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.
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 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 (DeltaHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of DeltaHU2a 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-1alpha 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.
Samoylenko
A
A
Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, Göttingen, Germany.
Roth
U
U
Jungermann
K
K
Kietzmann
T
T
D000818:Animals; D015687:Cell Hypoxia; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008099:Liver; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D014157:Transcription Factors / Q000235:genetics*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D017395:Plasminogen Activator Inhibitor 1; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat