REGULATION OF THE HUMAN PROTEIN-C INHIBITOR GENE-EXPRESSION IN HEPG2 CELLS - ROLE OF SP1 AND AP2

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 re gion (residues -1587 to +2) of the PCI gene showed an adequate transcr iptional activity, the maximum transcriptional activity being in a reg ion between residues -452 and -94, which contains an Sp1-binding site, two AP2-binding sites and an inverted AP2-binding site. Transient exp ression 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 -3 50 to -343) has a potent enhancer activity; no activity was detected i n the inverted (residues -413 to -404) and downstream (residues -136 t o -127) AP2-binding sites. In addition, a region of the PCI gene (resi dues -452 to -414) containing the STATx-binding site, the A-activator (PLA)-binding site, and the interferon alpha (IFN-alpha) response elem ent, and another region of the PCI gene (residues -176 to -147) contai ning the GATA-1 and the IFN-gamma response element showed potent silen cer activities. Gel mobility-shift assays with various DNA fragments i ndicated that the Sp1-binding site, the upstream AP2-binding site, the AA-binding site and the IFN-gamma response element interact with nucl ear protein(s) of HepG2 cells. These findings suggest that the Sp1-bin ding site is the promoter, the AP2-binding site (residues -350 to -343 ) the enhancer, and both the AA-binding site and the IFN-gamma respons e element are the silencers of human PCI gene expression in HepG2 cell s.