c-Src and Hydrogen Peroxide Mediate Transforming Growth Factor- 1–Induced Smooth Muscle Cell–Gene Expression in 10T1/2 Cells

Objective—Transforming growth factor1 (TGF1) 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 TGF1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. Methods and Results—TGF1 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 TGF1 was markedly attenuated in SYF cells (c-Src , Yes , and Fyn ) compared with Src cells (c-Src , Yes , and Fyn ). PP1 also inhibited the TGF1–induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF1–induced expression of SM22 . Furthermore, we demonstrate that TGF1 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 TGF1 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.)