Characterization of activating signal cointegrator-2 as a novel transcriptional coactivator of the xenobiotic nuclear receptor constitutive androstane receptor

Mol Endocrinol. 2005 Jul;19(7):1711-9. doi: 10.1210/me.2005-0066. Epub 2005 Mar 10.

Abstract

Activating signal cointegrator-2 (ASC-2) is a recently isolated transcriptional coactivator protein for a variety of different transcription factors, including many members of the nuclear receptor superfamily. In this report, we demonstrate that ASC-2 also serves as a coactivator of the xenobiotic nuclear receptor constitutive androstane receptor (CAR). First, transcriptional activation by CAR was enhanced by cotransfected ASC-2 in CV-1 and HeLa cells. In contrast, CAR transactivation was significantly impaired in HepG2 cells stably expressing specific small interfering RNA directed against ASC-2. Consistent with these results, chromatin immunoprecipitation experiments revealed that ASC-2 is recruited to the known CAR target genes in a ligand-dependent manner. Secondly, CAR specifically interacted with the first LXXLL motif of ASC-2, and these interactions were stimulated by CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene and repressed by CAR inverse agonist androstanol, suggesting that this motif may mediate the interactions of ASC-2 and CAR in vivo. In support of this idea, DN1, a fragment of ASC-2 encompassing the first LXXLL motif, suppressed CAR transactivation, and coexpressed ASC-2 but not other LXXLL-type coactivators such as thyroid hormone receptor-associated protein 220 reversed this repression. Finally, CAR was recently found to play a pivotal role in effecting the severe acetaminophen-induced liver damage. Interestingly, transgenic mice expressing DN1 were resistant to the acetaminophen-induced hepatotoxicity and expression of a series of the known CAR target genes was specifically repressed in these transgenic mice. Taken together, these results strongly suggest that ASC-2 is a bona fide coactivator of the xenobiotic nuclear receptor CAR and mediate the specific xenobiotic response by CAR in vivo.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetaminophen / metabolism
  • Acetaminophen / toxicity*
  • Amino Acid Motifs
  • Animals
  • Aryl Hydrocarbon Hydroxylases / genetics
  • Chromatin Immunoprecipitation
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / genetics
  • Gene Expression Regulation
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Intracellular Signaling Peptides and Proteins / physiology*
  • Liver / drug effects*
  • Mice
  • Mice, Transgenic
  • Nuclear Receptor Coactivators
  • Oxidoreductases, N-Demethylating / genetics
  • Protein Interaction Mapping
  • RNA Interference
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Trans-Activators / physiology*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Xenobiotics / metabolism
  • Xenobiotics / toxicity

Substances

  • Intracellular Signaling Peptides and Proteins
  • NCOA6 protein, human
  • Nuclear Receptor Coactivators
  • Receptors, Cytoplasmic and Nuclear
  • Trans-Activators
  • Transcription Factors
  • Xenobiotics
  • Acetaminophen
  • constitutive androstane receptor
  • Cytochrome P-450 Enzyme System
  • Aryl Hydrocarbon Hydroxylases
  • CYP2B6 protein, human
  • CYP3A protein, human
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 CYP3A
  • Oxidoreductases, N-Demethylating