Peptidoglycan enhances proinflammatory cytokine expression through the TLR2 receptor, MyD88, phosphatidylinositol 3-kinase/AKT and NF-kappaB pathways in BV-2 microglia

Abstract

In this study, we investigated the signaling pathways involved in inflammatory production caused by peptidoglycan (PGN), a cell wall component of the gram-positive bacterium, in BV-2 microglia. PGN caused a concentration- and time-dependent increase in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein levels. In addition, PGN also induced IL-1 beta, TNF-alpha and IL-6 mRNA up-regulation in a concentration-dependent manner. Moreover, PGN also increased Toll-like receptor 2 (TLR2) expression in BV-2 microglia. Administration of TLR2 neutralizing antibody effectively inhibited PGN-induced iNOS and COX-2 expression. On the other hand, PGN-induced iNOS and COX-2 up-regulation were attenuated by PI3-kinase inhibitors (LY294002 and wortmannin), and an AKT inhibitor. Treatment of BV-2 microglia with PGN caused a time-dependent activation of PI3-kinase (p85) and AKT. PGN-induced PI3-kinase/AKT activation, iNOS and COX-2 expression were also inhibited by MyD88 inhibitory peptide. Treatment of cells with NF-kappaB inhibitor (pyrrolidine dithiocarbamate), I kappaB alpha phosphorylation inhibitor (Bay 117082), or I kappaB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone) inhibited PGN-induced iNOS and COX-2 expression. Furthermore, stimulation of cells with PGN also activated IKK alpha/beta, I kappaB alpha phosphorylation, I kappaB alpha degradation, p65 phosphorylation at Ser(536), and increased kappaB-luciferase activity. PGN-induced IKK alpha/beta phosphorylation, I kappaB alpha phosphorylation, and I kappaB alpha degradation were further inhibited by pre-treatment with PI3-kinase inhibitors. Moreover, PGN-mediated increase of kappaB-luciferase activity was also inhibited by pre-transfection with dominant-negative mutants of p85, AKT, IKK alpha or IKK beta. Our data demonstrate that PGN-induced iNOS, COX-2 and proinflammatory cytokine expression was mediated through the TLR2/MyD88/PI3-kinase/AKT pathway, which in turn initiates IKK alpha/beta and NF-kappaB activation in BV-2 microglia.