RT Journal Article
SR Electronic
T1 Mechanistic Insights of Phenobarbital-mediated activation of Human but not Mouse Pregnane X Receptor
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.119.116616
DO 10.1124/mol.119.116616
A1 Linhao Li
A1 Matthew Welch
A1 Zhihui Li
A1 Bryan Mackowiak
A1 Scott Heyward
A1 Peter W Swaan
A1 Hongbing Wang
YR 2019
UL http://molpharm.aspetjournals.org/content/early/2019/07/10/mol.119.116616.abstract
AB Phenobarbital (PB), a broadly used anti-seizure drug, was the first to be characterized as an inducer of cytochrome P450 (CYP) by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of human CAR and PXR (hCAR, hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration-dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (KD = 1.42E-05). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.SIGNIFICANCE STATEMENT Phenobarbital, a selective mouse CAR activator, activates both human CAR and human PXR, which may contribute to its species-specific roles in energy homeostasis and cell proliferation.