Modulators of induction of plasminogen activator inhibitor type-1 in HepG2 cells by transforming growth factor-ß.

ABSTRACT

OBJECTIVE: An increased expression of plasminogen activator inhibitor type-1 (PAI-1) has been implicated in accelerating atherogenesis and coronary artery disease in patients with type 2 diabetes. Transforming growth factor (TGF)-ß increases its expression. An increased PAI-1 appears to predispose also to augmented fibrosis potentially contributing to negative left ventricular remodeling and heart failure after myocardial infarction. Diabetes is well known to induce oxidative stress. To elucidate molecular mechanisms underlying an increased PAI-1 production, the effects of TGF-ß and oxidative stress implicated as agonists of PAI-1 synthesis were characterized with the use of human liver-derived HepG2 cells. METHODS: PAI-1 mRNA was assayed by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-825 -+42 bp) activity was assessed with the luciferase assay. The role of the 3'-untranslated region was delineated with the use of luciferase constructs containing the 3'-untranslated region. Oxidative stress was measured after loading carboxy-2,7-dichlorodihydrofluorescein into cells. RESULTS: TGF-ß increased oxidative stress, which was accompanied by increases in NADPH oxidase 3 mRNA and membrane translocation of Rac proteins. TGF-ß-inducible increases in the PAI-1 promoter activity involved Smad-binding elements and a nuclear factor-κB-binding site. TGF-ß did not increase the activity of the PAI-1 mRNA 3'-untranslated region. TGF-ß-inducible PAI-1 expression was attenuated by simvastatin and curcumin, a natural polyphenol. CONCLUSION: TGF-ß can increase the expression of PAI-1 through multiple mechanisms involving Smad and nuclear factor-κB pathways and oxidative stress. As both oxidative stress and PAI-1 production were reduced by simvastatin and curcumin, modulation of oxidative stress and PAI-1 production are attractive targets for pharmacotherapy of cardiovascular disorders associated with an increased PAI-1 including type 2 diabetes and its associated consequences including accelerated coronary artery disease and an increased fibrosis that may exacerbate adverse left ventricular remodeling after myocardial infarction.