The Role of the Nuclear Receptors CAR and PXR in the Drug Induced Transcriptional Regulation of the Murine CYP2C Subfamily of Cytochrome P450 Monooxygenases

Abstract

The CYP2C subfamily of cytochrome P450 monoxygenases (P450) is responsible for the metabolism of approximately 20% of therapeutic drugs. Recently, phenytoin has been reported to induce the expression of the human genes CYP2B6, CYP3A4, and CYP2C8. Cytochrome P450 expression is often induced by prior exposure to xenobiotics often resulting in drug-drug interactions. Induction of the human CYP2C enzymes by xenobiotics occurs at the transcriptional level and is reported to involve the constitutive androstane receptor (CAR) and/or the pregnane X receptor (PXR). However, the molecular mechanisms regulating drug induction of the murine CYP2C enzymes remain unclear. The mouse is an excellent model system to investigate CYP2C drug induced transcription due to the availability of nuclear receptor knockout mice. Herein, we report the identification of two phenobarbital and phenytoin inducible murine CYP2C genes, Cyp2c29 and Cyp2c37. Quantitative RT-PCR demonstrates that hepatic CYP2C29 and CYP2C37 mRNA is induced by phenobarbital and phenytoin. Additionally, immunoblots indicated that phenytoin induced hepatic CYP2C29 and CYP2C37 protein. We utilized in vivo gene reporter assays of the Cyp2c29 promoter to delineate the phenytoin-response activity to a phenytoin responsive module (PHREM) located -1371 bp upstream of the Cyp2c29 translation start site. Similarly, using in vitro gene reporter assays of the Cyp2c37 promoter, we identified a single functional CAR-RE located -2791 bp upstream of translation start site of Cyp2c37. Mutagenesis studies demonstrated that these sites are essential for CAR transactivation of their respective gene promoters in HepG2 cells. Using quantitative RT-PCR, we demonstrated that induction of CYP2B10 mRNA by phenytoin was completely abolished in CAR-null mice, but only moderately reduced in PXR-null mice. Similarly, phenytoin induction of CYP2C29 and CYP2C37 mRNA was severely reduced in CAR-null mice and only modestly reduced in PXR-null mice. Taken together, these results indicate that the induction of Cyp2b10, Cyp2c29, and Cyp2c37 by phenytoin is predominately regulated by mCAR. However, induction of CYP3A11 mRNA was only partially decreased in either null mice strain. Studies have demonstrated that CYP3A11 mRNA is induced by CAR and PXR agonists, thus our results also indicate that phenytoin acts as an agonist of both CAR and PXR.