(Received for publication, December 5, 1996)
From the Departments of Molecular Endocrinology,
Metabolic Diseases, ¶ Molecular Sciences, and
Medicinal Chemistry, Glaxo Wellcome Research and
Development, Research Triangle Park, North Carolina 27709 and the
** Department of Chemistry, Dartmouth College,
Hanover, New Hampshire 03755
Accumulation of cholesterol causes both
repression of genes controlling cholesterol biosynthesis and cellular
uptake and induction of cholesterol 7-hydroxylase, which leads to
the removal of cholesterol by increased metabolism to bile acids. Here,
we report that LXR
and LXR
, two orphan members of the nuclear
receptor superfamily, are activated by
24(S),25-epoxycholesterol and
24(S)-hydroxycholesterol at physiologic concentrations. In
addition, we have identified an LXR response element in the promoter
region of the rat cholesterol 7
-hydroxylase gene. Our data provide
evidence for a new hormonal signaling pathway that activates
transcription in response to oxysterols and suggest that LXRs play a
critical role in the regulation of cholesterol homeostasis.
Cholesterol (CH)1 is a major structural constituent of cellular membranes and serves as the biosynthetic precursor for bile acids and steroid hormones. Animal cells can obtain CH endogenously through de novo synthesis from acetyl-CoA or exogenously through receptor-mediated endocytosis of low density lipoproteins. Cells must balance the internal and external sources of CH so as to maintain mevalonate biosynthesis while at the same time avoiding the accumulation of excess CH, which can result in diseases such as atherosclerosis, gallstones, and several lipid storage disorders (1).
CH homeostasis is maintained in part through feedback regulation of the low density lipoprotein receptor gene and at least two genes encoding enzymes in the CH biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase (1). Although increases in dietary CH lead to the inhibition of expression of these genes in vivo, it remains unclear whether CH or CH metabolites are responsible for this inhibition (2). Experiments performed in vitro using several different cell lines have indicated that derivatives of CH that are oxygenated on the CH side chain are significantly more potent in the suppression of sterol biosynthesis than CH (3). These oxysterols are produced through the actions of P450 enzymes in various metabolic pathways including bile acid synthesis in the liver and sex hormone synthesis in the adrenal glands. The in vitro activities of oxysterols together with their presence in vivo suggests that oxysterols may serve in metabolic feedback loops to regulate CH homeostasis.
Although CH and its oxysterol metabolites can repress gene
transcription, in at least one instance dietary CH has been shown to
stimulate gene expression. Expression of the cholesterol
7-hydroxylase (CYP7A) gene, which encodes the enzyme responsible for
the initial and rate-limiting step in the conversion of CH to bile
acids (4), is up-regulated in rats fed a CH-rich diet (5-7). This
stimulatory effect provides a regulatory mechanism whereby excess
dietary CH can be converted to more polar bile acids for subsequent
removal from the body. Although the molecular mechanism is unknown,
induction of CYP7A expression in the presence of CH occurs at the level of gene transcription (8, 9).
A variety of CH derivatives, including steroid hormones and vitamin D, exert effects on gene expression through interactions with members of the nuclear receptor superfamily (10). Members of this family function as ligand-activated transcription factors by binding to short stretches of DNA, termed hormone response elements, present in the regulatory regions of target genes. In addition to the nuclear receptors with known ligands, this superfamily includes a large number of structurally related members that contain DNA binding domains and putative ligand binding domains but lack identified ligands, the so-called "orphan receptors."
In this report, we have identified a binding site for the orphan
nuclear receptor LXR in the proximal promoter region of the rat
CYP7A gene. LXR
and the closely related orphan receptor LXR
are
broadly expressed and bind to DNA as heterodimers with the retinoid X
receptors (RXRs) (11-14). As part of an effort to identify natural LXR
ligands, we have found that the oxysterols 24(S),25-epoxycholesterol and
24(S)-hydroxycholesterol activate both LXR
and LXR
at
concentrations consistent with those found in tissue extracts. Our
results provide evidence for a novel oxysterol signaling pathway
regulating hepatic CH homeostasis that may also be important in
mediating other cellular and developmental effects of CH.
24(S),25-epoxycholesterol, 24(R),25-epoxycholesterol, 24(S)-hydroxycholesterol, and 24(R)-hydroxycholesterol were prepared as described previously (20, 21). 24-Ketocholesterol was synthesized from cholenic acid by conversion to the Weinreb amide and reaction with isopropyl magnesium chloride. All other compounds were obtained through commercial sources (Sigma; Steraloids).
PlasmidsTo generate the plasmids pSG5-mLXR,
pSG5-hLXR
, and pSG5-hRXR
, the cDNAs encoding the murine
LXR
, human LXR
, or human RXR
were inserted into the expression
vector pSG5 (Stratagene). The GAL4-LXR constructs contain in the pSG5
expression vector the translation initiation sequence and amino acids
1-76 of the glucocorticoid receptor fused to the DNA-binding domain of
the yeast transcription factor GAL4 (amino acids 1-147) and the SV40 large T antigen nuclear localization signal (APKKKRKVG). The cDNAs encoding amino acids 164-447 and 157-440 of the human LXR
and LXR
were amplified by polymerase chain reaction and inserted C-terminal to the nuclear localization sequence to generate the plasmids pSG5GAL4-hLXR
and pSG5GAL4-hLXR
, respectively. The reporter plasmids (CYP7-LXRE)4-tk-CAT and
(DR-4)4-tk-CAT were generated by inserting four copies of
the CYP7-LXRE (5
-gatcCCTTTGGTCACTCAAGTTCAAGTG) or the DR-4-LXRE
(5
-gatcCCTTAGTTCACTCAAGTTCAAGTG) into the BamHI restriction site of pBLCAT2 (29).
CV-1 cells were plated in 24-well
plates in Dulbecco's modified Eagle's medium supplemented with 10%
charcoal-stripped fetal calf serum at a density of 1.2 × 105 cells/well. In general, transfection mixes contained 33 ng of receptor expression vector, 100 ng of reporter plasmid, 200 ng of
-galactosidase expression vector (pCH110, Pharmacia Biotech Inc.),
and 166 ng of carrier plasmid. Cells were transfected overnight by
lipofection using Lipofectamine (Life Technologies Inc.) according to
the manufacturer's instructions. The medium was changed to Dulbecco's
modified Eagle's medium supplemented with 10% delipidated calf serum
(Sigma), and compound was added for 24 h. Cell extracts were
prepared and assayed for chloramphenicol acetyltransferase (CAT) and
-galactosidase activities as described previously (30).
LXR, LXR
, and RXR
were
transcribed and translated in vitro using pSG5-mLXR
,
pSG5-hLXR
, and pSG5-hRXR
as templates and the TNT coupled
transcription/translation system (Promega). Gel mobility shift assays
(20 µl) contained 10 mM Tris (pH 8.0), 40 mM
KCl, 0.1% Nonidet P-40, 6% glycerol, 1 mM dithiothreitol,
0.2 µg of poly(dI-dC), 2.5 µl each of in vitro
synthesized LXR
or LXR
, and RXR proteins. The total amount of
reticulocyte lysate was maintained constant in each reaction (5 µl)
through the addition of unprogrammed lysate. After a 10-min incubation
on ice, 1 ng of 32P-labeled oligonucleotide was added, and
the incubation continued for an additional 10 min. DNA-protein
complexes were resolved on a 4% polyacrylamide gel in 0.5 × TBE
(1 × TBE = 90 mM Tris, 90 mM boric
acid, 2 mM EDTA). Gels were dried and subjected to autoradiography at
70 °C. Liver nuclear extracts were prepared as
described previously (31). In experiments performed with liver nuclear
extracts, 5 µg of extract was used, the amount of poly(dI-dC) in each
reaction was increased to 8 µg, and DNA-protein complexes were
resolved on 8% polyacrylamide gels. The following double-stranded
oligonucleotides were synthesized and used in the gel mobility assay
(sense strand shown, mutated nucleotides underlined): CYP7-LXRE,
GATCCCTTTGGTCACTCAAGTTCAAGTGGATC; mCYP7-LXRE, GATCCCTTTGGTCACTCAAG
CAAGTGGATC; and DR-4-LXRE,
GATCCCTT
G
TCACTCAAGTTCAAGTGGATC.
For the antibody supershift assays, a pool of monoclonal antibodies was
used. The antibodies were generated by fusing spleenocytes from an SJL
mouse (male, 8 weeks old, Jackson, Bar Harbor, ME) immunized with
histidine-tagged recombinant human LXR protein (amino acids
164-447) with mouse myeloma cells as previously described (32).
Previous work had identified a region of the CYP7A promoter
that interacts with nuclear proteins and contains a motif that closely
resembles known binding sites for members of the nuclear receptor
family (15, 16). This putative response element is composed of a nearly
perfect tandem repeat of the nuclear receptor half-site recognition
sequence AG(G/T)TCA separated by four nucleotides, a so-called DR-4
motif (17). This type of DR-4 response element has been shown to
function as a high affinity binding site for heterodimers between RXR
and the orphan nuclear receptors LXR and LXR
(11-14).
To investigate whether the DR-4 motif in the CYP7A promoter could
function as an LXR response element, we performed a series of gel
retardation assays. First, to verify that the in vitro synthesized receptor proteins were functional, we used an
oligonucleotide in which two nucleotides in the 5 half-site of the
CYP7A direct repeat sequence were mutated to obtain an idealized,
tandem repeat of the AGTTCA motif (Fig. 1A,
DR4-LXRE). Consistent with previous findings (11-14), a
strong complex was formed when either LXR
or LXR
was added
together with RXR, indicating that high affinity DNA binding required
the formation of LXR-RXR heterodimers (Fig. 1B, lane
3). Interestingly, a weaker complex migrating slightly faster than
the LXR-RXR heterodimer was observed when LXR
was used in the
absence of RXR (Fig. 1B, lane 1), suggesting the
weak binding of an LXR homodimeric complex. In experiments performed with an oligonucleotide containing the CYP7A DR-4 element (Fig. 1A), strong binding was observed with both the LXR
-RXR
and LXR
-RXR heterodimers (Fig. 1B, lane 6).
This binding was sequence-specific because an excess of either the
unlabeled CYP7 oligonucleotide or the idealized DR-4-LXRE
oligonucleotide (Fig. 1B, lanes 7-10) competed
efficiently for binding to the probe, but no competition for binding
was seen with an oligonucleotide in which the 3
half-site had been
mutated to a consensus glucocorticoid receptor half-site (mCYP7-LXRE)
(Fig. 1, A and B, lanes 11 and
12). As observed with the DR-4-LXRE, a weak and slightly
faster migrating complex of a potential LXR
homodimer was seen when
LXR
was added alone to the CYP7A direct repeat element (Fig.
1B, lane 4). Based on these data, we refer to the
DR-4 of the CYP7A promoter as the CYP7-LXRE.
LXR is abundantly expressed in the liver (11, 12), the site of CYP7A
expression. To determine whether endogenously expressed LXR
bound to
the CYP7-LXRE, we prepared nuclear extracts from rat liver for use in
gel retardation assays. A strong, shifted complex was observed in
assays performed with the CYP7-LXRE and liver extracts (Fig.
2B, lane 3) that migrated at the
same position as the one obtained with in vitro synthesized
LXR and RXR protein (Fig. 2B, lane 1). A portion
of this complex was supershifted (Fig. 2B, lane
4) upon the addition of a pool of monoclonal antibodies that
specifically recognizes the LXR
but not the LXR
ligand binding
domain (Fig. 2A). An analogous supershifted complex was seen
in experiments performed with these antibodies and in vitro synthesized LXR
and RXR (Fig. 2B, lane 2).
These data show that LXR
is one of the components present in liver
extracts that binds to the CYP7-LXRE.
Oxysterols Activate LXR
The presence of a
binding site for LXR in the promoter of the CYP7A gene, which encodes
the rate-limiting enzyme responsible for the conversion of CH to bile
acids, prompted us to test a comprehensive set of CH precursors and
metabolites including bile acids, oxysterols, and steroids for their
ability to activate LXR or LXR
in cotransfection experiments
(Fig. 3A). The compounds were initially
tested using chimeric receptor proteins containing the ligand binding
domains of either LXR
or LXR
fused to the DNA binding domain of
the yeast transcription factor GAL4 (18). The use of chimeric receptors
allowed us to avoid complications in the interpretation of the results
caused by endogenous nuclear receptors as well as to identify compounds
that mediated their effects through the ligand binding domains of these
orphan receptors. Expression plasmids encoding the chimeric receptors
were cotransfected into CV-1 cells together with a reporter plasmid
containing five copies of the GAL4-binding site upstream of the minimal
tk promoter driving the expression of CAT. All compounds were tested on
the chimeric receptors at a concentration of 10 µM.
As shown in Fig. 3A, neither of the LXR chimeras was
activated in response to CH or its precursors lanosterol and
desmosterol. Interestingly, however, significant activation of both the
LXR and LXR
chimeras was observed in transfected cells treated
with the oxysterols 20(S)-, 22(R)-, and
24(S)-hydroxycholesterol (Fig. 3A). 25- and
27-hydroxycholesterol had relatively little or no effect on either LXR
chimera (Fig. 3A). Notably, activation of the LXRs was more
efficient with the naturally occurring 22(R)- and
24(S)-hydroxycholesterol enantiomers than with the synthetic 22(S)- and 24(R)-hydroxycholesterol
stereoisomers. We note that while this manuscript was in preparation,
several of these oxysterols were shown to activate LXR
(19).
24-Keto-cholesterol was also an efficacious activator of both LXRs
(Fig. 3A). In contrast, no activation of either LXR
or
LXR
was seen in the presence of additional, downstream intermediates
in the steroid hormone biosynthetic pathway or in the presence of
testosterone or progesterone (Fig. 3A).
7
-Hydroxycholesterol, its bile acid derivatives chenodeoxycholic acid and cholic acid, or bile salts also failed to activate the LXR
chimeras (data not shown). In addition, steroids with the 3
-hydroxyl
configuration were inactive (data not shown).
In addition to the various hydroxycholesterol derivatives, we also
examined the activity of 24(S),25-epoxycholesterol (Fig. 3B). 24(S),25-Epoxycholesterol is derived from a
shunt in the mevalonate pathway in which squalene epoxide, rather than
undergoing cyclization to lanosterol, is instead metabolized to
24(S),25-epoxycholesterol (20).
24(S),25-epoxycholesterol has been identified in cultured cells (21) and in tissue homogenates prepared from human and rat livers
(22, 23, 28). Because 24(S),25-epoxycholesterol is derived
from a biosynthetic pathway different from that of any of the other
oxysterols, it has been proposed that this epoxide might serve as a
signaling molecule in the feedback regulation of CH biosynthesis (23).
Indeed, we found that 24(S),25-epoxycholesterol was an
efficacious activator of both LXRs (Fig. 3A). Taken
together, the results of these transfecton studies indicate that the
optimal structural requirement for LXR activation is a 3-sterol with an oxygen substituent at C-24 capable of forming a hydrogen bond.
We next wished to determine whether the potencies of these oxysterols in the activation of the LXRs were consistent with their known abundance in vivo. Three of these compounds are particularly abundant in tissue extracts. 24(S)-hydroxycholesterol is present in brain extracts at concentrations of roughly 100 µM. Although its physiological relevance is not known, these high concentrations have led to 24(S)-hydroxycholesterol being termed "cerebrosterol." Although circulating levels of 24(S)-hydroxycholesterol are low in adults (~50 nM), this concentration can be elevated as much as 10-fold in children (24). 24(S),25-Epoxycholesterol has been isolated from human or rat livers at concentrations estimated to be 1-5 µM (22, 23, 28). Finally, 22(R)-hydroxycholesterol is present in adrenal extracts at micromolar concentrations. Other oxysterols are present in the circulation or in tissue extracts at concentrations that are nanomolar or lower (25).
We performed dose response analysis using expression plasmids for
full-length LXR or LXR
. Reporter constructs were generated that
contained four copies of either the CYP7-LXRE or the idealized DR-4-LXRE driving CAT gene expression. In preliminary experiments performed with 10 µM
24(S),25-epoxycholesterol, we found that LXR
induced
expression of both the CYP7A-LXRE-CAT and DR-4-LXRE-CAT reporter
constructs but that LXR
only efficiently induced expression of the
DR-4-LXRE-CAT reporter construct (data not shown). We speculate that
LXR
may not bind to the CYP7A-LXRE with sufficient affinity to
activate reporter expression. Subsequent dose response analyses with
LXR
and LXR
were performed with the CYP7A-LXRE-CAT and DR-4-LXRE-CAT reporter constructs, respectively. LXR
and LXR
responded to 24(S),25-epoxycholesterol with EC50
values of 7.5 and 1.5 µM, respectively (Fig.
4A). 24(R),25-Epoxycholesterol, which has not been found to occur naturally, was significantly less
active with an EC greater than 10 µM on both LXRs.
24(S)-Hydroxycholesterol had an activation profile similar
to that of 24(S),25-epoxycholesterol on the LXRs, activating
LXR
and LXR
with EC50 values of 7 and 1.5 µM, respectively (Fig. 4B). The synthetic
isomer 24(R)-hydroxycholesterol was at least 1 order of
magnitude less active. 20(R)-Hydroxycholesterol, 22(R)-hydroxycholesterol, and 24-ketocholesterol displayed
EC50 values for LXR activation that were greater than 10 µM (data not shown).
Based upon these data, we suggest that
24(S),25-epoxycholesterol, which is abundant in liver, and
24(S)-hydroxycholesterol, which is abundant in brain, may
function as endogenous activators of LXR and LXR
. We note that
both LXRs are expressed in the liver. Interestingly, Northern analyses
have demonstrated that LXR
is expressed in the brain (14). More
detailed in situ studies have shown that LXR
is broadly
expressed in fetal brain and that its expression becomes more
restricted in postnatal and adult brains (26). Taken together, these
findings support the suggestion that
24(S)-hydroxycholesterol may serve in the development and function of the brain via interactions with LXR
(22).
In summary, we have demonstrated that the LXRs are activated by the oxysterols 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at concentrations consistent with those found in tissues. It is well known that oxysterols suppress de novo CH biosynthesis as well as cellular uptake of CH (1). However, oxysterols have a number of other biological effects including marked effects on DNA synthesis and cell growth and proliferation (25). Our data suggest that some of these effects may be mediated through the LXRs. It is interesting that the nuclear receptor family member most closely related to the LXRs is the insect ecdysone receptor (12, 13, 15). Ecdysone is a sterol hydroxylated at the 22 and 25 positions that functions as a temporal signal to coordinate tissue-specific morphogenetic changes during insect development (27). The use of nuclear receptor pathways as a means to transduce the effects of oxygenated sterols thus appears to have been conserved throughout a large part of evolution, from insects to man.