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 ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 907-914 ◽  
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.

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 ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4177-4185 ◽  
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.

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 ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2657-2666 ◽  
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.

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

2007 ◽  
Vol 98 (08) ◽  
pp. 296-303 ◽  
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.

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 ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4177-4185 ◽  
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.

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

2005 ◽  
Vol 93 (06) ◽  
pp. 1176-1184 ◽  
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.

Critical Roles of Rho Kinase and Mek/Erk Pathways for Angiotensin Ii-Induced Plasminogen Activator Inhibitor-1 Gene Expression

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Kotaro Takeda ◽  
Toshihiro Ichiki ◽  
Tomotake Tokunou ◽  
Satoshi Fujii ◽  
Akira Kitabatake ◽  
...  
Keyword(s):  
Gene Expression ◽  
Angiotensin Ii ◽  
Plasminogen Activator ◽  
Rho Kinase ◽  
Plasminogen Activator Inhibitor ◽  
Luciferase Assay ◽  
Ang Ii ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

P157 Plasminogen activator inhibitor-1 (PAI-1) plays an integral role not only in the regulation of plasminogen activity and fibrinolytic system but also in the pathogenesis of atherosclerosis and hypertension. Because angiotensin II (Ang II) is also involved in these processes, we investigated its role in the intracellular signaling cascade leading to PAI-1 gene expression in vascular smooth muscle cells (VSMC). Ang II increased the PAI-1 mRNA and protein levels through Ang II type 1 receptor. Although PAI-1 mRNA stability was not increased by Ang II, PAI-1 gene promoter activity, which was measured by luciferase assay, was significantly increased by Ang II. This process did not require de novo protein synthesis. BAPTA-AM, genistein and AG1478 completely inhibited the Ang II-induced PAI-1 mRNA upregulation, suggesting that intracellular calcium, tyrosine kinase and epidermal growth factor (EGF) receptor transactivation were involved in this process. However, inhibition of protein kinase C (PKC) by calphostin C, GF109203, or prolonged exposure to PMA failed to abolish the Ang II-induced PAI-1 upregulation, suggesting PKC pathway was not involved. PD98059 suppressed Ang II-induced PAI-1 upregulation, whereas SB203580 did not, suggesting that MEK/ERK1/2 pathway rather than p38 MAP kinase pathway was crucial in this process. Furthermore, adenovirus-mediated expression of dominant negative form of Rho kinase or Rho kinase inhibitor Y27632 also completely suppressed PAI-1 induction by Ang II without affecting Ang II-induced ERK1/2 activation. These data suggest that activation of both MEK/ERK1/2 and Rho kinase pathways will be necessary for the upregulation of PAI-1 gene expression and these two pathways may act synergically to promote PAI-1 gene transcription at least at the downstream of ERK1/2 in VSMC. These findings are important biological and therapeutical implications for the evolution of arterial wall thrombus and the pathogenesis of atherosclerosis by Ang II.

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