Mutational Analysis of the CYP2B2 Phenobarbital Response Unit and Inhibitory Effect of the Constitutive Androstane Receptor on Phenobarbital Responsiveness

Yanick Paquet,Eric Trottier,Marie-Josée Beaudet,Alan Anderson

doi:10.1074/jbc.m005776200

Yanick Paquet, Eric Trottier + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m005776200

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Abstract

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.

Highlights

A 163-base pair enhancer in the CYP2B2 5؅ flank confers phenobarbital (PB) inducibility and constitutes a PB response unit (PBRU)
The PBRU and PB-responsive enhancer module (PBREM) Are Not Equivalent When Tested in the Natural Sequence Context—The mouse Cyp2b10 and rat CYP2B2 PBREMs differ by a single nucleotide in the NR2 spacer (Fig. 1A) and conferred identical PB responses when tested in the tk and natural sequence contexts
In accord with the conclusions of Honkakoski et al (8), the mouse Cyp2b10 PBRU and rat CYP2B2 PBREM gave essentially equivalent PB responses when tested in the tk sequence context (Fig. 2A)

Summary

Introduction

A 163-base pair enhancer in the CYP2B2 5؅ flank confers phenobarbital (PB) inducibility and constitutes a PB response unit (PBRU). CAR, in the form of a heterodimer with the retinoid X receptor (RXR) binds to the retinoic acid ␤2 response element (␤RARE) (16, 17) and, with lower affinity, to the NR1 and NR2 sites of the PBREM (18) and activates transcription of reporter genes driven by these enhancers in cultured cell lines (7, 9, 18).

Results

Conclusion