c-jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves Src-dependent epidermal growth factor receptor transactivation

The phenotypic properties of the endothelium are subject to modulation by o xidative stress, and the c-dun N-terminal kinase (JNK) pathway is important in mediating cellular responses to stress, although activation of this pat hway in endothelial cells has not been fully characterized. Therefore, we e xposed endothelial cells to hydrogen peroxide (H2O2) and observed rapid act ivation of JNK within 15 min that involved phosphorylation of JNK and c-Jun and induction of AP-1 DNA binding activity, Inhibition of protein kinase C and phosphoinositide 3-kinase did not effect JNK activation. In contrast, the tyrosine kinase inhibitors, genistein, herbimycin A, and 4-amino-5-(4-c hlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) significantly atten uated H2O2-induced JNK activation as did endothelial cell adenoviral transf ection with a dominant-negative form of Src, implicating Src as an upstream activator of JNK, Activation of JNK by H2O2 was also inhibited by AG1478 a nd antisense oligonucleotides directed against the epidermal growth factor receptor (EGFR), implicating the EGFR in this process. Consistent with this observation, H2O2 stimulated EGFR tyrosine phosphorylation and complex for mation with Shc-Grb2 that was abolished by PP2, implicating Src in H2O2-ind uced EGFR activation. Tyrosine phosphorylation of the EGFR by H2O2 did not involve receptor autophosphorylation at Tyr(1173) as assessed by an autopho sphorylation-specific antibody. These data indicate that H2O2-induced JNK a ctivation in endothelial cells involves the EGFR through an Src-dependent p athway that is distinct from EGFR ligand activation. These data represent o ne potential pathway for mediating oxidative stress-induced phenotypic chan ges in the endothelium.