title: Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase.
abstract: It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide ( [Formula: see text] ) production, respectively. The generation of both nitrate and [Formula: see text] in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated [Formula: see text] production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin.
authors.affiliation: Division of Digestive Diseases, University of Cincinnati, Cincinnati, OH 45267-0595, USA.
authors.forename: Gila
authors.initials: G
authors.lastname: Idelman
authors.affiliation: Division of Digestive Diseases, University of Cincinnati, Cincinnati, OH 45267-0595, USA.
authors.forename: Darcey L H
authors.initials: DLH
authors.lastname: Smith
authors.affiliation: Division of Digestive Diseases, University of Cincinnati, Cincinnati, OH 45267-0595, USA. Electronic address: zuckersd@ucmail.uc.edu.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Zucker
mesh_terms: D000818:Animals; D001663:Bilirubin / Q000494:pharmacology*; D002460:Cell Line; D016207:Cytokines / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism*; D009566:Nitrates / Q000032:analysis; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013482:Superoxide Dismutase / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D016207:Cytokines; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008070:Lipopolysaccharides; D009566:Nitrates; D000072317:Polychlorinated Dibenzodioxins; D017382:Reactive Oxygen Species; D018336:Receptors, Aryl Hydrocarbon; D051197:Toll-Like Receptor 4; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D001663:Bilirubin
keywords: Aryl hydrocarbon receptor (AhR); Bilirubin; Hypoxia-inducible factor (HIF); NADPH oxidase; Nitric oxide synthase; Superoxide; Toll-like receptor 4 (TLR4)