Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by cAMP in HepG2 liver cells.

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a physiologic fibrinolysis inhibitor, is implicated in atherosclerosis. Cyclic adenosine monophosphate (cAMP) alters PAI-1 expression in several cells. Nevertheless, posttranscriptional regulation of PAI-1 has not been elucidated. To determine whether cAMP affects PAI-1 expression at posttranscriptional level, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity and protein levels of PAI-1 using HepG2 cells. cAMP decreased PAI-1 promoter activity at 24 h and mRNA expression at 4 h while it increased mRNA expression and accumulation of PAI-1 protein into media at 24 h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2 kb), and cAMP increased baseline luciferase activity of 3'-UTR of the 3.2 kb PAI-1 mRNA [3'-UTR (+1358-3176)] and 1 kb fragment of 3'-terminus of 3'-UTR of 3.2 kb mRNA [3'-UTR (+2177-3176)]. cAMP increased PAI-1 protein expression despite decrease in promoter activity, presumably by regulating PAI-1 expression at the posttranscriptional level and thereby affecting mRNA stability. The 53-nt fragment in 3'-UTR (+2591 to +2643 nt) was involved in posttranscriptional regulation by cAMP. Thus, cAMP can stabilize 3.2 kb PAI-1 mRNA mediated by specific effects on 3'-UTR, and these effects are associated with increased expression of PAI-1 protein.

Increased expression of plasminogen activator inhibitor type-1 (PAI-1) in HEPG2 cells induced by insulin mediated by the 3'-untranslated region of the PAI-1 gene and its pharmacologic implications.

OBJECTIVE: Insulin increases, through several molecular mechanisms, expression of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis. This phenomenon has been implicated as a cause of accelerated coronary artery disease and the increased incidence of acute coronary syndromes associated with type 2 diabetes. We have previously reported that physiologic and pharmacologic concentrations of insulin induce PAI-1 synthesis in human HepG2 cells and that simvastatin can attenuate its effects. This study was performed to further elucidate mechanisms responsible for the insulin-induced PAI-1 production. METHODS: Concentrations of PAI-1 mRNA were determined by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-829 to +36 bp) activity was assayed with the use of luciferase reporter assays. The potential role of the 3'-untranslated region (UTR) in the PAI-1 gene was assayed with the use of luciferase constructs containing the 3'-UTR. Oxidative stress was measured by loading cells with carboxy-2,7 dichlorodihydrofluorescein. RESULTS: Insulin increased PAI-1 promoter activity, PAI-1 mRNA, and accumulation of PAI-1 protein in the conditioned media. Insulin-inducible PAI-1 promoter activity was attenuated by simvastatin. Experiments performed with luciferase reporters containing the 3'-UTR showed that insulin increased luciferase activity through this region. Insulin also increased oxidative stress. Both insulin-inducible luciferase activity through the 3'-UTR and oxidative stress were attenuated by simvastatin. CONCLUSION: Insulin can increase PAI-1 expression through multiple mechanisms including induction mediated by the 3'-UTR of the PAI-1 gene. Accordingly, beneficial pleiotropic effects of statins on coronary artery disease may be attributable, in part, to attenuation of overexpression of PAI-1 mediated by the 3'-UTR in syndromes of insulin resistance (such as the metabolic syndrome) and type 2 diabetes.