Oxidant-sensitive and phosphorylation-dependent activation of NF-kappa B and AP-1 in endothelial cells

Relatively low concentrations of reactive oxygen cause reversible alterations of endothelial cell signal transduction and gene transcription. The hypothesis that low levels of oxidant stress activate retention of trans-acting proteins in the nucleus was investigated by determining time and dose requirements for oxidant-stimulated nuclear protein binding to consensus DNA sequences for nuclear factor (NF)-kappa B or activator protein 1 (AP-1). Nuclear proteins were extracted from low passage porcine aortic endothelial cells 15 min to 24 h after addition of increasing concentrations of H2O2 Electrophoretic mobility shift assays demonstrated that protein binding to NF-kappa B and AP-1 sequences increases over 1-2 h after stress relative to time-matched controls and resolves by 24 h. The selective protein kinase C inhibitor, calphostin C, prevents similar to 30% of this increase. Inhibition of tyrosine kinase activity by herbimycin A (5 mu M) completely inhibits the response to H2O2. Exposure of intact cells to H2O2 increases substrate phosphorylation in pp60(src) immunoprecipitates. The activity of pp60(src) in immunoprecipitates from control cells or of recombinant pp60(src) increases after in vitro addition of H2O2. H2O2-stimulated pp60(src) activity is reduced by pretreatment of the enzyme preparation with N-acetylcysteine. These data indicate that oxidants increase nuclear levels of trans-acting factors in endothelial cells and that these increases require oxidant-sensitive changes in both tyrosine and serine/threonine phosphorylations.