Evaluation of a novel PXR-knockout in HepaRG™ cells.

Abstract

The nuclear pregnane X receptor (PXR) regulates the expression of genes involved in the metabolism, hepatobiliary disposition, and toxicity of drugs and endogenous compounds. PXR is a promiscuous nuclear hormone receptor (NHR) with significant ligand and DNA‐binding crosstalk with the constitutive androstane receptor (CAR); hence, defining the precise role of PXR in gene regulation is challenging. Here, utilising a novel PXR‐knockout (KO) HepaRG cell line, real‐time PCR analysis was conducted to determine PXR involvement for a range of inducers. The selective PXR agonist rifampicin, a selective CAR activator, 6‐(4‐chlorophenyl)imidazo[2,1‐b][1,3]thiazole‐5‐carbaldehyde O‐(3,4‐dichlorobenzyl)oxime (CITCO), and dual activators of CAR and PXR including phenobarbital (PB) were analyzed. HepaRG control cells (5F clone) were responsive to prototypical inducers of CYP2B6 and CYP3A4. No response was observed in the PXR‐KO cells treated with rifampicin. Induction of CYP3A4 by PB, artemisinin, and phenytoin was also much reduced in PXR‐KO cells, while the response to CITCO was maintained. This finding is in agreement with the abolition of functional PXR expression. The apparent EC50 values for PB were in agreement between the cell lines; however, CITCO was ~threefold (0.3 μmol/L vs. 1 μmol/L) lower in the PXR‐KO cells compared with the 5F cells for CYP2B6 induction. Results presented support the application of the novel PXR‐KO cells in the definitive assignment of PXR‐mediated CYP2B6 and CYP3A4 induction. Utilization of such cell lines will allow advancement in composing structure activity relationships rather than relying predominantly on pharmacological manipulations and provide in‐depth mechanistic evaluation.