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

Abstract

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...

The Transcription Factors HIF-1 and HNF-4 and the Coactivator p300 Are Involved in Insulin-regulated Glucokinase Gene Expression via the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway

“…The above mentioned findings are in line with studies showing that insulin enhances the binding of HIF-1 to HREs in HepG2 hepatoma cells, L6 rat skeletal muscle myoblasts, and primary hepatocytes (25)(26)(27). Furthermore, transfection studies with an HIF-1-dependent Luc reporter gene containing HRE sequences demonstrate an insulin-dependent enhancement of Luc activity, which also is in line with the present findings.…”

“…Furthermore, transfection studies with an HIF-1-dependent Luc reporter gene containing HRE sequences demonstrate an insulin-dependent enhancement of Luc activity, which also is in line with the present findings. In addition, the insulin-induced enhancement of HIF-1␣ protein levels is inhibited in a dose-dependent manner by specific PI3K inhibitors such as LY294002 and wortmannin in prostate carcinoma-derived cell lines (13) and hepatocytes (27), implicating the involvement of a PI3K-dependent pathway. Insulin appeared to act via a translationaldependent pathway, since cycloheximide inhibited the insulindependent enhancement of HIF-1␣ protein levels (50).…”

“…enolase-1 HRE, and plasminogen activator inhibitor-1 HRE have shown that HIF-1 binding activity is stimulated by insulin (25,27). Furthermore, it was shown that downstream of the EPO-HRE, the binding of HNF-4 to an HNF-4-responsive element enhances HIF-1-mediated EPO gene activation (32,33).…”

“…Since it has been shown that the expression of SREBP (28,49) and HIF-1␣ (26,27,50) can be stimulated by insulin, we examined whether SREBP and HIF-1␣ may regulate GK promoter activity via the putative HRE.…”

“…Two other HIF ␣-subunits, HIF-2␣ (EPAS/HLF/ HRF/MOP2) (18 -21), and HIF-3␣ (22,23) have been cloned from human, mouse, and rat, and together with other ARNT isoforms (ARNT2 and ARNT3/BMAL-1/MOP-3), they give rise to the existence of several HIF dimers composed of different HIF ␣-subunits and ARNT isoforms (18,24). Interestingly HIF-␣ protein levels and transcriptional activity have been shown to be enhanced by insulin (25)(26)(27).…”