Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells - PubMed (original) (raw)

Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells

Yasushi Mukai et al. Am J Physiol Heart Circ Physiol. 2007 Apr.

Abstract

Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with TNF-alpha, ANG II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with wortmannin or LY-294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly, overexpression of a dominant-negative mutant of PI3K or Akt increased TNF-alpha- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and posttranscriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF-alpha and insulin is mediated, in part, by ERK and p38 MAPK. PI3K inhibitors augmented TNF-alpha- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which is known to activate PI3K/Akt, blocks TNF-alpha- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF-alpha- and insulin-induced PAI-1 expression. These findings indicate that the PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors that activate the PI3K/Akt pathway in ECs may have therapeutic benefits for atherothrombotic vascular disease.

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Figures

Fig. 1

Fig. 1

Induction of plasminogen activator inhibitor type 1 (PAI-1) expression by TNF-α in endothelial cells (ECs). A: Northern blots showing dose-dependent induction of PAI-1 mRNA. B: Northern blots showing time-dependent induction of PAI-1 mRNA. C: Western blots showing time-dependent induction of PAI-1 protein expression.

Fig. 2

Fig. 2

Inhibition of phosphatidylinositol 3-kinase (PI3K) augments TNF-α-induced PAI-1 expression and activity. A: Northern blots showing effects of wortmannin (WM, 50 nmol/l) and LY-294002 (LY, 3 μmol/l) on TNF-α (10 ng/ml, 6 h)-induced PAI-1 mRNA expression. C, control. Values are means ± SE (n = 4). *P < 0.05 vs. TNF-α alone. B: Western blot showing effects of wortmannin or LY-294002 on TNF-α (10 ng/ml, 24 h)-induced PAI-1 protein expression. Values are means ± SE (n = 5). *P < 0.05 vs. TNF-α alone. C: Western blot showing effects of wortmannin or LY-294002 on insulin (1 μmol/l, 6 h)-induced PAI-1 protein expression. Values are means ± SE (n = 3). *P < 0.05 vs. insulin alone. D: Northern blots showing effects of PI3K inhibitors on ANG II- and FCS-induced PAI-1 mRNA expression. Cells were stimulated with ANG II or FCS in the presence or absence of PI3K inhibitors for 6 h. E: effect of wortmannin or LY-294002 on TNF-α-induced PAI-1 activity. At ∼12 h after TNF-α stimulation, PAI-1 activity was measured in conditioned media by ELISA. Values are means ± SE (n = 4). *P < 0.05 vs. TNF-α alone.

Fig. 3

Fig. 3

Effects of PI3K and Akt inhibition of PAI-1 expression. A and B: Northern and Western blots, respectively, showing effects of adenoviruses (Ad) encoding LacZ (Ad-LacZ), a dominant-negative (DN) mutant of PI3K (Ad-DN-PI3K), and a dominant-negative mutant of Akt (Ad-DN-Akt) on TNF-α (10 ng/ml)-induced PAI-1 mRNA expression. Blots were evaluated 6 and 24 h after TNF-α stimulation. Values are means ± SE (n = 4). *P < 0.05 vs. Ad-LacZ with TNF-α.

Fig. 4

Fig. 4

Transcriptional and posttranscriptional regulation of PAI-1 expression by PI3K. A: dual-luciferase reporter assay showing effects of wortmannin (50 nmol/l) and LY-294002 (3 μmol/l) on TNF-α (10 ng/ml)-induced PAI-1 promoter activity in ECs. *P < 0.05 vs. TNF-α alone. B: Northern blots with corresponding analysis showing half-life of PAI-1 mRNA in the presence of mRNA synthase inhibitor 5,6-dichlorobenzimidazole riboside (25 μmol/l), which was added after 6 h of stimulation with TNF-α (10 ng/ml). Ctl, control. Values are means ± SE (n = 4). *P < 0.05. **P < 0.01.

Fig. 5

Fig. 5

Role of MAPKs in PAI-1 expression. A: Northern blots showing effects of MAPK inhibitors on PAI-1 expression. Pretreatment with an ERK kinase (MEK) inhibitor (PD-98059, 10 μmol/l) or a p38 MAPK inhibitor (SB-203580, 10 μmol/l) suppressed TNF-α (10 ng/ml)-induced PAI-1 expression. Pretreatment with PD-98059 and SB-203580 almost completely suppressed TNF-α (10 ng/ml)-induced PAI-1 expression. B and C: Western blots showing effect of wortmannin (50 nmol/l) and LY-294002 (3 μmol/l) on phosphorylations of ERK1/2 and p38 MAPK. Cells were stimulated with TNF-α (10 ng/ml) or insulin (1 μmol/l) in the presence or absence of wortmannin (50 nmol/l) or LY-294002 (3 μmol/l) for 12 h.

Fig. 6

Fig. 6

Role of PI3K in the inhibitory effect of simvastatin on TNF-α-induced PAI-1 expression. A: Western blots showing activation (phosphorylation at Ser473) of Akt by simvastatin (Sim). B and C: Northern and Western blots, respectively, showing dose-dependent inhibition of TNF-α-induced PAI-1 expression by simvastatin and its reversal with LY-294002 (3 μmol/l).

Fig. 7

Fig. 7

Role of PI3K in the inhibitory effect of simvastatin on TNF-α-induced ERK and p38 MAPK phosphorylation. Western blots show dose-dependent inhibition of TNF-α-induced ERK1/2 and p38 MAPK phosphorylation by simvastatin and its reversal with LY-294002 (3 μmol/l).

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