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Molecular regulation of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1, and C/EBP

Hui Liao^*, Matthew C. Hyman, Daniel A. Lawrence^* and David J. Pinsky^*,,1

Departments of
^* Internal Medicine and

Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA

¹Correspondence: University of Michigan Health Systems, Cardiovascular Medicine, 7220 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0644, USA. E-mail: dpinsky{at}umich.edu

Hypoxia, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. Plasminogen activator inhibitor-1 (PAI-1) is an important factor suppressing fibrinolysis under conditions of low oxygen tension. We previously reported that hypoxia induced PAI-1 mRNA and antigen expression in murine macrophages secondary to increased de novo transcription as well as increased mRNA stability. We now show in RAW264.7 murine macrophages that the transcription factors early growth response gene-1 (Egr-1), hypoxia-inducible factor-1 (HIF-1), and CCAAT/enhancer binding protein (C/EBP) are quickly activated in hypoxia and are responsible for transcription and expression of PAI-1. Murine PAI-1 promoter constructs, including Egr, HIF-1, and/or C/EBP binding sites, were transfected into RAW 264.7 murine macrophages. To identify the relative importance of each of these putative hypoxia-responsive elements, cells were exposed to normobaric hypoxia, and transcriptional activity was recorded. At 16 h of hypoxic exposure, murine PAI-1 promoter deletion constructs that included Egr, HIF-1, and/or C/EBP binding sites demonstrated increased transcriptional activity. Mutation of each of these three murine PAI-1 promoter sites (or a combination of them) resulted in a marked reduction in hypoxia sensitivity as detected by transcriptional analysis. Functional data obtained using ³²P-labeled Egr, HIF-1 response element (HRE), and C/EBP oligonucleotides revealed induction of DNA binding activity in nuclear extracts from hypoxic RAW cells, with supershift analysis confirming activation of Egr-1, HIF-1, or C/EBP. ChIP analysis confirmed the authenticity of these interactions as each of these transcription factors binds to chromatin under hypoxic conditions. Further, the induction of PAI-1 by Egr-1, HIF-1, or C/EBP was replicated in primary peritoneal macrophages. These data suggest that although HIF-1 appears to dominate the PAI-1 transcriptional response in hypoxia, Egr-1 and C/EBP greatly augment this response and can do so independent of HIF-1 or each other. These studies are relevant to ischemic up-regulation of the PAI-1 gene and consequent accrual of microvascular thrombus under ischemic conditions.—Liao, H., Hyman, M. C., Lawrence, D. A., Pinsky, D. J. Molecular regulation of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1, and C/EBP.

Key Words: C/EBP overexpression • hypoxia sensitivity • HRE-1 and HRE-2 motifs • PAI-1 transcription

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