title: Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase.
abstract: 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-alpha, and IL-1beta, 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-1beta, or TNF-alpha 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 LPS-stimulated 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 IkappaB-alpha/epsilon and LPS-stimulated expression of IkappaB-alpha mRNA. Yet, only SP-600125 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.
authors.affiliation: Dept. of Cellular and Molecular Physiology, Penn State Univ. College of Medicine, Hershey Medical Center: H166, Hershey, PA 17033, USA. rfrost@psu.edu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Frost
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Nystrom
authors.forename: Charles H
authors.initials: CH
authors.lastname: Lang
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002454:Cell Differentiation; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D007375:Interleukin-1 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000097:blood / Q000235:genetics*; D048031:JNK Mitogen-Activated Protein Kinases; D007976:Leupeptins / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008809:Mice, Inbred C3H; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology; D032446:Myoblasts / Q000166:cytology / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000873:Anthracenes; D000893:Anti-Inflammatory Agents; D000970:Antineoplastic Agents; D015853:Cysteine Proteinase Inhibitors; D007375:Interleukin-1; D015850:Interleukin-6; D007976:Leupeptins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C432165:pyrazolanthrone; D003907:Dexamethasone; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde