Abstract
We tested the hypothesis that activation of protein kinase C (PKC) and generation of oxidants are critical sequential signals mediating tumor necrosis factor (TNF)-α-induced activation of nuclear factor-κB (NF-κB) and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Stimulation of human pulmonary artery endothelial (HPAE) cells with TNF-α (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-α stimulation, indicating that PKC activation mediated the production of oxidants in HPAE cells. In contrast, pretreatment of HPAE cells withN-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-α-induced activation of NF-κB 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-α-induced ICAM-1 mRNA expression. We conclude that TNF-α-induced oxidant generation secondary to the activation of a phorbol ester-insensitive PKC isozyme signals the activation NF-κB and ICAM-1 gene transcription.
Footnotes
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Send reprint requests to: Dr. Arshad Rahman, Department of Pharmacology, The University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612-7343. E-mail: ARahman{at}uic.edu
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↵1 Present address: Department of Pulmonary Medicine, Jichi Medical School, Tochigi, Japan 329-04.
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This work was supported by National Institute of Health Grants HL27016, HL46350, and HL45638.
- Abbreviations:
- ICAM-1
- intercellular adhesion molecule-1
- C-DCDHF-DA
- 5- (and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate bis(acetoxy-methyl) ester
- PMA
- phorbol-12-myristate-13-acetate
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- TNF-α
- tumor necrosis factor-α
- IL-1β
- interleukin-1β, HPAE, human pulmonary artery endothelial
- LUC
- luciferase
- NF-κB
- nuclear factor-κB
- EGM
- endothelial cell growth medium
- PMSF
- phenylmethylsulfonyl fluoride
- SSC
- standard saline citrate
- Received July 22, 1998.
- Accepted November 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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