Mutational analysis of the CYP2B2 phenobarbital response unit and inhibitory effect of the constitutive androstane receptor on phenobarbital responsiveness.
A 163-base pair enhancer in the CYP2B2 5' flank confers phenobarbital (PB) inducibility and constitutes a PB response unit (PBRU). By transfection of primary hepatocytes, we analyzed the function of elements comprising the PBRU and evaluated the role of the constitutive androstane receptor (CAR) in PB responsiveness. A 51-base pair PB-responsive enhancer module (PBREM) within the PBRU confers near-maximal PB response when fused to a tk promoter. However, replacing the PBRU with the PBREM in the CYP2B2 5' flank in the natural sequence context reduced PB responsiveness by approximately 4-fold. Mutational analysis also demonstrated that PBRU sequence elements outside the PBREM are essential for maximal PB responsiveness. The PBRU contains two putative nuclear receptor binding sites, NR1 and NR2. CAR binds to retinoic acid beta2 response elements (betaRARE) and to the NR1 and NR2 sites of the PBRU and activates transcription of reporter genes in cell lines. However, conversion of NR1 into betaRARE was the equivalent of an inactivating mutation, indicating that CAR does not activate PB-dependent transcription via NR1 in the natural sequence context. A betaRAREx2-tk reporter construct was inducible by all-trans-retinoic acid (at-RA) as expected and also responded to PB. The latter can be attributed to nuclear accumulation of CAR after PB exposure. Exogenous CAR increased both the basal and PB-induced response of betaRAREx2-tk but reduced PBRU-dependent PB response. Furthermore, exogenous CAR also reduced the at-RA response of the betaRAREx2-tk construct. Thus, CAR acts negatively on PB responsiveness mediated by the CYP2B2 PBRU just as it prevents maximal at-RA responsiveness mediated by betaRARE.
Paquet
Y
Y
Centre de recherche en cancérologie de l'Université Laval, L'H otel-Dieu de Québec, Centre hospitalier universitaire de Québec, Québec G1R 2J6, Canada.
Trottier
E
E
Beaudet
M J
MJ
Anderson
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004742:Enhancer Elements, Genetic; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology
D000090702:Constitutive Androstane Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D010634:Phenobarbital