Redox regulation of TNF-alpha biosynthesis: augmentation by irreversible inhibition of gamma-glutamylcysteine synthetase and the involvement of an IkappaB-alpha/NF-kappaB-independent pathway in alveolar epithelial cells.
The pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, contribute to the exacerbation of pathophysiological conditions in the lung. The regulation of cytokine gene transcription 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 hypothesised that the lipopolysaccharide (LPS)-induced biosynthesis of TNF-alpha in vitro is regulated by redox equilibrium. Furthermore, the likely involvement of the IkappaB-alpha/NF-kappaB signalling transduction pathway in regulating LPS-induced TNF-alpha biosynthesis was unravelled. In a model of alveolar epithelial cells, we investigated 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) biosynthesis, in regulating LPS-mediated TNF-alpha production and the IkappaB-alpha/NF-kappaB pathway. Pretreatment with BSO, prior to exposure to LPS augmented, in a dose-dependent manner, LPS-induced TNF-alpha biosynthesis. In addition, BSO blockaded 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 oxidant-initiated and redox-mediated mechanisms regulating TNF-alpha biosynthesis and that the IkappaB-alpha/NF-kappaB signal transduction pathway is redox-sensitive but differentially involved in redox-dependent regulation of cytokine signalling in the alveolar epithelium.
Haddad
John J
JJ
Oxygen Signalling Group, Center for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Scotland, Dundee, UK. jhaddad@itsa.ucsf.edu
Saadé
Nayef E
NE
Safieh-Garabedian
Bared
B
D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors*; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000166:cytology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D004268:DNA-Binding Proteins; D004151:Dipeptides; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; C017341:gamma-glutamylcysteine