ucn-1028-c and diacetyldichlorofluorescein

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.. 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.. 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. TGF-beta1 controls the expression of numerous genes, including SM22alpha and PAI-1. We investigated whether c-Src plays a role in TGF-beta1 signaling. TGF-beta1 induction of such genes was significantly reduced in Src family tyrosine kinase-deficient cells, and Csk and pharmacological inhibitors for Src family kinases or antioxidants inhibit the effects of TGF-beta1. These results indicate that c-Src and hydrogen peroxide are required for TGF-beta1 signaling.

Topics: Acetophenones; Acetylcysteine; Animals; Catalase; Cell Line; Ditiocarb; DNA-Binding Proteins; Fibroblasts; Flavonoids; Fluoresceins; Gene Expression Regulation; Gene Targeting; Hydrogen Peroxide; Mice; Mice, Inbred C3H; Microfilament Proteins; Muscle Proteins; NADPH Oxidases; Naphthalenes; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins pp60(c-src); Pyrazoles; Pyrimidines; Recombinant Fusion Proteins; Serum Response Factor; Signal Transduction; Smad3 Protein; Smad4 Protein; Smad6 Protein; src-Family Kinases; Sulfones; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1