TRANSCRIPTIONAL REGULATION OF HUMAN CYP2C GENES - FUNCTIONAL COMPARISON OF CYP2C9 AND CYP2C18 PROMOTER REGIONS

The cytochrome P4502C subfamily comprises a group of constitutive micr osomal hemoproteins which are expressed primarily in liver. In humans, this subfamily is responsible for metabolism of a variety of therapeu tic drugs such as warfarin, mephenytoin, omeprazole, and antiinflammat ory drugs. In the present study, we analyzed the promotor activity of the 5'-flanking region of two human CYP2C genes, CYP2C9 and CYP2C18. T he ability of the 2.2-kb 5'-flanking region of the CYP2C9 gene to dire ct expression of a luciferase reporter gene in HepG2 cells was 25 time s greater than that of the 1.3-kb 5'-flanking region of CYP2C18. Delet ional analysis of CYP2C9 indicated that the minimal promotor was locat ed between the translation start site and nucleotide -155, and an HPF- 1 domain consensus sequence was identified in this region. Gel shift a nalysis demonstrated that nuclear proteins from HepG2 cells had a high binding affinity for a 20-bp oligonucleotide containing the HPF-1 sit e of CYP2C9. Antiserum to rat HNF-4 supershifted this DNA-protein comp lex, and an oligonucleotide derived from an HNF-4 motif present in the human apolipoprotein CIII promotor competed for the supershifted comp lex, Cotransfection with an HNF-4 expression plasmid increased transcr iptional activity of the CYP2C9 minimal promotor (similar to 2-fold) i n HepG2 cells and elevated activity more substantially in nonhepatic N IH3T3 cells (26-fold) and Cos 1 cells (9-fold). A possible HPF-1 motif was identified 661 to 641 bases upstream of the translational start s ite of CYP2C18 which differed from the HPF-1 consensus sequence by the substitution of three cytosines for purines at positions 4-6 and one adenine residue at position 15. An oligonucleotide containing the CYP2 C18 HPF-1 motif bound nuclear proteins from HepG2 cells only weakly in gel shift assays, but replacement of the three tandem cytosine residu es in the HPF-1 site by guanines using site-directed mutagenesis cause d the formation of a complex whose mobility was supershifted by anti-H NF4. Similarly, mutation of the three guanines in the CYP2C9 HPF-1 sit e to cytosines prevented the formation of the specific DNA-protein com plex seen with this motif. However, cotransfection with an HNF-4 expre ssion plasmid did not increase transcriptional activity of CYP2C18 pro motor constructs containing the original CYP2C18 HPF-1 motif or the mu tated motif containing guanine residues in any of the cell lines teste d. We conclude that the HPF-1 site is an important cis-acting element directing hepatic expression of the CYP2C9 promoter but does not contr ibute to the weak transcriptional activity of the 1.3-kb upstream regi on of CYP2C18.