Lipopolysaccharide and proinflammatory cytokines stimulate ...

Am J Physiol Regul Integr Comp Physiol 285: R1153–R1164, 2003. First published July 3, 2003; 10.1152/ajpregu.00164.2003.

Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase Robert A. Frost, Gerald J. Nystrom, and Charles H. Lang Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Submitted 31 March 2003; accepted in final form 26 June 2003

Frost, Robert A., Gerald J. Nystrom, and Charles H. Lang. Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase. Am J Physiol Regul Integr Comp Physiol 285: R1153–R1164, 2003. First published July 3, 2003; 10.1152/ajpregu.00164.2003.—IL-6 is a major inflammatory cytokine that plays a central role in coordinating the acute-phase response to trauma, injury, and infection in vivo. Although IL-6 is synthesized predominantly by macrophages and lymphocytes, skeletal muscle is a newly recognized source of this cytokine. IL-6 from muscle spills into the circulation, and blood-borne IL-6 can be elevated ⬎100-fold due to exercise and injury. The purpose of the present study was to determine whether inflammatory stimuli, such as LPS, TNF-␣, and IL-1␤, could increase IL-6 expression in skeletal muscle and C2C12 myoblasts. Second, we investigated the role of mitogen-activated protein (MAP) kinases, and the Jun NH2-terminal kinase (JNK) in particular, as a mediator of this response. Intraperitoneal injection of LPS in mice increased the circulating concentration of IL-6 from undetectable levels to 4 ng/ml. LPS also increased IL-6 mRNA 100-fold in mouse fast-twitch skeletal muscle. Addition of LPS, IL-1␤, or TNF-␣ directly to C2C12 myoblasts increased IL-6 protein (6- to 8-fold) and IL-6 mRNA (5- to 10-fold). The response to all three stimuli was completely blocked by the JNK inhibitor SP-600125 but not as effectively by other MAP kinase inhibitors. SP-600125 blocked LPSstimulated IL-6 synthesis dose dependently at both the RNA and protein level. SP-600125 was as effective as the synthetic glucocorticoid dexamethasone at inhibiting IL-6 expression. SP-600125 inhibited IL-6 synthesis when added to cells up to 60 min after LPS stimulation, but its inhibitory effect waned with time. LPS stimulated IL-6 mRNA in both myoblasts and myotubes, but myoblasts showed a proportionally greater LPS-induced increase in IL-6 protein expression compared with myotubes. SP-600125 and the proteasomal inhibitor MG-132 blocked LPS-induced degradation of I␬B-␣/⑀ and LPS-stimulated expression of I␬B-␣ mRNA. Yet, only SP600125 and not MG-132 blocked LPS-induced IL-6 mRNA expression. This suggests that IL-6 gene expression is a downstream target of JNK in C2C12 myoblasts. skeletal muscle; tumor necrosis factor-␣; interleukin-1␤; mitogen-activated protein kinase

is a pleiotropic cytokine with multiple effects on both immune and nonimmune cells alike. IL-6 stimulates the acute-phase response in hepatocytes and plays an important role in regulating both protein and glucose homeostasis in response to tissue injury and infection (40). Recent work by Keller et al. (27) has demonstrated that the plasma concentration of IL-6 increases dramatically during exercise and that skeletal muscle is a major source of blood-borne IL-6. Skeletal muscle IL-6 is elevated by exercise (36), muscle contraction (25), and denervation (28). IL-6 is also elevated with aging (52), congestive heart failure, sepsis, and cancer (50). The plasma concentration of IL-6 is prognostic of mortality in septic patients (41). Elevated levels of IL-6 are associated with a reduction in muscle mass and muscle strength (52), and IL-6 blockade can be protective in some models of sepsis (44). The synthesis of IL-6 by skeletal muscle may also have beneficial effects. Pedersen et al. (37) have proposed that muscle-derived IL-6 has lipolytic and antiinflammatory effects and that it may function as a novel neuroendocrine hormone. These authors (37) and others (48) have found that IL-6 infusion increases circulating free fatty acids. Although this may be due to a concomitant increase in epinephrine with IL-6 infusion, even low levels of IL-6 reportedly increase plasma free fatty acids in humans (37). IL-6 may also inhibit the negative effects of TNF-␣ and thus indirectly promote glucose uptake and enhance insulin sensitivity in peripheral tissues such as skeletal muscle (10). Despite elegant studies examining the regulation of skeletal muscle IL-6 in vivo, little is known about how muscle IL-6 is regulated in response to inflammatory mediators and cytokines. Recently we have demonstrated that LPS stimulates IL-6 mRNA expression in mouse skeletal muscle via Toll-like receptor-4 (TLR-4) signaling. Mice that harbor a mutation in TLR-4 have a greatly reduced expression of IL-6 mRNA in skeletal muscle in response to LPS (17). Our laboratory (17) and others (21) have shown that human and mouse myoblasts produce IL-6

Address for reprint requests and other correspondence: R. A. Frost, Dept. of Cellular and Molecular Physiology, Penn State Univ. College of Medicine, Hershey Medical Center: H166, Hershey, PA 17033 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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0363-6119/03 $5.00 Copyright © 2003 the American Physiological Society

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REGULATION OF IL-6 IN SKELETAL MUSCLE

in response to inflammatory stimuli. In this regard, C2C12 myoblasts express a variety of LPS- and cytokine-responsive mRNAs, including IL-6, IL-1␣, IL-1Ra, IL-12, TNF-␣, and transforming growth factor (TGF)␤1, -␤2, and -␤3 (17). Myoblasts also express IL-6 in response to peptidoglycan from the cell wall of the Gram-positive bacteria, suggesting that a number of pathogen-associated molecules may influence muscle cytokine expression (17). LPS-stimulated IL-6 mRNA expression in C2C12 cells does not require new protein synthesis, but it can be blocked by the transcriptional inhibitor 5,6-dichloro␤-D-ribofuranosyl-benzimidazole (DRB). In addition, LPS does not change the half-life of IL-6 mRNA in C2C12 myoblasts. This suggests that LPS stimulates IL-6 transcription in myocytes as it does in monocytic cells (11). Because LPS and cytokines are known to activate stress-activated protein kinases, such as p38 and Jun NH2-terminal kinase (JNK), we have examined the role of these kinases in mediating the IL-6 response to LPS. The role of the JNK and nuclear factor-␬B (NF-␬B) pathways was also compared. Inhibitor studies suggest that the JNK pathway is partly responsible for LPS-induced IL-6 mRNA expression in C2C12 cells. MATERIALS AND METHODS

Experimental protocol for C3H/HeSnJ mice. C3H/HeSnJ mice were obtained from the Jackson Laboratories (Bar Harbor, ME). All mice were housed in a controlled environment and provided water and rodent chow ad libitum for 3 wk before their use. At the time of the study, mice were 8–9 wk old and weighed 21.4 ⫾ 0.3 g. In the experiment depicted in Fig. 1, C3H/HeSnJ mice were injected intraperitoneally with LPS derived from Escherichia coli 026:B6 (DIFCO Laboratories, Detroit, MI, 25 ␮g/mouse) or an equal volume of saline (250 ␮l/mouse). This dose was based on a preliminary doseresponse study and is similar to that used by other investigators (3). After 2, 6, and 18 h, mice were anesthetized with a mixture of ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (Bayer, Shawnee Mission, KS) at 90 and 9 mg/kg, respectively. Blood was collected from the inferior vena cava in heparinized syringes. Hindlimb skeletal muscle (gastrocnemius and plantaris) from both legs was dissected from each animal, wrapped in aluminum foil, and flash frozen in liquid nitrogen. Mice were killed by cardiac excision and subsequent exsanguination. Tissues were later powdered under liquid nitrogen by using a mortar and pestle and stored at ⫺70°C. All experiments were approved by the Institutional Animal Care and Use Committee at the Pennsylvania State University College of Medicine and adhere to the National Institutes of Health guidelines for the use of experimental animals. Cell culture. The C2C12 mouse myoblast cell line was purchased from the American Type Culture Collection (Manassas, VA) and used for all studies. Cells were grown in 100-mm Petri dishes (Becton Dickinson, Franklin Lakes, NJ) and cultured in minimal essential media (MEM) containing 10% bovine calf serum, penicillin (100 U/ml), streptomycin (100 ␮g/ml), and amphotericin B (25 ␮g/ml) (all from Sigma, St. Louis, MO). Cells were grown to confluence and switched to fresh serum-containing media before addition of LPS, cytokines, or other agents. In general, C2C12 cells were used AJP-Regul Integr Comp Physiol • VOL

Fig. 1. Effect of LPS on circulating IL-6 and IL-6 mRNA abundance in mouse skeletal (Sk) muscle. Wild-type (C3H/HeSnJ) mice were injected intraperitoneally with either saline (Sal) or a nonlethal dose of Esherichia coli LPS (LPS, 25 ␮g/mouse). Blood and tissue samples were collected after 2, 6, and 18 h. We measured plasma IL-6 by ELISA (A). Muscle was frozen in liquid nitrogen, powdered, and analyzed. RNA was isolated and hybridized to a cytokine mRNA RPA template as described in MATERIALS AND METHODS and run on a 5% acrylamide gel. All data are normalized to L32 mRNA and expressed as a fold increase relative to animals injected with saline alone. B: image of IL-6 and L32 mRNA in muscle at 2, 6, and 18 h. C: phosphorimager analysis of IL-6 mRNA in the dried gel. Values are means ⫾ SE. Bars with different lowercase letters are significantly different from each other (P ⬍ 0.05). Where absent, SE bars are within the bar.

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at the myoblast stage, but in some instances the cells were differentiated by incubation for 2 wk in medium containing 2% bovine calf serum. Experiments were performed with lipopolysaccharide B derived from Escherichia coli 026:B6 (DIFCO Laboratories). A variety of compounds was used to characterize the response to LPS, including PD-98059, SB202190, SP-600125, dicumarol, and MG-132 (all from Calbiochem, La Jolla, CA). Dexamethasone was obtained from Sigma Chemical. Additional experiments utilized the recombinant cytokines IL-1␤ and TNF-␣ (Peprotech, Rocky Hill, NJ). Doses of the cytokines and inhibitors were chosen based on both the literature (19) and preliminary dose-response curves to find effective doses (16, 17). RNA isolation and RPA. Total RNA, DNA, and protein were extracted from C2C12 cells or tissues in a mixture of phenol and guanidine thiocyanate (TRI Reagent, Molecular Research Center, Cincinnati, OH) using the manufacturer’s protocol. RNA was separated from protein and DNA by the addition of bromochloropropane and precipitation in isopropanol. After a 75% ethanol wash and resuspension in formamide, RNA samples were quantified by spectrophotometry. Ten micrograms of RNA was used for each assay. Riboprobes were synthesized from a custom multiprobe mouse template set containing a probe for IL-6 mRNA detection (Pharminigen). The labeled riboprobe was hybridized with RNA overnight using an RPA kit and the manufacturer’s protocol (Pharminigen). Protected RNAs were separated using a 5% acrylamide gel (19:1 acrylamide/bisacrylamide). Gels were transferred to blotting paper and dried under vacuum on a gel dryer. Dried gels were exposed to a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA), and the resulting data were quantified using ImageQuant software and normalized to the mouse ribosomal protein L32 mRNA signal in each lane. Western blot analysis and IL-6 ELISA. Cell extracts were electrophoresed on denaturing polyacrylamide gels and electrophoretically transferred to nitrocellulose with a semidry blotter (Bio-Rad Laboratories, Melville, NY). The resulting blots were blocked with 5% nonfat dry milk for 1.5 h and incubated with antibodies against either I␬B-␣ or -⑀, phosphorylated or total ERK, p38, or JNK as previously described (17). Unbound primary antibody was removed by washing with Tris-buffered saline containing 0.05% Tween-20, and blots were incubated with anti-rabbit or anti-mouse immunoglobulin conjugated with horseradish peroxidase. Blots were briefly incubated with the components of an enhanced chemiluminescent detection system (Amersham, Buckinghamshire, UK). Dried blots were used to expose X-ray film for 1–3 min. Conditioned media from C2C12 cells were collected at various time points and frozen at ⫺20oC until assay. Mouse IL-6 in plasma and conditioned media was measured with a sandwich ELISA consisting of two anti-mouse IL-6 antibodies and a strepavidin and horseradish peroxidase (HRP)linked secondary antibody (Pharminigen, San Diego, CA). Conditioned media were diluted with an equal volume of assay diluent, whereas plasma was diluted 1:12 before assay. Antigen and antibody complexes were detected with tetramethylbenzidine (TMB, an HRP substrate), and the reaction was stopped with 2 N H2SO4. Ninety-six well plates were read at the absorption maximum for TMB (450 nm). Statistics. Values are means ⫾ SE. Unless otherwise noted, each experimental condition was tested in triplicate, and each experiment was repeated two times. Data were analyzed by ANOVA followed by Student-Newman-Keuls test for multiple comparisons. Statistical significance was set AJP-Regul Integr Comp Physiol • VOL

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at P ⬍ 0.05. For animal studies, the number of mice per group was four (control) and six (LPS). IL-6 mRNA half-life was calculated from the slope of the regression line using the formula t1/2 ⫽ 0.5/m, where m is the slope of the line in arbitrary units (AU) per hour. Half-lives were compared by t-test where t ⫽ [(m1 ⫺ m2) /冑(SE12⫹SE22)]. Statistical significance was set at P ⬍ 0.05. RESULTS

LPS rapidly stimulates IL-6 expression in mouse plasma and skeletal muscle. Mice were injected with a nonlethal dose of LPS, and blood was obtained 2, 6, and 18 h thereafter. This time course corresponds to the early, middle, and later stages of the acute-phase response. LPS increased the plasma concentration of IL-6 from the detection limit of the assay (⬍0.01 ng/ml) to 2 ng/ml after 2 h. IL-6 in the plasma peaked at 6 h and was still significantly elevated 18 h after LPS administration (Fig. 1A). IL-6 mRNA was detected in mouse skeletal muscle 2 h after LPS administration (Fig. 1B) where its level was elevated 100-fold compared with mice receiving saline. IL-6 mRNA in skeletal muscle was elevated 20-fold compared with control mice after 6 h but returned to baseline 18 h after LPS administration (Fig. 1C). LPS stimulates IL-6 protein and mRNA expression in C2C12 myoblasts. Because skeletal muscle contains many cell types that could potentially respond to LPS and synthesize IL-6, we examined whether LPS could directly stimulate IL-6 protein and mRNA expression in C2C12 myoblasts. LPS increased IL-6 protein (Fig. 2A) and mRNA (Fig. 2B) time dependently. Maximal IL-6 mRNA expression in C2C12 cells occurred after 3 h, whereas IL-6 protein continued to be secreted for up to 16 h (Fig. 2, A and C). Changes in the steadystate level of IL-6 mRNA were independent of the mRNA level of two housekeeping genes (L32 and GAPDH, Fig. 2B), which were essentially unchanged over the course of the experiment. The JNK inhibitor SP-600125 blocks cytokine- and LPS-stimulated IL-6 synthesis. We previously demonstrated that LPS does not alter the half-life of IL-6 mRNA and that LPS-stimulated IL-6 mRNA expression can be inhibited by pretreatment with DRB, a transcriptional inhibitor (17). Mutation and sequencing studies have shown that activating protein-1 (AP-1) response elements in the human (12) and rat (46) IL-6 promoters are important for IL-6 promoter activity. Therefore, we inhibited AP-1 activation by treating C2C12 cells with a JNK inhibitor (SP-600125). This compound has previously been shown to inhibit c-Jun phosphorylation (4). Pretreatment of C2C12 cells with SP-600125 greatly attenuated TNF-␣-, IL-1␤-, and LPS-induced IL-6 protein expression (Fig. 3, A–C, respectively). PD-98059, a MEK1 inhibitor that prevents ERK-1/2 activation, attenuated LPS-induced IL-6 synthesis but failed to reduce IL-1␤- and TNF-␣induced IL-6 synthesis. SB-202190, a p38 kinase inhibitor, was effective at inhibiting IL-1␤- and LPS-stimulated IL-6 synthesis but was less effective at blocking 285 • NOVEMBER 2003 •

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ylation, we could not detect anisomycin-induced changes in the phosphorylation of either JNK or its substrate c-Jun in this cell type. SP-600125 inhibited LPS-induced IL-6 protein and mRNA expression dose dependently with an ED50 of 50 ␮M (Fig. 5, A and B). IL-6 protein and mRNA expression was completely inhibited at 100 ␮M SP-600125. Dicumarol, a second inhibitor of the JNK pathway, also

Fig. 2. Effect of LPS on IL-6 protein secretion and mRNA expression in C2C12 myoblasts. C2C12 myoblasts were grown in serum containing media and stimulated with 1 ␮g/ml LPS for 1, 2, 3, 6, and 16 h. Conditioned media were collected and assayed for IL-6 by ELISA (A). Data points with different lowercase letters are significantly different from each other (P ⬍ 0.05). IL-6 mRNA was determined by RPA as described in MATERIALS AND METHODS. A phosphorimage of the dried gel is shown for cells treated with either saline (Sal) or LPS at each of the time points. B: IL-6, L32, and GAPDH mRNA images. C: IL-6 mRNA was also quantified. The area under the LPS and saline curves are 84 ⫾ 2 and 18 ⫾ 1, respectively. Data are normalized to L32 mRNA as described in MATERIALS AND METHODS and expressed as a fold increase relative to cells treated with saline at the start of the experiment.

the TNF-␣-induced increase. The specificity of the three MAP kinase inhibitors was examined in C2C12 cells treated with anisomycin to stimulate the ERK and p38 kinases. Anisomycin stimulated the phosphorylation of ERK and p38 as detected with phosphospecific antibodies, and this was inhibited by PD-98059 and SB-202190, respectively (Fig. 4). SP-600125 did not inhibit anisomycin-stimulated ERK or p38 phosphorylation, and all of the above changes in kinase phosphorylation were independent of changes in the total amount of each kinase in the cell extracts. Although anisomycin stimulated ERK and p38 phosphorAJP-Regul Integr Comp Physiol • VOL

Fig. 3. SP-600125 [SP; a Jun NH2-terminal kinase (JNK) inhibitor] prevents TNF-␣-, IL-1␤-, and LPS-stimulated IL-6 synthesis in C2C12 myoblasts. C2C12 cells were grown as described in MATERIALS AND METHODS and treated with TNF-␣ (A, 40 ng/ml), IL-1␤ (B, 40 ng/ml), or LPS (C, 1 ␮g/ml from E. coli). Some cells received MAP kinase inhibitors: PD-98059 (PD, 20 ␮M), SB-202190 (SB, 20 ␮M), or SP-600125 (100 ␮M). Conditioned media were collected after 2 h for IL-1-treated and LPS-treated cells and 4 h for cells treated with TNF. IL-6 protein was assayed by ELISA. Values are means ⫾ SE. Bars with different lowercase letters are significantly different from each other (P ⬍ 0.05). 285 • NOVEMBER 2003 •

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Fig. 4. PD-98059 and SB-202190 inhibit their respective kinases in C2C12 myoblasts. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with anisomycin (Ans, 10 ␮M) to stimulate MAP kinases. Cell extracts were prepared 15 min after addition to the cells. Anisomycin stimulated the phosphorylation of ERK (pERK) and p38 (pp38) as detected by Western blotting with phosphospecific antibodies. Some cells were treated with PD-98059 and SB-202190 to inhibit ERK and p38 phosphorylation, respectively. Cells were also treated with SP-600125 (SP) to demonstrate that this compound did not inhibit either the phosphorylation or the total amount of ERK (Total ERK) or p38 (Total p38) in C2C12 cells.

inhibited LPS-induced IL-6 protein and mRNA synthesis dose dependently (Fig. 5, C and D, respectively). SP-600125 and dexamethasone inhibit TNF-␣-, IL1␤-, and LPS-induced IL-6 expression similarly. TNF-␣, IL-1␤, and LPS all stimulated IL-6 protein and mRNA expression. SP-600125 inhibited IL-6 protein and mRNA coordinately by completely blocking the ability of TNF-␣, IL-1␤, and LPS to stimulate IL-6 expression (Fig. 6, A and B). Similar results were seen with the anti-inflammatory glucocorticoid dexamethasone (Fig. 6, C and D).

SP-600125 but not dexamethasone alters IL-6 mRNA half-life. To determine if SP-600125 altered IL-6 mRNA half-life, we performed experiments with the transcriptional inhibitor DRB. We have previously shown that pretreatment of C2C12 cells with DRB completely blocks LPS-induced IL-6 mRNA expression (17). When cells were treated with LPS to maximize IL-6 mRNA levels, followed by DRB, there was a rapid decay in IL-6 mRNA (t1/2 ⫽ 64 min). SP-600125 significantly accelerated the loss of IL-6 mRNA (t1/2 ⫽ 41 min, P ⬍ 0.001) (Fig. 7A). In contrast, dexamethasone did not change the half-life of IL-6 mRNA (Fig. 7B). SP-600125 inhibits IL-6 expression up to 60 min after LPS addition to C2C12 cells. The response to LPS and proinflammatory cytokines in vivo and in vitro is relatively rapid. In addition, agents that interrupt the inflammatory insult after it has begun would be more clinically useful than those requiring a pretreatment. It was therefore of interest to determine whether SP600125 could inhibit LPS-induced IL-6 expression not only as a pretreatment but also after exposure to LPS. Addition of SP-600125 to C2C12 cells at the same time as LPS completely inhibited LPS-induced IL-6 expression (Fig. 8A). SP-600125 continued to inhibit IL-6 expression by at least 50% when it was added up to 90 min after LPS exposure. SP-600125 was ineffective in C2C12 cells if it was added 2 h or more after LPS exposure (Fig. 8A). The potent anti-inflammatory dexamethasone also inhibited IL-6 expression, but it was less effective than SP-600125 when added to the cells at later time points (Fig. 8B). The efficacy of SP-600125 wanes with time in C2C12 cells. Although SP-600125 completely inhibited LPSinduced IL-6 synthesis in most experiments, we did notice there was some variability in its efficacy. One possibility was that the effectiveness of SP-600125

Fig. 5. SP-600125 and dicumarol inhibit LPS-induced IL-6 protein and mRNA accumulation dose dependently. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with LPS (1 ␮g/ml) for 3 h. Some cells received SP-600125 at a final concentration of 10, 50, or 100 ␮M per well. Conditioned media were collected and assayed for IL-6 by ELISA (A). RNA was isolated in Tri-Reagent and hybridized to a cytokine mRNA RPA template as described in MATERIALS AND METHODS and run on a 5% acrylamide gel. The IL-6 band was quantified with ImageQuant software (B). Additional cells were treated with LPS and dicumarol, and IL-6 protein (C) and RNA (D) were quantified as above. All data are normalized to L32 mRNA as described in MATERIALS AND METHODS and expressed as a fold increase relative to time-matched cells treated with saline alone. Values are means ⫾ SE. Bars and data points with different lowercase letters are significantly different from each other (P ⬍ 0.05).

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Fig. 6. Dexamethasone (Dex) and SP-600125 inhibit TNF-␣-, IL-1␤-, and LPS-induced increases in IL-6 protein and mRNA in C2C12 cells. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with TNF-␣ (40 ng/ml), IL-1␤ (40 ng/ml), or LPS (1 ␮g/ml). Some cells were pretreated with SP-600125 (100 ␮M) or dexamethasone (1 ␮M) before addition of LPS. Conditioned media were collected after 3 h and assayed for IL-6 by ELISA (A and C). RNA was isolated in Tri-Reagent and hybridized to a cytokine mRNA RPA template as described in MATERIALS AND METHODS and run on a 5% acrylamide gel. The IL-6 band was quantified with ImageQuant software (B and D). All data are normalized to L32 mRNA as described in MATERIALS AND METHODS and expressed as a fold increase relative to time-matched cells treated with saline alone. Values are means ⫾ SE. Bars with different lowercase letters are significantly different from each other (P ⬍ 0.05).

wanes with time and that conditioned media collected from experiments performed over a longer time frame might contain more IL-6. To test this hypothesis, we treated C2C12 cells with either LPS alone or LPS and SP-600125, and we collected conditioned media at 2, 3, 4, 6, 8, and 22 h. LPS stimulated IL-6 very rapidly. After 3 h, LPS stimulated IL-6 synthesis 10-fold, and SP-600125 completely blocked LPS-induced IL-6 synthesis (Fig. 9A). SP-600125 continued to suppress LPSinduced IL-6 synthesis, but its effect waned such that by 6, 8, and 22 h there was significant LPS-induced IL-6 synthesis in the presence of SP-600125. To test whether SP-600125 could continue to inhibit LPS-induced IL-6 if it were added to the cells every 3 h, we treated C2C12 cells with SP-600125 at the start of the experiment, or at the start and at 3 h, or at the start and at 3 and 6 h. (Fig. 9B). Media were collected 9 h after exposure of the cells to LPS. LPS increased IL-6 eightfold, and this was completely inhibited in cells that received SP-600125 every 3 h to maintain active SP-600125 in the cultures (Fig. 9C, 3⫻). When SP600125 was given only as a pretreatment, it inhibited LPS-induced IL-6 synthesis by 40% (Fig. 9C, 1⫻). When SP-600125 was given twice, it inhibited LPSinduced IL-6 synthesis by 70%. Differentiation of C2C12 cells alters the secretion of IL-6. C2C12 cells were allowed to fully differentiate into myotubes for 2 wk or kept as myoblasts in growth media containing a high concentration of serum. Cells were stimulated with LPS, and conditioned media and RNA were collected after 3 h. LPS stimulated IL-6 synthesis and secretion in both myoblasts and myotubes. The net increase in IL-6 mRNA in both cell types was similar (Fig. 10A, 14-fold in myoblasts and 10-fold in myotubes). In contrast, the net increase in IL-6 AJP-Regul Integr Comp Physiol • VOL

protein secreted into the media by myotubes was greatly reduced compared with myoblasts (Fig. 10B, myoblasts 12-fold, myotubes 3-fold). SP-600125 and MG-132 inhibit LPS-induced I␬B␣/⑀ degradation and subsequent mRNA synthesis but differentially regulate IL-6 mRNA. Because LPS stimulates multiple signal transduction pathways on binding to TLR4 it was of interest to see if SP-600125 was specific for the JNK pathway or if it also inhibited other pathways such as the activation of NF␬B. We examined one of the earliest responses to LPS in C2C12 cells, which is the degradation of I␬B-␣ and -⑀. LPS decreased I␬B protein by 70% after 30 min (Fig. 11A). I␬B-␣ and -⑀ degradation were blocked by SP600125 and the proteasomal inhibitor MG-132. I␬B-␣ mRNA was transiently increased by LPS with a maximal (10-fold) stimulation after 1 h (Fig. 11B). Pretreatment of C2C12 cells with SP-600125 or MG-132 blunted LPS-induced I␬B-␣ mRNA expression (Fig. 11C). In contrast, MG-132 did not inhibit LPS-induced IL-6 mRNA expression (17). Indeed, MG-132 induced IL-6 mRNA expression over an 8-h period, and this was blunted by SP-600125 (area under the curve: MG-132 14.7 ⫾ 0.3 vs. MG-132 ⫹ SP 7.8 ⫾ 0.2, Fig. 11D). DISCUSSION

The molecular mechanisms regulating IL-6 mRNA expression in skeletal muscle have remained largely obscure due to the lack of a good model system for examining positive and negative regulators of IL-6 expression. Recently, our laboratory and others have shown that myoblasts express IL-6 mRNA and that its abundance can be positively regulated by LPS and inflammatory cytokines (17, 21). It is likely that a 285 • NOVEMBER 2003 •

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is very similar to our findings reported herein for mouse skeletal muscle. The steady-state level of IL-6 protein in the plasma was relatively sustained. IL-6 was still elevated in the plasma 18 h after LPS. In contrast, the expression of IL-6 mRNA in skeletal muscle was more transient. IL-6 mRNA peaked at 2 h, was still elevated at 6 h, but returned to baseline 18 h after intraperitoneal injection of LPS. IL-6 mRNA expression in skeletal muscle is associated with a concomitant increase in the level of IL-6 protein in the rat (29). The continued presence of IL-6 protein in the plasma after 18 h but a lack of IL-6 mRNA in skeletal muscle at this time point suggests that blood-borne IL-6 is derived from a non-skeletal muscle source such as inflammatory cells in the spleen. Despite the transient local exposure, muscle cytokines may have a prolonged effect on muscle function and protein synthesis in muscle cells (16). In vivo studies in mice are limited in that skeletal muscle is composed of multiple cell types, and, as a result, IL-6 mRNA may be derived not only from muscle cells but also from sequestered blood-borne immune

Fig. 7. SP-600125, but not dexamethasone, increases IL-6 mRNA turnover. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with LPS (1 ␮g/ml) to maximize expression of IL-6 mRNA. Cells were subsequently treated with a transcriptional inhibitor [5,6-dichloro-␤-D-ribofuranosyl-benzimidazole (DRB)] to examine the half-life of IL-6 mRNA. RNA was isolated between 0 and 60 min after the addition of DRB. The mRNA half-life curves for cells treated with LPS and LPS ⫹ the JNK inhibitor (inhib) were compared (A). Similar half-life curves were generated for cells treated with either LPS alone or LPS and dexamethasone (B). All data are normalized for L32 mRNA as described in MATERIALS AND METHODS. Values are means ⫾ SE of triplicate dishes. IL-6 mRNA had a half-life of 64 min, and SP-600125 significantly accelerated the loss of IL-6 mRNA (41 min, P ⬍ 0.001). In contrast, dexamethasone did not change the half-life of IL-6 mRNA.

number of other cytokines and serum hormones can also influence IL-6 synthesis by skeletal muscle and muscle cells (32, 33, 47, 55). These factors may have direct effects on muscle protein content and/or indirectly influence muscle function by altering the endocrine and autocrine synthesis of anabolic hormones such as insulin-like growth factor-I (IGF-I) (14, 20). Chronic inflammatory diseases and aging are also associated with a decline in muscle function, a decrease in IGF-I, and the overexpression of IL-6 (2, 52). In this study, we demonstrate that LPS is a potent stimulus for both the systemic expression of IL-6 in blood and the specific expression of IL-6 mRNA in mouse skeletal muscle. Plasma levels of IL-6 in C3H/ HeSnJ mice followed a time course similar to that seen in other studies (43) where LPS was given intraperitoneally. Few studies have examined the temporal expression of cytokine mRNAs in skeletal muscle after LPS. In a recent study using rats, we found muscle IL-6 mRNA to be elevated as early as 30 min after LPS with the peak of expression occurring at 4 h (29). This AJP-Regul Integr Comp Physiol • VOL

Fig. 8. SP-600125 inhibits LPS-induced IL-6 synthesis up to 60 min after LPS exposure. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with either LPS alone or pretreated with SP-600125 (A, bars) or dexamethasone (B, bars) before LPS exposure. At various times after LPS (0, 30, 60, 90, 120 min), SP-600125 or dexamethasone was added to the cells. Conditioned media were collected 3 h after LPS exposure in all cases and assayed for IL-6 by ELISA (A and B). Values are means ⫾ SE. Bars and data points with different lowercase letters are significantly different from each other (P ⬍ 0.05). 285 • NOVEMBER 2003 •

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detected by ELISA. IL-6 mRNA was also elevated in response to cytokines and LPS. The cytokine and LPSinduced increase in IL-6 mRNA was not due to a generalized increase in the abundance of mRNA in C2C12 cells. We have previously shown that RNA transcribed from a number of genes, including GAPDH, L32, and 18S ribosomal RNA, remained stable in response to LPS, and that has been reconfirmed here (20). The magnitude of the increase in IL-6 mRNA in C2C12 myoblasts was similar to that found in mouse skeletal muscle 6 h after LPS. The precise cell type(s) that synthesize IL-6 in muscle are unknown. Ostrowski et al. (36) have shown that IL-6 mRNA is detected in skeletal muscle only after and not before intense exercise. Muscle injury also increases IL-6 mRNA, and this tends to be present in damaged muscle fibers and satellite cells as detected by in situ hybridization (26). We have found that C2C12 myoblasts and myotubes respond equally well to LPS at the level of IL-6 mRNA expression but that

Fig. 9. Effectiveness of SP-600125 wanes with time. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with either LPS alone or pretreated with SP-600125. At various times after LPS exposure (2, 3, 4, 6, 8, 22 h) conditioned media were collected and assayed for IL-6 by ELISA (A). To determine whether multiple doses of SP-600125 could suppress IL-6 synthesis better than a single dose, some cells received SP-600125 not only before LPS exposure but also after 3 h or after 3 and 6 h as outlined in B. Conditioned media were collected at 9 h and assayed for IL-6 by ELISA (C). Values are means ⫾ SE. Bars and data points with different lowercase letters are significantly different from each other (P ⬍ 0.05).

cells (1) or endothelial cells that compose the muscle’s vasculature (31). We subsequently used C2C12 myoblasts as a model system to examine the mechanism by which LPS regulates IL-6 mRNA in skeletal muscle. These cells resemble satellite cells that are resident in mature muscle (5). In addition, we have found that the C2C12 cell line is responsive to multiple proinflammatory molecules at the myoblast stage, including LPS from Gram-negative bacteria, peptidoglycan from Gram-positive bacteria, IL-1␤, and TNF-␣ (17). C2C12 myoblasts responded to LPS, TNF-␣, or IL-1␤ with a five- to eightfold increase in IL-6 protein as AJP-Regul Integr Comp Physiol • VOL

Fig. 10. Differentiation of C2C12 cells alters IL-6 secretion. C2C12 myoblasts were grown as described in Fig. 2 but also allowed to differentiate in low serum containing media until they formed multinucleated myotubes. Myoblasts (Blast) and myotubes (Tube) were treated with LPS for 4 h. Conditioned media and RNA were isolated for quantification of IL-6 mRNA by RPA (A) and IL-6 protein by ELISA (B). All data were normalized to L32 mRNA as described in MATERIALS AND METHODS and expressed as a fold increase relative to cells treated with saline alone. Values are means ⫾ SE. Bars with different lowercase letters are significantly different from each other (P ⬍ 0.05). 285 • NOVEMBER 2003 •

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Fig. 11. LPS regulates I␬B and IL-6 mRNA expression through a JNK pathway. C2C12 myoblasts were grown as described in MATERIALS AND METHODS and treated with LPS for 30 min. Some cells were pretreated with SP-600125 (100 ␮M) or the proteasomal inhibitor MG-132 (40 ␮M) for 30 min. Cell extracts were isolated in sample buffer and run on a 12% SDS-PAGE gel. After transfer to nitrocellulose, the blots were probed for I␬B-␣ and -⑀ protein with antibodies from Santa Cruz Biotechnology (␣, SC371 and ⑀, SC-7156). A representative blot is shown, but similar results were attained in 3 experiments performed in triplicate (A). Additional cells were treated with LPS for between 0 and 24 h and RNA isolated and quantified as described in MATERIALS AND METHODS for I␬B-␣ mRNA (B). Some cells were pretreated with either SP-600125 or MG-132 for 30 min to see if these agents could block I␬B-␣ mRNA expression (C). Finally, some cells were treated with MG-132 for up to 8 h in the presence or absence of SP-600125, and IL-6 mRNA was quantified by RPA (D). Phosphorimages of the dried gels were quantified and are plotted in B–D. All data are normalized for L32 mRNA as described in MATERIALS AND METHODS. Values are means ⫾ SE of triplicate dishes. Bars with different lowercase letters are significantly different from each other (P ⬍ 0.05).

myotubes secrete less IL-6 protein than myoblasts. These data suggest that LPS receptor number and signaling are equivalent in myoblasts and myotubes but that differentiation alters either the translation of IL-6 mRNA or the secretory capacity of the cells. Although only in situ hybridization studies can identify the muscle cell type(s) that synthesizes IL-6 in vivo, our in vitro data predict that myoblasts and satellite cells may be one of the major sources of LPS-inducible IL-6 synthesis in skeletal muscle. A major finding of this paper is that SP-600125, a JNK inhibitor, completely prevents cytokine- and LPSinduced expression of IL-6 in C2C12 cells. This response is fairly specific because LPS-induced IL-6 was not inhibited as effectively by two other MAP kinase inhibitors, PD-98059 and SP-202190. In addition, dicumarol, a compound that has previously been shown to inhibit the JNK pathway (8, 34), was a potent inhibitor of LPS-induced IL-6 mRNA and protein expression in C2C12 cells much like SP-600125. We have also previously reported that LPS-induced IL-6 mRNA expression is not inhibited by PDTC (an NF␬B inhibitor). MG-132, which prevents NF␬B activation by preventing the degradation of its inhibitor (I␬B), also did not block IL-6 mRNA expression. MG132 failed to inhibit IL-6 mRNA expression despite the fact that MG-132 did block LPS-induced I␬B degradation and the expression of I␬B-␣ mRNA in C2C12 cells. The overall role of the NF␬B and c-Jun transcription factors during infection is poorly characterized, but in a rat model of sepsis the expression of NF␬B is transient, whereas AP-1 activation is more sustained (38). This suggests that AP-1 may regulate the long-term negative sequelae of infection, whereas NF␬B may regulate predominantly early responses to LPS. Surprisingly, MG-132 stimulated IL-6 synthesis on its own. It is likely that this increase represents a AJP-Regul Integr Comp Physiol • VOL

stress response to prolonged inhibition of the proteasome and perhaps the accumulation of misfolded proteins. This suspicion is supported by data indicating that MG-132 mimics the heat shock response in some cells (39) and can also generate free radicals (54) that stimulate IL-6 synthesis. These results suggest that there are NF␬B-independent mechanisms of increasing IL-6 biosynthesis (23). MG-132 may also indirectly stimulate the JNK pathway (54). We demonstrated that, in addition to LPS and cytokines, SP-600125 also inhibits MG-132-induced IL-6 synthesis. This suggests that JNK activity is critical for IL-6 synthesis under a variety of conditions. SP-600125 decreased the half-life of IL-6 mRNA in C2C12 cells, and this may, in part, explain the ability of the JNK inhibitor to decrease the accumulation of IL-6 mRNA. The p38 inhibitor SB-202190 has also been shown to alter IL-6 mRNA in LPS-stimulated monocytes (53). It is likely that both the p38 and JNK pathways regulate RNA binding proteins such as HuR (9) that bind to adenine and uracil (AU)-rich sequences in the 3⬘-untranslated region of IL-6 mRNA. Ectopic expression of MAP kinase phosphatase (MKP-1) in RAW264.7 cells also prevented LPS-induced IL-6 synthesis and JNK and p38 activation, and this also involved changes in IL-6 gene expression and protein and mRNA stability (7). Interestingly, the half-life of IL-6 mRNA is much shorter in myoblasts than in LPSstimulated macrophage, suggesting that IL-6 may be even more rapidly regulated in muscle cells (35). SP-600125 may be an effective inhibitor of the inflammatory response in C2C12 cells because it inhibits the activation of both c-Jun and NF␬B. SP-600125 may inhibit other kinases such as I␬B kinase or kinases that phosphorylate regulatory components of the 20S proteasome (24). The human IL-6 promoter has an AP-1 consensus-binding site, and mutation of this site 285 • NOVEMBER 2003 •

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in other cell types diminishes TGF-␤-stimulated IL-6 synthesis (12). TGF-␤ also stimulates IL-6 synthesis in human skeletal myoblasts (32), and we have found that SP-600125 inhibits TGF-␤-induced IL-6 mRNA expression in C2C12 cells (Frost, unpublished observation). SP-600125 inhibited LPS-induced IL-6 protein and mRNA expression in the same concentration range, suggesting that IL-6 protein levels are regulated primarily by changes in the steady-state levels of translatable IL-6 mRNA. SP-600125 also inhibited IL-6 expression at a similar concentration to that used to inhibit phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (45) and cytokine synthesis in PMA-activated CD4⫹ T-cells (4). The clinical usefulness of inhibitors of cytokine synthesis depends on how long after the initial insult the inhibitor can be administered. Many inhibitors, such as TNF binding proteins and IL-1 receptor antagonists, function optimally when given in combination and for a prolonged period of time (42). SP-600125 inhibited IL-6 synthesis in C2C12 cells when given either at the time of LPS exposure or up to 90 min after LPS. Dexamethasone, by comparison, was only effective for ⬃30 min after LPS exposure. Two hours after LPS, SP-600125 was ineffective at blocking IL-6 synthesis. Our previous experience suggests that by this time point the majority of the newly synthesized IL-6 mRNA has been translated into protein and is actively secreted from the cells. Although SP-600125 is a potent inhibitor of LPSinduced cytokine expression, its efficacy wanes with time. The JNK inhibitor may be inherently unstable in aqueous solutions, or SP-600125 may be metabolized. In our hands, SP-600125 inhibited LPS-induced IL-6 for 4 h, but by 6 h C2C12 cells could make a significant amount of IL-6 in response to LPS. Other kinase inhibitors have also been shown to lose their effectiveness over time (18, 51). Multiple doses of SP-600125 were more effective at inhibiting LPS-induced IL-6 synthesis over a 9-h period than a single prophylactic dose. It is likely therefore that SP-600125 would have to be infused or stabilized in some way to be a useful inhibitor of IL-6 synthesis in vivo. SP-600125 is a small molecule that inhibits JNK-1, -2, and -3 with similar potency. SP-600125 exhibits 300-fold selectivity against related MAP kinases such as Erk and p38 (4). We cannot exclude the possibility that SP-600125 inhibits kinases other than JNK that may be responsible for IL-6 synthesis. However, other kinase inhibitors including PD-98059 (a MEK inhibitor), SB-202190 (a p38 inhibitor), and H-89 (a protein kinase A inhibitor, data not shown) were less effective at blocking LPS-, IL-1␤-, and TNF-␣-induced IL-6 expression. While this paper was under review, Luo et al. (30) also showed that IL-1␤ can increase IL-6 synthesis in C2C12 cells and that this response was only partially blocked by inhibitors of the ERK and p38 pathways. SP-600125 did not inhibit anisomycin-stimulated ERK or p38 phosphorylation, suggesting that it is fairly specific for the JNK line of the MAP kinase family. Yet our initial results suggest that SP-600125 AJP-Regul Integr Comp Physiol • VOL

may also inhibit a kinase that lies upstream of I␬B-␣ and -⑀ since SP-600125 can inhibit the degradation of this key immunoregulatory protein. Thus we cannot completely rule out that SP-600125 has more than one site of action. JNK activation is not only associated with LPSinduced cytokine expression in muscle but is also strongly induced by eccentric exercise (6). Oxidative stress in patients with mitochondrial dysfunction also stimulates JNK activity in muscle fibers (15). Regimens that limit oxidative damage and JNK activity are likely to improve skeletal muscle function and protein synthesis. An IGF-I/IGFBP-3 complex restores the rate of muscle protein synthesis in septic rats to that found in control animals (49). IGF-I also inhibits apoptosis signal-regulating kinase 1 (ASK1), which is a MAP kinase kinase kinase (MAPKKK) required for JNK activation in response to TNF and oxidative stress (22). IGF-I and antioxidants may therefore have beneficial effects on muscle by indirectly inhibiting JNK activity. In contradistinction, activation of JNK by TNF-␣ may downregulate IGF-I mRNA expression in skeletal muscle and thereby contribute to muscle wasting (13, 20). In summary, our data show that LPS stimulates IL-6 mRNA expression in both mouse skeletal muscle and muscle cells with a similar time course. LPS-induced IL-6 synthesis was preferentially inhibited by SP-600125 (a JNK inhibitor). This compound inhibited IL-6 mRNA and protein similarly, suggesting that it changes steady-state levels of IL-6 mRNA but not translation of the protein. SP-600125 inhibited TNF␣-, IL-1␤-, and LPS-induced IL-6 synthesis similar to the anti-inflammatory glucocorticoid dexamethasone. SP-600125, but not dexamethasone, slightly accelerated IL-6 mRNA turnover and also prevented I␬B-␣/⑀ degradation and the subsequent LPS-induced expression of I␬B-␣ mRNA. Thus the efficacious nature of SP-600125 at inhibiting IL-6 synthesis in C2C12 myoblasts suggests the JNK inhibitor has potential usefulness in modulating the inflammatory response in skeletal muscle and other tissues. DISCLOSURES This work was supported in part by National Institutes of Health Grants GM-38032 and AA-11290. REFERENCES 1. Andersson U and Matsuda T. Human interleukin-6 and tumor necrosis factor-␣ production studied at a single-cell level. Eur J Immunol 19: 1157–1160, 1989. 2. Barbieri M, Ferrucci L, Ragno E, Corsi A, Bandinelli S, Bonafe M, Olivieri F, Giovagnetti S, Franceschi C, Guralnik JM, and Paolisso G. Chronic inflammation and the effect of IGF-I on muscle strength and power in older persons. Am J Physiol Endocrinol Metab 284: E481–E487, 2003. 3. Baumgarten G, Knuefermann P, Nozaki N, Sivasubramanian N, Mann DL, and Vallejo JG. In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J Infect Dis 183: 1617–1624, 2001. 4. Bennett BL, Sasaki DT, Murray BW, O’Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, and Anderson DW. SP600125, 285 • NOVEMBER 2003 •

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