Peptidoglycan-Induced IL-6 Production in RAW 264.7 Macrophages Is Mediated by Cyclooxygenase-2, PGE2/PGE4 Receptors, Protein Kinase A, IκB Kinase, and NF-κB

Abstract

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, PGE2, and cAMP production. PGN-mediated IL-6 production was inhibited by a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), a PGE2 (EP2) antagonist (AH6809), a PGE4 (EP4) antagonist (AH23848), and a protein kinase A (PKA) inhibitor (KT5720), but not by a nonselective NO synthase inhibitor (NG-nitro-l-arginine methyl ester). Furthermore, PGE2, an EP2 agonist (butaprost), an EP2/PGE3 (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 IκB kinase αβ (IKKdβ) and p65 phosphorylation at Ser276, and these effects were inhibited by NS398 and KT5720. Both PGE2 and 8-bromo-cAMP also caused IKKdβ kinase αβ phosphorylation. PGN resulted in two waves of the formation of NF-κB-specific DNA-protein complexes. The first wave of NF-κB activation occurred at 10–60 min of treatment, whereas the later wave occurred at 2–12 h of treatment. The PGN-induced increase in κB 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 PGE2, activation of the EP2 and EP4 receptors, cAMP formation, and the activation of PKA, protein kinase C, p38 MAPK, IKKdβ, kinase αβ, p65 phosphorylation, and NF-κB. However, PGN-induced NO release is not involved in the signaling pathway of PGN-induced IL-6 production.