© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-
B in human endothelial cells
aDepartment of Cardiovascular Medicine, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
bDepartment of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
* Corresponding author. Tel.: +81-96-373-5076; fax: +81-96-373-5078 hideyuki{at}gpo.kumamoto-u.ac.jp
Objective: Nuclear factor-kappa B (NF-
B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-B, the signaling pathway of oxygen free radical-induced NF-B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-B activation in human umbilical vein endothelial cells (HUVECs). Methods: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against IB
, phosphorylated by IB kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-B luciferase reporter gene assay was also performed. Results: The treatment of the cells with superoxide anion for 60 min increased the NF-B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of IB within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of IB. Pretreatment of the cells with calphostin C (100–400 nmol/l) and chelerythrine chloride (5–10 µmol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). Conclusion: These results suggest that PKC is involved in the activation of NF-B by superoxide anion in human endothelial cells.
KEYWORDS Atherosclerosis; Endothelial function; Free radicals; Protein kinases; Signal transduction
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