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.

Myb-binding protein 1a augments AhR-dependent gene expression.

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein 1a, whereas a mutant domain (F542A, I569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein 1a into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein 1a is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein 1a associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein 1a to increase AhR-dependent gene expression.