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-␤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 . 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.

Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts

“…Myostatin acted differently from TGF-b1 in this respect, and in its inability to induce (at least in 10 days) the smooth muscle cell lineage, since TGF-b1 stimulates considerably the appearance of smooth muscle markers in this cell line at an earlier stage, acting through the Smad2/3 pathway (Sato et al 2005.…”

Vasoprotective effects of neurocompensatory response to balloon injury during diabetes involve the improvement of Mas signaling by TGFβ1 activation

“…This event resulted from ALK5 activation by TGFβ 1 since the blockade of TGFβ 1 /ALK5 signaling by GW788388 inhibited the local Nox4 activity. In fact, TGFβ 1 is able to acutely trigger NAD(P)H oxidase-driven H 2 O 2 generation upon the activation of c-Src [35], which is required to p47 phox phosphorylation and translocation during NAD(P)H oxidase assembly [36]. Moreover, transcriptional effects of TGFβ 1 also enhance Nox4-driven ROS generation due to the induction of Nox4 expression by Smad2/3 signaling-dependent mechanisms [37].…”

TGF-β1-induced PAI-1 expression is E box/USF-dependent and requires EGFR signaling

“…Discussion pp60 c-src is a critical intermediate in a complex TGF-β1-initiated transduction cascade leading to MEK signaling, PAI-1 transcription and the subsequent phenotypic response [34,35,47,48]. PP1 and DN pp60 c-src effectively reduced TGF-β1-induced PAI-1 levels in various cell types (e.g., T2, R22, HaCaT) ( [34]; this paper), moreover, confirming the generality of src kinase involvement in PAI-1 gene regulation.…”