Redox regulation of TNF-alpha biosynthesis: Augmentation by irreversible inhibition of gamma-glutamylcysteine synthetase and the involvement of an I kappa B-alpha/NF-kappa B-independent pathway in alveolar epithelial cells

The pro-inflammatory cytokincs, 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-(SR)-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-a 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. (C) 2002 Elsevier Science Inc. All rights reserved.