Abstract
Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function.
Footnotes
↵
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by a grant from the National Institutes of Health National Institute of General Medicine [Grant GM066411].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- NR
- nuclear receptor
- AF
- activation function
- ANDRO
- 5α-androstan-3α-ol
- CAR
- constitutive androstane receptor
- CITCO
- 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime
- CMV
- cytomegalovirus
- DAX-1
- dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1
- DBD
- DNA-binding domain
- DMSO
- dimethyl sulfoxide
- CAS
- Chemical Abstract Service
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- HNF4α
- hepatocyte nuclear factor 4α
- LBD
- ligand-binding domain
- LXR
- liver X receptor
- PB
- phenobarbital
- PBREM
- phenobarbital response enhancer module
- RID
- receptor interaction domain
- RXR
- retinoid X receptor
- SHP
- small heterodimer protein
- SRC-1
- steroid receptor coactivator 1
- TSD
- transcription silencing domain
- VP16
- virus protein 16
- XREM
- xenobiotic response enhancer module
- NFDM
- nonfat dried milk
- HRP
- horseradish peroxidase
- TTBS
- Tween Tris-buffered saline.
- Received June 21, 2012.
- Accepted August 15, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|