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 approximately 30% of this increase. Inhibition of tyrosine kinase activity by herbimycin A (5 microM) completely inhibits the response to H2O2. Exposure of intact cells to H2O2 increases substrate phosphorylation in pp60src immunoprecipitates. The activity of pp60src in immunoprecipitates from control cells or of recombinant pp60src increases after in vitro addition of H2O2. H2O2-stimulated pp60src 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.
Barchowsky
A
A
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Munro
S R
SR
Morana
S J
SJ
Vincenti
M P
MP
Treadwell
M
M
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D002352:Carrier Proteins; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D010447:Peptide Hydrolases