Stimulation of the type I IGF-I receptor by either IGF-I or IGF-II and subsequent downstream signaling cascades stimulates fusion of myoblasts into myotubes. Introduction of a mutant type I IGF-I receptor that is unable to autophosphorylate reduces myoblast differentiation upon serum withdraw (40). Conversely, increasing expression of the type I IGF-I receptor promotes spontaneous differentiation of myoblasts (41). Therefore, we chose to examine the possibility that TNF{alpha} interferes with tyrosine autophosphorylation of the IGF-I receptor and activation of the immediate downstream docking proteins. Even the highest concentration of 10 ng/ml, TNF{alpha} did not diminish autophosphorylation of the IGF-I receptor ß-chain following ligand stimulation (Fig. 3Go). This finding agrees with earlier results from our laboratory using human epithelial cells (20). These results establish that TNF{alpha} induces resistance of the IGF-I receptor not at the level of the receptor but rather by targeting some of the earliest signaling proteins that associate with the IGF-I receptor. Importantly, this differs from the model of insulin resistance, in which TNF{alpha} impairs insulin signaling in muscle (42, 43), at least in part by reducing autophosphorylation of the insulin receptor. Consistent with this conclusion, blocking the activity of downstream signals from the IGF-I receptor by using either pharmacological inhibitors (44, 45) or dominant negative constructs (45, 46) prevents myoblast differentiation.

Activation of the IGF-I receptor results in tyrosine phosphorylation of IRS-1, IRS-2, and Src homologous and collagen (Shc) docking molecules (reviewed in Ref. 18). Tyrosine phosphorylation of IRS-1 and its subsequent activation of downstream kinases is needed for IGF-I-dependent expression of myogenin and myogenic differentiation (47). Inhibition of IGF-I signaling through the IRS proteins could have significant negative consequences for muscle growth. For example, IRS-1 is indispensable for the growth-promoting effects of IGF-I in skeletal muscle (48, 49). Mice genetically modified to delete IRS-1 are retarded in postnatal growth, with the greatest reduction in skeletal muscle (50), despite normal concentrations of plasma IGF-I and IGF-II (48). Overexpression of IGF-I slightly improved the growth in IRS-1-deficient mice, but skeletal muscle mass and postnatal growth remained greatly attenuated (50). Importantly, growth of homozygous IRS-1 knockout mice is greater than that of IGF-I receptor knockout mice, providing strong evidence for both IRS-1-dependent and -independent pathways for IGF-I signaling (48). For example, in the absence of IRS-1, IRS-2 tyrosine phosphorylation is markedly enhanced in skeletal muscle (49).

There are several potential mechanisms by which TNF{alpha} may negatively regulate IRS-1 and IRS-2. These include, but are not limited to, tyrosine phosphatase activity and site-specific serine phosphorylation (reviewed in Refs. 51 and 52). For example, protein tyrosine phosphatase 1B is well known to dephosphorylate tyrosine residues on IRS-1 (53). However, if this were the case, this tyrosine phosphatase would need to preferentially target the IRS proteins rather than the receptor. However, tyrosine phosphorylation of the IGF-I receptor was not affected by TNF{alpha} (Fig. 3Go). More recently, it has been demonstrated that TNF{alpha}, acting via c-Jun NH2-terminal kinase, stimulates serine phosphorylation of IRS-1 at Ser307 (54). This event leads to a reduction in insulin-induced tyrosine phosphorylation of IRS-1, which is likely to occur by blocking IRS-1 interaction with the insulin receptor (55). Phosphorylation of Ser312 on IRS-1 also results in a decrease in IRS-1 tyrosine phosphorylation (56). New evidence has also established that serine phosphorylation of IRS-1 by mammalian target of rapamycin (mTOR) targets IRS-1 for ubiquitination and subsequent degradation (57). However, more than 100 potential serine phosphorylation sites exist on IRS-1 and IRS-2, and extensive research is now underway to identify the consequences of phosphorylation at these serine residues.

IGF-I induces protein synthesis in myoblasts and increases expression of key myogenic transcription factors that are required for myoblast fusion (reviewed in Ref. 58). These transcription factors ultimately control expression of muscle-specific proteins, such as myosin heavy chain. The myogenic transcription factor, myogenin, is required for the early stages of myoblast differentiation (32). Myogenin knockout mice have severe muscle deficiency and experience perinatal death (31, 59, 60). Here we confirm the early finding of Florini (33) that IGF-I increases protein synthesis and expression of myogenin. Conversely, TNF{alpha} impairs myoblast differentiation (37, 38) by an unknown mechanism. Here we establish that TNF{alpha} acts by inhibiting the ability of IGF-I to increase expression of myogenin (Fig. 5Go).

During the past 10 yr, receptors for a number of protein hormones and cytokines have been shown to use common intracellular messenger pathways. The use of common messengers at least partially explains some of the redundancies between hormones and cytokines. Here we studied muscle cells that express receptors for both a cytokine (TNF{alpha}) and a growth factor (IGF-I). Surprisingly, we could not detect any effect of TNF{alpha} on the ability of myoblasts to synthesize protein in the absence of IGF-I. Yet, when the IGF-I receptor was stimulated, concentrations of TNF{alpha} as low as 0.1 ng/ml inhibited de novo protein synthesis by 50%. These data therefore begin to provide a molecular basis for the cross-talk that exists between receptors for ligands of both the immune and endocrine systems.


   Footnotes
 
This work was supported by NIH Grant AI-50442 (to K.W.K.).

Abbreviations: IRS, Insulin receptor substrate; PVDF, polyvinylidene difluoride.

Received January 17, 2003.

Accepted for publication March 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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