c-Src and Hydrogen Peroxide Mediate Transforming Growth Factor-β1–Induced Smooth Muscle Cell–Gene Expression in 10T1/2 Cells

Abstract

Objective— Transforming growth factor-β1 (TGF-β1) 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-β1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. Methods and Results— TGF-β1 induction of the SMC contractile protein SM22α 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 SM22α by TGF-β1 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-β1–induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-β1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-β1–induced expression of SM22α. Furthermore, we demonstrate that TGF-β1 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-β1 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 SM22α and PAI-1.