Aims: 1) To investigate whether known orphan receptors or nuclear receptors such as CAR and PXR are responsible for the constitutive and inducible regulation of the human CYP2Cs and to determine which compounds induce each of the CYP2Cs. To identify upstream elements responsible for regulation and induction of these enzymes. To determine which classes of drugs induce each of these enzymes (CYP2C8, CYP2C9, CYP2C19). 2) To investigate whether liver/kidney/gut transporters are inducible and determine which orphan receptors and nuclear factors regulate the tissue specificity and possible inducibility 1a)CYP2C9 regulation. Experiments using HepG2 cells stably transfected with CAR demonstrated that CAR constitutively activates endogenous levels of CYP2C9 transcripts monitored by RT-PCR. This activation is inducible by PB-type inducers. CYP2C9 appears to be at least partly regulated by the PB responsive receptor CAR. However, CYP2C8 and CYP2C9 are differentially induced by different compounds such as phenobarital and rifampicin. Transfection studies with luciferase reporter constructs containing the first 3 kb of CYP2C9 have shown that CAR can activate transcription constitutively and inducibility . We have analyzed the sequence of promoter regions of CYP2C9 and and identified two regulatory elements (one proximal and one distal) which bind CAR and appear to be involved in at least the constitutive regulation of CYP2C9. Gel shift assays showed that both elements bind CAR, but sequential mutation indicates that although both elements are responsible for CAR activation (simultaneous mutation abolishes CAR activation), the distal element is the most important. Transfection studies with this element driving a thymidine kinase promoter showed that hCAR>hPXR>mCAR constitutively activated this element. These studies show that there are at least two CAR binding sites which regulate the constitutive expression and could be involved in drug inducibility of CYP2C9.1b)We have identified two putative CAR binding sites in the promoter of a gene, CYP2C19, which has high sequence homology with CYP2C9. However, due to a one base mutation in a distal putative CAR binding element versus the active element in CYP2C9, the distal element of CYP2C19 does not bind CAR in gel shift assays. However, a proximal element of CYP2C19 (present in both CYP2Cs) does bind CAR. We are further examing whether CYP2C19 mRNA is inducible in primary hepatocytes and whether the element we have identified is important in this induction. It is possible that the presence of a distal CAR-binding element in CYP2C9 which is absent in CYP2C19 may be one reason why CYP2C9 is expressed in human liver at much higher levels than the closely related CYP2C19. This could also affect the relative inducibility of the two genes.
| Ferguson, Stephen S; LeCluyse, Edward L; Negishi, Masahiko et al. (2002) Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site. Mol Pharmacol 62:737-46 |
