The adaptor protein Ruk/CIN85 activates plasminogen activator inhibitor-1 (PAI-1) expression via hypoxia-inducible factor-1α

SummaryIncreased levels of plasminogen activator inhibitor-1 (PAI-1) indicate an enhanced risk of ischaemic/hypoxic cardiovascular events and a poor prognosis. The expression of PAI-1 can be induced by various stimuli including hypoxia, insulin and insulin-like growth factor 1 (IGF-1). The hypoxia-inducible factor-1 (HIF-1) is critical for hypoxia or insulin/IGF-1 mediated PAI-1 induction, but the components involved in merging the signals are not known so far. The adaptor/scaffold protein Ruk/CIN85 may be a candidate since it plays important roles in the regulation of processes associated with cardiovascular and oncological diseases such as downregulation of receptor tyrosine kinases, apoptosis, adhesion and invasion. Therefore, it was the aim of this study to investigate the involvement of Ruk/CIN85 in the regulation of PAI-1 expression. It was found that Ruk/CIN85 induced PAI-1 mRNA and protein expression both under normoxia and hypoxia. The induction of PAI-1 expression by Ruk/CIN85 occurred at the transcriptional level since the half-life of PAI-1 mRNA was not affected in cells overexpressing Ruk/ CIN85 and reporter gene assays using wild-type and mutant human PAI-1 promoter luciferase constructs showed that the hypoxia responsive element was responsible for Ruk/CIN85 effects. Further, knocking down HIF-1α abolished not only the hypoxia-dependent but also the Ruk/CIN85-dependent PAI-1 induction. In addition, transient or stable overexpression of Ruk/CIN85 also induced HIF-1α protein levels and HIF-1 activity and knocking down Ruk/CIN85 reversed these effects. Thereby, Ruk/CIN85 interfered with the proline hydroxylation-dependent HIF-1α protein destabilisation. Together, these results provide the first evidence that Ruk/CIN85 induces PAI-1 expression via modulation of HIF-1α stability.

Download Full-text

Transcriptional regulation of plasminogen activator inhibitor-1 expression by insulin-like growth factor-1 via MAP kinases and hypoxia-inducible factor-1 in HepG2 cells

Thrombosis and Haemostasis ◽

10.1160/th04-11-0761 ◽

2005 ◽

Vol 93 (06) ◽

pp. 1176-1184 ◽

Cited By ~ 27

Author(s):

Ulrike Möller ◽

Stephan Herzig ◽

Trine Fink ◽

Vladimir Zachar ◽

Peter Ebbesen ◽

...

Keyword(s):

Growth Factor ◽

Hepg2 Cells ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Dominant Negative ◽

Plasminogen Activator Inhibitor 1 ◽

Protein Levels ◽

Hypoxia Inducible Factor 1 ◽

Activator Inhibitor ◽

Pai 1

SummaryInsulin-like growth factor 1 (IGF-1) and plasminogen activator inhibitor-1 (PAI-1) appear to play a crucial role in a number of processes associated with growth and tissue remodelling. IGF-1 was shown to enhance PAI-1 expression in primary hepatocytes and HepG2 hepatoma cells, but the molecular mechanisms underlying this effect have not been fully elucidated. In this study, we investigated the transcriptional mechanism and the signaling pathway by which IGF-1 mediates induction of PAI-1 expression in HepG2 cells. By using human PAI-1 promoter reporter gene assays we found that mutation of the hypoxia responsive element (HRE), which could bind hypoxia-inducible factor-1 (HIF-1), nearly abolished the induction by IGF-1. We found that IGF-1-induced up-regulation of PAI-1 expression was associated with activation of HIF-1α. Furthermore, IGF-1 enhanced HIF-1α protein levels and HIF-1 DNA-binding to each HRE, E4 and E5 as shown by EMSA. Mutation of the E-boxes, E4 and E5, did not affect the IGF-1-dependent induction of PAI-1 promoter constructs under normoxia but abolished the effect of IGF-1 under hypoxia. Inhibition of either the PI3K by LY294002 or ERK1/2 by U0126 reduced HIF-1α protein levels while both inhibitors together completely abolished the IGF-1 effect on HIF-1α. Remarkably, transfection of HepG2 cells with vectors expressing a dominant-negative PDK1 or the PKB inhibitor, TRB3, did not influence while dominant-negative Raf inhibited the IGF-1 effect on HIF-1α. Thus, IGF-1 activates human PAI-1 gene expression through activation of the PI3-kinase and ERK1/2 via HIF-1α.

Download Full-text

Plasminogen Activator Inhibitor-1 Regulates LPS Induced Inflammation in Rat Macrophages through Autophagy Activation

The Scientific World JOURNAL ◽

10.1155/2014/189168 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 7

Author(s):

Zhong-Hui Wang ◽

Wei-Ying Ren ◽

Lei Zhu ◽

Li-Juan Hu

Keyword(s):

Western Blot ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Mrna Levels ◽

Plasminogen Activator Inhibitor 1 ◽

Inflammatory Reactions ◽

Protein Levels ◽

Nr8383 Cells ◽

Activator Inhibitor ◽

Pai 1

Background. The mechanisms by which plasminogen activator inhibitor-1 (PAI-1) regulates inflammation, especially in acute respiratory distress syndrome (ARDS), are largely unknown.Objective. To assess the relationship between PAI-1 and autophagy in inflammatory reactions induced by LPS in rat NR8383 cells.Methods. ELISA was used to assess the amounts of TNF-α, IL-1β, and PAI-1 in cell culture supernatants; TLR4, MyD88, PAI-1, LC3, Beclin1, and mTOR protein and mRNA levels were determined by western blot and quantitative RT-PCR, respectively; western blot was used to determine NF-κB protein levels. To further evaluate the role of PAI-1, the PAI-1 gene was downregulated and overexpressed using the siRNA transfection technology and the pCDH-PAI-1, respectively. Finally, the GFP Positive Expression Rate Method was used to determine the rate of GFP-LC3 positive NR8383 cells.Results. In LPS-induced NR8383 cells, TNF-α, IL-1β, and PAI-1 expression levels increased remarkably. Upon PAI-1 knockdown, TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1 levels were decreased, while mTOR increased. Conversely, overexpression of PAI-1 resulted in increased amounts of TNF-α, IL-1β, PAI-1, TLR4, MyD88, NF-κB, LC3, and Beclin1. However, no significant change was observed in mTOR expression.Conclusions.In NR8383 cells, PAI-1 contributes in the regulation of LPS-induced inflammation, likely by promoting autophagy.

Download Full-text

Induction of the Plasminogen Activator Inhibitor-1 Gene Expression by Mild Hypoxia Via a Hypoxia Response Element Binding the Hypoxia-Inducible Factor-1 in Rat Hepatocytes

Blood ◽

10.1182/blood.v94.12.4177.424k14_4177_4185 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4177-4185 ◽

Cited By ~ 7

Author(s):

Thomas Kietzmann ◽

Ulrike Roth ◽

Kurt Jungermann

Keyword(s):

Plasminogen Activator ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Rat Hepatocytes ◽

Plasminogen Activators ◽

Plasminogen Activator Inhibitor 1 ◽

Hypoxia Response ◽

Activator Inhibitor ◽

Pai 1 ◽

Mild Hypoxia

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O2tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O2). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5′-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1 vector and the PAI-1–LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O2deficiency.

Download Full-text

The upstream stimulatory factor-2a inhibits plasminogen activator inhibitor-1 gene expression by binding to a promoter element adjacent to the hypoxia-inducible factor-1 binding site

Blood ◽

10.1182/blood.v97.9.2657 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2657-2666 ◽

Cited By ~ 34

Author(s):

Anatoly Samoylenko ◽

Ulrike Roth ◽

Kurt Jungermann ◽

Thomas Kietzmann

Keyword(s):

Plasminogen Activator ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Wild Type ◽

Upstream Stimulatory Factor ◽

Plasminogen Activator Inhibitor 1 ◽

Hypoxia Inducible Factor 1 ◽

Stimulatory Factor ◽

Activator Inhibitor ◽

Pai 1

Abstract Plasminogen activator inhibitor-1 (PAI-1) expression is induced by hypoxia (8% O2) via the PAI-1 promoter region −175/−159 containing a hypoxia response element (HRE-2) binding the hypoxia-inducible factor-1 (HIF-1) and an adjacent response element (HRE-1) binding a so far unknown factor. The aim of the present study was to identify this factor and to investigate its role in the regulation of PAI-1 expression. It was found by supershift assays that the upstream stimulatory factor-2a (USF-2a) bound mainly to the HRE-1 of the PAI-1 promoter and to a lesser extent to HRE-2. Overexpression of USF-2a inhibited PAI-1 messenger RNA and protein expression and activated L-type pyruvate kinase expression in primary rat hepatocytes under normoxia and hypoxia. Luciferase (Luc) gene constructs driven by 766 and 276 base pairs of the 5′-flanking region of the PAI-1 gene were transfected into primary hepatocytes together with expression vectors encoding wild-type USF-2a and a USF-2a mutant lacking DNA binding and dimerization activity (ΔHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of ΔHU2a did not influence Luc activity. Mutation of the HRE-1 (−175/−168) in the PAI-1 promoter Luc constructs decreased USF-dependent inhibition of Luc activity. Mutation of the HRE-2 (−165/−158) was less effective. Cotransfection of a HIF-1α vector could compete for the binding of USF at HRE-2. These results indicated that the balance between 2 transcriptional factors, HIF-1 and USF-2a, which can bind adjacent HRE sites, appears to be involved in the regulation of PAI-1 expression in many clinical conditions.

Download Full-text

CREB binding to the hypoxia-inducible factor-1 responsive elements in the plasminogen activator inhibitor-1 promoter mediates the glucagon effect

Thrombosis and Haemostasis ◽

10.1160/th07-02-0140 ◽

2007 ◽

Vol 98 (08) ◽

pp. 296-303 ◽

Cited By ~ 15

Author(s):

Elitsa Dimova ◽

Malgorzata Jakubowska ◽

Thomas Kietzmann

Keyword(s):

Plasminogen Activator ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Plasminogen Activator Inhibitor 1 ◽

Hypoxia Inducible Factor 1 ◽

E Box ◽

Activator Inhibitor ◽

Pai 1

SummaryPlasminogen activator inhibitor-1 (PAI-1) controls the regulation of the fibrinolytic system in blood by inhibiting both urokinase-type and tissue-type plasminogen activators. Enhanced levels of PAI-1 are related to pathological conditions associated with hypoxia or hyperinsulinemia. In this study, we investigated the regulation of PAI-1 expression by glucagon and the cAMP/ PKA/CREB signalling pathway in the liver. Stimulation of the cAMP/PKA/CREB signalling cascade by starvation in vivo or glucagon in vitro induced PAI-1 gene expression in liver. Furthermore, this response was associated with enhanced phosphorylation of CREB. By using EMSAs we found that three promoter elements, the HRE2, E-box 4 and E-box 5, were able to bind CREB but only the HRE2 and E5 appeared to be functionally active. Reporter gene assays confirmed that cAMP induced PAI-1 gene transcription via the same element in both human and rat promoters. Interestingly, although the HRE2 was involved, the glucagon/cAMP pathway had no influence on hypoxia-inducible factor-1 (HIF-1) mRNA and protein levels. Thus, CREB binding to the HIF-1 responsive elements in PAI-1 promoter mediates the glucagon effect in the liver.

Download Full-text

Vascular Heme Oxygenase-1 Induction Suppresses Tissue Factor and Plasminogen Activator Inhibitor-1 Expressions

Blood ◽

10.1182/blood.v112.11.5447.5447 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5447-5447

Author(s):

Eriko Morishita ◽

Keiko Maruyama ◽

Akiko Sekiya ◽

Shigeki Ohtake ◽

Shinji Nakao ◽

...

Keyword(s):

Endothelial Cell ◽

Tissue Factor ◽

Plasminogen Activator ◽

Heme Oxygenase ◽

Plasminogen Activator Inhibitor ◽

Heme Oxygenase 1 ◽

Plasminogen Activator Inhibitor 1 ◽

Protein Levels ◽

Activator Inhibitor ◽

Pai 1

Abstract Objective - Heme oxygenase-1(HO-1), the rate-limiting enzyme of heme degradation, has recently been considered to have protective roles against various pathological conditions. 10 years have passed since we lost the first and the only patient of HO-1 deficiency. Since the patient of HO-1 deficiency showed endothelial cell injury and extremely enhanced coagulation and fibrinolytic parameters, we examined the effect of HO-1 modulation on tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression on endothelial cells. Methods and Results - Human umbilical vein endothelial cell (HUVEC) was stimulated with hemin (100mM), HO-1 inducer, and mRNA and protein levels for HO-1, TF and PAI-1 were examined. Total RNA was extracted from HUVEC, and was analyzed by real time RT-PCR. Protein expression levels of HO-1, TF and PAI-1 were measured by ELISA. Hemin stimulation increased HO-1 mRNA levels by 20 times. On the other hand, TF mRNA and antigen levels were minimum even after 8 hours of stimulation. Importantly, hemin stimulation reduced PAI-1 mRNA more than half after 4 hours. After HO-1 induction by hemin (100 mM) for 6 hours, HUVEC cultures were exposed to 10 ng/ml tumor necrosis factor (TNF). Prior exposure to hemin significantly increased HO-1 mRNA by 60 times in 30 minutes after stimulation with TNF. However, TNF alone could not induce HO-1 mRNA and protein levels in HUVEC. Although stimulation with TNF enhanced expressions of both TF and PAI-1 mRNA, they were significantly inhibited more than half by prior treatment with hemin. TF antigen levels were similarly decreased (5.0 to 0.7 pg/ml). PAI-1 antigen levels were also inhibited by prior treatment with hemin (1.8 to 0.1 ng/ml)(3) To see if hemin effect on HUVEC is due to HO-1 production, HO-1 inhibitor tin-protoporphyrin IX (SnPP-IX) was added to the cultures. The inhibitor effect of hemin on TF and PAI-1 productions was cancelled when HUVEC was cocultured with SnPP-IX. Conclusions - These results indicate that hemin exert inhibitory effect on TF and PAI-1 expressions through HO-1 production. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with inflammation.

Download Full-text

Regulation of the hypoxia-dependent plasminogen activator inhibitor 1 expression by MAP kinases

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613573 ◽

2003 ◽

Vol 89 (04) ◽

pp. 666-673 ◽

Cited By ~ 40

Author(s):

Kurt Jungermann ◽

Agnes Görlach ◽

Thomas Kietzmann

Keyword(s):

Plasminogen Activator ◽

Map Kinases ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Mrna Levels ◽

Plasminogen Activator Inhibitor 1 ◽

Hypoxic Response ◽

Pi3k Inhibitor Ly294002 ◽

Activator Inhibitor ◽

Pai 1

SummaryMitogen-activated protein kinases (MAPKs) and protein kinase B (PKB) mediate growth and stress signals and have been implicated in the hypoxic response. Under hypoxic conditions, the expression of plasminogen activator inhibitor-1 (PAI-1) is mainly controlled by the hypoxia-inducible factor HIF-1. However, the role of MAPKs and PKB in HIF-1-mediated PAI-1 regulation is not clear.Treatment with the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, but not with the MEK1 inhibitor PD98059, abrogated hypoxia-dependent PAI-1 induction in HepG2 cells. Consistently, overexpression of PKB or of the p38 upstream kinases MKK6 and MKK3 and of JNK, but not of ERK, enhanced PAI-1 mRNA levels. In MKK3-,MKK6- and PKB-expressing cells luciferase (Luc) activities from a hypoxia-inducible PAI-1-Luc construct or from a HIF-dependent Luc construct and, concomitantly, HIF-1α protein levels were enhanced. These findings indicate that p38- and PKB-dependent signalling pathways contribute to enhanced PAI-1 levels in the hypoxic response.Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.

Download Full-text

Hypoxia-inducible factor-1 and hypoxia response elements mediate the induction of plasminogen activator inhibitor-1 gene expression by insulin in primary rat hepatocytes

Blood ◽

10.1182/blood-2002-06-1693 ◽

2003 ◽

Vol 101 (3) ◽

pp. 907-914 ◽

Cited By ~ 72

Author(s):

Thomas Kietzmann ◽

Anatoly Samoylenko ◽

Ulrike Roth ◽

Kurt Jungermann

Keyword(s):

Gene Expression ◽

Plasminogen Activator ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activator Inhibitor 1 ◽

Hypoxia Response ◽

Response Elements ◽

Hypoxia Inducible Factor 1 ◽

Activator Inhibitor ◽

Pai 1

AbstractThe expression of the plasminogen activator inhibitor-1(PAI-1) gene is enhanced by insulin both in vivo and in various cell types. Because insulin exerts a number of its biologic activities via the phosphatidylinositol 3-kinase and protein kinase B (PI3K/PKB) signaling pathway, it was the aim of the present study to investigate the role of the PI3K/PKB pathway in the expression of the PAI-1 gene and to identify the insulin responsive promoter sequences. It was shown that the induction of PAI-1 mRNA and protein expression by insulin and mild hypoxia could be repressed by the PI3K inhibitor wortmannin. Overexpression of a constitutively active PKB led to induction of PAI-1 mRNA expression and of luciferase (Luc) activity from a gene construct containing 766 bp of the rat PAI-1 promoter. Mutation of the hypoxia response elements (HRE-1 and HRE-2) in rat PAI-1 promoter, which could bind hypoxia inducible factor-1 (HIF-1), abolished the induction of PAI-1 by insulin and PKB. Insulin and the constitutive active PKB also induced Luc expression in cells transfected with the pGl3EPO-HRE Luc construct, containing 3 copies of the HRE from the erythropoietin gene in front of the SV40 promoter. Furthermore, insulin and the active PKB enhanced all 3 HIF α-subunit protein levels and HIF-1 DNA-binding activity, as shown by electrophoretic mobility shift assays (EMSAs). Thus, the insulin-dependent activation of the PAI-1 gene expression can be mediated via the PI3K/PKB pathway and the transcription factor HIF-1 binding to the HREs in the PAI-1 gene promoter.

Download Full-text

Induction of the Plasminogen Activator Inhibitor-1 Gene Expression by Mild Hypoxia Via a Hypoxia Response Element Binding the Hypoxia-Inducible Factor-1 in Rat Hepatocytes

Blood ◽

10.1182/blood.v94.12.4177 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4177-4185 ◽

Cited By ~ 173

Author(s):

Thomas Kietzmann ◽

Ulrike Roth ◽

Kurt Jungermann

Keyword(s):

Plasminogen Activator ◽

Hypoxia Inducible Factor ◽

Plasminogen Activator Inhibitor ◽

Rat Hepatocytes ◽

Plasminogen Activators ◽

Plasminogen Activator Inhibitor 1 ◽

Hypoxia Response ◽

Activator Inhibitor ◽

Pai 1 ◽

Mild Hypoxia

Abstract Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O2tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O2). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5′-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1 vector and the PAI-1–LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O2deficiency.

Download Full-text