Published ahead of print on March 23, 2004, doi:10.1165/rcmb.2003-0296OC Am. J. Respir. Cell Mol. Biol., Volume 31, Number 2, August 2004, 209-215 A more recent version of this article appeared on August 1, 2004
Submitted on August 11, 2003 The PAI-1 Gene As a Direct Target of Endothelial PAS-domain Protein-1 in Adenocarcinoma A549 cellsMahito Sato1,1 Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan; Department of General Medicine, Gunma University School of Medicine, Maebashi, Japan, 2 Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan, 3 Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan, 4 Department of General Medicine, Gunma University School of Medicine, Maebashi, Japan * To whom correspondence should be addressed. E-mail: mkuraba{at}med.gunma-u.ac.jp.
EPAS1 (Endothelial PAS domain protein-1) regulates transcription of the genes encoding erythropoietin, vascular endothelial growth factor, which are important for maintaining oxygen homeostasis. We have previously shown that PAI-1 (plasminogen activator inhibitor-1) gene expression is induced by hypoxia. In this study, we sought to determine whether PAI-1 gene expression is directly regulated by EPAS1 in cancer cells because activities of proteases and their inhibitors are tightly regulated for tumor invasion. Hypoxia increased the PAI-1 mRNA levels in A549 cells, human adenocarcinoma cells. Overexpression of EPAS1 significantly increased the PAI-1 mRNA and protein levels. Transient transfection assays revealed that EPAS1 increased PAI-1 gene transcription through a sequence containing 5'-CACGTACA-3' located at -194 (we refer to it as site HREPAI-1) and GT-box located at -78. Electrophoretic gel mobility shift assays revealed that HREPAI-1 serves as a binding site for EPAS1, and Sp1 constitutively binds to GT-box. In conclusion, PAI-1 expression is induced by EPAS1 through HREPAI-1 and through an Sp1-binding site. These results indicate that the PAI-1 gene is a direct target of EPAS1 and suggest the role of EPAS1 and Sp1 in the hypoxic response of cancer cells.
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