Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-kappa B in human endothelial cells.
OBJECTIVE
Nuclear factor-kappa B (NF-kappa 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-kappa B, the signaling pathway of oxygen free radical-induced NF-kappa 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-kappa 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 kappa B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against I kappa B alpha, phosphorylated by I kappa B alpha kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-kappa B luciferase reporter gene assay was also performed.


RESULTS
The treatment of the cells with superoxide anion for 60 min increased the NF-kappa B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of I kappa B alpha within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of I kappa B alpha. Pretreatment of the cells with calphostin C (100-400 nmol/l) and chelerythrine chloride (5-10 mumol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-kappa B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-kappa 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).


CONCLUSIONS
These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion in human endothelial cells.
Ogata
N
N
Department of Cardiovascular Medicine, Kumamoto University School of Medicine, Japan.
Yamamoto
H
H
Kugiyama
K
K
Yasue
H
H
Miyamoto
E
E
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000704:Analysis of Variance; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D003864:Depression, Chemical; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D016328:NF-kappa B / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D018384:Oxidative Stress; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D016328:NF-kappa B; D009281:Naphthalenes; D011506:Proteins; D013481:Superoxides; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D004247:DNA; D011493:Protein Kinase C; C058819:calphostin C