title: 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.
authors.affiliation: Institute of Medical Science, China Medical University, Taichung, Taiwan.
authors.forename: Hsiao-Yun
authors.initials: HY
authors.lastname: Lin
authors.forename: Chih-Hsin
authors.initials: CH
authors.lastname: Tang
authors.forename: Yi-Hung
authors.initials: YH
authors.lastname: Chen
authors.forename: I-Hua
authors.initials: IH
authors.lastname: Wei
authors.forename: Jia-Hong
authors.initials: JH
authors.lastname: Chen
authors.forename: Chih-Ho
authors.initials: CH
authors.lastname: Lai
authors.forename: Dah-Yuu
authors.initials: DY
authors.lastname: Lu
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D002461:Cell Line, Transformed; D002867:Chromones / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D009025:Morpholines / Q000494:pharmacology; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010457:Peptidoglycan / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D013211:Staphylococcus aureus; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D019138:Antibodies, Blocking; D002867:Chromones; D016207:Cytokines; D018836:Inflammation Mediators; D009025:Morpholines; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D010457:Peptidoglycan; D000081082:Phosphoinositide-3 Kinase Inhibitors; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D052247:Nitric Oxide Synthase Type II; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D000077191:Wortmannin