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

Cheng Hsien Chen, Tzu Hurng Cheng, Heng Lin, Neng Lang Shih, Yen Ling Chen, Yee Shiuan Chen, Ching Feng Cheng, Wei Shiung Lian, Tzu Ching Meng, Wen Ta Chiu, Jin Jer Chen

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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 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 ET A 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 p47 phox 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-ET A-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.

Original languageEnglish
Pages (from-to)1347-1355
Number of pages9
JournalMolecular Pharmacology
Volume69
Issue number4
DOIs
Publication statusPublished - Apr 2006
Externally publishedYes

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Endothelin-1
Phosphoric Monoester Hydrolases
Epidermal Growth Factor Receptor
Transcriptional Activation
Reactive Oxygen Species
Fibroblasts
Cysteine
Epidermal Growth Factor
Heparin
Signal Transduction
Phosphorylation
2-tyrosine
Protein Tyrosine Phosphatases
Matrix Metalloproteinase Inhibitors
NADPH Oxidase
Neutralizing Antibodies
Matrix Metalloproteinases
Small Interfering RNA
Fibrosis

ASJC Scopus subject areas

  • Pharmacology

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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. / 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.

In: Molecular Pharmacology, Vol. 69, No. 4, 04.2006, p. 1347-1355.

Research output: Contribution to journalArticle

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. / 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. In: Molecular Pharmacology. 2006 ; Vol. 69, No. 4. pp. 1347-1355.
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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 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 ET A 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 p47 phox 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-ET A-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.",
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AU - Lin, Heng

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AU - Chen, Yen Ling

AU - Chen, Yee Shiuan

AU - Cheng, Ching Feng

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AU - Chen, Jin Jer

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AB - 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 ET A 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 p47 phox 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-ET A-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.

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