Regulation of human CYP2C9 expression by electrophilic stress involves activator protein 1 activation and DNA looping.
Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes.
Human Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (N.L.M., S.S., J.A.G.); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky (R.A.P.); and Research Centre for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary (K.M.).
Ngome L
NL
Makia
Human Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (N.L.M., S.S., J.A.G.); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky (R.A.P.); and Research Centre for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary (K.M.).
Sailesh
S
Surapureddi
Human Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (N.L.M., S.S., J.A.G.); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky (R.A.P.); and Research Centre for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary (K.M.).
Katalin
K
Monostory
Human Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (N.L.M., S.S., J.A.G.); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky (R.A.P.); and Research Centre for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary (K.M.).
Russell A
RA
Prough
Human Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (N.L.M., S.S., J.A.G.); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky (R.A.P.); and Research Centre for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary (K.M.) goldste1@niehs.nih.gov.
Joyce A
JA
Goldstein
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D004247:DNA / Q000235:genetics*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics* / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D018384:Oxidative Stress / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics* / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D015262:Xenobiotics / Q000378:metabolism
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D015262:Xenobiotics; D004247:DNA; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D048049:Extracellular Signal-Regulated MAP Kinases