Reactive Oxygen Species Generation Is Involved in Epidermal Growth Factor Receptor Transactivation through the Transient Oxidization of Src Homology 2-Containing Tyrosine Phosphatase in Endothelin-1 Signaling Pathway in Rat Cardiac Fibroblasts
Resource
MOLECULAR PHARMACOLOGY v.69 n.4 pp.1347-1355
Journal
MOLECULAR PHARMACOLOGY
Journal Volume
v.69
Journal Issue
n.4
Pages
1347-1355
Date Issued
2006
Date
2006
Author(s)
CHEN, CHENG-HSIEN
CHENG, TZU-HURNG
LIN, HENG
SHIH, NENG-LANG
CHEN, YEN-LING
CHEN, YEE-SHIUAN
CHENG, CHING-FENG
LIAN, WEI- SHIUNG
MENG, TZU-CHING
CHIU, WEN-TA
CHEN, JIN-JER
Abstract
Endothelin-1 (ET-1)is implicated in fibroblast proliferation ,which results in cardiac fibrosis. Both reactive oxygen species (ROS) generation and epidermal growth factor receptor (EGFR) transactivation play critical roles in ET-1 signal transduction. In this study, we used rat cardiac fibroblasts critical treated with ET-1 to investigate the connection between ROS generation and EGFR transactivation. ET-1 treatment was found to stimulate the phosphorylation of EGFR and ROS generation, which were abolished by ETA receptor antagonist N-(N-(N-((hexahydro-1H-azepin-1-yl) carbonyl)-L-leucyl)-D- tryptophyl)-D-tryptophan (BQ485). NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI), ROS scavenger N-acetyl cysteine (NAC), and p47phox small interfering RNA knockdown all inhibited the EGFR transactivation induced by ET-1. In contrast, EGFR inhibitor 4-(3 `-chloroanilino)-6,7- dimethoxyquinazoline (AG-1478) cannot inhibit intracellular ROS generation induced by ET-1. Src homology 2-containing tyrosine phosphatase (SHP-2) was shown to be associated with EGFR during ET-1 treatment by EGFR coimmunoprecipitation. ROS have been reported to transiently oxidize the catalytic cysteine of phosphotyrosine phosphatases to inhibit their activity. We examined the effect of ROS on SHP-2 in cardiac fibroblasts using a modified malachite green phosphatase assay. SHP-2 was transiently oxidized during ET-1 treatment, and this transient oxidization could be repressed by DPI or NAC treatment. In SHP-2 knockdown cells, ET-1-induced phosphorylation of EGFR was dramatically elevated and is not influenced by NAC and DPI. However, this elevation was suppressed by GM6001[a matrix metalloproteinase (MMP) inhibitor] and heparin binding( HB)-epidermal growth factor( EGF) neutralizing antibody. Our data suggest that ET-1-ETA- mediated ROS generation can transiently inhibit SHP-2 activity to facilitate the MMP-dependednt and HB-EGF- stimulated EGFR transactivation and mitogenic signal transduction in rat cardiac fibroblasts.
Subjects
ROS (Reactive Oxygen Species )
EGFR (Transactivation)
Endothelin-1 (ET-1)
Cardiac Fibroblast
Type
journal article