Rodent-specific hypoxia response elements enhance PAI-1 expression through HIF-1 or HIF-2 in mouse hepatoma cells.
Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of numerous pathophysiological processes such as inflammation, thrombosis, angiogenesis and tumor metastasis. Its expression is induced by hypoxia at the transcriptional level, via the hypoxia inducible factor-1 (HIF-1) or -2 (HIF-2). In this study, we elucidated the mechanism of transcriptional regulation of mouse PAI-1 gene by hypoxia in mouse hepatoma cells. We searched for hypoxia response elements (HREs) of murine PAI-1 promoter using several molecular biological assays. DNAse I hypersensitivity assay first suggested that PAI-1 gene expression is up-regulated by protein-DNA interactions at the -3.6- and -3-kb upstream regions of the PAI-1 gene transcription start site. An approximately 6.4-kb region of DNA containing the 5'-flanking promoter region of the PAI-1 gene was isolated, mapped, and cloned into reporter gene assay vectors and sequenced. Luciferase reporter gene assay subsequently identified two functional HREs, located around -3.6 kb of the 5'-flanking promoter region of PAI-1 gene that were responsible for the enhancement of luciferase reporter gene activity. Mutation of the HREs in this fragment abolished luciferase reporter gene activity. Finally, in vitro and in vivo protein-DNA interaction assays confirmed binding of the two HREs to HIF-1 or HIF-2 protein. Our results show that two HREs located around -3.6 kb of the 5'-flanking promoter region of the mouse PAI-1 gene function as hypoxia enhancers, which, alongside other regulatory regions, control PAI-1 gene transcription by HIF-1 or HIF-2 under hypoxic environments in mouse hepatoma cells.
Departments of Pediatrics, Stanford University School of Medicine, 300 Pasteur Dr Stanford, CA 94305, USA.
Yong-Tae
YT
Ahn
Mei-Sze
MS
Chua
James P
JP
Whitlock
Yong-Chil
YC
Shin
Woon-Heung
WH
Song
Yongkuk
Y
Kim
Chi-Yong
CY
Eom
Won Gun
WG
An
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia / Q000235:genetics; D002478:Cells, Cultured; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism / Q000502:physiology*; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D020218:Response Elements / Q000502:physiology*; D012377:Rodentia / Q000235:genetics*; D058258:Serpin E2 / Q000235:genetics*; D013045:Species Specificity; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation / Q000235:genetics
C485502:HIF-2 protein, mouse; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D058258:Serpin E2; C511869:Serpine2 protein, mouse; D014157:Transcription Factors