Department of Cellular and Molecular Physiology, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033
Submitted 8 January 2004 ; accepted in final form 23 July 2004
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ABSTRACT |
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interleukin-1 receptor-associated kinase; myotube; interleukin; dominant negative
Skeletal muscle contains three forms of NOS. Two of these isoforms, NOS1 and NOS3, are constitutively expressed. Because NOS2 is stimulated by bacterial cell wall components, it is also referred to as inducible NOS (iNOS). NOS2 is expressed in skeletal muscle in response to injury and repair. Dystrophin-deficient muscle has a reduction in the constitutive expression of NOS, and it undergoes a repetitive cycle of damage and repair because of the presence of tissue macrophages. This damage can be prevented by expression of a NOS transgene in muscle (45). NO may therefore be cytoprotective in this model of muscle damage. Yet, in mdx mice, there is overexpression of NOS2 that is reversed by somatic gene transfer of dystrophin (32). These data suggest that the NOS enzymes may play distinct roles in muscle damage and repair and that NO may influence both congenital (22) and acquired muscle-wasting diseases (10, 38).
Multiple investigators have examined the expression of NOS2 in the diaphragm and skeletal muscle after administration of lipopolysaccharide (LPS) (6, 25, 43). Boczkowski et al. (6) and Hussain et al. (25) found NOS2 protein to be abundant in skeletal muscle fibers between 6 and 48 h after LPS. In addition, Thompson et al. (43) found that muscle explants incubated with LPS exhibited NOS2 immunostaining in myocytes. Sambe et al. (40) demonstrated that LPS induces contractile dysfunction in respiratory muscles and that diaphragmatic contraction could be restored by either dexamethasone or a NOS inhibitor.
We (17) and others (21) have shown that human and murine myoblasts produce IL-1, IL-6, and tumor necrosis factor-
(TNF
) in response to various inflammatory stimuli. Furthermore, investigators at our laboratory recently demonstrated that LPS stimulates cytokine expression in murine skeletal muscle via Toll-like receptor-4 (TLR-4) signaling. Mice that harbor a mutation in TLR-4 (C3H/HeJ mice) have greatly reduced expression of IL-6 mRNA in skeletal muscle in response to LPS (17). Because LPS stimulates NOS2 expression in macrophages, we were interested to know whether NOS2 is also a downstream target of TLR4 in skeletal muscle and muscle cells. To address this question, we first challenged wild-type and C3H/HeJ mice with LPS to examine whether skeletal muscle NOS2 expression was TLR4 dependent. Second, LPS was added directly to C2C12 muscle cells to determine whether this stimulus was capable of increasing NOS2 protein and mRNA. A dominant negative form of TLR4 was used to ascertain whether LPS signals through the TLR4 receptor to increase NF-
activity in C2C12 cells. We also examined whether NOS2 is regulated at the transcriptional level and whether MAP kinase and stress-activated protein kinases regulate NOS2 expression. Finally, exogenous NO was added to myocytes to demonstrate the presence of a putative negative feedback loop for NOS2 expression.
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MATERIALS AND METHODS |
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Cell culture.
The C2C12 murine myoblast cell line used for all studies was purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in 100-mm petri dishes (Becton Dickinson, Franklin Lakes, NJ) and cultured in Eagle's minimum essential medium containing 10% bovine calf serum (BCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (25 µg/ml) (all purchased from Sigma, St. Louis, MO). Cells were grown to confluence and switched to fresh serum-containing medium before LPS, cytokines, or other agents were added. C2C12 cells were used at the myoblast stage. In some experiments, the cells were switched to medium containing 2% serum and allowed to differentiate into myotubes. Experiments were performed with E. coli LPS (026:B6; DIFCO Laboratories). A variety of compounds purchased from Calbiochem (La Jolla, CA), including SP-600125, PD-98059, SB-202190, MG-132, cycloheximide, and 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB), were used to characterize the response to LPS.
Transient transfection assays.
C2C12 cells were plated at 50% confluence in 24-well plates. Cells were switched to serum-free medium and transfected with a pNF-Luc reporter vector (BD Biosciences, Palo Alto, CA) and pSV-
-galactosidase control vector (Promega, Madison, WI). Both plasmids were added as a preformed complex with Lipofectamine 2000 at a 5:1 ratio of lipid to DNA. After 2 h, cells were allowed to recover in serum-containing medium for 16 h. Cell extracts were isolated at various times after the addition of LPS in reporter lysis buffer (Promega) and frozen until assay. In some transfection studies, the cells also received a third plasmid expressing a dominant negative form of the TLR4 receptor (pZERO-mTLR4; InvivoGen, San Diego, CA) in which the Toll interleukin-1 receptor (TIR) domain was deleted from the murine TLR4 gene. Luciferase reporter activity was measured with firefly luciferase assay reagents (Promega) on a Turner Biosystems luminometer (Sunnyvale, CA).
-Galactosidase activity was measured with a commercially available kit (Promega) and used to normalize for transfection efficiency.
RNA isolation and ribonuclease protection assay. Total RNA, DNA, and protein were extracted from C2C12 cells or tissues in a mixture of phenol and guanidine thiocyanate (TriReagent; Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol. RNA was separated from protein and DNA by the addition of bromochloropropane and precipitation in isopropanol. After being washed in 75% ethanol and resuspended in formamide, RNA samples were quantified by spectrophotometry. RNA (10 µg) was used for each assay. Riboprobes were synthesized from a custom multiprobe mouse template set containing a probe for NOS2, IL-6, and suppressor of cytokine signaling-3 (SOCS-3) mRNA detection (Pharmingen, San Diego, CA). The labeled riboprobe was hybridized with RNA overnight using a ribonuclease protection assay (RPA) kit according to the manufacturer's protocol (Pharmingen). Protected RNA were separated using a 5% acrylamide gel (19:1 dilution of acrylamide to bisacrylamide). Gels were transferred to blotting paper and dried under vacuum on a gel dryer. Dried gels were exposed to a phospho-imager screen (Molecular Dynamics, Sunnyvale, CA), and the resulting data were quantified using ImageQuant software and normalized to the murine ribosomal protein L32 mRNA signal in each lane.
Immunohistochemistry and nitrate measurements. NOS2 and myocyte enhancer factor (MEF)-2 were detected by performing immunohistochemistry. Briefly, cells were either grown in 10% BCS alone (control) or stimulated with LPS for 6 h and then fixed in 100% methanol for 10 min at 20°C. Fixed cells were washed and blocked with normal goat serum before primary antibodies were added to either NOS2 or MEF-2 (Santa Cruz Biotechnology, Santa Cruz, CA). The specificity of each antibody was determined by exclusion of the primary antibody. Antibodies were detected by adding a complex of biotin-labeled anti-rabbit IgG and horseradish peroxidase (HRP)-labeled avidin (Vectastain Elite; Vector Laboratories, Burlingame, CA). Slides were incubated for equal amounts of time with the HRP substrate 3-amino-9-ethylcarbazole (AEC). AEC formed a red-colored reaction product to demark the amount and position of each antigen.
For the detection of NO, conditioned medium from the cells was incubated with nitrate reductase to convert nitrate to nitrite, and total nitrite was measured colorimetrically as a colored azo dye product of the Griess reaction in a mircrotiter plate at 540 nm. The nitrite concentration of triplicate tissue culture wells was determined by comparing the absorbance of samples against a nitrite standard curve as supplied in a kit purchased from Stressgen Biotechnology (Victoria, BC, Canada).
Western blot analysis. Cell extracts were electrophoresed onto 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 NOS2, interleukin-1 receptor-associated kinase (IRAK-1), myosin heavy chain (MHC), or Erk kinase as previously described (18, 19). 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 HRP. Blots were briefly incubated with the components of an enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK). Dried blots were used to expose x-ray film for 13 min.
Statistics.
Values are means ± SE. Unless otherwise noted, each experimental condition was tested in triplicate and each experiment was repeated twice. Data were analyzed using analysis of variance followed by Student-Newman-Keuls test. Statistical significance was set at P < 0.05. For animal studies, we used four mice in the control group and six in the LPS group. NOS2 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 per hour. Half-lives were compared using a t-test where t = (m1 m2)/[)]. Statistical significance was set at P < 0.05.
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RESULTS |
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LPS also stimulated NOS2 protein expression in rat skeletal muscle 6 h after LPS but not saline administration (Fig. 2A). NOS2 coimmunoprecipitated with the muscle-specific caveolin, caveolin-3, in muscle homogenates when rats underwent injection with LPS. LPS did not alter the total amount of caveolin-3 in muscle homogenates (Fig. 2B). Differentiated C2C12 myotubes also expressed NOS2 protein as detected using immunohistochemistry. Myotubes exhibited diffuse staining throughout the cytoplasm (Fig. 2C, middle). The NOS2 pattern of expression was different from that observed for MEF2, a transcription factor that exhibits predominantly nuclear localization (Fig. 2C, right). Immunostaining for both antigens was dependent on the presence of the primary detection antibodies.
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DISCUSSION |
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Under in vivo conditions, NO has beneficial effects in skeletal muscle because it increases blood flow and enhances glucose uptake. However, if NO synthesis is sustained or excessive, it may also have damaging effects. It is not known how NOS2 is regulated in muscle cells. We have demonstrated for the first time that LPS stimulates the expression of NOS2 mRNA in skeletal muscle in vivo and in C2C12 myoblasts in vitro and that TLR4 is necessary for LPS to signal under both conditions. Mice with a mutation in TLR4 (C3H/HeJ) failed to express NOS2 mRNA in skeletal muscle after an intraperitoneal injection of LPS. By comparison, wild-type C3H/HeSnJ mice responded to LPS with a marked increase in the skeletal muscle expression of NOS2. The increase in NOS2 mRNA was not the result of a generalized increase in muscle mRNA, because the skeletal muscle mRNA levels of two housekeeping genes (GAPDH and L32) were unchanged. The LPS-induced expression of IB mRNA in murine skeletal muscle was also TLR4 dependent. Wild-type C3H/HeSnJ mice responded to LPS with a marked increase in the skeletal muscle expression of I
B, whereas C3H/HeJ mice failed to respond to LPS. Although LPS stimulated NOS2 expression fourfold, the response was transient and therefore most likely was not damaging to muscle. Experimental models that more closely mimic infection, such as cecal ligation and puncture or the infusion of whole bacteria, may produce a greater and/or more sustained rise in NOS2 mRNA (9).
Previous studies using immunohistochemistry have demonstrated that LPS stimulates the expression of NOS2 specifically in muscle fibers (6, 25, 43). In this study, we showed that NOS2 coimmunoprecipitates with caveolin-3. Caveolin-3 is a muscle-specific caveolin that may localize NOS2 to caveolae within the cell membrane. C2C12 myoblasts were also used to determine whether skeletal muscle cells per se respond to LPS or whether the increase in NOS2 expression in vivo resulted from lymphocytes or tissue macrophages that were sequestered in skeletal muscle at the time muscle was removed from the animal. LPS increased NOS2 immunostaining in C2C12 myotubes throughout the cytoplasm, and this staining pattern contrasted with the staining for a muscle transcription factor, MEF2, that exhibited predominantly nuclear localization.
LPS increased NOS2 mRNA expression dose and time dependently in C2C12 cells. NOS2 was also increased by peptidoglycan from S. aureus, suggesting that TLR2 receptors on the myocytes can also signal. Maximal NOS2 expression in response to both PAMP occurred 34 h after exposure and within a time frame consistent with that found for NOS2 activation in vivo. NOS2 protein was detected by performing Western blotting of C2C12 cell extracts from both myoblasts and myotubes. LPS also increased total nitrate in the conditioned medium, suggesting that NOS2 mRNA is transcribed and translated and that the enzyme is biologically active in myocytes.
The transient nature of NOS2 mRNA expression in vitro and in vivo suggests that NOS2 transcription is tightly regulated. We (17, 18) previously reported that LPS stimulates the degradation of I and I
in C2C12 cells but that these proteins are rapidly resynthesized to moderate the NF-
response. Although it is likely that continual activation of NF-
is necessary for sustained expression of NOS2, NO may also downregulate NOS2 expression by nitrosylating and inactivating NF-
(33). We speculate that in other, more severe models of sepsis, NOS2 expression may be more prolonged. However, it remains possible that even a transient exposure of muscle to NO and/or reactive nitrogen intermediates might have a prolonged effect on muscle function if critical transcription factors, enzymes, and/or proteins are irreversibly modified.
LPS from gram-negative bacteria binds to serum proteins and proteins on the cell surface. These proteins present LPS to TLR4, which initiates multiple signal transduction pathways (14). We found that LPS increased NOS2 mRNA in both C2C12 myoblasts and differentiated myotubes, suggesting that TLR4 is present on both cell types. Indeed, we detected TLR4 mRNA in both myoblasts and differentiated myotubes, and this finding is consistent with the detection of the TLR mRNA in rat skeletal muscle (29). TLR2 also was expressed in C2C12 myocytes and was markedly increased by LPS. These data suggest that LPS may prime muscle for a subsequent response to gram-positive bacteria, because these bacteria display the TLR2 ligand peptidoglycan as a component of the cell wall.
TLR4 receptor binding activates tyrosine and serine/threonine kinase cascades that ultimately impinge on transcription factors that activate the expression of inflammatory cytokines and the NOS2 gene. The most extensively studied signaling cascade for LPS involves the activation of NF-. This transcription factor is bound to an inhibitory protein (I
) that, upon phosphorylation and ubiquitination, is targeted for degradation by the 20S proteasome. Release of NF-
allows for nuclear translocation of the transcription factor and subsequent gene activation. We report that LPS-stimulated NOS2 protein expression in C2C12 cells is dependent on a functional proteasome because, when administered prophylactically, the proteasomal inhibitor MG-132 completely blocked LPS-induced NOS2 protein expression. In contrast, MG-132 was ineffective when added at a time point after which NF-
would have been presumed to be activated. In addition, LPS also stimulated the expression of an NF-
reporter plasmid in C2C12 cells, and this effect was prevented by pretreatment of the cells with MG-132. These results suggest that NF-
activation is necessary for LPS-induced NOS2 expression in C2C12 cells.
We transfected C2C12 cells with a dominant negative form of TLR4 to determine whether TLR4 mediates the affects of LPS on myocytes. The TLR4 dominant negative construct lacks its TIR domain and therefore cannot form an active complex with other TIR domain-containing proteins such as the adapter protein MyD88 (41). MyD88 recruits IRAK-1 to TLR4 and initiates at least two signaling cascades that lead to the activation of NF- and the JNK pathway. LPS stimulated the expression of an NF-
reporter plasmid in C2C12 cells, and this effect was completely abolished by cotransfection with dominant negative TLR4. Hence, TLR4 appears to be necessary for the initiation of a signaling cascade that activates NF-
-responsive genes in C2C12 cells and skeletal muscle. In contrast, IL-1
-stimulated NF-
reporter activity was not inhibited by coexpression of the dominant negative form of TLR4 in C2C12 cells, thus demonstrating the specificity of the response.
LPS stimulated the activation of IRAK both in vivo and in vitro in our study. In C2C12 cells, LPS increased the phosphorylation of IRAK-1 rapidly and transiently. The significance of this response is not known, because the IRAK-1 kinase domain has been found to be dispensable. IRAK-1 with a point mutation in the ATP binding pocket or even complete deletion of the kinase domain continues to activate NF- and JNK (26, 31). This suggests that other domains within the protein facilitate its activity. In addition, other IRAK family members, such as IRAK-4, are thought to be at least partially functionally redundant with IRAK-1 and can transphosphorylate the kinase. IRAK is both phosphorylated and degraded in response to LPS. Current studies suggest that the degradation of IRAK restrains IL-1 and TLR signaling and may be involved in the development of LPS tolerance. Noubir et al. (36) found that CD14 and TLR4 mediate a rapid degradation of IRAK-1, whereas complement receptor type III mediates a more sustained degradation of IRAK-1. The mechanism by which IRAK-1 is decreased in C2C12 cells, and the significance of this response to the metabolic and mechanical activity of skeletal muscle remains to be determined.
LPS-stimulated NOS2 mRNA expression in C2C12 cells required ongoing transcription and translation and was completely blocked by the transcriptional inhibitor DRB as well as by cycloheximide (an inhibitor of protein synthesis). These data suggest that an intermediary protein, such as a transcription factor that binds to the NOS2 promoter, must be synthesized at the outset for NOS2 mRNA to be expressed. It is noteworthy that NOS2 expression is regulated differently from IL-6 and TNF expression in this respect, because cycloheximide enhances the LPS-induced expression of these cytokines (17). In the current study, we show that cycloheximide potentiates IL-6 mRNA expression by stabilizing the message and extending its half-life approximately threefold. This response is similar to the ability of cycloheximide to stabilize IL-6 mRNA in lung epithelial cells and human fibroblasts (1, 39). Such a response is not observed for NOS2 mRNA.
MAP kinase pathways that include ERK, p38, and JNK can also be activated during infection or after exposure of immune cells to LPS (11). The JNK pathway is especially noteworthy because it is also activated by eccentric exercise (7) and oxidative stress that can damage skeletal muscle. JNK phosphorylates c-Jun on serine 63, which promotes the heterodimerization of c-Jun with other activator protein-1 (AP-1) transcription factors. The JNK pathway may be important for NOS2 mRNA expression in C2C12 cells, because the JNK inhibitor SP-600125 dose dependently inhibited NOS2 mRNA expression. SP-600125 also inhibited IL-6 synthesis, and therefore it is likely that SP-600125 is a broad inhibitor of the innate immune response and that JNK and the AP-1 transcription factors regulate a diverse set of inflammatory genes.
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 Erk1 and p38 (4). We cannot exclude the possibility that SP-600125 inhibits kinases other than JNK that may be responsible for NOS2 expression. Yet, other kinase inhibitors including PD-98059 (a MEK inhibitor) and SB-202190 (a p38 inhibitor) were less effective or completely ineffective at blocking LPS-induced NOS2 expression. Importantly, we also previously showed that the three inhibitors used in the present study are selective for their respective kinase pathways in C2C12 cells (18).
NO negatively regulates JNK activity by nitrosylating cysteine 116, a critical cysteine in the redox regulation of the kinase (37). Nitrosylation of JNK may be a component of a negative feedback loop in which generation of excess NO by NOS2 inhibits JNK and AP-1 transcription factors. Because JNK activity is important for NOS2 gene expression, excess NO hypothetically could downregulate NOS2 similarly to the end product inhibition observed with some enzymes. It is not known whether endogenous NO downregulates NOS2 in C2C12 cells, but we report that the NO donor SNP blunted LPS-induced NOS2 expression. SNP also blunted LPS-stimulated IL-6 mRNA but not SOCS-3 expression, suggesting that there are both NO-sensitive and -insensitive genes. Further experiments are needed to examine whether SNP alters the binding of c-Jun and NF- to their respective consensus DNA binding sites in the NOS2 promoter.
Lanone et al. (30) reported that overexpression of NOS2 during sepsis may have a negative impact on skeletal muscle function. They also found that NOS2 protein levels in skeletal muscle correlated with the severity of sepsis in humans. In addition, NO3 stably modified muscle proteins with nitrotyrosine and was associated with reduced contractile activity.
In summary, the results of the present study indicate that C2C12 myoblasts are a good model for examining the effects of LPS on NOS2 expression in skeletal muscle. LPS stimulates NOS2 mRNA expression in both murine skeletal muscle and muscle cells with a similar temporal pattern. LPS-induced NOS2 expression in skeletal muscle is TLR4 dependent. LPS-induced NF- activity in C2C12 myoblasts also requires TLR4. NOS2 expression requires ongoing transcription and translation, an active proteasome, and the p38 and JNK pathways. Sustained activation of NOS2 expression in skeletal muscle may participate in the erosion of lean body mass that occurs in inflammatory diseases such as sepsis and acquired immunodeficiency syndrome.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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