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.

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor

“…HepaRG cells have been validated as a promising surrogate for HPHs, and importantly, fully differentiated HepaRG cells exhibit proper CAR cellular localization and maintain physiologically relevant metabolic capacity, which are not present in most immortalized cell models (Jackson et al, 2016). The PXR-KO HepaRG cell line obtained from Sigma-Aldrich is a newly generated cell line that does not express functional PXR (Williamson et al, 2016). As expected, PK11195 and other known CAR/PXR modulators induced the expression of CYP2B6 and CYP3A4 mRNA and protein in wild-type HepaRG cells in a trend that mirrors what was observed in HPHs (Fig.…”

Targeting the pregnane X receptor using microbial metabolite mimicry

“…In PXR‐transfected LS180 cells, FKK6 (and to a lesser extent FKK5) induced CYP3A4 (Fig C middle panel) and MDR1 mRNA (Fig C, bottom panel). In HepaRG cells harboring loss of PXR or AhR, there is diminished target gene induction when compared to the wild‐type control cell line (Williamson et al , ; Brauze et al , ). TCDD (2,3,7,8—tetrachlorodibenzo‐ p ‐dioxin), a known AhR ligand, induces CYP1A1 mRNA in HepaRG™ control 5F clone and PXR‐KO (PXR‐knockout) cells.…”

Functional polarization of human hepatoma HepaRG cells in response to forskolin

“…S5b ). PXR role in FSK-mediated BC formation was further studied using PXR-KO HepaRG cells and corresponding control F5 HepaRG cells 34 . PXR-KO cells failed to express PXR as demonstrated by Western-blotting (Supplementary Fig.…”