The orphan nuclear receptor DAX-1 functions as a potent corepressor of the constitutive androstane receptor (NR1I3)

Mol Pharmacol. 2012 Nov;82(5):918-28. doi: 10.1124/mol.112.080721. Epub 2012 Aug 15.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Aryl Hydrocarbon Hydroxylases / biosynthesis
  • Aryl Hydrocarbon Hydroxylases / genetics
  • Co-Repressor Proteins / metabolism*
  • Constitutive Androstane Receptor
  • Cytochrome P-450 CYP2B6
  • DAX-1 Orphan Nuclear Receptor / genetics
  • DAX-1 Orphan Nuclear Receptor / metabolism*
  • Genes, Reporter
  • Hepatocytes / metabolism
  • Humans
  • Immunoprecipitation
  • Luciferases / genetics
  • Molecular Sequence Data
  • Mutation
  • Oxidoreductases, N-Demethylating / biosynthesis
  • Oxidoreductases, N-Demethylating / genetics
  • Oximes / pharmacology
  • Primary Cell Culture
  • Protein Isoforms / metabolism
  • Receptors, Cytoplasmic and Nuclear / agonists
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Thiazoles / pharmacology
  • Transcription, Genetic
  • Transcriptional Activation
  • Two-Hybrid System Techniques

Substances

  • Co-Repressor Proteins
  • Constitutive Androstane Receptor
  • DAX-1 Orphan Nuclear Receptor
  • NR1I3 protein, human
  • Oximes
  • Protein Isoforms
  • Receptors, Cytoplasmic and Nuclear
  • Thiazoles
  • 6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime
  • Luciferases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2B6 protein, human
  • Cytochrome P-450 CYP2B6
  • Oxidoreductases, N-Demethylating