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). CONCLUSION: These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion 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). CONCLUSION: These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion in human endothelial cells.
Authors: Dongze Zhang; Jinxu Liu; Huiyin Tu; Robert L Muelleman; Kurtis G Cornish; Yu-Long Li Journal: Hypertension Date: 2013-10-07 Impact factor: 10.190