Plasminogen Activator Inhibitor Type-1 Promoter Activity Research Articles

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3′-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3′ terminus (+2177 to 3176nt) of the 3′-UTR of the 3.2kb PAI-1 mRNA [3′-UTR (+2177–3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3′-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3′-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.

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

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)-β 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-β and oxidative stress implicated as agonists of PAI-1 synthesis were characterized with the use of human liver-derived HepG2 cells. 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. TGF-β increased oxidative stress, which was accompanied by increases in NADPH oxidase 3 mRNA and membrane translocation of Rac proteins. TGF-β-inducible increases in the PAI-1 promoter activity involved Smad-binding elements and a nuclear factor-κB-binding site. TGF-β did not increase the activity of the PAI-1 mRNA 3'-untranslated region. TGF-β-inducible PAI-1 expression was attenuated by simvastatin and curcumin, a natural polyphenol. TGF-β 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.

Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.

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

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. 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. 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. 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.

Impact of statins on modulation by insulin of expression of plasminogen activator inhibitor type-1

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic inhibitor of fibrinolysis and a known determinant of cardiovascular risk. That its expression is stimulated by insulin in HepG2 human hepatoma cells has been shown earlier. Others have found that increased expression of PAI-1 is a risk factor for acute myocardial infarction. Pleiotropic (noncholesterol lowering) effects of statins seem to reduce cardiovascular risk. This study was designed to determine whether insulin stimulation of PAI-1 expression is attenuated by statins, and if so to explore mechanisms that are responsible for the attenuation. PAI-1 protein in the conditioned media was assayed by western blotting, and PAI-1 mRNA expression was measured by real-time reverse transcriptase polymerase chain reactions. Insulin (0.001-10 micromol/l) increased accumulation of PAI-1 protein in the conditioned media and PAI-1 mRNA expression in HepG2 cells. A transient transfection assay of the human PAI-1 promoter-luciferase construct demonstrated that insulin increased PAI-1 promoter activity. Increased PAI-1 mRNA was attenuated significantly by U0126 and PD98059, specific inhibitors of mitogen-activated protein kinase. In contrast, GF109203X, an inhibitor of the protein kinase C pathway, and LY294002, an inhibitor of phosphatidylinositol 3-kinase, exerted no effects. Simvastatin (10 micromol/l), known to translocate membrane-bound sterol regulatory element-binding protein to nuclei, attenuated PAI-1 mRNA expression induced by insulin. It did not affect baseline PAI-1 mRNA expression. Intraperitoneal injection of insulin (0.1 IU/kg) increased concentrations of PAI-1 antigen in plasma in mice within 3 h, correlating with hepatic PAI-1 mRNA expression. Insulin-mediated augmented expression of PAI-1 may be amenable to suppression attributable to pleiotropic effects of statins, potentially diminishing cardiovascular risk in patients with insulin resistance.

Glutathione suppresses TGF-β-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

Transforming growth factor (TGF)-beta upregulates plasminogen activator inhibitor type 1 (PAI-1) in a variety of cell types, and PAI-1 is considered to be an essential factor for the development of fibrosis. Our previous studies demonstrated that TGF-beta decreased intracellular glutathione (GSH) content in murine embryonic fibroblasts (NIH/3T3 cells), whereas treatment of the cells with GSH, which restored intracellular GSH concentration, inhibited TGF-beta-induced collagen accumulation by blocking PAI-1 expression and enhancing collagen degradation. In the present study, we demonstrate that GSH blocks TGF-beta-induced PAI-1 promoter activity in NIH/3T3 cells, which is associated with an inhibition of TGF-beta-induced JNK and p38 phosphorylation. Interestingly, although exogenous GSH does not affect phosphorylation and/or nuclear translocation of Smad2/3 and Smad4, it completely eliminates TGF-beta-induced binding of transcription factors to not only AP-1 and SP-1 but also Smad cis elements in the PAI-1 promoter. Decoy oligonucleotides (ODN) studies further demonstrate that AP-1, SP-1, and Smad ODNs abrogate the inhibitory effect of GSH on TGF-beta-induced PAI-1 promoter activity and inhibit TGF-beta-induced expression of endogenous PAI-1. Furthermore, we show that GSH reduces TGF-beta-stimulated reactive oxygen species (ROS) signal. Blocking ROS production with diphenyleneiodonium or scavenging ROS with a superoxide dismutase and catalase mimetic MnTBaP dramatically reduces TGF-beta-induced p38 and JNK phosphorylation as well as PAI-1 gene expression. In composite, these findings suggest that GSH inhibits TGF-beta-stimulated PAI-1 expression in fibroblasts by blocking the JNK/p38 pathway, probably by reducing ROS, which leads to an inhibition of the binding of transcription factors to the AP-1, SP-1, and Smad cis elements in the PAI-1 promoter.

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

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.

Cell Type-dependent Regulation of the Hypoxia-responsive Plasminogen Activator Inhibitor-1 Gene by Upstream Stimulatory Factor-2

Transcriptional regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene is an important issue since PAI-1 plays a crucial role in various pathological conditions. The transcription factor USF-2 was shown to be a negative regulator for rat PAI-1 expression, and therefore it was the aim of this study to evaluate the role of USF-2 for human PAI-1 expression. We found in human hepatoma cells (HepG2) that USF-2 induced human PAI-1 expression via two classical E-boxes and the hypoxia-responsive element (HRE) within the promoter. Gel-shift analyses showed that E-box 4 and E-box 5 bound USFs, and although the HRE contributed to the USF-dependent effects, it did not bind them. By contrast, USF-2 inhibited PAI-1 promoter activity in primary rat hepatocytes suggesting that PAI-1 expression depends on either the promoter context or USF activity which might be cell type-specific. Cotransfection of human or rat PAI-1 promoter luciferase constructs with expression vectors for wild-type USF-2 or USF-2 mutants in human HepG2 and rat H4IIE cells as well as in primary rat hepatocytes revealed that the effects of USF on PAI-1 expression depend on the cell type rather than the promoter context and that the USF-specific region domain of USF accounts for the observed cell type-specific effects.

Mast Cell–Derived Exosomes Activate Endothelial Cells to Secrete Plasminogen Activator Inhibitor Type 1

Previous studies supported the contribution of exosomes to an acellular mode of communication, leading to intercellular transfer of molecules. In this study we provide evidence that mast cell-derived exosomes induce plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells, detectable at the level of PAI-1 mRNA and protein synthesis. The stimulating effect was also measured at the level of PAI-1 promoter activity. To identify components responsible for this activity, exosome proteins were separated by 2-dimensional PAGE, and protein spots were identified by microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometer. Components of 3 independent systems that can be involved in activation of endothelial cells, namely the prothrombinase complex, tumor necrosis factor-alpha, and angiotensinogen precursors were identified. Procoagulant activity of exosomes was confirmed by a thrombin generation assay using a specific chromogenic substrate. Because the potential of mast cell-derived exosomes to induce PAI-1 expression was completely abolished by hirudin, thrombin generated on exosomes seems to be responsible for this activity. It can be concluded that mast cell-derived exosomes via significant upregulation of PAI-1 secretion from endothelial cells may provide feedback between the characteristically increased PAI-1 levels and procoagulant states, both observed in diverse syndromes manifesting as endothelial cell dysfunction.

Differential regulation of plasminogen activator and inhibitor gene transcription by the tumor suppressor p53.

The ability of p53 to activate or repress transcription suggests that its biological function as tumor suppressor is in part accomplished by regulating a number of genes including such required for inhibition of cell growth. We here give evidence that p53 also may regulate genes responsible for the proteolytic degradation of the extracellular matrix, which is considered a crucial feature for local invasion and metastasis of neoplastic cells. An important and highly regulated cascade of such proteolytic events involves the plasminogen activator system. We show that wild-type p53 represses transcription from the enhancer and promoter of the human urokinase-type (u-PA) and the tissue-type plasminogen activator (t-PA) gene through a non-DNA binding mechanism. Oncogenic mutants lost the repressing activity. In contrast, wild-type but not mutant p53 specifically binds to and activates the promoter of the plasminogen activator inhibitor type-1 (PAI-1) gene. Interestingly, one of the p53 mutants (273his) inhibited PAI-1 promoter activity. Our results suggest that altered function of oncogenic forms of p53 may lead to altered expression of the plasminogen activators and their inhibitor(s) and thus to altered activation of the plasminogen/plasmin system during tumor progression.