Reactive oxygen species mediate oxidized low-density lipoprotein-induced endothelin-1 gene expression via extracellular signal-regulated kinase in vascular endothelial cells.
BACKGROUND
Oxidized low-density lipoprotein (oxLDL) promotes expression and secretion of endothelin-1 (ET-1), however, the precise mechanism involved is unclear. This study was designed to identify the regulatory mechanism of oxLDL-induced ET-1 expression in endothelial cells.


METHODS
ET-1 mRNA expression, secretion and promoter activity were evaluated by reverse transcriptase-PCR (RT-PCR), enzyme immunometric and luciferase assays, respectively.


RESULTS
oxLDL (35 microg/ml) significantly enhanced reactive oxygen species (ROS), mRNA expression, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), all of which were nullified by the antioxidant N-acetyl cysteine (NAC). oxLDL stimulated the extracellular signal-regulated kinase (ERK) phosphorylation in HUVECs, which was blocked by NAC and the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD98059. NAC and PD98059 stopped oxLDL-elicited increase in mRNA expression, secretion and promoter activity of ET-1. Fusion plasmids with decreasing length of 5'-flanking sequence of ET-1 from -566 bpLuc to -250 bpLuc displayed increased luciferase activity after 24 h of oxLDL treatment. Interestingly, fusion plasmid from -233 and -185 bpLuc significantly reduced the luciferase activity in control and oxLDL-treated HUVECs. In addition, transfection of the reporter construct -250Luc, which contains a 2 bp mutation at activator protein-1 site, abolished both basal and oxLDL-stimulated ET-1 promoter activities.


CONCLUSIONS
Collectively, our data favor the notion that oxLDL stimulates ERK phosphorylation via ROS accumulation, which in turn stimulates vascular endothelial transcriptional factor activator protein-1 and ET-1 expression as well as secretion.
Xu
Haishan
H
Faculty of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, China.
Duan
Jinhong
J
Wang
Wen
W
Dai
Shunling
S
Wu
Yunqing
Y
Sun
Renyu
R
Ren
Jun
J
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D019332:Endothelin-1 / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D008077:Lipoproteins, LDL / Q000502:physiology*; D010766:Phosphorylation; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D014471:Umbilical Veins / Q000378:metabolism
D019332:Endothelin-1; D008077:Lipoproteins, LDL; D017382:Reactive Oxygen Species; C099252:oxidized low density lipoprotein; D048049:Extracellular Signal-Regulated MAP Kinases