Characterization of the porcine constitutive androstane receptor (CAR) and its splice variants

Abstract

1. Porcine constitutive androstane receptor (CAR; NR1I3) was cloned and compared for homology and activity with mouse and human CAR (mCAR, hCAR). Porcine CAR (pgCAR) was 86% and 75% homologous to hCAR at the nucleotide and protein levels.

2. Five alternatively spliced variants of pgCAR were identified, each of which generated a truncated protein product. Real-time polymerase chain reaction (PCR) analyses showed that these variants were present in pig liver cDNA samples from 4.61% to 9.20% of total pgCAR.

3. pgCAR and hCAR responded similarly to more ligands than did hCAR and mCAR. The known hCAR agonist (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) activated pgCAR, while the murine agonist 1,4 bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) had no effect. 5β-dihydrotestosterone was identified as a novel inverse agonist of both pgCAR and hCAR.

4. pgCAR splice variant 2 (SV2) had a dose-dependent dominant negative effect on the activity of wild-type pgCAR in dual luciferase assays. SV2 had no effect against pgPXR (pregnane X receptor) or pgFXR (farnesoid X receptor) activity when using PXR- or FXR-specific reporters.

Keywords::

• Nuclear receptor
• splice variants
• dominant negative
• gene expression

Acknowledgements

The authors thank Dr Masahiko Negishi (NIEHS, NIH) for supplying the expression plasmids for mCAR, hCAR, the (NR1)₅-tk-luciferase reporter plasmid, and the XREM-3A4-tk-luciferase reporter plasmid. They also thank Dr David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, Texas) for supplying the IR-1-tk-luciferase reporter plasmid. This was part of the MSc work of J. N. Peacock and M. A. Gray who contributed equally to this work.

Declaration of interest: This work was supported by NSERC Discovery Grant Number 400066 and funding from the Ontario Ministry of Agriculture and Food (Grant Number 026398) to E. J. Squires. The authors report no conflict of interest. The authors alone are responsible for the content and writing on the paper.