Kurabayashi M: c-Src and hydrogen peroxide mediate transforming growth factor-β1-induced smooth muscle cell-gene expression in 10 T1/2 cells. Arterioscler Thromb Vasc Biol 2005

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 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 Src cells (c-Src, 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. (Arterioscler Thromb Vasc Biol. 2005;25:341-347.