c-Src and hydrogen peroxide mediate transforming growth factor-β1-induced smooth muscle cell -: Gene expression in 10T1/2 cells

Objective - Transforming growth factor-beta1 ( TGF-beta1) 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-beta1 signaling in mouse embryonic fibroblast C3H10T1/ 2 cells. Methods and Results - TGF-beta1 induction of the SMC contractile protein SM22alpha 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 SM22alpha by TGF-beta1 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-beta1 - induced expression of serum response factor ( SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-beta1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD( P) H oxidase inhibitors such as apocynin inhibited the TGF-beta1 - induced expression of SM22alpha. Furthermore, we demonstrate that TGF-beta1 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-beta1 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 SM22alpha and PAI- 1.