Protein kinase C-activated oxidant generation in endothelial cells signalsintercellular adhesion molecule-1 gene transcription

We tested the hypothesis that activation of protein kinase C (PKC) and gene ration of oxidants are critical sequential signals mediating tumor necrosis factor (TNF)-alpha-induced activation of nuclear factor-kappa B (NF-kappa B) and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Stimulation of human pulmonary artery endothelial (HPAE) cells with TNF-alp ha (100 U/ml) induced the activation of PKC and, subsequently, generation o f oxidants. Pretreatment with calphostin C, a specific PKC inhibitor, preve nted oxidant generation after TNF-alpha stimulation, indicating that PKC ac tivation mediated the production of oxidants in HPAE cells. In contrast, pr etreatment of HPAE cells with N-acetytcysteine, an antioxidant and a precur sor of glutathione, failed to prevent PKC activation, indicating that PKC a ctivation was not secondary to the oxidant production. These findings sugge st that oxidant generation in endothelial cells occurs downstream of PKC ac tivation. However, both PKC activation and oxidant generation were necessar y for ICAM-1 mRNA expression because the pretreatment of HPAE cells with ei ther calphostin C or N-acetylcysteine inhibited the TNF-alpha-induced activ ation of NF-kappa B and prevented the activation of ICAM-1 promoter. Prolon ged exposure of HPAE cells to the phorbol ester, phorbol-12-myristate-13-ac etate, which is known to deplete all except atypical PKC isozymes, failed t o prevent TNF-alpha-induced ICAM-1 mRNA expression. We conclude that TNF-al pha-induced oxidant generation secondary to the activation of a phorbol eat er-insensitive PKC isozyme signals the activation NF-kappa B and ICAM-1 gen e transcription.