Antioxidant/pro-oxidant equilibrium regulates HIF-1 alpha and NF-kappa B redox sensitivity - Evidence for inhibition by glutathione oxidation in alveolar epithelial cells

The O-2 and redox-sensitive transcription factors hypoxia inducible factor- 1 alpha (HIF-1 alpha) and nuclear factor-kappa B (NF-kappa B) are different ially regulated in the alveolar epithelium over fetal to neonatal oxygen te nsions. We have used fetal alveolar type II epithelial cells to monitor the ir regulation in association with redox responsiveness to antioxidant pretr eatment in vitro. N-Acetyl-L-cysteine, a glutathione (GSH) precursor and a potent scavenger of reactive oxygen species, induced HIF-1 alpha and amelio rated NF-kappa B nuclear abundance and DNA binding activity, respectively, in a dose-dependent manner. Analysis of variations in glutathione homeostas is at ascending Delta pO(2) regimen with N-acetyl-(L)-cysteine reveals incr eased GSH at the expense of the oxidized form of glutathione (GSSG;), there by shifting GSH/GSSG into reduction equilibrium. Pyrrolidine dithiocarbamat e (PDTC), which exerts both antioxidant and pro-oxidant effects, provoked a substantial increase in HIF-1 alpha nuclear abundance, with no apparent ef fect on its activation. PDTC reduced NF-kappa B nuclear abundance and its i nhibitory effects on binding activity are dose-dependent. Assessment of glu tathione homeostasis with PDTC shows increasing levels of GSSG at the expen se of GSH, lowering GSH/GSSG in favor of an oxidative equilibrium. Our resu lts indicate the hypoxic activation of HIF-1 alpha and the hyperoxic induct ion of NF-kappa B in the fetal epithelium is redox-sensitive and, thus, tig htly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key regulatory component for determining genetic responsiveness to oxidan t/antioxidant imbalance in normal lung development and pathophysiological c onditions.