Abstract
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 ETA 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 importantcis-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.
Footnotes
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This study was in supported with grants from National Science Council (91-2314-B-038-255-) and Taipei Medical University (TMU 91-Y05-A151), Taiwan, R.O.C.
- Abbreviations:
- ET
- endothelin
- AP-1
- activator protein-1
- CAT
- chloramphenicol acetyltransferase
- DCF
- dichlorofluorescein
- DCF-DA
- 2′,7′-dichlorofluorescein diacetate
- DPI
- diphenylene iodonium
- ECE
- endothelin-converting enzyme
- ERK
- extracellular signal-regulated kinase
- JNK
- c-Jun N-terminal kinase
- MAPK
- mitogen-activated protein kinase
- MEK
- mitogen-activated protein kinase kinase
- NAC
- N-acetylcysteine
- ROS
- reactive oxygen species
- BQ485
- (hexahydro-1H-azepinyl)carbonyl-Leu-d-Trp-d-OH
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole
- DMEM
- Dulbecco's modified Eagle's medium
- PBS
- phosphate-buffered saline
- BQ788
- cis-2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine
- Bp
- base pair(s)
- Received September 23, 2002.
- Accepted February 6, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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