A gene superfamily of heme-proteins, the cytochromes P450 (CYP), encodes the main enzymatic system for metabolism of structurally diverse lipophilic substrates 1. A subset of these CYPs can be activated or inhibited in the liver by a variety of xenobiotic and endobiotic compounds. Transcriptional activation of these CYPs is part of an adaptive response to exposure to drugs and other xenobiotics and has major clinical and toxicological implications. The enzymatic capacities of the affected CYPs are changed, leading to an altered metabolic profile in the liver 2. The barbiturate phenobarbital (PB) is prototypical for a class of compounds that induce or repress hepatic CYPs and many other genes 3. PB-responsive enhancer units (PBRU) have been identified in the 5'-flanking regions of several of these CYPs and transcription factors binding to those units could be isolated (reviewed in 4567). In mammals, the pregnane X receptor (PXR, official nomenclature NR1I2) and the constitutive androstane receptor (CAR, NR1I3), both belonging to the gene superfamily of nuclear receptors, have been identified to be involved in hepatic drug-induction 89101112.

Strikingly, in contrast to the two xenobiotic-sensing nuclear receptors in mammals, only one xenosensor has been found in non-mammalian species, e.g. chicken 13, fish (fugu Fugu rubripes 14 and zebrafish Danio rerio 15) or the nematode Caenorhabditis elegans 16. The amino acid sequence of the full-length chicken xenobiotic receptor (CXR, NR1I3) is about equally related to those of mammalian PXRs and CARs 17. Moreover, chicken CXR and mammalian PXR and CAR as well as drug-inducible CYP enhancer elements from these species could be freely interchanged in transactivation and electrophoretic mobility shift assays suggesting evolutionary conservation of the fundamental hepatic drug-induction mechanisms from birds to man 18.

In this report, we studied the evolutionary aspects of these findings. Despite using various methods and techniques, we were unable to isolate further genes that encode chicken xenobiotic-sensing nuclear receptors confirming the hypothesis that non-mammalian genomes only have one xenosensor gene. Since PXR and CAR exhibit different typical features concerning their activation, localization and regulation 619, we examined the properties of CXR to see whether on the functional and structural level, the chicken xenosensor shares common aspects with one or both of the mammalian receptors. Our findings give important insights the evolution of hepatic detoxification systems that protect different species from toxic compounds in their particular diet and environment.

Orthologs of PXR and CAR have been isolated from man, monkey, pig, dog, rabbit, mouse and rat 15. In non-mammalian species, only one xenosensor gene is found and sequence-wise, the corresponding receptors from chicken, zebrafish, fugu fish and C. elegans are about equally related to the mammalian PXRs and CARs (Fig. 1A). Of the 18 nuclear receptors in the fruitfly Drosophila melanogaster genome, DHR96 shares considerable similarity to the xenosensors but the functions of this receptor have not been elucidated yet. Although the African clawed frog Xenopus laevis has two nuclear receptors, benzoate X receptor α and β (BXRα/β, NR1I2), that are related to the xenobiotic-sensing nuclear receptors, the BXRs are pharmacologically distinct from PXR and CAR and do not respond to xenobiotics 1520. No drug-sensing nuclear receptors have thus been isolated in amphibians so far. Figure 1A shows the phylogeny of the xenobiotic-sensing nuclear receptors from different species. The completion of the rat genome allowed a global analysis of the nuclear receptors from three mammalian species, man, mouse and rat. In the nuclear receptor subfamily NR1I which includes the 1,25-dihydroxyvitamin D₃ receptor (VDR, NR1I1) in addition to PXR and CAR, intron-exon junctions are highly conserved 21. Human and rodent CARs and PXRs have the same number of introns. Moreover, apart from one intron which is found in the variable region 5' of the DNA-binding domain, all other seven introns are located in the same position on the corresponding genes, even in the ligand-binding domains that in the case of CAR and PXR are unusually divergent for nuclear receptor orthologs 22. Using a chicken genomic library, we isolated the gene encoding CXR and analyzed its structure. Again, the number of introns in the CXR coding sequence was the same as those in the mammalian xenosensors and the intron-exon junctions occur at the same locations (Figure 1B). The apparent conservation of gene structures between the single chicken xenosensors and the two mammalian orthologs suggest a close relationship between these receptors and supports the hypothesis that CXR constitutes an ancestral gene in chicken from which two receptors diverged in mammals.

To further test this hypothesis, we used different experimental approaches in order to isolate additional chicken xenobiotic-sensing nuclear receptors. Neither high- and low-stringency screening of a chicken liver cDNA library using CXR, CAR and PXR fragments as probes nor PCR-based strategies with degenerate primers designed on CAR and PXR alignments or degenerate primers based on generic nuclear receptor DNA-binding domains 23 resulted in the identification of novel chicken xenobiotic-sensing receptors (data not shown). The sequences of the previously unknown chicken orthologs for estrogen-related receptor γ (ERRγ, NR3B3) and a partial fragment of ear2 (NR2F6) that were found in these screens have been deposited (Genbank accession numbers AY702438 and AY702439, respectively).

If CXR in fact is the only chicken xenobiotic-sensing nuclear receptor, ablation of CXR expression or function is predicted to drastically reduce drug-induction of CYPs and other target genes. To reduce CXR expression, we designed RNAi oligonucleotides targeting CXR and stably expressed those in the chicken hepatoma cell line leghorn male hepatoma (LMH). LMH cells express endogenous CXR and retain induction of genes by PB-type inducer compounds and other drugs 18. As shown in Figure 2A, endogenous mRNA levels of CXR were reduced about 60% by the RNAi. LMH cells expressing either control vector or CXR RNAi were subsequently transfected with drug-responsive enhancer elements from CYP2H1 17, CYP3A37 24, CYP2C45 25 and δ-aminolevulinate synthase (ALAS-1) 26 and treated with vehicle or 400 μM PB for 16 hours. ALAS-1 is the first and rate-limiting enzyme in heme biosynthesis and its transcription is regulated by a variety of factors and stimuli, including PB-type inducers and other drugs 2627. In the case of ALAS-1, the 2-fold PB-induction was completely abolished by the CXR RNAi (Figure 2E). In contrast, PB-activation of the CYP2H1, CYP3A37 and CYP2C45 PBRUs was only partially reduced by 50 to 60% (Figure 2B,2C,2D). In these cases, reduction of CXR levels by 60% might not be enough. Alternatively, these findings could also be explained by the presence of additional drug-sensing signalling mechanism independent of CXR.

Thus, we used an alternative method that aimed at reducing CXR activity by designing dominant-negative CXR alleles. These CXR mutants were then tested in reporter gene assays on drug-responding enhancer elements. In our case, we generated three different CXR alleles (Figure 3A): first, we deleted the N-terminus since in some nuclear receptors, this part harbours a ligand-independent activation domain AF-1 2829. Second, site-directed mutagenesis of the cysteine residues in the zinc-fingers of the DNA-binding domain results in a CXR mutant that is expected to lack DNA-binding but to retain its ability to bind activators and to heterodimerize with its partner retinoid X receptor (RXR, NR2B1/2/3). Third, helix 12 in the ligand-binding domain was deleted which harbours a ligand-dependent activation domain AF-2. Nuclear receptors that act as dominant-negative alleles due to the absence of a functional AF-2 domain have been observed in some diseases (e.g. see refs. 3031). These findings were subsequently used to generate various dominant-negative nuclear receptor mutants for cellular assays 32.

First, the three CXR mutants were tested for their ability to bind to and activate a 264-bp PBRU isolated from the 5'-flanking region of chicken CYP2H1 1733. As shown in electrophoretic mobility shift assays (Figure 3B), CXR can heterodimerize with RXR and bind to the 264-bp PBRU as wild-type, full-length receptor and when the N-terminal region from amino acid 1–29 (called ΔN-term) or the C-terminal region from amino acid 383–391 (referred to as ΔAF-2) are deleted, respectively (Figure 3B, lanes 8, 10 and 12). As expected, site-directed mutagenesis of four cysteine within the DNA-binding domain into alanine residues (denominated DBD) that participate in forming the zinc-finger domains abolishes protein-DNA interaction (lane 11). These results show that removal of the N-terminus or the C-terminus of CXR does not influence its binding to DNA. Subsequently, the CXR mutants were tested in CV-1 transactivation assays for functionality. The CV-1 monkey kidney cells constitute an excellent tool to study nuclear receptor function in a cellular system which does not express endogenous xenosensors, is not drug-inducible and thus has a very low background in these assays. Neither CXR lacking its C-terminal activation domain AF-2 (ΔAF-2) nor CXR with the mutated DNA-binding domain (DBD) are able to transactivate the CYP2H1 264-bp PBRU in CV-1 cell assays (Figure 3C). In contrast, removal of the N-terminus of CXR (ΔN-term) has no effect on its transactivation potential suggesting that no activation function AF-1 is present in these 29 amino acids. Finally, the test whether any of these CXR mutant alleles acts in a dominant-negative fashion is performed in the LMH cells which do express endogenous CXR and which are drug-inducible 18. When co-transfected with the 264-bp PBRU, the CXR allele lacking a functional AF-2 domain (ΔAF-2) drastically decreases PB-induction of the PBRU (Figure 3D). In contrast, the DNA-binding domain (DBD) and the N-terminal truncated (ΔN-term) mutants have no effect. Similar results were obtained with PBRUs from other drug-responsive genes (data not shown). Together, the RNAi experiments and the findings using the dominant-negative CXR mutants show that functionally, CXR is the major drug-sensing nuclear receptor in chicken.

A significant difference between PXR and CAR in mammals is their mode of activation and their cellular localization 19. PXR is strongly activated by a huge number of compounds. In contrast, CAR exhibits less promiscuity but high constitutive activity in most cellular assays 34. However, CAR activity can be modulated by inverse agonists, agonists and different protein phosphorylation events 35. In terms of activation, CXR is also highly promiscuous and normally has a low basal activity, thus pharmacologically more resembling a PXR-type than a CAR-type receptor 1315. Regulation of CAR activity can in part be explained by its unusual cellular localization. Both PXR and CAR undergo cytoplasmic-nuclear shuttling upon activation 3637383940. However, in contrast to PXR, CAR translocates after activation by PB, other xenobiotics or bilirubin for which no direct binding to the ligand-binding pocket was found. Although some progress in identifying CAR-interaction partners have been made recently 4142, the mechanisms controlling the cytosolic-nuclear translocation are not clear. Interestingly, CAR translocation is independent of the C-terminal AF-2 function but instead requires the xenochemical response signal (XRS) LXXLXXL located between leucine 313 and leucine 319 in the human CAR sequence 37. In contrast, cytoplasmic-nuclear translocation of VDR, the glucocorticoid receptor (GR, NR3C1) and the progesterone receptor (PR, NR3C3) is dependent on AF-2 suggesting a different mechanism for CAR shuttling 3743. A putative XRS (LLLLTEL) is also found in the CXR sequence between leucine 356 and leucine 362. Thus, to assess the relatedness of CXR to PXR and CAR in terms of cellular localization, we engineered different CXR-green fluorescent protein (GFP) fusion proteins. These were subsequently tested for functionality in CV-1 cell transactivation assays using the 264-bp PBRU as drug-sensitive enhancer. CXR with N-terminal, but not C-terminal GFP is activated by 400 μM PB and 10 μM clotrimazole after 16 hours of incubation (Figure 4A). Site-directed leucine to glycine mutagenesis in the CXR XRS reduces its ability to confer drug activation in these assays (Figure 4A).

Subsequently, N-terminal GFP-CXR was transiently expressed in LMH cells, the cells were counterstained with DAPI to stain the nuclei and GFP-CXR localization was compared to that of GFP-expression vector without insert. GFP was found to be evenly distributed throughout the cell (Figure 4B). As depicted in Figure 4C, GFP-CXR in vehicle-treated LMH cells is exclusively in the nucleus. Treatment of transiently transfected LMH cells with 400 μM PB for 16 hours leads to an increase of GFP-staining in the cytosol (Figure 4D). Similar observations have been made for a variety of nuclear receptors where activation stimulates their export from the nucleus and subsequent degradation in the cytosol 4445. Accordingly, PB-treatment of LMH cells results in decreased CXR protein levels in total cell lysates (Figure 5, lanes 1 and 2) and even more dramatic in nuclear extracts (Figure 5, lanes 3 and 4) suggesting that activated CXR protein is more rapidly exported from the nucleus and degraded of this receptor. Most nuclear receptors that are exported and degraded upon activation share a conserved KXFF^K/_RR motif between the two zinc-fingers in the DNA-binding domain that can serve as binding site for calreticulin which is involved in the nuclear export 45. PXR, CAR and CXR also contain a KGFFRR-motif but whether calreticulin plays a role in nuclear export of these receptors remains to be investigated. The protein phosphatase inhibitor okadaic acid inhibits PB-induction of mammalian and chicken PBRUs 13184647. In transiently transfected LMH cells, 100 nM okadaic acid prevents nuclear localization of CXR after 16 hours (Figure 4E). Moreover, protein levels of the GFP-CXR fusion protein were reduced. Okadaic acid treatment prevents the drug-induced cytosolic-nuclear translocation of CAR 36. Our findings regarding CXR are therefore very reminiscent of those results. Furthermore, site-directed mutagenesis of the XRS reduces the nuclear localization of CXR (Figure 4F) but not as completely as XRS mutations of CAR in mouse primary hepatocyte cultures 37. The nuclear-cytoplasmic redistribution of this CXR mutant correlates with the decrease in its ability to activate the 264-bp PBRU in transactivation assays (Figure 4A). Thus, although CXR is normally found in the nucleus like PXR, it shares some features with CAR concerning its localization after treatment with okadaic acid or when its XRS is mutated.

In primary human hepatocytes, glucocorticoids have a dual effect on the expression of the drug-inducible CYP3A4 that is regulated by both PXR and CAR 48. At low concentrations, these compounds activate GR which subsequently induces transcript levels of PXR and CAR 4950 whereas higher concentrations of glucocorticoids directly activate PXR 951. We thus wanted to test whether the chicken CXR is regulated in the same way as the mammalian xenobiotic-sensing receptors. Treatment of LMH with 50 μM dexamethasone (Dex) for 16 hours did not alter CXR expression (Figure 6). Moreover, dexamethasone does not activate CXR directly, at least at this concentration 13. In contrast, dexamethasone increases transcription of the chicken peroxisome-proliferator activated receptor α (PPARα, NR1C1), the chicken liver X receptor (LXR, NR1H3) and the chicken farnesoid X receptor (FXR, NR1H4). These receptors play important roles in maintaining hepatic bile acid, cholesterol and lipid homeostasis, respectively 52. PXR, CAR and CXR have been found to be activated by bile acids and thus are involved in the regulation of the intrahepatic levels of lipid soluble compounds by stimulating metabolism and subsequent excretion of these compounds 125354. Therefore, activation of one of these receptors leads to changes in intrahepatic lipid levels which then potentially affects transcription of the other receptors. However, the regulatory network of these receptors is still under investigation.

Molecular modelling studies confirm the close relationship between chicken and fish xenosenors to mammalian PXR. X-ray structures of human PXR revealed several peculiarities of the PXR ligand-binding domain which are not found in other nuclear receptors 5556. First, PXR has an expanded β-sheet with two more strands. Moreover, helix 6 is completely and helix 7 partially unwinded which leaves a solvent-accessible hole in the ligand-binding pocket that is capped by an extension in helix 1–3. Although an extended β-sheet is not obvious in chicken and fish xenosensors, both receptors have long helix 1–3 inserts which could potentially induce partial unwinding of helix 6 and 7. Thus, molecular modelling of aligned amino acid sequences suggest enlarged ligand binding pockets for both fish and chicken xenobiotic-sensing receptors which could explain their high promiscuity 15. In striking contrast, CARs not only lack an extended β-sheet but also have a much shorter helix 1–3 resulting in a more rigid and less promiscuous ligand binding pocket 155758. Therefore, the relatively high degree of promiscuity of CAR could at least partially be due to the ability of different compounds to trigger cytoplasmic-nuclear translocation of this receptor independent of direct binding 35. The loop connecting helix 11 and 12 is much shorter in the CAR sequence than most other nuclear receptors 1557. This short loop might reduce the ability of helix 12 and AF-2 to reach an inactive conformation and thus could explain the constitutive activity of CAR 57. CAR also has a shorter helix 12 than most other nuclear receptors 57. Interestingly, helix 12 of CXR is very conserved to that of mammalian CARs in terms of amino acid composition and of length whereas the length of the zebrafish xenosensor helix 12 is intermediate between CARs and PXRs.

In summary, our results confirm that in contrast to mammals which have two xenobiotic-sensing receptor PXR and CAR, the genome of other species encodes for only one xenosensor. This hypothesis is supported by analysis in the fugu fish genome (data not shown), unsuccessful attempts to isolate further xenosenors in chicken and functional assays showing that ablation of CXR function drastically reduces drug-inducibility in a chicken hepatoma cell line. Our findings presented here and those of other laboratories imply that PXR and CAR origin from one ancestral gene which diverged into two genes in mammals. This ancestral gene, in chicken coding for CXR, is a promiscuous, PXR-like receptor. Thus, CXR and related receptors from fish are activated by a variety of different compounds 1315. Interestingly, in a comprehensive study of different classes of ligands on xenosensors from man, monkey, pig, dog, mouse, chicken and fish, CXR was one of the most promiscuous receptors in regard to the compounds tested 15. Therefore, the ancestral xenosensors in non-mammalian species might have a broader substrate spectrum than their mammalian counterparts where the task for detoxification is split between two receptors 59. On the other hand, CXR also shares some features with CAR that are not found in PXR: its short helix 12, the xenochemical response signal and in part its cellular localization after okadaic acid treatment. Finally, in contrast to both PXR and CAR, CXR is not regulated by glucocorticoid treatment in the chicken LMH cells suggesting that this regulation was acquired only after birds and mammals diverged from a common ancestor.

Evolution of drug-metabolizing CYPs and xenobiotic-sensing nuclear receptors is influenced by diet and exposure to other environmental chemicals. Accordingly, drug-induction is very species specific. This is reflected in the unusually divergent ligand-binding domains of PXRs and CARs orthologs 22. When comparing PXRs and CARs from human, mouse and rat, nonsynonymous nucleotide substitution rates are considerably higher in comparison to any other nuclear receptor 21 and reflect the different evolutionary adaptations of these species to their specific environment. It is thus extremely puzzling why in non-mammalian species, one xenosensor is sufficient whereas two xenobiotic-sensing nuclear receptors have evolved in mammals. Furthermore, it is unclear why in addition to the ligand-activated PXR, mammalian genomes encode CAR, a nuclear receptor that is unorthodox in many ways. On one hand, CAR and PXR might just share the workload in hepatic detoxification of xenobiotics. On the other hand, evidence accumulated in recent years that both PXR and CAR have functions that go beyond detoxification. As example, PXR and CAR form an intricate network with other nuclear receptors and transcription factors to regulate hepatic cholesterol and bile acid homeostasis 60. It is thus conceivable that these receptors have so-far unidentified functions in mammals which require two receptors and that are thus absent in non-mammalian species. Therefore, further insights into the evolution of drug-sensing nuclear receptors are extremely important in order to gain novel insights into the role of these factors in the physiology and pathophysiology of the liver.

CYP, cytochrome P450; PB, phenobarbital; PXR, pregnane X receptor; CAR, constitutive androstane receptor; CXR, chicken xenobiotic receptor; PBRU, phenobarbital-responsive enhancer unit; ALAS-1, δ-aminolevulinate synthase; AF-1/2, activation function-1/2; LXR, liver X receptor; PB, phenobarbital; XRS, xenochemical response signal; GFP, green fluorescent protein; PPAR, peroxisome-proliferator activated receptor; FXR, farnesoid X receptor.

The authors declare that they have no competing interests.

CH carried out the cellular localization assays, cloned the various CXR mutants, performed the reporter gene and the electrophoretic-mobility shift assays as well as the transcriptional regulation studies. SB did the various screens for further chicken xenobiotic-sensing nuclear receptors. AR performed the RNAi experiments. RL and MO isolated the CXR antibody and carried out the protein stabilization and localization assays. MRK helped with the RNAi experiments. MP and CG helped with the CV-1 cell transactivation assays. UAM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.