Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site.
The CYP2C subfamily metabolizes many clinically important drugs. These genes respond to prototypical inducers such as phenobarbital and rifampicin, yet little has been reported on the mechanisms of induction. This report examines the regulation of CYP2C9 with respect to two specific receptors thought to be involved in phenobarbital (PB) induction, the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). Transfection of either mouse CAR (mCAR) or human CAR (hCAR) into HepG2 cells results in increased CYP2C9 mRNA content. Inducers further increased this response in CAR transfected cells. mCAR but not hCAR conferred drug inducibility to the proximal -2145 bp of the CYP2C9 promoter in luciferase assays. Further examination of a -2925-bp promoter construct revealed that hCAR cotransfection increased activity 20-fold. Gel shift assays confirmed the presence of a distal PB-responsive enhancer module-like enhancer module, CAR-responsive enhancer (CAR-RE), between -2900 and -2841 bp consisting of two DR-5 nuclear receptor binding motifs capable of binding hCAR, mCAR, and, to a lesser extent, human PXR. The majority of binding and hCAR activation is derived from the NR1 portion of the CAR-RE. PB treatment did not further increase the hCAR activation in any of the constructs. In summary, a new CAR/PXR binding site was identified in the CYP2C9 promoter, and this site seems to constitutively regulate transcription via a CAR-dependent mechanism; however, it could not be shown to account for PB inducibility of the gene.
Ferguson
Stephen S
SS
Laboratory of Pharmacology & Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
LeCluyse
Edward L
EL
Negishi
Masahiko
M
Goldstein
Joyce A
JA
D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D000090702:Constitutive Androstane Receptor; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000090702:Constitutive Androstane Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase