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The PAI-1 Gene as a Direct Target of Endothelial PAS Domain Protein-1 in Adenocarcinoma A549 Cells

Second Department of Internal Medicine, and Department of General Medicine, Gunma University School of Medicine, Gunma; and Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan

Address correspondence to: Masahiko Kurabayashi, M.D., Ph.D., Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, 371-8511, Japan. E-mail: mkuraba{at}med.gunma-u.ac.jp

Endothelial PAS domain protein-1 (EPAS1) regulates transcription of the genes encoding erythropoietin and vascular endothelial growth factor, which are important for maintaining oxygen homeostasis. We have previously shown that plasminogen activator inhibitor-1 (PAI-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 human adenocarcinoma A549 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.

Abbreviations: cAMP response element, CRE • CRE binding protein, CREB • electrophoretic mobility shift assay, EMSA • endothelial PAS domain protein-1, EPAS1 • erythropoietin, EPO • hypoxia-inducible factor-1, HIF-1 • HIF-1–like factor, HLF • hypoxia response element, HRE • kilobases, kb • messenger RNA, mRNA • multiplicity of infection, MOI • plasminogen activator inhibitor-1, PAI-1 • phosphate-buffered saline, PBS • vascular endothelial growth factor, VEGF