Identification of intracellular pathways through which TGF-β1 upregulates PAI-1 expression in endothelial cells

Upregulation of plasminogen activator inhibitor type 1 (PAI-1) expression is a critical mechanism through which transforming growth factor-β1 (TGF-β1) accelerates intimal growth. The aim of this study was to identify signaling pathways through which TGF-β1 upregulates PAI-1 expression in endothelial cells (EC) and test interventions for blocking these pathways. We transduced cultured bovine EC with an adenoviral vector containing the PAI-1 promoter fused to a β-galactosidase reporter gene. We used these cells, along with vectors expressing potential modifiers of TGF-β1 signaling and pharmacologic antagonists of mitogen-activated protein kinase (MAPK) pathways to identify key mediators of basal and TGF-β1-regulated PAI-1 expression. Basal activity of the PAI-1 promoter was directly correlated with Ras activation and was blocked by a dominant negative (DN) type I TGF-β receptor. TGF-β1-stimulated activity of the PAI-1 promoter did not require Ras activation, and was lessened or eliminated by expression of either DN type I or type II TGF-β receptors and by inhibition of either of two MAPKs: MEK and p38. Our results suggest unanticipated pathways of TGF-β1 signaling in EC and point to new strategies to limit TGF-β1-induced vascular disease.