title: Regulation of the human protein C inhibitor gene expression in HepG2 cells: role of Sp1 and AP2.
abstract: Protein C inhibitor (PCI) is the plasma inhibitor of activated protein C, which is the main protease of the anticoagulant protein C pathway. In this study the transcriptional regulation of human PCI gene in the human hepatoma cell line, HepG2, was characterized by evaluating the transient expression of a luciferase reporter gene. The 5' flanking region (residues -1587 to +2) of the PCI gene showed an adequate transcriptional activity, the maximum transcriptional activity being in a region between residues -452 and -94, which contains an Sp1-binding site, two AP2-binding sites and an inverted AP2-binding site. Transient expression assays with various deletion mutants and site-directed mutants showed that the Sp1-binding site (residues -302 to -294) has a potent promoter activity and that the upstream AP2-binding site (residues -350 to -343) has a potent enhancer activity; no activity was detected in the inverted (residues -413 to -404) and downstream (residues -136 to -127) AP2-binding sites. In addition, a region of the PCI gene (residues -452 to -414) containing the STATx-binding site, the A-activator (AA)-binding site, and the interferon alpha (IFN-alpha) response element, and another region of the PCI gene (residues -176 to -147) containing the GATA-1 and the IFN-gamma response element showed potent silencer activities. Gel mobility-shift assays with various DNA fragments indicated that the Sp1-binding site, the upstream AP2-binding site, the AA-binding site and the IFN-gamma response element interact with nuclear protein(s) of HepG2 cells. These findings suggest that the Sp1-binding site is the promoter, the AP2-binding site (residues -350 to -343) the enhancer, and both the AA-binding site and the IFN-gamma response element are the silencers of human PCI gene expression in HepG2 cells.
authors.affiliation: Department of Molecular Pathobiology, Mie University School of Medicine, Tsu-city, Mie 514, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
authors.forename: M
authors.initials: M
authors.lastname: Usui
authors.forename: J
authors.initials: J
authors.lastname: Nishioka
authors.forename: Z X
authors.initials: ZX
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis / Q000502:physiology*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D018046:Protein C Inhibitor / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051704:Sp2 Transcription Factor; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018046:Protein C Inhibitor; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051704:Sp2 Transcription Factor