NF-κB activation in endothelial cells treated with oxidized high-density lipoprotein

We first determined whether oxidized high-density lipoprotein (ox-HDL) activates transcription factor nuclear factor-kappaB (NF-kappaB) in cultured human umbilical vein endothelial cells (HUVECs). Treatment for 7 h with 100 mug/ml ox-HDL elicited a marked downregulation of IkappaBalpha and upregulation of the phosphorylated form Of IkappaBalpha in HUVECs in a manner dependent on the dose of ox-HDL. Electrophoretic mobility shift assay in nuclear fraction from HUVECs showed translocation of NF-icB to the nucleus and binding of NF-kappaB to NF-kappaB consensus oligonucleotides during ox-HDL exposure for 7h, suggesting that ox-HDL brings about NF-kappaB activation in endothelial cells. To clarify the mechanism of NF-kappaB activation in HUVECs treated with ox-HDL, we investigated the effect of ox-HDL treatment on intracellular production of reactive oxygen species (ROS) in HUVECs. Ox-HDL induced a significant dose-dependent increase in ROS production during 4 h incubation and this enhanced production of ROS was inhibited in the presence of probucol or diphenylene iodonium (DPI), an inhibitor of NADPH oxidase. In addition, pretreatment with probucol or DPI suppressed the phosphorylation and degradation of IkappaBalpha protein induced by ox-HDL, demonstrating that increased generation of ROS by ox-HDL may be associated with NF-kappaB activation. Pretreatment with antibody against oxidized low-density lipoprotein receptor-1 (LOX-1) significantly suppressed the ox-HDL-induced downregulation of IkappaBalpha, suggesting that LOX-1 mediates NF-kappaB activation in endothelial cells stimulated with ox-HDL. Taking all of the above findings together, ox-HDL activates NF-kappaB via binding to LOX-1 on the cell surface, followed by enhancement of intracellular ROS production in endothelial cells. (C) 2003 Elsevier Science (USA). All rights reserved.