The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals
© Handschin et al; licensee BioMed Central Ltd. 2004
Received: 08 September 2004
Accepted: 12 October 2004
Published: 12 October 2004
Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals.
To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR.
Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.
A gene superfamily of heme-proteins, the cytochromes P450 (CYP), encodes the main enzymatic system for metabolism of structurally diverse lipophilic substrates . 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 . The barbiturate phenobarbital (PB) is prototypical for a class of compounds that induce or repress hepatic CYPs and many other genes . 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 [4–7]). 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 [8–12].
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 , fish (fugu Fugu rubripes  and zebrafish Danio rerio ) or the nematode Caenorhabditis elegans . The amino acid sequence of the full-length chicken xenobiotic receptor (CXR, NR1I3) is about equally related to those of mammalian PXRs and CARs . 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 .
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 [6, 19], 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.
Results and Discussion
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  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).
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 [17, 33]. 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 . 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.
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 [55, 56]. 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 . 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 [15, 57, 58]. 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 . The loop connecting helix 11 and 12 is much shorter in the CAR sequence than most other nuclear receptors [15, 57]. 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 . CAR also has a shorter helix 12 than most other nuclear receptors . 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 [13, 15]. 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 . 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 . 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 . When comparing PXRs and CARs from human, mouse and rat, nonsynonymous nucleotide substitution rates are considerably higher in comparison to any other nuclear receptor  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 . 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.
LMH and CV-1 cell culture, transfection and reporter gene assays
Culture and transfection of LMH cells with FUGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Rotkreuz, Switzerland) were performed as published [17, 33]. Before transfections, LMH cells were kept in serum-free medium for 24 hours. CV-1 cell transactivation assays have been described in detail [17, 33]. Sixteen or twenty-four hours after drug-treatment, cells were harvested and assays for CAT expression using a CAT ELISA Kit (Roche Molecular Biochemicals, Rotkreuz, Switzerland). CAT concentrations were normalized against β-galactosidase activities to compensate for different transfection efficiencies.
Isolation of the CXR gene
Chicken BAC filters (UK Human Genome Mapping Project Resource Center, UK) were hybridised with a probe encoding for CXR. Positive clones were purchased, digested with different restriction enzymes and Southern blots obtained using the same probe. Bands hybridising with the CXR probe were isolated, subcloned and CXR genomic information obtained by PCR using primers designed after the CXR mRNA sequence.
Mutagenesis was carried out using overlapping primers as described . Mutated fragments were excised, cloned into new vectors and verified by sequencing.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays have been described in detail . Proteins were expressed using the TNT in vitro transcription/translation kit (Promega, Wallisellen, Switzerland) before being subjected to non-denaturing SDS-polyacrylamide gel electrophoresis with [32P]-radiolabeled CYP2H1 264-bp PBRU.
Targeting of CXR in LMH cells by RNAi
Expression of CXR in LMH cells was repressed by RNAi as described . In brief, a 19 bp fragment ranging from position 857 to 875 in the open reading frame of CXR was chosen for targeting. A double-stranded oligonucleotide containing this sequence and compatible ends for cloning into pSUPER was obtained by annealing single stranded oligonucleotides for the sense (GATCCCCGGATGGGGCTCTGGCCGGCTTCAAGAGAGCCGGCCAGAGCCCCATCCTTTTTGGAAA) and the anti-sense strand (AGCTTTTCCAAAAAGGATGGGGCTCTGGCCGGCTCTCTTGAAGCCGGCCAGAGCCCCATCCGGG) and subsequent ligation into pSUPER cut with BglII and HindIII (underlined letters refer to CXR-specific targeting sequence). After verification of the ligation product the pSUPER-CXR-RNAi expression cassette was cut out using BamHI and XhoI and subcloned into BglII/XhoI-digested pcDNA3 (Invitrogen, Carlsbad, USA). The ScaI-linearised construct was transfected into LMH cells using FUGENE 6 (Roche Molecular Biochemicals, Rotkreuz, Switzerland). Stable transfectants were selected by addition of 175 μg/ml G418 (PAA Laboratories, Pasching, Austria) to the cell culture medium. A control cell line was selected in parallel which was stably transfected with pcDNA3 carrying the empty pSUPER expression cassette. Reporter gene assays in LMH cells using the CXR-RNAi clones were performed using reporter constructs for CYP2H1, CYP3A37, CYP2C45 and ALAS-1 described previously [17, 24–26].
Cellular localization studies
LMH cells were cultivated on glass cover slips and subsequently transfected with pEGFP-C1 or pEGFP-N1 expression plasmids (Clontech, Allschwil, Switzerland) before cells were either treated with vehicle, 400 μM PB or 0.1 μM okadaic acid for 16 hours. Cells were washed with PBS, fixed in 3% formaldehyde for 30 minutes, washed again with PBS, stained with 300 nM DAPI and subsequently mounted on glass slides. Digital images were captured using a Leica DC 300F camera (Leica, Nidau, Switzerland) mounted on a Leitz DMRB microscope with the Leica IM50 Image Manager program version 1.20. Figures were assembled with Adobe Photoshop version 5.0.
CXR antibodies, nuclear extracts and Western blots
CXR ligand-binding domain was expressed in bacteria, purified and injected into rabbits for antibody production according to standard procedures. Anti-CXR-ligand-binding domain antibody from rabbit serum was subsequently used in Western blots. LMH cells were grown under standard conditions and treated with vehicle or 400 μM PB overnight. Cells were subsequently washed with PBS and protein extracts prepared using RIPA buffer. As control, CV-1 cells were transfected with empty pSG5 expression vector or vector expressing CXR and subsequently lysed with RIPA buffer. Nuclear extracts were prepared as published .
LMH cells were treated with the indicated compounds for 16 hours before total RNA was isolated using the TRIZOL Reagent (Life Technologies, Basel, Switzerland). Twenty μg of total RNA were subjected to electrophoresis and analysed in Northern hybridisations as described [17, 33].
List of Abbreviations
pregnane X receptor
constitutive androstane receptor
chicken xenobiotic receptor
phenobarbital-responsive enhancer unit
liver X receptor
xenochemical response signal
green fluorescent protein
peroxisome-proliferator activated receptor
farnesoid X receptor.
This work was supported by the Swiss National Science Foundation.
The mRNA sequences for chicken estrogen-related receptor γ (ERRγ) and a partial fragment of ear2 have been submitted to Genbank under accession numbers AY702438 and AY702439, respectively.
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