Journal
MOLECULAR PHARMACOLOGY
Volume 96, Issue 3, Pages 345-354Publisher
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/mol.119.116616
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Funding
- National Institutes of Health National Institute of General Medicine [GM107058, GM121550]
- National Institute of Diabetes and Digestive and Kidney Diseases [DK61425]
- University of Maryland's Center of Excellence in Regulatory Science and Innovation Scholars Program - Food and Drug Administration [1U01FD005946]
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Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 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 (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (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 (K-D = 1.42 x 10(-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.
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