CITCO Directly Binds to and Activates Human Pregnane X Receptor

Area of Science:

• Pharmacology
• Molecular Biology
• Biochemistry

Background:

• The xenobiotic receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate gene expression and have overlapping functions.
• Receptor-selective agonists are vital for dissecting these overlapping roles.
• CITCO was previously identified as a selective human CAR (hCAR) agonist, widely used to differentiate hCAR from human PXR (hPXR) activity.

Purpose of the Study:

• To investigate the selectivity of CITCO in human liver cell models.
• To determine if CITCO activates human PXR (hPXR) in addition to hCAR.
• To provide a clearer understanding of CITCO's activity for interpreting existing and future research on PXR and CAR.

Main Methods:

• CITCO's binding and activation of hPXR in HepG2 cells and primary human hepatocytes were assessed.
• The role of tryptophan-299 in hPXR activation by CITCO was investigated.
• Recruitment of steroid receptor coactivator 1 (SRC1) to hPXR was measured.
• hPXR activation by CITCO was examined in HepaRG cells with knocked-out hCAR.
• hPXR activation was confirmed using an hPXR-specific antagonist (SPA70).

Main Results:

• CITCO directly binds to and activates hPXR in human liver cell models (HepG2, HepaRG, primary hepatocytes).
• hPXR activation by CITCO is dependent on tryptophan-299 and involves the recruitment of SRC1.
• CITCO activates hPXR even when hCAR is absent, confirming its dual agonism.
• CITCO does not activate mouse PXR.

Conclusions:

• CITCO is a dual agonist, activating both hCAR and hPXR.
• The previous assumption of CITCO's selectivity for hCAR is incorrect in human liver cell contexts.
• These findings necessitate a re-evaluation of studies using CITCO and emphasize the importance of considering dual receptor activation for accurate data interpretation and experimental design.