Differential Regulation of Gene Expression by CAR and PXR Nuclear Receptors

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 338-365-Metabolic & Stress Receptors in Energy Homeostasis
Basic/Translational
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-356
William Chi-shun Ho*, Adam Avery and Maria Ines Morano
Originus Inc, Ann Arbor, MI
Nuclear hormone receptors (NHR) are transcription factors that work in concert with other modulators to regulate gene expression. PXR (pregnane X receptor) and CAR (constitutive androstane receptor) form the NR1I subfamily of NHR. They were initially considered regulators of drug metabolizing enzymes such as Cytochrome (CYP) P450 enzymes. In addition to xenobiotic disposition, both receptors are implicated in the metabolism of carbohydrates, lipids, steroid, and bile acids, some of which constitute activators of these receptors. More than 20 splice variants of CAR transcripts have been identified. The main isoform, CAR1, showed strong constitutive activity in transfected cell lines, while the isoforms CAR2 and CAR3 were shown to transactivate the CYP enzymes CYP2B6 and CYP3A4 upon ligand binding. In this study, secreted alkaline phosphatase (SEAP) reporter gene constructs were assembled using native promoter sequences from CYP2B6 and CYP3A4 genes. These reporters were co-transfected in HepG2 cells with combinations of CAR, PXR, and HNF4α using a solid-phase transfection approach called Surface Transfection and Expression Protocol (STEP). In the presence of PXR, both CYP2B6 and CYP3A4 reporters were induced by 10μM Rifampicin (6.88±0.91 and 21.82±3.94 fold at 96hr) and 3μM Paclitaxel (14.66±0.92 and 11.14±1.14 fold at 96hr). In the presence of CAR1, the CYP2B6 reporter exhibited a strong constitutive activity (14.88±2.47 fold over no CAR control) which masked any potential induction, while CYP3A4 reporter showed little constitutive activity by CAR1 but a 2.90±0.28 fold induction by the ligand CITCO at 1µM. In the presence of CAR2 or CAR3, no constitutive activity was observed and 1µM CITGO induced the CYP2B6 reporter (1.44±0.08 and 2.09±0.30 fold at 72hr). When both PXR and CAR1 were present, the PXR-mediated Rifampicin response of the CYP3A4 reporter was moderately hampered (4.37±0.24 fold) while the CAR1-mediated CITCO effect was not affected (2.90±0.24 fold). Interestingly, the co-expression of PXR with CAR1 drastically suppressed the basal constitutive activation of CYP2B6 reporter gene by CAR1 and unmasked the effect of CITCO (3.85±0.85 fold). This study emphasizes the significance of nuclear receptor interactions in the regulation of gene expression. In addition, it illustrates the importance of using native promoter sequences instead of a short synthetic regulatory element in assembling reporter genes for nuclear receptor assays.

Nothing to Disclose: WCSH, AA, MIM

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Supported by NIH/NIEHS Grant 1R44ES019807.