Abstract-The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22 phox -containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-␤ 1 (TGF-␤ 1 ) upregulated HIF-1␣ protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1␣ as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1␣, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22 phox antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1␣, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22 phox -containing NADPH oxidase is crucially involved in this response. Key Words: oxygen radicals Ⅲ p22 phox Ⅲ platelets Ⅲ vascular endothelial growth factor Ⅲ plasminogen activator inhibitor-1

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 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 re- IntroductionThe broad-spectrum serine protease plasmin is activated by the proteases, tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. 1 The tPA and uPA activity is regulated, in part, by plasminogen activator inhibitors (PAIs) that are glycoproteins of the serine protease inhibitor (serpin) superfamily. 2 Among 2 identified inhibitors, PAI-1 and PAI-2, PAI-1 is the primary physiologic inhibitor of both tPA and uPA. It can be produced by platelets, vascular endothelial cells, 3 vascular smooth muscle cells, 4 and several nonvascular cell types, among them hepatocytes. 5,6 PAI-1 regulates fibrinolysis in many normal and pathologic conditions such as atherosclerosis, coronary heart disease, wound healing, and cancer metastasis. [7][8][9] It was shown that hyperinsulinemia especially associated with obesity, hypertension, and diabetes type 2 could increase PAI-1 levels in blood. [10][11][12][13] Furthermore, insulin induced PAI-1 expression in vitro in various cell types, including primary human hepatocytes, 14,15 HepG2 cells, 16,17 and arterial endothelial cells. 18,19 Insulin signaling involves second messengers, including members of the phosphatidylinositol 3-kinase (PI3K) and mitogenactivated protein kinase (MAPK) cascades. 20 The PI3K, which generates phosphatidylinositol-3,4,5-phosphate (PI3,4,5P 3 ), has a key role in the metabolic actions of insulin. 21 PI3,4,5P 3 regulates the activity or subcellular localization of a variety of signaling molecules such as phosphatidylinositol-dependent kinase (PDK) and protein kinase B (PKB) known as Akt, which are also involved in the transmission of the insulin signal. 22,23 The expression of the PAI-1 gene and many other genes, expression of which can be induced by insulin, such as genes for the glucose transporters, 24 several glycolytic enzymes, 25 nitric oxide (NO) synthase, 26 erythropoietin (EPO), 27 and vascular endothelial growth factor (VEGF), 28 can also be induced by hypoxia. [29][30][31][32][33] The rat PAI-1 gene was induced by hypoxia via an O 2 responsive promoter sequence (Ϫ175/Ϫ158) containing 2 hypoxia response elements (HRE-1, Ϫ175/Ϫ168; HRE-2, Ϫ165/Ϫ158) 34 binding the transcription factor hypoxia inducible factor-1 (HIF-1). HIF-1 is a dimer of HIF-1␣ and HIF-1␤ (arylhdrocarbon receptor nuclear translocator [ARNT]) both belonging to the basic helix-loophelix (bHLH) PAS (Per-ARNT-Sim) transcription factor family. Two other HIF ␣-subunits (HIF-2␣/EPAS/HRF/HLF and HIF-3␣) a...

Glucokinase plays a key role in the regulation of glucose utilization in liver and its expression is strongly enhanced by insulin and modulated by venous pO 2 . In primary rat hepatocytes, pO 2 modulated insulindependent glucokinase (GK) gene expression was abolished by wortmannin an inhibitor of phosphatidylinositol 3-kinase (PI3K). Transfection of vectors encoding the p110 catalytic subunit of PI3K or constitutively active proteinkinase B (PKB) stimulated GK mRNA and protein expression. The transfection of GK promoter constructs together with expression vectors for p110 or constitutively active PKB revealed that the GK promoter region ؊87/؊80 mediates the response to PI3K/PKB. Transfection experiments and gel shift assays show that this element is able to bind hypoxia-inducible factor-1 (HIF-1) in a hypoxia-and PKB-dependent manner. The ability of HIF-1␣ to activate the GK promoter was enhanced by hepatocyte nuclear factor-4␣ (HNF-4␣), acting via the sequence ؊52/؊39, and by the coactivator p300. Stimulation of the GK promoter by insulin was dependent on the intact ؊87/؊80 region and maximal stimulation was achieved when HIF-1␣, HNF-4, and p300 were cotransfected with the ؊1430 GK promoter Luc construct in primary hepatocytes. Maximal stimulation of GK promoter activity by insulin was inhibited when a p300 vector was used containing a mutation within a PKB phosphorylation site. Thus, a regulatory transcriptional complex consisting of HIF-1, HNF-4, and p300 appears to be involved in insulin-dependent GK gene activation.

Liver glucokinase (GK) is localized predominantly in the perivenous zone. GK mRNA was induced by insulin maximally under venous O2 partial pressure (pO2) and only half-maximally under arterial pO2. CoCl2 and desferrioxamine mimicked venous pO2 and enhanced the insulin-dependent induction of GK mRNA under arterial pO2. H2O2 mimicked arterial pO2 and reduced insulin-induced GK mRNA under venous pO2 to the lower arterial levels. Thus the zonal O2 gradient in liver seems to have a key role in the heterogenous expression of the GK gene.

Plasminogen activator inhibitor-1 (PAI-1) expression is induced by hypoxia (8% O 2 ) 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 IntroductionThe tissue-type and the urokinase-type plasminogen activators (tPA and uPA) are serine proteases converting the inactive zymogen plasminogen to the active endopeptidase plasmin. 1 The tPA and uPA activity is regulated, in part, by plasminogen activator inhibitors (PAIs). 2 Among 2 identified inhibitors, PAI-1 and PAI-2, PAI-1 is the primary physiologic inhibitor of both tPA and uPA. 3 PAI-1 is a 50-kd glycoprotein from the serpin superfamily. 4 It can be produced by platelets, vascular endothelial cells, 5 vascular smooth muscle cells, 6 and several nonvascular cell types, 7,8 including hepatocytes. 9 PAI-1 has also been identified as a component of the extracellular matrix. 10 The plasminogen activator inhibitors are involved in many functions, both under normal and pathological conditions, including fibrinolysis, extracellular matrix turnover, and fibrosis. 11 PAI-1 also participates in wound healing and cancer metastasis. 12,13 Certain pathophysiologic processes in which PAI-1 levels increase (eg, prethrombotic events, hemorrhage, and thrombus formation) are associated with hypoxia. PAI-1 gene expression was induced by mild hypoxia (8% O 2 ) via an O 2 -responsive promoter sequence (Ϫ175/Ϫ158) containing hypoxia response element-1 (HRE-1, Ϫ175/Ϫ168) and hypoxia response element-2 (HRE-2, Ϫ165/Ϫ158) in primary cultured rat hepatocytes. 14 HRE-2 was shown to bind the hypoxia-inducible factor-1 (HIF-1) and thus to be most critical for the increase in PAI-1 gene expression under hypoxia. The HRE-1 sequence was shown to bind a factor other than HIF-1.The HRE-1 and also HRE-2 include a CACGTG-like sequence that can be recognized by transcription factors containing basic helix-loop-helix (bHLH) domains. Besides HIF-1 consisting of the bHLH-PAS (Per-ARNT-Sim) domain proteins HIF-1␣ and HIF-1␤ (ARNT, or arylhdrocarbon receptor nuclear translocator), 15 the bHLH leucine zipper (bHLH-zip) proteins, upstream stimulatory factors (USF), 16 or the Myc/Max 16,17 transcription factors also can bind to the CACGTG sequence. USF was initially identified in HeLa cells as a protein binding the CACGTG sequence located immediately upstream of the TATA sequence of the adenovirus major late promoter, 16 thereby activating transcription. 18 Two different ubiquituously expressed forms of USF (USF-1 and USF-2) with different molecular weights have been identified; however, their relative abundance varies in different cell types. 19,20 The US...

Glucokinase (GK) is a key enzyme for glucose utilization in liver and shows a higher expression in the perivenous zone. In primary rat hepatocytes, the GK gene expression was activated by HNF (hepatic nuclear factor)-4alpha via the sequence -52/-39 of the GK promoter. Venous pO2 enhanced HNF-4 levels and HNF-4 binding to the GK-HNF-4 element. Thus, HNF-4alpha could play the role of a regulator for zonated GK expression.

Glucokinase (GK) is the key enzyme of glucose utilization in liver and is localized in the less aerobic perivenous area. Until now, the O2-responsive elements in the liver-specific GK promoter are unknown, and therefore the aim of this study was to identify the O2-responsive element in this promoter. We found that the GK promoter sequence -87/-80 matched the binding site for hypoxia inducible factor 1 (HIF-1) and upstream stimulatory factor (USF). In primary rat hepatocytes we could show that venous pO2 enhanced HIF-1alpha and USF-2a levels, both of which activated GK expression. Furthermore, transfection experiments revealed that the GK sequence -87/-80 mediated the HIF-1alpha- or USF-2-dependent activation of the GK promoter. The binding of HIF-1 and USF to the GK-HRE was corroborated by electrophoretic mobility shift assay (EMSA). However, the maximal response to HIF-1alpha or USF was only achieved when constructs with the -87/-80 sequence in context with a 3'-36 bp native GK promoter sequence containing a hepatocyte nuclear factor 4 (HNF-4) binding site were used. HIF-1alpha and HNF-4 additively activated the GK promoter, while USF-2 and HNF-4 together did not show this additive activation. Thus, HIF-1 and USF may play differential roles in the modulation of GK expression in response to O2.