Transcriptional Regulation of Human CYP2C Genes: Functional Comparison of CYP2C9 and CYP2C18 Promoter Regions

• Published in: Biochemistry (Easton) Vol. 34; no. 25; pp. 8028 - 8036
• Main Authors: Ibeanu, Gordon C, Goldstein, Joyce A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 1995
• Subjects: Animals; Aryl Hydrocarbon Hydroxylases; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Binding Sites; Carcinoma, Hepatocellular; Cytochrome P-450 CYP2C9; Cytochrome P-450 Enzyme System - genetics; DNA - chemistry; DNA - metabolism; DNA-Binding Proteins; Gene Deletion; Gene Expression Regulation; Haplorhini; Hepatocyte Nuclear Factor 4; Humans; Liver Neoplasms; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphoproteins; Promoter Regions, Genetic; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases - genetics; Transcription Factors - metabolism; Transcription Factors - pharmacology; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00025a008

The cytochrome P4502C subfamily comprises a group of constitutive microsomal hemoproteins which are expressed primarily in liver. In humans, this subfamily is responsible for metabolism of a variety of therapeutic drugs such as warfarin, mephenytoin, omeprazole, and antiinflammatory drugs. In the present study, we analyzed the promoter activity of the 5'-flanking region of two human CYP2C genes, CYP2C9 and CYP2C18. The ability of the 2.2-kb 5'-flanking region of the CYP2C9 gene to direct expression of a luciferase reporter gene in HepG2 cells was 25 times greater than that of the 1.3-kb 5'-flanking region of CYP2C18. Deletional analysis of CYP2C9 indicated that the minimal promoter was located between the translation start site and nucleotide -155, and an HPF-1 domain consensus sequence was identified in this region. Gel shift analysis demonstrated that nuclear proteins from HepG2 cells had a high binding affinity for a 20-bp oligonucleotide containing the HPF-1 site of CYP2C9. Antiserum to rat HNF-4 supershifted this DNA--protein complex, and an oligonucleotide derived from an HNF-4 motif present in the human apolipoprotein CIII promoter competed for the supershifted complex. Cotransfection with an HNF-4 expression plasmid increased transcriptional activity of the CYP2C9 minimal promoter (approximately 2-fold) in HepG2 cells and elevated activity more substantially in nonhepatic NIH3T3 cells (26-fold) and Cos 1 cells (9-fold).