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Vol. 55, Issue 3, 575-583, March 1999
Department of Pharmacology, College of Medicine, University of
Illinois, Chicago, Illinois
We tested the hypothesis that activation of protein kinase C (PKC) and
generation of oxidants are critical sequential signals mediating tumor
necrosis factor (TNF)-
-induced activation of nuclear factor-
B
(NF-B) and transcription of the intercellular adhesion molecule
(ICAM)-1 gene. Stimulation of human pulmonary artery endothelial (HPAE)
cells with TNF- (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-
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--induced activation of NF-B 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--induced ICAM-1 mRNA expression. We conclude that TNF--induced oxidant generation secondary to the activation of a
phorbol ester-insensitive PKC isozyme signals the activation NF-B
and ICAM-1 gene transcription.
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