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Department of Pathology and Committee on Clinical Pharmacology and Pharmacogenomics (M.D.K.), University of Chicago, Chicago, Illinois 60637; Department of Pharmaceutical Sciences (K.Y., E.G.S.), St. Jude Children’s Research Hospital, Memphis, Tennessee 38105; Zoological Society of San Diego (L.R.H.), San Diego, California 92112; and Department of Medicine (L.R.H.), University of California San Diego, San Diego, California 92093
Address all correspondence and requests for reprints to: Matthew D. Krasowski, M.D., Ph.D., University of Pittsburgh, Department of Pathology, 200 Lothrop, Pittsburgh, Pennsylvania 15213. E-mail: kra3{at}uchicago.edum or erin.schuetz{at}stjude.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PXR genes have been cloned and functionally characterized from a variety of vertebrate species, including zebrafish, frog, chicken, rat, mouse, rabbit, dog, pig, rhesus monkey, and human (1, 7, 9, 19, 20, 21). PXR has the typical NR organization of an N-terminal DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD); however, in comparing PXR sequences between vertebrate species, a striking feature is high cross-species sequence divergence in the LBD. The LBD of PXR shares amino acid identities of only 75% between human and rodent sequences and only 50% between human and chicken or fish sequences. These sequence identities are unusually low when compared with other NRs, for which the corresponding amino acid sequence identities tend to be at least 10–15% higher than the values above (7, 22).
The variation in the LBD of PXR is more striking when DNA sequences are analyzed, in particular by comparing the rate of nonsynonymous (i.e. changes amino acid sequence) and synonymous (does not change amino acid sequence) nucleotide substitution rates. When human and rodent sequences are compared, the LBD of PXR has nonsynonymous substitution rates (dN) 3.7 times higher than the average for all other NRs, even though the synonymous substitution rates (dS) for PXR are average for NRs. The ratios of dN/dS for the PXR are 4 times higher than the average for other NR genes (22). Elevated dN/dS (
) ratios suggest positive natural selection due to the accumulation of advantageous amino acid substitutions (23).
The high sequence divergence of the PXR LBD has been speculated to be an adaptation to xenobiotic ligands (7, 22), although the nature of such ligands is currently unknown. The xenobiotics currently known to activate PXR are all synthetic ligands or herbal products; dietary ligands for PXR have not yet been described. An alternative hypothesis is that cross-species variation in endogenous ligands has influenced PXR evolution (11). Of the endogenous ligands known to activate PXR, biliary bile salts vary the most across vertebrate species (Table 1
) (24, 25, 26). For example, most mammals produce predominantly 24-carbon (C24) bile acids such as cholic acid and chenodeoxycholic acid, conjugated to either glycine or taurine, from end metabolism of 27-carbon cholesterol. In contrast, cartilaginous fish and many early bony fish typically synthesize C27 bile alcohols, conjugated with sulfate, and, in fact, do not produce C24 bile acids (Table 1 and Figs. 1 and 2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The bile salts of many nonmammalian species differ from the typical bile salts found in mammals and are generally commercially unavailable. To allow for the study of these compounds, the following chemically diverse bile salts were isolated from animals by extraction and Flash column chromatography (see Table 2 for chemical formulae): myxinol disulfate from the Atlantic hagfish (Myxine glutinosa); 5
-cyprinol sulfate from the Asiatic carp (Cyprinus carpio); 5ß-scymnol sulfate from the Spotted eagle ray (Aetobatus narinari); and 3,7,12-trihydroxy-5ß-cholestan-27-oic acid, taurine conjugated, from the American alligator (Alligator mississippiensis). The isolation and purification of cyprinol and cyprinol sulfate have been previously described in detail (27). Bile alcohol sulfates were chemically deconjugated. Completeness of deconjugation and assessment of purity were performed by thin-layer chromatography using known standards.
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, A and C, shows that, as previously described (7, 8, 28), the mammalian bile acid lithocholic acid activated human PXR and VDR in the micromolar range. This activation had low efficacy relative to that produced by rifampicin and 1,25-(OH)2-vitamin D3, respectively (see Supplemental Table Ia, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org, for maximal activators used for each receptor). Lithocholic acid did not activate zebrafish PXR (Fig. 3B). In contrast, the major digestive detergent of zebrafish bile, 5-cyprinol sulfate, was a robust activator of human and zebrafish PXRs, but not human VDR (Fig. 3). None of the three receptors were affected by unconjugated cyprinol, a poorly water-soluble compound that is present in fish mainly as a precursor to the secreted cyprinol sulfate (27). Both lithocholic acid and cyprinol sulfate also activated a GAL4-LBD fusion construct for human PXR. The EC50 values for activation of this fusion construct were similar to those determined for the full-length human PXR; the efficacies of lithocholic acid and cyprinol sulfate relative to rifampicin were higher than seen for the full-length receptor although in either case cyprinol sulfate was more efficacious than lithocholic acid as a human PXR activator (Table 2
). These results indicate that the activation of the human PXR by cyprinol sulfate is a function of the LBD and not of another region of the receptor.
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and 4 and Table 2). Zebrafish PXR was only activated by four of 47 bile salts tested. In addition to cyprinol sulfate, zebrafish PXR was also activated by 5ß-scymnol sulfate (a C27 hexa-hydroxylated bile alcohol sulfate derived from the Spotted eagle ray, a cartilaginous fish) and lithocholic acid 3-sulfate, indicating a preference for sulfated bile salts; zebrafish PXR was also activated weakly by the synthetic bile acid derivative lithocholic acid acetate. None of the unconjugated bile alcohols, glycine-conjugated bile acids, or taurine-conjugated bile acids tested activated zebrafish PXR (Figs. 3B and 4 and Table 2). Human and mouse VDRs were activated by a narrow range of bile acids, mainly lithocholic acid and its metabolites or synthetic derivatives, and not by any of the C27 bile acids, sulfated bile alcohols, or unconjugated bile alcohols (Figs. 3C and 4 and Table 2). Mouse VDR was not activated by specific rodent primary or secondary biliary bile acids including -muricholic acid, ß-muricholic acid (unconjugated and taurine conjugated), -muricholic acid, and murideoxycholic acid (Table 2).
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Although the lamprey VDR was activated well by 1,25-(OH)2-vitamin D3, no other compounds tested were able to activate this receptor to more than 10% of the maximal response achieved with 300 nM 1,25-(OH)2-vitamin D3. Unlike the mouse and human VDRs, recent bile acids, particularly lithocholic acid, did not activate the lamprey VDR (supplemental Table Ib). Several early bile salts, such as petromyzonol sulfate, 3-ketopetromyzonol sulfate, and cyprinol sulfate, produced weak but concentration-dependent and reproducible increases in reporter activity; however, for all three compounds, the maximal effects were only less than 5–6% of the maximal effect seen with 300 nM 1,25-(OH)2-vitamin D3. The EC50 value for activation of the lamprey VDR by petromyzonol sulfate and 3-ketopetromyzonol sulfate was approximately 1 µM whereas that for cyprinol sulfate was approximately 20 µM (supplemental Table Ib). The small effects produced by these bile alcohol sulfates require correlation with in vivo findings in sea lampreys.
Activation of Other Vertebrate PXRs by Bile Salts
Rats and mice have a similar bile acid profile although they use slightly different primary bile acids (- and ß-muricholic acid, respectively; Table 1 and Fig. 2). PXRs from both rodents were activated by micromolar concentrations of murideoxycholic acid (the secondary bile acid metabolite formed by bacterial 7-dehydroxylation of - or ß-muricholic acids), lithocholic acid, and -muricholic acid (Table 3 and Fig. 4).
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and Fig. 4).
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and Fig. 2). The chicken PXR was activated by chenodeoxycholic acid, glycochenodeoxycholic acid, cholic acid, and lithocholic acid in the micromolar range (Table 4 and Fig. 4).
Interestingly, chicken, mouse, rabbit, rat, and human PXRs were all activated by 5-cyprinol sulfate and 5ß-scymnol sulfate, bile alcohol sulfates derived from carp and skate bile, respectively (Figs. 3A and 4 and Tables 2–4). Because these or similar bile salts are not the major bile salts of chickens, rabbits, mice, rats, or humans (although they can be found in trace amounts in healthy animals or in higher levels in certain disease states; see Discussion), activation by these compounds likely represents an ancestral property conserved in chicken and mammalian PXRs.
Benzoate X Receptors (BXR and BXRß) Are Insensitive to All Bile Salts
Bile from Xenopus laevis frogs and some other amphibians contain a mixture of C27 bile alcohols and bile acids (Hagey, L. R., unpublished data) (24, 25, 26). In this study, we tested a variety of bile alcohol sulfates, unconjugated bile alcohols, and bile salts, including myxinol disulfate (from hagfish), cyprinol sulfate (from carp), scymnol sulfate (from skate), petromyzonol sulfate (from sea lamprey), and taurine-conjugated 3,7,12-trihydroxy-5ß-cholestan-27-oic acid (from alligator; same as the C27 bile acid found in X. laevis), on BXR and BXRß. None of these compounds activated BXR or BXRß (supplemental Table Ic and Fig. 4), suggesting that these receptors do not play a role in sensing bile salts. As previously reported (21), benzoate compounds robustly activated these two receptors (supplemental Table Ic).
The effects of five benzoate compounds on zebrafish, chicken, and mammalian PXRs have been previously reported (7). Interestingly, two of the benzoate compounds (n-butyl-p-aminobenzoate and n-propyl-p-hydroxybenzoate) tested by Moore et al. (7) activated a number of the PXRs. The endogenous benzoate isolated by Blumberg et al. (21) from X. laevis embryos, 3-aminoethylbenzoate, however, only activated chicken PXR in addition to BXR (7). We confirmed the sensitivity of human PXR to n-butyl-p- aminobenzoate and also show activation of zebrafish PXR by n-propyl-p-hydroxybenzoate (Table 2). In contrast, n-butyl-p-aminobenzoate, n-propyl-p-aminobenzoate, and 3-aminoethylbenzoate all did not activate human, mouse, or lamprey VDRs (Tables 2 and 4 and supplemental Table Ib).
Activation of PXRs by the Bile Salt Precursor 5ß-Cholestan-3,7,12-triol
The bile salt biosynthetic pathways for all of the vertebrates considered in this study except zebrafish include 5ß-bile alcohol precursors (zebrafish use an early 5-bile alcohol pathway). One of these precursors is 5ß-cholestan-3,7,12-triol, a compound that accumulates in the rare disease cerebrotendinous xanthomatosis (OMIM accession no. 213700), an inborn error of metabolism caused by deficiency of CYP27A1. In humans, CYP27A1 deficiency leads to pathological accumulations of C27 bile alcohols and the hallmark symptoms of cerebrotendinous xanthomatosis, namely xanthomas, gallstones, and neurological dysfunction (31).
Tables 2–4 show that whereas human and zebrafish PXRs were not activated by 5ß-cholestan-3,7,12-triol, mouse, rabbit, rat, and chicken PXRs were all activated by low micromolar concentrations of this bile alcohol. Rat and mouse PXRs were activated particularly efficaciously by this bile alcohol (Table 3), as previously described for the mouse PXR (3, 4). BXR and BXRß were not activated by 5ß-cholestan-3,7,12-triol (supplemental Table Ic). The lack of activation of zebrafish PXR by 5ß-cholestan-3,7,12-triol may be a result of these fish utilizing the earlier 5-bile alcohol biosynthetic pathway also found in jawless fish; as a result, 5ß-cholestan-3,7,12-triol is not an intermediate in the synthesis of 5-cyprinol sulfate, the major zebrafish bile salt (27). None of the PXRs or VDRs were activated by 7-hydroxycholesterol, the earliest precursor in the biotransformation of cholesterol to bile salts (Tables 2–4).
The lack of activation of human PXR by the bile salt precursor 5ß-cholestan-3,7,12-triol detected in the transactivation assay is consistent with previous data by Dussault et al. (3) showing that 5ß-cholestan-3,7,12-triol is a very low efficacy partial agonist of human PXR. In that report, 5ß-cholestan-3,7,12-triol weakly transactivated a human PXR construct but was unable to induce CYP3A4 mRNA expression in primary human hepatocytes; 5ß-cholestan-3,7,12-triol did, however, competitively displace [3H]SR12813 from binding to human PXR, demonstrating that this bile alcohol does bind to human PXR (3). The significance of the lack of efficacy for 5ß-cholestan-3,7,12-triol for activating human PXR is uncertain, although it may be of interest that studies with primary human hepatocytes show that this bile alcohol precursor is practically nonexistent in the formation of bile acids such as cholic or chenodeoxycholic acid, a finding that contrasts with the previously assumed pathways of bile acid biosynthesis in humans (32). Comparative studies in other species have not been performed to study whether this finding is unique to humans or common throughout mammals.
Evidence for Nonneutral Evolution of the PXR LBD
Previous phylogenetic investigations of PXR genes have only looked at two-sequence comparisons in calculating dN/dS () ratios: one study focused on human, mouse, and rat sequences (22) and the other on human, chimpanzee, and mouse sequences (33). Table 5 shows the results from phylogenetic analysis by maximum likelihood (PAML) analysis of sequence data for PXR and VDR genes. A total of six analyses were performed for each gene: full-length sequence, restricted to DBD, and restricted to LBD for datasets of either all available vertebrate species or mammals only.
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-ratios, consistent with purifying (negative) selection as the dominant evolutionary force for this gene. The PXR analyses, on the other hand, for the full-length sequence and LBD show a minority of codons (sites) with -ratios approaching or even exceeding 1 (results in bold in Table 5). In contrast, the -ratios for the DBD are low for the PXR analyses. These results are consistent with positive selection acting on the PXR LBD.
The analysis for the LBD of PXR restricted to mammalian species, in which 7% of codons were associated with an -ratio of 1.23, also highlighted four amino acid residues as likely targets of positive selection: 187S, 231S (H1–H3 insert), 312A (helix 6), and 360R (helix 9) [amino acid numbers for human PXR as in Moore et al., (7); complete sequence alignment of the LBD of VDRs and PXRs available as supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Posterior probabilities for the positive selection of the individual codon sites are greater than 0.99 for 360R and 0.90–0.98 for 187S, 231S, and 312A.
We also used PAML to detect whether evidence of molecular adaptation occurs along specific lineages. With regard to ligand specificity, the most divergent of the PXRs are clearly the BXRs (Fig. 5). If the assignment of BXR and BXRß as true orthologs of other PXRs is correct, then these receptors have lost the ability to respond to endogenous steroids and bile salts and also the broad specificity required to bind structurally diverse xenobiotics. Using a PAML model that allows for two discrete classes of for all sequences in a phylogeny and an additional class of for defined lineage(s), (model B of Ref. 34 ; see Materials and Methods for more details), we analyzed the entire set of available PXRs. For the full-length, DBD-only, and LBD-only sequences, we separately analyzed four specific sets of lineages for positive selection: the branch from all other PXRs to the BXRs (branch a of Fig. 5), the branch to BXR (branch b of Fig. 5), the branch to BXRß (branch c of Fig. 5), and branches a, b, c together.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PXR activation by bile salts parallels the evolution of complexity of the bile salt biosynthetic pathway. Unlike most forms of evolution, bile salt evolution has proceeded by elongation of an enzyme pathway. This long pathway is now shared by different hepatic organelles, with its start in the mitochondria, steroid ring modification in the endoplasmic reticulum and cytoplasm, and side-chain modifications in the peroxisomes (38). Jawless fish, cartilaginous fish, and some bony fish (e.g. zebrafish) utilize relatively simple bile alcohol pathways that generally leave the side chain of cholesterol intact. The insensitivity of zebrafish PXR to recent 5ß-bile acids is perhaps not surprising, as these fish would never encounter such compounds (nor would their ancestors). PXR activation by bile salts has thus expanded from narrow specificity for C27 bile alcohol sulfates (early fish pattern) to a broader specificity for recent bile acids and their synthetic precursors (birds and mammals). PXR selectivity for bile salts has therefore paralleled the elongation and increasing complexity of the bile salt synthetic pathway during vertebrate evolution.
The evolution of the bile salt biosynthesis pathway and its relation to PXR evolution contrasts with that for the classical steroid hormones and their cognate receptors. In a detailed analysis of the vertebrate NR3 family (estrogen, androgen, progesterone, glucocorticoid, and mineralocorticoid receptors), Thornton (39) has proposed that "... the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances." Thornton also concluded that the estrogen receptor was likely the most ancient classical steroid receptor (39), an idea supported by an elegant reconstruction and functional expression of the ancestral estrogen receptor (40). The bile salt biosynthetic pathway has evolved in a considerably different fashion from the adrenocortical and sex steroid pathways, starting off from a relatively simple pathway to produce C27 bile alcohol sulfates and steadily elongating to a more complex pathway that results in the production of C24 bile acids. Therefore, the evolution of PXR in mammals and birds has adapted not to the intermediate products of bile salt biosynthesis but to the species-specific, specialized, terminal products of a biosynthetic pathway elongating over evolutionary time.
Biliary bile salts show a striking amount of diversity between different vertebrate species (24, 25). This contrasts with hormones or neurotransmitters, which tend to be well conserved (41). The PXRs have adapted to cross-species variation in bile composition. The zebrafish PXR has narrow selectivity toward bile salts, being activated by only two sulfated bile alcohols, one sulfated bile acid, and one synthetic bile acid derivative out of a panel of 47 bile compounds tested. The most efficacious activation of zebrafish PXR was by its dominant bile detergent, 5-cyprinol sulfate. In contrast, mammalian PXRs, in particular human and rabbit PXRs, are activated efficaciously by a wide variety of bile acids (conjugated or unconjugated) and sulfated bile alcohols. Rat and mice PXRs are activated efficaciously by -muricholic acid as well as murideoxycholic acid, the secondary bile acid of rodent - and ß-muricholic acids. In contrast, murideoxycholic acid does not activate PXRs from humans or chickens, two species in which this bile acid would not be present. This suggests that PXRs have adapted to subtle species-specific differences in bile salt biosynthesis and metabolism.
An unexpected finding was that all PXRs except frog BXRs were activated by 5-cyprinol sulfate and 5ß-scymnol sulfate. The most ancient vertebrates, hagfish and lampreys (jawless fish), utilize 5-bile alcohol sulfates similar to cyprinol sulfate as their major bile detergents; these compounds are assumed to be the first class of bile detergents synthesized by ancestral vertebrates (42, 43). Consequently, activation of PXR by the earliest bile constituents, 5-bile alcohol sulfates, is an ancestral property conserved in comparatively recent fish (like zebrafish), birds, and mammals. This pattern of conservation is remarkable considering the extensive divergence between the LBDs of zebrafish and mammalian PXRs (only 50% amino acid sequence identity) and the more than 400 million years of time since the last common ancestor of tetrapods and telost fish (44).
Interestingly, even though C24 bile acids are the dominant digestive detergents of most mammals, low concentrations of 5ß-cyprinol or similar C27 bile alcohols are found in humans and other mammals (45, 46, 47). This is a reflection of the evolution of the bile salt biosynthetic pathway described above, in which the synthesis of a recent bile acid such as cholic acid results from extra processing and enzymatic steps added onto the basic pathway that produces C27 bile alcohols (38). As a result, all mammals produce some C27 bile alcohols, although the efficiency of conversion to C24 bile acids in healthy animals normally reduces bile alcohols to trace levels. Detailed analysis of urinary bile alcohols in healthy humans reveals a range of bile alcohols including 5ß-cyprinol; in patients with cholestasis and certain inborn errors of bile metabolism, levels of urinary bile alcohols can rise dramatically (46, 48). In addition, a small number of mammals including elephants and rock hyraxes produce C27 bile alcohol sulfates as their only bile detergents; in essence, these mammals have retained the basic bile detergent synthetic pathway found in ancestral fishes (24, 25). The persistence of C27 bile alcohols even in healthy animals and their elevation in certain disease states may provide some selective advantage for mammalian PXRs to maintain their ancestral sensitivity to early bile salts such as cyprinol sulfate and scymnol sulfate.
Physiological Importance of Bile Salt Activation of Vertebrate PXRs
Detection of bile salts by PXR may not simply be a mechanism to increase removal of bile salts but part of a more general protective mechanism. In fact, with a few exceptions (e.g. lithocholic acid), bile salts are not highly toxic compounds (49). However, elevated intrahepatic levels of bile acids are a sentinel marker of cholestasis and herald impaired biliary elimination of other toxic compounds. Activation of PXRs by abnormally high bile salt concentrations would be beneficial by inducing alternative routes of metabolism and elimination of toxins normally cleared in the bile.
Lithocholic acid activated chicken PXR and all mammalian PXRs. Numerous studies have demonstrated the toxicity of this mono-hydroxylated, secondary bile acid, generated by bacterial 7-dehydroxylation of primary bile acids such as chenodeoxycholic acid in the intestine (49). Lithocholic acid and its metabolites were also activators of mouse and human VDRs, as previously reported (28). For animals using C24 bile acids such as chenodeoxycholic acid, mechanisms to detect and reduce the toxicity of lithocholic acid would be advantageous. In the case of mammals, two related NRs, PXR and VDR, both detect lithocholic acid and activate pathways to detoxify this compound (28, 49). The studies with sea lamprey VDR show that activation by lithocholic acid and its derivatives is not a conserved property of all VDRs.
Whereas the physiological importance of PXR activation by bile acids in mammals has been demonstrated, the situation is less clear in nonmammalian species. The metabolism and toxicology of fish bile salts has been little studied, and the genes affected by PXR activation in fish have not been defined. 5-Cyprinol sulfate, the bile detergent of zebrafish and other cypriniform fish (e.g. carp, goldfish, zebrafish), is quite toxic to a number of mammals (27, 50), including humans, and can cause renal and hepatic failure in individuals who ingest raw goldfish or carp gallbladders (51), a delicacy in East Asia.
Relation of PXR Activation to Physiologically Relevant Bile Salt Concentrations
The concentrations of species-specific bile salts that activate the vertebrate PXRs in this study are within the range encountered in disease affecting the hepatobiliary system. In healthy humans, plasma concentrations of bile acids are generally low micromolar (see Materials and Methods). In subjects with cholestasis from a variety of etiologies, plasma bile acid concentrations can rise dramatically and exceed 100 µM, well within the levels that activate human PXR (10).
Plasma concentrations of bile acids in healthy chickens, mice, rats, and rabbits are similar to humans, with normal total plasma bile acid concentrations generally in the low micromolar range (see Materials and Methods). The concentrations required to activate PXRs in these species are generally about 1 order of magnitude or more higher than would be seen in healthy animals and would be encountered in animals with hepatobiliary disorders such as cholestasis. As in humans, diseases affecting the hepatobiliary systems can dramatically elevate plasma bile acid concentrations in other animals. Concentrations of plasma bile acids exceeding 1 mM have been achieved in experimental animals, such as in mice following bile duct ligation (52). Unfortunately, at the present time, intrahepatocyte or plasma concentrations of bile salts, or their degree of binding to plasma proteins, have not been reported for fish or amphibians.
Lack of Activation of Frog BXRs by Bile Salts
BXR and BXRß, the putative PXRs from X. laevis, are clearly different from other vertebrate PXRs. Although the complete genome of X. laevis has not yet been sequenced (leaving open the possibility of discovering additional PXR-like genes), sequence similarity and phylogeny strongly support the classification of these receptors as PXRs (7, 37, 53). Nevertheless, the tissue distribution and functional properties of these receptors are quite different from other PXRs. For example, BXRs are highly expressed in ovary and testis but poorly expressed in xenobiotic metabolizing organs such as liver, lung, or kidney (37). BXR and BXRß do not appear to function as xenobiotic sensors, and previous reports have shown these receptors to be insensitive to a wide range of steroidal compounds (7, 21, 37). The present study finds these two receptors to be completely insensitive to amphibian bile salts. BXR and BXRß are both activated well by benzoates, compounds that have unique roles in frog development (21). Assuming that these two receptors are indeed orthologous to other PXRs, they have evidently diverged extensively to perform specific benzoate-mediated developmental functions in frogs (21, 37).
In this report, we confirmed the findings of Moore et al. (7) that certain benzoate compounds activate a variety of PXRs other than the BXRs, including those from zebrafish, chicken, and human, and also show that activation by benzoates is not shared by human, mouse, or lamprey VDRs. The significance of activation of PXRs other than BXR and BXRß by synthetic benzoates is unknown; physiological actions of this class of endogenous compounds has only been described in amphibians (21, 37). Even if benzoates do not have a physiological role in modern-day fish, birds, or mammals, it is possible that ancestors to these species may have used benzoate compounds and, thus, that sensitivity to these compounds is a shared ancestral trait of PXRs. Alternatively, low-affinity activation of nonamphibian PXRs by benzoates, compounds with low molecular weight relative to other PXR ligands, may simply be a consequence of the broad specificity of PXRs and may not have physiological or evolutionary significance. BXRs have not been cloned and characterized from any species other than X. laevis (21, 37). Whether receptors similar to the Xenopus BXRs exist in other amphibians (e.g. the salamander axolotl), reptiles, or any other species remains to be discovered. Complete sequencing of the Xenopus genome and of other species will be critical in determining whether BXRs are indeed orthologous to other PXRs and also whether amphibians have PXR-like xenobiotic receptors that have not yet been identified.
Evolution of the NR1I Subfamily of Nuclear Hormone Receptors
The NR1I subfamily includes VDR (NR1I1), PXR (NR1I2), and the constitutive androstane receptor (CAR; NR1I3) (6, 7, 54). VDR and PXR are found in diverse vertebrates from bony fish to mammals. CAR, on the other hand, appears restricted to mammals, although there is some uncertainty in classifying the chicken PXR, as this receptor has about equal sequence and functional similarity to both PXRs and CARs (7, 20). Recent evidence suggests that the CAR gene arose from a duplication of a premammalian PXR gene (55). This is consistent with the demonstration of two xenobiotic-responsive NRs in mammals, but only one in chickens and fish (53, 55). The complete genome of the fugu fish contains a single PXR gene and shows no evidence for a CAR gene (53).
Based on current genetic data, distinct NR1I subfamily members have been found only in vertebrates. A VDR was cloned and functionally expressed from the sea lamprey (P. marinus), a surprising finding as this jawless vertebrate lacks calcified tissue (29). PXR has not yet been identified in jawless vertebrates, and our attempts to clone a PXR gene from the sea lamprey have been unsuccessful. The draft genome of the urochordate Ciona intestinalis, the closest invertebrate relative of vertebrates for which complete genome information is available, revealed a single NR gene equally related to VDR and a Drosophila NR (56). The functional properties of this invertebrate NR remain uncharacterized.
An excellent study of the evolutionary genomics of the NR superfamily provides convincing evidence that a single ancestral NR duplicated and diverged to form VDR and PXR. This paper also proposes that the NR complement in the ancestor to Bilateria (animals) possessed nine genes in the NR1 family (57). Thus, distinct PXR and VDR genes are likely vertebrate innovations although complete genome information is not yet available for a cephalochordate such as amphioxus, an invertebrate chordate more closely related to vertebrates than Ciona.
Figure 5 shows a hypothetical phylogenetic tree of the NR1I subfamily that incorporates functional characteristics of the subfamily members. Pharmacological features shown are activation by endogenous compounds [bile acids, bile alcohol sulfates, steroid hormones, benzoates, and 1,25-(OH)2-vitamin D3] and xenobiotics (pregnenolone 16-carbonitrile, rifampicin). Figure 5 shows the progressive broadening of ligand specificity for the PXRs, with human, dog, and pig PXRs having the broadest ligand specificity, at least for the ligands that have been tested to date (7).
Several reports have suggested that the most ancient function of the NR1I family is to recognize toxic levels of exogenous and/or endogenous compounds (7, 11, 28, 29). The detoxification function of VDR in humans has only been recently recognized and is exemplified by the ability of VDR to sense toxic levels of lithocholic acid in the intestine and up-regulate metabolic enzymes to detoxify this secondary bile acid (17, 28). A further description of the NR1I subfamily origins awaits complete genome information in jawless fish and cephalochordates and functional characterization of the NR1I subfamily members in these organisms, if present.
Structural Basis for Differing Ligand Selectivities between the PXRs
The mechanism of ligand activation of human PXR has been demonstrated by high-resolution, crystallographic, three-dimensional structures of the LBD alone (35) and in combination with the hypocholesterolemic compound SR12813 (35, 58), hyperforin (active component of St. John’s wort) (36), or the human steroid receptor coactivator-1 (SRC-1) (58). The human PXR LBD shares structural features with other NR LBDs, including an -helical sandwich of helices 1/3, 4/5/8, and 7/10 in one hemisphere of the molecule and the ligand-binding cavity in the other hemisphere (35, 59). The human PXR ligand-binding cavity is smooth, hydrophobic, and large (1150 Å3), second in size for NRs only to peroxisome proliferator-activated receptor-
(1500 Å3) (60). This is in contrast to most NRs, which have a compact ligand-binding cavity that closely resembles the shape of their specific ligand(s) (59). There is also considerable flexibility in the human PXR ligand-binding cavity, which increases in size by 250 Å3 following binding of hyperforin (36). There are a number of additional features of the human PXR LBD that contribute to its broad ligand specificity: a variable four-residue turn between helices 1 and 3, replacement of 6 by a large, flexible loop, and two additional ß-strands not observed in other NRs (35, 36, 58, 59).
A total of 18 amino acid residues in the human PXR LBD have been identified as contacting either SR12813, hyperforin, or both (see supplemental Fig. 1 for sequence alignments and highlighting of ligand-binding residues); one residue contacts hyperforin only and five residues contact SR12813 only (35, 36). No three-dimensional structures of human PXR bound to an endogenous ligand have been reported. The generally low affinity of these compounds may make this difficult to achieve. In comparing human and zebrafish PXR sequences, 12 of the 18 amino acid residues identified in binding SR12813 and/or hyperforin differ between the two sequences, in some cases dramatically (supplemental Fig. 1, A and B). In fact, alignment of human PXR, chicken PXR, BXR, BXRß, and zebrafish PXR shows that only three of these 18 ligand-contacting amino acid residues are completely conserved among these five sequences (supplemental Fig. 1B). A combination of molecular modeling and site-directed mutagenesis will probably be required to elucidate the structural basis of ligand selectivity differences between these PXRs.
Contrasting Levels of PXR Nucleotide Diversity within Humans and between Vertebrates
The marked diversity of the PXR LBD between vertebrate species contrasts with a detailed resequencing study of the human PXR gene that showed very low nucleotide diversity and no nonsynonymous substitutions in the PXR LBD in genomic DNA of approximately 100 individuals from diverse ethnic populations. The total nucleotide diversity was 1 in 21,607 bp for the LBD and 1 in 22,053 for the DBD (61). This low level of nucleotide diversity within humans differs markedly with the high levels of divergence of PXR genes between vertebrate species. This suggests that important ligand(s) for PXR do not vary significantly across human populations but do vary across vertebrate species. We propose biliary bile salts as a group of endogenous ligands that fit this qualification.
In summary, this study shows that activation by species-specific bile salts is a conserved property of PXRs from teleost fish to mammals and that recognizing high concentrations of bile salts is likely an ancestral function of PXRs. More strikingly, the specificity of PXRs to bile salts has expanded during vertebrate evolution, mirroring the complex evolution of bile salt biosynthetic pathways across vertebrate species. Given evidence for positive selection acting on the PXR LBD, we propose that differences in biliary bile salts between vertebrate species have been a selective force for PXR evolution.
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for chemical formulae) were: myxinol disulfate from the Atlantic hagfish (M. glutinosa); 5-cyprinol sulfate from the Asiatic carp (C. carpio); 5ß-scymnol sulfate from the Spotted eagle ray (A. narinari); and 3,7,12-trihydroxy-5ß-cholestan-27-oic acid, taurine conjugated, from the American alligator (A. mississippiensis). The isolation and purification of cyprinol and cyprinol sulfate have been previously described in detail (27). Bile alcohol sulfates were deconjugated using a solution of 2,2-dimethoxypropane-1.0 N HCl, 7:1 (vol/vol), and incubating 2 h at 37 C, followed by the addition of water and extraction into ether. Completeness of deconjugation and assessment of purity was performed by thin-layer chromatography using known standards.
Reagents
The sources of the chemicals were as follows: rifampicin, n-propyl-4-hydroxybenzoate, n-butyl-4-aminobenzoate, pregnenolone 16-carbonitrile, nifedipine, 1,25-(OH)2-vitamin D3, glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid 3-sulfate, taurolithocholic acid 3-sulfate, and glycolithocholic acid 3-sulfate (Sigma Chemical Co., St. Louis, MO); 5-petromyzonol, petromyzonol sulfate, 3-ketopetromyzonol, 3-ketopetromyzonol sulfate, 3-keto-7,12-dihydro-5-cholanic acid, and allocholic acid (Toronto Research Chemicals, Inc., North York, Ontario, Canada). All other bile salts, as well as 5-androstan-3-ol and 5ß-pregnane-3,20-dione, were obtained from Steraloids (Newport, RI).
Construction of HepG2 Cell Line Stably Expressing Human Na+-Taurocholate Transporter (hNTCP)
HepG2 (human liver) cell lines stably expressing hNTCP (62), which is required for effective uptake of conjugated bile compounds into cells, were used for the transactivation experiments. To construct this line, hNTCP cDNA was subcloned into pcDNA3. HepG2 cells (104 cells per P60 dish, plated on d 1) were transfected on d 2 by calcium phosphate coprecipitation with 5 µg of pcDNA3-hNTCP plasmid. HepG2 cells stably transfected with pcDNA3-hNTCP were grown in MEM- medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and 1% penicillin/streptomycin. The HepG2-hNTCP cells were grown under selective pressure with 1000 µg/ml G418. The cell lines were grown at 37 C in 5% CO2.
To determine the hNTCP expression, RNA was isolated from HepG2-hNTCP cells using Trizol reagent (Invitrogen). Total RNA (5 µg) was reverse transcribed according to instructions. Primers used to amplify were 5'-gggacatgaacctcagcatt-3' (forward) and 5'-cgtttggatttgaggacgat-3' (reverse). PCRs contained 1 µl of cDNA, 5 pmol of primers, 1.5 mmol MgCl2, 0.2 mmol deoxynucleotide triphosphates, and 2.5 U of Taq polymerase (Promega Corp., Madison, WI). PCR consisted of an initial denaturation at 92 C for 5 min followed by 34 cycles of denaturation at 92 C for 30 sec, annealing at 60 C for 30 sec, and synthesis at 72 C for 30 sec, with a final extension at 72 C for 10 min.
Transactivation Assays and Cell Culture
Plasmids containing cDNAs for eight PXRs from seven species [human, mouse, rat, rabbit, chicken (chicken PxR), frog (BXR and BXRß), zebrafish], human vitamin D receptor (VDR; NR1I1), human Na+-taurocholate cotransporter (NTCP; SLC10A1), human organic anion transporting polypeptide (SLC21), as well as the reporter constructs tk-UAS-Luc and CYP3A4-PXRE-Luc, and empty vectors pCDNA, pSG5, and PM2 were generously provided by S. A. Kliewer, J. T. Moore, and L. B. Moore (GlaxoSmithKline, Research Triangle Park, NC). The sea lamprey VDR clone (the insertless full-length cDNA) was generously provided by G. K. Whitfield (University of Arizona College of Medicine, Tucson, AZ) (29). Mouse VDR (IMAGE clone 3710866) and pCMV-sport6 vectors were obtained from Invitrogen. The zebrafish RXRß cDNA clone (IMAGE clone 5410111) was obtained from ATCC (Manassas, VA). The expression vectors were either full-length receptors (i.e. containing both a DBD and LBD; human, mouse, rat, rabbit, and chicken PXRs, as well as human, mouse, and sea lamprey VDRs) or GAL4/PXR chimeras that contain only the LBD of the PXR (human SXR, BXR, BXRß, and zebrafish PXR) (7). For the full-length expression vectors, the reporter plasmid was CYP3A4-PXRE-Luc, a construct that contains a promoter element from CYP3A4 (recognized by PXR and VDR DBDs) driving luciferase expression. For the GAL4/LBD expression constructs, the reporter plasmid was tk-UAS-Luc, which contains GAL4 DNA binding elements driving luciferase expression. The sea lamprey VDR cDNA was cotransfected with zebrafish RXRß (15 ng/well) for more robust expression.
It should be pointed out that cross-species differences in the DBDs of various PXRs could impact the ability of a particular PXR to activate the human CYP3A4-based promoter driving luciferase expression. However, this is unlikely to affect the pharmacology of the various ligands studied in this report, particularly as ligand activation of a particular receptor was normalized to a specific maximal activator. The most distantly related PXRs to the human PXR, zebrafish PXR and the frog BXRs, were studied using GAL4-LBD fusion constructs; therefore, issues of cross-species differences in the DBD do not affect those receptors in this study. Also, the transactivation assays used will not detect compounds that bind to PXRs but do not transactivate (or transactivate very poorly). Other than for human PXR, suitable radioligands for other PXRs have not yet been developed.
The cell line used for the assays was HepG2 (human liver) cells stably expressing human NTCP, a transporter that can uptake conjugated bile acids. For experiments involving sulfated compounds, human organic anion transporting polypeptide was cotransfected at 10 ng/well to facilitate uptake of sulfated steroids or bile salts. On d 1, 30,000 cells per well were seeded onto 96-well white opaque plates (Corning-Costar, Corning, NY). On d 2, cells were transfected using the calcium phosphate precipitation method with expression vector or empty control vector and luciferase reporter plasmid. On d 3, the cells were washed and then incubated with medium containing charcoal-dextran-treated fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) and drugs or vehicle. On d 4, the cells were washed and the medium replaced with serum-free medium. Luciferase reagent with passive lysis buffer (Steady-Glo, Promega Corp.) was added. Two hours later, luminescence was measured in a 96-well luminometer (Analyst AD, Molecular Devices, Sunnyvale, CA). An aliquot of lysed cells was taken to measure total protein content (RC DC Protein Assay; Bio-Rad Laboratories, Inc., Hercules, CA). Experiments were performed in quadruplicate and repeated for a total of at least three times.
Activation of receptor by ligand was compared with receptor exposed to identical conditions without ligand (vehicle control). In general, dimethyl sulfoxide (Sigma) was used as vehicle and was adjusted to be 1% (vol/vol) in all wells. A control was also run with transfection of empty vector (i.e. lacking the receptor cDNA) and reporter vector to control for activation of reporter vector by endogenous receptor(s). In experiments with a variety of activators, activation by endogenous receptors was not seen. Concentration-response curves were fitted using Kaleidagraph software (Synergy Software, Reading, PA). Data are presented throughout as mean ± SEM. In combining data from multiple experiments, the pooled variance was calculated by the formula spooled = {[(n1 – 1)s12 + (n2 – 1)s22 + ... + (nk – 1)sk2]/[N – k]}–1/2, where there are N total data points among k groups, with n replicates in the ith group.
Determination of Maximal Activators for the Various PXRs
Using previous comparative studies of vertebrate PXRs as a guide (6, 7), each PXR construct was tested with compounds previously shown to be robust activators of the respective receptors. Supplemental Table Ia shows the receptors tested, the type of reporter construct used, the amount of reporter construct and receptor cDNA transfected per well, and the activation of luciferase activity, relative to vehicle control, produced by a maximal activator. As can be seen, maximal induction of luciferase activity varies over almost 2 orders of magnitude. This is a result of differing levels of baseline (constitutive) activation and intrinsic differences in receptor properties. Mouse and chicken PXRs show high levels of constitutive activation and low maximal activation by ligand; in contrast, human VDR, mouse VDR, BXR, BXRß, and zebrafish PXR have low levels of constitutive activity and show greater than 10-fold maximal activation (6, 7, 20, 21, 37). This variability in activation makes it difficult to perform cross-species comparisons of differences in ligand efficacy.
To facilitate more reliable cross-species comparisons, complete concentration-response curves for ligands were determined in the same microplate as determination of response to a maximal activator. This allows for determination of relative efficacy, 
, defined as the maximal response to test ligand divided by the maximal response to a reference maximal activator (note than can exceed 1). Compounds with less than 0.10 were designated "inactive." The maximal activators and concentrations eliciting maximal response are listed in supplemental Table Ia. Figure 3 shows concentration-response curves for human PXR, zebrafish PXR, and human VDR. The ordinate represents activation of the PXR or VDR, relative to vehicle control, and normalized to the maximal activator. For the human PXR data in Fig. 3A, rifampicin is the reference maximal activator (and hence has = 1.0 by definition) whereas lithocholic acid has lower relative efficacy (
= 0.15). Similarly, 5-androstan-3-ol and 1,25-(OH)2-vitamin D3 are the reference maximal compounds for zebrafish PXR and human VDR in Fig. 3, B and C, respectively.
Plasma or Serum Concentrations of Bile Salts in Vertebrates
Circulating total serum concentrations of deoxycholic acid, chenodeoxycholic acid, and cholic acid in humans are 0.23–0.89 µM, 0–1.61 µM, and 0.08–0.91 µM, respectively (63). Total serum bile acids exceed 100 µM in patients with severe cholestasis (10). Bile acids are bound tightly to plasma proteins (70–95%); therefore, the corresponding free plasma bile acid concentrations will be lower than the values above (64, 65, 66).
Total bile acid concentrations in Sprague Dawley rat plasma are 8.26 ± 6.13 nmol/100 g body weight (67). With rats weighing approximately 400 g and assuming an estimated plasma volume of 5 ml/100 g body weight (68), this corresponds to 0.413 ± 0.31 µM total bile acid concentration in plasma. Concentration of total bile acids in mouse plasma is slightly higher than that in rats (52). Similarly, total bile acid concentrations in New Zealand rabbit plasma were 1.08 ± 0.44 nmol/100 g body weight (67). With rabbits weighing approximately 2000 g and assuming an estimated plasma volume of 20 ml/100 g body weight (69), this corresponds to 0.0027 ± 0.0011 µM. Total bile acid concentrations in hens are 0.075–0.15 µM (70). Concentrations of bile salts in fish or amphibian plasma or serum have not been reported.
Phylogenetic Analysis
Sequences were aligned with Clustal X. Estimation of dN/dS () ratios was carried out by maximum likelihood using a codon-based substitution model in PAML (Phylogenetic Analysis by Maximum Likelihood) version 3.13 (71). The input to PAML is a tree file of the phylogeny of the sequences to be studied and a file with aligned sequences. The phylogeny is based on the known phylogenetic relationships between the species to be studied, determined by a consensus of morphological and molecular data (44).
PAML can determine estimates of for models of varying complexity. The most commonly applied models are as follows (the PAML model numbers are shown in parentheses; the "sites" refers to codons) (72, 73): model M0 (null model with a single -ratio among all sites), M3 (discrete model, with two or more categories of sites with the -ratio free to vary for each site), M7 (ß-model, 10 categories of sites, with 10 -ratios in the range 0–1 taken from a discrete approximation of the ß- distribution), and M8 (ß + model, 10 categories of sites from a ß- distribution as in M7 plus an additional category of sites with an -ratio that is free to vary from 0 to >1). PAML estimates the -ratios that are allowed to vary under these models, as well as the proportion of sites (codons) with each ratio. We also used a combined branch-site model (model B of Ref. 34) in PAML to test for positive selection in specific lineages of the phylogeny. This model allows for a branch or branches of a phylogeny to be selected as foreground and the remaining branches as background. Model B is compared statistically to the simpler model of M3 with two site classes and no selection of specific lineages.
Of the PAML models listed above, only M3 and M8 can detect positive selection (i.e. > 1). Each PAML model generates a log likelihood, indicating how well the models fit the input data. Some PAML models are nested within each other (e.g. M0 within M3, M7 within M8). In those cases, twice the log likelihood difference between the two models is compared with a 
2 distribution with degrees of freedom equal to the difference in degrees of freedom between the two models. P values for sites potentially under positive selection are obtained using a Bayesian approach in PAML (34). The accuracy and power of PAML models increase with more sequences and longer length sequences (<
), lithocholic acid (LCA, 
), androstanol (
), 5
), 5
), and 1,25-(OH)2-vitamin D3 (
). A, Human PXR is activated efficaciously by rifampicin, androstanol, and 5
