Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors.
The constitutive androstane receptor (CAR) regulates mouse and human CYP2B genes through binding to the direct repeat-4 (DR4) motifs present in the phenobarbital-responsive enhancer module (PBREM). The preference of PBREM elements for nuclear receptors and the extent of cross-talk between CAR and other nuclear receptors are currently unknown. Our transient transfection and DNA binding experiments indicate that binding to DR4 motifs does not correlate with the activation response and that mouse and human PBREM are efficiently 'insulated' from the effects of other nuclear receptors despite their substantial affinity for DR4 motifs. Certain nuclear receptors that do not bind to DR4 motifs, such as peroxisome proliferator-activated receptor-alpha and farnesoid X receptor, can suppress PBREM function via a coactivator-dependent process that may have relevance in vivo. In competition experiments, mouse PBREM is clearly more selective for CAR than human PBREM. Pregnane X, vitamin D, and thyroid hormone receptors can potentially compete with human CAR on human PBREM. In contrast to the selective nature of PBREM, CYP3A enhancers are highly and comparably responsive to CAR, pregnane X receptor, and vitamin D receptor. In addition, the ligand specificities of human and mouse CAR were defined by mammalian cotransfection and yeast two-hybrid techniques. Our results provide new mechanistic explanations to several previously unresolved aspects of CYP2B and CYP3A gene regulation.
Department of Pharmaceutics, University of Kuopio, Kuopio, Finland.
Janne
J
Mäkinen
Christian
C
Frank
Johanna
J
Jyrkkärinne
Jukka
J
Gynther
Carsten
C
Carlberg
Paavo
P
Honkakoski
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000187:drug effects*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D051379:Mice; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics; D010634:Phenobarbital / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D020239:Receptor Cross-Talk; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
D000090702:Constitutive Androstane Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D010634:Phenobarbital