Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene...

We tested the hypothesis that activation of protein kinase C (PKC) and generation of oxidants are critical sequential signals mediating tumor necrosis factor (TNF)-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Stimulation of human pulmonary artery endothelial (HPAE) cells with TNF-alpha (100 U/ml) induced the activation of PKC and, subsequently, generation of oxidants. Pretreatment with calphostin C, a specific PKC inhibitor, prevented oxidant generation after TNF-alpha stimulation, indicating that PKC activation mediated the production of oxidants in HPAE cells. In contrast, pretreatment of HPAE cells with N-acetylcysteine, an antioxidant and a precursor of glutathione, failed to prevent PKC activation, indicating that PKC activation was not secondary to the oxidant production. These findings suggest that oxidant generation in endothelial cells occurs downstream of PKC activation. However, both PKC activation and oxidant generation were necessary for ICAM-1 mRNA expression because the pretreatment of HPAE cells with either calphostin C or N-acetylcysteine inhibited the TNF-alpha-induced activation of NF-kappaB and prevented the activation of ICAM-1 promoter. Prolonged exposure of HPAE cells to the phorbol ester, phorbol-12-myristate-13-acetate, which is known to deplete all except atypical PKC isozymes, failed to prevent TNF-alpha-induced ICAM-1 mRNA expression. We conclude that TNF-alpha-induced oxidant generation secondary to the activation of a phorbol ester-insensitive PKC isozyme signals the activation NF-kappaB and ICAM-1 gene transcription.