Crucial role of extracellular signal-regulated kinase pathway in reactive oxygen species-mediated endothelin-1 gene expression induced by endothelin-1 in rat cardiac fibroblasts.
Endothelin-1 (ET-1) has been implicated in fibroblast proliferation. However, the mechanism involving ET-1 is not clear. The present study was performed to examine the role of endogenous ET-1 in ET-1-stimulated fibroblast proliferation and to investigate the regulatory mechanism of ET-1-induced ET-1 gene expression in cardiac fibroblasts. Both ET(A) receptor antagonist [(hexahydro-1H-azepinyl)carbonyl-Leu-D-Trp-D-OH (BQ485)] and endothelin-converting enzyme inhibitor (phosphoramidon) inhibited the increased DNA synthesis caused by ET-1. ET-1 gene was induced by ET-1, as revealed with Northern blotting and ET-1 promoter activity assay. ET-1 increased intracellular reactive oxygen species (ROS), which were significantly inhibited by BQ485 and antioxidants. Antioxidants suppressed ET-1 gene expression and DNA synthesis stimulated by ET-1. ET-1 activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase, which were significantly inhibited by antioxidants. Only ERK inhibitor U0126 could inhibit ET-1-induced transcription of the ET-1 gene. Cotransfection of dominant-negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced increase in ET-1 transcription, suggesting that the Ras-Raf-ERK pathway is required for ET-1 action. Truncation and mutational analysis of the ET-1 gene promoter showed that the activator protein-1 (AP-1) binding site was an important cis-element in ET-1-induced ET-1 gene expression. Antioxidants attenuated the ET-1-stimulated AP-1 binding activity. Our data suggest that ROS were involved in ET-1-induced fibroblast proliferation and mediated ET-1-induced activation of ERK pathways, which culminated in ET-1 gene expression.
Cheng
Cheng-Ming
CM
Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan, ROC.
Hong
Hong-Jye
HJ
Liu
Ju-Chi
JC
Shih
Neng-Lang
NL
Juan
Shu-Hui
SH
Loh
Shih-Hurng
SH
Chan
Paul
P
Chen
Jin-Jer
JJ
Cheng
Tzu-Hurng
TH
D000818:Animals; D000831:Animals, Newborn; D002455:Cell Division; D004247:DNA / Q000096:biosynthesis; D019332:Endothelin-1 / Q000235:genetics*; D005347:Fibroblasts / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000378:metabolism; D014158:Transcription, Genetic
D019332:Endothelin-1; D017382:Reactive Oxygen Species; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D004247:DNA; D020928:Mitogen-Activated Protein Kinases