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 respo nse element (HRE-2) binding the hypoxia-inducible factor-1 (HIF-1) and an a djacent response element (HRE-1) binding a so far unknown factor. The aim o f 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 t hat the upstream stimulatory factor-2a (USF-2a) bound mainly to the HRE-1 o f 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-ty pe 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 (Delta HU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by abou t 8-fold, whereas cotransfection of Delta HU2a did not influence Luc activi ty. 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-1 alpha vector coul d 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 bin d adjacent HRE sites, appears to be involved in the regulation of PAI-1 exp ression in many clinical conditions. (Blood, 2001;97:2657-2666) (C) 2001 by The American Society of Hematology.