Redox regulation of TNF-α biosynthesis: Augmentation by irreversible inhibition of γ-glutamylcysteine synthetase and the involvement of an IκB-α/NF-κB-independent pathway in alveolar epithelial cells

Abstract

The pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, contribute to the exacerbation of pathophysiological conditions in the lung. The regulation of cytokine gene transcription involves the reduction–oxidation (redox)-sensitive nuclear factor-κB (NF-κB), the activation of which is mediated through an upstream kinase that regulates the phosphorylation and subsequent degradation of inhibitory-κB (IκB)-α, the major cytosolic inhibitor of NF-κB. It was hypothesised that the lipopolysaccharide (LPS)-induced biosynthesis of TNF-α in vitro is regulated by redox equilibrium. Furthermore, the likely involvement of the IκB-α/NF-κB signalling transduction pathway in regulating LPS-induced TNF-α 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 γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in glutathione (GSH) biosynthesis, in regulating LPS-mediated TNF-α production and the IκB-α/NF-κB pathway. Pretreatment with BSO, prior to exposure to LPS augmented, in a dose-dependent manner, LPS-induced TNF-α biosynthesis. In addition, BSO blockaded the phosphorylation of IκB-α, reduced its degradation, thereby allowing its cytosolic accumulation, and subsequently inhibited the activation of NF-κB. These results indicate that there are oxidant-initiated and redox-mediated mechanisms regulating TNF-α biosynthesis and that the IκB-α/NF-κB signal transduction pathway is redox-sensitive but differentially involved in redox-dependent regulation of cytokine signalling in the alveolar epithelium.