Role of reactive oxygen species-sensitive extracellular signal-regulated kinase pathway in angiotensin II-induced endothelin-1 gene expression in vascular endothelial cells.
BACKGROUND
Circulating angiotensin II (Ang II) increases vascular endothelin-1 (ET-1) tissue levels, which in turn mediate a major part of Ang II-stimulated vascular growth and hypertension in vivo. Ang II also stimulates the generation of reactive oxygen species (ROS) within vascular endothelial cells. However, whether ROS are involved in Ang II-induced ET-1 gene expression, and the related intracellular mechanisms occurring within vascular endothelial cells remain unclear.


METHODS
Cultured endothelial cells were stimulated with Ang II, and the thus elicited ET-1 gene expression was examined by Northern blotting and a promoter activity assay. Antioxidant pretreatment of endothelial cells was performed prior to Ang II-induced extracellular signal-regulated kinase (ERK) phosphorylation in order to elucidate the redox-sensitive pathway for ET-1 gene expression.


RESULTS
The ET-1 gene was induced with Ang II, which was inhibited with Ang II type 1 receptor antagonist (irbesartan). Ang II-enhanced intracellular ROS levels were inhibited by irbesartan and several antioxidants, and antioxidants also suppressed Ang II-induced ET-1 gene expression. Further, Ang II-activated ERK phosphorylation was also significantly inhibited by certain antioxidants. An ERK inhibitor, U0126, inhibited Ang II-induced ET-1 expression completely. Cotransfection of the dominant negative mutant of Ras, Raf and MEK1 (ERK kinase) attenuated the Ang II-enhanced ET-1 promoter activity, suggesting that the Ras/Raf/ERK pathway is required for Ang II-induced ET-1 gene expression. Ang II-induced activator protein-1 (AP-1) reporter activities were inhibited by antioxidants. Moreover, mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important CIS element in Ang II-induced ET-1 gene expression.


CONCLUSIONS
Our data suggest that ROS are involved in Ang II-induced ET-1 gene expression within endothelial cells. The redox-sensitive ERK-mediated AP-1 transcriptional pathway plays an important role in Ang II-induced ET-1 gene expression.
Hsu
Yung-Ho
YH
Department of Medicine, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC.
Chen
Jin-Jer
JJ
Chang
Nen-Chung
NC
Chen
Cheng-Hsien
CH
Liu
Ju-Chi
JC
Chen
Tso-Hsiao
TH
Jeng
Cherng-Jye
CJ
Chao
Hung-Hsing
HH
Cheng
Tzu-Hurng
TH
D000804:Angiotensin II / Q000494:pharmacology; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D019332:Endothelin-1; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D020928:Mitogen-Activated Protein Kinases