Conservation of Signaling Pathways of Xenobiotic-Sensing Orphan Nuclear Receptors, Chicken Xenobiotic Receptor, Constitutive Androstane Receptor, and Pregnane X Receptor, from Birds to Humans
Christoph Handschin,
Michael Podvinec,
Jacqueline Stöckli1,
Klaus Hoffmann2 and
Urs A. Meyer
Division of Pharmacology/Neurobiology, Biozentrum of the University
of Basel, CH-4056 Basel, Switzerland
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ABSTRACT
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Chicken xenobiotic receptor, pregnane X receptor, and constitutive
androstane receptor are orphan nuclear receptors that have recently
been discovered to regulate drug- and steroid-mediated induction of
hepatic cytochromes P450 (CYP). This induction is part of an
adaptive response involving numerous genes to exposure to drugs and
chemicals and has major clinical and toxicological implications. Here
we report experiments in the chicken hepatoma cell line LMH that
suggest evolutionary conservation of the signaling pathways triggered
by pregnane X receptor, constitutive androstane receptor, and chicken
xenobiotic receptor. Thus, the phenobarbital-inducible enhancer units
of the mouse Cyp2b10, rat CYP2B2, and human CYP2B6 genes were activated
in reporter gene assays by the same compounds that activate the chicken
CYP2H1 phenobarbital-inducible enhancer units. Chicken xenobiotic
receptor, pregnane X receptor, and constitutive androstane receptor
all bound to the CYP2H1 phenobarbital-inducible enhancer units in
gel-shift experiments. In CV-1 cell transactivation assays, mammalian
pregnane X receptors activate the chicken phenobarbital-inducible
enhancer units to the same extent as does chicken xenobiotic receptor,
each receptor maintaining its species-specific ligand spectrum. To
assess the reported role of protein phosphorylation in drug-mediated
induction, we treated LMH cells with okadaic acid and observed
increased mRNA of
-aminolevulinate synthase and CYP2H1 whereas
expression of CYP3A37 was decreased. The effects of okadaic acid and
other modifiers of protein phosphorylation in LMH cells are comparable
to those seen on CYP2Bs and CYP3As in mammalian primary hepatocyte
cultures. These results indicate that closely related nuclear
receptors, transcription factors, and signaling pathways are mediating
the transcriptional activation of multiple genes by xenobiotics in
chicken, rodents, and man.
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INTRODUCTION
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A GENE SUPERFAMILY of heme proteins, the
cytochromes P-450 (CYP), encodes for the main enzymatic system for
metabolism of lipophilic compounds of diverse structures
(1). A common feature of most of these CYP substrates,
which include drugs, fatty acids, cholesterol precursors, and
metabolites such as steroid hormones or bile acids, is their
hydrophobicity, which enables direct diffusion into cells and binding
to CYPs as well as intracellular receptors (2, 3, 4, 5, 6, 7, 8). A
subset of these CYPs can be induced or inhibited in the liver by a
variety of substances, including their own substrates. Foreign
compounds inducing CYPs can be categorized into different classes
represented by the prototypical substrates dioxin, phenobarbital (PB),
dexamethasone, clofibrate, and ethanol. For example, PB and PB-like
inducers affect predominantly the CYP2B, CYP2C, and CYP3A subfamilies
(2, 5, 6, 7, 8, 9). Apart from these CYPs, at least 50 other genes
are influenced, triggering a pleiotropic hepatic reaction characterized
by an increase in liver weight, proliferation of smooth endoplasmic
reticulum, and tumor promotion as well as many other effects
(10). PB induction of CYP has been described in a variety
of species from Bacillus megaterium to man. Nevertheless,
marked differences in induction or inhibition potentials of different
drugs have been observed in different species. For instance, whereas
the antiglucocorticoid 5-pregnen-3ß-ol-20-one-16
-carbonitrile
(PCN) is a strong inducer of CYP3As in rat and mouse, it barely changes
CYP3A levels in man or rabbit (7, 11, 12). In contrast,
5ß-pregnane-3,20-dione and RU486 (mifepristone) are potent CYP3A
inducers in man and mouse but induce to a lesser extent in rabbit or
rat (7, 11, 12).
Although the inducing effect of PB and other drugs was discovered more
than 40 yr ago, progress in understanding the molecular mechanisms of
induction was limited until recently, when transfectable primary
hepatocyte cultures of mice, rat, and chicken were developed. Reporter
gene assays in these in vitro systems allowed the
characterization of drug-responsive elements in the flanking regions of
several inducible genes and transactivation assays in conjunction with
EMSAs identified transcription factors that bind to these elements
(13, 14, 15, 16, 17, 18, 19, 20, 21, 22). However, primary cultures of hepatocytes have
significant drawbacks that hinder the elucidation of molecular details
linking the drug-induced signal to the transcription machinery. For
instance, in primary hepatocyte cultures, the response to PB is often
delayed, attenuated, and terminated rapidly. In addition, cultured
hepatocytes may express an abnormal CYP profile. Primary hepatocytes
are difficult to transfect, and the time needed for transfection and
drug treatment is excessively long. Moreover, hepatoma-derived cell
lines such as HepG2 do not express a complete set of CYPs and have lost
the response to PB (for reviews, see Refs. 23 and
24).
In search of a convenient culture system, we discovered the leghorn
male hepatoma cell line (LMH) to respond to PB and PB-like inducers to
the same extent as primary chicken hepatocytes or chick embryo liver
in ovo. The LMH cell line was established by inducing a
hepatocellular carcinoma with diethylnitrosamine in a male leghorn
chicken (25). Characteristically, the LMH cells feature a
well differentiated morphology and biochemistry (25, 26).
The biochemical and chromosomal properties, a triploid karyotype with
six marker chromosomes, remain constant over a prolonged period of
propagation in culture (25).
Here, we describe the characterization of PB-type induction in the LMH
cells using CYP2H1 and CYP3A37 as well as
-aminolevulinate synthase
(ALAS) as representative genes regulated by PB. CYP2H1 is one of the
major PB-inducible enzymes in chicken liver (27). CYP3A37,
the first avian CYP of the 3A subfamily, was discovered in our
laboratory and shown to be PB inducible (28). ALAS is the
first and rate-limiting enzyme of heme biosynthesis (29).
It is activated to meet the increased need of heme for CYP
heme-proteins (30).
The flanking region of one of these genes, chicken CYP2H1, has recently
been analyzed in detail and a PB-responsive enhancer unit (PBRU) has
been defined (17, 19). Interestingly, the arrangement of
certain putative transcription factor recognition sites, in particular
a nuclear receptor consensus sequence arranged as a direct repeat of
two hexamers separated by four nucleotides (DR-4) in the vicinity of a
nuclear factor-1 site on the chicken PBRU, is highly conserved in
comparison to PBRUs of mammalian PB-inducible CYPs (19, 31). This avian 264-bp enhancer element could be activated by
the recently discovered chicken drug-sensing orphan nuclear receptor,
chicken xenobiotic receptor (CXR) (22). Sequence
comparisons revealed that CXR is closely related to both mammalian
xenobiotic-activated receptors [constitutive androstane receptor (CAR)
and pregnane X receptor (PXR)] showing between 61% and 67% amino
acid identity in the DNA-binding domains and between 49% and 56%
amino acid identity in the ligand-binding domains, respectively
(22). We therefore wanted to test whether these orphan
nuclear receptors interact with the avian PBRU.
Both drug-induction and orphan nuclear receptor signaling have been
reported to be highly influenced by phosphorylation and
dephosphorylation events (32, 33, 34, 35). We studied the effects
of the protein phosphatase inhibitor okadaic acid and the cAMP
modulator forskolin on CYP2H1, CYP3A37, and ALAS as well as the effects
of inducers on cAMP levels in the LMH cells to determine whether
phosphorylation and dephosphorylation events elicit the same effects in
LMH cells as those previously reported for primary cultures of rat and
mouse hepatocytes (36, 37, 38, 39, 40).
The data presented here demonstrate the interchangeability of
drug-responsive elements and xenobiotic-activated nuclear receptors in
chicken and mammals and also suggest conservation of protein
phosphorylation and dephosphorylation effects on induction in these
different species.
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RESULTS
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PB Induction of CYP2H1 in the Chicken Hepatoma Cell Line LMH
To assess the inducibility of the LMH cell line by xenobiotics,
CYP2H1 transcript and protein levels were measured after drug
treatment. Induction of CYP2H1 mRNA was dose dependent with maximal
transcript levels after treatment with 1 mM PB as shown by
both semiquantitative PCR and Northern blot (Fig. 1A
). Higher PB concentrations produced
significant cell toxicity. In Northern blots, a band of about 3.5 kb
corresponding to CYP2H1 was observed (Fig. 1A
). As shown in Fig. 1B
, transcript levels of CYP2H1 continued to rise even after 30 h of
exposure to 600 µM PB. PB induction of CYP2H1 was dose
dependent and reversible at the mRNA level. Similar results were
obtained with mRNA of
-aminolevulinic acid synthase (data not shown)
and CYP3A37 (28). When comparing protein levels of CYP2H1
in chicken embryo liver (in ovo) with those of chicken
primary hepatocytes and LMH cells, we observed that control and
PB-treated protein levels in the LMH cells reflected the in
ovo situation better than the chicken primary hepatocytes (Fig. 1C
). LMH cells were exposed to 600 µM PB for
24 h, chicken embryo hepatocytes were exposed to 1.2
mM PB for 48 h, and chick embryos were
treated with 3 mg PB per egg for 48 h. Basal levels in
ovo and in the LMH cells were lower than those in chicken
embryo hepatocytes, but consistent induction could be seen in all
three systems. The identities of the upper bands in the Western blot
are not yet clear. We have found members of the CYP2C subfamily in
chicken (data not shown), and these bands might represent CYP2Cs
that are recognized by the polyclonal CYP2H1 antibody due to the close
relationship of CYP2Cs and CYP2Hs. Nevertheless, these bands are
clearly distinguishable from the band representing CYP2H1 by their
higher molecular weight. Induction of CYP3A37 protein by PB in the LMH
cells has previously been demonstrated (28).

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Figure 1. Inducibility of LMH Cells
A, LMH cells were exposed to different concentrations of PB for 24
h. RNA was isolated and reverse-transcribed, and the level of CYP2H1
and ß-actin cDNA was measured by semiquantitative PCR and Northern
blot. CYP2H1 levels were adjusted relative to ß-actin levels. Data
represent the average of three independent experiments with
error bars representing standard deviations. B, LMH
cells were treated with 600 µM PB for the indicated time
period, and transcript levels of CYP2H1 and ß-actin were measured.
Data represent the average of three independent experiments with
error bars representing standard deviations. C, LMH
cells were exposed to 600 µM PB for 24 h, chicken
embryo hepatocytes (CEH) were exposed to 1.2 mM PB for
48 h, and chicken embryos in ovo were treated with
3 mg PB per egg for 48 h before preparation of liver homogenate.
Protein from chicken liver homogenate, primary cultures of chicken
hepatocytes, and LMH cells was isolated and a Western blot performed
with CYP2H1 antibodies as described in Materials and
Methods. D, LMH cells were transfected with the 4.8-kb
PB-responsive enhancer from CYP2H1 in pBLCAT5 containing an
enhancerless tk-promoter. After exposure to the indicated
concentrations of PB for 48 h, a CAT ELISA was performed. The
relative CAT expression was standardized against untreated control
cells and expressed as fold induction. Data represent the average of
three independent experiments with error bars
representing standard deviations.
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A 4.8-kb fragment of the 5'-flanking region of CYP2H1, which has been
shown to be a PB-responsive enhancer element (41), was
cloned into a pBLCAT5-reporter vector containing an enhancerless
thymidine kinase (tk)-promoter and transfected into LMH cells.
Nonradioactive chloramphenicol acetyltransferase (CAT) reporter gene
assays were performed, and a dose-dependent increase in CAT protein was
observed amounting to a 60-fold induction at 200 µM PB
after 48 h (Fig. 1D
). This response is higher than the induction
levels obtained in chicken primary hepatocytes after a 48 h
exposure to 400 µM PB, resulting in a 20-fold increase
(data not shown). In experiments reported by another laboratory
(17, 41), the 4.8-kb enhancer fragment cloned into a
pCAT-reporter vector containing the weak, enhancerless SV40 promoter
gave a 7- to 14-fold induction after treatment of chicken primary
hepatocytes with 500 µM PB for 48 h. Thus, LMH cells
provide a reliable tool that is as highly drug-responsive as are
in ovo systems (Fig. 1C
).
Mammalian Drug-Responsive Enhancer Elements Are Activated in the
Chicken LMH Cells
In previous experiments, we have observed that a 51-bp PBREM from
the mouse Cyp2b10 5'-flanking region (15) was activated by
PB-like inducers in the LMH cell line (19). To extend
these studies, we tested the response elements of the rat CYP2B2
(13, 16), the human CYP2B6 (18), and the
mouse Cyp2b10 (15) gene with different inducers in LMH
cells and compared the results to the findings of the chicken 264-bp
PBRU (19). The 163-bp PBRU of rat CYP2B2, the 51-bp PBREM
of human CYP2B6, the 51-bp PBREM of mouse Cyp2b10, and the 264-bp PBRU
of chicken CYP2H1 subcloned in reporter gene vectors were transfected
into LMH cells, and reporter gene assays were performed after applying
different inducers for 16 h (Fig. 2
). We selected the same drugs as those
used in previous studies of the chicken 264-bp PBRU and the mouse
Cyp2b10 PBREM (19). As prototypical CYP2B gene activators,
we used PB (400 µM) and the PB-like inducers
propylisopropylacetamide (PIA) (250 µM) and glutethimide
(500 µM). The two CYP3A inducers dexamethasone (50
µM) and metyrapone (400 µM) were also
examined. Rifampicin (100 µM) and PCN (50
µM) have species-specific effects on CYP3As and were
therefore of interest. Furthermore, LMH cells were treated with the
prototypical CYP1A1-inducer ß-naphtoflavone (10 µM). In
general, both the human and the rat response element reacted in the
same way to the different inducers varying only in the extent of the
inductions (Fig. 2
, A and B). PB-induction of the rat 163-bp PBRU was
6-fold in contrast to the human 51-bp PBREM that was 1.5-fold induced.
High induction on both elements was observed when using PIA,
glutethimide, or metyrapone. PIA conferred a 68-fold induction on the
rat PBRU and a 6.1-fold induction on the human PBREM, slightly higher
than the 34-fold induction of the rat PBRU and the 4.2-fold induction
of the human PBREM by glutethimide. Metyrapone induced the rat PBRU
46-fold and the human PBREM 8.1-fold. Dexamethasone, PCN, rifampicin,
and ß-naphtoflavone had no or only very minor effects on both
elements (Fig. 2
, A and B). These results demonstrate that the rat
163-bp PBRU and the human 51-bp PBREM react comparably to the
chemicals. Moreover, these inductions correspond to the reporter gene
activations by the chicken 264-bp PBRU and the mouse 51-bp PBREM
in LMH cells (Fig. 2
, C and D). The different relative magnitude of the
responses to different drugs might be explained by recent findings
demonstrating that the 163-bp PBRUs and the 51-bp PBREMs are not
equivalent in their response to different inducers and that additional
transcription factor-binding sites on the 163-bp PBRU contribute to
maximal induction (42). The mouse and the chicken enhancer
reacted almost identically to drug treatment as observed previously
(19). Consequently, the same or very similar receptors in
the chicken LMH cells can bind to and activate the different response
elements from human, rat, mouse, and chicken after drug treatment.

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Figure 2. Trans-species Activation of Rat CYP2B2 163-bp PBRU,
Human CYP2B6 51-bp PBREM, Mouse Cyp2b10 51-bp PBREM, and Chicken CYP2H1
264-bp PBRU in LMH Cells
The PB-responsive elements from human CYP2B6 (A), rat CYP2B2 (B), mouse
Cyp2b10 (C), and chicken CYP2H1 (D) genes were cloned into the pBLCAT5
reporter vector containing a tk promoter as described in
Materials and Methods and Ref. 19 . These
constructs were transfected into LMH cells in suspension, and the cells
were induced for 16 h with PB (400 µM), PIA (250
µM), glutethimide (500 µM), dexamethasone
(50 µM), metyrapone (400 µM), PCN (50
µM) rifampicin (100 µM), or
ß-naphtoflavone (10 µM). Cells were harvested and a
CAT-ELISA was performed. The relative CAT expression was standardized
against untreated control cells and expressed in fold induction. Values
represent the average of three independent experiments with
error bars representing standard deviations.
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Human PXR and Human CAR Bind to the Chicken CYP2H1 264-bp PBRU in
Gel-EMSAs
Since the drug-response elements are exchangeable between chicken,
mouse, rat, and human, we wanted to test whether the different
xenobiotic-sensing nuclear receptors PXR, CAR, and CXR from human and
chicken bind to the CYP2H1 264-bp PBRU. None of the in vitro
transcribed/translated receptors bound to radiolabeled 264-bp PBRU as
monomers (Fig. 3
, lanes 25).
Heterodimerized with chicken RXR
, both human PXR and human CAR
formed a complex on the CYP2H1 PBRU as did the CXR/RXR
complex (Fig. 3
, lanes 6, 8, and 10, region b). These complexes could be supershifted
when using an antibody against RXR (Fig. 3
, lanes 7, 9, and 11, region
c). Thus, all three drug-activated nuclear receptors, PXR, CAR, and
CXR, could be shown to interact with the chicken PBRU.

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Figure 3. Human PXR and Human CAR Bind to the Chicken 264-bp
PBRU in EMSAs
Radiolabeled 264-bp PBRU was incubated with in vitro
transcribed/translated CXR (lanes 3, 6, and 7), human PXR (lanes 4, 8,
and 9), human CAR (lanes 5, 10, and 11), chicken RXR (lanes 2 and
611), and anti-RXR antibody (lanes 7, 9, and 11). The
arrow depicts unbound probe (a). Complexes of CXR, human
PXR, and human CAR with chicken RXR result in shifts (b) and with
addition of anti-RXR antibody in supershifts (c).
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Human and Mouse PXR Transactivate the Chicken 264-bp PBRU in CV-1
Cells
As mammalian xenobiotic-sensing orphan nuclear receptors are
binding to the chicken 264-bp PBRU, functional tests were performed to
determine whether these receptors also activate the avian response
element. In contrast to CXR and PXR, CAR activation by PB is not
measurable in CV-1 cell transactivation assays for the following
reasons: 1) CAR is a constitutively active transcription factor
(43, 44); 2) PB apparently is not a ligand of CAR but
induces by an indirect mechanism involving cytoplasmic-nuclear transfer
of CAR (35, 45); 3) transiently or stably expressed CAR is
exclusively nuclear in cultured cells (35, 45). Due to
these limitations, we restricted the transactivation experiments to PXR
and CXR in this study. Monkey kidney epithelial CV-1 cells were
cotransfected with the reporter gene construct containing the 264-bp
PBRU and the expression plasmid for CXR, human PXR, and mouse PXR,
respectively. Cells were treated for 24 h with different inducer
compounds and reporter gene levels were measured. Mammalian PXRs and
chicken CXR all were able to transactivate the chicken 264-bp PBRU.
Moreover, activation patterns corresponded to the relative potencies of
induction observed with a CYP3A4-responsive enhancer module and
recapitulated the typical species-specific differences between human
and mouse PXR (46). Thus, in CV-1 cells, human PXR was
strongly activated by PB, glutethimide, metyrapone, RU486, rifampicin,
and clotrimazole whereas dexamethasone, RU486, and PCN activated mouse
PXR (Fig. 4
, A and B). The CYP2H1 264-bp
PBRU is also activated by the chicken drug-activated orphan nuclear
receptor CXR that was recently discovered in this laboratory
(22). When comparing the activation patterns of mouse PXR,
human PXR, and chicken CXR, CXR had almost identical specificity
for ligands as human PXR concerning PB, dexamethasone, metyrapone, PCN,
clotrimazole, and TCPOBOP (Fig. 4
, A and C). Overlap between CXR and
mouse PXR activation patterns was also observed after treatment with
glutethimide and rifampicin (Fig. 4
, B and C). In contrast, CXR was the
only receptor that was activated by ß-naphtoflavone and by PIA and
the only receptor not affected by RU486 (Fig. 4C
). These data indicate
overlapping ligand specificity for these evolutionary distant
receptors. Moreover, all three receptors could bind to the chicken
enhancer element and transactivate this PBRU upon drug treatment using
the transcriptional machinery of the monkey epithelial kidney
cells.

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Figure 4. Human and Mouse PXR and Chicken CXR Activate the
Avian 264-bp PBRU in CV-1 Transactivation Assays
CV-1 cells were cotransfected with expression plasmids for either human
PXR (A), mouse PXR (B), or chicken CXR (C) together with a CAT reporter
gene plasmid containing the CYP2H1 264-bp PBRU as described in Ref.
22 . Cells were then treated with vehicle (0.1% DMSO), PB
(400 µM), PIA (250 µM), glutethimide (500
µM), TCPOBOP (10 µM), dexamethasone (50
µM), metyrapone (400 µM), RU486 (10
µM), PCN (50 µM), rifampicin (100
µM), ß-naphtoflavone (10 µM), or
clotrimazole (10 µM) for 24 h. Cell extracts were
analyzed for CAT expression normalized against ß-galactosidase
levels. Values are the average of three independent experiments and the
error bars represent standard deviations.
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The DR-4 Element Within the CYP2H1 264-bp PBRU is the Main Binding
and Activation Site for PXR, CAR, and CXR
Nuclear receptor binding sites consisting of two direct repeats of
nucleotide hexamers with the consensus sequence AGT/GTCA separated by
four nucleotides (DR-4) have been identified to constitute the binding
sites for xenobiotic-sensing orphan nuclear receptors on mammalian and
chicken PBRUs and to confer drug inducibility of those (15, 16, 18, 19, 21). To assess the importance of this DR-4 in
cross-species experiments, we compared the effects of the CYP2H1 264-bp
PBRU containing mutated hexamer half-sites in its DR-4 (called
"double," described in Ref. 19) to those of wild-type
CYP2H1 264-bp PBRU (wt). EMSAs revealed that the mutated CYP2H1 264-bp
PBRU was no longer able to bind either CXR, human PXR, or human CAR
heterodimerized to chicken RXR
(Fig. 5A
, lanes 4, 6, and 8) in contrast to the
wild-type 264-bp PBRU (Fig. 5A
, lanes 3, 5, and 7). Moreover, the
mutated 264-bp PBRU was also compared with its wild-type counterpart in
CV-1 cell transactivation assays with human PXR, mouse PXR, and CXR.
After transfection, CV-1 cells were treated with either
vehicle [0.1% dimethylsulfoxide (DMSO)], RU486 (10
µM), PCN (50 µM), or clotrimazole (10
µM) for 24 h. Cell extracts were analyzed for CAT
expression normalized against ß-galactosidase levels. These compounds
were chosen because of their ability to strongly induce either human
PXR (RU486, clotrimazole), mouse PXR (RU486, PCN), or CXR
(clotrimazole) as shown in Fig. 4
. Activation levels of all three
receptors were severely reduced on the 264-bp PBRU DR-4 double mutant
compared with the wild-type PBRU (Fig. 5
, B, C, and D). The
species-specific induction levels of human PXR by RU486 and
clotrimazole, mouse PXR by RU486 and PCN, and CXR by clotrimazole were
strongly reduced on the 264-bp CYP2H1 PBRU DR-4 double mutant in
comparison with the wild-type 264-bp PBRU. Both the EMSA and the
transactivation experiments thus underline the importance of the DR-4
element within the CYP2H1 264-bp PBRU in drug induction.

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Figure 5. The DR-4 Element Within the CYP2H1 264-bp PBRU
Confers Binding and Inducibility by PXR, CAR, and CXR
A, Radiolabeled wild-type 264-bp PBRU (wt, lanes 1, 3, 5, and 7) or
264-bp PBRU mutated in both hexamers of the DR-4 element (double, lanes
2, 4, 6, and 8) were incubated with in vitro
transcribed/translated CXR (lanes 3 and 4), human PXR (lanes 5 and 6),
and human CAR (lanes 7 and 8) together with chicken RXR (lanes
38). The arrow depicts unbound probe (a) and the
complexes of CXR, human PXR, and human CAR with chicken RXR
resulting in a shifts (b). B, C, and D, CV-1 cells were cotransfected
with expression plasmids for either human PXR (B), mouse PXR (C) or
chicken CXR (D) together with a CAT reporter gene plasmid containing
the wild-type CYP2H1 264-bp PBRU (wt) or the CYP2H1 264-bp PBRU
containing mutated hexamer half-sites in its DR-4 element (double).
Cells were then treated with vehicle (0.1% DMSO), RU486 (10
µM), PCN (50 µM), or clotrimazole (10
µM) for 24 h. Cell extracts were analyzed for CAT
expression normalized against ß-galactosidase levels. Values are the
average of three independent experiments, expressed as percent of
maximal induction levels, and the error bars represent standard
deviations.
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Okadaic Acid Affects Drug-Induction of CYP2H1, CYP3A37, and
ALAS
In diverse in vitro systems including rat and mouse
primary hepatocyte cultures, both nuclear receptor activity and drug
induction of CYPs have been shown to be modulated by diverse protein
kinases and protein phosphatases (for reviews, see Refs.
32 and 34). Okadaic acid (OA) is an inhibitor
of various protein phosphatases (PP) of which PP-1, PP-2A, and PP-2B
are the best studied among those thought to be affected
(47). We used OA as a tool to inhibit PPs and measure
their effect on induction of ALAS, CYP2H1, and CYP3A37 by
semiquantitative RT-PCR using the Taqman system. In preliminary
dose-response experiments, OA induced CYP2H1 and ALAS mRNA levels at
concentrations of about 100 nM whereas CYP3A37
mRNA levels were inhibited by 10 nM OA (data not
shown). We therefore used 1 µM OA in the
following coincubation experiments to obtain clear results for all
three genes. To get a general overview of induction pathways affected
by PP, we tested the effect of OA treatment on inducers of three
different types, namely PB, dexamethasone, a synthetic glucocorticoid,
and metyrapone, a substituted pyridine. LMH cells were induced with 400
µM PB, 50 µM
dexamethasone, and 400 µM metyrapone alone and
together with 1 µM OA or 1
µM 1-nor-okadaone, an inactive analog of OA.
After 16 h, transcript levels of ALAS, CYP2H1, and CYP3A37 were
determined (Fig. 6
). PB, dexamethasone,
and metyrapone induced ALAS, CYP2H1, and CYP3A37 with metyrapone being
the strongest inducer, most impressive in the case of CYP3A37 (Fig. 6C
). In contrast to 1-nor-okadaone, OA had profound effects on all
three enzymes, i.e. increase of ALAS and CYP2H1 transcript
level in contrast to complete inhibition of CYP3A37 induction by PB,
dexamethasone, or metyrapone (Fig. 6
). When OA was added in combination
with PB, dexamethasone, or metyrapone, there was no increase in
transcript levels of ALAS and CYP2H1 over the levels achieved with OA
alone, suggesting that OA affects the mechanism by which drugs activate
transcription (Fig. 6
). This effect is best seen in coexposures to OA
and metyrapone for ALAS, where transcript levels were even lowered by
combined treatment compared with metyrapone alone. The effects of OA
were confirmed at the protein level by Western blotting for CYP2H1 and
CYP3A37 (data not shown). Thus, the inhibition of PB induction of CYPs,
as observed for rat CYP2B2 and mouse Cyp2b10 in primary cultures of
hepatocytes, could also be shown for chicken CYP3A37 on mRNA levels in
LMH cells. The apparently paradoxical CYP2H1 mRNA induction can be
explained by previous findings showing that PB induction on the
CYP2H1 264-bp PBRU is inhibited by OA and the observed overall
induction of CYP2H1 is due to activation of its promoter by OA
(19).

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Figure 6. OA Affects PB-, Dexamethasone-, and Metyrapone
Induction of ALAS, CYP2H1, and CYP3A37
LMH cells were treated with 1 µM OA or 1 µM
1-nor-okadaone for 16 h in combination with either 400
µM PB, 50 µM dexamethasone, or 400
µM metyrapone. Total RNA was isolated and reverse
transcribed. Relative mRNA levels of ALAS (A), CYP2H1 (B), and CYP3A37
(C) from treated cells against control cells were determined with a
Taqman ABI PRISM 7700 Sequence Detection System (Applied Biosystems,
Rotkreuz, Switzerland) and normalized against GAPDH mRNA level
as described in Materials and Methods. Values are the
average of three independent experiments and the error
bars represent standard deviations.
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PB Does Not Change cAMP Levels in LMH Cells
To study drug-mediated changes in second messenger systems
involved in phosphorylation and dephosphorylation events, we measured
the intracellular cAMP levels in the LMH cells after treatment with PB
and modulators of cellular cAMP levels using a nonradioactive,
competitive immunoassay. In rat and mouse primary hepatocytes, PB
modulated neither cAMP levels nor PKA activities (39, 40).
In LMH cells, forskolin, an activator of adenylate cyclase, elevated
intracellular cAMP 30 min after addition to the LMH cells from 0.9
pmol/ml to 1,323 pmol/ml (Fig. 7
).
Sixteen hours later, cAMP levels were not higher than in control cells,
comparable to the kinetics described previously in primary rat and
mouse hepatocytes (36, 39, 40). Treatment with PB
or 1,9-dideoxyforskolin, a negative analog of forskolin, had no effect
on intracellular cAMP level. As expected, none of these three compounds
could raise cAMP levels in the medium to a detectable concentration.
The cAMP analog (Bu)2cAMP could be detected in
the cells, both after 30 min and after 16 h, at a high
concentration of approximately 9,500 pmol/ml whereas another cAMP
analog, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), was less potent (Fig. 7
). Both analogs were designed for easy penetration in cell culture
systems, and (Bu)2cAMP was apparently more stable
compared with CPT-cAMP in LMH cell cultures. These and previous results
demonstrate that PB treatment of chicken LMH cells and of primary
hepatocytes of rat and mouse has no effect on the second messenger
cAMP.

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|
Figure 7. Effect of Drug Treatment on cAMP Levels in
the LMH Cells
LMH cells were treated with 400 µM PB, 100
µM forskolin, 100 µM 1,9-dideoxyforskolin,
100 µM (Bu)2cAMP or 100 µM
CPT-cAMP for either 30 min or 16 h. Cells were harvested, and a
competitive immunoassay was used to measure intracellular cAMP and cAMP
in the medium. Values are the average of three independent experiments
and the error bars represent standard deviations.
Concentrations below detection limits are designated n.d.
(nondetectable).
|
|
Forskolin Activates CYP2H1, CYP3A37, and ALAS Independently of
Adenylate Cyclase and cAMP
No effect of forskolin on CYP2b10 was observed in primary cultures
of mouse hepatocytes (39), but forskolin affected rat
CYP2B1/2 and rat CYP3A1 transcription in primary hepatocytes in
different ways. In contrast to the inhibition of CYP2B1/2
(48), an induction of CYP3A1 by forskolin was proposed to
be due to adenylate cyclase-independent effects since the analog
1,9-dideoxyforskolin, which shows no adenylate cyclase-activation, had
the same inducing effect (36). Transcript levels of
CYP2H1, CYP3A37, and ALAS were determined after treating LMH cells for
16 h with 400 µM PB, 100 µM forskolin,
100 µM 1,9-dideoxyforskolin, or 100 µM
(Bu)2cAMP. No effect of
(Bu)2cAMP on any of the three enzymes was
observed (Fig. 8
) whereas both forskolin
and 1,9-dideoxyforskolin elevated mRNA levels of ALAS, CYP2H1, and
CYP3A37 (Fig. 8
). Thus, the effect of forskolin and
1,9-dideoxyforskolin is apparently independent of adenylate cyclase
activation and cAMP levels in the chicken LMH cells, similar to
regulation of rat CYP3A1 in primary hepatocyte cultures
(36). Whereas CYP2B2 and CYP3A1 in rat both were inhibited
by cAMP (50), our results correlate with the lack of
effect of cAMP on mouse Cyp2b10 in primary hepatocytes
(39) showing no effect of cAMP on PB-inducible enzymes.
Thus, comparisons of our data with previous results in primary cultures
of rat and mouse hepatocytes show a high conservation of protein
phosphorylation and dephosphorylation events in enzyme induction
triggered by drug treatment between chicken and rodents (36, 37, 39, 40).

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|
Figure 8. Influence of Forskolin and cAMP on ALAS, CYP2H1,
and CYP3A37
LMH cells were treated with 100 µM forskolin, 100
µM 1,9-dideoxyforskolin, or 100 µM
(Bu)2cAMP for 16 h with or without 400
µM PB. Total RNA was isolated and reverse transcribed.
Relative mRNA levels of ALAS (A), CYP2H1 (B), and CYP3A37 (C) from
treated cells against control cells were determined with a Taqman ABI
PRISM 7700 Sequence Detection System and normalized against GAPDH mRNA
level as described in Materials and Methods. Values are
the average of three independent experiments and the error
bars represent standard deviations.
|
|
 |
DISCUSSION
|
---|
Drug-Responsive Enhancer Units and Xenobiotic-Sensing Receptors Are
Conserved in Chicken, Rodents, and Man
CYP induction by drugs and xenobiotics has been observed in a wide
range of species (2, 4, 5, 6, 7, 8, 9). However, there are major
interspecies differences in the spectrum of inducer drugs and the
pattern of activated genes (7, 11, 12). The molecular
origin of this divergence has remained unclear. The results summarized
in this report demonstrate conservation of the basic mechanism of drug
induction between different species and suggest that divergent
ligand-binding domains of orphan nuclear receptors account for the
observed species differences. These conclusions are deduced from the
following observations: First, drug-responsive elements from the human
CYP2B6, the rat CYP2B2, the mouse Cyp2b10, and the chicken CYP2H1 could
be activated in the chicken LMH cells. The response elements showed
similar activation patterns after treatment with different drugs that
closely resembled the CXR-mediated activation pattern of the chicken
CYP2H1 PBRU in CV-1 cell transactivation assays as well. These data
suggest that mammalian enhancers also interact with CXR. Similarly, all
elements are highly conserved in their structure of transcription
factor binding sites of which a direct repeat of hexamer half-sites
separated by four nucleotides (DR-4) is responsible for binding of the
xenobiotic-sensing orphan nuclear receptor heterodimers
(19). Second, the three different members of drug-sensing
orphan nuclear receptors CXR, PXR, and CAR bound the chicken 264-bp
PBRU in EMSAs. Moreover, in CV-1 cell transactivation assays, the
mammalian PXRs could activate the chicken CYP2H1 PBRU to the same
extent as the chicken CXR. Thus, in monkey CV-1 cells, the dynamic
multiprotein complex required for the induction machinery is able to
cross-react with the chicken, human, and mouse receptors and the
chicken response element. We interpret these data as support for the
concept that species differences in drug induction are due to divergent
ligand-binding domains of the xenobiotic-sensing orphan nuclear
receptors and not to fundamental differences in the induction
mechanism. The 60% to 80% amino acid sequence similarity in the
ligand-binding domain of drug-activated orphan nuclear receptor
orthologs is significantly lower than the 90% amino acid similarity
observed in principle between nuclear receptor orthologs. Accordingly,
mice devoid of endogenous PXR that are rescued with human PXR exhibit a
humanized xenobiotic response (49). These findings
indicate that it is exclusively the ligand-binding domain of
drug-sensing orphan nuclear receptors that independently diverged
during evolution of species as a reaction to different environmental
challenges (7, 12, 49).
Drug-Induction Mechanisms in Birds and Mammals Are Similarly
Affected by Protein Phosphorylation and Dephosphorylation Events
The conservation of xenobiotic-triggered signaling pathways and
the protein-protein interactions involved therein are further supported
by our observations on the effect of modifiers of protein
phosphorylation on drug induction. Although only CYP3A37 mRNA levels
were reduced by OA, we showed previously that induction of CYP2H1 by OA
is predominantly mediated by a 205-bp core promoter fragment whereas
drug induction of the 264-bp PBRU is abolished by OA (19).
Thus, the inhibition of CYP induction by OA correlates with the CYP
inhibitions in rat and mouse. Moreover, nuclear translocation of the
drug-sensing orphan nuclear receptor CAR triggered by drug treatment
could be inhibited by OA leading to repression of CYP induction in
mouse liver (35). These results suggest that PPs play an
important role in the molecular mechanism of CYP induction. In
contrast, cAMP levels were not affected by drug treatment, and the CYP
induction triggered by the adenylate-cyclase activator forskolin
apparently is not linked to changes in cAMP in the cell. Forskolin
mediates a multitude of physiological effects that cannot be reproduced
by cAMP analogs, i.e. inhibiting a variety of membrane
proteins such as membrane transporters, voltage-sensitive channels, or
P-glycoprotein. The diterpenic structure of forskolin is related to
steroids, and forskolin might therefore exhibit steroid-like properties
(see Ref. 36 and references therein). More recently,
forskolin was found to activate the farnesoid X receptor (FXR), the
bile acid receptor in liver, kidney, and intestine that is involved in
regulation of CYP7A, a key enzyme in cholesterol homeostasis
(50, 51, 52). Overlapping ligand affinities to different
orphan nuclear receptor also have been observed for
troglitazone, a drug used for treatment of type 2 diabetes
that activates both the PPAR and PXR. Similarly, SR12813, a compound
that lowers cholesterol levels in a number of species, activates both
FXR and PXR (12). These findings indicate that most, if
not all, xenobiotic inducer compounds affect more than one nuclear
receptor system and that pronounced cross-talk exists between receptors
that bind lipophilic drugs, fatty acids, steroids, and cholesterol
derivatives such as CXR, PXR, CAR, FXR, and PPAR. In summary, these
results in LMH cells demonstrate that both the orphan nuclear receptors
and the signaling mechanisms affecting drug-induction pathways are
similar in different species in spite of previous findings that
proposed different proteins and transcription factors to be responsible
for CYP induction in chicken compared with mammals
(17).
The studies reported in this manuscript were possible by using a new,
drug-inducible experimental system, the chicken hepatoma cell line LMH.
The LMH cells offer the advantages of a continuously dividing culture
system, including the availability of frozen stocks of clonal origin,
long-term culture, high comparability, and transfection efficiency. LMH
cells therefore allow the development of new experimental tools to
study the molecular details of the effects of xenobiotics on gene
expression, such as subclones with permanently expressed or mutated
transcription factors or induction-resistant cell mutants suitable for
complementation experiments.
 |
MATERIALS AND METHODS
|
---|
Reagents
OA sodium salt, 1-nor-okadaone, forskolin, and
1,9-dideoxyforskolin were purchased from Alexis Biochemicals
(Läufelfingen, Switzerland). Dexamethasone, metyrapone
(2-methyl-1,2-di-3-pyridyl-1-propanone), PCN, rifampicin,
clotrimazole (1-[o-chlorotrityl]-imidazole), CPT-cAMP, and
(Bu)2cAMP) were obtained from Sigma
(Buchs, Switzerland). PIA was generously provided by Dr. P. Sinclair
(VA Hospital, White River Junction, VT). Glutethimide and
ß-naphtoflavone were purchased from Aldrich (Buchs,
Switzerland). RU486 was obtained from Roussel-UCLAF SA (Paris, France).
TCPOBOP (1, 4-bis[2-(3, 5-dichloropyridyloxy)]benzene) was a gift
from Dr. U. Schmidt (Institute of Toxicology, BAYER AG, Wuppertal,
Germany). PB sodium salt (5-ethyl-5-phenyl-barbituric acid sodium salt)
was purchased from Fluka Chemical Co. (Buchs,
Switzerland). All other reagents were from standard suppliers. Cell
culture media, sera, and tissue culture reagents were purchased from
Life Technologies, Inc. (Basel, Switzerland) unless noted
otherwise.
Plasmids
The rat CYP2B2 163-bp PBRU was amplified from rat genomic DNA,
and the PCR product was digested with Sau3AI and subcloned
into a BamHI-digested pBlueScript vector. From a clone
containing two copies in head-to-tail orientation, the insert was
excised with XbaI and HindIII and ligated into
the pBLCAT5 reporter gene vector. The coding regions of human and mouse
CAR were amplified from human and mouse cDNA, respectively, followed by
subcloning into the expression vector pSG5 (Stratagene,
Basel, Switzerland). The expression vectors for human and mouse PXR,
pSG5-hPXR, and pSG5-mPXR.1, kindly provided by Dr. S. A. Kliewer
(Department of Molecular Endocrinology, Glaxo Wellcome Inc. Research and Development, Research Triangle Park, NC), were
described in previous publications (11, 20). The reporter
gene vector containing the human CYP2B6 51-bp PB-responsive enhancer
module (PBREM), a kind gift of Dr. M. Negishi (NIEHS, NIH, Research
Triangle Park, NC), was described previously (18).
Culture and Transfection of LMH Cells
LMH cells were obtained from the American Type Culture Collection (Manassas, VA) and thawed immediately after arrival.
Cultivation in Williams E medium and transfection with FuGENE 6
Transfection Reagent (Roche Molecular Biochemicals,
Rotkreuz, Switzerland) were performed as described previously
(22).
cAMP Determination
The levels of cAMP were determined by using the Format A Cyclic
AMP Enzyme Immunoassay Kit (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) according to the instructions of the
manufacturer. Briefly, cells were incubated with the drugs for 30 min
or 16 h and lysed. Intracellular cAMP and cAMP levels in the
medium were measured using a competitive immunoassay.
Analysis of Reporter Gene Expression
Sixteen hours after drug treatment, the cells were harvested and
nonradioactive chloramphenicol acetyltransferase (CAT) assays were
performed using the CAT-ELISA kit according to the manual of the
supplier (Roche Molecular Biochemicals). Cell extracts
were also used for the determination of protein concentration using the
ESL protein assay for normalization of specific CAT expression to total
protein content (Roche Molecular Biochemicals).
RNA Isolation and SQRT-PCR Analysis
RNA purification and cDNA synthesis were performed as described
previously (30). The primers for each cDNA amplification
were designed to be specific and selective for the predicted sequences.
For chicken CYP2H1, the primers used for RT-PCR analysis were the
following: forward 5'-GAC ACT TGA CAT CTC TTC CTC-3', and reverse
5'-CTG GGC ATT GAC TAT CAT T-3', and amplified a 1,572-bp fragment. In
parallel, we analyzed chicken ß-actin as an internal control for
normalization. The forward and reverse primers, 5'-CCC TGA ACC CCA AAG
CCA AC-3' and 5'-GAC TCC ATA CCC AAG AAA GA-3', respectively, produced
a predicted 487-bp fragment between positions 394 and 880. The
conditions for cDNA amplification have been described previously
(30).
RNA Isolation and Taqman Analysis
RNA from LMH cells was isolated with the RNeasy Kit
(QIAGEN AG, Basel, Switzerland). One microgram of total
RNA was reverse-transcribed with the Moloney murine leukemia
virus reverse transcriptase (Roche Molecular Biochemicals). PCR was performed using the Taqman PCR Core
Reagent Kit (PE Applied Biosystems, Rotkreuz, Switzerland)
and the transcript level quantitated with an ABI PRISM 7700 Sequence
Detection System (PE Applied Biosystems, Rotkreuz,
Switzerland) according to the manufacturers protocol. Briefly, relative
transcript levels in induced cells and nontreated control cells were
determined using the relative quantitation method measuring the

Ct. The following primers and fluorescent probes were used in
these PCR reactions: CYP2H1: probe, 5'-TCG CAG TTG CCT CCA GGT CTC
CC-3'; forward primer, 5'-AGG GTG GTG AGG GCA AAT C-3'; reverse primer,
5'-ACA GGC ATT GTG ACC AGC AA-3'; CYP3A37: probe, 5'-TTG GCC CAG GAA
TGC CCA GCT-3'; forward primer, 5'-GTC CCA AAG AAA GGC AAT GGT 3';
reverse primer, 5'-GGC CAT TTG GGT TGT TCA AG-3'; ALAS: probe, 5'-TTC
CGC CAT AAC GAC GTC AAC CAT CTT- 3'; forward primer, 5'-GCA GGG TGC CAA
AAC ACA T-3'; reverse primer, 5'-TCG ATG GAT CAG ACT TCT TCA ACA-3';
glyceraldehyde 3-phosphate dehydrogenase (GAPDH): probe, 5'-TGG CGT GCC
CAT TGA TCA CAA GTT T-3'; forward primer, 5'-GGT CAC GCT CCT GGA AGA
TAG T-3'; reverse primer, 5'-GGG CAC TGT CAA GGC TGA GA-3'. CYP2H1,
CYP3A37, ALAS, and GAPDH transcript levels were measured in separate
tubes, and GAPDH was used for normalization of the CYP2H1, the CYP3A37,
and the ALAS values.
Western Blot Analysis
Chicken embryo livers were frozen in liquid nitrogen and crushed
in a mortar. The tissue was resuspended in 10 mM HEPES, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride (lysis buffer) and sonicated three times
for 10 sec on ice. The extract was centrifuged at 4 C for 20 min at
14,000 rpm. Chick embryo hepatocytes were cultured and prepared as
previously described (53, 54). Primary hepatocytes and LMH
cells were washed twice with PBS, resuspended in lysis buffer,
sonicated, and centrifuged as described above. The protein
concentration of the supernatant was determined using the ESL protein
assay (Roche Molecular Biochemicals). Equal amounts of
protein were separated on 10% Tricine-SDS-PAGE. The proteins were
blotted onto a NYTRAN 13 N membrane (Schleicher & Schuell, Inc., Dassel, Germany) using a Multiphor II Nova Blot (Pharmacia
LKB, Dübendorf, Switzerland) and 39 mM
glycine, 48 mM Tris, 0.0375% SDS, and 20% methanol for
semidry transfer of the proteins at 0.8 mA/cm2
for 1 h. Proteins were visualized according to the enhanced
chemiluminescence protocol (Amersham Pharmacia Biotech,
Zürich, Switzerland) using an anti-CYP2H1 antibody. This
polyclonal rabbit antibody was generously provided by Dr. P.
Sinclair (VA Hospital, White River Junction, VT).
Northern Blot Analysis
Total RNA (10 µg) was subjected to electrophoresis on a 1%
agarose/formamide gel. RNA was transferred to a NYTRAN 13 N membrane
(Schleicher & Schuell, Inc.) overnight in 10x SSC (1.5
M NaCl, 150 mM sodium citrate) and was
cross-linked to the membrane by UV exposure for 12 sec.
Prehybridization was carried out in 50% formamide, 5x SSC, 5x
Denhardts solution, 1% SDS, and 10% (wt/vol) dextran sulfate.
Hybridization probes were generated by PCR amplification using the
primers mentioned above and labeled with
[
-32P]dATP using the Random Primed DNA
Labeling Kit (Roche Molecular Biochemicals). The probes
were boiled for 5 min in 500 µl of 10 mg/ml salmon sperm DNA and
chilled on ice. Hybridization was carried out for 1620 h at 42 C.
Washes were performed in 2x SSC/0.1% SDS at room temperature for 30
min and in 2x SSC/0.1% SDS at 65 C for 20 min. Membranes were exposed
to x-ray film using intensifying screens or to PhosphorImager screens
for 1248 h.
EMSAs
Chicken CXR and chicken RXR
were synthesized in
vitro using the TNT T7 Quick Coupled Transcription/Translation
System (Promega Corp., Catalys AG, Wallisellen,
Switzerland) according to the manufacturers instructions. Chicken
RXR
was chosen for these experiments because RXR
is the only
chicken RXR ortholog known so far. Probes were labeled with Klenow
enzyme in the presence of radiolabeled
32P-
-ATP, and the probe was purified over a
Biospin 30 Chromatography Column (Bio-Rad Laboratories, Inc., Glattbrugg, Switzerland). A volume of labeled
oligonucleotide corresponding to 100,000 cpm was used for each reaction
in 10 mM Tris (pH 8.0), 40
mM KCl, 0.05% NP-40, 6% glycerol, 1
mM dithiothreitol, 0.4 µg/µl BSA, 0.2 µg
poly(dI-dC)1poly(dI-dC), and 2.5 µl of each of the in
vitro synthesized proteins as described previously
(22). To test for supershifts, 0.5 µl of monoclonal
antimouse-RXR rabbit antibody (kindly provided by Dr. P. Chambon,
IGBMC, Université Louis Pasteur, Illkirch, France) was added to
the reaction mix. This antibody has been positively tested for
cross-reaction with the chicken RXR
in Western blots (data not
shown). The mix was incubated for 20 min at room temperature and
subsequently electrophoresed on a 6% polyacrylamide gel in 0.25x
Tris-Borate-EDTA buffer followed by autoradiography at -70 C.
Transcriptional Activation Assays
To perform transactivation assays, CV-1 cells were kept in
DMEM/F12 medium without phenol red, supplemented with 10%
charcoal-stripped FBS, and plated in six-well dishes at a density of
625,000 cells per well. A total of 2.5 µg DNA per well, including 150
ng of receptor expression vector, 400 ng of CAT reporter gene plasmid,
800 ng pSV-ß-galactosidase expression vector (Promega Corp.) and carrier plasmid were transfected and cells were
exposed to drugs. Cell extracts were prepared and assayed for CAT using
a CAT-ELISA kit (Roche Molecular Biochemicals).
ß-Galactosidase activities were determined. CAT concentrations were
then normalized against ß-galactosidase values to compensate for
varying transfection efficiencies as described previously
(22).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Urs A. Meyer, Division of Pharmacology/Neurobiology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. E-mail:
Urs-A.Meyer{at}unibas.ch
This work was supported by the Swiss National Science Foundation.
1 Present address: Friedrich Miescher Institute,
Maulbeerstrasse 66, CH-4058 Basel, Switzerland. 
2 ProteinGenesys Ltd., Rheinstrasse 28, CH-4302 Augst,
Switzerland. 
Abbreviations: ALAS,
-aminolevulinate synthase; CAR,
constitutive androstane receptor; CAT, chloramphenicol
acetyltransferase; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; CXR, chicken
xenobiotic receptor; CYP, cytochrome P-450; FXR, farnesoid X receptor;
LMH, leghorn male hepatoma; PB, phenobarbital; PBRU, PB-responsive
enhancer unit; PCN, 5-pregnen-3ß-ol-20-one-16
-carbonitrile; PIA,
propylisopropylacetamide; PP, protein phosphatase; PXR, pregnane X
receptor; SSC, NaCl-sodium citrate; tk, thymidine kinase.
Received for publication December 20, 2000.
Accepted for publication June 1, 2001.
 |
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