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.
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 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-dependent and HB-EGF-stimulated EGFR transactivation and mitogenic signal transduction in rat cardiac fibroblasts.
Chen
Cheng-Hsien
CH
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China.
Cheng
Tzu-Hurng
TH
Lin
Heng
H
Shih
Neng-Lang
NL
Chen
Yen-Ling
YL
Chen
Yee-Shiuan
YS
Cheng
Ching-Feng
CF
Lian
Wei-Shiung
WS
Meng
Tzu-Ching
TC
Chiu
Wen-Ta
WT
Chen
Jin-Jer
JJ
D000818:Animals; D019332:Endothelin-1 / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics*; D047468:Immunoprecipitation; D010084:Oxidation-Reduction; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation; D018909:src Homology Domains
D019332:Endothelin-1; D017382:Reactive Oxygen Species; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases