200 - NADPH de novo Biosynthesis Regulates NOX4-Induced Cell Growth and Cellular Bioenergetics

Abstract

Reactive oxygen species play an important role in cellular events controlling growth, differentiation and adaptation to stress. NADPH oxidase 4 (NOX4) is an inducible enzyme belonging to the family of NADPH oxidases (NOX), expressed in several cell types. As opposed to other members of NADPH oxidases family, NOX4 does not produce superoxide radical anion but releases hydrogen peroxide (H2O2). We postulated that NOX4 regulates cell resistance to injury by mechanisms dependent on de novo NADPH biosynthesis coupled to stimulated mitochondrial bioenergetics. NAD depletion by FK866 (10 nM, 24h) was followed by 44% decrease in NADPH in HEK-NOX4 cells while only a 20% decrease was detected in HEK cells. This result indicated a greater dependence of HEK-NOX4 cells activity and consumption than HEK on de novo NADPH synthesis. Extracellular flux analysis was used to monitor cellular oxygen consumption rates. HEK-NOX4 consistently showed a higher basal rate (72.7 vs 42.7 pmol/min/mg protein) than HEK cells, which was offset by pre-treatment with FK866. Since the extracellular flux assay media contained pyruvate, this inhibition was taken as indication of differences in mitochondrial mass. Consistently, western blot analysis revealed lower mitochondria proteins VDAC and COX4 in HEK than HEK-NOX4 cells. Depletion of NADPH content in FK866-treated HEK-NOX4 diminished H2O2 release as verified with coumarin boronate (CBA) assay. Supplementation of cells with nicotinamide mononucleotide, a precursor of both NAD and NADPH, 24 h prior assays increased both the release of H2O2 and mitochondrial respiration in HEK-NOX4 but not HEK cells. In combination, these results indicate that de novo NAD/NADPH by regulating NOX4 derived H2O2 improves cell growth and bioenergetics, which may be an important mechanism conferring cell resistance to injury.