Activation of human CYP2C9 promoter and regulation by CAR and PXR in mouse liver.
The activity of various genomic segments at the 5'-flanking region of the human CYP2C9 gene in driving gene expression and their involvement in pregnane X receptor (PXR) and constitutive androstane receptor (CAR) mediated activation were evaluated in mouse hepatocytes. Using the genomic sequence of human CYP2C9 as a template, segments covering different regions of CYP2C9 5'-flanking sequences starting from the translation start site were amplified by PCR and inserted into a pGL-3 luciferase vector. Plasmid DNA containing the 0.2K, 1K, 2K, 3K, 5K, or 10K upstream sequences of the CYP2C9 gene were transfected into mouse liver by hydrodynamic delivery, and the activity of each fragment in driving reporter gene expression was assessed. With the exception of the 10K fragment, the level of luciferase activity in transfected mouse liver was similar among the constructs examined. Cotransfection of these reporter constructs with the pCMX-PXR or pCMX-CAR plasmids resulted in a slight increase in luciferase gene expression that could be significantly enhanced by chemical inducers. In mice cotransfected with pCMX-PXR, pregnenolone-16 alpha-carbonitrile (PCN) induced a 20-fold increase in the luciferase level compared to a 70-fold increase induced by rifampicin. Similarly, when animals were cotransfected with the pCMX-CAR plasmid, phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene enhanced luciferase gene expression by 10- and 57-fold, respectively. The element responsible for PXR- and CAR-mediated activation of luciferase gene expression by chemical inducers was found to reside in the -2000 to -1000 bp region of the 5'-flanking sequence of the CYP2C9 gene. These results prove that PXR and CAR are transcription factors regulating CYP2C9 gene expression.
Al-Dosari
Mohammed S
MS
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Knapp
Joseph E
JE
Liu
Dexi
D
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D000090702:Constitutive Androstane Receptor; D065729:Cytochrome P-450 CYP2C9; D018628:Gene Dosage; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D000090702:Constitutive Androstane Receptor; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases