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Induction of PAI‐1 expression by tumor necrosis factor α in endothelial cells is mediated by its responsive element located in the 4G/5G site
Author(s) -
Swiatkowska Maria,
Szemraj Janusz,
Cierniewski Czeslaw S.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04979.x
Subject(s) - microbiology and biotechnology , electrophoretic mobility shift assay , tumor necrosis factor alpha , transfection , proinflammatory cytokine , plasminogen activator inhibitor 1 , reporter gene , promoter , transcription factor , biology , plasminogen activator , gene expression , chemistry , inflammation , gene , immunology , endocrinology , biochemistry
Plasminogen activator inhibitor type 1 (PAI‐1) is induced by many proinflammatory and pro‐oxidant factors. Among them, tumor necrosis factor α (TNFα), a pivotal early mediator that regulates and amplifies the development of inflammation, is one of the strongest PAI‐1 synthesis activators. Location of the TNFα response element in the PAI‐1 promoter is still ambiguous. In this study, we attempted to evaluate the significance of the element located in the 4G/5G site of the PAI‐1 promoter in the TNFα stimulation of PAI‐1 expression in endothelial cells. PAI‐1 expression was monitored at: (a) the level of mRNA using real‐time PCR, (b) PAI‐1 gene transcription by transfection reporter assays, and (c) protein synthesis using the enzyme immunoassay. NF‐κB activity was monitored using the electrophoretic mobility shift assay. Its activity was modified by either antisense oligonucleotides or transfection of endothelial cells with the wild‐type or mutated IκBα. We have shown that TNFα‐induced expression and gene transcription of PAI‐1 involves a regulatory region present in segment −664/−680 of the PAI‐1 promoter. This reaction involves the TNFα‐induced generation of superoxide leading to activation of NF‐κB, and can be abolished by antioxidants and by overexpression of a super‐suppressor phosphorylation‐resistant IκBα. Stimulation of PAI‐1 under these conditions involves the motif of the PAI‐1 promoter adjacent to the 4G/5G site, which can directly interact with NF‐κB. We show that activation of PAI‐1 gene by TNFα and reactive oxygen species is mediated by interaction of NF‐κB with the cis ‐acting element located in the −675 4G/5G insertion/deletion in the PAI‐1 promoter.