Induction of PAI-1 promoter-dependent luciferase activity by insulin and activated PKB is conferred by the HREs.
(A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites (A-G). In the “C-site” the 2 HIF-1 binding elements (HRE) matching the HIF-1 consensus sequence BACGTSSK50 where B = G/C/T, S = G/C, and K = G/T are underlined. B, S, or K is shown only if the actual PAI-1 sequence does not match any of the bases allowed by the consensus. (B) Hepatocytes were transiently cotransfected with either the myrPKB expression plasmid or the empty control vector 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, or Luc gene construct containing 3 copies of the EPO HRE element in front of the SV40 promoter (pGl3EPO-HRE). The cells were treated with 10 nM insulin, as indicated. In each experiment the percentage of Luc activity was determined relative to the pGl3PAI-766, pGl3PAI-766M1, pGl3PAI-766M2, or pGl3EPO-HRE controls that were set equal to 100%. In pGl3PAI-766M1 and pGl3PAI-766M2 the wild-type PAI-1 sequence is shown on the upper strand, mutated bases are indicated by * and are shown in lowercase letters. The values are represented as means ± SEMs of 3 independent experiments. Student t test for paired values: *significant difference 16% O2 versus 8% O2, **significant difference 16% O2 + insulin versus 16% O2 (control), ***significant difference 16% O2 + myrPKB versus 16% O2 (control),P ≤ .05.
