INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

INTERLEUKIN-6 (IL-6) is an important pleiotropic cytokine with both pro- and anti-inflammatory properties (19, 36). Increased circulating levels of IL-6 have been reported in a number of pathophysiological conditions, including injury, sepsis, cancer, and tissue ischemia-reperfusion (23, 38, 39). Although the majority of systemic IL-6 may be produced by cells in classical immune tissues, such as the liver and spleen, there is growing evidence that other tissues as well may be an important source of IL-6. In particular, recent studies suggest that skeletal muscle may be a significant site of IL-6 production in various conditions. Thus increased gene expression and production of IL-6 in skeletal muscle were reported during exercise (14, 21), muscle inflammation (2), hypoperfusion (31), and after denervation (16) or local muscle injury (1).

IL-6 produced in skeletal muscle may have both local and systemic biological effects. For example, previous studies suggest that IL-6 released from muscle during exercise can stimulate lipolysis in adipose tissue and glycogenolysis in liver (22). IL-6 is a major regulator of acute-phase protein synthesis in the liver, and although the Kupffer cells are probably the most important source of IL-6 regulating hepatocyte protein synthesis, it is possible that IL-6 from muscle may contribute to the regulation of acute-phase protein production during inflammation. Locally, IL-6 has trophic effects and may participate in tissue repair after injury and in regeneration of muscle tissue in dystrophy and after denervation (12, 16, 17). There is also evidence that IL-6 may regulate muscle protein degradation in various muscle-wasting conditions (29, 32), although this is somewhat controversial (10, 35). Considering the multiple important biological effects of IL-6, an increased understanding of the regulation of muscle IL-6 production has significant clinical implications.

Previous studies suggest that IL-6 production is regulated by IL-1 in various cell types, including enterocytes (18, 20), smooth muscle cells (15), monocytes (3), and fibroblasts (9). In contrast, the influence of IL-1 on IL-6 production in skeletal muscle cells has not been reported, and the molecular regulation of muscle IL-6 production is poorly understood. In the present study, we tested the hypothesis that IL-1 stimulates IL-6 production in cultured myotubes and tested whether mitogen-activated protein (MAP) kinase signaling and NF-B activation are involved in IL-1-induced IL-6 production.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. C2C12 cells, a mouse skeletal muscle cell line (37), were seeded between passages 5 and 25 at a density of 100,000 cells/cm² onto six-well culture plates for ELISA and luciferase assays or onto 10-cm tissue culture plates for electrophoretic mobility shift assay (EMSA), Western blot analysis, and real-time PCR (see below). The cells were grown in 5% CO₂ in DMEM supplemented with 10% FBS, nonessential amino acids, 6 mM glutamine, 10 mM HEPES, 10 µg/ml apotransferrin, 1 mM pyruvate, 24 mM NaHCO₃, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from GIBCO-BRL, Grand Island, NY). When cells achieved confluence, the concentration of FBS was reduced to 2% to induce differentiation into myotubes. This typically took 4-5 days during which time the medium was exchanged on a daily basis. The cultures were examined under a light microscope daily, and experiments were not performed until myotubes had formed.

Determination of cell viability. Cell viability was determined by measuring mitochondrial respiration, assessed by the mitochondrial-dependent reduction of 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide to formazan as described previously (26). Cell viability was not influenced by any of the experimental conditions in the present study (data not shown).

Preparation of cytoplasmic and nuclear extracts. For extraction of cytoplasmic and nuclear fractions, myotubes were harvested by scraping into ice-cold PBS and pelleted by centrifugation at 3,800 g for 5 min. Cells were then suspended in one packed-cell volume of lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.2% (vol/vol) Nonidet P-40, 1 mM DTT, 0.5 mM PMSF, 100 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1.5 µM pepstatin A, 1.4 µM transepoxysuccinyl-L-leucylamidol, 4 µM bestatin, 2.2 µM leupeptin, 0.08 µM aprotinin, 0.0045 µM microcystin LR, 0.46 µM cantharidin, and 0.2 µM ()-p-bromotetramisole. After incubation on ice for 5 min with intermittent vortexing, the nuclear pellet was isolated by centrifugation at 3,800 g for 5 min. The supernatant was removed and saved as the cytoplasmic fraction. The pellet was resuspended in 1 cell volume of extract buffer containing 20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% glycerol (vol/vol), 1 mM DTT, 0.5 mM PMSF, 100 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1.5 µM pepstatin A, 1.4 µM transepoxysuccinyl-L-leucylamidol, 4 µM bestatin, 2.2 µM leupeptin, 0.08 µM aprotinin, 0.0045 µM microcystin LR, 0.46 µM cantharidin, and 0.2 µM ()-p-bromotetramisole and incubated on ice for 30 min with intermittent vortexing. The nuclear debris was pelleted by centrifugation at 16,000 g for 20 min, and the supernatant was saved as the nuclear fraction. Protein concentrations in the nuclear and cyptoplasmic extracts were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as a standard.

Western blot analysis. Western blot analysis was performed for determination of cytoplasmic levels of IB as described in detail previously (24). A rabbit polyclonal anti-IB antibody (Santa Cruz Laboratories, Santa Cruz, CA) was used as primary antibody, and a peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody. A molecular weight marker (Kaleidoscope Prestained Standards, Bio-Rad Laboratories) was included in the experiments. The membranes were incubated in enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Uppsala, Sweden) and exposed on radiographic film (Eastman Kodak, Rochester, NY).

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed as described in detail previously (25). In short, NF-B gel shift oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Santa Cruz Laboratories) was end-labeled with [-³²P]ATP using polynucleotide kinase T4 (GIBCO BRL). End-labeled probe was purified from unincorporated [-³²P]ATP using a purification column (Bio-Rad Laboratories) and recovered in Tris-EDTA buffer, pH 7.4. Labeled probe was added to nuclear extracts (7.5 µg protein), and the samples were incubated for 30 min on ice. Where indicated in RESULTS, an excess (20 ng) of unlabeled NF-B consensus oligonucleotide or unlabeled mutant NF-B oligonucleotide (5'-AGT TGA GGC GAC TTT CCC AGG C-3'; 1 base pair substitution underlined) (Santa Cruz Laboratories) was added for competition reaction. Samples were then subjected to electrophoretic separation on a nondenaturing 5% polyacrylamide gel at 30 mA using Tris borate EDTA buffer (0.45 M Tris borate, 0.001 M EDTA, pH 8.3). Blots were dried at 80°C for 3 h and analyzed by exposure to PhophorImager screen (Molecular Dynamics, Sunnyvale, CA).

Plasmids. A luciferase reporter plasmid containing a promoter construct consisting of three tandem repeats of an NF-B binding site was purchased from Stratagene, LaJolla, CA. Wild-type and NF-B-mutant IL-6 promoter luciferase reporter plasmids were kindly provided by Dr. O. Eickelberg (Yale Univ., New Haven, CT). The wild-type IL-6 promoter plasmid has an NF-B binding sequence located at 72 to 63, 5'-GGG ATT TTC C-3' with the mutant binding sequence having the underlined mutations 5'-CTC ATT TTC C-3' as a result of site-directed mutagenesis (8).

Cell transfection and luciferase assays. C2C12 cells were seeded onto six-well culture dishes and grown to 50% confluence before transfection. The Lipofection (GIBCO-BRL) transfection method was used for cell transfection. LipofectAmine Plus was incubated with serum-free OPTIMEM and 1 µg of plasmid at room temperature for 15 min. The myocytes were washed three times with serum-free medium, and 4:1 lipid:DNA complexes were added to the cells. An expression vector containing the pCMV-SPORT-Gal plasmid was used as a control for transfection efficiency. After incubation at 37°C for 4 h, the culture medium was changed to DMEM supplemented with 10% FBS and incubated for an additional 24 h at 37°C. After the cells had been washed with serum-free DMEM, they were treated with IL-1 (1 ng/ml) in serum-free medium for 8 h. For luciferase assays, cells were washed twice with PBS, and 250 µl of Luciferase Cell Culture Lysis Reagent (Promega, Madison, WI) were added to each well for 15 min, after which the cells were harvested and stored at 70°C. The next day, the samples were thawed and centrifuged at 14,000 g for 2 min. Supernatant (30 µl) was combined with 100 µl of Luciferase Assay Substrate (Promega) in Sarstedt 12- × 75-mm tubes in duplicate and read for 10 s on a Berthold AutoLumat LB953 luminometer (PerkinElmer, Gaithersburg, MD). Results were expressed as arbitrary units after normalization to -galactosidase activity.

Determination of IL-6 levels. IL-6 levels were determined by ELISA using a commercially available kit specific for mouse IL-6 (Endogen, Cambridge, MA). The limit of detection was 1 pg/ml according to the manufacturer.

Real-time PCR. RNA was isolated from myotybes according to Chomczynski and Sacchi (5). RNA was resuspended in RNase/DNase-free water (GIBCO/Invitrogen, Carlsbad, CA) and quantified with an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA). First-strand cDNA synthesis was performed using SuperScript first-strand synthesis system for real-time PCR (Life Technologies, Rockville, MD) with oligo(dT) as the primer. As an additional quality control, Arabidopsis thaliana mRNA was added to each RNA sample before cDNA synthesis. Real-time PCR was performed in a Smart Cycler (Cepheid, Sunnyvale, CA) using the Pre-Developed Taqman Assay Reagent (Applied Biosystems, Langen, Germany) for mouse IL-6. Measurements were taken at the end of the 72°C extension step in each cycle, and the second-derivative method was used to calculate the threshold cycle. Real-time PCR was also performed with primers specific for A. thaliana. Fluorescence growth curves and threshold cycle for A. thaliana mRNA were equal for all samples ensuring equal cDNA loading.

Statistical analysis. Results are expressed as means ± SE. Student's t-test or ANOVA followed by Tukey's test was used for statistical analysis. Significance was set at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Treatment of cultured C2C12 cells for 24 h with different concentrations of IL-1 up to 2 ng/ml resulted in a dose-dependent stimulation of IL-6 production with a maximal effect seen at an IL-1 concentration of 1 ng/ml (Fig. 1A). When myotubes were treated with this concentration of IL-1 for various periods of time, an increased IL-6 production was noticed already after 2 h (Fig. 1B).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   A: effect of different concentrations of IL-1 on IL-6 production in cultured C2C12 myotubes. Myotubes were treated with IL-1 for 24 h whereafter IL-6 was determined in the culture medium by ELISA. Results are means ± SE with n = 6 or 7 for each condition. B: influence of different times of treatment with IL-1 (1 ng/ml) on IL-6 production in cultured C2C12 myotubes. Filled bars, cells cultured in the absence of IL-1; open bars, cells cultured in medium containing IL-1. C: IL-6 mRNA levels determined by real-time PCR in C2C12 myotubes cultured for 8 h in the absence (control) or presence of IL-1 (1 ng/ml).

To test whether the increased IL-6 production was associated with increased expression of the IL-6 gene, IL-6 mRNA levels were determined by real-time PCR. Results from that experiment showed a >25-fold increase in IL-6 mRNA levels in C2C12 myotubes treated for 8 h with 1 ng/ml of IL-1 (Fig. 1C).

Cytokine-induced IL-6 production is regulated by the transcription factor NF-B in many cell types, but the involvement of NF-B in muscle IL-6 production is not known. NF-B is typically sequestered in the cytoplasm as an inactive complex by a member of the inhibitory IkB family, most commonly IB (11). In the present study, treatment of the myotubes with IL-1 resulted in a rapid (15-30 min) and transient degradation of cytoplasmic IB (Fig. 2A), consistent with the response to inflammatory stimulus in other cell types. The degradation of IB was accompanied by nuclear translocation and increased DNA binding activity of NF-B determined by EMSA (Fig. 2B). In this experiment, NF-B DNA binding activity was measured 2 h after addition of IL-1 to the myotubes. This time point was based on preliminary experiments in which a maximal increase in NF-B DNA binding was noticed after treatment of C2C12 myotubes for 1-2 h (data not shown). The results are consistent with a model in which IB is first degraded followed by a subsequent translocation of NF-B to the nucleus with activation of various genes, including IB. Note that the IB levels at 90 min were higher than control levels (Fig. 2A), consistent with upregulated IB production at this point, most likely regulated by NF-B. The specificity of the EMSA was confirmed by adding an excess amount of unlabeled NF-B oligonucleotide in a competition reaction (Fig. 2B, 2 right lanes).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   A: IB cytoplasmic levels determined by Western blotting in cultured C2C12 myotubes treated with 1 ng/ml of IL-1 for 0-90 min as indicated below the blot. The band on the Western blot represented a protein with a molecular mass of ~37 kDa as determined by the molecular mass markers included in the experiment. B: NF-B DNA binding activity determined by electrophoretic mobility shift assay (EMSA) in cultured C2C12 myotubes treated for 2 h with 1 ng/ml of IL-1. Addition of an excess amount of unlabeled wild-type NF-B (comp), but not mutated NF-B (mut) oligonucleotide, competed out the NF-B band, confirming the specificity of the EMSA. The experiments shown in A and B were performed 3 times with almost identical results. C: luciferase activity in cultured C2C12 myotubes treated for 8 h with 1 ng/ml of IL-1. Control cells were cultured in medium without addition of IL-1. Before experiments, cells were transiently transfected with a plasmid containing a luciferase reporter construct with an upstream promoter region of 3 tandem repeat NF-B binding sites. Results are means ± SE with n = 6 in each group. CTR, control. * P < 0.05.

To test whether the increased NF-B DNA binding determined by EMSA was associated with gene activation, C2C12 cells were transiently transfected with a luciferase reporter plasmid containing a promoter construct of three tandem repeats of NF-B-responsive elements. Treatment of the transfected cells with IL-1 resulted in an ~2-fold increase in luciferase activity, suggesting that NF-B activation resulted in increased gene transcription under the present experimental conditions (Fig. 2C).

We next wanted to determine whether the increased NF-B activity was involved in IL-6 production in IL-1-stimulated muscle cells. This was done by transfecting C2C12 cells with a luciferase reporter plasmid containing an IL-6 wild-type promoter or an IL-6 promoter in which the NF-B binding site had been mutated. Treatment of the cells with IL-1 resulted in increased luciferase activity in the cells transfected with a plasmid containing the wild-type IL-6 promoter (Fig. 3). This effect of IL-1 was abolished in cells transfected with the NF-B-mutated IL-6 promoter, providing support for the role of NF-B in the regulation of the IL-6 gene in IL-1-treated muscle cells.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Luciferase activity in C2C12 myotubes cultured for 8 h in the absence (filled bars) or presence (open bars) of 1 ng/ml of IL-1. Before IL-1 treatment, cells were transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter construct or an IL-6 promoter construct in which the NF-B binding site had been mutated as described in MATERIALS AND METHODS. Results are means ± SE with n = 6 or 7 in each group. * P < 0.05 vs. all other groups by ANOVA.

Previous studies suggest that the MAP kinase signaling pathway mediates multiple responses to cytokine stimulation. We examined whether the MAP kinase pathway is involved in the regulation of IL-6 production in IL-1-stimulated myotubes by treating the cells with the specific p38 inhibitor SB-208350 or the p42/44 (ERK) inhibitor PD-98059. Both of these treatments significantly reduced IL-6 production in IL-1-treated myotubes, suggesting that the MAP kinase signaling pathway at least in part regulates IL-6 production under the present experimental conditions (Fig. 4).

View larger version (9K): [in this window] [in a new window]   Fig. 4.   IL-6 production in C2C12 myotubes treated for 24 h with 1 ng/ml of IL-1 in the absence or presence of SB-208350 (10 µM) or PD-98059 (10 µM). Results are means ± SE with n = 6 or 7 in each group. * P < 0.05 vs. all other groups;  P < 0.05 vs. IL-1 and vs. control (no additions to the medium).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results in the present study support the concept that skeletal muscle is a significant site for IL-6 production, confirming previous reports in the literature (1, 2, 14, 16, 21, 31). In addition, our study provides the novel information that the expression of IL-6 in skeletal muscle cells is regulated by the proinflammatory cytokine IL-1 and that this effect of IL-1 is at least in part regulated by MAP kinase signaling pathways and NF-B. In related studies, we observed increased IL-6 gene expression and IL-6 production in incubated intact rat extensor digitorum longus muscles treated with IL-1 (unpublished observations), suggesting that the present results were not cell or species specific.

The regulation of IL-6 production by IL-1 was examined in the present study because previous reports suggest that this cytokine is an important regulator of the IL-6 gene in several other cell types (3, 9, 15, 18, 20). In previous research in our laboratory, IL-1 stimulated IL-6 production in cultured enterocytes (20). Interestingly, the IL-6 production noticed in the present study in IL-1-stimulated C2C12 myotubes was ~100 times higher than noticed for the same number of cultured enterocytes, further supporting the concept that skeletal muscle is a significant source of IL-6.

Exercise was reported previously to be an important stimulus for increased gene expression and production of IL-6 in skeletal muscle (14, 21), and it was calculated that the majority of circulating IL-6 during exercise originated from contracting muscle (28). Although there is evidence that reduced muscle glycogen levels augments IL-6 production (27), the mediators and cellular mechanisms of muscle IL-6 production during exercise are not fully understood. It is interesting to note, however, that circulating levels of IL- have been reported during exercise (4), and based on the results reported here it is tempting to speculate that IL-1 may be involved in the upregulation of IL-6 in muscle during exercise.

In addition to exercise, the expression of IL-6 was increased in skeletal muscle in other conditions as well. For example, IL-6 mRNA levels were increased in hypoperfused muscles of patients with peripheral vascular disease (31). Interestingly, the expression of IL-1 was increased in the same muscles, raising the possibility of an autocrine regulation of IL-6 by IL-1 in ischemic muscle. Denervation of muscle and muscle dystrophy are additional examples of conditions associated with upregulated expression of muscle IL-6 (1, 16).

It should be noted that although the present study is the first report of IL-1-induced IL-6 production in muscle cells, the influence of other cytokines on the expression of IL-6 in muscle cells was reported previously by De Rossi et al. (7). In that study, treatment of cultured human myoblasts with TNF- or IFN- resulted in increased gene expression of IL-6. The molecular mechanisms by which the cytokines upregulated the expression of IL-6 were not examined in that study.

Signaling pathways and transcription factors involved in the regulation of the IL-6 gene in skeletal muscle have not been reported previously. The present results suggest that NF-B and the MAP kinase signaling pathway regulate the expression of IL-6 in IL-1-stimulated myotubes. The IL-6 gene promoter is under the regulation of multiple transcription factors, including activator protein (AP)-1, CCAAT/enhancer binding protein (C/EBP), cAMP response element-binding protein (CREB), and NF-B (34). There is evidence that the role of the transcription factors varies in different cell types and with different stimuli (33). It should be noted that although the present result of abolished increase in luciferase activity in cells transfected with a plasmid containing an NF-B-mutated IL-6 promoter suggests that NF-B is an important regulator of the IL-6 gene under the present experimental conditions, the result does not rule out the possibility that other transcription factors participate in the regulation of IL-6 production in muscle cells. It will be important in future studies to determine the involvement of AP-1, C/EBP, and CREB in the regulation of the IL-6 gene in skeletal muscle.

Activation of the MAP kinase signaling pathway by proinflammatory cytokines, including IL-1, was reported previously in other cell types (30). In studies in our laboratory, IL-6 production in IL-1-treated cultured enterocytes was partly regulated by MAP kinase activity (13). Interestingly, in a recent study, treatment of cultured cardiac myocytes with TNF- resulted in upregulated gene expression and production of IL-6, and this response to TNF- was blocked by p38 inhibition (6). In addition, mutation of the NF-B binding site in the IL-6 gene resulted in a loss of p38-inducible IL-6 reporter activity, suggesting that NF-B played an important role in IL-6 production in the cardiocytes. Taken together, the authors of that report interpreted their results as indicating that induction of IL-6 gene transcription in cardiac myocytes depends on NF-B activation and that NF-B activity was further stimulated by the p38 pathway at the level of IkB kinase , consistent with a p38/NF-B cross-talk (6). In contrast to the present study, the IL-6 production in stimulated cardiac myocytes was not affected by the p42/44 inhibitor PD-98059, suggesting that the involvement of different MAP kinase pathways may vary in different cell types. Alternatively, the results may reflect differential activation of the MAP kinase pathways by different stimuli, i.e., IL-1 in the present study and TNF- in the cardiac myocyte study.

The potential biological effects of muscle-derived IL-6 were reviewed recently by Pedersen et al. (22). There is evidence that IL-6 stimulates lipolysis in adipose tissue with release of free fatty acids and glycogenolysis in liver with release of glucose, systemic effects of IL-6 that are particularly pertinent to exercise-induced release of muscle IL-6. In addition to systemic effects, IL-6 probably has important biological effects in muscle tissue as well. Previous studies provided evidence that IL-6 has trophic effects in muscle and is important for repair of muscle injury (1, 12). Also in cardiac myocytes, evidence was found that IL-6 may have cell-protective effects (6). Although the role of IL-6 in the regulation of muscle protein breakdown is controversial (10, 29, 32, 35), the present result of increased muscle IL-6 production after stimulation with a proinflammatory cytokine raises the possibility that locally produced IL-6 may regulate protein metabolism in atrophying muscle.