Peptidoglycan-induced IL-6 production in RAW 264.7 macrophages is mediated by cyclooxygenase-2, PGE2/PGE4 receptors, protein kinase A, I kappa B kinase, and NF-kappa B.
In this study, we investigated the signaling pathway involved in IL-6 production caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused concentration- and time-dependent increases in IL-6, PGE(2), and cAMP production. PGN-mediated IL-6 production was inhibited by a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), a PGE(2) (EP2) antagonist (AH6809), a PGE(4) (EP4) antagonist (AH23848), and a protein kinase A (PKA) inhibitor (KT5720), but not by a nonselective NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester). Furthermore, PGE(2), an EP2 agonist (butaprost), an EP2/PGE(3) (EP3)/EP4 agonist (misoprostol), and misoprostol in the presence of AH6809 all induced IL-6 production, whereas an EP1/EP3 agonist (sulprostone) did not. PGN caused time-dependent activations of IkappaB kinase alphabeta (IKKdbeta) and p65 phosphorylation at Ser(276), and these effects were inhibited by NS398 and KT5720. Both PGE(2) and 8-bromo-cAMP also caused IKKdbeta kinase alphabeta phosphorylation. PGN resulted in two waves of the formation of NF-kappaB-specific DNA-protein complexes. The first wave of NF-kappaB activation occurred at 10-60 min of treatment, whereas the later wave occurred at 2-12 h of treatment. The PGN-induced increase in kappaB luciferase activity was inhibited by NS398, AH6809, AH23848, KT5720, a protein kinase C inhibitor (Ro31-8220), and a p38 MAPK inhibitor (SB203580). These results suggest that PGN-induced IL-6 production involves COX-2-generated PGE(2), activation of the EP2 and EP4 receptors, cAMP formation, and the activation of PKA, protein kinase C, p38 MAPK, IKKdbeta, kinase alphabeta, p65 phosphorylation, and NF-kappaB. However, PGN-induced NO release is not involved in the signaling pathway of PGN-induced IL-6 production.
Chen
Bing-Chang
BC
School of Respiratory Therapy, Graduate Institute of Biomedical Technology, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Liao
Chiao-Chun
CC
Hsu
Ming-Jen
MJ
Liao
Yi-Ting
YT
Lin
Chia-Chin
CC
Sheu
Joen-Rong
JR
Lin
Chien-Huang
CH
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D051546:Cyclooxygenase 2 / Q000378:metabolism / Q000502:physiology*; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D015850:Interleukin-6 / Q000096:biosynthesis*; D008264:Macrophages / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D010457:Peptidoglycan / Q000494:pharmacology*; D010766:Phosphorylation; D018078:Receptors, Prostaglandin E / Q000037:antagonists & inhibitors / Q000502:physiology*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D013211:Staphylococcus aureus / Q000276:immunology; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000378:metabolism
D052246:Cyclooxygenase 2 Inhibitors; D015850:Interleukin-6; D016328:NF-kappa B; D010457:Peptidoglycan; C545651:Ptger4 protein, mouse; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D051996:Transcription Factor RelA; D012694:Serine; D000242:Cyclic AMP; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D051550:I-kappa B Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone