Phenobarbital-responsive Unit Research Articles

Dexamethasone Induction of MurineCYP2BGenes Requires the Glucocorticoid Receptor

Hepatic cytochrome P450 (P450) enzymes metabolize exogenous and endogenous compounds, and many are inducible by xenobiotics. Their synthesis is tightly regulated, particularly through nuclear receptors. Expression of murine CYP2B genes is strongly activated by treatment with phenobarbital or phenobarbital-like inducers, and a detectable response requires the presence of the constitutive androstane receptor (CAR). However, other compounds can also induce murine CYP2B proteins. For example, dexamethasone is known to induce rat CYP2B1 and CYP2B2 and mouse CYP2B10. Using human HepG2 and rat H4IIEC3 hepatoma cell lines, we found that dexamethasone induction of CYP2B2 and Cyp2b10 luciferase reporters required the glucocorticoid receptor. Given the well known observation that CYP2B genes are not phenobarbital-responsive in cultured cell lines, the dexamethasone responsiveness of CYP2B reporter constructs in cell lines demonstrates in itself that the mechanism of dexamethasone induction is distinct from that of phenobarbital. We also analyzed the relative importance of the phenobarbital response unit (PBRU) and of a known glucocorticoid response element in this response. Both sites contributed to the response, but other sites were required for maximal induction. CAR was also found to act as an accessory factor to stimulate the response to dexamethasone by the glucocorticoid receptor. Furthermore, in H4IIEC3 cells, CAR activated the PBRU in the natural sequence context of the CYP2B2 and Cyp2b10 5' flanks. In summary, there are at least two independent mechanisms of CYP2B induction: one involving phenobarbital and phenobarbital-like inducers and another involving glucocorticoids that induce via the glucocorticoid receptor with CAR acting as an accessory factor.

The CYP2B2 5′ flank contains a complex glucocorticoid response unit

Rat CYP2B1 and CYP2B2 and mouse CYP2B10 are dramatically induced by phenobarbital (PB) in liver. PB responsiveness requires the constitutive androstane receptor (CAR). However, dexamethasone treatment can also induce CYP2B genes in both rat and mouse liver. Three regions have been shown to be involved in conferring dexamethasone responsiveness on CYP2B2 reporter constructs. They are the PB response unit, a functional glucocorticoid response element at −1.3 kb in the 5′ flank and a weak element in the basal promoter. We report here the identification, by deletion analysis of the CYP2B2 5′ flank, of new glucocorticoid response elements or accessory factor sites. Moreover, we show that CAR acts as an accessory factor in the dexamethasone response in vivo of CYP2B10 protein in mice, by increasing both the basal and induced levels. We propose a model to explain the dexamethasone responsiveness of the CYP2B2 gene in which induction is mediated by a complex glucocorticoid response unit.

Effects of TK Promotor and Hepatocyte Nuclear Factor‐4 in CAR‐Mediated Transcriptional Activity of Phenobarbital Responsive Unit of CYP2B Gene in Monkey Kidney Epithelial‐Derived Cell Line COS‐7

Previous studies reported that constitutive androstane receptor (CAR) does not transactivate phenobarbital responsive unit (PBRU)2C1luciferase reporter gene in COS cells in which endogenous CYP2B1 gene is not induced with PB. In order to understand molecular mechanism(s) whereby PBRU is transactivated, this article determined if the use of strong thymidine kinase (TK) promotor rather than the minimal CYP2C1 promotor, and hepatocyte nuclear factor-4 (HNF-4) can affect CAR-mediated transactivation of PBRU in the monkey kidney epithelial-derived COS-7 cells. To examine CAR-mediated transactivation, cultured COS-7 cells were transfected with CAR expression plasmid, pEGFP-mCAR1, and confirmed for high level of the protein expression. In COS-7 cells, TK promotor induced CAR-mediated PBRU transactivation in a dose-dependent manner. Whereas expression of HNF-4 slightly promoted PBRU transactivation with low amount of CAR transfected, it repressed PBRU transactivation in a dose-dependent manner with high amount of CAR. Consistent with the previous reports in Hep G2 cells, CAR transactivated PBRU2C1luciferase in a dose-dependent manner and this CAR-mediated transactivation required functional NR-1 and NF-1 sites. However, HNF-4 did not affect CAR-mediated PBRU transactivation in Hep G2 cells. These results suggest that proximal promotor and a trans-acting factor, HNF-4, can affect CAR-mediated transactivation of PBRU in COS-7 cells.

Analysis of multiple nuclear receptor binding sites for CAR/RXR in the phenobarbital responsive unit of CYP2B2

The phenobarbital (PB) responsive enhancers in CYP2B genes contain a core of two direct repeat-4 nuclear receptor binding sites, NR-1 and NR-2, which flank an NF-1 site and appear to be most important for PB responsiveness. Additional sequences outside the core are required for maximal PB responsiveness, including a third direct repeat-4 site, NR-3. The PB response is mediated by constitutive androstane receptor (CAR) which binds as a CAR/RXR heterodimer to the NR sites. To determine the relative importance of the third NR site, each of the NR sites was mutated individually and in all combinations in the rat PB responsive unit (PBRU). Mutation of NR-3 resulted in similar effects on transactivation of the PBRU by CAR in HepG2 cells as did mutations of NR-1 and NR-2. The recruitment of GRIP1/SRC-2 by CAR/RXR to the PBRU assessed by gel shift assays was cooperatively enhanced if more than one NR site in the PBRU was occupied by CAR/RXR. NR-3 in combination with NR-1 or NR-2 was equal to NR-1 and NR-2 in mediating this cooperative recruitment. Recruitment of SRC-1 and GRIP1/SRC-2 was similar for all NR sites, while some selectivity of NR-1 for SRC-3 was observed. SRC-3 also exhibited CAR-independent activation of the PBRU in HepG2 cells. Micrococcal nuclease mapping of nucleosomes revealed that the NR-1/NR-2 core of the PBRU is present in a nucleosome while NR-3 is present in the linker adjacent to the nucleosome. In the linear sequence NR-3 is further from NR-1 than NR-2 is, but in a nucleosomal structure, NR-3 is well positioned for cooperative recruitment of GRIP1/SRC-2 by CAR/RXR that is bound to NR-3 and either NR-1 or NR-2, while NR-1 and NR-2 are on opposite sides of the nucleosome separated by the histone core. These results demonstrate that NR-3 is functionally similar to NR-1 and NR-2 in CAR transactivation of the PBRU in vitro and suggest that NR-3 may have a greater role in a chromatin context in vivo than is apparent from transient transfection studies.

The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX–PREP1, the thyroid hormone receptor β and the liver X receptor

A 163 bp enhancer in the CYP2B2 5' flank confers PB (phenobarbital) inducibility and constitutes a PBRU (PB response unit). The PBRU contains several transcription factor binding sites, including NR1, NR2 and NR3, which are direct repeats separated by 4 bp of the nuclear receptor consensus half-site AGGTCA, as well as an ER (everted repeat) separated by 7 bp (ER-7). Constitutive androstane receptor (CAR)-RXR (retinoic X receptor) heterodimers are known to bind to NR1, NR2 and NR3. Electrophoretic mobility-shift analysis using nuclear extracts from livers of untreated or PB-treated rats revealed binding of several other proteins to different PBRU elements. Using supershift analysis and in vitro coupled transcription and translation, the proteins present in four retarded complexes were identified as TRbeta (thyroid hormone receptor beta), LXR (liver X receptor), HNF-4 (hepatocyte nuclear factor 4) and heterodimers of PBX-PREP1 (pre-B cell homoeobox-Pbx regulatory protein 1). LXR-RXR heterodimers bound to NR3 and TRbeta bound to NR3, NR1 and ER-7, whereas the PBX-PREP1 site is contained within NR2. The HNF-4 site overlaps with NR1. A mutation described previously, GRE1m1, which decreases PB responsiveness, increased the affinity of this site for HNF-4. The PBRU also contains a site for nuclear factor 1. The PBRU thus contains a plethora of transcription factor binding sites. The profiles of transcription factor binding to NR1 and NR3 were quite similar, although strikingly different from, and more complex than, that of NR2. This parallels the functional differences in conferring PB responsiveness between NR1 and NR3 on the one hand, and NR2 on the other.

GABA A/central benzodiazepine receptor and peripheral benzodiazepine receptor ligands as inducers of phenobarbital-inducible CYP2B and CYP3A

A sequence critical for phenobarbital (PB) induction, the PB response unit (PBRU), situated upstream of the rat CYP2B1 and CYP2B2 genes, includes two nuclear receptor binding sites, NR1 and NR2. When NR1 and NR2 are mutated PB responsiveness is abolished. While no nuclear receptor for which PB is an agonist ligand has yet been identified, PB is a ligand of GABA A receptors and it can displace [ 3H] 1-(2-chlorophenyl)- N-methyl- N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) from its binding site on the peripheral benzodiazepine receptor (PBR). We assessed CYP2B levels in primary rat hepatocytes following treatment with 10 ligands of either or both of these receptors. All compounds tested were found to be CYP2B1/CYP2B2 inducers and most were CYP3A inducers. Five had not previously been described as CYP2B1/CYP2B2 inducers: bicuculline, flunitrazepam, 4′-chlorodiazepam (Ro5-4864), N, N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN 1–27) and 7-(dimethylcarbamoyloxy)-6-phenylpyrrolo-[2,1- d][1,5]benzothiazepine (DCPPBT). Reporter gene analysis demonstrated that CYP2B induction by these agents and other PBR or GABA A receptor ligands is mediated through the PBRU and the NR1/NR2 sites, suggesting a molecular mechanism similar to that for PB induction. The potencies for PBRU-dependent induction by 11 ligands of PBR or the GABA A receptor was evaluated. FGIN-127, DCPPBT and PK 11195 exhibited EC 50 values for PBRU-dependent transcription activation about three orders of magnitude higher than the reported affinities of the PBR for these agents, arguing against the involvement of the PBR in PB induction. However the EC 50 values found for the agents tested encourage further investigation on the possible involvement of the GABA A receptor in PB induction.

Repression of CAR-mediated transactivation of CYP2B genes by the orphan nuclear receptor, short heterodimer partner (SHP).

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.

Functional analysis of the phenobarbital-responsive unit in rat CYP2B2

An 163-bp fragment of the rat cytochrome P450 gene, CYP2B2 has been shown to contain sequences that mediate phenobarbital (PB) responsiveness of this gene. In studies on this rat gene and the orthologous mouse gene, Cyp2b10, the minimal fragment required for near full PB responsiveness has varied from about 50 to 80 bp depending on the gene used and the number of copies of the PB responsive sequences assessed. Since there is a single copy of the CYP genes in the genome, we have evaluated deletion and block mutations across an 84-bp region of the PB responsive unit (PBRU), by in situ transfection in rat liver using single copies of the PBRU sequences. From the 5′ end, deletions to −2243 retained more than 50% responsiveness to PB compared to the 163-bp fragment. The fragment −2237 to −2155 retained less than 20% responsiveness even though it contained the nuclear receptor (NR)-1, NR-2, and NF-1 motifs which are present in the core of the PBRU. From the 3′ end, deletions from −2170 to −2194 eliminated PB responsiveness indicating that the 74-bp sequence from −2243 to −2170 is able to mediate full PB responsiveness. Block mutations within the NR-1 and NF-1 regions reduced responsiveness most dramatically, but did not abolish it, and mutations 3′ of the NF-1 site modestly reduced responsiveness. Protein binding was not affected by mutations in the NR-1 region as assessed by DNase I footprinting in vitro but mutations within the NR-2 region reduced binding to the NF-1 site. Mutations of the 5′ half or the 3′ half of the bipartite NF-1 site, resulted in loss of protection of the NF-1 site and new footprints to the 3′ or 5′ side, respectively, of the NF-1 site. These results indicate that sequences in addition to the NR-1 and -2 and the NF-1 sites are required for full responsiveness to PB and suggest that proteins which bind to these sites may interact.

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.

A Conserved Nuclear Receptor Consensus Sequence (DR-4) Mediates Transcriptional Activation of the Chicken CYP2H1 Gene by Phenobarbital in a Hepatoma Cell Line

Phenobarbital-responsive DNA elements were identified in the 5'-flanking region of the chicken CYP2H1 gene by in reporter gene assays in a chicken hepatoma cell line (leghorn male hepatoma (LMH)). A 264-base pair (bp) enhancer sequence (phenobarbital-responsive unit (PBRU)) responded to phenobarbital and a variety of phenobarbital-type inducers. Analysis of putative transcription factor binding sites within the 264-bp element revealed a nuclear receptor half-site repeat (DR-4) neighboring a putative nuclear factor-1 site. This motif resembles phenobarbital response elements in the flanking regions of three phenobarbital-inducible genes, rat CYP2B2, mouse Cyp2b10, and human CYP2B6. Activation of the 264-bp element was eliminated after site-directed mutagenesis of the DR-4 hexamer half-sites. Evidence for evolutionary conservation of this recognition site was indicated by activation in LMH cells of a mouse Cyp2b10 phenobarbital-responsive enhancer by the same spectrum of inducers that activate the CYP2H1 264-bp PBRU. Inhibition of this activation by okadaic acid may explain the reported inhibitory effects on induction of CYP2B1/2 and Cyp2b10 by this phosphatase inhibitor. We show that this inhibition occurs directly on the 264-bp PBRU, whereas the proximal promoter of CYP2H1 is induced by okadaic acid in reporter gene assays. These experiments exploit the unique phenobarbital inducibility of the hepatoma-derived cell line LMH.

Tissue-specific chromatin structure of the phenobarbital-responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital.

Phenobarbital induction of transcription of CYP2B genes is mediated by an enhancer, termed a phenobarbital responsive unit (PBRU), approximately 2000 bp 5' of the transcription start site. To further delineate the mechanism of phenobarbital induction, protein binding in native chromatin and the nucleosomal structure of the PBRU and proximal promoter were examined in liver and kidney, in which the CYP2B1/2 genes are expressed and not expressed, respectively. Protein binding to the PBRU in kidney chromatin was not detected even though in vitro DNase I footprints were not detectably different with nuclear extracts from liver and kidney. Likewise, protein binding to regulatory motifs was not detected in the proximal promoter region in kidney chromatin. In liver chromatin, however, DNase I hypersensitivity and partial protection of the regulatory motifs from DNase I digestion or reaction with dimethyl sulfate was observed and phenobarbital treatment increased the hypersensitivity but only modestly affected protection. Low resolution Southern analysis of micrococcal nuclease-digested chromatin from untreated rats revealed micrococcal nuclease hypersensitive regions in the proximal promoter and PBRU regions in liver, but not in kidney. Phenobarbital treatment increased hyper-sensitivity in liver in both regions. Micrococcal nuclease hypersensitivity in the PBRU was largely restricted to a linker region between phased nucleosomes while in the proximal promoter hypersensitivity extended over approximately 200 bp suggesting disruption of a nucleosome in this region. These data indicate that in liver phenobarbital treatment substantially alters protein binding to regulatory motifs in the PBRU, while not greatly affecting such binding in the proximal promoter, and substantially alters chromatin structure in both regions, presumably as a result of chromatin modifying factors recruited to the PBRU. In the kidney, chromatin is probably in a closed conformation that prevents binding of regulatory factors.

Positive regulation of the rat CYP2B2 phenobarbital response unit by the nuclear receptor hexamer half-site·nuclear factor 1 complex

Abstract. A distal 163-bp fragment mediates phenobarbital responsiveness of the rat CYP2B2 gene. Multiple cis-acting elements in this fragment cooperate to form a phenobarbital response unit (PBRU). A nuclear factor 1 binding site and an associated nuclear receptor hexamer half-site are present in both the rat CYP2B2 PBRU and the homologous mouse Cyp2b10 sequence. Based on mutational analyses, the hexamer half-site has been reported to act positively in CYP2B2 and negatively in Cyp2b10. However, the specific mutations introduced into the rat and mouse hexamer half-sites were different, raising the possibility that the different roles attributed to the element may be a consequence of the different mutations used. We introduced into the rat CYP2B2 hexamer half-site the specific mutational change previously introduced into the Cyp2b10 sequence, where its effect was to increase the basal level of expression and to abolish phenobarbital responsiveness. In the rat context, this mutation reduced but did not abolish phenobarbital responsiveness and decreased, rather than increased, the basal level of expression. The residual phenobarbital responsiveness of the hexamer half-site mutant, as well as that of nuclear factor 1 mutants, indicates that these elements behave as positive accessory sites, suggesting that factors binding to them function as activators of phenobarbital-dependent transcription.

Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.

Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific mutations within the 2.5kb 5'-flanking region of the rat CYP2B2 gene. Protein-DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between -2500 and -1700bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800bp of 5'-flanking CYP2B2 sequence are not PB responsive. DNA affinity enrichment techniques and immunoblotting and electromobility shift assays were used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at -2200bp in the PB responsive unit (PBRU) of CYP2B2. To test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type -2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA-protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.

The CYP2B2 Phenobarbital Response Unit Contains an Accessory Factor Element and a Putative Glucocorticoid Response Element Essential for Conferring Maximal Phenobarbital Responsiveness

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.

Phenobarbital Alters Protein Binding to the CYP2B1/2Phenobarbital-responsive Unit in Native Chromatin

Phenobarbital is a classical inducer of the drug metabolizing cytochrome P450 genes, but the molecular mechanism of induction has not been elucidated. Functional analyses have identified a phenobarbital-responsive unit in the rat CYP2B1/2 and mouse Cyp2b10 genes about -2.3 kilobase pairs from the transcriptional start site, but little or no changes in protein binding to this region were observed in vitro. To examine the role of chromatin structure, protein binding to the phenobarbital-responsive unit assessed by in vitro DNase I footprinting was compared with that assessed by DNase I in vivo footprints in native chromatin. A region centered on a putative nuclear factor-1 site was the major protected region in in vitro footprints, and there were no detectable differences in binding between extracts from control and phenobarbital-treated animals. In contrast, phenobarbital treatment dramatically altered the protection pattern in native chromatin. In control samples a core region of about 25 base pairs (bp) centered on the nuclear factor-1 site was protected. However, after phenobarbital treatment, the protection of this core region was increased, and more dramatically the region of protection was extended 20 bp to either side so that a total of about 60 bp were protected. These results provide the first evidence that phenobarbital treatment alters the composition or architecture of proteins binding to the phenobarbital-responsive unit region and indicate that chromatin structure is important in this process. Because proteins are bound to the region in the untreated animal, the mechanism of induction involves the activation of proteins bound to the region and possibly recruitment of additional regulatory proteins rather than conversion of a closed chromatin structure to an open one that can bind regulatory factors.