NF-kappa B 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-kappa B (NF-kappa B) in cultured human umbilical vein endothelial cells (HUVECs). Treatment for 7h with 100 microg/ml ox-HDL elicited a marked downregulation of I kappa B alpha and upregulation of the phosphorylated form of I kappa B alpha 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-kappa B to the nucleus and binding of NF-kappa B to NF-kappa B consensus oligonucleotides during ox-HDL exposure for 7h, suggesting that ox-HDL brings about NF-kappa B activation in endothelial cells. To clarify the mechanism of NF-kappa B 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 4h 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 I kappa B alpha protein induced by ox-HDL, demonstrating that increased generation of ROS by ox-HDL may be associated with NF-kappa B activation. Pretreatment with antibody against oxidized low-density lipoprotein receptor-1 (LOX-1) significantly suppressed the ox-HDL-induced downregulation of I kappa B alpha, suggesting that LOX-1 mediates NF-kappa B activation in endothelial cells stimulated with ox-HDL. Taking all of the above findings together, ox-HDL activates NF-kappa B via binding to LOX-1 on the cell surface, followed by enhancement of intracellular ROS production in endothelial cells.
Matsunaga
Toshiyuki
T
Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan. t_matsu@saitama-med.ac.jp
Hokari
Shigeru
S
Koyama
Iwao
I
Harada
Tsuyoshi
T
Komoda
Tsugikazu
T
D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D016328:NF-kappa B; D017382:Reactive Oxygen Species; C099252:oxidized low density lipoprotein; D010100:Oxygen