Inhibition of Luc activity by USF-2a is conferred by HRE-1 and HRE-2 in the rat PAI-1 promoter.

(A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites [A-G]. In the “C-site” the 2 potential USF-2a binding elements (HRE) matching the E-box consensus sequence are underlined. S (G/C) is shown because the actual PAI-1 sequence does not match bases allowed by the consensus. (B) Hepatocytes were transiently cotransfected with either USF-2a or ΔHU2a expression plasmids and Luc gene constructs driven by a wild-type 276-bp rat PAI-1 promoter (pGl3PAI-276) or the 276 bp promoter mutated at either the HRE-1 (pGl3PAI-276M1) or HRE-2 (pGl3PAI-276M2) site. In control experiments Luc constructs were cotransfected with pCMV plasmid. In each experiment the percentage of Luc activity was determined relative to the pGl3PAI-276 + pCMV control, which was set equal to 100%. In pGl3PAI-276M1 and pGl3PAI-276M2 the wild-type PAI-1 sequence is shown on the upper strand, and mutated bases are indicated by an asterisk and are shown in lowercase letters. The values represent means ± SEM of 3 independent experiments. Statistics, Student t test for paired values: *, significant difference pGl3PAI-276 + USF-2a versus pGl3PAI-276 + pCMV control; pGl3PAI-276M1 + USF-2a versus pGl3PAI-276M1 + pCMV control; pGl3PAI-276M2 + USF-2a versus pGl3PAI-276M2 + pCMV control; P < .05.