Peptidoglycan induces interlukin-6 expression through the TLR2 receptor, JNK, c-Jun, and AP-1 pathways in microglia

We recently reported that peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, induces NF-κB activation and microglia activation. However, PGN-regulated AP-1 activation and cytokine expression in microglia remains unclear. This study investigated how PGN influences the signaling pathway involved in IL-6 production in microglia. IL-6 mRNA and protein level up-regulation were increased by PGN in a concentration- and time-dependent manner. In addition, PGN increased toll-like receptor-2 (TLR2) expression, but not TLR4 receptor up-regulation. Administration of TLR2 siRNA or TLR2 neutralized antibody effectively inhibited PGN-induced IL-6 expression. In contrast, PGN-induced IL-6 mRNA and protein up-regulation were attenuated by the SAPK/JNK (c-Jun N-terminal kinases) inhibitor SP600125. Treatment of microglia with PGN increased levels of JNK phosphorylation and c-Jun phosphorylation, and up-regulated of JNK kinase activity. Treatment of microglia with AP-1 inhibitors (Tanshinone IIA and curcumin) effectively reduced PGN-induced IL-6 expression. PGN also significantly increased c-Fos and phospho-c-Jun translocation to nucleus. In line with this, PGN also increased AP-1-DNA complexes formation, as determined by the electrophoretic mobility shift assay. Furthermore, PGN also increased IL-6 transcription activity determined by transfection with IL-6 promoter construct plasmid. Co-transfection with dominant negative mutant of JNK (DN-JNK), or treatment with SP600125, curcumin, or Tanshinone IIA effectively antagonized PGN-increased IL-6 transcription activity. Our data demonstrate that PGN-induced IL-6 expression is mediated by AP-1 activation through the TLR2 and JNK/c-Jun pathways in microglia.