Figure 3
Cdk5 activation attenuates PXR-mediated gene expression. (A) Overexpression of Cdk5 reduces PXR-mediated CYP3A4 promoter activity. HepG2 was transiently co-transfected with indicated plasmids as well as CMV-renilla luciferase plasmid (as a transfection control). In cases wherein Cdk5, FLAG-PXR, or CYP3A4-luc constructs were not used, a pcDNA3 vector was used instead. Equal amount of each plasmid (for a total of 1 μg combined) was used to transfect 8 × 105 cells seeded in 6-well plates. Cells were treated with 5 μM rifampicin or DMSO for 24 h after transfection before Dual-glo luciferase assay. CYP3A4 promoter activity (expressed as relative luciferase unit, or RLU) was normalized by using activity of the CMV-renilla. The values represent the average of 8 independent experiments, with the standard deviation denoted as bars. The significance of the difference between datasets was determined by using the Student's t test. (B) Expression of PXR and Cdk5. Transfections were performed as described in (A). Western blotting shown was from a representative experiment. (C) Downregulation of Cdk5 enhances the activity of PXR. HepG2 was transfected with siRNA specific for Cdk5 (siRNA-Cdk5) or control siRNA (siRNA-Control) in addition to indicated plasmids and CMV-renilla as described in Methods. Cells were treated with 5 μM rifampicin. CYP3A4 promoter activity was expressed as RLU as described in (A). The values represent the average of 6 independent experiments. The efficiency of Cdk5 knockdown was verified in (D).