Evaluation of the Ligand-Dependent Coactivator Recruitment of the Nuclear Receptor Pregnane X Receptor Using Its Three-Alanine Residues-Inserted Mutant

The pregnane X receptor (PXR) is a nuclear receptor that regulates the expression of drug-metabolizing enzymes in response to various chemicals. Ligand binding to PXR induces a conformational alteration of the C-terminal coactivator–binding motif activation function 2 (AF2), leading to the recruitment of coactivators. The contribution of each coactivator to PXR-mediated transcription may vary depending on the ligand. Therefore, identifying ligand-dependent differences in coactivator recruitment is important for understanding the biological functions of PXR. In this study, we investigated the ligand-dependent recruitment of coactivators using a PXR mutant containing a three-alanine insertion immediately upstream of AF2 (PXR-3A). Mammalian two-hybrid assays showed that PXR-3A recruited coactivators in a ligand-dependent manner. Treatment with the PXR ligand SR12813 induced the interaction of PXR-3A with coactivator peptides. Using reporter assays with the PXR-binding motifs from CYP3A4, CYP2C19, and CYP2C9 promoters, we evaluated the influences of coactivator overexpression on ligand-dependent reporter activity. In assays with the CYP3A4 promoter, rifampicin, rifaximin, or SR12813 induced reporter activity, and overexpression of peroxisome proliferator–activated receptor γ coactivator-1α (PGC1α) or nuclear receptor coactivator 1 (NCoA1) enhanced rifaximin- and SR12813-dependent transactivation. In assays using the CYP2C19 promoter, rifampicin and rifaximin induced reporter activity, whereas SR12813 did not. PGC1α enhanced rifaximin- or SR12813-induced transcription, whereas NCoA1, NCoA2, and activating signal cointegrator-2 (ASC2) did not exhibit clear ligand-dependent effects. NCoA3 knockdown in HepaRG cells attenuated SR12813-dependent induction of CYP2C19. Taken together, using the PXR-3A mutant, we demonstrated ligand-dependent recruitment of coactivators by PXR.