PKCβ/Egr-1Involved In Ox-LDL-induced Pro-inflammatory Mechanism In Vascular Endothelial Cells

Objective：To investigate the effect of oxidized low density lipoprotein(ox-LDL) onintercellular cell adhesion molecular (ICAM-1) expression in vascular endothelialcells, and explore the possible mechanism involved in PKCβ/MAKP/Egr-1signaling.Methods： Human umbilical vein endothelial cell line EA. hy926were used in thisstudy. Cells were incubated with various concentrations of ox-LDL for6h, theexpression of ICAM-1protein was tested by western blot to screen the optimizedintervention concentration. Cells then were stimulated with the optimizedconcentration of ox-LDL for different periods, ICAM-1, PKCβⅡ,MAPKs and Egr-1protein expression were detected by western blot, phosphorylation of intracellularPKCβⅡ by immunofluorecence, and the expression of Egr-1mRNA by real-timePCR. In order to determine the roles of PKCβⅡ on theabove effects induced byox-LDL, cells were pre-incubated with100nmol/L PKCβ inhibitor (LY333531) for1hbefore exposure to10μg/ml ox-LDL, the phosphorylation level of MAPKs and theexpression of Egr-1and ICAM-1were evaluated at the peak duration respectively.Results: Ox-LDL induced ICAM-1expression in a dose-dependent manner, themaximal effect was at10μg/ml.10μg/ml ox-LDL induced ICAM-1protein expressionin a time-dependent manner, with the peak at6h after stimulation, which is2.1foldscompared to the control group (p<0.05).10μg/ml ox-LDL increased the levels ofp-PKCβⅡ,p-ERK1/2and p-JNK, the peaks were at15min,15min/45min, and30minrespectively, and the increase was3.6,1.4/1.5, and1.2folds compared to the controlgroup respectively（p<0.05）.10μg/ml ox-LDL increased Egr-1mRNA and proteinexpression2.5and1.5folds at45min after stimulation respectively(p<0.05）；.Blockage of the activation of PKCβⅡ with PKCβ inhibitor(LY333531) successfullyinhibited the phosphorylation level of PKCβⅡ induced by ox-LDL. Moreover,LY333531decreased the ox-LDL-induced phosphorylation of ERK1/2/JNK（25.1%and60%）, the activation of Egr-1（mRNA54.6%, protein32.8%）and the expression of ICAM-1（50%）.Conclusion：PKCβ is involved in the mechanism of ox-LDL-induced ICAM-1expression in vascular endothelial cells. ERK1/2、JNK and Egr-1are downstreamregulatory molecules of PKCβ, and probably involved in the signaling pathway ofox-LDL-induced ICAM-1expression.