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 PB RU and evaluated the role of the constitutive androstane receptor (CAR) in PB responsiveness. A 51-base pair PR-responsive enhancer module (PBREM) wit hin the PBRU confers near-maximal PB response when fused to a th promoter. However, replacing the PBRU with the PBREM in the CYP2B2 5' flank in the na tural sequence context reduced PB responsiveness by approximately C-fold. M utational analysis also demonstrated that PBRU sequence elements outside th e 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 (beta RARE) and to the NR1 and NR2 sites of t he PBRU and activates transcription of reporter genes in cell lines. Howeve r, conversion of NR1 into beta RARE was the equivalent of an inactivating m utation, indicating that CAR does not activate PB-dependent transcription v ia NR1 in the natural sequence context. A beta RARE x 2-tk reporter constru ct was inducible by all-trans-retinoic acid (at-RA) as expected and also re sponded to PB, The latter can be attributed to nuclear accumulation of CAR after PB exposure. Exogenous CAR increased both the basal and PB-induced re sponse of beta RAREx2-tk but reduced PBRU dependent PB response. Furthermor e, exogenous CAR also reduced the at-RA response of the beta RARE x 2-tk co nstruct, Thus, CAR acts negatively on PB responsiveness mediated by the CYP 2B2 PBRU just as it prevents maximal at-RA responsiveness mediated by beta RARE.