title: L-Buthionine-(S,R)-sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, augments LPS-mediated pro-inflammatory cytokine biosynthesis: evidence for the implication of an IkappaB-alpha/NF-kappaB insensitive pathway.
abstract: The pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, contribute to the exacerbation of pathophysiological conditions in the lung. The regulation of cytokines involves the reduction-oxidation (redox)-sensitive nuclear factor-kappaB (NF-kappaB), the activation of which is mediated through an upstream kinase that regulates the phosphorylation and subsequent degradation of inhibitory-kappaB (IkappaB)-alpha, the major cytosolic inhibitor of NF-kappaB. It was hypothesized that lipopolysaccharide (LPS)-induced biosynthesis of TNF-alpha and IL-6 in vitro is tightly regulated by redox equilibrium. Furthermore, the likely involvement of the IkappaB-alpha/NF-kappaB signalling transduction pathway in mediating redox-dependent regulation of LPS-induced cytokine biosynthesis was revealed. Using alveolar epithelial cells, the role of L-buthionine-(S,R)-sulfoximine (BSO), a specific and irreversible inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in glutathione (GSH - an antioxidant thiol) biosynthesis, in regulating LPS-mediated TNF-alpha and IL-6 production and the IkappaB-alpha/NF-kappaB signalling pathway was investigated. Pre-treatment with BSO, prior to exposure to LPS augmented, in a dose-dependent manner, LPS-induced TNF-alpha and IL-6 biosynthesis, an effect associated with the induction of intracellular accumulation of reactive oxygen species (ROS). Interestingly, BSO blocked the phosphorylation of IkappaB-alpha, reduced its degradation, thereby allowing its cytosolic accumulation, and subsequently inhibited the activation of NF-kappaB. These results indicate that there are ROS and redox-mediated effects regulating pro-inflammatory cytokines, and that the IkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially involved in mediating redox-dependent regulation of cytokine signaling.
authors.affiliation: Neuroscience Research Laboratory, University of California at San Francisco, CA 94143-0542, USA. haddadj@anesthesia.ucsf.edu [corrected]
authors.forename: J J
authors.initials: JJ
authors.lastname: Haddad
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors*; D020672:I-kappa B Proteins; D015850:Interleukin-6 / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology*; D011247:Pregnancy; D011650:Pulmonary Alveoli / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D015342:DNA Probes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase