on December 9, 2004
Published online before print December 9, 2004, doi: 10.1161/01.ATV.0000152608.29351.8f
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Submitted on June 1, 2004
Accepted on November 16, 2004
c-Src and Hydrogen Peroxide Mediate Transforming Growth Factor-
1-Induced Smooth Muscle Cell-Gene Expression in 10T1/2 Cells
Mahito Sato ;
From the Departments of Medicine and Biological Science (M.S., K.K.-K., H.S., Y. Oyama, H.K., Y. Ohyama, T.S., T.M., Y.A., M.K.) and General Medicine (M.S., J.T., H.S.), Gunma University Graduate School of Medicine, Japan; and the Department of Cardiovascular Medicine (R.N.), Graduate School of Medicine, University of Tokyo, Japan.
* To whom correspondence should be addressed. E-mail: mkurabayashi{at}med.gunma-u.ac.jp.
Objective--Transforming growth factor-
1 (TGF-1) controls the expression of numerous genes, including smooth muscle cell (SMC)-specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-1 signaling in mouse embryonic fibroblast C3H10T1/2 cells.
Methods and Results--TGF-1 induction of the SMC contractile protein smooth muscle protein 22-
(SM22) gene expression was inhibited by PP1 (an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src). Induction of SM22 by TGF-1 was markedly attenuated in SYF cells (c-Src, Yes, and Fyn) compared with Src2+ cells (c-Src2+, Yes, and Fyn). PP1 also inhibited the TGF-1-induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-1-induced expression of SM22. Furthermore, we demonstrate that TGF-1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin.
Conclusion--Our results suggest that TGF-1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22 and PAI-1.
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