Benzoate X receptors alpha and beta are pharmacologically distinct and do not function as xenobiotic receptors.

Abstract

The Xenopus benzoate nuclear hormone receptors, BXRalpha and BXRbeta, share 82% identity within their ligand-binding domains and are classified as members of the NR1I2 subfamily that includes the mammalian steroid and xenobiotic receptor, SXR/PXR. Although alkyl benzoates have been identified as endogenous ligands, the exact role of the benzoate receptors in amphibian physiology has not been established. In this report, we show that BXRalpha and BXRbeta are pharmacologically distinct from each other: BXRalpha is more promiscuous than BXRbeta with respect to both ligand specificity and co-activator recruitment. BXRalpha can be transactivated by a number of benzoate derivatives including 4-amino-butylbenzoate (4-ABB), 4-hydroxy-butylbenzoate (4-HBB), 3-hydroxy ethyl benzoate (3-HEB), and benzyl benzoate, but only 4-HBB acts as an agonist for both receptors. Furthermore, BXRalpha-specific agonists such as 4-ABB, chlorpyrifos, and trifluralin act as antagonists on BXRbeta. BXRs are widely distributed in adult tissues but do not show any enrichment in liver and intestine, major sites of SXR/PXR expression that are critical in xenobiotic metabolism. Neither BXR shows the broad specificity toward steroids or xenobiotics exhibited by SXR/PXR. Therefore, we conclude that the BXRs are pharmacologically distinct from each other and unlikely to serve as xenobiotic sensors.

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Benzoate X Receptors ? and ? Are Pharmacologically Distinct and
Do Not Function as Xenobiotic Receptors*
Received for publication, July 2, 2002, and in revised form, August 26, 2002
Published, JBC Papers in Press, August 26, 2002, DOI 10.1074/jbc.M206553200
Felix Gru ¨n, Ranga N. Venkatesan‡, Michelle M. Tabb, Changcheng Zhou, Junran Cao,
Daniel Hemmati, and Bruce Blumberg§
From the Department of Developmental and Cell Biology, University of California, Irvine, California 92697-2300
The Xenopus benzoate nuclear hormone receptors,
BXR? and BXR?, share 82% identity within their ligand-
binding domains and are classified as members of the
NR1I2 subfamily that includes the mammalian steroid
and xenobiotic receptor, SXR/PXR. Although alkyl ben-
zoates have been identified as endogenous ligands, the
exact role of the benzoate receptors in amphibian phys-
iology has not been established. In this report, we show
that BXR? and BXR? are pharmacologically distinct
from each other: BXR? is more promiscuous than BXR?
with respect to both ligand specificity and co-activator
recruitment. BXR? can be transactivated by a number of
benzoate derivatives including 4-amino-butylbenzoate
(4-ABB), 4-hydroxy-butylbenzoate (4-HBB), 3-hydroxy
ethyl benzoate (3-HEB), and benzyl benzoate, but only
4-HBB acts as an agonist for both receptors. Further-
more, BXR?-specific agonists such as 4-ABB, chlorpyri-
fos, and trifluralin act as antagonists on BXR?. BXRs are
widely distributed in adult tissues but do not show any
enrichment in liver and intestine, major sites of SXR/
PXR expression that are critical in xenobiotic metabo-
lism. Neither BXR shows the broad specificity toward
steroids or xenobiotics exhibited by SXR/PXR. There-
fore, we conclude that the BXRs are pharmacologically
distinct from each other and unlikely to serve as xeno-
biotic sensors.
Nuclear receptors are ligand-modulated transcription factors
that respond to steroids, retinoids, and thyroid hormones to
control development and body physiology. Orphan nuclear re-
ceptors possess apparent DNA and ligand-binding domains but
lack identified ligands (1–3). Each orphan has the potential to
regulate a distinct signaling pathway. The promise of orphan
receptors is that the identification of novel and perhaps unsus-
pected classes of ligands may offer insight into potentially new
principles of development and physiology. In recent years, a
number of orphan receptors have been adopted or matched
with physiological ligands (3, 4). Consequently, new insights
into cholesterol and bile acid metabolism and transport (4)
have been gained.
Previously, we identified a Xenopus orphan nuclear receptor
that represented a distinct branch of the nuclear receptor su-
perfamily and named it benzoate “X” receptor (BXR)1(5) (also
known as xONR1 (6)). The name reflects its activation by alkyl
esters of amino and hydroxyl benzoic acids, one of which is
found endogenously in the Xenopus embryo (5). The identifica-
tion of BXR as a receptor for benzoate ligands illustrates the
potential of uncovering previously unsuspected signaling path-
ways through orphan receptor characterization. Recently, a
second Xenopus BXR cDNA was described (7). This BXR shares
only 88% nucleic acid sequence identity and 83% amino acid
sequence identity (see Fig. 1 and Ref. 7) with the BXR we
characterized previously (5). Therefore, the two receptors have
been designated as BXR? and BXR? (7).
BXR is most closely related to the human steroid and xeno-
biotic receptor, SXR (8) (also known as pregnane X receptor,
PXR (9), and pregnane activated receptor, PAR (10)). SXR and
its rodent ortholog PXR function as xenobiotic sensors in the
liver and intestine. They mediate the breakdown and elimina-
tion of steroids, drugs, and xenobiotic compounds by activating
the expression of degradative cytochrome P450 enzymes and
members of the ABC family of organic molecule transporters.
BXR and SXR/PXR have been assigned to the NR1I2 family by
the Nuclear Receptor Nomenclature Committee (11). This in-
dicates that these receptors are orthologous, i.e. the same gene
from different species. During our characterization of SXR, we
noted that none of the compounds that activated SXR was able
to activate BXR? (8).2This led us to question whether BXR and
SXR are functionally equivalent, i.e. do BXRs function as xe-
nobiotic sensors? As described below, we found that neither
BXR? nor BXR? is activated by the types of xenobiotic com-
pounds that activate SXR/PXR. In addition, BXRs are ubiqui-
tously expressed at varying levels in different tissues rather
than showing the high level expression only in the liver and
intestine characteristic of SXR/PXR. We infer that BXRs are
unlikely to be functioning as xenobiotic sensors and are there-
fore functionally distinct from their mammalian relatives.
Lastly, we show that BXR? is activated only by 4-hydroxyl
benzoates as compared with BXR?, which can also be activated
by other related compounds including amino benzoates, benzyl
benzoate, chlorpyrifos, and trifluralin. Interestingly, several of
the BXR?-selective activators function as antagonists for
BXR?, thus suggesting that the two BXRs are also pharmaco-
logically distinct from each other.
* This work was supported by Grant GM60572 from the National
Institutes of Health and a gift from Eisai Co. Ltd. (Japan). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Present address: Dept. of Pathology, University of Washington
Medical School, Seattle, WA, 98195.
§ To whom correspondence should be addressed: Dept. of Develop-
mental and Cell Biology, University of California, 5205 McGaugh Hall,
Rm. 5205, Irvine, CA 92697-2300. Tel.: 949-824-8573; Fax: 949-824-
4709; E-mail: blumberg@.uci.edu.
1The abbreviations used are: BXR, benzoate X receptor; SXR, steroid
and xenobiotic receptor; PXR, pregnane X receptor; 4-ABB, 4-amino-
butylbenzoate; 4-HBB, 4-hydroxy-butylbenzoate; 3-HEB, 3-hydroxy
ethyl benzoate; DMEM, Dulbecco’s modified Eagle’s medium; RT, re-
verse transcription; ABC, ATB-binding cassette; PBP, peroxisome pro-
liferator-activated receptor (PPAR)-binding protein; luc, luciferase;
DBD, DNA-binding domain.
2B. Blumberg, unpublished observations.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 46, Issue of November 15, pp. 43691–43697, 2002
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
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EXPERIMENTAL PROCEDURES
Cell Culture and Transfection—COS-7 cells were cultured in phenol
red-free DMEM supplemented with 10% FBS. For transient transfec-
tion experiments, COS-7 cells were seeded into 96-well plates at a
density of 5000 cells/well. 4–5 h after seeding, the cells were transfected
with CMX-GAL4-xBXR? (5), CMX-GAL4-xBXR?, or CMX-GAL4 (con-
trol) together with tk(MH100)4-luc reporter (12) and CMX-?-galacto-
sidase transfection control plasmids using standard calcium phosphate
precipitation methodology. 22–24 h after transfection, the cells were
washed twice with phosphate-buffered saline supplemented with 1 mM
MgCl2or DMEM-ITLB (DMEM containing 5 ?g/ml insulin, 5 ?g/ml
holo-transferrin, 5 ?g/ml selenium, 0.5% defined lipid mix (Invitrogen),
0.12% w/v delipidated bovine serum albumin (Sigma)) (13). Ligands
were next added in DMEM-ITLB, and the cells were incubated for an
additional 24–48 h. Ligands were typically purchased from Sigma,
ChemService (West Chester, PA), or Roche Molecular Biochemicals and
made freshly from powder in Me2SO as 0.1 M stocks, diluted in Me2SO
to appropriate concentrations and added to media with vigorous vortex
mixing. The cells were incubated with ligands for 24–48 h and then
lysed in situ. Extracts were prepared and assayed for ?-galactosidase
and luciferase activity as described (8). Reporter gene activity was
normalized to the ?-galactosidase transfection controls, and the results
were expressed as normalized relative luciferase units per OD of ?-ga-
lactosidase per minute to facilitate comparisons between plates. Fold
induction was calculated relative to solvent controls. Each data point
represents the average of triplicate experiments ? S.E. and was repli-
cated in independent experiments.
Isolation of Total RNA—Tissues were obtained from adult Xenopus
laevis males and females, dissected into small pieces, flash-frozen in
liquid N2, and stored at ?80 °C. Total RNA was isolated from the
tissues using standard guanidine thiocyanate procedure (14). Northern
blots were performed using the ligand-binding domain of BXR?, BXR?,
or EF-1? according to standard methods (15). For RT-PCR analysis, 1
?g of total RNA was reverse-transcribed using Superscript II reverse
transcriptase according to the manufacturer-supplied protocol (Invitro-
gen). Quantitative real time RT-PCR was performed using the following
primer sets: BXRa (5?-CTGTCCTGGTAGGGCAATGT-3?, 5?-AATGGG-
ACTGAAGCAACGTC-3?), BXRb (5?-CAGCCGGTGAATTGTCTTCT-3?,
5?-AGTTGTGGGGCTTGATTTTG-3?), or EF1a (5?-CCTGAATCACCCA-
GGCCAGATTGGTG-3?, 5?-GAGGGTAGTCTGAGAAGCTCTCCACG-
3?) using the SYBR green PCR kit (Applied Biosystems) in a DNA
Engine Opticon continuous fluorescence detection system (MJ Re-
search). All samples were quantitated by the comparative cycle thresh-
old (Ct) method for relative quantitation of gene expression, normalized
to EF-1? (16).
Isolation of Xenopus BXR?—Xenopus BXR? was isolated by RT-PCR
based on the published sequence (GenBankTMaccession number
AF305201) (7). 1 ?g of Xenopus total RNA was primed with
oligo(dT)12–18and reverse-transcribed with Superscript II reverse tran-
scriptase according to the manufacturer-supplied protocol (Invitrogen).
The cDNA was PCR-amplified using Pwo polymerase (Roche Molecular
Biochemicals) and the specific primers (5?-TCCGTGCTCACCTGGTTC-
CGT-3?) and (5?-CCTATCCATGTAGGTATCCCAGAT-3?) that annealed
in the 5?- and 3?-untranslated regions of BXR?. The amplified product
was gel-purified, and the ligand-binding domain of BXR? (amino acids
104–388) was amplified using nested primers (5?-TCGCCGGAATTCA-
GGAAAGAGCTGATCATGTCA-3?) and (5?-TGGCCAGGATCCCTATC-
ACTCATTCAGGGATCC-3?). The resulting product was purified and
ligated into pCMX-GAL4 to generate a GAL4-DBD-BXR?-ligand-bind-
ing domain fusion protein.
Similarly, GAL4 coactivator plasmids were generated by cloning the
receptor interaction domains of human TIF2 (GenBank™ accession
number NM006540, amino acids 563–790), human F-SRC-1 (Gen-
Bank™ accession number U59302, amino acids 600–800), or human
ACTR (GenBank™ accession number AF036892, amino acids 600–788)
into pCMX-GAL4. The GAL4-PBP construct was a gift from B. Forman
(City of Hope Medical Center). To construct Herpesvirus VP16 activa-
tion domain fusion proteins, full-length BXR? and BXR? were PCR-
amplified and ligated in-frame into pCMX-VP16 vector. All constructs
were sequenced to verify that no errors were introduced in the PCR.
RESULTS
Comparative Expression of BXR? and BXR?—Recently, a
second Xenopus BXR cDNA was described (7). This BXR shares
only 88% nucleic acid sequence identity and 83% amino acid
sequence identity (Fig. 1A and Ref. 7) with the BXR we char-
acterized previously (5). This is more than would be expected
for the divergence between two duplicated genes in the
pseudotetraploid X. laevis genome (17). Accordingly, the new
receptor was called BXR? (7). To gain insight into the possible
target tissues for BXR action, we examined the expression
patterns of BXR? and BXR? in adult frogs by Northern blot
and quantitative real time RT-PCR analysis. Both genes en-
code ubiquitously expressed single transcripts of ?3.2 kb (data
not shown) and are found at very high levels in the ovary (Fig.
1B). BXR? is expressed at high levels in the brain and skin
with moderate levels in the testis, stomach, and intestines and
lower levels in the lung, kidney, liver, and heart (Fig. 1B).
BXR? is expressed at comparable levels to BXR? in the intes-
tines, lung and kidney with slightly higher levels in the testis
and heart and lower levels in the liver, skin and brain (Fig. 1B).
It is notable that these ubiquitous expression patterns for BXR
differ considerably from those of its putative human ortholog,
the steroid and xenobiotic receptor SXR (8). SXR functions as a
xenobiotic sensor and is expressed primarily in the liver and
intestine, where it modulates the levels of cytochrome p450
enzymes and ABC family transporters (8, 9). Since BXRs are
ubiquitously expressed, they do not show the tissue distribu-
tion expected for a xenobiotic sensor.
BXR? and BXR? Are Pharmacologically Distinct—Our pre-
vious work showed that alkyl esters of amino and hydroxyl
benzoic acids specifically bind to and activate BXR? (5). There-
fore, it was surprising that BXR? was reported to be activated
FIG. 1. BXRs are expressed in different patterns in adult tis-
sues. A, comparison of BXR sequences to related receptors in an opti-
mal sequence alignment. Percentages indicate the degree of homology
conservation in the receptor DNA- and ligand-binding domains relative
to BXR?. hVDR, human vitamin D receptor; hCAR, human constitutive
androstane receptor. B, tissue-specific expression of BXR? and BXR? in
adult tissues as determined by real time PCR analysis of reverse-
transcribed total cellular RNA. Data are shown as the RNA expression
levels normalized to X. laevis EF1? controls. Values represent the
average of duplicates ? range.
BXRs Are Not Promiscuous Xenobiotic Receptors
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only very weakly by 4-amino butyl benzoate (7), which strongly
activates BXR? (5). We tested the activation profiles of these
receptors to determine whether the two BXRs might exhibit
different ligand specificity. BXR? and BXR? were transiently
transfected into COS-7 cells, and then a panel of benzoates and
related compounds was tested for their ability to activate tran-
scription of a luciferase reporter gene (Fig. 2). Activation of
BXR? paralleled our published results (Fig. 2A) in that both
hydroxyl and amino benzoates were robust activators. In addi-
tion, we observed that benzyl benzoate, trifluralin, and chlor-
pyrifos also activated BXR? (Fig. 2A). In contrast, only 4-hy-
droxyl butyl benzoate was able to activate BXR? (Fig. 2B). It is
particularly notable that 3-hydroxyl ethyl benzoate, which was
identified as an endogenous embryonic activator of BXR?,
could activate BXR? but was inactive on BXR? (Fig. 2, A and
B). BXR? was considerably more active in response to ligand
than was BXR? (Fig. 2, A and B). A similar trend was also
observed in dose-response experiments (Fig. 2, C and D). BXR?
was strongly activated by 10 ?M 4-ABB, 4-HBB, chlorpyrifos,
and trifluralin (Fig. 2C). BXR? was only activated by 4-HBB
among the many compounds tested (Fig. 2, B and D). The
activation of BXR? was very robust with 50 ?M of 4-HBB
yielding between 50- and 100-fold activation of the reporter
gene (Fig. 2, B and D).
Co-activator Recruitment by BXRs—To confirm that BXR?
and BXR? show distinct pharmacological responses to ligand,
we conducted co-activator recruitment studies to determine the
ability and preferences of the various ligands to support the
FIG. 2. Comparative activation of BXRs by benzoates and related compounds. Cells were transfected with GAL-BXR? (A and C) or
GAL-BXR? (B and D) reporter and control plasmids as described under “Experimental Procedures.” Ligands were added at a fixed concentration
of 50 ?M (A and B) or as indicated (C and D). DMSO, Me2SO solvent control; 4-AEB, 4-amino-ethylbenzoate; 3-AEB, 3-amino-ethylbenzoate; NBB,
4-nitro-butylbenzoate; BHA, butyl-hydroxyanisol; BB, benzylbenzoate; CP, chlorpyrifos; TF, trifluralin; RLU, relative luciferase units. Cells were
further incubated for 24 h, harvested, and assayed for luciferase and ?-galactosidase activity. Data were normalized to ?-galactosidase activity and
plotted as relative luciferase units (RLU) against concentration. Points represent the means of triplicates ? S.E. from a representative experiment.
The compounds were cytotoxic at concentrations greater than 100 ?M as measured by reduced activity for the ?-galactosidase transfection controls.
BXRs Are Not Promiscuous Xenobiotic Receptors
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formation of specific active transcriptional complexes. This
mammalian two-hybrid assay utilized VP16-BXR? and VP16-
BXR? together with fusions between the GAL4-DNA-binding
domain and the receptor-interacting domain of the nuclear
hormone receptor co-activators, TIF-2, ACTR, SRC-1, and PBP
(18) to investigate the ability of BXR? agonists to promote
productive transcriptional interactions.
VP16-BXR? was able to interact with all four co-activators in
the presence of agonistic ligands (Fig. 3A). VP16-BXR? showed
the strongest interaction with SRC-1 irrespective of the ligand
tested (4-HBB, 12-fold; 4-ABB, 8-fold; chlorpyrifos and triflu-
ralin 6-fold), although, a rank order of co-activator response
could be identified (SRC-1 ? TIF-2 ? PBP ? ACTR) (Fig. 3A).
This is notably different from the rank order of potency of SXR
for the same coactivators (SRC1 ? PBP ? TIF-2/GRIP ?
ACTR) (19). Overall, the rank order of potency of compounds in
the co-activator recruitment assay paralleled their potency in
the activation assays (Fig. 2).
Co-activator interaction with BXR? was much more re-
stricted in response to agonist (Fig. 3B). Consistent with the
dose-response experiments (Fig. 2D), only 4-HBB possessed
any strong ability to promote co-activator interaction. The re-
sponse observed with SRC-1 was approximately equivalent to
that seen with BXR? (10- versus 12-fold). The level of interac-
tion with PBP was weaker (4-fold) although comparable with
that seen with BXR? (Fig. 3A). The activation responses with
ACTR and TIF-2 were poor. The rank order was SRC-1 ?
PBP ? ACTR ? TIF-2. The data suggest that SRC-1 is a strong
co-activator for both BXR? and BXR?. BXR? is promiscuous
both in its choice of ligand and in its choice of co-activator,
whereas BXR? is not promiscuous for either (Fig. 3).
BXR?-specific Activators Are Antagonists for BXR?—The ob-
servation that most BXR? activators could not activate BXR?
leads to two possible inferences. One is that the compounds
specifically bind only to BXR?. In this case, we would not
expect to discern any effect of these compounds on BXR? acti-
vation. Alternatively, the compounds might bind to both recep-
tors but only activate BXR?. In this scenario, the BXR? acti-
vators could act as antagonists for BXR?. Accordingly, we
conducted antagonism experiments using the BXR? agonist
4-HBB and BXR?-specific agonists. Two types of experiments
were employed.
First, we tested the ability of BXR?-specific agonists to di-
rectly interfere with BXR?-mediated activation. COS-7 cells
were transfected with GAL4-BXR?, and inhibitory dose-re-
sponse curves were derived. Cells were treated with increasing
doses of 4-ABB, chlorpyrifos, or trifluralin in the presence of a
fixed series of agonist 4-HBB concentrations in the range of
1–50 ?M. Transcriptional activation of BXR? by 4-HBB alone
gave a derived mean EC50of 33 ? 3.2 ?M (n ? 4) (Table I).
Titration with either chlorpyrifos or trifluralin resulted in a
dose-dependent inhibition of 4-HBB-mediated transcriptional
activation of the reporter gene (Fig. 4A). In contrast, 4-ABB
showed a small additive effect up to 10 ?M and was inhibitory
at 100 ?M under these conditions (Fig. 4A). Data were fitted by
non-linear regression analysis, and inhibitory constants (Ki) for
BXR?-mediated transcriptional activation were calculated us-
ing the Cheng-Prusoff equation (20) and shown in Table I.
Chlorpyrifos yielded a Kiof 0.5 ? 0.2 ?M (n ? 13), trifluralin
yielded a Kiof 5.2 ? 0.8 ?M (n ? 8), and 4-ABB yielded a Kiof
?82 ? 9 ?M (n ? 15). By comparison with the EC50value of 33
?M for 4-HBB on BXR?, the data suggest that both chlorpyrifos
and trifluralin can act as potent competitive antagonists of
BXR? activation, whereas 4-ABB demonstrates weaker antag-
onism on this receptor. 3-HEB did not antagonize the activa-
tion of BXR? (data not shown).
We next tested the ability of the BXR?-specific agonists to
FIG. 3. BXR? and BXR? co-activator recruitment. COS-7 cells
were transiently transfected with a GAL4 reporter and VP16-BXR? (A)
or VP16-BXR? (B) together with expression vectors for the GAL4 DNA-
binding domain (vector) or the GAL4 DNA-binding domain linked to the
receptor interaction domains of the indicated nuclear receptor co-acti-
vators. Cells were treated with 50 ?M 4-ABB, 4-HBB, chlorpyrifos (CP),
or trifluralin (TF) or with vehicle (ethanol) alone.
TABLE I
BXR? agonists are BXR? antagonists
Compound EC50
Kia
n
?M
?M
4-HBB
4-ABB
Chlorpyrifos
Trifluralin
33 ? 3.2b
4
82 ? 9
0.5 ? 0.2
5.2 ? 0.8
15
13
8
aKivalues were derived from inhibition curves at constant HBB
concentrations in the range of 1–50 ?M 4-HBB using the Cheng-Prusoff
equation (20). Values represent the mean ? S.E. calculated from the
indicated number of inhibition curves (n).
bEC50for BXR? activation by 4-HBB was determined from non-
linear regression analysis of dose-response curves.
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interfere with 4-HBB-mediated co-activator recruitment in the
mammalian two-hybrid assay as described above. COS-7 cells
were transfected with VP16-BXR?, GAL-SRC1, and CMX-?-
galactosidase and treated with 50 ?M 4-HBB in combination
with a dose series of 4-ABB, chlorpyrifos, trifluralin, or solvent
controls. Fig. 4B shows that 4-ABB, chlorpyrifos, and triflura-
lin were each able to impair co-activator recruitment by 4-HBB.
Therefore, we conclude that these BXR?-selective activators
are able to act as antagonists for BXR?, supporting the conten-
tion that BXR? and BXR? are pharmacologically distinct from
each other.
BXRs Are Not Xenobiotic Sensors—The very different tissue
distributions of BXRs and SXR/PXRs led us to suspect that
these receptors might be functionally different. To test this
hypothesis, we examined a panel of compounds for their ability
to activate BXR?, BXR?, and human SXR. We found that
neither BXR? nor BXR? was activated by any of the classic
SXR/PXR activators (e.g. rifampicin, pregnenolone-16 ?-carbo-
nitrile, phenobarbitals, or clotrimazole) (Fig. 5). The only xeno-
biotic compounds that activated BXR?, chlorpyrifos and triflu-
ralin, have chemical structures similar to benzoates. We also
evaluated other known SXR activators including steroids and
bile acids for their ability to activate BXR. None of these SXR
activators were able to activate BXRs (data not shown). We also
note that two compounds reported previously to activate BXR?,
dexamethasone and methylprednisolone (7), do not activate
either BXR in our experiments. The reason for this discrepancy
is unknown at present but may relate either to the different
experimental systems used or to the very low levels of activa-
tion originally observed (?2-fold) (7).
DISCUSSION
Despite a flood of genomic and expressed sequence tag se-
quence information in recent years, there is still little informa-
tion available that suggests a function for BXR or confirms its
presence in animals other than X. laevis. The recent identifi-
cation of BXR? (7) and its activation by 4-HBB (Figs. 2 and 3)
suggests that both BXRs are receptors for endogenous hydroxyl
benzoates or closely related compounds. This narrows the
range of compounds expected to activate both BXR? and BXR?
to a relatively small group and suggests that a detailed focus on
these structures will lead to the elucidation of endogenous
ligands. It is particularly interesting that BXR? and BXR? are
pharmacologically distinct and that several potent BXR? acti-
vators are BXR? antagonists. This means that treating early
embryos with compounds such as 4-ABB would simultaneously
activate BXR? while inhibiting the activity of BXR?. This could
explain why such treatments do not have obvious adverse
effects on the embryos at subtoxic doses.2To sort out the
biology of the two receptor subtypes, it will be necessary to
FIG. 4. BXR? activators are BXR? antagonists. In A, COS-7 cells
were transfected with GAL-BXR?, reporter, and control plasmids as
described under “Experimental Procedures.” Cells were incubated at a
constant concentration of 4-HBB within the range of 1–50 ?M (10 ?M
shown in figure). The BXR? agonists 4-ABB (white square), trifluralin
(black circle), or chlorpyrifos (white circle) were then titrated from 0.1 to
100 ?M. Cells were incubated with ligands for 24 h, harvested, and
assayed for luciferase and ?-galactosidase activity. Data are from a
typical experiment and plotted as the percent of relative luciferase units
obtained with 10 ?M 4-HBB alone. Data points are the means of trip-
licates; S.E. was less than 15%. In B, COS-7 cells were transfected with
a GAL4 reporter together with VP16-BXR? and GAL4-SRC1 expression
vectors. Cells were then treated with 50 ?M 4-HBB in the absence
(none) or presence of 50 ?M 4-ABB, 50 ?M chlorpyrifos (CP), or 10 ?M
trifluralin (TF).
FIG. 5. BXRs are not xenobiotic receptors. COS-7 cells were
transfected with BXR? or BXR?, reporter, and control plasmids as
described under “Experimental Procedures.” Cells were treated with
vehicle only (Me2SO (DMSO)) or the indicated xenobiotic ligands at 50
?M for 24 h. Data represent the means of triplicates ? S.E. from a
representative experiment.
BXRs Are Not Promiscuous Xenobiotic Receptors
43695