Abstract
Urotensin II (U-II) is implicated in cardiomyocyte hypertrophy, which results in cardiac remodeling. We recently demonstrated that both reactive oxygen species (ROS) generation and epidermal growth factor receptor (EGFR) transactivation play critical roles in U-II signal transduction. However, the detailed intracellular mechanism(s) underlying cardiac hypertrophy and remodeling remain unclear. In this study, we used rat cardiomyocytes treated with U-II to investigate the association between ROS generation and EGFR transactivation. U-II treatment was found to stimulate cardiomyocyte hypertrophy through phosphorylation of EGFR and ROS generation. Apocynin, an NAD(P)H oxidase inhibitor, and N-acetyl cysteine (NAC), an ROS scavenger, both inhibited EGFR transactivation induced by U-II. In contrast, 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478, an EGFR inhibitor) failed to inhibit intracellular ROS generation induced by U-II. Src homology 2-containing tyrosine phosphatase (SHP-2), but not protein tyrosine phosphatase 1B (PTP 1B), was shown to be associated with EGFR during U-II treatment by EGFR coimmunoprecipitation. ROS have been reported to transiently oxidize the catalytic cysteine of phosphotyrosine phosphatases, subsequently inhibiting their activity. We examined the effect of U-II on SHP-2 and PTP 1B in cardiomyocytes using a modified malachite green phosphatase assay. SHP-2, but not PTP 1B, was transiently oxidized during U-II treatment, which could be repressed by NAC treatment. In SHP-2 knockdown cells, U-II-induced phosphorylation of EGFR and myocyte hypertrophy were dramatically elevated, and these effects were not influenced by NAC. Our data suggest that U-II-mediated ROS generation can transiently inhibit SHP-2 activity, thereby facilitating EGFR transactivation and hypertrophic signal transduction in rat cardiomyocytes.
Footnotes
This work was supported by the National Science Council [Grant NSC 95-2314-B-039-048-MY2], the New Century Health Care Promotion Foundation Medical Research Award [Grant 97-TMU-IAC-021], and the China Medical University [Grant CMU97-233], Taiwan, R.O.C.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.058297
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ABBREVIATIONS:
- U-II
- urotensin II
- ROS
- reactive oxygen species
- EGFR
- epidermal growth factor receptor
- ERK
- extracellular signal-regulated kinase
- PTP
- protein tyrosine phosphatase
- AG-1478
- 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline
- NAC
- N-acetyl-cysteine
- DAPI
- 4′,6-diamidino-2-phenylindole
- PBS
- phosphate-buffered saline
- DCFH
- 2′,7′-dichlorodihydrofluorescein
- DCF
- 2′,7′-dichlorofluorescein
- IAA
- iodoacetic acid
- siRNA
- small interfering RNA
- GFP
- green fluorescent protein
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene
- SHP-2
- Src homology 2-containing tyrosine phosphatase.
- Received June 3, 2009.
- Accepted September 15, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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