7-Ketocholesterol Is an Endogenous Modulator for the
Arylhydrocarbon Receptor*
Jean-Francois
Savouret
§,
Monica
Antenos¶
,
Monique
Quesne
,
Jing
Xu¶,
Edwin
Milgrom
, and
Robert Frederick
Casper¶
From the ¶ Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, Toronto, Ontario M5G 1X5, Canada, the
Department of
Zoology, University of Toronto, Toronto, Ontario M5G 1A1, Canada, and
the
INSERM Unit 135, Hopital de Bicetre, CHU Niveau 3, 78 rue du Général Leclerc, Le Kremlin-Bicetre
94270, France
Received for publication, July 7, 2000, and in revised form, October 20, 2000
 |
ABSTRACT |
We have identified 7-ketocholesterol (7-KC) as an
endogenous modulator that inhibits transactivation by the
arylhydrocarbon receptor (AhR) through competitive binding against
xenobiotic ligands. 7-KC binds AhR and displaces labeled dioxin
(2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD)).
IC50 is 5 × 10
7
M in vivo and 7 × 10
6 M in vitro. These
figures are consistent with its concentration in human blood plasma and
tissues. Association with 7-KC prevents AhR binding to DNA. 7-KC blocks
the TCDD-mediated transactivation of stably expressed reporter gene
constructs in T47-D cells as well as the expression of the endogenous
CYP 1A1 gene in HepG2 cells and in primary porcine aortic endothelial
cells. Injection of 7-KC to rats blocks the induction of CYP 1A1
messenger RNA and protein in endothelial cells from myocardial blood
vessels. The differential sensitivity of mammalian species to toxic
effects of AhR ligands, especially dioxin (TCDD), correlates with the expression of 7-hydroxycholesterol dehydrogenase, which synthesizes 7-KC from 7-hydroxycholesterol. The documented involvement of AhR
ligands in cardiovascular diseases through lipid peroxidation and
endothelium dysfunction can now be examined in the context of
displacement of this protective modulator.
 |
INTRODUCTION |
Extreme interspecies variations in toxicity are a striking feature
of xenobiotics that bind the arylhydrocarbon receptor
(AhR)1. Such compounds
comprise dioxins (including the prototypical dioxin:
2,3,7,8-tetrachlorodibenzo(p)dioxin, TCDD),
benzo(a)pyrene (B(a)P), and
polychlorobiphenyls. A recent review discusses the numerous
publications reporting that hamsters are very resistant to TCDD
(LD50 > 3 mg/kg), humans seem less sensitive than most laboratory animals, while guinea pigs, rats, and mice are most sensitive (LD50 around 1 µg/kg), with detectable
interstrain variations (1). This phenomenon is not restricted to AhR
ligands: in most cases, the variable occurrence of a specific enzyme
accounts for the variations in drug or hormone sensitivity (1). We
recently described the binding of the trihydroxystilbene resveratrol to the AhR and the subsequent antagonization of toxic effects of AhR
ligands (2). We observed the parallel between the alleged anti-inflammatory and cardioprotective role of resveratrol and the
increasing amount of evidence for the involvement of AhR ligands in
cardiovascular diseases (3-5). A variety of data suggest that these
effects occur through their oxidative and atherogenic properties mediated by the AhR in the vascular endothelium (6, 7). Due to the
structural requirements of the AhR for ligand accommodation, putative
endogenous AhR ligand(s) or modulator(s) also involved in these
mechanisms could be oxidized lipidic compound(s), preferably polycyclic hydrocarbons.
7-Hydroxycholesterol dehydrogenase (7-HCD), the enzyme that synthesizes
7-ketocholesterol (7-KC) from 7-
,
-hydroxycholesterols, is
expressed in large amounts in various tissues of the hamster, moderately expressed in humans and bovines, and is totally absent in
rabbits, rats, and mice (8-10). This distribution closely correlates with the observed degrees of resistance to TCDD (reviewed in Ref. 1).
Accordingly, by testing a panel of the most frequent oxysterols present
in blood, we have identified 7-KC as an endogenous, physiological modulator for AhR. We describe here the parameters of this interaction and its inhibitory effects on the gene transactivation properties of
the AhR in vitro and in vivo.
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EXPERIMENTAL PROCEDURES |
Chemicals--
TCDD was a generous gift from Dr S. Safe (Texas
A & M University, College Station, TX).
2,3,7,8-Tetrachloro(1,6-3H)dibenzo-p-dioxin, 28 Ci/mmol was purchased from Terrachem (Lenexa, KS). Dioxin stock
solutions were initially dissolved in dimethyl sulfoxide and handled
under a fume hood. TCDD stock was subsequently diluted in ethanol for
use in experiments described below. Steroids were purchased from
Steraloids (Wilton, NH). All other chemicals were purchased from Sigma.
Cell Culture and CAT Assay--
All culture media were from Life
Technologies, Inc. T47-D and HepG2 cells lines were routinely grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 1 g/liter glucose, 2 mM sodium pyruvate, 50 nM sodium selenite, and 0.6 unit/ml
(10
6 M) insulin. Unless stated
otherwise, cells were established 24 h before any experiment in a
modified form (stripped condition) of the medium: 1% charcoal-stripped
newborn calf serum and 0.5 g/liter glucose. All other components
remained unchanged. The stable cell line 47DRE bearing the
TCDD-responsive CAT construct was described in Ref. 2. Porcine aortic
endothelial cells (PAEC) (a gift from Dr. Langille, Toronto General
Hospital, Toronto, Ontario, Canada) of passage 4-7 were maintained in
199 medium supplemented with 5% fetal bovine serum.
Northern Blot Experiments--
RNA from rat heart tissues were
extracted using the TRIzol reagent (Life Technologies, Inc.). 10 µg
of RNA were electrophoresed on a 1% agarose-formaldehyde gel and
transferred overnight to a nylon membrane (Nytran, Keene, NH).
The membrane was dried at room temperature and then hybridized
overnight at 60 °C with a 32P-labeled rat CYP 1A1
cDNA probe. The blot was exposed to a PhosphorImager screen
and scanned using Image Quant software (version 5.0). The blot was
later hybridized with rat
-actin cDNA, which served as a sample
control. Scan generated values were used to calculate for each group
the mean and standard error of the mean (S.E.) values.
Western Blot Experiments--
Cells were treated with drugs in
stripped condition as described in the figures legends and processed as
described previously (2). The rabbit polyclonal antibody against CYP
1A1 and the CYP 1A1 protein standard were from Daiichi Pure Chemicals
Co. (Tokyo, Japan). The mouse monoclonal antibody against the
AhR was from Affinity Bioreagents Inc. (Golden, CO). All were used at 1 µg/ml.
Whole Cell Binding Assay--
T47-D cells were plated in
six-well culture dishes in standard passage conditions. At 70%
confluence, cells were established in 2 ml of Opti-MEM I medium
supplemented with 0.5 mg/ml bovine serum albumin for 24 h. Binding
was performed the next day after a change of medium, in the
CO2 incubator, at 37 °C for 20 min with labeled TCDD (5 nM) alone or challenged with unlabeled competitors. Cells
were then taken at 4 °C and washed four times 10 min with 2 ml of
phosphate-buffered saline (PBS) containing 0.5 mg/ml bovine serum
albumin at 4 °C. Cells were lysed for 30 min at room temperature in
800 µl of cytosol buffer (20 mM Hepes, pH 7.6, 100 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, with Complete protease mixture
(Roche Molecular Biochemicals, Meylan, France) containing 1% Nonidet
P-40. Protein contents were measured, and aliquotes were counted for
radioactivity: 500 µl of the supernatants were counted in 5 ml of
Ultima Gold mixture (Packard, Meridien, CT) in a Beckman liquid
scintillation counter (45% counting efficiency). Binding competition
assays were repeated twice for each competitor and each point performed
in duplicate.
In Vitro Binding Assay--
Binding competition was performed
using female New Zealand rabbit liver cytosol as the receptor source.
The cytosol was prepared at 4 °C in 20 mM Hepes, pH 7.6, 1.5 mM EDTA, 10% glycerol, 10 mM
-mercaptoethanol, with Complete protease mixture (Roche
Molecular Biochemicals, Meylan, France) by homogenization in an
Ultra-Turax homogenizer (Bioblock Scientific, Illkirch, France)
followed with 20 strokes in a Dounce homogenizer with the tight pestle.
The homogenate was centrifuged 30 min at 20,000 × g.
The supernatant was centrifuged at 105,000 × g for 65 min. The cytosol was aliquoted, snap-frozen in liquid nitrogen, and
kept at
80 °C. Cytosol aliquotes (4.5 ml) were thawed on ice,
diluted 10-fold in 20 mM Hepes, pH 7.6, 0.1 mM
EDTA, containing Complete protease mixture and 10 mM
-mercaptoethanol, and recentrifuged 30 min at 105,000 × g. The cleared cytosol dilution was adjusted to 1 mM CaCl2 and 0.85 mg of protein/ml. 930 µl of
diluted cytosol were preincubated with the desired amounts of unlabeled
competitors in ethanol solutions for 1 h at 4 °C. Subsequently,
2,3,7,8-tetrachloro(1,6-3H)dibenzo-p-dioxin (28 Ci/mmol) was added at 0.2 nM and incubation continued for
3 h at 4 °C. Ethanol volume was adjusted in all tubes at 0.8%
to optimize 7-KC solubilization without hampering specific binding.
Nondisplaceable binding was assessed by incubating the aliquotes with
70 µl of a 2% activated charcoal suspension in 20 mM
Hepes, pH 7.6, for 90 min at 4 °C, followed by centrifugation at
15,000 × g for 10 min. 500 µl of the supernatants
were counted in 5 ml of Ultima Gold mixture (Packard, Meridien,
CT) in a Beckman liquid scintillation counter (45% counting
efficiency). Binding competition assays were repeated at least twice
for each competitor, and each point was performed in triplicate.
In Vitro DNA Binding--
T47-D cells were established in
stripped condition for 48 h. When they reached 90% confluence,
they were treated with drugs for 90 min in the incubator, as described
in the legend to Fig. 4. The probe used for gel retardation was
a 35-base pair double-stranded oligonucleotide bearing a single copy of
the CYP 1A1 gene DRE: '-AGCTTAGCTAGGCGTTGCGTGAGAAGGACCG-3'.
Cell extracts were prepared and gel retardation assays done as
described previously (2). Another oligonucleotide was also used in
chase experiments, bearing a copy of the sterol responsive element
(SRE) from the low density lipoprotein receptor gene promoter (11):
5'-GATCCATTTGAAAATCACCCCACTGCAAACTC-3'.
In Vivo Antagonism Experiments--
36 male Harlan
Sprague-Dawley rats were used for this 11-day study: group 1 were
controls (n = 6). Animals were subcutaneously injected
with olive oil (vehicle) on days 1, 4, and 7. Group 2 (n = 10) were injected with 1 mg/kg
B(a)P/DMBA on days 1 and 7. Group 3 (n = 10)
were injected with 1 mg/kg 7-KC on days 1, 4, and 7. Group 4 (n = 10) were injected with mixtures of
B(a)P/DMBA and 7-KC in a 1:1:1 ratio on days 1, 4 (7-KC
only), and 7. Animals were sacrificed on day 11. Tissues were harvested
and snap-frozen in liquid N2. Protein, mRNA, and
immunocytochemical studies were then performed.
Immunocytochemistry--
Rat tissue samples were fixed in
phosphate-buffered paraformaldehyde (4%), dehydrated, and embedded in
paraffin. Paraffin sections were deparaffinated in xylene, rehydrated
in alcohol series to water, and washed in PBS with 1% Triton X-100.
Sections were incubated in 0.3% H2O2 for 30 min to quench endoperoxidase activity. Following PBS wash, sections
were preincubated in 10% horse serum and 2% bovine serum albumin
diluted in PBS for 30 min and then incubated with rabbit polyclonal
antibody against CYP 1A1 at 1:500 overnight at 4 °C. Subsequent
antibody detection was performed with biotinylated anti-rabbit mouse
IgG and the Vectastain ABC kit (Vectastain, Burlingame, CA) with
3,3-diamidobenzidine as peroxidase substrate. Sections were then
counterstained with hematoxylin, dehydrated, and mounted in DPX
Mountant (Fluka, Toronto, Canada).
 |
RESULTS |
In the search for AhR-modulating oxysterols, we focused primarily
on 7-KC and on other molecular species identified in human plasma
lipoproteins at concentrations compatible with efficient receptor
interactions. We expected the active compound to behave as antagonists
on TCDD-like effects such as CYP 1A1 or interleukin-1
overexpression
as well as on TCDD-mediated transactivation in general. From a panel of
nine oxysterols present in human plasma, including those able to
inhibit TCDD-mediated transactivation, only 7-KC was able to displace
labeled TCDD from the AhR in an in vivo competition assay
using T47-D cells (Fig. 1). Side chain hydroxylated cholesterol derivatives (20-, 22-, and 25-OH) as well as
6- and 7-ketocholestanols were ineffective (not shown). The in
vivo competition assay (Fig.
2A) shows that 7-KC displaced labeled TCDD from its receptor with an IC50 of 5 × 10
7 M, in close correlation with
its circulating concentration (12). These results were confirmed with
an in vitro competition assay (Fig. 2B): in these
conditions 7-KC displaced labeled TCDD with an IC50 of
7 × 10
6 M.

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Fig. 1.
7-KC displaces TCDD in whole cell binding
assay. Nine oxysterols present in human plasma were tested for
their ability to displace labeled [3H]TCDD from the AhR
in in vivo whole cells uptake competition assays. Of those
tested, only 7-KC was capable of displacing approximately 80% of the
labeled TCDD from the receptor at 5 × 10 6 M. Briefly, T47-D cells were
incubated with unlabeled oxysterols (5 × 10 6 M) and [3H]TCDD
(5 × 10 9 M) for 20 min in a
37 °C CO2 incubator. Cells were extensively washed and
lysed in a cytosolic buffer containing 1% Nonidet P-40. Protein
aliquots were then quantitated and counted in a Beckman liquid
scintillation counter. Assays were repeated twice, and each point was
performed in triplicate. Mean ± S.E. are shown.
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Fig. 2.
7-KC displaces labeled TCDD in binding
competition assays for the arylhydrocarbon receptor. Whole cell
and in vitro cytosol binding competition assays with labeled
TCDD are described under "Experimental Procedures." Competitors are
unlabeled TCDD (squares) or 7-KC (open circles).
A, in vivo binding competition assay: 7-KC
displaces labeled TCDD from the arylhydrocarbon receptor (AhR) of T47-D
cells at physiological concentrations found in human blood plasma. The
assay was performed as in Fig. 1. The IC50 for 7-KC
competition of TCDD binding in vivo is 5 × 10 7 M. B, in
vitro binding competition assay for rabbit liver cytosol AhR: 7-KC
displaces labeled TCDD with an IC50 of 7 × 10 6 M. Specific binding in
cytosol was 913 ± 35 dpm/mg protein (mean ± S.E.).
Nonspecific binding amounted to 50% of total binding. With a specific
activity of 4937 dpm/pmol, the specific binding corresponds to 183 ± 7 fmol of AhR per mg of rabbit liver cytosolic protein in these
nonsaturating conditions (n = 6).
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We then performed a dose-response analysis of the transcriptional
repression ability of 7-KC in the 47DRE stable cell line. Fig.
3 shows that 7-KC counteracts
TCDD-mediated transactivation with an IC50 of 2 × 10
6 M, a value consistent with
its binding ability to AhR. The gel retardation experiment in Fig.
4 revealed that 7-KC prevents AhR from
binding to its cognate DNA binding element in T47-D cells extracts, in
contrast to TCDD and resveratrol, which elicit complex formation. The
binding was efficiently competed by a 50-fold excess of unlabeled DRE
oligonucleotide as well as a 300-fold excess of a SRE oligonucleotide,
showing that the AhR displays a moderate affinity for these regulatory
elements. Binding of AhR to the SRE was not directly amenable to gel
retardation electrophoresis, probably due to the observed weaker
affinity. Preincubation with a polyclonal antibody against AhR
inhibited complex formation as previously shown (2-13).

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Fig. 3.
7-KC blocks TCDD-mediated transactivation in
a dose-response manner. 47DRE cells were established in stripped
condition for 24 h then challenged with
10 9 M TCDD alone or in
combination with increasing concentrations of 7-KC. CAT assay was
performed after 48 h. IC50 was 2 × 10 6 M. Assays were repeated
twice, and each point was performed in triplicate. Mean ± S.E. are shown.
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Fig. 4.
Gel retardation assay shows that 7-KC
prevents the AhR from binding to DNA. T47-D cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 1%
charcoal-stripped newborn calf serum and 0.5 g/liter glucose
for 48 h. Cells were treated with ethanol (control) (0.1%), TCDD
(5 × 10 9 M), 7-KC (5 × 10 6 M), or resveratrol (5 × 10 6 M) for 90 min. Cells
extracts were prepared as described (2) and incubated with a
32P-labeled 35-base pair double-stranded oligonucleotide
bearing a single copy of the CYP 1A1 DRE. The incubate was run on a
nondenaturing polyacrylamide gel. Lane 1, control (untreated
cytosol); lane 2, TCDD (5 × 10 9 M); lane 3, TCDD
plus unlabeled DRE oligonucleotide (50-fold excess); lane 4,
TCDD plus unlabeled SRE oligonucleotide (300-fold excess); lane
5, TCDD plus polyclonal antibody against AhR; lane 6,
TCDD and antibody against SRE-binding protein 1; lane 7,
control (ethanol 0.1%); lane 8, 7-KC, 5 × 10 6 M; lane 9,
resveratrol, 5 × 10 6 M. The
arrow points to the specific AhR-containing band.
Arrowheads depict nonspecific bands.
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We confirmed these in vitro data with endogenous CYP 1A1
expression experiments. Western blot experiments showed that 7-KC efficiently blocks the induction by TCDD of the endogenous CYP 1A1
protein in HepG2 cells (Fig. 5). When
administered alone, 7-KC is able to suppress the basal level of CYP 1A1
expression observable in this cell line. This supports our hypothesis
of 7-KC as a protective, inhibitory modulator of AhR. We also analyzed the inhibition of B(a)P/DMBA-induced CYP 1A1 protein in
primary cultures of PAEC by Western blot analysis to eliminate the
possibility that our results could be limited to the above mentioned
established cell lines. 7-KC efficiently reduced CYP 1A1 protein levels
induced by B(a)P/DMBA in PAEC. In contrast, resveratrol was
ineffective in PAEC (Fig. 6). This
discrepancy could be due to a faster metabolization of resveratrol in
these cells or to the inability of resveratrol to bind the porcine
AhR.

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Fig. 5.
7-KC competes CYP 1A1 induction by TCDD in
HepG2 cells. HepG2 cells were treated with ethanol (0.1%), TCDD
(10 9 M), resveratrol (5 × 10 6 M), or 7-KC (5 × 10 6 M). Proteins were extracted
from HepG2 cells following a 48-h treatment and separated by
SDS-polyacrylamide gel electrophoresis. The resolved proteins were
transferred to membrane and detected with a polyclonal antibody against
CYP 1A1. As with resveratrol, 7-KC reduced the protein levels of CYP
1A1 induced by a 48-h treatment with TCDD. 7-KC alone elicited
suppression of the basal levels of CYP 1A1 protein in this cell
line.
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Fig. 6.
7-KC reduces CYP 1A1 levels in PAEC induced
by B(a)P/DMBA exposure. PAEC (a gift from Dr.
Langille, Toronto General Hospital, Toronto, Ontario, Canada) at
passages 4-7 were maintained in 199 medium supplemented with
5% fetal bovine serum. Cells were treated with B(a)P/DMBA
(10 7 M) and 7-KC (in varying
concentrations) and resveratrol (10 6
M) for 48 h. Protein extracts were harvested as
described previously, and CYP 1A1 protein levels were examined by
Western blot. As was observed in the rat, 7-KC reduced CYP 1A1 protein
levels induced by B(a)P/DMBA. In contrast, resveratrol did
not affect the protein levels of CYP 1A1 in this experiment. Lane
1, CYP 1A1 protein standard; lane 2, control (ethanol
0.1%); lane 3, B(a)P/DMBA; lane 4,
B(a)P/DMBA and resveratrol; lane 5, resveratrol;
lane 6, 7-KC; lane 7, B(a)P/DMBA and
7-KC (10 5 M); lane 8,
B(a)P/DMBA and 7-KC (10 6
M); lane 9, B(a)P/DMBA and 7-KC
(10 7 M).
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Finally, we ascertained whether these results would concur with the
in vivo situation. In keeping with our previous work (2) and
to focus on the mechanism of tobacco-related cardiovascular diseases,
rats were treated by subcutaneous injections of B(a)P/DMBA (1 mg/kg) alone or in conjunction with 7-KC (1 mg/kg) as described under "Experimental Procedures." The ability of 7-KC to prevent B(a)P-mediated induction of CYP 1A1 was demonstrated by
Northern blot followed by scanning densitometry of the bands (Fig.
7A), Western blot (Fig.
7B), and immunocytochemistry (Fig.
8). As can be seen on the
photomicrographs in Fig. 8, CYP 1A1 induction by B(a)P/DMBA
was limited to the endothelial cells of myocardial blood vessels and
absent from myocardial tissue. 7-KC efficiently suppressed the
overexpression of CYP 1A1.

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Fig. 7.
7-Ketocholesterol antagonizes
B(a)P/DMBA in the heart tissue of Harlan
Sprague-Dawley rats. In vivo antagonism experiments
using male rats were performed to ascertain whether the ex
vivo results obtained would concur with the in vivo
situations. 36 male rats were utilized for this 11-day study: group 1 were controls (n = 6). Animals were subcutaneously
injected with olive oil (vehicle) on days 1, 4, and 7. Group 2 (n = 10) were injected with 1 mg/kg of
B(a)P/DMBA on days 1 and 7. Group 3 (n = 10)
were injected with 1 mg/kg 7-KC on days 1, 4, and 7. Group 4 (n = 10) were injected with mixtures of
B(a)P/DMBA and 7-KC in a 1:1:1 ratio on days 1, 4 (7-KC
only), and 7. Animals were sacrificed on day 11. Tissues were harvested
and snap-frozen in liquid N2. Protein, mRNA, and
immunocytochemical studies were then performed. A, Northern
analysis of mRNA in heart tissue showed that 7-KC challenges
B(a)P/DMBA induction of CYP 1A1 and reduced CYP 1A1 mRNA
levels by 45%. Mean ± S.E. are shown for each group.
B, CYP 1A1 protein was also significantly reduced in the
heart by cotreatment of 7-KC and B(a)P/DMBA. Representative
results obtained from two different animals are shown. Lane
1, CYP 1A1 protein standard; lane 2, control (vehicle);
lanes 3 and 6, B(a)P/DMBA; lanes
4 and 7, 7-KC; lanes 5 and 8,
B(a)P/DMBA and 7-KC.
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Fig. 8.
Immunocytochemistry for CYP 1A1 expression in
the rat heart. Following B(a)P/DMBA exposure, CYP 1A1
expression was induced in endothelial cells of rat myocardial vessels
but not in the myocardial tissue. CYP 1A1 overexpression was decreased
in endothelial cells of animals treated with B(a)P/DMBA and
7-KC. Arrows point at the outer boundaries of the
endothelial vessels.
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DISCUSSION |
The search for a physiological, endogenous ligand for AhR has been
ongoing for several years and has produced very dissimilar contenders
such as lipoxin A4, bilirubin, and tryptophan or vitamin B2
metabolites (14-17). None of these compounds shed any light on the
various features of AhR ligands toxicity and especially the
interspecies variation in sensitivity to AhR ligands. Our observation,
from literature analysis, that species-specific 7-HCD expression
was correlated with TCDD resistance led us to describe this inhibitory
interaction between 7-KC and the AhR.
The known examples of species-specific variation in sensitivity to
drugs are linked to the variable occurrence of enzymes that either
metabolize the xenobiotic (aflatoxin B1 resistance of mice) or convert
it into a more toxic compound (rapid acetylator phenotype for
heterocyclic amines and generation of carcinogenic diol-epoxides from
benzo(a)pyrene), as was reviewed in Ref. 1. Hamsters are
also more resistant to digitoxin due to hyperexpression of cytochrome
P450 3A (which catalyzes testosterone 6-hydroxylation) (18). In
contrast, we present here a different mechanism, i.e. the
production of an endogenous antagonistic compound as a protective agent
against xenobiotics. The possibility that AhR is redundant with other
more recent lipid-dependent modulators such as adipocyte differentiation factor 1 (19) or oxysterol receptors (20) or represents
a specialized subdomain in lipid metabolism remains open. The 7-HCD
gene has not been described yet, nor has the regulation of its
expression in 7-HCD-expressing mammals. In any case, it will be
interesting to search for correlations between predisposition to
certain cancers or diseases (especially cardiovascular diseases) and
genetic polymorphism or mutations occurring in this gene.
Cholesterol and oxysterols are basic elements in cellular homeostasis
for the constitution of membranes as well as steroidogenesis (reviewed
in Refs. 21 and 22). It was initially believed that oxysterols were
mainly cytotoxic molecules, but cytotoxicity assays for oxysterols were
usually conducted with nonphysiological concentrations (10
4 to 10
5
M), utilizing rather crude end points (21, 22). However, as
first proposed by Kandutsch and Chen (23), oxysterols are now more and
more recognized as bona fide regulatory molecules in various
physiological domains (reviewed in Ref. 24). The suspicion for
cytotoxicity is now aimed at more oxidized molecules such as
cholesterol peroxides (25, 26). 7-KC may be only a precursor of these
more toxic molecules, synthesized in an uncontrolled way during cell
culture experiments using high levels of 7-KC. However, this
controversy is still fueled by a wealth of data on cytotoxic and
apoptotic effects of 7-KC and other oxysterols (reviewed in Ref. 27).
Oxysterols originate from the oxidation of dietary cholesterol but also
from endogenous pathways involving 7-hydroxylases and 7-HCD (8-10).
Their ability to repress enzymatic activities has been known for 20 years, but their direct impact on gene expression through the
activation of nuclear receptors is a more recent concept. Receptors
from the steroid superfamily existing as heterodimers with retinoid X
receptor have been identified and shown to mediate
transactivation either under the control of bile acids (FXR) or
of a variety of hydroxysterols substituted in 4, 7, 20, 22, or
25 (LXR) (reviewed in Ref. 20). These receptors have a
low affinity for their ligands (Kd values
around 10
6 to 10
5
M), but the values are in the range of the blood plasma
concentrations of these oxysterols.
We were unable to assay a complete panel of 7-ketosterols and
7-ketosteroids, due to the lack of availability of many of them, especially derivatives of sex hormones. Nevertheless, most of these
compounds are present in minute amounts and could hardly participate in
a regulatory mechanism involving the AhR. 7-KC, on the contrary, is a
major sterol constituent of plasma in humans: Dzeletovic et
al. (12), using mass spectrometry, report a plasma concentration
of 0.55 × 10
7 M (range
0.2-2) in healthy humans. This was correlated with a concentration in
low density lipoproteins (LDLs) of 211 ± 86 ng/mg LDL proteins.
These values are in good agreement with previous data obtained by gas
chromatography (28). In keeping with our determination of an
IC50 of 5 × 10
7
M for the in vivo binding competition of labeled
TCDD by 7-KC, these values are in a range that allows 7-KC to act as a
physiological AhR modulator. We confirmed these in vivo data
with an in vitro competition assay. This showed that 7-KC
displaces labeled TCDD with a lower IC50 (7 × 10
6 M) in vitro. This
14-fold difference is reminiscent of a comparable difference observed
between in vivo and in vitro competitions in our
resveratrol studies (2). We were unable to achieve complete competition
beyond 41% due to the lack of solubility of higher 7-KC
concentrations. Setting up the assay revealed that efficient competition of TCDD binding to the AhR by 7-KC required preincubation of the competitor before addition of the labeled TCDD as well as the
presence of 1 mM calcium chloride in the incubation medium. Such specific conditions, unnecessary for our classic assay (2), suggest that TCDD and 7-KC may have different structural requirements for binding to the AhR if not distinct binding sites. Hence our use of
the word "modulator" instead of "endogenous ligand." This hypothesis is currently under scrutiny in our laboratory. Other oxysterols reported in the Dzeletovic study (12) are present in similar
concentrations in humans: 10
9 to
10
7 M for 7
- and
7
-hydroxycholesterol and 0 to 10
7
M for cholesterol epoxides and cholestanetriol (12). In
contrast to 7-KC, these compounds are not known to be restricted to any particular mammalian species, and they do not bind the AhR. 7- and
6-ketocholestanols are also unable to bind. This high structural specificity of the interaction further supports the relevance of our
findings. The physiological importance of our proposed interaction
between AhR and 7-KC is further supported by the phenotype observed in
in AhR
/
animals (29, 30). The animals showed several hepatic
defects including microvesicular fatty metamorphosis together with a
delayed generation of peripheral monocytes and concomitant reduction in
number. This latter trait may be related to the well documented
immunomodulatory activities of oxysterols (31-35) and the impact of
oxidized low density lipoproteins on macrophage metabolism (36).
Our observations shed new light on the documented effects of AhR on the
metabolism of lipids as well as on pre-adipocyte differentiation (37,
38). The regulatory network created by LXRs, FXR, sterol responsive element-binding proteins (such as adipocyte differentiation factor 1) and AhR should be reconsidered at the double level of ligand
preference and cross-talk between receptors. AhR has been shown to be
able to counteract several members of the steroid receptor superfamily,
such as the estrogen receptor (Ref. 39 and references within) or the
progesterone receptor.2 As an
example of the multiple levels of possible interplay, cholesterol 7-hydroxylases are induced by LXR activated by 22-OH
cholesterol. This enzyme is the first step toward 7-KC biosynthesis.
7-KC will in turn repress the expression of these hydroxylases
(reviewed in Refs. 21 and 22). The involvement of AhR in human diseases can now be scrutinized under multiple new approaches.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: INSERM Unit 135, Hopital de Bicetre, CHU Niveau 3, 78 rue du Général
Leclerc, Le Kremlin-Bicetre 94270, France. Tel.: 33-1-45-21-33-29; Fax: 33-1-45-21-27-51; E-mail: savouret@kb.inserm.fr.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M005988200
2
J.-F. Savouret, M. Antenos, M. Quesne, J. Xu, E. Milgrom, and R. F. Casper, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
AhR, arylhydrocarbon
receptor;
TCDD, 2,3,7,8-tetrachlorodibenzo(p)dioxin;
DRE, dioxin
responsive element;
SRE, sterol responsive element;
CAT, chloramphenicol acetyltransferase;
CYP 1A1, cytochrome P450 1A1;
B(a)P, benzo(a)pyrene;
DMBA, 7,12-dimethyl-benzanthracene;
LDL, low density lipoprotein;
7-KC, 7-ketocholesterol.
 |
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