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.

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.

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

2010 ◽  
Vol 103 (05) ◽  
pp. 901-909 ◽  
Author(s):  
Anatoly Samoylenko ◽  
Elitsa Dimova ◽  
Nina Kozlova ◽  
Lyudmyla Drobot ◽  
Thomas Kietzmann
Keyword(s):  
Plasminogen Activator ◽  
Tyrosine Kinases ◽  
Hypoxia Inducible Factor ◽  
Adaptor Protein ◽  
Plasminogen Activator Inhibitor ◽  
Transcriptional Level ◽  
Plasminogen Activator Inhibitor 1 ◽  
Protein Levels ◽  
Activator Inhibitor ◽  
Pai 1

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.

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.

Abstract 1331: Myocardial Function Alterations Correlate to Cardiac Fibrosis and Upregulation of Profibrotic Signaling in Plasminogen Activator Inhibitor-1 Deficient Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
William Bradham ◽  
Linda Gleaves ◽  
Mousumi Medda ◽  
Douglas Vaughan
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Cardiac Fibrosis ◽  
Plasminogen Activator Inhibitor ◽  
Rt Pcr ◽  
Plasminogen Activator Inhibitor 1 ◽  
Internal Dimension ◽  
Functional Consequences ◽  
Activator Inhibitor ◽  
Pai 1

Cardiac fibrosis is a common sequelae of cardiac injury and has deleterious functional consequences impacting cardiac filling, function and rhythm. Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of tissue fibrosis in mice. To investigate the longitudinal effect of PAI-1 on cardiac structure and function, M-mode echocardiography was employed to examine cardiac function in PAI-1 deficient (PAI-1 −/− ) and wild-type (WT) control mice in four age groups (6,12,18, 24 months). Eighteen month old PAI-1 −/− mice exhibited reduced left ventricular (LV) diastolic internal dimension ( p =0.0118) and a trend towards increased LV posterior wall (LVPW) thickness, compared to WT. Two year old PAI-1 −/− mice showed increased diastolic and systolic LVPW thickness ( p =0.0127 and p =0.0212, respectively), reduced diastolic and systolic LV internal dimension ( p =0.0486 and p =0.0124), but with preserved LV fractional shortening compared to WT. Histological examination of cardiac sections revealed fibrosis on the anterior epicardial surface of the hearts in 18 month old PKO, which in 26 month old mice had become confluent with extensive (10 –17% by area) epicardial, perivascular, and interstitial distribution (compared to none in WT). Real time polymerase chain reaction (RT-PCR) revealed upregulation of transforming growth factor beta (TGF-β) and fibroblast growth factor 2 in PAI-1 −/− compared to WT ( p =0.0234 and p =0.037, respectively). Immunofluoresence confirmed this finding with bright TGF-β staining localized in the media of intra-myocardial arterioles, and phosphorylated SMAD2/3, the downstream TGF-β signaling mediator, in areas of fibrosis. Thoracic aortic cells from aged (18 –24 month) PKO and WT mice were grown in culture, with RT-PCR revealing 4 fold increased TGF-β and 17 fold increased SMAD3 ( p <0.05 for both) RNA levels in PAI-1 −/− , supplying additional evidence for upregulation of a profibrotic TGF-β/SMAD tissue signaling pathway. The present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways related to aging and PAI-1 deficiency mediated cardiac fibrosis.

scholarly journals Fibroblast-specific plasminogen activator inhibitor-1 depletion ameliorates renal interstitial fibrosis after unilateral ureteral obstruction

2019 ◽  
Vol 34 (12) ◽  
pp. 2042-2050 ◽  
Author(s):  
Lan Yao ◽  
M Frances Wright ◽  
Brandon C Farmer ◽  
Laura S Peterson ◽  
Amir M Khan ◽  
...  
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Ureteral Obstruction ◽  
Interstitial Fibrosis ◽  
Plasminogen Activator Inhibitor ◽  
Unilateral Ureteral Obstruction ◽  
Tubular Epithelium ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Background Plasminogen activator inhibitor-1 (PAI-1) expression increases extracellular matrix deposition and contributes to interstitial fibrosis in the kidney after injury. While PAI-1 is ubiquitously expressed in the kidney, we hypothesized that interstitial fibrosis is strongly dependent on fibroblast-specific PAI-1 (fbPAI-1). Methods Tenascin C Cre (TNC Cre) and fbPAI-1 knockdown (KD) mice with green fluorescent protein (GFP) expressed within the TNC construct underwent unilateral ureteral obstruction and were sacrificed 10 days later. Results GFP+ cells in fbPAI-1 KD mice showed significantly reduced PAI-1 expression. Interstitial fibrosis, measured by Sirius red staining and collagen I western blot, was significantly decreased in fbPAI-1 KD compared with TNC Cre mice. There was no significant difference in transforming growth factor β (TGF-β) expression or its activation between the two groups. However, GFP+ cells from fbPAI-1 KD mice had lower TGF β and connective tissue growth factor (CTGF) expression. The number of fibroblasts was decreased in fbPAI-1 KD compared with TNC Cre mice, correlating with decreased alpha smooth muscle actin (α-SMA) expression and less fibroblast cell proliferation. TNC Cre mice had decreased E-cadherin, a marker of differentiated tubular epithelium, in contrast to preserved expression in fbPAI-1 KD. F4/80-expressing cells, mostly CD11c+/F4/80+ cells, were increased while M1 macrophage markers were decreased in fbPAI-1 KD compared with TNC Cre mice. Conclusion These findings indicate that fbPAI-1 depletion ameliorates interstitial fibrosis by decreasing fibroblast proliferation in the renal interstitium, with resulting decreased collagen I. This is linked to decreased M1 macrophages and preserved tubular epithelium.

IL-1 and IL-6 induce hepatocyte plasminogen activator inhibitor-1 expression through independent signaling pathways converging on C/EBPδ

2007 ◽  
Vol 292 (1) ◽  
pp. C209-C215 ◽  
Author(s):  
Jie Dong ◽  
Satoshi Fujii ◽  
Shogo Imagawa ◽  
Shuichiro Matsumoto ◽  
Michiaki Matsushita ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Plasminogen Activator ◽  
Hepg2 Cells ◽  
Plasminogen Activator Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Plasminogen Activator Inhibitor 1 ◽  
Mobility Shift ◽  
Primary Mouse ◽  
Activator Inhibitor ◽  
Pai 1

To elucidate signaling pathways activated by IL-1 and IL-6 that contribute to increased expression of plasminogen activator inhibitor-1 (PAI-1), we studied human hepatoma (HepG2) cells and primary mouse hepatocytes. HepG2 cell PAI-1 mRNA increased in response to IL-1β, IL-6, and IL-1β plus IL-6 as shown by real-time PCR. Activity of the transiently transfected PAI-1 promoter (−829 to +36 bp) increased as well. Systematic promoter deletion assays showed that the region from −239 to −210 bp containing a putative CCAAT-enhancer binding protein (C/EBP) binding site was critical. Point mutations in this region abolished the IL-1β and IL-6 responses. Antibody interference electrophoretic mobility shift assays showed that C/EBPδ (but not C/EBPα or C/EBPβ) binding and protein were increased by IL-1β, IL-6, and IL-1β plus IL-6 in HepG2 cells. IL-1β and IL-6 increased expression of both PAI-1 mRNA and C/EBPδ mRNA in mouse primary hepatocytes as well. Downregulation of C/EBPδ induced with small interfering RNA (siRNA) decreased secretion of PAI-1. As judged from results obtained with inhibitors, signal transduction in all three of the mitogen-activated protein kinase pathways was involved in IL-1-inducible PAI-1 expression. By contrast, JAK signaling was responsible for the IL-6-induced inducible expression. Thus IL-1 and IL-6 exert directionally similar effects on PAI-1 expression, but the induction involves distinct signaling pathways with a final common mediator, C/EBPδ.

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