Smirlis, Despina; (2001) Transcriptional regulation of rat CYP2B genes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cytochromes P450 (CYPs) are a large superfamily of haemoproteins involved in the metabolism of numerous endogenous and exogenous compounds. CYP2B1 and CYP2B2 expression is increased by a variety of xenobiotics including the anti-epileptic drug phenobarbital. The expression of CYP2B genes declines during hepatocyte culture. The profiles of liver-enriched and stress-induced transcription factors were examined during cell culture. No absolute correlation was found between the profiles of transcription factors (C/EBP, SP1, NFκB, AP1) and CYP2B mRNA amounts. The binding activity of SP1 varied in a manner similar to CYP2B mRNA expression up to 24 hours of cell culture. This led to the identification of two putative SP1 elements in the CYP2B1 promoter. Co-transfection studies of primary hepatocytes with an SP1 expression vector and a luciferase reporter gene linked to the CYP2B1 promoter (-2/-179) indicates that SP1 has a functional role in the regulation of CYP2B1 gene expression. Phenobarbital and other CYP2B inducers, such as the plant product picrotoxin, stimulate constitutive androstane receptor (CAR) activation of a reporter gene linked to the CYP2B1 gene PBRE (phenobarbital response element) in transfected, primary hepatocytes and liver, but not in the transfected, transformed cell lines, HEPG2, CV-1 and HeLa, which support only constitutive transactivation by CAR. Co-transfection experiments in primary hepatocytes demonstrated that the co-activator SRC-1, mediates the action of CAR. Gel shift assays show that phenobarbital is a direct ligand of CAR. Different classes of inducers were shown to mediate induction of CYP2B genes via different receptors. Pregnenolone 16a-carbonitrile and glucocorticoids mediate their actions on the CYP2B1 gene through the Pregnane X Receptor (PXR). Both CAR and PXR bind to the same PBRE direct repeat (DR)4 element to mediate xenobiotic-regulated CYP2B1 expression. Our results suggest that CAR also binds to the DR3 PXRE of the CYP3A1 promoter to mediate the phenobarbital response of this gene.

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