![Fig. 3. Regulation of transfected rat PAI-1 promoter LUC gene constructs by hypoxia and cotransfected HIF-1 in transiently transfected rat hepatocyte cultures. Enhancement of PAI-1 production by HIF-1. (A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites [A-G]. In the “C-site”, the 2 potential HIF-1 binding elements (HRE) matching the HIF-1 consensus sequence BACGTSSK32 were B=G/C/T, S=G/C and K=G/T are underlined. B, S, or K are 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 pcDNAHIF-1 containing the full-length rat HIF-1 cDNA 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. After 24 hours, the transfected cells were cultured for 18 hours under hypoxia, additionally pGl3PAI-766-transfected cells were treated with CHX (10 μg/mL). In each experiment, the fold stimulation of LUC activity by hypoxia (8% O2) was determined relative to the normoxic (16% O2) control, which was set equal to 1. The values represent means ± SEM of 3 independent experiments. Statistics, Student'st-test for paired values: * significant differences 16% O2 versus 8% O2, P ≤ .05; ** significant differences 16% O2 versus 16% O2+ HIF-1, P ≤ .05. 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 lower case letters. (C) Western blot analysis of PAI-1, HIF-1, and ARNT in cultured hepatocytes transfected with a HIF-1 vector. A total of 50 μg of protein from the medium and 100 μg of total cellular protein of the transfected hepatocytes were subjected to Western analysis (cf, Materials and Methods) with antibodies against rat PAI-1, rat HIF-1, and ARNT, respectively. Blots were scanned by videodensitometry, and in each experiment, the PAI-1 level and HIF-1 level measured under normoxia (16% O2) were set equal to 1. Values represent the fold induction of PAI-1 and HIF-1 of 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/94/12/10.1182_blood.v94.12.4177/5/blod42414003w.jpeg?Expires=1735844593&Signature=DAwdC7WyOFXEQ1YbMUTy8OknslTpA3y5XbKnEugupuNUp1Bf5Ny0kK4rw7v4NDF~T6OpuJhtrQ6plxyj6kS9E-hwRBA64PXPhF6x6Zq2oeWJ3a2IPQV8gohIXeex8BslJgq88HNGpsiUfQUxlZo8NnW48jX5Pbuh0iOlUWRMurpgHMSDpgpcPRU6EGvseXQXokxX0p6CbsJGTI61YPxXaBGnQTeiIRqGY4ZN9coy9Mww~zYEEZA5SH3ko8qFeDIL02nhjbtThKKrlITK2cRrKV3hRQhkcle9HREclGqAKLMpNvHeFEEhxxOOTsKgHiLty4xZg6074mLOiwhlMPCAxg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Regulation of transfected rat PAI-1 promoter LUC gene constructs by hypoxia and cotransfected HIF-1 in transiently transfected rat hepatocyte cultures. Enhancement of PAI-1 production by HIF-1. (A) The 5′-flanking region of the rat PAI-1 gene with its footprinted sites [A-G]. In the “C-site”, the 2 potential HIF-1 binding elements (HRE) matching the HIF-1 consensus sequence BACGTSSK32 were B=G/C/T, S=G/C and K=G/T are underlined. B, S, or K are 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 pcDNAHIF-1 containing the full-length rat HIF-1 cDNA 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. After 24 hours, the transfected cells were cultured for 18 hours under hypoxia, additionally pGl3PAI-766-transfected cells were treated with CHX (10 μg/mL). In each experiment, the fold stimulation of LUC activity by hypoxia (8% O2) was determined relative to the normoxic (16% O2) control, which was set equal to 1. The values represent means ± SEM of 3 independent experiments. Statistics, Student'st-test for paired values: * significant differences 16% O2 versus 8% O2, P ≤ .05; ** significant differences 16% O2 versus 16% O2+ HIF-1, P ≤ .05. 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 lower case letters. (C) Western blot analysis of PAI-1, HIF-1, and ARNT in cultured hepatocytes transfected with a HIF-1 vector. A total of 50 μg of protein from the medium and 100 μg of total cellular protein of the transfected hepatocytes were subjected to Western analysis (cf, Materials and Methods) with antibodies against rat PAI-1, rat HIF-1, and ARNT, respectively. Blots were scanned by videodensitometry, and in each experiment, the PAI-1 level and HIF-1 level measured under normoxia (16% O2) were set equal to 1. Values represent the fold induction of PAI-1 and HIF-1 of 3 independent experiments.
