title: Oxidative stress reprograms lipopolysaccharide signaling via Src kinase-dependent pathway in RAW 264.7 macrophage cell line.
abstract: Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-kappa B translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-kappa B translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-kappa B translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-kappa B translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-kappa B translocation in oxidant-treated macrophages, this strategy had no effect on NF-kappa B translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion.
authors.affiliation: Department of Surgery, University of Toronto and University Health Network, Toronto, Ontario M5G 2C4, Canada.
authors.forename: Rachel G
authors.initials: RG
authors.lastname: Khadaroo
authors.forename: Andras
authors.initials: A
authors.lastname: Kapus
authors.forename: Kinga A
authors.initials: KA
authors.lastname: Powers
authors.forename: Myron I
authors.initials: MI
authors.lastname: Cybulsky
authors.forename: John C
authors.initials: JC
authors.lastname: Marshall
authors.forename: Ori D
authors.initials: OD
authors.lastname: Rotstein
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011233:Precipitin Tests; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015427:Reperfusion Injury; D015398:Signal Transduction; D050857:Synaptotagmins; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; C000606205:Nfkbia protein, mouse; D016877:Oxidants; D050857:Synaptotagmins; D000072000:NF-KappaB Inhibitor alpha; D014443:Tyrosine; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D010100:Oxygen