Competition by HIF-1α via HRE-2 of the USF-2a–dependent inhibition of pGl3PAI-766 Luc expression in hepatocytes.

(A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites [A-G]. In the “C-site” the USF and HIF-1 binding element14 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 expression plasmids for either USF-2a, HIF-1α, or both and Luc gene constructs driven by a wild-type 766-bp rat PAI-1 promoter (pGl3PAI-766) or the 766-bp promoter mutated at either the HRE-1 (pGl3PAI-766M1) or HRE-2 (pGl3PAI-766M2) 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-766 + pCMV control, which was set equal to 100%. In pGl3PAI-766M1 and pGl3PAI-766M2 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-766 + USF-2a versus pGl3PAI-766 + pCMV control; pGl3PAI-766M1 + USF-2a versus pGl3PAI-766M1 + pCMV control; pGl3PAI-766M2 + USF-2a versus pGl3PAI-766M2 + pCMV control; **, significant difference pGl3PAI-766 + HIF-1α versus pGl3PAI-766 + pCMV control; pGl3PAI-766M1 + HIF-1α versus pGl3PAI-766M1 + pCMV control; ***, significant difference pGl3PAI-766 + USF-2a + HIF-1α versus pGl3PAI-766 + USF-2a; pGl3PAI-766M1 + USF-2a + HIF-1α versus pGl3PAI-766M1 + USF2a; P < .05.