Hepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5'-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2 requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit.
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...
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