title: Reactive oxygen species mediate oxidized low-density lipoprotein-induced endothelin-1 gene expression via extracellular signal-regulated kinase in vascular endothelial cells.
abstract: 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.
authors.affiliation: Faculty of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, China.
authors.forename: Haishan
authors.initials: H
authors.lastname: Xu
authors.forename: Jinhong
authors.initials: J
authors.lastname: Duan
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.forename: Shunling
authors.initials: S
authors.lastname: Dai
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Wu
authors.forename: Renyu
authors.initials: R
authors.lastname: Sun
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
mesh_terms: 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
chemical_list: D019332:Endothelin-1; D008077:Lipoproteins, LDL; D017382:Reactive Oxygen Species; C099252:oxidized low density lipoprotein; D048049:Extracellular Signal-Regulated MAP Kinases