title: Reactive oxygen species mediate cyclic strain-induced endothelin-1 gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in endothelial cells.
abstract: Endothelin-1 (Et-1) is a peptide synthesized by endothelial cells (ECs) both in culture and in vivo. Cyclic strain induces gene expression of Et-1, however, the molecular mechanisms remain unclear. Since cyclic strain induces a sustained increase in intracellular reactive oxygen species (ROS), we hypothesized that the ROS could be a modulator in strain-induced Et-1 gene expression. Human umbilical vein ECs (HUVECs) subjected to cyclic strain had increased Et-1 secretion. Pretreatment of HUVECs with antioxidants, catalase (300 U/ml) or 1,3-dimethyl-2-thiourea (DMTU, 0.1 mm), abolished the strain-induced Et-1 release. ECs strained for 6 h had elevated Et-1 mRNA levels. In contrast, ECs treated with catalase or DMTU did not have increase Et-1 mRNA levels stimulated by cyclic strain. Bovine aortic ECs (BAECs) transfected with fusion plasmid containing Et-1 5'-flanking sequence (4.4 kb) and chloramphenicol acetyltransferase reporter gene produced a maximal Et-1 promoter activity after undergoing strain for 6 h, whereas pretreatment with catalase decreased this activity. BAECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or catalytically inactive mutant of extracellular signal-regulated kinase (mERK2) had inhibited strain-induced Et-1 promoter activity, indicating the Ras/Raf/ERK pathway was involved; moreover, ERK phosphorylation was induced in ECs which were strained. This strain-activated ERK phosphorylation was attenuated in the presence of catalase. Functional analysis of the Et-1 promoter with site-directed mutagenesis indicates that the activator protein-1 (AP-1) binding site had to be within 143 base-pairs upstream of transcription initiation site for strain-induced promoter activity. Pretreatment of ECs with catalase also decreased the strain-induced promoter activity in the minimal construct (-143 bp). Our data demonstrate that strain-induced Et-1 gene expression is modulated by ROS via Ras/Raf/ERK signaling pathway, and indicate the responsiveness of the AP-1 binding site for strain-induced Et-1 expression.
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: T H
authors.initials: TH
authors.lastname: Cheng
authors.forename: N L
authors.initials: NL
authors.lastname: Shih
authors.forename: S Y
authors.initials: SY
authors.lastname: Chen
authors.forename: S H
authors.initials: SH
authors.lastname: Loh
authors.forename: P Y
authors.initials: PY
authors.lastname: Cheng
authors.forename: C S
authors.initials: CS
authors.lastname: Tsai
authors.forename: S H
authors.initials: SH
authors.lastname: Liu
authors.forename: D L
authors.initials: DL
authors.lastname: Wang
authors.forename: J J
authors.initials: JJ
authors.lastname: Chen
mesh_terms: D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D002374:Catalase / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D019332:Endothelin-1 / Q000096:biosynthesis* / Q000235:genetics*; D004730:Endothelium, Vascular / Q000201:enzymology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006439:Hemodynamics; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014471:Umbilical Veins / Q000201:enzymology*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D019332:Endothelin-1; D010455:Peptides; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D002701:Chloramphenicol; C038983:1,3-dimethylthiourea; D002374:Catalase; D015500:Chloramphenicol O-Acetyltransferase; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D013890:Thiourea