Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor–1

Abstract Plasminogen activator inhibitor–1 (PAI-1) plays a key role in control of coagulation and tissue remodeling and has been shown to be regulated by a number of cell stimuli, among those hypoxia. In this study we characterize the hypoxia-mediated induction of PAI-1 in human hepatoma cell line HepG2. We found that PAI-1 is tightly regulated in a narrow oxygen gradient. After incubation at oxygen concentrations of 1% to 2%, a 60-fold increase in PAI-1 messenger RNA levels was observed, whereas mild hypoxic conditions of more than 3.5% did not appear to induce transcription. Moreover, increased levels of PAI-1 protein were observed after incubation at low oxygen tensions. Through sequence analysis, several putative hypoxia-response elements (HREs 1-5) were identified in the human PAI-I promoter. Reporter gene assays showed that the HRE-2 (−194 to −187) was necessary and sufficient for the hypoxia-mediated response. By electrophoretic mobility assay we observed hypoxia-dependent binding of a protein complex to the HRE-2 motif. Further analysis demonstrated that HRE-2 was specifically recognized by the hypoxia-inducible transcription factor 1α–arylhydrocarbon nuclear translocator complex. Taken together, our data demonstrate that hypoxia-induced transcription is mediated through HIF-1 interaction with the HRE-2 site of the human PAI-1 promoter.

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Plasminogen activator inhibitor-1 synthesis in the human hepatoma cell line Hep G2. Metformin inhibits the stimulating effect of insulin.

Journal of Clinical Investigation ◽

10.1172/jci116445 ◽

1993 ◽

Vol 91 (5) ◽

pp. 2185-2193 ◽

Cited By ~ 57

Author(s):

F Anfosso ◽

N Chomiki ◽

M C Alessi ◽

P Vague ◽

I Juhan-Vague

Keyword(s):

Cell Line ◽

Plasminogen Activator ◽

Hepatoma Cell ◽

Plasminogen Activator Inhibitor ◽

Hepatoma Cell Line ◽

Human Hepatoma ◽

Hep G2 ◽

Plasminogen Activator Inhibitor 1 ◽

Human Hepatoma Cell ◽

Activator Inhibitor

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Developmental Expression of Plasminogen Activator Inhibitor-1 Associated With Thrombopoietin-Dependent Megakaryocytic Differentiation

Blood ◽

10.1182/blood.v94.2.475 ◽

1999 ◽

Vol 94 (2) ◽

pp. 475-482 ◽

Cited By ~ 12

Author(s):

Seiji Madoiwa ◽

Norio Komatsu ◽

Jun Mimuro ◽

Kouzoh Kimura ◽

Michio Matsuda ◽

...

Keyword(s):

Cord Blood ◽

Mrna Expression ◽

Progenitor Cells ◽

Plasminogen Activator ◽

Messenger Rna ◽

Plasminogen Activator Inhibitor ◽

Developmental Expression ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

Abstract Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet -granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34+/CD41− cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-typec-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor- (TNF-), and interleukin-1β (IL-1β) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34+/CD41− cells to CD34−/CD41+ cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.

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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.

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Developmental Expression of Plasminogen Activator Inhibitor-1 Associated With Thrombopoietin-Dependent Megakaryocytic Differentiation

Blood ◽

10.1182/blood.v94.2.475.414k38_475_482 ◽

1999 ◽

Vol 94 (2) ◽

pp. 475-482

Author(s):

Seiji Madoiwa ◽

Norio Komatsu ◽

Jun Mimuro ◽

Kouzoh Kimura ◽

Michio Matsuda ◽

...

Keyword(s):

Cord Blood ◽

Mrna Expression ◽

Progenitor Cells ◽

Plasminogen Activator ◽

Messenger Rna ◽

Plasminogen Activator Inhibitor ◽

Developmental Expression ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet -granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34+/CD41− cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-typec-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor- (TNF-), and interleukin-1β (IL-1β) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34+/CD41− cells to CD34−/CD41+ cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.

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Immunoaffinity Purification of HTC Rat Hepatoma Cell Plasminogen Activator-Inhibitor-1

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646047 ◽

1987 ◽

Vol 58 (04) ◽

pp. 1017-1023 ◽

Cited By ~ 23

Author(s):

Ron Zeheb ◽

Usha M Rafferty ◽

Miguel A Rodriguez ◽

Peter Andreasen ◽

Thomas D Gelehrter

Keyword(s):

Plasminogen Activator ◽

Hepatoma Cell ◽

Plasminogen Activator Inhibitor ◽

Hepatoma Cells ◽

Tissue Type ◽

Plasminogen Activator Inhibitor 1 ◽

Major Band ◽

Activator Inhibitor ◽

Pai 1 ◽

Rat Hepatoma

SummaryIncubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45° C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.

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Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone

Blood ◽

10.1182/blood.v79.10.2636.2636 ◽

1992 ◽

Vol 79 (10) ◽

pp. 2636-2642 ◽

Cited By ~ 1

Author(s):

BA Konkle ◽

SJ Schuster ◽

MD Kelly ◽

K Harjes ◽

DE Hassett ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Messenger Rna ◽

Plasminogen Activator Inhibitor ◽

Acute Injury ◽

Plasminogen Activator Inhibitor 1 ◽

Sinusoidal Endothelial Cells ◽

Activator Inhibitor ◽

Pai 1

Abstract Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue plasminogen activator (tPA), plays a crucial role in the regulation of fibrinolysis. Both hepatocytes and endothelial cells have been implicated as major sources of plasma PAI-1. To study the relative contribution of these cell types to hepatic PAI-1 production, we have separated hepatocytes and hepatic sinusoidal endothelial cells by fractionation of freshly isolated rat livers using metrizamide density gradients and centrifugal elutriation. In untreated animals, PAI-1 messenger RNA (mRNA) was detected only in the purified endothelial cell fraction, and not in the hepatocyte fraction or in unfractionated liver. However, when the animals were treated with dexamethasone, PAI-1 mRNA expression was transiently induced in the liver. This induction paralleled the appearance of PAI-1 mRNA in purified hepatocytes, while PAI-1 expression in sinusoidal endothelial cells was unchanged. Four hours after dexamethasone treatment, plasma PAI-1 levels were increased approximately twofold over levels measured in animals treated with the diluent alone. These data suggest that PAI- 1 production by hepatocytes may contribute to elevated plasma PAI-1 levels in the setting of acute injury and stress.

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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.

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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.

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Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone

Blood ◽

10.1182/blood.v79.10.2636.bloodjournal79102636 ◽

1992 ◽

Vol 79 (10) ◽

pp. 2636-2642 ◽

Cited By ~ 35

Author(s):

BA Konkle ◽

SJ Schuster ◽

MD Kelly ◽

K Harjes ◽

DE Hassett ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Messenger Rna ◽

Plasminogen Activator Inhibitor ◽

Acute Injury ◽

Plasminogen Activator Inhibitor 1 ◽

Sinusoidal Endothelial Cells ◽

Activator Inhibitor ◽

Pai 1

Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue plasminogen activator (tPA), plays a crucial role in the regulation of fibrinolysis. Both hepatocytes and endothelial cells have been implicated as major sources of plasma PAI-1. To study the relative contribution of these cell types to hepatic PAI-1 production, we have separated hepatocytes and hepatic sinusoidal endothelial cells by fractionation of freshly isolated rat livers using metrizamide density gradients and centrifugal elutriation. In untreated animals, PAI-1 messenger RNA (mRNA) was detected only in the purified endothelial cell fraction, and not in the hepatocyte fraction or in unfractionated liver. However, when the animals were treated with dexamethasone, PAI-1 mRNA expression was transiently induced in the liver. This induction paralleled the appearance of PAI-1 mRNA in purified hepatocytes, while PAI-1 expression in sinusoidal endothelial cells was unchanged. Four hours after dexamethasone treatment, plasma PAI-1 levels were increased approximately twofold over levels measured in animals treated with the diluent alone. These data suggest that PAI- 1 production by hepatocytes may contribute to elevated plasma PAI-1 levels in the setting of acute injury and stress.

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