HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY 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% O2) via the PAI-1 promoter region 2175/2159 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 Ltype 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 (DHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of DHU2a did not influence Luc activity. Mutation of the HRE-1 (2175/2168) in the PAI-1 promoter Luc constructs decreased USF-dependent inhibition of Luc activity. Mutation of the HRE-2 (2165/ 2158) was less effective. Cotransfection of a HIF-1a 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. (Blood. 2001;97:2657-2666)