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

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 ␤2 response elements (␤RARE) and to the NR1 and NR2 sites of the PBRU and activates transcription of reporter genes in cell lines. However, conversion of NR1 into ␤RARE was the equivalent of an inactivating mutation, indicating that CAR does not activate PB-dependent transcription via NR1 in the natural sequence context. A ␤RARE؋2-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 ␤RARE؋2-tk but reduced PBRU-dependent PB response. Furthermore, exogenous CAR also reduced the at-RA response of the ␤RARE؋2-tk construct. Thus, CAR acts negatively on PB responsiveness mediated by the CYP2B2 PBRU just as it prevents maximal at-RA responsiveness mediated by ␤RARE. Hepatic cytochrome P450s (CYPs)1 play a critical role in the metabolism of hydrophobic xenobiotics, and many CYPs are selectively inducible by xenobiotic compounds. Recently progress has been made toward understanding the molecular mechanisms underlying phenobarbital (PB) inducibility of the homologous rat CYP2B2 (and CYP2B1) and mouse Cyp2b10 genes (Refs. 1-9; reviewed in Refs. 10 -13) and of the chicken CYP2H1 gene (14). We identified a 163-bp Sau3AI fragment at coordinates Ϫ2317/Ϫ2155 in the CYP2B2 5Ј-flank that confers PB inducibility on the heterologous tk promoter and has the properties of a transcriptional enhancer when cat reporter constructs are transfected into primary rat hepatocytes (1). The homologous region of the 5Ј-flank of the PB-inducible mouse Cyp2b10 gene was found to contain a 162-bp segment 92% identical to the rat CYP2B2 163-bp fragment (there is a 1-bp deletion in the mouse sequence with respect to the rat; see Fig. 1A) that also possesses the properties of a transcriptional enhancer and confers PB inducibility on the heterologous tk promoter in primary mouse hepatocytes (3). Further analysis of the rat 163-bp Sau3AI fragment led us to conclude that it is a multicomponent enhancer constituting a PB response unit or PBRU (4). The localization of the PBRU at coordinates Ϫ2317/ Ϫ2155 in the CYP2B2 5Ј-flank is in agreement wit...

Inhibitory Cross-talk between Estrogen Receptor (ER) and Constitutively Activated Androstane Receptor (CAR)

“…CAR is promiscuous in binding to a variety of nuclear receptor-binding sites (17,31), so it was not too surprising that we found that transfected CAR/RXR from HepG2 nuclear extracts, as well as purified CAR/RXR, was able to bind to the consensus ERE. However, the binding affinity of CAR for the ERE was much less than that of the ER, so that a several hundred-fold excess of CAR did not affect binding of the ER to the ERE in vitro, whereas ER competed efficiently for CAR binding.…”

Modulation of Mouse and Human Phenobarbital-Responsive Enhancer Module by Nuclear Receptors

“…Despite previous observations that mouse CAR/RXR␣ heterodimer binds to NR1 and NR2 sites with equal efficiency in vitro (Tzameli et al, 2000), the present functional studies indicated that NR1 site is the stronger of these DR4 motifs. Paquet et al (2000) have also suggested that NR1 and NR2 in rat CYP2B2 gene are not identical. Among many NRs capable of DR4 binding, only hPXR and mPXR have been reported to bind to the NR1 site with affinity similar to CAR (Xie et al, 2000b;Goodwin et al, 2001;Smirlis et al, 2001).…”

Les récepteurs nucléaires CAR et PXR contrôlent l’induction des cytochromes P450 par le phénobarbital

“…Récemment, il a été montré que deux éléments de séquence additionnels sont nécessaires, au moins dans les hépatocytes de rat en culture primaire, pour conférer une induction maximale. Il s'agit d'un GRE (glucocorticoid response element) immé-diatement en 5' du PBRU, et d'une répétition opposée de type ER7 légèrement plus en amont (positions -2282 à -2264 pb du gène CYP2B2) [31].…”