Hypoxia-inducible factor-1 and hypoxia response elements mediate the induction of plasminogen activator inhibitor-1 gene expression by insulin in primary rat hepatocytes.
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 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 alpha-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.
Kietzmann
Thomas
T
Institut für Biochemie und Molekulare Zellbiologie, Göttingen, Germany. tkietzm@gwdg.de
Samoylenko
Anatoly
A
Roth
Ulrike
U
Jungermann
Kurt
K
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D000860:Hypoxia / Q000235:genetics*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D020218:Response Elements; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism
D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007328:Insulin; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017395:Plasminogen Activator Inhibitor 1; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D014157:Transcription Factors; C497444:Hif1a protein, rat